PMID- 11884248 TI - Reporting of measures of accuracy in systematic reviews of diagnostic literature AB - Abstract | Background | There are a variety of ways in which accuracy of clinical tests can be summarised in systematic reviews. Variation in reporting of summary measures has only been assessed in a small survey restricted to meta-analyses of screening studies found in a single database. Therefore, we performed this study to assess the measures of accuracy used for reporting results of primary studies as well as their meta-analysis in systematic reviews of test accuracy studies. Methods | Relevant reviews on test accuracy were selected from the Database of Abstracts of Reviews of Effectiveness (1994 --2000), which electronically searches seven bibliographic databases and manually searches key resources. The structured abstracts of these reviews were screened and information on accuracy measures was extracted from the full texts of 90 relevant reviews, 60 of which used meta-analysis. Results | Sensitivity or specificity was used for reporting the results of primary studies in 65/90 (72%) reviews, predictive values in 26/90 (28%), and likelihood ratios in 20/90 (22%). For meta-analysis, pooled sensitivity or specificity was used in 35/60 (58%) reviews, pooled predictive values in 11/60 (18%), pooled likelihood ratios in 13/60 (22%), and pooled diagnostic odds ratio in 5/60 (8%). Summary ROC was used in 44/60 (73%) of the meta-analyses. There were no significant differences in measures of test accuracy among reviews published earlier (1994 --97) and those published later (1998 --2000). Conclusions | There is considerable variation in ways of reporting and summarising results of test accuracy studies in systematic reviews. There is a need for consensus about the best ways of reporting results of test accuracy studies in reviews. PMID- 11884248_Background TI - AB - The manner in which accuracy of clinical tests is mathematically summarised in the biomedical literature has important implications for clinicians. Appropriate accuracy measures would be expected to sensibly convey the meaning of the study results with scientifically robust statistics without exaggerating or underestimating the clinical significance of the findings. Lack of use of appropriate measures may lead authors of primary accuracy studies to draw biased conclusions. In systematic reviews of test accuracy literature, there are many ways of synthesising results from several studies, not all of which are considered to be scientifically robust. For example, measures such as sensitivity and specificity commonly used in primary studies are not considered suitable for pooling separately in meta-analysis. Variations in reporting of summary accuracy and use of inappropriate summary statistics may increase the risk of misinterpretation of clinical value of tests. A recent study evaluated a small sample of meta-analytical reviews of screening tests to demonstrate the variety of approaches used to quantitatively summarise accuracy results. This study confined itself to a limited Medline search. It exclusively examined meta-analytical studies so reviews not using quantitative synthesis were excluded. It did not look at accuracy measures used to report results of primary studies separately from those used for meta-analyses. In order to address these issues, we undertook a comprehensive search to survey systematic reviews (with and without meta-analysis) of test accuracy literature to assess the measures used for reporting results of included primary studies as well as their quantitative synthesis. PMID- 11884248_Methods TI - AB - We manually searched for relevant reviews in the Database of Abstracts of Reviews of Effectiveness (DARE). In order to limit the impact of human error inherent in manual searching, we complemented it with electronic searching. DARE was searched electronically with word variants of relevant terms (diagnostic, screening, test, likelihood ratio, sensitivity, specificity, positive and negative predictive value) combined using OR. From 1994 to 2000 DARE has identified 1897 reviews of different types by regular electronic searching of several bibliographic databases, hand searching of key major medical journals, and by scanning grey literature (search strategy and selection criteria can be found at ). The structured abstracts of these reviews were screened independently by the authors to identity systematic reviews of test accuracy. The full texts were obtained of those abstracts judged to be potentially relevant. Reviews addressing test development and diagnostic effectiveness or cost effectiveness were excluded. Any disagreements about review selection were resolved by consensus. Information from each of the selected reviews was extracted for the measures of test accuracy used to report the results of the primary studies included in the review. If a meta-analysis was conducted, information was also extracted for the summary accuracy measures. The various accuracy measures are shown in Table . We sought the following in the primary studies: sensitivity or specificity, predictive values, likelihood ratios and diagnostic odds ratio. For meta-analysis, we sought the summary measures pooling the above results and summary receiver operating characteristics (ROC) plot or values. All extracted data were double-checked. We divided the reviews into two groups arbitrarily according to time of publication; one group covering the period 1994 --97 (50 reviews) and another covering 1998 --2000 (40 reviews). This allowed us to assess whether there were any significant differences in measures being used to report test accuracy results among reviews published earlier and those published later. As the approaches to summarising results are not mutually exclusive, we evaluated and reported the most commonly used measures and their most common combinations. We used chi-squared statistical test for comparison of differences between proportions. Table 1 | Measures of accuracy of dichotomous test results PMID- 11884248_Results TI - AB - Of the abstracts available in DARE, 150 were considered to be potentially relevant. Excluding reviews that addressed test development and diagnostic effectiveness or cost, 90 reviews of test accuracy were left for inclusion in our survey. There were 45 reviews of dichotomous test results, 42 reviews of continuous results dichotomised by the original authors, and 3 reviews that contained both result types. Meta-analysis was used in 60/90 (67 %) reviews, 50 in 1994 --97 and 40 in 1998 --2000. (See : BMC_IncludedRefList_04032002 for a complete listing of the 90 reviews included in our study). As shown in Table , sensitivity or specificity was used for reporting the results of primary studies in 65/90 (72%) reviews, predictive values in 26/90 (28%), and likelihood ratios in 20/90 (22%). For meta-analysis, independently pooled sensitivity or specificity was used in 35/60 (58%) reviews, pooled predictive values in 11/60 (18%), pooled likelihood ratios in 13/60 (22%), and pooled diagnostic odds ratio in 5/60 (8%). Summary ROC was used in 44/60 (73%) of the meta-analyses. There were no significant differences between reviews published earlier and those published later as shown in Table . Table 2 | Measures of test accuracy reported in review of diagnostic literature (1994 --2000) PMID- 11884248_Discussion TI - AB - Our study showed that sensitivity and specificity remain in frequent use, both for primary studies and for meta-analyses over the time period surveyed. Sensitivity and specificity are considered inappropriate for meta-analyses, as they do not behave independently when they are pooled from various primary studies to generate separate averages. In our survey, separate pooling of sensitivities or specificity was used frequently in meta-analyses where summary ROC would have been more appropriate. . Our findings about reporting of summary accuracy measures in meta-analyses are different to those reported previously. We found a higher rate of use of summary ROC, though use of independent summaries of sensitivity, specificity and predictive values were similar. These differences may be due to differences in searching strategies (databases and time frames) and selection criteria. Our search was more recent and comprehensive, using DARE, which has covered seven different databases (Medline, CINAHL, BIOSIS, Allied and Alternative Medicine, ERIC, Current Contents clinical medicine and PsycLIT), and hand-searched 68 peer-reviewed journals and publications from 33 health technology assessment centres around the world since February 1994. Moreover, as we did not restrict our selection to meta-analytical reviews only, we were able to examine reviews summarising accuracy results of primary studies without quantitative synthesis, which constituted 33% (30/90) of our sample. Therefore, compared to the previous publication on this topic, our survey provided a broader and more up-to-date overview of the state of reporting of accuracy measure in test accuracy reviews. PMID- 11884248_Conclusions TI - AB - The use of inappropriate accuracy measures has the potential to bias judgement about the value of tests. Of the various approaches to reporting accuracy of dichotomous test results, likelihood ratios are considered to be more clinically powerful than sensitivities or specificities. Crucially, it has been empirically shown that authors of primary studies may overstate the value of tests in the absence of likelihood ratios. There is also evidence that readers themselves may misinterpret test accuracy measures following publication. It is conceivable that the problem of inconsistent usage of test accuracy measures in published reviews, as found in our survey, may contribute to misinterpretation by clinical readership. The reason for variation in reported accuracy measures may, in part, be attributed to a lack of consensus regarding the best ways to summarise test results. It is worth noting that despite authoritative publications about appropriate summary accuracy measures in the past, (we have only quoted a few references) inconsistent and inappropriate use of summary measures has remained prevalent in the period 1994 --2000. Our paper highlights the need for consensus to support change in this field of research. PMID- 11884248_Competing interest TI - AB - None declared PMID- 11914161 TI - A randomised controlled trial of a patient based Diabetes recall and Management system: the DREAM trial: A study protocol [ISRCTN32042030] AB - Abstract | Background | Whilst there is broad agreement on what constitutes high quality health care for people with diabetes, there is little consensus on the most efficient way of delivering it. Structured recall systems can improve the quality of care but the systems evaluated to date have been of limited sophistication and the evaluations have been carried out in small numbers of relatively unrepresentative settings. Hartlepool, Easington and Stockton currently operate a computerised diabetes register which has to date produced improvements in the quality of care but performance has now plateaued leaving substantial scope for further improvement. This study will evaluate the effectiveness and efficiency of an area wide 'extended' system incorporating a full structured recall and management system, actively involving patients and including clinical management prompts to primary care clinicians based on locally-adapted evidence based guidelines. Methods | The study design is a two-armed cluster randomised controlled trial of 61 practices incorporating evaluations of the effectiveness of the system, its economic impact and its impact on patient wellbeing and functioning. PMID- 11914161_Background TI - AB - Delivering care to people with diabetes | There is broad, international agreement over what constitutes high quality health care for people with diabetes . This will be enshrined in a National Service Framework for people with diabetes, due in summer 2002. However, in the face of poor current performance the most efficient method of delivering care remains unclear . Following a 1994 systematic literature review suggesting structured care improved patient care, an editorial in the British Medical Journal concluded that more evaluative research was needed before widespread adoption of any of the models could be recommended . A subsequent systematic review of routine surveillance of patients with diabetes by Griffin and Kinmonth concluded "Computerised central recall, with prompting for patients and their family doctors, can achieve standards of care as good or better than hospital outpatient care, at least in the short term. The evidence supports provision of regular prompted recall and review of people with diabetes by willing general practitioners and demonstrates that this can be achieved, if suitable organisation is in place'. However, the evidence base on which these conclusions are based is limited in several ways. Firstly there are only five randomised controlled trials (RCTs) involving 1058 patients. All of these studies are 'patient randomised" trials, thus potentially under-estimating the effectiveness of the intervention (see Study Design). They were all evaluating more or less selected patients and general practices and none of them were explicitly evaluating a UK National Health Service (NHS) service area wide intervention. Only one of the four UK based studies evaluated patient based outcomes and included an economic assessment and this study only involved patients from three general practices . Thus, the effectiveness of an area wide, patient focussed, structured recall and management system (in terms of process of care, patient outcome and economic impact) remains unknown. The current system | The current computerised diabetes management system runs in Hartlepool, Easington and Stockton, three Primary Care Group (PCG) areas, in the Northern and Yorkshire Region. It was introduced to all 36 general practices in Hartlepool and Easington Districts in mid-1995. Stockton (25 practices) agreed to join the system in 1999 and it was operational there by October 2000. There are three key components to the current system: 1. A central register of patients with diabetes. 2. A structured minimum dataset to be completed and returned to the central register. 3. The provision of both patient specific and aggregated data to both patients and clinicians. The system (developed by Westman Medical Software) allows three methods of collection of data at each contact with a patient with diabetes who is registered on the database. Two methods use a standard form completed by clinicians to collect data concordant with the UK minimum data set . Within secondary care, forms are completed at every new patient or annual review. In primary care, forms are completed by the practice nurse (usually) or general practitioner, either opportunistically or at practice diabetic clinics. In both cases, the completed data forms are sent to the Diabetes Register Facilitator for data entry. Thirdly, the hospital laboratory provides a monthly download of laboratory test details (e.g. HbA1c). A patient can be identified as having diabetes and added to the register by any permutation of one or more of these three routes. Feedback of individual patients' data, including review status, is provided to general practices quarterly. This feedback is 'passive' in that it does not explicitly prompt either patients or doctors as to required actions. Audit packages within the software can audit on every variable collected. District wide audit is provided on anonymised aggregated data; individual practice audits (with comparisons to other practices) are provided to participating practices at least annually. Feedback of the data to the patient (for hospital patients only) is by a patient information sheet and to the GP as a standardised letter. A Diabetes Register Facilitator co-ordinates and updates the register. A steering group composed of GH, the Diabetes Register Facilitator and representatives of the PCGs and patients, oversees the register and deals with issues such as confidentiality. Impact of the system to date | Measures of the impact of the system to date relate only to Hartlepool, Easington and Stockton. The main impact on patient registration was in its first 12 to 18 months of operation: during 1995, 747 patients were registered on the system (0.4% prevalence) which had increased to 3867 (1.8% prevalence) by the end of 1996. The increase in registration has stabilised since then, reaching 4324 (2% prevalence) by 1999. During 1999, 70% of registered patients attended a clinic; 52% had their feet examined and 51% had their eyes examined. Seventy three per cent had an HbA1c result recorded and 69% a blood pressure measurement. These figures are similar to those reported by other centres using the same system . The need for an extended system | Recording of clinical measures increased during the first few years of operation of the system but began to plateau more recently (for example, 50% of patients had an HbA1c recorded during 1996, compared to 60% in 1997 and 63% in 1998). This plateauing of performance has been reported by others . We believe that this is due to a lack of coordination (patients being lost to follow up) and lack of prompting of clinicians to deliver appropriate clinical interventions. Furthermore, given that most patients with diabetes are primarily seen in primary care the greatest potential impact is from optimising and extending the system in primary care. In order to address these shortcomings the additional key components, over and above those already in the system, will be: 1. Locally adapted evidence based guidelines for the management and follow up of patients with diabetes. 2. Automated prompts to patients and primary care clinicians that a review consultation is necessary. 3. A structured management sheet (including patient specific management suggestions based on (1)). 4. An enhanced monitoring system to follow up reasons for non-attendance from both patients and clinicians and to re-schedule appointments, based on nonreturn of a completed management sheet. 5. Patient feedback for patients in primary care. There is some limited supportive trial evidence for these developments, although the existing studies involved small sample sizes and may not be generalisable to the NHS . In evaluating the system with these extended features this study will also address the design shortcomings of previous studies of shared care in diabetes . It will be tailored to each practice, PCG defined areas will be studied, rather than an unrepresentative sample of general practices; and the system will be transparent and replicable in other areas. PMID- 11914161_Methods TI - AB - Design of the study | The study design is a pragmatic two-arm cluster randomised controlled trial. The unit of randomisation will be the general practice. Simple patient randomised trials are rightly considered the most robust method of assessing most health care innovations . This design, however, cannot be regarded as the gold standard for evaluating systematic approaches to chronic disease management, an essentially behavioural field of research . If both intervention and control patients were to be cared for within the same practice there is the risk that the management of control patients would be influenced by the practitioners knowledge of the care of intervention patients. This would result in an underestimation of the effect of the intervention . Therefore, practices rather than patients are the appropriate unit of randomisation and analysis. As the current system has been in place for different lengths of time within the three participating PCGs, we will stratify the randomisation by PCG. Randomisation will be performed by a statistician independent of the research team using computer generated numbers to avoid allocation bias . Study setting and recruitment of practices | The study will be based in the general practices of the three PCGs of Easington, Hartlepool and Stockton. Since the recent merger of Hartlepool and North Tees Acute Trusts all three PCGs are now exclusively served by one secondary care diabetes service (and thus the one diabetes register). GH is the lead clinician for diabetes services in the new Trust. The 61 general practices in the three PCGs constitute the target practices for the study and we will attempt to recruit all practices. The PCG diabetes leads or the PCG clinical governance leads in all three PCGs have provided letters confirming their support for the project. We do not envisage major difficulties with recruitment, given the need to agree local guidelines as part of the process involved in the Trust merger, the likely requirements in the forthcoming National Service Framework for diabetes, and the 100% practice coverage with the current diabetes system. We will (through the PCGs) write to all practices, giving information about the project to the senior partner or diabetes lead and practice manager of practices. Practices will be invited to opt out if they do not wish to be included in the study -- this is an approach we have used successfully before. The PCG diabetes lead, clinical governance lead and GH will be co-signatories of this letter. If practices do decline we will collect data on characteristics of non-participating practices to assess the impact on the generalisability of the trial's findings. Finally, if there are significant problems with recruitment, there are other practices which could be approached in a nearby PCG (South Tyneside) which uses the same software for its diabetes register. Details of the intervention | Local guidelines and management prompts | A guideline development group will be established to develop local guidelines for the management of diabetes, based upon available evidence based guidelines (Scottish Intercollegiate Guidelines Network (SIGN, 1996, 1997a, 1997b, 1997c), and Effective Care Bulletins . They will also use the forthcoming national diabetes guidelines as these become available. The group will be multidisciplinary and contain primary and secondary care doctors and nurses, patients and the Diabetes Register Facilitator . The group will define review periods for specified patient groups (e.g. patients with diabetes satisfactorily controlled on diet alone should be reviewed every 12 months), referral criteria for patients moving from primary to secondary care and back and simple decision rules for the management prompts. These would be of two types. The first would prompt for actions to be performed and only require their performance to be documented (e.g. asking for a foot examination to be performed in a patient who does not have a recorded foot examination). The second would be more complex and suggest alterations to clinical management on the basis of data in the database (e.g. patients with persistently raised blood pressure should have their anti-hypertensive medication increased). These decision rules will be integrated into the recall and management system. Running the system | The proposed enhancements to the system are designed to require the primary care team to perform no additional work over and above the current configuration. The current database has a patient identifier, a minimum dataset and retrieval systems to support the structured recall of patients. Westman Medical Software has agreed to amend the system as required. A 'circle of information exchange' will be established between the participating general practices and the database. The local guidelines will be used to adapt the current centralised database, along with the practices' preferred method of following up patients (for example, within consultations in routine surgeries or within special clinics). The central database system will identify when patients are due for review (based upon the local guidelines) and will generate a letter to the patient asking them to make an appointment for a review consultation. Patient information or educational materials could be included with the letter. At the same time, the central database will generate a letter to the practice stating that the patient should be making a review appointment in the near future. The letter to the practice will include a management sheet (to be held in the patient's record) to capture an agreed minimum data set to be collected during the consultation. This management sheet will also contain the relevant prompts (as described above). When the patient is seen in the practice, the primary care professional (currently this is usually done by the practice nurse) will complete the management sheet and return a copy for entry onto the central register within a designated period of time. This circle of information is broken if the patient does not visit the general practice as planned or the general practice does not return the management sheet to the central register. If this happens, the central register would alert the Diabetes Register Facilitator who will ascertain the reason for failure and take appropriate action, (e.g. send a reminder to the patient, prompt the practice to return the management sheet). A range of educational activities will be provided for intervention practices, as part of the usual local structures for contact with practices, with some additions, These will include: distribution of information about the trial in local newsletters; meetings with practice clinical governance leads; evening meetings for practice nurses (with small group discussion of the practical implications for intervention practices); and a telephone meeting with the practice diabetes lead (usually the practice nurse) in each intervention practice. Practices in the control arm will continue to receive the recall system as currently configured. Logistical considerations | From the prevalence of patients with diabetes on the current register, there will be about 7500 patients on the system if 61 practices are recruited. Half of these will be in intervention practices. On current patterns of usage, we anticipate there being the need for 1.5 recalls per annum per patient on the register, resulting in about 6000 recalls per year for the intervention group. Assuming a 40 week working year, the system will need to dispatch, receive and process about 150 forms per group per week. Identification of patients | Patients for the structured recall and management system are already identified on the Hartlepool and North Tees database. As some practices have children registered on the system, who are under the care of an exclusively secondary care adolescent service, an age limit of 18 years or over will be set for inclusion. Practices will be asked to check lists of their patients on the database regularly throughout the study. The central database will remove patients from the recall system who are known to have died or moved away. Patient consent | Patients have already consented, or are being consented, to their data being held within the current diabetes register. The study will involve no extra 'routine' data being collected, and this data will be anonymised before being sent for analysis; all data held for analysis will be held in accordance with the Data Protection Act. For the patient-based questionnaire study, we will seek additional patient consent to complete one survey. The three relevant Local Research Ethics Committees have approved the trial. Data collection | The main study outcome measures will be rates of performance of process of care and the patient based measures of functional and psychosocial wellbeing. Data will be collected for 15 months after the start of the intervention. Fifteen months was chosen to allow for patients who are reviewed every 12 months but fail to attend on initial invitation. Process of care variables | Process of care variables will be collected via the computerised database. The exact data to be collected will be determined by both the current content of the database and the guidelines but will include such data items as rates of attendance at clinics and annual reviews, conduct of eye and feet examinations, performance of investigations and prescribing. We will also collect data on clinical measures (e.g. HbA1c, and blood pressure levels). Outcome of care measures | Outcome of care data will be collected, by postal questionnaire, 15 months after commencement of the study. A portfolio of validated and responsive generic and disease specific instruments will be used to measure functional and psychosocial variables that will be potentially influenced by the intervention. These will include: i) The SF36 health status profile which we will use to generate Mental (MCS) and Physical Component Summary Scales (PCS) . ii) The Newcastle Diabetes Symptoms Questionnaire . iii) The Bradley Treatment Satisfaction Questionnaire . Patient costs questions will be developed by the study health economist. We have successfully used such packages of questionnaires within trials before and have achieved response rates in excess of 70% in similar surveys in this region. . Sample size considerations | On the basis of previous work we have made the following assumptions. The mean number of patients per practice for whom we will be able to collect process data will be 30 and the ICC (a measure of the lack of independence of responses from patients from the same practice) calculated from our local data is 0.14 for measures of process (whether a blood pressure measurement and whether an HbA1c measurement has been recorded in a 12 month period). Standard methods for determining the sample size requirements for a cluster randomised trial indicate that we need 60 practices to detect a difference of 15% (42.5% v 57.5%) with 80% power assuming a significance level of 5%. Assessment of outcome of care will be based on health status scales such as the SF-36. Previous work has shown that this type of intervention is likely to produce an effect size of approximately 0.25 in such measures and that the ICCs for such measures will be approximately 0.07. The most efficient study design (that minimises the number of patients required) is one that makes use of all the available practices. A sample of 27 patients from each of 61 practices will give us 85% power to detect an effect size of 0.25 assuming a significance level of 5%. With a predicted response rate of approximately 70% (based on our experience in the COGENT study ) after two reminders, our starting sample size will need to be 2379 patients (approximately 39 patients per practice). Principles of data analysis | Analysis will be by intention to treat. Multilevel modelling (using the MlwiN package ) will be used to take into account the clustering of patients within practices . Both binary variables (when a process was undertaken or not) and continuous variables (such as the physical health component of the SF-36) can be analysed using these techniques. For both types of variable, variation between practices will be fitted as a random effect and the difference between intervention and control practices will be fitted as a fixed effect. In the case of binary variables, a logit link function will be used. Economic evaluation | The economic impact of implementing the new structured recall and management system will be evaluated in terms of the marginal costs of adapting and running the system; the costs of developing and disseminating the guidelines; the educational activities for intervention practices; the implications for the use of health care services; and the costs to the patients and their carers. The benefits will be measured as described earlier on in the clinical study. The estimation of health service resource use will relate to diabetes-specific clinical visits, tests, investigations, and procedures. This data will be routinely collected as part of the management system implementation and subsequent costing, using health service pay and price data, will be undertaken using a mixed approach based on micro-costing and gross-costing methods . Use of drugs, referrals to secondary care and the impact of the intervention on the change of use of patients' and their carers' time will also be monitored through postal questionnaires at the end of the follow-up period. A sensitivity analysis will be undertaken to test the robustness of the results to the uncertainty not related to sampling variations and to enhance the generalisability of the results . We are aware that the costs of the system might be balanced only in the longer term against the cost savings related to averted complications . However, the assessment of the benefits in terms of final outcomes (e.g lives saved, or QALYs) and long term costs is beyond the objective of the present study. PMID- 11914161_Competing interests TI - AB - None declared PMID- 11914161_Pre-publication history TI - AB - The pre-publication history for this paper can be accessed here: PMID- 11914164 TI - Inter-rater agreement in the scoring of abstracts submitted to a primary care research conference AB - Abstract | Background | Checklists for peer review aim to guide referees when assessing the quality of papers, but little evidence exists on the extent to which referees agree when evaluating the same paper. The aim of this study was to investigate agreement on dimensions of a checklist between two referees when evaluating abstracts submitted for a primary care conference. Methods | Anonymised abstracts were scored using a structured assessment comprising seven categories. Between one (poor) and four (excellent) marks were awarded for each category, giving a maximum possible score of 28 marks. Every abstract was assessed independently by two referees and agreement measured using intraclass correlation coefficients. Mean total scores of abstracts accepted and rejected for the meeting were compared using an unpaired t test. Results | Of 52 abstracts, agreement between reviewers was greater for three components relating to study design (adjusted intraclass correlation coefficients 0.40 to 0.45) compared to four components relating to more subjective elements such as the importance of the study and likelihood of provoking discussion (0.01 to 0.25). Mean score for accepted abstracts was significantly greater than those that were rejected (17.4 versus 14.6, 95% CI for difference 1.3 to 4.1, p = 0.0003). Conclusions | The findings suggest that inclusion of subjective components in a review checklist may result in greater disagreement between reviewers. However in terms of overall quality scores, abstracts accepted for the meeting were rated significantly higher than those that were rejected. PMID- 11914164_Background TI - AB - Interest in the peer review process and research aimed at determining the method of obtaining the best quality reviews has grown in recent years. Checklists have been developed that aim to guide reviewers when assessing the quality of papers, but little evidence exists concerning the extent of agreement between two referees when evaluating the same paper. In addition, little is known about which dimensions of a checklist are likely to result in greater agreement between referees. There were two aims of this study: (1) to examine inter-rater agreement of the quality of abstracts submitted to a primary care research conference (Annual Meeting of the South West Association of University Departments of General Practice, Exeter 2000, UK), and (2) to compare the scores of abstracts accepted and rejected for the meeting. PMID- 11914164_Materials and Methods TI - AB - Abstracts were anonymised and scored using a structured assessment comprising seven categories: (1) importance of the topic (2) originality (3) overall quality of the study design (4) appropriateness of the design used (5) achievement of aim (6) contribution to academic primary care (7) likelihood of provoking discussion. For comparison purposes, we have classified the assessment of categories 1, 2, 6 and 7 as more 'subjective' in nature, and categories 3, 4 and 5 as more 'objective'. Between one (poor) and four (excellent) marks were awarded for each category, giving a maximum possible score of 28 marks. Every abstract was assessed independently by two referees (AM and AG). Agreement between referees was assessed using intraclass correlation coefficients (ICC), a chance corrected measure of agreement. The ICC indicates perfect agreement only if the two assessments are numerically equal and is preferable to the more usual (Pearson) correlation coefficient. The crude ICC is lowered by any systematic differences between referees' scores. In terms of a plot of the two referees' scores, a line with a non-zero intercept will further lower the ICC irrespective of any disagreement, represented by deviation of the slope of the line away from unity and scatter around the line. In a further analysis, this effect was investigated by subtracting the mean difference for each component from the higher of the two referees' scores. The ICCs were then recalculated, giving estimates of agreement corrected for both systematic differences and chance. There are no universally applicable standard values for the ICC that represent adequate agreement, but the following convention is used here to aid interpretation: ICC <0.20 'slight agreement'; 0.21 --0.40 'fair agreement'; 0.41 --0.60 'moderate agreement'; 0.61 --0.80 'substantial agreement'; >0.80 'almost perfect agreement'. Scores from referees from three different institutions were summed to give each abstract an overall score. Abstracts were ranked by this overall score and the top 45 were accepted for oral presentation at the meeting. Of the 52 abstracts refereed by AM and AG, mean total scores of those accepted and rejected for the meeting were compared using an unpaired t test. PMID- 11914164_Results TI - AB - Chance corrected agreement between the two referees' scores measured using crude ICCs was greater for the three components relating to design and execution of the study (Table : items 3 to 5) compared to those relating to more subjective elements of the abstract (Table : items 1, 2, 6, 7). After adjustment for systematic differences in referees' scores, ICCs for items 3 to 5 remained highest, demonstrating fair to moderate agreement. Table 1 | Inter rater agreement between two referees for 52 abstracts submitted for a primary care research conference A total of 76 abstracts were submitted for the meeting. Of 52 received by the authors for assessment, 26 were accepted for oral presentation . Abstracts accepted for the meeting had a significantly higher mean score than those that were rejected (95% CI for difference 1.3 to 4.1, p = 0.0003) . Table 2 | Summary statistics of abstracts accepted and rejected for oral presentation at a primary care research conference PMID- 11914164_Discussion TI - AB - This study has shown that when using a structured assessment form, two independent reviewers were more likely to agree on design or methodological components of a checklist than on subjective components of abstracts submitted for an annual research meeting. Abstracts accepted for the meeting had significantly higher total scores, but overlapped considerably with rejected abstracts. This was due to acceptance for the meeting being determined by an overall aggregate of scores awarded by referees from three institutions. While the subject of inter-reviewer agreement on different components of a checklist is relatively under-researched, some previous studies offer support for our finding that agreement is better when reviewers can be more objective in their assessments. Among a group of reviewers asked to rate a series of review articles, agreement on scientific quality of the papers was very high (60% of ICCs > 0.7) both within and between groups with varying levels of research training and expertise. All 10 dimensions of the checklist that reviewers rated could be regarded as objective. Divergent reviewers have been identified in a study comparing an overall rating score that indicated a recommendation to publish rather than individual dimensions of a review checklist. This study does have limitations. Importantly, we assessed agreement between only two reviewers on a relatively small number of abstracts. This could be addressed by having more abstracts assessed by a greater number of reviewers. However the study was conducted pragmatically within the time and administrative constraints of a small annual scientific meeting rather than submissions to a journal over an extended period. Another limitation is that the reviewer checklist was constructed prior to conceiving the study. If future meetings are to be used to investigate the content of structured reviewer assessments, such checklists should be constructed with specific hypotheses in mind. Characteristics associated with good peer review are age under 40 years and training in epidemiology or statistics, characteristics that applied to both reviewers in the present study. Structured assessment forms that ask the reviewer for their opinion of a paper's interest, originality or likelihood of provoking discussion may be more likely to result in scores that reflect the reviewer's own research interests. This is not necessarily a criticism -- it is perhaps only natural that individuals will differ in their opinions of how interesting they find, and think others will find, a particular paper. It is interesting that the two components with the lowest agreement, importance of the topic and originality of the study, both require more knowledge about a specific subject area than either of the other two subjective questions. Journal editors and meeting organisers should be aware that including subjective components in review checklists may result in greater disagreement between reviews. PMID- 11914164_Conclusions TI - AB - This study provides some evidence that inclusion of subjective components in a review checklist may result in greater disagreement between reviewers. An interesting area for further research would be to investigate the effects of attaching different weights to subjective and objective components of a checklist, or to exclude subjective components altogether from overall quality scores and simply use them a guide to acceptance or rejection. PMID- 11914164_Competing interests TI - AB - None declared. Figure 1 | Difference between referees' scores versus mean score Difference between referees' scores versus mean score PMID- 11914164_Pre-publication history TI - AB - The pre-publication history for this paper can be accessed here: PMID- 11943069 TI - The Caenorhabditis elegans Y87G2A.14 Nudix hydrolase is a peroxisomal coenzyme A diphosphatase AB - Abstract | Background | The number of Nudix hydrolase family members varies widely among different organisms. In order to understand the reasons for the particular spectrum possessed by a given organism, the substrate specificity and function of different family members must be established. Results | The Y87G2A.14 Nudix hydrolase gene product of Caenorhabditis elegans has been expressed as a thioredoxin fusion protein in Escherichia coli and shown to be a CoA diphosphatase with catalytic activity towards CoA and its derivatives. The products of CoA hydrolysis were 3',5'-ADP and 4'-phosphopantetheine with Km and kcat values of 220 muM and 13.8 s-1 respectively. CoA esters yielded 3',5'-ADP and the corresponding acyl-phosphopantetheine. Activity was optimal at pH 9.5 with 5 mM Mg2+ and fluoride was inhibitory with a Ki of 3 muM. The Y87G2A.14 gene product has a potential C-terminal tripeptide PTS1 peroxisomal targeting signal -- SKI. By fusing a Y87G2A.14 cDNA to the C-terminus of yeast-enhanced green fluorescent protein, the enzyme appeared to be targeted to peroxisomes by the SKI signal when transfected into yeast cells. Deletion of SKI abolished specific targeting. Conclusions | The presence of related sequences with potential PTS1 or PTS2 peroxisomal targeting signals in other organisms suggests a conserved peroxisomal function for the CoA diphosphatase members of this group of Nudix hydrolases. PMID- 11943069_Background TI - AB - The Nudix hydrolase family comprises enzymes that hydrolyse predominantly the diphosphate (pyrophosphate) linkage in a variety of nucleoside triphosphates, dinucleoside polyphosphates, nucleotide sugars and nucleotide cofactors having the general structure of a nucleoside diphosphate linked to another moiety, X . They are found in archaea, eubacteria, animal, plants, and fungi and all possess the Nudix box sequence signature motif Gx5Ex5 [UA]xREx2EExGU (where U is an aliphatic hydrophobic amino acid) . The proposed functions of this family are to eliminate potentially toxic nucleotide metabolites from the cell and to regulate the concentrations of nucleotide cofactors and signalling molecules for optimal cell growth and survival. The number of genes encoding Nudix hydrolases varies widely, from zero in Mycoplasma genitalium to 22 in Deinococcus radiodurans. This variation presumably reflects the growth or environmental adaptability, stress tolerance and metabolic capacity of the different organisms. The Nudix hydrolases thus offer an ideal system with which to study the evolution of a largely inessential protein family and its contribution to the individual biology of an organism. Understanding such variation requires a combination of detailed biochemical, genetic and cellular studies to reveal the individual functions of family members within the set in any given system. In the case of multicellular eukaryotes, the nematode Caenorhabditis elegans offers a genetically amenable model system with which to carry out such studies. There are 11 members of the Nudix hydrolase family in C. elegans. So far only two of these have been characterized -- a diadenosine tetraphosphate pyrophosphohydrolase (the orthologue of human NUDT2) and an NADH diphosphatase . Sequence comparisons would predict the existence of an ADP-sugar diphosphatase (NUDT5 orthologue) , an ADP-ribose diphosphatase (NUDT9 orthologue) , a diphosphoinositol polyphosphate pyrophosphohydrolase (NUDT3/4 orthologue) , two probable coenzyme A diphosphatases, one of which is highly similar to the mouse Nudt7 CoA diphosphatase , and 4 proteins of unknown function, including one with a strong similarity to the Saccharomyces cerevisiae PSU1/DCP2 protein and another similar to the developmentally-regulated mouse RP2 protein . Recent characterization of the S. cerevisiae NADH and CoA diphosphatases and the mouse Nudt7 CoA diphosphatase has revealed that they are located in peroxisomes. The function of these peroxisomal enzymes may be to regulate the concentration of these essential nucleotide cofactors for peroxisomal metabolism or, by analogy with the E. coli MutT 8-oxo-dGTPase, to eliminate toxic modified cofactor metabolites from the highly oxidizing peroxisomal environment. In order to investigate these possibilities in the C. elegans model system, we have cloned and characterised the putative C. elegans Y87G2A.14 CoA diphosphatase and shown that it displays the expected enzymatic activities and that it appears to be targetted to peroxisomes by a C-terminal PTS1 targeting signal. PMID- 11943069_Results and discussion TI - AB - Cloning, expression and purification of Y87G2A.14 | The C. elegans Y87G2A.14 gene encodes a 234 amino acid protein with an expected molecular weight of 26,601 Da. It was amplified by PCR from a C. elegans cDNA library. The PCR fragment was inserted into the pET-32b(+) expression vector and the nucleotide sequence of the insert was determined to be exactly the same as that submitted to GenBank under accession no. CAB54476. The recombinant plasmid pETY87G2A.14 was then used to transform E. coli BL21 (DE3) cells to generate a His-tagged thioredoxin fusion protein with an expected molecular mass of 43,731 Da. When the Trx-Y87G2A.14 fusion protein was expressed at 37C, it was confined to inclusion bodies, so the induction temperature was decreased to 25C to enhance protein solubility. As the expression level was low at this temperature, the induction time was increased to 8 h. These conditions markedly increased the solubility of Trx-Y87G2A.14 which was then purified from the soluble fraction (Fig , lane 2) to apparent homogeneity on NiCAMTM-HC resin (Fig , lane 3). To determine the molecular weight of the Y87G2A.14 protein itself, the Trx-Y87G2A.14 fusion was cleaved with thrombin, which generated Y87G2A.14 with an apparent molecular weight of 27 kDa (expected molecular weight, 29,807 Da) and thioredoxin (15 kDa, Fig , lane 4). Figure 1 | Purification and cleavage of Trx-Y87G2A.14 fusion protein. Purification and cleavage of Trx-Y87G2A.14 fusion protein. Samples were analysed by SDS-PAGE (15% polyacrylamide) and stained with Coomassie Brilliant blue R 250. Lane 1, 2 mug protein standards: bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde 3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (20 kDa) and alpha-lactalbumin (14.2 kDa); lane 2, soluble cell extract of BL21 (DE3) cells transformed with recombinant plasmid pETY87G2A.14 and induced with 1 mM IPTG for 8 hours at 25C before applying to a column of NiCAMTM-HC resin ; lane 3, 3 mug purified Trx-Y87G2A.14 fusion protein; lane 4, 3 mug purified Trx-Y87G2A.14 fusion protein after cleavage with thrombin. Substrate specificity and product analysis | Purified Trx-Y87G2A.14 was inactive towards the following nucleotides when assayed at a fixed concentration of 0.5 mM: NADH, NAD+, NDP-sugars, 5'-(d)NTPs, 5'-NDPs, 5'-NMPs and diadenosine polyphosphates. High activity was found with CoA and its derivatives. HPLC analysis of CoA hydrolysis by Trx-Y87G2A.14 showed that the enzyme was a CoA diphosphatase, cleaving the diphosphate linkage in CoA to yield adenosine 3',5'-bisphosphate (3',5'-ADP) and 4'-phosphopantetheine . Figure 2 | Identification of reaction products of CoA hydrolysis. Identification of reaction products of CoA hydrolysis. Reaction mixtures containing 0.5 mM CoA were incubated at 37C for 20 min with or without 0.1 mug Trx-Y87G2A.14 fusion protein and the products separated by HPLC as described in Materials and methods. Without enzyme (------), with enzyme , gradient ( --- --- --- --- ---). Positions of authentic standards are indicated. Reaction requirements and kinetic parameters | Trx-Y87G2A.14 displayed optimal activity with 0.5 mM CoA as a substrate at pH 9.5. A divalent metal ion was absolutely required for activity, with optimal activity at 5 mM MgCl2. In common with all other Nudix hydrolases tested, fluoride was a strong inhibitor with a Ki value of approximately 3 muM (results not shown). Km, and kcat values for CoA, CoA esters and oxidized CoA were calculated by non-linear regression from data obtained by HPLC analysis . A graphical example of the data for CoA in the form of a hyperbolic plot and double reciprocal plot show that the enzyme obeys simple Michaelis-Menten kinetics. The kcat / Km ratios show that the enzyme prefers reduced forms of CoA to oxidized CoA with CoA itself the best substrate of those tested. Figure 3 | Lineweaver-Burk and Michaelis-Menten (inset) plots for the hydrolysis of CoA. Lineweaver-Burk and Michaelis-Menten (inset) plots for the hydrolysis of CoA. Reaction mixtures containing various concentrations of CoA (0.05 --0.7 mM) were incubated at 37C for up to 20 min with 0.1 mug Trx-Y87G2A.14 fusion protein. Initial rates of hydrolysis were determined after separation of the products by HPLC as described in Materials and methods. Table 1 | Kinetic parameters for the hydrolysis of CoA and CoA derivatives by Trx-Y87G2A.14 fusion protein Subcellular localization | Y87G2A.14 has the C-terminal tripeptide sequence SKI. This conforms to the pattern typical of PTS1 peroxisomal targeting signals found in many peroxisomal matrix proteins, suggesting that Y87G2A.14 may be targeted to these organelles . However, possession of a potential PTS1 sequence is not always sufficient on its own to result in peroxisomal targeting and other elements of the protein sequence may also be involved. Since targeting of animal peroxisomal proteins expressed in yeast has often been observed, yeast cells were transformed with expression plasmids encoding C-terminal or N-terminal fusions of Y87G2A.14 to yeast-enhanced green fluorescent protein (yEGFP) in order to determine the subcellular location of Y87G2A.14. The cells were then examined by confocal microscopy. Cells transformed with pY87G2A.14-yEGFP, in which the C-terminus of Y87G2A.14 is fused to the N-terminus of yEGFP, showed a diffuse, cytoplasmic fluorescence with no clear subcellular localization . In contrast, cells transformed with pyEGFP-Y87G2A.14, in which the C-terminal tripeptide SKI is free to act as a targeting signal showed the clear punctate fluorescence that is indicative of yeast peroxisomes . The identity of SKI as the targeting signal was confirmed by transformation of cells with pyEGFP-Y87G2A.14Delta SKI, in which the C-terminal tripeptide was deleted during construction. This again showed a diffuse, cytoplasmic, fluorescence . Together, these results strongly suggest that Y87G2A.14 is targeted to peroxisomes by its C-terminal tripeptide, SKI. Figure 4 | Subcellular localization of Y87G2A.14 by fluorescence confocal microscopy. Subcellular localization of Y87G2A.14 by fluorescence confocal microscopy. yEGFP fluorescence of yeast cells transformed with (a) pY87G2A.14-yEGFP; (b) pyEGFP-Y87G2A.14 and (c) pyEGF-Y87G2A.14DeltaSKI PMID- 11943069_Conclusions TI - AB - On the basis of its sequence, the C. elegans Y87G2A.14 gene product was predicted to be a peroxisomal coenzyme A diphosphatase. In addition to the Nudix motif, Y87G2A.14 possesses the PROSITE UPF0035 motif , which we have previously suggested confers a specificity for coenzyme A and its derivatives , and a C-terminal tripeptide, SKI, that conforms to the pattern typical of PTS1 peroxisomal targeting signals. The experiments described here confirm these predictions. Fig shows a multiple sequence alignment of the motif-containing region of Y87G2A.14 with related sequences from other organisms. Those marked with a tick have been experimentally shown to be coenzyme A diphosphatases . In most cases, higher organisms possess two related sequences, e.g. mouse Nudt7 and Nudt8, one of which encodes a peroxisomal enzyme (e.g. Nudt7). However, S. cerevisiae has only one sequence containing the UPF0035 motif while Arabidopsis thaliana has three, and the second of the two Drosophila melanogaster sequences, RH61317, is currently only represented in GenBank by a single expressed sequence tag, so its status is still questionable. For the peroxisomal enzymes, either a putative C-terminal PTS1 or an N-terminal PTS2 targeting signal is present. Interestingly, in each case, the putative PTS2 signal is contained within or near a predicted mitochondrial targeting or chloroplast transit peptide sequence , suggesting a possible dual location for these proteins. Such a possibility has not yet been experimentally observed; however, mutation of a glutamate five residues to the C-terminal side of the PTS2 of rat peroxisomal 3-ketoacyl-CoA thiolase to a neutral or basic amino acid has been shown to result in partial mitochondrial targeting, suggesting that the negative charge on glutamate may normally block translocation to the mitochondria . Whether or not a system exists in vivo to regulate dual targeting is clearly a topic requiring further investigation. The non-peroxisomal sequences provide no clear indication of possible subcellular location, hence they are likely to be cytoplasmic. Given the existence of mitochondrial, peroxisomal and cytoplasmic pools of CoA and CoA esters , it would not be surprising to find CoA diphosphatase activity in all these locations. However, the precise substrate specificities of the "cytoplasmic" activities remain to be determined. Figure 5 | Partial sequence alignment of Y87G2A.14 and related sequences. Partial sequence alignment of Y87G2A.14 and related sequences. The partial sequence of Y87G2A.14 containing the UPF0035 and Nudix motifs (arrowed) was aligned using the Clustal W program with related sequences from other organisms retrieved from a BLAST search. Organisms and database accession numbers are: Caenorhabditis elegans Y38A8.1, Q23236; Homo sapiens NUDT7, XP_058753; H. sapiens NUDT8, AI743601; Mus musculus Nudt7, Q99P30; M. musculus Nudt8, AK009700; Drosophila melanogaster CG11095, Q9VY79; D. melanogaster RH61317, BI631687; Schizosaccharomyces pombe YDH5, Q92350; S. pombe YDZA, 013717; Ambidopsis thaliana At2g33980, 022951; A. thaliana At1g28960, Q9SHQ7; A. thaliana At5g45940, BAB09322; Saccharomyces cerevisiae PCD1, Q 12524; Escherichia coli YeaB, P43337; Deinococcus radiodurans DR1184, Q9RV46. Sequences encoding experimentally confirmed CoA diphosphatases are marked with a tick. Columns on the right indicate whether the full sequence contains a putative peroxisomal targeting signal (PTS1 or PTS2) and/or a putative mitochondrial targeting peptide (mTP) or chloroplast transit peptide (cTP). Regarding the possible function of these enzymes in general, and the C. elegans peroxisomal enzyme in particular, a recent functional genomic analysis by RNA-mediated interference of C. elegans chromosome I, on which the Y87G2A.14 gene is located, revealed no phenotype in relation to growth, survival, fecundity or morphology when the expression of Y87G2A.14 was ablated . This would indicate that, within the limitations of RNAi, the CoA diphosphatase activity of Y87G2A.14 is not essential. However, now that the biochemical properties of this protein have been established, a more detailed and targeted biochemical analysis can be undertaken that should reveal its cellular function and benefit to the organism. Nudix hydrolases are believed to regulate the concentrations of nucleotides for optimal cell performance and also to eliminate potentially toxic nucleotide metabolites from the cell. With regard to regulation, CoA diphosphatase activity is associated with the 400 kDa CoA synthesizing protein complex from S. cerevisiae, in which it forms part of an alternative pathway for CoA biosynthesis that differs from the principal route of 3'-dephospho-CoA and CoA synthesis by this complex . This CoA/4'-phosphopantetheine cycle involves hydrolysis of CoA to 3',5'-ADP and 4'-phosphopantetheine, which then reacts with ATP to give 3'-dephospho-CoA then CoA. Whether such a pathway operates in peroxisomes and whether the C. elegans Y87G2A.14 protein is involved remain to be established. With regard to the elimination of toxic nucleotide metabolites, the 13-fold higher kcat / Km ratio for oxidized CoA (CoASSCoA) compared to CoA for the S. cerevisiae PCD1 CoA diphosphatase previously suggested to us that this enzyme might preferentially remove non-functional and potentially toxic oxidized CoA and CoA esters from within the oxidizing environment of the peroxisomes . However, neither the mouse Nudt7 nor the C. elegans Y87G2A.14 proteins show this preference. Nevertheless, the potential production of adenine ring-oxidized derivatives of CoA by reactive oxygen species generated in the peroxisomes analogous to the 2-oxo-dATP and 8-oxo-dATP substrates of the mammalian MTH1 Nudix hydrolase suggests that such species could be more relevant substrates for peroxisomal CoA diphosphatases in vivo. The amenability of C. elegans to studies of cellular and molecular stress will allow the question of the biological function of these enzymes to be addressed. PMID- 11943069_Materials and methods TI - AB - S. cerevisiae strain BY4741 (MAT a; his3D1; leu2D0; met15D0; ura3D0) was from Research Genetics. Calf intestinal alkaline phosphatase, yeast inorganic pyrophosphatase, EcoR1 and BamH1 were from Roche while BspH1 (Pag1) was from Helena Biosciences. Pfu DNA polymerase was from Stratagene. All other chemicals and nucleotides were from BDH or Sigma. The E. coli expression vector pET-32b(+) was from Novagen and the yeast-enhanced green fluorescent protein (yEGFP) fusion vectors pUG35 and pUG36 were a gift from J.H. Hegemann, Institute of Microbiology, University of Dusseldorf, Germany. The C. elegans cDNA library was prepared from adult nematodes by H. M. Abdelghany, School of Biological Sciences, University of Liverpool, U.K. Cloning of Y87G2A.14 from C.elegans | A cDNA corresponding to the C. elegans Y87G2A.14 gene on chromosome 1 (GenBank accession no. CAB54476) was amplified from a cDNA library by PCR using as forward and reverse primers 5' GCAAATCATGAAGTGTGTGGTTAGCCGAGCTG 3' and 5' TAAATGAATTCACTAAATTTTGGATTTCGGTTC 3' respectively. These primers provided a BspH1 restriction site at the start of the amplified gene and an EcoR1 site at the end. After amplification with Pfu DNA polymerase, the DNA was recovered by phenol/chloroform extraction and digested with BspH1 and EcoR1. The digest was gel-purified and the restriction fragment ligated into the Nco1 and EcoR1 restriction sites of pET-32b(+) as both BspH1 and Nco1 form compatible ends with each other. The resulting construct, pETY87G2A.14, yielded Y87G2A.14 downstream of the 109-amino acid thioredoxin (Trx) fusion and His-tag and S-tag sequences under the control of an IPTG-inducible promoter. The structure of the insert was confirmed by sequencing. The construct was propagated by transformation of E. coli XL1-Blue cells. Expression of Y87G2A.14 in E. coli and protein purification | E. coli strain BL21(DE3) was transformed with pETY87G2A.14. A single colony was picked from an LB agar plate containing 50 mug/ml ampicillin and inoculated into 10 ml LB medium containing 50 mug/ml ampicillin and incubated at 37C. When the cells reached an A600 of 0.5, they were transferred to 1 litre of fresh LB medium containing 50 mug/ml ampicillin and grown to an A600 of 0.3 at 37C, then transferred to an incubator at 25C. Isopropyl-1-thio-beta-D-galactopyranoside (IPTG) was added to 1 mM at an A600 of 0.8, and the cells induced for 8 h. The induced cells (4 g) were harvested, washed and resuspended in 20 ml breakage buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM DTT). The cell suspension was sonicated and the cell lysate was then cleared by centrifugation at 10,000 x g at 4C for 15 min. The supernatant was applied to a 15 x 50 mm column of NiCAMTM-HC resin (Sigma) equilibrated with 50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1 mM 2-mercaptoethanol at a flow rate of 0.5 ml/min. After eluting the unbound proteins, a linear gradient of 0 --40 mM histidine in the same equilibration buffer was applied at flow rate of 1 ml/min and 1 ml fractions collected and analysed by SDS-PAGE. Those containing pure Trx-Y87G2A.14 fusion protein were collected, dialysed overnight at 4C against 1 litre of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, ImM DTT and then concentrated by ultraflltration (Amicon) and stored at -20C in 50% glycerol. Enzyme assays | Potential substrates were screened by measuring the Pi released after nucleotide hydrolysis in presence of inorganic pyrophosphatase or alkaline phosphatase . The standard assay (200 mul) for phosphodiester substrates was incubated at 37C for 30 min and contained 50 mM l,3-bis [tris(hydroxymethyl)-methylamino]propane-HCl (BisTrisPropane-HCl), pH 8.0, 5 mM MgCl2, 1 mM DTT, 0.5 mM substrate, 0.1 mug of Trx-Y87G2A.14 fusion protein and 0.5 mug (1 unit) alkaline phosphatase. Assays with phosphomonoester substrates were as above, except 0.5 mug (100 mU) inorganic pyrophosphatase was used instead of alkaline phosphatase. The Pi released in each case was measured colorimetrically. Chromatographic analysis | Kinetic parameters and reaction products generated from hydrolysis of CoA and its derivatives were measured by high performance anion-exchange chromatography. The reaction mixtures (100 mul) contained 50 mM BisTrisPropane-HCl, pH 9.5, 5 mM MgCl2, 1 mM DTT (in cases of substrates requiring reducing conditions), substrate in the range of 0.05 --0.7 mM, (0.1 --1 mM in the case of oxidized CoA) and were incubated at 37C for up to 20 min (during which time the reaction rates remained linear) with 0.1 mug Trx-Y87G2A.14 fusion protein. A 90 mul sample of each reaction mixture was applied to a 1 ml Resource-Q column (Amersham Pharmacia Biotech) equilibrated with 0.045 M CH3COONH4 (pH 4.6, adjusted with H3PO4), and eluted with a linear gradient from 0 to 0.45 M NaH2PO4 (pH 2.7 adjusted with CH3COOH) for 10 min at a flow rate of 2 ml/min . Elution was monitored at 259 nm and peaks identified with the aid of standards and quantified by area integration. GFP fusion constructs and subcellular localization | Expression plasmids encoding C-terminal and N-terminal fusions of Y87G2A.14 to yeast-enhanced green fluorescent protein (yEGFP) were constructed by amplification of the coding region of Y87G2A.14 from C. elegans cDNA by PCR using the same forward primer 5' CGACGGATCCATGAAGTGTGT 3' and one of the reverse primers 5' TAAATGAATTCACTAAATTTTGGATTTCGGTTC 3', 5' CACTAAGAATTCTATTTCGGTTCAAATTTCCTACTTGC 3', or 5' GCTCGAATGAATTCAATTTTGGATTTCGGTTC 3' to give PCR products "C", "CDeltaSKI" or "N" respectively. These primers provided a BamH1 restriction site at the start of the amplified gene and EcoR1 sites at the end. PCR products "C" and "CDeltaSKI" were cloned as C-terminal fusion proteins to yEGFP, while PCR product "N" with a deletion of the Y87G2A.14 termination codon was cloned as an N-terminal fusion to yEGFP. After amplification with Pfu DNA polymerase, the DNA products were recovered by phenol/chloroform extraction and digested with BamH1 and EcoR1. The digested PCR products "C", and "CDeltaSKI" were gel purified and the restriction fragments ligated between the BamH1 and EcoR1 restriction sites of pUG36 (yEGFP-C-fusion) to give pyEGFP-Y87G2A.14 and pyEGFP-Y87G2A.14DeltaSKI respectively. The digested PCR product "N" was gel purified and the restriction fragment ligated between the BamH1 and EcoR1 restriction sites of pUG35 (yEGFP-N-fusion) to give pY87G2A.14-yEGFP. The structures of the inserts were confirmed by sequencing. The plasmids were propagated by transformation of E. coli XL 1-Blue cells. For microscopy, S. cerevisiae strain BY4741 was transformed with pyEGFP-Y87G2A.14, pyEGFP-Y87G2A.14DeltaSKI or pY87G2A.14-yEGFP and grown on solid SC-Ura medium containing 2% glucose. Cells were viewed by conventional and confocal fluorescent microscopy on a Zeiss LSM510 confocal microscope with a 100 x 1.4 NA objective. Other methods | Protein concentrations were estimated by the Coomassie blue binding dye-based colorimetric method using equal weights of bovine serum albumin, conalbumin, cytochrome c and myoglobin as standards . PMID- 11914163 TI - Outcomes research in the development and evaluation of practice guidelines AB - Abstract | Background | Practice guidelines have been developed in response to the observation that variations exist in clinical medicine that are not related to variations in the clinical presentation and severity of the disease. Despite their widespread use, however, practice guideline evaluation lacks a rigorous scientific methodology to support its development and application. Discussion | Firstly, we review the major epidemiological foundations of practice guideline development. Secondly, we propose a chronic disease epidemiological model in which practice patterns are viewed as the exposure and outcomes of interest such as quality or cost are viewed as the disease. Sources of selection, information, confounding and temporal trend bias are identified and discussed. Summary | The proposed methodological framework for outcomes research to evaluate practice guidelines reflects the selection, information and confounding biases inherent in its observational nature which must be accounted for in both the design and the analysis phases of any outcomes research study. PMID- 11914163_Background TI - AB - The development of practice guidelines | In clinical medicine, variations exist that do not appear to be related to variations in the clinical presentation and severity of disease . In response, practice guidelines have been developed in an attempt to reduce the wide practice variations and, through this process, to increase the appropriateness and quality of medical care and to reduce health care costs . Despite the publication and dissemination of practice guidelines , there has been relatively little evaluation of the application and impact of clinical practice guidelines . Some of the difficulty in the evaluation of these guidelines relates to the methods that were used to develop them . Guidelines have often have been developed before adequate data have been available to assess the relationship between clinical practice patterns and desired clinical outcomes. Nevertheless, there have been some reviews of practice guideline evaluation . While epidemiological designs are commonly used to evaluate the effectiveness of health care interventions, never has this been discussed in the context of outcomes research. We propose the use of a methodological framework for outcomes research to evaluate practice guidelines. Methodological issues with the measurement of practice variations | In the debate about reasons to promote the development of practice guidelines, few have questioned whether the variations are real, or alternatively, whether they are simply a function of methodological flaws in the measurement of medical practices themselves, the result of variations in practice patterns across groups of patients with a similar diagnosis, or both. Furthermore, few studies have addressed whether practice variations, in fact, lead to outcome variations. Finally, little attention has been paid to the identification and measurement of initial conditions, that is, the potentially confounding factors and effect modifiers of the practice patterns outcomes relationship. Measurement of practice pattern variation | The measurement of medical practice patterns is susceptible to error. Measurement error may affect the validity of medical practice measurement in three major ways. First, it may lead to selection bias, in that subjects are selected to belong to a certain group based on an erroneous diagnosis. Secondly, it may lead to misclassification of exposure (information bias), in that patients treated with a specific practice pattern are classified in the wrong diagnostic group. Thirdly, it may lead to misclassification of outcomes, in that patients with a given outcome are classified in the wrong diagnostic group. Potential problems with the measurement of practice variations relate to the mechanisms that underlie the choice of groups that are compared in studies of practice variations. These mechanisms must be defined clearly to minimize selection bias. In many studies of practice variations, populations are arbitrarily divided according to hospitals, regions, counties, or countries. Little information is available about the factors that lead these groups to go to a particular hospital, live in a particular region, go to a particular doctor, etc. The population base from which each comparison group is derived should, in principle, be quite similar for all groups. Basically, if the groups are drawn from a similar population, unmeasurable and potentially confounding variables are more likely to be equally distributed between groups. In addition, the measurement of practice variations cannot be valid without information on relevant "initial conditions". Initial conditions are all confounding factors and effect modifiers, other than the treatment/practice patterns, that may cause or influence the clinical outcomes of interest. These factors may explain practice variations among groups that do not share similar initial conditions. To evaluate practice patterns-outcomes associations, potential confounders must be identified and controlled for in the analysis. Aside from clinical presentation and severity of illness; the initial conditions to be identified and characterized as completely as possible include physician, patient, and practice environment factors . Measurement of such factors is essential to minimize the chance of a systematic error following confounding biases and effect modification . Figure 1 | Table 1 | Initial conditions to be taken into account when making inferences about practice patterns-outcomes associations Identification and measurement of outcomes of interest | Limitations to the development and evaluation of practice guidelines also include the absence of a clear concept of the targeted outcomes and the paucity of outcomes data to support these guidelines . There appears to be only a weak relationship between the purpose of guidelines and many of the outcomes usually measured in clinical research, that is, the source of evidence for guideline development (evidence-based). The initial goals of establishing practice guidelines -- to reduce costs and enhance the quality and appropriateness of treatment -- are, in fact, rarely the basis for guideline development, since little data is available for these outcomes. To some degree, the development of guidelines has been driven by the availability of data on clinical outcomes, such as morbidity and mortality, rather than those outcomes related to the primary goals of the guidelines. The evaluation of practice guidelines | Throughout the development of practice guidelines, the major deficiency has been the lack of an evaluative method . Thus, we suggest a methodological framework for outcomes research to be applied to evaluate practice guidelines. Outcomes research evaluates practice patterns as they occur in actual clinical settings. This type of research can describe practice patterns, evaluate their divergence from practice guidelines and determine the effect of practice variations on outcomes. Outcomes research is necessarily observational in nature and, although observational studies have been used to evaluate health care interventions, the proposed methodological framework has yet to be applied to outcomes research. Why should outcomes research be used to evaluate and validate practice guidelines? The primary goal of practice guidelines is the consistent adherence by physicians to practice patterns that achieve the "best" outcomes at the lowest cost. Outcomes research evaluates practice patterns as they occur in actual clinical settings, and is thus the logical method to evaluate practice guidelines. In fact, outcomes research and practice guidelines are connected through concepts that relate to efficacy and effectiveness research . Efficacy studies, which normally complement practice guideline development, are those performed in highly selected groups of patients to investigate if a particular intervention works under controlled conditions set by the study investigators. In contrast, outcomes research evaluates practice as it occurs in actual clinical settings . Research in these settings is called effectiveness research because the investigators have limited control over the conditions that qualify the practice settings. The difference between efficacy and effectiveness research can be summarized as follows: does it work at all (efficacy) or does it work in the real world (effectiveness)? Thus, there exists a dynamic process in which evidence from both effectiveness and efficacy studies feeds into the development and evaluation of practice guidelines, as depicted in Figure . Figure 2 | Relationship between outcomes research and practice guidelines Relationship between outcomes research and practice guidelines Most practice guidelines are derived from efficacy studies rather than effectiveness studies. Therefore, it is not surprising that practice guidelines are not fully applicable in actual clinical practice. We suggest that effectiveness studies be used not only as a method to evaluate practice guidelines but also as a basis for their development. These could include both observational studies and effectiveness trials. Outcomes research better reflects practice in the real world and may make guidelines more likely to be applied. However, to date, little attention has been paid to the epidemiological underpinnings of the methods used to conduct outcomes research. PMID- 11914163_Discussion TI - AB - We will first propose a methodological framework for outcomes research. Then, we will show how it can be used to evaluate practice guidelines. Finally, we will address the limitations of the proposed methodological framework. Generic epidemiological issues in outcomes research | In the proposed methodological framework, the generic issues related to outcomes research will be discussed in sequential order. In outcomes research, the first step is to identify the study population and the groups (hospitals, providers, regions, etc.) that will be compared. The next step is the measurement of practice patterns and outcomes. After groups are compared on the basis of the treatment they receive and outcomes of interest, associations are sought between practice patterns and the various measures of outcome. This step of the methodological framework raises issues of confounding bias because not all factors that can confound these associations are measured and controlled or even known. The presence or absence of confounding bias can be affected by the other sources of bias namely selection and information biases. Lastly, we discuss the issue of temporal trends. In the evaluation of practice guidelines, the measurement of practice patterns may not be contemporaneous with the publication of practice guidelines. This may explain and even lead to the frequently observed discrepancy between the actual practice and what the guidelines state that it should be. Finally, two particularities of outcomes research 1) the presence of ecological exposures in individual level studies and 2) the common use of large administrative databases are discussed. Specification of the model | Definition of the elements of the proposed epidemiological model for outcomes research | In the proposed model for outcomes research designed to evaluate practice guidelines, the outcome of interest can be a disease . For example, if the practice patterns that are being studied pertain to coronary revascularization, complications such as mortality and reinfarction after acute myocardial infarction may constitute the outcome of interest. Finally, the consequences of different practice patterns on medical resources (cost, quality and appropriateness) may be another possible outcome of interest. Table 2 | Epidemiological model for outcomes research to evaluate practice guidelines In the studies of outcome research, practice patterns, (which constitute the exposure in the proposed model), range from the use of medication, diagnostic tests and therapeutic procedures to the length of hospital stay, transfer to other facilities and/or scheduled physicians visits. The primary goal of outcomes research is the evaluation of the effects of the selected practice patterns on the outcomes of interest. Consequently, any inference made about this association must be evaluated as a function of the potential selection, information (measurement error) and confounding biases. A limitation of outcomes research as it is most often performed is the lack of attention given to the measurement of each of the elements of the epidemiological model shown in Table 3. The basis of the proposed methodological framework will be the identification of generic sources of potential bias that relate to each element of the proposed model. Selection bias | Since outcomes research is observational in nature, the choice of the study population and of the compared groups is highly susceptible to selection bias. As applied to outcomes research, selection bias is defined as a distortion in the estimate of the practice patterns outcomes association due to the way that subjects are selected for inclusion in the study population and in the different groups to be compared . A major consequence of selection bias is the potential confounding of inferences made about practice patterns-outcomes associations. This occurs when some characteristics of the subjects related to practice patterns or clinical outcomes influence the selection or exclusion of individual subjects, groups of subjects or practice environments. The selection process should be such that patients included in the study population come from the same target population . Furthermore, patients or study members should have a similar probability of being selected and included in the actual population. Inclusion and exclusion criteria must be clearly defined in order to characterize the actual population as precisely as possible. Judging the internal validity of a study is more feasible when there is a detailed account of how the individuals were selected to become members of the actual population. Finally, the study population, also needs to be carefully characterized so that the inferences derived from the analysis of the study population can be evaluated for both internal validity (based on the data analyzed in the study) and external validity (the extent to which results obtained from the data analyzed in a particular study can be generalized to populations outside of the study). Any systematic differences between those actually studied and the source (target) population could result in biased estimates of the impact of a practice pattern on a clinical outcome. In many studies of outcomes research, groups exposed to different practice patterns are compared. The identification of such groups of patients is sought to assess the impact of different practice patterns on various outcomes in actual clinical settings and, as previously mentioned, can be used to assess practice guidelines. Because of such study design, it becomes unclear as to what the target population precisely is. Is it the group (the set of patients in a given environment) or is it the individuals receiving the various practice patterns within each group? For example, in a study of regional variations in the treatment of acute myocardial infarction in the U.S., the treatment of patients (practice patterns) was compared across different regions of the U.S. In this study, one wishes to generalize the findings about practice patterns-outcomes associations to all individuals with acute myocardial infarction (individual level). One also wishes to generalize the effect of the exposure, which is in this case practice patterns, to those prevalent in a given region (ecological level). The presence of these two levels, the individual and the ecological levels, introduces an added level of complexity in terms of the assessment of the effect of the exposure on outcome. When comparing practice patterns across regions using individual data, there is a certain degree of correlation brought about by the clustering of practice patterns that needs to be taken into account. Such a correlation is very difficult to quantify. In contrast, when assessing the effect of the exposure at the individual level, there are ecological factors (initial conditions particular to a given region) that need to be taken into account. The data originating from studies with mixed design, which are often the design of outcomes research studies, need to be analyzed with special attention to the degree of correlation between the individual covariates and to the presence of ecological exposure variables. Another potential source of selection bias is the choice of the groups to be compared, which depends on the criteria used to divide the groups. Individuals included in the groups to be compared should have the same probability of being included in these groups. Not infrequently in outcomes research, geographic criteria (such as country, regions, hospitals) are used because such criteria allow the identification of clinically comparable groups that receive very different treatments, whose resulting outcomes can then be assessed. However, such a process must be scrutinized for the possibility of selection bias other than the treatments that are being evaluated. Such selection bias would make groups not comparable as to clinical and other factors that could affect outcomes. The presence of a biased selection process could lead to confounding bias when practice patterns-outcomes associations are assessed. Such a situation may occur when the study groups are not comparable with regard to some characteristics of the subjects related to practice patterns or clinical outcomes that influenced the selection or exclusion of individual subjects, groups of subjects or practice environments. For example, in the same study of regional variations in the treatment of acute myocardial infarction, census regions of the U.S. were arbitrarily chosen as a basis for comparison. In this example, patients with similar risk of developing the outcome of interest, which is defined here as a complication after acute myocardial infarction, may not have had the same probability of being included in the different groups to be compared. Confounders may then bias the practice patterns/outcomes association if the selection of different risk groups is related to practice patterns. Selection bias can also affect the assessment of outcomes. Potential sources of this bias include loss to follow-up or missing data. Follow-up data is difficult to obtain in outcomes research studies, which often rely on administrative databases for data acquisition. Linkage, either of different databases or of the same database over time, is often performed . A failure to link the databases for a number of individuals presents a problem equivalent to having data missing for these individuals. Information bias | The second step in outcomes research studies is the measurement of practice patterns and of the outcomes of interest. Here, issues of information bias must be considered. Information bias can be defined as a distortion of the potential practice patterns outcomes association due to misclassification of subjects with regard to practice patterns, outcome measures or both, or due to measurement error . There are two major ways in which practice patterns can be misclassified. They relate to the sensitivity and specificity of the tests that are used for the diagnosis for which practice patterns are being evaluated and for the classification of the outcomes of interest. The measurement of the different practice patterns and their related outcomes largely depend on the identification of a group of patients who have a given diagnosis and require a given treatment. The characteristics that make a diagnosis more amenable to outcomes research are the following: 1) a precise diagnostic definition, 2) a diagnostic test with high sensitivity and specificity, 3) reproducibility among different individuals and locations, 4) easily coded, 5) related to a procedure, and 6) common and costly, so that it is likely to be collected in large, administrative databases frequently used in outcomes research. Because of such requirements, only a limited number of clinical conditions are amenable to outcomes research. Acute myocardial infarction is an example of a diagnosis that can be made with a high level of certainty because it has a precise diagnostic definition and well-defined diagnostic criteria, which, when taken together, have high sensitivity and specificity for the correct classification of patients. Therefore, it is easy to identify a study population that, in fact, has this disease and to describe their treatment. Thus, in order to minimize the misclassification of relevant practice patterns, the methods used to classify the disease and the outcomes that relate to the practice patterns under investigation must have high sensitivity and specificity . Given the principles underlying the measurement of practice patterns and outcomes, how are the measurements generally made in outcomes research studies? The measurement of the exposure (practice patterns) in outcomes research is valid only if it corresponds to the "true" practice as performed in the clinical setting. Again, practice can only be "true" if the diagnosis is correct. The identification of both patients with the disease of interest and their treatment requires a source of information that has the features of a diagnostic test. In outcomes research, administrative databases are often used as an information source to identify a study population and to obtain data on exposure. The database coding of diagnoses and procedures can be used as a "diagnostic test" to identify the clinical condition for which practice patterns will be described and to classify the practice patterns themselves and the outcomes of interest. Such a "diagnostic test" will have higher sensitivity and specificity values for some diagnoses than for others. For example, administrative database coding will have higher sensitivity and specificity for procedure-related diagnoses (such as hip fracture) because the diagnostic code is related to a major operation and is likely to be recorded for administrative purposes. In contrast, a diagnostic criterion for osteoarthritis can be quite vague and administrative coding is likely to have very low sensitivity and specificity for this diagnosis. The use of databases as a diagnostic test must be validated in all outcomes research studies, especially those using administrative databases. Methods to validate these databases include chart reviews, a priori coding systems or both. These validation methods ensure that coding is as accurate and reproducible as possible, thus allowing the database to be used as a diagnostic test to identify the study population and the practice patterns and the outcomes in outcomes research. However, these validation methods are rarely used. Finally, appropriate measures of outcomes that will serve to evaluate practice guidelines must be identified. This presents a problem because most practice guidelines aim to reduce practice variations, which will, in turn, lead to improved appropriateness and quality of care. However, how appropriateness and quality of care are measured is controversial and will not be discussed here . Nevertheless, defining the outcomes that will be used to evaluate practice guidelines is a crucial step in this process. Quality of life and functional status measures constitute another group of outcome measures that should be included for the evaluation of practice guidelines. These dimensions of outcomes have received more attention from health providers, while consumers have become more concerned about outcomes of care. However, these outcomes also are difficult to measure, because they rely heavily on patient interviews and questionnaires. They are likely to vary with patient expectations, culture, and climate and are thus potentially to be measured with error and be misclassified. A few reliable, valid instruments have been developed to assess health-related quality of life , but such instruments are not easily used to collect this information from large databases. There is a need to develop instruments to measure these types of outcomes, whether they are conversion factors for existing databases (such using length of stay as a proxy for cost) or new measures that could easily be integrated in administrative databases. Such measures could include estimates of functional class or severity of illness. At present, many outcomes research studies measure mortality and disease-specific morbidity. The validity of the measurement of these outcomes is limited by the type of database that is used. For example, using death registries to obtain causes for death is a notoriously invalid source for this type of information. There are many examples of poor correlation between cause of death as established by death registries versus disease registries. Death certificates in New York City during 1992 were assessed to determine the accuracy and frequency of reporting tuberculosis as a cause of death. Of 310 persons who died with active tuberculosis in 1992 (based on a disease-specific registry), only 34% had tuberculosis listed on their death certificate. Thus, in this example, as in many others like it, using death certificates led to an inaccurate measure of disease burden . Confounding bias | In outcomes research terms, confounding bias is present when the effect of the practice variations on the outcomes of interest is distorted because of the effects of extraneous variables (variables that are causally associated with the practice variations and the outcomes of interest) . This issue is crucial in outcomes research because, while outcomes research shares the purpose of a clinical trial (to evaluate different treatments), it primarily uses observational methods -- investigators conducting outcomes research have limited control over potentially confounding factors (the initial conditions of individual groups of patients). Because outcomes research builds on existing practice variations and analyses the natural ongoing experiment, there is ample opportunity for confounding bias to invalidate any inference made about practice patterns outcome associations . For example, variations in practice patterns could reflect variation not only in the use of a given procedure but also in the severity of disease. Assignment of patients to certain procedures on the basis of the severity of illness makes sense clinically, but in outcomes research, it is a common and important source of confounding if the procedure is either efficacious or particularly harmful in high-risk patients. Many indices have been developed to measure the severity of illness when using existing databases to correct for such confounding, but one can never be sure that this type of confounding has been entirely controlled . This presents an intrinsic limitation of outcomes research. Avoidance of confounding bias is limited by the source of data used to describe practice patterns, particularly when observational data, such as the large Medicare administrative databases, are used to compare outcomes among patients who receive different treatments. The potential for confounding bias arises because many factors other than the treatment under evaluation may affect patient outcomes. These factors include comorbid diseases, severity of illness, and patient, physician and environmental factors. Such factors are likely to influence treatment decisions but are difficult to capture fully in recorded data. Researchers cannot adjust for imbalances in prognostic factors that are unmeasured or poorly categorized and administrative data, in particular, may lack the precise and accurate coverage of clinical details needed to permit full and fair adjustments. Further data collection might solve this issue, but it is not always possible to collect additional information. Standard statistical modeling can attempt to adjust for the known differences between the groups, but this might not be sufficient for unmeasured differences. Several alternative methods have been suggested. One method is subgroup analysis to adjust for unmeasured differences between groups of individuals who differ on known risk factors. Another method consists of the use of instrumental variables . Instrumental variables are observable factors that influence treatments but do not directly affect patient outcomes. This approach uses the so-called instrumental variables to mimic a randomization of patients to different likelihoods of receiving alternative treatments. McClellan et al. applied this methodology to assess whether more aggressive use of invasive cardiac procedures improved outcomes in the elderly. In this study, the instrumental variable was the distance of the patient's residence from the nearest hospital with on-site angiography. The authors noted lower mortality among elderly individuals who received more aggressive treatment than among those treated more conservatively. Temporal trend bias | We propose a bias called a "temporal trend bias" that is particular to the use of outcomes research to evaluate practice guidelines. This bias results from the inability to control for secular trends. It reflects the fact that by the time practice guidelines are published and disseminated, new treatments and technology are being incorporated into clinical practice. Thus, it is difficult to identify a pure application of a practice guideline whose application is not undermined by recent advances in medicine and technology. For example, we evaluated the effect of a specific set of guidelines on return to work after acute myocardial infarction. The use of these guidelines had been successful in a university setting; this study assessed their use in a community setting. During the 5 years that elapsed between these two studies, practices changed. The use of guidelines was less successful in the community not only because they did not influence practice but also because usual care had grown closer to the proposed guidelines . Ecological exposure in individual level studies | A frequently encountered particularity of outcomes research study design is the presence of both ecological exposure and individual level covariates in the same analysis. Because the unit of analysis is a group, but inferences are made about the impact of a given practice pattern on individual outcomes, many outcomes research analyses have elements of both individual and ecological analyses . In our study of regional variations in the treatment of acute myocardial infarction, measures describing practice patterns at the regional level, ecological exposure, (proportion of patients receiving angiography, angioplasty, and coronary artery bypass surgery) were linked to the outcome measures of mortality adjusting for individual level variables that measured severity of disease. Then, inferences were made about the use of these procedures at the patient level. Although the unit of analysis is the region, which would demand an ecological analysis, there are individual level covariates, which are likely to be correlated within each region, that need to be taken into account. When group measures are used that contain individual-level variability with some degree of correlatedness (within region) and aggregate-level variability (between regions), specific analytic tools must be used. It has been suggested that hierarchical logistic regression modeling be used to examine the interplay between sources of variation in the use of health-care services, that is, between ecological-level and individual-level sources. This type of modeling is designed to separate true variability across areas from observed variability. An application of this method is the work by Gatsonis et al. who found that practice variations across regions of the U.S. in the use of angiography after acute myocardial infarction were largely explained by differences in patient characteristics and geographic region. However, states that had more on-site availability of angiography still tended to have higher angiography rates after accounting for between-region and within-region variability. After analysis for sources of variability, more reliable inferences about the associations between practice patterns and outcomes can be made. Sources of data | The application of the proposed methodological framework for outcomes research largely depends on the sources of data that are used to evaluate the effect of the practice variations on outcomes . Most commonly, the study design is a retrospective cohort analysis and the dataset that is used has been obtained either for administrative purposes (discharge databases) or for a randomized clinical trial that addressed a different question . Less often, a prospective cohort study is designed to evaluate a particular set of practice guidelines . Although a prospective design provides more control in data collection than a retrospective analysis, both designs are subject to selection, information and confounding biases. The ideal database to use for the evaluation of practice guidelines is one that allows the precise measurement of the practice patterns (exposure) and outcomes (disease) as well as the measurement of potential confounders (severity of illness, precision of diagnosis, socioeconomic characteristics). Unfortunately, such a database probably does not exist. The strength of administrative databases, such as that of Medicare is that they allow the observation of large numbers of patients for which practice patterns can be evaluated as they occur in actual clinical practice. Furthermore, administrative databases allow the observation of practice patterns outcomes associations in large numbers of unselected patients. However, the limitations of such databases include the missing information about potential confounding factors, such as severity of illness, and the limited ability to measure exposure and outcome accurately. Many databases that are not designed for clinical research either mismeasure patient outcomes or fail to capture outcomes that are important to both physicians and patients (such as quality of life and functional status). The control of these biases was the basis of the methodological framework for outcomes research proposed in this chapter. The application of outcomes research methods to practice guideline evaluation | The application of outcomes research methods to practice guideline evaluation can accomplish several goals. One important goal is the evaluation of practice guidelines, that is, to determine to what extent the guidelines accomplished their primary goals after their dissemination. We have suggested the model of chronic disease epidemiology as the methodological framework for outcomes research to evaluate practice guidelines. The steps to evaluate practice guidelines using outcomes research when the basic design is a retrospective cohort study are summarized in Figure Some limitations to the application of this model exist. The reasons for the inability of the proposed methodological framework to deal completely with the intrinsic biases in outcomes research are listed in Figure . They relate mostly to the databases usually used in studies of outcomes research. Figure 3 | Steps to evaluate practice guidelines using outcomes research Steps to evaluate practice guidelines using outcomes research Figure 4 | Reasons for the inability of the proposed methodological framework to deal with biases in outcomes research Reasons for the inability of the proposed methodological framework to deal with biases in outcomes research PMID- 11914163_Summary TI - AB - The proposed methodological framework for outcomes research to evaluate practice guidelines reflects the selection, information and confounding biases inherent in its observational nature which must be accounted for in both the design and the analysis phases of any outcomes research study. Indeed, a major limitation of outcomes research is the inability to account for unobserved heterogeneity that directly correlates with practice patterns and/or health outcomes. This may lend bias to any inferences made about practice variations and outcomes. "Researchers cannot correct for the subtle reason doctors choose one treatment over another for a particular patient. That bias, in turn, can undermine the entire premise of outcomes research" . These are intrinsic properties of outcomes research that can be dealt with only in part, by applying the principles of chronic disease epidemiology. Thus, this proposed methodology can serve as a framework for the conduct of outcomes research in the evaluation of practice guidelines but its application will be limited. PMID- 11914163_Competing interests TI - AB - none declared PMID- 11914163_Pre-publication history TI - AB - The pre-publication history for this paper can be accessed here: PMID- 11914162 TI - Organization specific predictors of job satisfaction: findings from a Canadian multi-site quality of work life cross-sectional survey AB - Abstract | Background | Organizational features can affect how staff view their quality of work life. Determining staff perceptions about quality of work life is an important consideration for employers interested in improving employee job satisfaction. The purpose of this study was to identify organization specific predictors of job satisfaction within a health care system that consisted of six independent health care organizations. Methods | 5,486 full, part and causal time (non-physician) staff on active payroll within six organizations (2 community hospitals, 1 community hospital/long-term care facility, 1 long-term care facility, 1 tertiary care/community health centre, and 1 visiting nursing agency) located in five communities in Central West Ontario, Canada were asked to complete a 65-item quality of work life survey. The self-administered questionnaires collected staff perceptions of: co-worker and supervisor support; teamwork and communication; job demands and decision authority; organization characteristics; patient/resident care; compensation and benefits; staff training and development; and impressions of the organization. Socio-demographic data were also collected. Results | Depending on the organization, between 15 and 30 (of the 40 potential predictor) variables were found to be statistically associated with job satisfaction (univariate analyses). Logistic regression analyses identified the best predictors of job satisfaction and these are presented for each of the six organizations and for all organizations combined. Conclusions | The findings indicate that job satisfaction is a multidimensional construct and although there appear to be some commonalities across organizations, some predictors of job satisfaction appear to be organization and context specific. PMID- 11914162_Background TI - AB - There appears to be no one commonly accepted definition for quality of work life. In healthcare organizations, quality of work life (QWL) has been described as referring to the strengths and weaknesses in the total work environment . Characteristics that describe the overall organization are viewed as part of the behaviour and reward system of the staff working in that setting. Organizational features such as policies and procedures, leadership style, operations, and general contextual factors of the setting, all have a profound effect on how staff view the quality of their work life. QWL is an umbrella term which includes many concepts. Therefore, concentrating on only one job characteristic, whether it is wages or management style, is an inadequate approach to assessing QWL. Because the perceptions held by employees play an important role in their decisions to enter, stay with or leave an organization, it is important that staff perceptions be included when assessing QWL. And although job satisfaction is not QWL, perception of QWL is often assessed using job satisfaction surveys. Previous studies have shown that low job satisfaction is a major cause of turnover among health care providers . In addition, job satisfaction may affect the quality of service and organizational commitment and may be a contributing factor associated with shortages of health care providers . Such findings have recently increased interest in studying job satisfaction among health care providers . The results of a 1993 meta-analysis of 48 studies looking at work satisfaction in over 15,000 nurses revealed that job satisfaction was associated strongly with reduced work stress, organizational commitment, communication with supervisors, autonomy, employee recognition, fairness, locus of control, years of experience, education, and professionalism. This study also found a strong relationship between job satisfaction and QWL for nurses . After reviewing the literature on QWL and job satisfaction, and considering the wide variety of health care settings, situational contexts, and organizational structures (including management styles, reporting structures, staffing complements, and levels of training and experience) in which employees work, we hypothesized that the predictors of job satisfaction would vary depending on the organization. The purpose of this study was to identify organization specific predictors of job satisfaction within a health care system that consisted of six independent and distinct organizations located in five communities in Central West Ontario, Canada. PMID- 11914162_Methods TI - AB - Setting | The settings for this study included six independent and distinct health care organizations providing varying levels and types of care. All six organizations were affiliated with the St. Joseph's Health System (SJHS) located in five Central West Ontario communities. Collectively, the SJHS is one of the largest corporations in Canada devoted to health care. At the time of the study (2000), the SJHS employed 5,486 full, part and casual time (non physician) staff. Additional information about of each of the six organizations and their respective communities is provided in Table . Table 1 | Characteristics of the Organizations within the St. Joseph's Health System. Questionnaire development | Items included in the "Quality of Work Life Survey 2000" were selected after a review of the literature and extensive consultation between research team members and the QWL Task Force (a management group consisting of representatives from each of the six SJHS organizations). The initial selection of items was influenced by a recently published Canadian study and reports from two meta-analyses . The QWL Task Force then refined these items to consider, among other things, issues of accuracy, relevance, readability, grammar, potential for offensiveness, and appearance of cultural or gender bias. After several months of development, the instrument was pretested on a small group of staff at two of the participating organizations (Site 2 and Site 4 -- see Table ). This pretesting was done to ensure that individuals could follow the instructions associated with the format, to obtain estimates of the time required to complete the survey instrument, to identify items that were poorly written or ambiguous, and to identify an appropriate implementation strategy. The questionnaire and implementation strategies were revised accordingly. The final 65-item survey contained nine sections representing topic areas considered relevant to assessing QWL in the SJHS. Eight scale scores were developed from the individual items (see below and : Statistically Significant Organization Specific (Univariate) Predictors of Job Satisfaction). The Co-worker and supervisor support section included 10 closed-ended and 1 open-ended questions. A 3-item supervisor social support scale included questions about supervisor helpfulness, concern about the welfare of employees, and ability to facilitate effective interaction among employees. Co-worker support was measured by a 7-item scale reflecting the extent to which co-workers were seen as competent, understanding, and supportive of employees. Both scales where adapted from Woodward et al. (1999) . The Teamwork and Communication section included 9 closed-ended and 1 open-ended questions. For determining teamwork, a 7-item scale was adapted from Taylor and Bowers (1972) to measure the extent to which one's work unit coordinates efforts, solves problems and works together effectively . A 2-item scale developed for this project measured how communication was practiced within the organization. The Job Demands and Decision Authority section included 15 closed-ended and 1 open-ended questions. It included a 4-item scale adapted from Brosnan and Johnson (1980) to measure clarity regarding responsibilities, workloads and conflicting demands . There was also a 9-item scale adapted from Karasek et al. (1998) to measure the extent to which respondents' jobs gave them autonomy or decision-making latitude , and 2 questions which reflected the demands of one's work . The Characteristics of Your Organization section included 6 closed-ended and 1 open-ended questions. This section was adapted from Woodward et al. (1999) and included a 4-item scale that inquired about the extent to which the organization encouraged the best efforts from staff, and how employees were treated . Two additional questions examined the extent to which staff were kept informed, and organizational recognition of employee contributions. The Patient/Resident Care section included 5 closed-ended and 1 open-ended questions. The questions (developed for this project) were used to measure employees' perceptions of the quality and timeliness of care provided for patients and residents at their respective organizations. The Compensation and Benefits section included 10 closed-ended and 1 open-ended questions. These questions were developed for this project to determine employee satisfaction concerning a number of employee benefits and level of pay. The Staff Training and Development section included 6 closed-ended and 1 open-ended questions. These questions (developed for this project) measured the extent to which each organization supports its staff in training, educational development and opportunities for advancement. The Overall Impressions of Your Organization section included 4 closed-ended and 4 open-ended questions. All of the questions (developed for this project) assessed staffs' impressions of and overall satisfaction with their organization. The question "Overall, how satisfied are you with your job?" was used as the outcome variable in this study. The Staff Socio-Demographic Information section included 10 closed-ended questions (developed for this project) to collect information on gender, age, marital status, education, length of employment, supervisory status, time spent on job activities, job status and job classification. Within each of the first 8 sections, employees were asked to circle the response that best described their feelings using 5-point Likert scales. Employees were also asked for written comments pertaining to each of the sections and were provided space to comment on other issues they felt were important. Survey Procedure | Because of the diversity of organizations and staff within the SJHS, it was decided by the QWL Task Force, organization administrators and researchers that the implementation of the survey would be customized to best fit each of the organizations. It was felt that a varied approach would be more feasible for the organizations and that this would help maximize response rates. Although the procedures were not identical, all of the organizations provided as a minimum: advance notification (written or voice mail) of the survey to all staff (eligibility was based on whether the worker was active on the organization's pay roll at the time of the study and was not a physician); access to questionnaires for all staff (the QWL Task Force felt that each staff member in the SJHS should have the opportunity to complete a questionnaire); one or more reminder notices (e.g., letters, newsletters, voice mail, personal communication); and sealed drop off boxes for completed questionnaires. Pilot testing of the questionnaire revealed that employees felt that tracking individual employees for the purpose of follow-up (i.e., to increase response rate), violated the perception of anonymity and confidentiality. Therefore, to help ensure anonymity and confidentiality, follow-up attempts were limited to general reminder notices to all staff. Analysis | All closed-ended (or quantitative) responses were entered directly from the questionnaires into SPSS (version 10.0.5 for Windows, SPSS, Inc., Chicago, 1999). Prior to data analysis, most of the survey questions were re-coded. Questions which asked participants to select one response within a five point scale (never to always; very dissatisfied to very satisfied; very poor to very good; no, definitely not to yes, definitely) were collapsed into two categories. For example, for the response scale (1=very dissatisfied, 2=dissatisfied, 3=not sure, 4=satisfied, 5=very satisfied) those who indicated they were either satisfied or very satisfied were re-coded as "satisfied" while all others were re-coded "not satisfied" by default. In several instances, it was appropriate to combine two or more of the questions into a composite scale score. See "Questionnaire Development" section and : Statistically Significant Organization Specific (Univariate) Predictors of Job Satisfaction for additional details on how the composite scale scores were calculated. In total, there were eight scale scores (supervisor social support; co-worker support; teamwork; communication; role clarity; decision latitude; organization/staff relations; patient/resident care). Scale scores were generated by summing the participant responses (i.e. one to five) for all questions that made up the scale. In the rare situation where a participant failed to answer one or more of the questions that made up a scale score, missing values were replaced with mean values for that organization. Scale scores were categorized into meaningful dichotomous categories prior to analysis (e.g., satisfied or not satisfied). For the purpose of this study, QWL was operationally defined using the global question "Overall, how satisfied are you with your job?". Employees rated job satisfaction from very dissatisfied to very satisfied using a five point scale (very dissatisfied, dissatisfied, not sure, satisfied, very satisfied). For the analysis, however, those indicating they were either satisfied or very satisfied were considered to be "satisfied" with their jobs. All others were considered "not satisfied" with their jobs. Prior to analysis, study researchers reached a consensus on which survey questions to include as potential predictors of job satisfaction. In total, there were eight scale scores and 32 questions that were rationalized a priori as potential predictors of job satisfaction. Data from each of the organizations, as well as all of the organizations combined (representing the SJHS), were analyzed separately to identify predictors of job satisfaction. T-test, chi-square analyses and, when appropriate, Fisher exact tests were used to determine which of the variables were statistically associated with job satisfaction i.e., were potential predictors of job satisfaction. Descriptive information (numbers and percentages) for each of the variables was calculated by whether or not staff were satisfied with their jobs. In addition, p-values, odds ratios, and 95% confidence intervals for the odds ratios were calculated for each potential predictor of job satisfaction. Separate logistic regression analyses were used to identify the best predictors of job satisfaction for each organization and for all organizations combined (SJHS). Only variables which had a statistically significant association with job satisfaction were included in these analyses. Adjusted odds ratios and corresponding 95% confidence intervals are reported for each organization and the SJHS. The logistic regression analyses produces odds ratios which have been simultaneously adjusted for all other variables in their respective final models. The goodness of fit of the logistic regression models were assessed using the rho-squared statistic . A rho-square value between 0.20 and 0.40 suggests a very good fit of the model. A probability level of <0.05 was used to determine statistical significance. SPSS and Epi-Info (version 6.04a, Centers for Disease Control and Prevention, Atlanta, 1995) were used for statistical computations. PMID- 11914162_Results TI - AB - Table provides additional information about each of the six health care organizations, including the type of organization, number of staff, number of beds or visits/year, and the size of the community where the organization was located. Respondent participation rate | Response rates are often used as an indicator of the representativeness of a sample of respondents. Of the combined 5,486 staff, 1,819 (33.2%) returned a completed questionnaire. Organization specific response rates varied from 25.3% to 55.3% . In an attempt to assess the representativeness of respondents, a comparison was made of available socio-demographic information between respondents and all staff within each of the organizations. Overall, female employees were more likely to respond than male employees (it should be noted, however, that the vast majority of staff (82% to 98%), were females within each of these organizations), as were full-time employees compared to part-time, casual or temporary employees. There were also some differences in respondents, across organizations, based on job classification. All organizations, however, had respondents within each job classification. A statistical estimating procedure was also used to assess how accurately respondents represent staff at each of the organizations . This calculation suggests that the organization specific findings were accurate plus or minus 3.6% to 8.8%, 19 times out of 20 . Table 2 | Response rates and accuracy of responses by organization. Potential predictors of job satisfaction | Organization specific and combined SJHS (univariate) analyses (t-test, chi-square analyses and, when appropriate, Fisher exact tests) were used to determine which of the potential predictor variables were statistically associated with job satisfaction. Included in these analyses were the 40 potential predictor variables (8 scale scores and 32 individual questions). See : Statistically Significant Organization Specific (Univariate) Predictors of Job Satisfaction for a list of all variables. The number of statistically significant variables ranged from 15 to 30 depending on the organization and 32 for all organizations (SJHS) combined (see : Statistically Significant Organization Specific (Univariate) Predictors of Job Satisfaction). Best predictors of job satisfaction | Separate logistic regression analyses were then used to identify the best predictors of job satisfaction for each organization and for all organizations combined (SJHS). All variables found to be statistically associated with job satisfaction from the univariate analyses were entered into these logistic regressions analyses. The best predictors of job satisfaction are presented in Table . The ranking assigned to these variables relates to the order in which variables were added to the logistic regression models. For example, the rank "1" refers to the first variable that was added to the model i.e., the variable which best improved the fit of the model (or the most important variable). A more detailed description of the magnitude (as represented by the size of the odds ratios) and statistical significance (as represented by the 95% confidence intervals of the odds ratios) of the association between each of these predictors and job satisfaction is presented below for each organization and all organizations combined (SJHS). The best predictors of job satisfaction are again ranked according to their importance. All of the odds ratios presented below have been simultaneously adjusted for all other variables in their respective final logistic regression models. All logistic regression models achieved a rho-square between 0.20 and 0.40 suggesting they were very good (fitting) models for predicting job satisfaction. Table 3 | Best Predictors of Job Satisfaction1 Ranked by Organization2. Site 1 (community hospital) | The most important predictors of job satisfaction were: 1) being satisfied with the organization's recognition of employee contributions (OR 5.01, 95% CI 1.59 to 15.81), 2) good decision authority (OR 7.91, 95% CI 1.46 to 42.92), 3) being satisfied with patient resident care (OR 4.66, 95% CI 1.36 to 15.97), and 4) good role clarity (OR 4.24, 95% CI 1.16 to 15.49). The final model achieved a rho-square of 0.30. Site 2 (community hospital/long-term care facility) | The most important predictors of job satisfaction were: 1) good open communication between staff (OR 2.55, 95% CI 1.03 to 6.35), 2) good supervisor social support (OR 6.27, 95% CI 1.36 to 29.00), 3) organization keeps staff informed (OR 3.73, 95% CI 1.51 to 9.20), 4) good decision authority (OR 3.49, 95% CI 1.25 to 9.73), and 5) being satisfied with pay level (OR 2.47, 95% CI 1.14 to 5.34). The final model achieved a rho-square of 0.24. Site 3 (visiting nurse organization) | The most important predictors of job satisfaction were: 1) less frequently (never/seldom/sometimes) asked to do an excessive amount of work (OR 7.22, 95% CI 2.22 to 23.46), 2) being satisfied or very satisfied that the organization keeps employees informed (OR 4.52, 95% CI 1.43 to 14.32), 3) belief the organization carries out its Mission statement (OR 11.17, 95% CI 2.04 to 61.14, and 4) good decision authority (OR 5.29, 95% CI 1.32, to 21.22). The final model achieved a rho-square of 0.34. Site 4 (long-term care facility) | The most important predictors of job satisfaction were: 1) belief the organization carries out its Mission statement (OR 4.63, 95% CI 1.77 to 12.51), 2) good supervisor social support (OR 3.32, 95% CI 1.22 to 9.04), 3) good decision latitude (OR 11.61, 95% CI 1.33 to 101.8), 4) often or always given enough time to get the job done (OR 3.05, 95% CI 1.00 to 9.35), and 5) spending 38 hours or more on the job or job related activities (OR 3.55, 95% CI 1.32 to 9.59). The final model achieved a rho-square of 0.34. Site 5 (community hospital) | The most important predictors of job satisfaction were: 1) belief the organization carries out its Mission statement (OR 3.42, 95% CI 1.82 to 6.43), 2) satisfied that the organization keeps staff informed (OR 2.62, 95% CI 1.48 to 4.65), 3) not being asked frequently to do an excessive amount of work (OR 2.41, 95% CI 1.36 to 4.27), 4) good decision latitude (OR 5.65, 95% CI 2.09 to 15.25), 5) being satisfied with pay level (OR 2.41, 95% CI 1.37 to 4.23), 6) being female (OR 2.99, 95% CI 1.29 to 6.90), and 7) good role clarity (OR 2.45, 95% CI 1.02 to 5.86). The final model achieved a rho-square of 0.25. Site 6 (tertiary care hospital/community health centre) | The most important predictors of job satisfaction were: 1) belief the organization carries out its Mission statement (OR 3.99, 95% CI 2.52 to 6.31), 2) good communication (OR 3.00, 95% CI 1.85 to 4.88), 3) being given enough time to get the job done (OR 2.63, 95% CI 1.58 to 4.40), 4) being a member of the nursing staff (OR 2.73, 95% CI 1.75 to 4.26), 5) good organization support for training and development (OR 3.51, 95% CI 1.59 to 7.76), 6) good decision latitude (OR 2.57, 95% CI 1.30 to 5.09) and 7) being satisfied with the organization's recognition of employee contributions (OR 2.05, 95% CI 1.07 to 3.91). The final model achieved a rho-square of 0.25. All sites combined (SJHS) | The most important predictors of job satisfaction after adjusting for site were: 1) belief the organization carries out its Mission statement (OR 2.79, 95% CI 2.07 to 3.77), 2) good communication (OR 1.87, 95% CI 1.33 to 2.62), 3) less frequently being asked to do an excessive amount of work (OR, 1.80, 95% CI 1.33 to 2.43), 4) good decision latitude (OR 3.28, 95% CI 2.09 to 5.17), 5) being satisfied with pay level (OR 1.61, 95% CI 1.21 to 2.15), 6) being satisfied with the organization's recognition of employee contributions (OR 1.57, 95% CI 1.07 to 2.29), 7) being female (OR 2.83, 95% CI 1.81 to 4.42), 8) good role clarity (OR 1.73, 95% CI 1.17 to 2.56), 9) being satisfied that the organization keeps employees informed (OR 1.35, 95% CI 1.00 to 1.85), 10) good teamwork (OR 1.45, 95% CI 1.01 to 2.09), 11) being given enough time to get the job done (OR 1.57, 95% CI 1.10 to 2.23), and 12) good organization/staff relations (OR 2.02, 95% CI 1.13 to 3.62). The final model achieved a rho-square of 0.26. PMID- 11914162_Discussion TI - AB - The results of this survey were intended to assist decision-makers in identifying key workplace issues, as perceived by employees, in order to develop strategies to address and improve the quality of working conditions for staff within each of the individual health care organizations and the SJHS as a whole. This research represents the first step of an ongoing process to ensure better QWL for employees. In addition to the findings presented here, information from the survey's open-ended written comments have also been summarized for each of the six organizations (L Lohfeld, K Brazil, P Krueger, G Edward, D Lewis, E Tjam, E., personal communication, 2001) and the SJHS as a whole (St. Joseph's Health System Quality of Work Life Technical Reports 2000). This open-ended information provides additional and complementary information to that which is provided in this report. Together, these findings are currently being used by decision-makers at each of the organizations, and the SJHS, in an effort to improve employee QWL. It should be noted that at the time of this survey, all of the hospitals included in this study (as well all other hospitals within the Province of Ontario) were operating in an environment of restructuring and change. This was a time of anxiety for many health care professionals, hospital staff and the general public. In 1996, the Ontario government created a Health Services Restructuring Commission (HSRC) with a four year mandate to restructure Ontario's hospitals and health services system. The HSRC was given authority under the Public Hospitals Act and The Ministry of Health Act to direct public hospitals to change their roles, transfer services and programs, amalgamate or close. The HSRC completed its mandate, announced its decisions and was terminated in March 2000. The timing for this study was after the decisions of the HSRC were announced. All of the organizations included in this study were impacted to varying degrees either directly or indirectly the HSRC decisions. The most notable impacts occurred at Site 1 and Site 2. Site 1 (a community hospital) was ordered closed effective March 2001 with programs and services to be transferred to the other local community hospital while site 2 (a community hospital/long-term care facility) was ordered to transfer its acute care services to the other local hospital in its community thereby becoming a long-term care facility. During the time of the survey, a new building (adjacent to the current facility) for the new long-term care facility was under construction and was scheduled to open in 2002. These contextual issues could have influenced employee responses and therefore the predictors of job satisfaction for all of these organizations, particularly for site 1 and site 2. There are several positive attributes of this study. First, to our knowledge, it is the largest QWL investigation of health care workers in Canada with 1,819 completed interviews. Second, it is also unique in that we collected information from staff at six distinct and functionally diverse health care organizations. Third, because we could not find an "off-the-shelf" QWL instrument that suited our needs and collected all the information desired by key stakeholders, we developed (through a combination of modifying existing instruments and creating our own questions and scales) our own questionnaire. Finally, although the response rates were not as high as we would have hoped, the findings: appear to be consistent with what we expected a priori (the study's investigators had offices within 5 of the 6 organizations thus having inside knowledge about these organizations); appear consistent with the published literature; and were judged credible by management and staff at each of the sites. The statistical estimating procedure to assess how accurately respondents represent staff at each of the organizations also suggest that our findings were fairly representative of staff within these organizations, particularly the larger organizations. PMID- 11914162_Conclusions TI - AB - The results of this research show that job satisfaction is a multidimensional construct and is a product of the global evaluation of one's work place and context. This report provides valuable information about how employees in specific health care settings view their work environment. A number of organization specific predictors of job satisfaction were identified as a result of this study. The implications of these findings are currently being deliberated as they relate to improving QWL within each of the six health care organizations that make up the SJHS. These findings, may also be of relevance and value to employees, researchers, evaluators, human resource planners and administrators of similar health care organizations. The results of this survey can also be used as baseline measures against which the findings of future job satisfaction surveys can be compared. Such comparisons place this type of research within a continuous quality improvement framework. PMID- 11914162_Competing interests TI - AB - None declared. PMID- 11914162_Pre-publication history TI - AB - The pre-publication history for this paper can be accessed here: PMID- 12022922 TI - Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos AB - Abstract | Background | The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro. Results | We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B), alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues. Conclusion | The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase. PMID- 12022922_Background TI - AB - Mos belongs to a small family of cytoplasmic protein serine/threonine kinases having oncogenic activity . It is highly expressed in germ cells but barely detectable in a variety of somatic tissues . Studies in Xenopus oocytes have established a role for c-mos in a) initiation of the maturation process and the meiosis I / meiosis II transition and b) in metaphase II arrest in mature oocytes . In mouse c-Mos is apparently not required for initiation of maturation, however, like in Xenopus it is absolutely essential for the metaphase II arrest . The 124-v-mos oncogene represents one of several transforming gene isolates of the moloney murine sarcoma virus and shows unique constitutive protein kinase activity and enhanced transforming activity when compared to other v-Mos proteins or to c-Mos [,-]. The transforming mechanism of Mos involves signalling through the MAP kinase pathway as phosphorylation of MEK by c-Mos has been demonstrated and mapping analyses have shown that Mos and Raf phosphorylate identical sites on MEK . The upstream events of the Mos/MEK/MAPK signalling cascade have not as yet been identified. In earlier studies we have shown that an activating mechanism of c-Mos is likely to involve a conformational change which is mimicked when a single amino acid is exchanged in the alpha-helix C loop of the kinase domain (Arg145-Gly) resulting in constitutive active c-Mos . Recently Fisher and co-workers proposed an activating mechanism of c-Mos by sequential association with Hsp70 and Hsp90, in addition to phosphorylation . Presence of the activating Arg145-Gly amino acid substitution in 124-v-Mos does not change kinase specificity but is sufficient for constitutive kinase activity . Hence the kinase activity of 124-v-Mos is independent of upstream effectors and we have used this oncogenic Mos derivative to identify substrates for the Mos protein kinase in vitro. Using the baculo virus expression system we have expressed active 124-v-Mos protein kinase, as demonstrated by its ability to auto-phosphorylate, predominantly on serine residues, and to phosphorylate vimentin in vitro. We have analysed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified two novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B and alpha/beta-casein. PMID- 12022922_Results TI - AB - Three tryptic 124-v-Mos peptides include target sites for auto-phosphorylation | We have expressed 124-v-Mos with the baculovirus system in Sf9 insect cells and immunopurified 124-v-Mos using the anti-Mos N13 antiserum . As a control, a Mos-unrelated protein, a synthetic kinase-inactive construct of PKC, PKCgammaK380R, was expressed in Sf9 cells. Mos kinase assays, completed in the presence of [gamma-32P]ATP, were resolved using SDS-PAGE and the Coomassie blue staining of the protein gel showed visible amounts of immunopurified 124-v-Mos (fig. , arrowhead). The corresponding autoradiograph in figure demonstrates that 124-v-Mos is expressed as a constitutive active protein kinase indicated by its ability to auto-phosphorylate in vitro. Further, a parallel kinase reaction was used for phosphoamino acid analyses which confirmed that 124-v-Mos auto-phosphorylation occurred predominantly on serine residues (fig. ) and a two-dimensional resolution of a tryptic digest of auto-phosphorylated 124-v-Mos showed that three tryptic peptides include auto-phosphorylation target sites (fig. ), demonstrating that auto-phosphorylation occurs on multiple sites of the Mos protein . Figure 1 | Constitutive kinase activity of immunopurified 124-v-Mos from baculovirus expressing Sf9 insect cells. Constitutive kinase activity of immunopurified 124-v-Mos from baculovirus expressing Sf9 insect cells. Auto-phosphorylation of immunopurified 124-v-Mos expressed in Sf9 cells is shown in B (Coomassie stained 10% SDS-PAGE) and A (corresponding autoradiograph). Parallel 124-v-Mos kinase assays were subjected to a two-dimensional phosphoamino acid analysis (C) or a tryptic digestion followed by a two-dimensional resolution (D). Arrowheads indicate the origin of sample application in (C,D) and the position of 124-v-Mos (A,B). 124-v-Mos phosphorylates vimentin but not tubulin in vitro | Initially, we tested the kinase activity of 124-v-Mos using previously identified Mos substrates. It has been shown that 124-v-Mos, derived from mos-transformed fibroblasts, phosphorylates vimentin in vitro and as presented here in figure , in vitro kinase assays using immunopurified 124-v-mos from Sf9 insect cells showed strong vimentin phosphorylation. In contrast, tubulin which has been shown to be phosphorylated in vivo and in vitro by Xenopus c-Mos was not a substrate for 124-v-Mos in vitro (fig. ). We have tested tubulin purified from various organs (mouse brain, testis and spleen) either polymerised, unpolymerised or pretreated with phosphatases but in none of these states found tubulin to be phosphorylated by 124-v-Mos (data not shown). Figure 2 | 124-v-Mos phosphorylates vimentin but not tubulin. 124-v-Mos phosphorylates vimentin but not tubulin. In vitro 124-v-Mos kinase assays with either vimentin (C,D) or purified tubulin from brain (A,B) as substrates were electrophoresed using 10% SDS-PAGE and Coomassie stained (B,D), the corresponding autoradiographs are shown in (A,C). Immunoprecipitates of Sf9 cells expressing the kinase-inactive PKCgammaK380R were indicated as controls. Demonstration of alpha and beta-casein phosphorylation by 124-v-Mos | In search of further substrates for the 124-v-Mos protein kinase we tested MBP; histone HI, H2AS, H3; protamine; protaminsulphate; purified PKC-alpha/-beta II/gamma and alpha- and beta-casein. With the exception of alpha- and beta-casein (fig. ) none of these substrates were phosphorylated by 124-v-Mos (data not shown). The possibility that factors other than 124-v-Mos in the immunoprecipitate might be responsible for the observed casein phosphorylation was eliminated by including a synthetic kinase-inactive construct of 124-v-Mos, 124-v-MosK121R, as a control in addition to the Mos-unreleated protein, PKC_K380R. A comparison of background phosphorylation on beta-casein in the immunoprecipitates of both controls and 124-v-Mos specific phosphorylation showed that 124-v-Mos phosphorylates beta-casein 7fold relative to background (fig. ). Critically, a tryptic digest of phosphorylated beta-casein revealed that 124-v-Mos phosphorylates a specific tryptic peptide in beta-casein which shows no background phosphorylation in either controls (fig. , arrowhead) strongly supporting that 124-v-Mos is able to phosphorylate beta-casein. Further, a two-dimensional phosphoamino acid analysis (fig. ) showed that 124-v-Mos phosphorylates alpha- and beta-casein on serine and threonine residues at a ratio of 1:1. Figure 3 | 124-v-Mos phosphorylates alpha- and beta-casein in vitro. 124-v-Mos phosphorylates alpha- and beta-casein in vitro. Mos kinase assays, in the presence of alpha- and beta-casein, were resolved using 10% SDS-PAGE; the Coomassie stained protein gel shown in 3A, right panel and the corresponding autoradiograph on the left panel. Arrowheads indicate the position of 124-v-Mos, alpha- and beta-casein and the antibody. Using two control immunoprecipitates of Sf9 cells expressing the synthetic kinase-inactive constructs, 124-v-MosK121R or PKCgammaK380R, Mos-specific beta-casein phosphorylation was demonstrated in 3B and 3C: Mos kinase assays were blotted on nylon-membrane, the phospho-beta-casein bands (B, arrowhead) excised and 32P-Cerenkov counts recorded (B). Alternatively, the excised phospho-beta-casein bands were digested with trypsin and electrophoresed using 16% SDS-PAGE (C). The arrowhead in 3C indicates the tryptic beta-casein peptide phosphorylated by wild-type 124-v-Mos only. Further, two-dimensional phosphoamino acid analyses of 124-v-Mos phosphorylated alpha-casein (D, left panel) and beta-casein (D, right panel) were completed, the arrowheads indicating the origins of sample application. The protein tyrosine phosphatase 1B is a novel in vitro substrate for 124-v-Mos | Protein tyrosine phosphatases constitute a diverse family of enzymes that can be divided into several subgroups, including receptor and non-receptor PTPs . The non-transmembrane protein tyrosine phosphatase PTP-1B, a major intracellular PTP is widely expressed. PTP-1B has been demonstrated to be phosphorylated on multiple sites in a cell cycle specific manner whereby mitotic hyper-phosphorylation occurs, reflected by a protein mobility shift in SDS-PAGE analyses . Using purified PTP-1B as a substrate, we show here that 124-v-Mos can phosphorylate PTP-1B in vitro (fig. ). We controlled this result by using immunoprecipitates from Sf9 cells expressing the synthetic kinase-inactive 124-v-Mos construct or purified PTP-1B alone in parallel kinase assays (fig. ). Other kinases such as PKC and CKII that phosphorylate PTP-1B in vitro are unable to induce a mobility shift of PTP-1B as observed in mitotic cells . Likewise, as shown in figure , a Mos-dependent phosphorylation did not result in a mobility shift of PTP-1B. Figure 4 | PTP-1B is a substrate for 124-v-Mos in vitro. PTP-1B is a substrate for 124-v-Mos in vitro. In vitro Mos kinase assays, using purified PTP-1B as a substrate, were resolved using 10% SDS-PAGE and the autoradiograph is shown in 4A. Immunoprecipitates of Sf9 cells expressing the kinase-inactive 124-v-MosK121R variant or PTP-1B alone were included as controls (A,B). A parallel kinase assay was blotted on nylon-membrane and PTP-1B was detected (B) using the PTP-1B-specific antiserum FG6 , arrowheads indicate the position of 124-v-Mos and PTP-1B. PMID- 12022922_Discussion TI - AB - In this study we have expressed constitutive active 124-v-Mos using the baculovirus expression system and identified novel in vitro substrates for Mos by immunocomplex kinase assays. It has been shown that 124-v-Mos from mos-transformed mouse fibroblasts phosphorylates vimentin in vitro and that v-Mos is physically associated with vimentin in transformed cells . We have used vimentin as a positive control for 124-v-Mos kinase assays in vitro to demonstrate protein kinase activity of baculovirus expressed 124-v-Mos (fig. ). It is known that the kinase activity of c-Mos is regulated by cellular factors and therefore we have chosen the oncogenic variant of c-Mos, 124-v-Mos, in our study since it is independent of activating mechanisms. Recently it has been shown that Hsp70 and Hsp90 physically interact with c-Mos in Xenopus oocytes and are required for c-Mos activation . Another factor controlling c-Mos kinase activity in Xenopus oocytes was identified by Chen and colleagues to be CKII, a tetrameric holoenzyme composed of two catalytic alpha-subunits and two regulatory beta-subunits . In Xenopus oocytes c-Mos kinase activity is inhibited by binding to the C-terminus of CKII beta-subunit and by over-expression of the alpha-subunit of CKII this effect can be neutralized suggesting a binding competition between c-Mos and the alpha-subunit of CKII . Another protein that interacts with c-Mos in Xenopus oocytes is tubulin. Tubulin not only co-precipitates with c-Mos but also serves as an in vivo and in vitro substrate . In contrast, tubulin was not a substrate for 124-v-Mos in our immunocomplex kinase studies (fig. ). Possibly, this indicates that a cellular factor present in Xenopus oocytes and co-precipitating with c-Mos might be necessary for tubulin phosphorylation by the Mos protein kinase. This factor might not interact with the v-Mos protein, be absent in Sf9 insect cells or unable to interact with v-Mos. Interestingly, we have not detected any co-precipitation of the _-subunit of CKII from Sf9 cells with 124-v-Mos in our immunoprecipitates (data not shown). However, as previously mentioned, Hsp70 is known to interact also with 124-v-Mos . Having established that our recombinant 124-v-Mos protein is active in vitro, we tested a variety of molecules in immunocomplex kinase assays and identified alpha- and beta-casein as very good substrates in vitro (fig. ). This phosphorylation was specific to active 124-v-Mos as the overall phosphorylation on casein was significantly reduced using the synthetic kinase-inactive construct 124-v-MosK121R and more importantly, a tryptic peptide of casein was identified to be phosphorylated by 124-v-Mos only and not by either of the controls used in this study. As expected, casein phosphorylation occured on serine and threonine residues. The Mos-specific consensus phosphorylation site has not as yet been identified and only the mos-phosphorylation sites on MAP kinase kinase have been mapped revealing them to be identical to raf-phosphorylation sites . Using the mos substrates identified in this study, it may be possible to determine the specific consensus phosphorylation site for the mos protein kinase as a basis for developing Mos-specific inhibitors. We have also identified protein tyrosine phosphatase 1B (PTP-1B) as a substrate for 124-v-mos in vitro (fig. ). PTP-1B is phosphorylated on multiple sites in vivo and during mitosis becomes hyper-phosphorylated resulting in a mobility shift in SDS-PAGE . Protein kinase C and CKII phosphorylate PTP-1B in vitro but neither is responsible for the observed mitotic hyper-phosphorylation in vivo . We show here that likewise PTP-1B phosphorylation by 124-v-mos is insufficient to effect a mobility shift (fig. ). PTP-1B phosphorylation occurs on serine 386, a phosphoacceptor site for Cdc2/cyclin B in vitro and serine 352, phosphorylated by an unknown kinase. The serine 352 phosphorylation site either might not be a target for Mos in vitro or PTP-1B may be sequentially phosphorylated by multiple kinases in vivo. Interestingly, it has been shown that PTP-1B hyper-phosphorylation does not occur uniquely in mitosis but also during osmotic shock and is induced by several other stress stimuli . Given that activation of c-Mos is dependent on its interaction with the heatshock proteins, Hsp70 and Hsp90, it is tempting to speculate that the Mos kinase may phosphorylate PTP-1B also in vivo. PMID- 12022922_Conclusions TI - AB - The crucial biological functions of c-mos during meiosis have been analysed by antisense experiments in Xenopus lavis and by generating mos-deficient mice establishing mos as the main player in metaphase II arrest. In contrast, not much is known about activating mechanisms of mos and biochemical properties such as the mos-specific consensus phosphorylation site. In this study we immunopurified an oncogenic and constitutive active variant of mos, 124-v-Mos, and identified novel phosphorylation substrates, PTP1B and alpha- and beta-casein. Our substrates represent a basis to determine the consensus mos-specific phosphorylation site and further, to analyze this phosphorylation ability functionally in vivo. PMID- 12022922_Materials and Methods TI - AB - Protein expression and in vitro immunocomplex protein kinase assays | The construction and isolation of recombinant baculoviruses expressing active 124-v-Mos and the synthetic kinase-inactive variant of 124-v-Mos, 124-v-MosK121R, is described in detail elsewhere . According to the standard procedure published by Summers & Smith , recombinant proteins were expressed at 27C in Sf9 cells for 48 hrs. and mos was immunopurified using the anti-Mos N13 antiserum as stated in . Mos kinase assays were carried out in 50 _l kinase reaction buffer (10 mM HEPES pH 7.3, 150 mM NaCl, 0.1% Triton X-100, 2 mM DTT, 15 mM MnCl2, 5 mM MgCl2, 2.5 mM beta-glycerophosphate, 2.5 mM NaF, 20 muM ATP/ 10 muCi [_gamma32P]ATP), incubated for 20 min. at 25C and stopped by the addition of Laemmli buffer. For in vitro substrate kinase assays, 2 mug of substrate was added to each kinase reaction. Phosphoproteins were resolved using 10% SDS-PAGE, Coomassie stained, dried and compared with the corresponding autoradiograph. Immunodetection of western blots were performed using the ECL system and protocol (Amersham). Substrates for in vitro immunocomplex kinase assays | alpha- and beta-casein (dephosphorylated, bovine origin) were purchased from Sigma and vimentin from Roche. Purified PTP-1B and the PTP-lB-specific antiserum FG6 were provided by N. Tonks, Cold Spring Harbor . Tubulin was purified from either mouse brain, testis or spleen by F. Propst, Vienna. Two-dimensional phosphoamino acid analyses | Two-dimensional phosphoamino acid analyses were completed according to Boyle and colleagues . Briefly, phosphoproteins were separated using SDS-PAGE, blotted on nylon-membrane and the desired protein bands were excised. The membrane strips were washed sequentially with 100% methanol and water and the phosphoproteins hydrolysed for 60 min. at 110C in 5.7 N HCl. The hydrolysed samples were lyophilised, resuspended in 2.5% formic acid, 7.8% acetic acid and mixed at 15:1 with a non-radioactive amino acid standard (1 mg/ml of each phospho-serine, -threonine, -tyrosine; Sigma). Finally, samples were spotted on thin-layer chromatography plates and separated in two dimensions using the HTLE-7000 apparatus and manufacture's procedure (Two-Dimensional Peptide Mapping And Phosphoamino Acid Analysis, Featuring The Hunter Thin Layer Plate Electrophoresis System. B. Boyle & T. Hunter, C.B.S. Scientific Company, Del Mar, USA). First dimension: 20 min. electrophoresis at 0.8 bar, 1 kV in 2.5% formic acid, 7.8% acidic acid. Second dimension: 16 min. at 0.8 bar, 1.3 kV in 5% acidic acid, 0.5 % pyridine. The phosphoamino acids were fixed for 10 min. at 65C and the standard non-radioactive amino acids visualised by spraying the chromatography plates with 0.25% ninhydrin followed by incubation for 15 min. at 65C. The phosphoamino acids were located by comparing the autoradiograph with the stained standard amino acids. Tryptic digests and one- or two-dimensional separation of tryptic phosphopeptides | According to Boyle and colleagues phosphorylated proteins were proteolytically digested with trypsin by incubating twice for 2 hrs. at 37C, on each occasion with 10 mug trypsin (Promega, modified trypsin, sequencing grade) in 200 mul 50 mM NH4HCO3 and a two-dimensional separation of tryptic phosphopeptides was completed using the HTLE-7000 apparatus and manufacture's protocol: electrophoretic separation was performed on thin layer chromatography plates for 25 min. at 0.8 bar and 1 kV, followed by conventional chromatography in 39.25% n-butanol, 30.25% pyridine, 6.1% acetic acid. One-dimensional separation of tryptic phosphopeptides was achieved using 16% SDS-PAGE according to Schagger and von Jagow . PMID- 12022922_List of Abbreviations used TI - AB - Sf9, Spodoptera frugiperda cell line; MAPK, mitogen-activated protein kinase; MEK, MAP and erk kinase; Hsp, heat-shock protein; PTP, protein tyrosine phosphatase; MBP, myelin basic protein; PKC, protein kinase C; CKII, casein kinase II. PMID- 12014993 TI - Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate AB - Abstract | Background | Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. Results | Here we examine ThDP uptake by mitochondria from several human cell types, including cells from patients with thiamine-responsive megaloblastic anemia (TRMA) that lack a functional thiamine transporter of the plasma membrane. Although mitochondria from normal lymphoblasts took up thiamine in the low micromolar range, surprisingly mitochondria from TRMA lymphoblasts lacked this uptake component. ThDP was taken up efficiently by mitochondria isolated from either normal or TRMA lymphoblasts. Uptake was saturable and biphasic with a high affinity component characterized by a Km of 0.4 to 0.6 muM. Mitochondria from other cell types possessed a similar high affinity uptake component with variation seen in uptake capacity as revealed by differences in Vmax values. Conclusions | The results suggest a shared thiamine transporter for mitochondria and the plasma membrane. Additionally, a high affinity component of ThDP uptake by mitochondria was identified with the apparent affinity constant less than the estimates of the cytosolic concentration of free ThDP. This finding indicates that the high affinity uptake is physiologically significant and may represent the main mechanism for supplying phosphorylated thiamine for mitochondrial enzymes. PMID- 12014993_Background TI - AB - Thiamine is a water-soluble, B-complex vitamin that cannot be synthesized by mammals, and thus thiamine can be obtained only from dietary intake. This can lead to severe consequences in humans when thiamine is limiting; thiamine deficiency may result in beriberi and the Wernike-Korsakoff syndrome . Being positively charged and present in relatively low plasma concentrations, thiamine movement across cellular membranes requires transporters. Upon being taken up by a cell, thiamine is rapidly diphosphorylated by thiamine diphosphokinase to give thiamine diphosphate (ThDP) . Thus, thiamine represents only a few percent of the total cellular thiamine/thiamine phosphate derivatives. ThDP serves as a cofactor for several enzymes that are found both in the cytosol (transketolase) and mitochondria (alpha-ketoglutarate dehydrogenase complex being the most studied example). The intracellular concentration of ThDP has been estimated at 30 muM, with only about 7 percent being free cytosolic and the remainder being enzyme-bound with much of this within mitochondria . Previous findings indicate a complex, cell-type dependent regulation of compartmentalization and intracellular pools of thiamine and its phosphorylated derivatives in response to fluctuating extracellular thiamine levels . Hence, thiamine transport, including that by mitochondria, is of interest. Thiamine entry into mammalian cells occurs by a saturable, high affinity transporter that is deficient in humans with thiamine-responsive megaloblastic anemia (TRMA) . Thiamine uptake by mitochondria has been demonstrated , yet thiamine diphosphokinase is cytosolic and mitochondria cannot convert thiamine to ThDP . Barile and coworkers demonstrated saturable uptake of ThDP by rat liver mitochondria characterized by a Km of around 20 muM. Although the estimated concentration, in mice and human cells, of intracellular ThDP is about 30 muM, much of this is found in a low turnover pool representing enzyme-bound ThDP . The estimated concentration of free cytosolic ThDP available for intracellular transport is about 10% of the total concentration and thus 2 to 3 muM . Hence the physiological significance of the mitochondrial ThDP uptake just decribed is uncertain. Within mitochondria, ThDP can be converted to thiamine monophosphate . Thiamine or ThDP entry into mitochondria from TRMA cells has not been studied. Interestingly, ThDP-utilizing enzymes in mitochondria are much less affected (as revealed by loss of activity) upon progressive depletion of thiamine available to TRMA cells than are ThDP-utilizing enzymes of the cytosol . For these reasons, we have examined the uptake of thiamine and especially ThDP by mitochondria from several human cell types, including cells from TRMA patients. PMID- 12014993_Results TI - AB - Uptake of thiamine by cells and mitochondria | Although our interests primarily were in mitochondrial uptake of thiamine and its derivatives, we first examined cellular uptake of thiamine by the lymphoblast cell lines and found thiamine uptake properties typical of other mammalian cells. Figure indicates thiamine uptake by normal lymphoblasts and lymphoblasts derived from a TRMA patient. The high affinity transport of thiamine by normal lymphoblasts is abolished in the presence of a 100 fold excess of unlabeled thiamine. Under such conditions, some uptake continues from a low affinity (Km in the mM range) transport mechanism and/or from diffusion that characterizes thiamine uptake in all mammalian cells examined to date. Using an expanded range of thiamine concentrations from that shown in fig. in multiple experiments resulted in a Km of 1.0 +- 0.9 muM for the high affinity transport by normal lymphoblasts. As expected, lymphoblasts derived from the TRMA patient showed no high affinity thiamine transport as revealed by thiamine uptake being the same in the absence and presence of excess unlabelled thiamine. Figure 1 | Uptake of radioactive thiamine by normal and TRMA lymphoblasts and mitochondria isolated from the lymphoblasts. Uptake of radioactive thiamine by normal and TRMA lymphoblasts and mitochondria isolated from the lymphoblasts. A. Late log phase lymphoblasts from normal (squares) or TRMA individuals (circles) were incubated for 30 minutes with various concentrations of radioactive thiamine. Incubations were carried out in the absence (unfilled symbols) or presence (filled symbols) of a 100 fold excess of non-radioactive thiamine (at each concentration). Cell-associated counts per minute were determined, and the velocity (V) (pmol thiamine per 2 x 106 cells per min.) is plotted versus the concentration (in micromolar) of radioactive thiamine.). Error bars represent SEM for two independent experiments. B. Mitochondria were isolated from lymphoblasts derived from normal (squares) or TRMA individuals (circles) were incubated for 15 minutes with various concentrations of radioactive thiamine. Incubations were carried out in the absence (unfilled symbols) or presence (filled symbols) of a 100 fold excess of non-radioactive thiamine (at each concentration). Mitochondrial-associated counts per minute were determined, and the velocity (V) (pmol thiamine per mg mitochondrial protein per min.) is plotted versus the concentration (in micromolar) of thiamine.). Error bars represent +- SEM for two independent experiments. C. Western anaylsis indicating the presence of the thiamine transporter in plasma membrane fractions and in mitochondrial fractions. Equivalent volumes of subcellular fractions were electrophoretically separated, blotted to a filter, and probed using antisera specific for the human thiamine transporter that is mutated in TRMA individuals. Lane 1, plasma membrane fraction; 2, initial mitochondrial fraction; 3 and 4, successive washes of the mitochondrial fraction; 5, final mitochondrial fraction. 75 micrograms of protein were loaded into each lane with the exception of the lanes containing the washes (3 and 4) which were not quantitated. A faint but reproducible (using different preparations) band was found in the final mitochondrial fraction. Mitochondria isolated from normal lymphoblasts also were found to take up thiamine (fig. ) in a manner similar to that of cellular uptake, with both a high and low affinity component. Using an expanded range of thiamine concentrations in multiple experiments resulted in a Km of 2.1 +- 0.4 muM for high affinity thiamine uptake by normal lymphoblast mitochondria. Interestingly, mitochondria from TRMA lymphoblasts did not possess a "high affinity" thiamine transport capacity as did mitochondria form normal lymphoblasts. No difference in uptake of thiamine by TRMA mitochondria was found in the presence and absence of excess unlabelled thiamine (fig. ). This finding suggests that cellular and mitochondrial uptake of thiamine may be mediated by the same transporter since TRMA is defined by mutation within the thiamine transporter located on the plasma membrane . Using antiserum specific for the human thiamine transporter that is mutated in TRMA individuals, western analysis consistently resulted in a faint but detectable band within the isolated mitochondrial suspension (fig. ), even after extensive and multiple washing. Uptake of ThDP by mitochondria | Although mitochondria from lymphoblasts (above) and other mammalian cells were found to take up thiamine, the physiological significance of the uptake is unknown given that thiamine diphosphokinase is cytosolic and mitochondria cannot convert thiamine to ThDP . We thus were interested in uptake of ThDP by mitochondria, a possibility demonstrated previously with rat liver . Mitochondria from normal human lymphoblasts were able to take up ThDP in a saturable, biphasic manner (fig. ) with a first saturation in the submicromolar range (described below) and a second at much higher concentrations of ThDP. Time course experiments indicated ThDP uptake was linear for at least 20 minutes (data not shown), and uptake was linear with the amount of mitochondrial protein added (inset of fig. ). Figure 2 | The rate of uptake of ThDP by mitochondria isolated from normal lymphoblasts. The rate of uptake of ThDP by mitochondria isolated from normal lymphoblasts. Mitochondria were isolated from normal lymphoblasts and were incubated for 15 minutes with various concentrations of radioactive ThDP. Mitochondrial-associated counts were determined, and the velocity (V) (pmol ThDP per mg mitochondrial protein per min.) is plotted versus the concentration in micromolar of ThDP ([S]). Error bars represent SEM for four independent experiments. The inset shows uptake (V, pmol ThDP per mg mitochondrial protein per min.) versus varying amounts of resuspended mitochondria (mug of protein) in the presence of 2 M radioactive ThDP. Although the biphasic nature of the uptake is readily seen in various plots of the data, the existence of the high affinity component perhaps is best illustrated in fig. in which the uptake at submicromolar concentrations of ThDP is illustrated (open squares). Uptake of ThDP was repeated in the presence of 30 muM non-radioactive ThDP, a concentration that is 100 to about 40 fold excess over the concentration of radioactive ThDP that was used. Given the Km values for the high and low affinity components (see below), this excess should abolish most of the high affinity uptake but have little to no effect on the low affinity uptake. The uptake due solely to the low affinity component was calculated using the kinetic parameters determined for this component and was plotted as open triangles. As seen in fig. , uptake in the presence of 30 muM non-radioactive ThDP (open circles) was essentially identical to that calculated for the low affinity component and abolition of the high affinity uptake indeed was observed. Figure 3 | The rate of uptake of ThDP at submicromolar concentrations by mitochondria from normal lymphoblasts. The rate of uptake of ThDP at submicromolar concentrations by mitochondria from normal lymphoblasts. Mitochondria were isolated from normal lymphoblasts and were incubated for 15 minutes with various concentrations of radioactive ThDP in the absence (open squares) or presence (open circles) of 30 muM nonradioactive ThDP. Mitochondrial-associated counts were determined, and the velocity (V) (pmol ThDP per mg mitochondrial protein 15 min.) is plotted versus the concentration in micromolar of radioactive ThDP ([S]). The calculated V versus [S] for the low affinity component only, using the kinetic parameters for that component, also is plotted (open triangles). Error bars represent +- SEM for four independent experiments in the absence and three experiments in the presence of non-radioactive ThDP. Using the data from 4 independent experiments resulted in the determination of a Km of 0.38 muM for the high affinity (table ) and 115 muM for the low affinity uptake components. The high affinity Km value compares favorably with the estimated 2 to 3 muM intracellular concentration of free ThDP. Table 1 | Kinetic constants for the high affinity component of ThDP uptake by mitochondria isolated from various cell types. Mitochondria isolated from TRMA lymphoblasts took up ThDP in an essentially identical saturable, biphasic fashion as uptake by normal mitochondria (fig. ). The inset compares the Lineweaver-Burk plot of the high affinity uptake component for mitochondria of both cell types. A high affinity Km of 0.60 (table ) was calculated for mitochondrial uptake of ThDP for TRMA lymphoblasts, a value that is essentially the same as that for mitochondria isolated from normal lymphoblasts. The results indicate that although mitochondria from TRMA lymphoblasts cannot take up thiamine with high affinity, they can efficiently import ThDP, the active form of thiamine. Figure 4 | The rate of uptake of ThDP by mitochondria isolated from normal and TRMA lymphoblasts. The rate of uptake of ThDP by mitochondria isolated from normal and TRMA lymphoblasts. Mitochondria were isolated from normal (open squares) or TRMA (open circles) lymphoblasts and were incubated for 15 minutes with various concentrations of radioactive ThDP. Mitochondrial-associated counts were determined, and the velocity (V) (pmol ThDP per mg mitochondrial protein per min.) is plotted versus the concentration in micromolar of ThDP ([S]). The inset shows the Lineweaver-Burk plot (1/V vs. 1/S) of the high affinity component of ThDP uptake for normal (open squares) and TRMA (open circles) derived mitochondria. Previous work has indicated that different cell types may differentially regulate intracellular pools of thiamine and/or its phosphorylated derivatives . Thus, we examined ThDP uptake by several other cell types. As shown in table , high affinity uptake of ThDP by mitochondria from fibroblasts and neuroblastoma cells was essentially the same as that for normal and TRMA lymphoblasts as revealed by the similar Km values. The apparent affinity for ThDP characteristic of the high affinity uptake component was essentially identical for all cell types examined, however variation was seen in transport capacity, as revealed by substantially different values for Vmax (table ). Mitochondria from all of the cell types examined also possessed the low affinity uptake characterized by Km's similar to that of normal lymphoblasts but with a greater range of values being found (20 to 115 muM). The final entry in table is for ThDP uptake by mitochondria isolated from glyB cells. GlyB cells are a Chinese hamster ovary cell line that is deficient in the transport of folate into mitochondria. For reasons discussed below, ThDP uptake was examined in these cells. As seen in table , high affinity uptake was found with kinetic constants similar to those of the human cell types. The low affinity uptake component was also similar to that by human mitochondria (data not shown). PMID- 12014993_Discussion + Conclusion TI - AB - As has been reported for mitochondria of rat liver , mitochondria from human lymphoblasts were found herein to take up thiamine in a saturable manner characterized by a Km of 2.1 muM. Upon entry into a cell, thiamine is rapidly diphosphorylated to ThDP, resulting in a low intracellular thiamine concentration . The Km determined here is about an order of magnitude greater than the estimated intracellular thiamine concentration , raising questions about the efficiency of such uptake. Surprisingly, mitochondria derived from TRMA lymphoblasts lacked the high affinity uptake of thiamine. TRMA is caused by mutations which destroy the high affinity thiamine transporter of the plasma membrane . The similar Km values found for cellular and mitochondrial uptake of thiamine for normal lymphoblasts and the lack of such uptake by TRMA mitochondria and TRMA cells suggests that high affinity thiamine import into mitochondria may be carried out by the same transporter or a variant form, perhaps generated by differential splicing, of that serving on the plasma membrane. Although western analysis using anti-human transporter (that is mutated in TRMA individuals) antiserum supports this interpretation, further experiments need to be carried out to substantiate the suggestion of a shared thiamine transporter between these two membrane systems. Even if true, the physiological significance of thiamine uptake by mitochondria is unknown since mitochondria cannot form ThDP from thiamine . We find that mitochondria from a variety of human cell types efficiently take up ThDP. Uptake is biphasic with a high and a low affinity component. The Km values characteristics of the high affinity uptake component (all around 0.4 muM) are comparable to the estimated intracellular concentration of free (non-enzyme bound) cytosolic ThDP of around 3 muM . This suggests that the high affinity uptake system is the physiologically relevant mechanism responsible for ThDP entry into mitochondria. Earlier work with rat liver mitochondria identified a ThDP uptake system with an estimated Km of around 20 muM . This is of the same order of magnitude that we find for the low affinity component in the human cells examined herein. The previous work used a less sensitive procedure of examining ThDP uptake and did not examine uptake below 10 muM. This would explain the lack of identification in the previous work of the high affinity uptake component. There is a high degree of amino acid similarity among folate transporters and the thiamine transporter of the plasma membrane . Recently, it was found that in murine cells there can be a substantial efflux of ThDP mediated by the reduced folate carrier protein . GlyB cells are a Chinese hamster ovary cell line derivative that are deficient in the transport of folates into mitochondria, and the responsible mitochondrial transporter has recently been identified and its gene cloned . We wondered if the mitochondrial folate transporter was responsible for uptake of ThDP into mitochondria, making analogies to the ability of the plasma membrane folate transporter being able to transport ThDP. However, this is not the case as glyB cells that lack the mitochondrial folate transporter were found to take up ThDP with high affinity kinetics similar to that of mitochondria of human cells. Mitochondria from three human cell types -- lymphoblasts, fibroblasts, and neruoblastoma cells -- all possessed a high affinity ThDP uptake component characterized by equivalent apparent affinity for ThDP as revealed by essentially identical Km values. Previous studies indicate the existence of a complex, cell-type dependent regulation of compartmentalization and intracellular pools of thiamine and/or its phosphorylated derivatives in response to fluctuating extracellular thiamine levels . The cell lines used here were also used in the studies leading to this conclusion. Clearly, differences in mitochondrial transporter affinities do not contribute to the cell-type dependent regulation of ThDP compartmentalization. However, we did find significant differences in ThDP uptake capacity with respect to cell type as revealed by variation in the Vmax values. Neuroblastoma mitochondria possessed the largest uptake capacity, having a Vmax 4 to 30 fold higher than that of the other cell types examined. Interestingly, of the three cell types neuroblastoma cells also are the most resistant to changes in mitochondrial ThDP-utilizing enzyme activity upon progressive depletion of the thiamine made available to the cells . This suggests that cell-dependent variation in ThDP uptake capacity by mitochondria may contribute to the cell-dependent regulation of ThDP compartmentalization. As such regulation was most clearly revealed upon progressively depleting thiamine from cells , it will be of interest to examine possible changes in mitochondrial transport capacity in response to thiamine depletion. Studies on the sensitivity of ThDP-utilizing enzymes to progressive depletion of thiamine that is available to the cell indicate that such enzymes in mitochondria are significantly less sensitive than cytosolic enzymes in TRMA cells . This could be interpreted as efficient import of thiamine/ThDP into mitochondria in TRMA cells even though thiamine inefficiently enters these cells due to the lack of the high affinity thiamine transporter. Although we found a lack of mitochondrial uptake of thiamine in TRMA cells, our finding of an intact, high affinity ThDP transport mechanism for TRMA mitochondria is consistent with and offers an explanation for such an interpretation. PMID- 12014993_Materials and Methods TI - AB - Radiochemicals | [3H]thiamine (1 Ci/mmol, radiochemical purity greater than 97%) and [3H]thiamine diphosphate (1.4 Ci/mmol, radiochemical purity greater than 98 %) were purchased from Moravek Biochemical Inc (Brea, CA). Cell culture | Normal lymphoblasts, TRMA lymphoblasts and fibroblasts cell lines were obtained and have been characterized as described . The human neuroblastoma cells were an SY-SY5Y cell line, a thrice-cloned subline of SK-N-SH . GlyB cells, a Chinese hamster ovary K1 subline, are deficient in the transport of folate into mitochondria and were a kind gift from L. Chasin (Columbia University). All cell types were growth at 37C in the presence of 10 muM thiamine in RPMI 1640 medium supplemented with 10% heat-inactive fetal calf serum, 2 mM L-glutamine and 1 g/L penicillin/streptomycin, except glyB cells which were grown in MEM medium. Cells were grown and used at late log phase or at 80 --90% confluency. Cellular thiamine transport | Cells were harvested, washed four times with 40 ml of ice-cold transport buffer (145 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4), titered, and preincubated for 30 min. at 37C after resuspending 3 x 107 cells in 1 ml of transport buffer. Various amounts of [3H]thiamine (to give submicromolar and micromolar final concentrations) were added and the reactions were incubated for 30 min. The specific activity of the radioactive thiamine was the same for each concentration used. The cells were collected by rapid filtration onto glass fiber filters (type A/E, Gelman Sciences, Ann Arbor, MI) and washed via filtration with 10 ml of cold transport buffer. After thorough drying (overnight at 60C), the amount of labeled thiamine taken up by the cells was determined by scintillation counting . For each concentration, the uptake in the presence of a 100-fold excess of unlabelled thiamine was performed to assess the contribution to uptake from a low affinity (Km in the mM range) component and/or from diffusion . Isolation of mitochondria | Mitochondria were isolated from about 3 x 108 cells according to published procedures . The final mitochondrial pellet was suspended in suspension buffer (140 mM KCl, 0.3 mM EDTA, 5 mM MgCl2, 10 mM HEPES, pH 7.4) to give a protein concentration of 3 --4 mg/ml. Protein concentration was determined using the Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). The isolation and purity of the mitochondrial preparations were monitored by western analysis using anti-cytochrome C antiserum and by microscopy. Western analysis also was performed on the subcellular fractions using antiserum raised against a human thiamine transporter-specific peptide. The antiserum detects a protein of 55 KD (predicted size of the plasma membrane thiamine transporter) that is not detected in cells from TRMA individuals that possess a premature stop codon within the transporter gene (unpublished results). Uptake of thiamine and ThDP by mitochondria | Uptake of thiamine and ThDP by mitochondria was determined by a rapid filtration procedure . Incubations were performed at 37C by rapidly mixing 30 mul of mitochondrial suspension (ca. 100 micrograms of protein) with 220 mul of incubation buffer (140 mM KCl, 0.3 mM EDTA, 5 mM MgCl2, 10 mM Mes, pH 6.5) containing labeled thiamine or ThDP at various concentrations. The uptake was stopped at 15 min. by the addition of 2 ml of ice-cold stop buffer (100 mM KCl, 100 mM mannitol, 10 mM potassium phosphate, pH 7.4) and the mitochondria were collected by rapid filtration on 0.45 muM Millipore membrane filters. The filters were immediately washed with 5 ml of stop buffer via filtration, and they were subsequently dried at 60C overnight. The amount of labeled thiamine or ThDP taken up by the mitochondria was determined by scintillation counting. Background binding was determined by using a 100 fold excess of unlabelled thiamine or ThDP in parallel reactions. PMID- 12014993_Authors' Contributions TI - AB - QS carried out most of the experiments and participated in writing the manuscript. CKS conceived of the study, participated in its design and coordination, performed a few of the thiamine uptake by cells experiments, and participated in writing the manuscript. PMID- 12014993_Abbreviations TI - AB - thiamine diphosphate (ThDP); thiamine-responsive megaloblastic anemia (TRMA) PMID- 11972899 TI - Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis AB - Abstract | Background | Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. Results | Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. Conclusions | We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed. PMID- 11972899_Background TI - AB - Recently, homologs of the well-known green fluorescent protein (GFP) from jellyfish Aequorea victoria were discovered in Anthozoa species . These proteins can be subdivided into two main types. First type, fluorescent proteins (FPs), emit a significant portion (25 --80%) of the absorbed photons. Second type, chromoproteins (CPs), effectively absorb but practically do not emit light. Peculiarities of structure that make each GFP-like protein fluorescent or non-fluorescent are poorly understood to date. Only the importance of position 148 (we will use numbering in accordance to GFP, see Fig. ) was demonstrated in experiments on appearance of fluorescence in CPs . Introduction of Ser148 into several CPs made them clearly fluorescent, although the emission brightness of these mutants was significantly lower in comparison with wild type FPs. Figure 1 | Sequence alignment of asCP, GFP, and DsRed proteins. Sequence alignment of asCP, GFP, and DsRed proteins. The numbering is based on GFP. Introduced gaps are represented by dashes. The residues whose side chains form the interior of the beta-can are shaded. Mutations introduced in asCP and DsRed are designated under and below their sequences, respectively. Due to a great and still growing popularity of GFP and novel FPs in biotechnology, a comprehension of structure-function correlations in GFP-like proteins has both a scientific and a practical significance, showing novel possibilities to achieve desirable protein properties artificially. Here, we applied mutagenesis to a chromoprotein asFP595 (asCP) and a red fluorescent protein drFP583 (DsRed) to study transformation of a chromoprotein into a fluorescent protein and vise verse. PMID- 11972899_Results TI - AB - Although sequence comparison of known GFP-like proteins does not reveal absolutely invariable differences between FPs and CPs, one can draw attention to three positions, specifically, 148, 165, and 203, which are occupied by noticeably different residues in the two types of proteins (Fig. , Table ). Since residues at these positions are in a close proximity to chromophore , it is reasonable to presume that they can participate in the determination of the state (fluorescent or non-fluorescent) of a particular protein. Figure 2 | Schematic outline of the chromophores and selected neighboring residues in GFP (A), DsRed (B, D), and DsRed-NF (C, E, F) in "sticks" and "spacefill" representation. Schematic outline of the chromophores and selected neighboring residues in GFP (A), DsRed (B, D), and DsRed-NF (C, E, F) in "sticks" and "spacefill" representation. Carbon atoms are gray, nitrogen atoms are blue, and oxygen atoms are red. Images were generated by RasMol 2.6 software. Computer modeling for DsRed-NF was performed using Swiss-PdbViewer and HyperChem 5.01 software. Table 1 | Amino acids occupying positions 148, 165, and 203 (GFP numbering) in known GFP-like proteins. Random mutagenesis of asCP at position 148 | Earlier, we demonstrated for several CPs that Ser-148 containing mutants possess red fluorescence . To check other residue we fulfilled mutagenesis using degenerated primers encoding any amino acid at position 148. Visual inspection of about 50 recombinant clones and sequence analysis of the selected clones showed the following. Only Ser148 ensured clear fluorescence. Several intensively colored non-fluorescent clones contained Ala, Cys, Asn, or Gly at position 148 (remarkably, known wild type CPs carry the very Ala, Cys, or Asn at this position). All other substitutions of Ala148 appeared to be intolerable for proper protein folding and chromophore maturation. Mutagenesis of asCP at position 165 | First of all, we tested a substitution S165V because several FPs carry Val at this position. This mutation resulted in the appearance of a clearly visible red fluorescence with a maximum at 620 nm (Fig. , Table ). Interestingly, in comparison with the wild type asCP, the mutant asCP-S165V showed a strongly modified absorption spectrum which included an additional peak at 390 nm. Absorption at this wavelength produced a very weak (about 10-fold less than the red fluorescence) blue fluorescence at 465 nm. Figure 3 | Normalized spectra for selected mutants of asCP and DsRed. Normalized spectra for selected mutants of asCP and DsRed. Absorption (black solid lines), excitation (colored dashed lines), and emission (colored solid lines) spectra are shown for each mutant. Blue, green, or red excitation-emission lines correspond to color of fluorescence. (A) asCP-S165V. Blue fluorescence is about tenfold weaker than red. (B) asCP-S165A. (C) asCP-S165C. (D) asCP-S165T. Green emision is about twofold stronger than red. (E) DsRed-NF. Green emission peak is about threefold lower than red one. Table 2 | Spectral characteristics for some mutants of asCP and DsRed. To reveal other substitutions at position 165 that could lead to fluorescence appearance we exploited randomization at this position. As a result, several red fluorescent clones of different brightness were selected. The most bright clones carried the already known substitution S165V. All other fluorescent mutants were considerably (5 --10 fold) dimmer and contained Ala, Cys, or Thr165 (in decreasing brightness order). Absorption spectra for these mutants have a characteristic peak at about 390 nm, but it produces no detectable blue fluorescence . An interesting feature of these low fluorescent mutants is that their excitation spectra for red emission do not coincide with the absorption spectra. This phenomenon implies the existence of different spectral forms within a spectrally heterogeneous population of the mutant protein molecules. Red emitting spectral forms are underrepresented or they possess a very low extinction coefficient. At the same time, the major red light-absorbing spectral forms are non-fluorescent. Random mutagenesis of asCP | To extend the search of amino acid substitutions that are able to convert asCP into a fluorescent protein, we used random mutagenesis of the whole asCP gene. Visual screening of about 5000 recombinant clones revealed only one brightly fluorescent colony. Sequence analysis showed that this fluorescent mutant contained the already known substitution A148S. After a more thorough visual inspection we found several very weakly fluorescent clones containing the following substitutions: S68G; I72N; H176R/K219I; H203R; H203Q; Q220L . Importantly, two independent clones carrying different substitutions at position 203 were collected. Summing up, this experiment has not highlighted novel important sites, because all random mutants were considerably (3 --5 fold) dimmer than the mutants at positions 148 and 165 mentioned above. One can conclude that positions 148, 165 are probably the most important sites that influence the state of asCP. Mutagenesis of DsRed | Finally, we attempted to transform the fluorescent DsRed into a chromoprotein. First of all, mutation S148A was tested. Unexpectedly, this substitution did not exert a strong influence on the fluorescence -- quantum yield for DsRed-S148A mutant decreased by a factor of 1.5 only in comparison to the wild type protein . Then, on the base of this mutant, a series of mutants carrying substitutions I165S, K167M, and S203A,L in different combinations was generated. Position 167 was added to mutagenesis considering the crystallographic studies that revealed a direct interaction between Lys167 and chromophore's Tyr66 . This bond appeared to stabilize the ionized form of the DsRed fluorophore. Mutant proteins containing Leu203 were colorless because of unsatisfactory protein folding in E. coli. Following the spectral properties of other mutants, one can notice a gradient of emission intensity and conclude that all positions mentioned above are important for DsRed fluorescence . However, even a quadruple mutant S148A/I165S/K167M/S203A displayed a clearly visible fluorescence comparable to that of some asCP fluorescent mutants (e.g., asCP-S165V). Thus, this DsRed mutant can not be regarded as a true chromoprotein, although it is very close to the CP state because it possesses hundredfold decreased fluorescence in comparison to DsRed. Then, we tested Cys and Asn that are characteristic for some other known CPs at positions 148 and 165, respectively. Triple mutant DsRed-S148C/I165N/S203A possessed a low quantum yield similarly to the mutant S148A/I165S/K167M/S203A mentioned above. When a substitution K167M was added, the final quadruple mutant S148C/I165N/K167M/S203A became practically non-fluorescent . At the same time, this mutant named DsRed-NF intensively absorbed light. Altogether, these properties make DsRed-NF practically indistinguishable from wild type CPs. Spectra for DsRed-NF are shown in Fig. . An extremely weak dual-color fluorescence can be detected at high protein concentration only. Similarly to the low fluorescent mutants of asCP mentioned (see Fig. ), absorption and excitation spectra for DsRed-NF strongly differ from each other. Interestingly, excitation spectrum for green emission displays 2 peaks: a major peak at 410 nm and a minor peak at 490 nm. Such a shape of the excitation curve is similar to that of wild type GFP and has never been detected for DsRed mutants (to date, only EGFP-like single-peak excitation spectra were described for green-emitting mutants of DsRed ). Probably, the short-wave excitation peak corresponds to a neutral (protonated) form of GFP-like chromophore within DsRed-NF. PMID- 11972899_Discussion TI - AB - Great diversity of fluorescent and non-fluorescent colors in the family of GFP-like proteins poses an challenging problem of understanding its structural background. Mutagenetic studies lately demonstrated various transitions of fluorescence color in Anthozoa proteins: from red to green , from yellow to green, from green to yellow, and from green to red . Also, red and far-red fluorescent mutants of non-fluorescent CPs were generated . Basic investigation of relationship between fluorescent and non-fluorescent GFP-like proteins was the main goal of the present work. However, some practical applications of the results obtained can be considered. The first part of our work, attempts to convert asCP into FP, revealed importance of position 165 for fluorescence appearance. This finding can be applied on other CPs. To date, mutagenesis of natural CPs is the only way to generate a far-red FPs that are in high demand for various applications. Additional far-red fluorescence color broadens abilities of multicolor labeling and assays based on fluorescence resonance energy transfer (FRET). Knowledge about the ways of transforming CPs into FPs could help to generate novel far-red FPs when novel CPs with red-shifted absorption spectra are found. The second part of our work was to transform DsRed into CP. At first glance, such fluorescence quenching can not be used in practice. However, we found that DsRed-NF mutant can be used to resolve a problem of DsRed tetramerization that is the main disadvantage of this tag . When DsRed is fused with a target protein, especially with oligomeric protein, it often results in improper folding and functioning of the tagged partners as well as intensive aggregation of the fusion protein. To neutralize injurious consequences of DsRed tetramerization we suggest to use a simultaneous co-expression of DsRed-tagged proteins with excess free DsRed-NF. In this case mixed heterotetramers are formed so that DsRed becomes a "monomeric" tag (this approach will be published elsewhere). It was recently demonstrated that DsRed and asCP carry chemically distinct chromophores . Theoretically, spectral differences between DsRed and asCP and generally between FPs and CPs may be explained by the diversity of their chromophores. If so, the appearance of fluorescence in asCP mutants and the disappearance of fluorescence in DsRed mutants should resulted from formation of altered chromophores within these mutants. Alternatively, one may suggest that each chromophore type in GFP-like proteins can be fluorescent or non-fluorescent depending on the protein environment. Some observations speak in favor of this hypothesis. First, all key residues mentioned above (positions 148, 165, 167, and 203) are grouped in a close proximity to the phenolic ring of Tyr66 . Thus, they can more likely participate in stabilization and positioning of the chromophore but not in chromophore cyclization events that result in the diversity of chromophores. Second, asCP demonstrates a striking phenomenon of light-induced reversible increasing of fluorescence . This photoconversion clearly shows that an initially non-fluorescent protein molecule can be switched into a fluorescent state due to some conformation changes. It is well-known that GFP-like chromophores and other chromophores that are capable of cis-trans isomerization are practically non-fluorescent in solution because of fast relaxation of the excited state through chromophore isomerization . Probably, chromophore in FPs must be strongly stabilized by the amino acid environment to ensure high quantum yield, while chromophore surrounding within CPs should be more relaxed to allow energy of absorbed light to dissipate into heat. From this point of view, we can draw the following scheme of DsRed chromophore stabilization. According to the crystal structure of DsRed Ser148 and Lys167 hold the chromophore by a direct interaction with phenolate oxygen . Bulky Ile165 supports the ring of Tyr66 and prevents its movement required for the chromophore isomerization . Although Ser203 has no direct H-bonds with the chromophore in the wild type DsRed, such bonds could be formed in mutants with altered 148, 165 and 167 positions. Possibly, Ser203 in DsRed mutants can turn similarly to GFP Thr203 that forms an H-bond with chromophore's phenolate oxygen . Quantitative data on the influence of each substitution on fluorescence intensity speak in favor of this scheme. Comparing in pairs quantum yields for the available DsRed mutants that differ from each other by one substitution (see Table ), one can note the following. The contribution of each substitution strongly depends on mutation order: the later the substitution is introduced the stronger the impact is. For instance, the mutant S203A demonstrates the same quantum yield as the wild type protein. At the same time, an addition of S203A to the mutant S148A leads to a 1.5-fold decrease in quantum yield. Then, introducing Ala-203 into a double mutant S148A/K167M results in a 2.4-fold decreased fluorescence. Analogously, mutation K167M leads to 2-, 3.2-, or 8.6-fold decrease of quantum yield when Met167 is introduced as second, third or fourth substitution, respectively. Also, 4.8- or 12.9-fold decrease of fluorescence intensity is associated with substitution I165S added to S148A/S203A or S148A/K167M/S203A mutants, respectively. The model of several chromophore-stabilizing interactions mentioned above implies such tendency because the importance of each interaction must progressively increase in absence of one, two or more other bonds. Computer modeling of the chromophore environment within DsRed-NF showed the following . In contrast to Ser148 and Lys167 in DsRed, Cys148 and Met167 in DsRed-NF are incapable of stabilizing the chromophore by H-bonds with phenolate oxygen. Moreover, substitution I165N generates a vacant space near the chromophore (compare Fig. and ). We believe that this space is sufficient to ensure the chromophore cis-trans isomerization after light absorption . Thus, absence of phenolate-stabilizing interactions together with free space around the chromophore can explain an extremely low fluorescence quantum yield of DsRed-NF. Unfortunately, no protein structures for CPs were published to date. Obviously, further crystallographic studies of FPs, CPs, and their mutants are required to make valid conclusions about the structural background of differences between FPs and CPs. PMID- 11972899_Conclusions TI - AB - The ability for fluorescence of GFP-like proteins depends to a great extent on the surrounding of the phenolic ring of the chromophore. For asCP chromoprotein, mutations at positions 148 and 165 can lead to red fluorescence appearance. For DsRed red fluorescent protein, fluorescence can be quenched by mutagenesis at positions 148, 165, 167, and 203. This knowledge can be applied to other GFP-like proteins in effort of customizing spectral characteristics of FPs and CPs. PMID- 11972899_Materials and Methods TI - AB - Mutagenesis and protein expression | Site-directed mutagenesis was performed by PCR with primers containing target substitution using the overlap extension method . The Diversity PCR Random Mutagenesis kit (Clontech) was used for random mutagenesis of asCP, in conditions optimal for 4 --5 mutations per 1000 bp. All mutants were cloned into pQE30 vector (Qiagen), so that recombinant proteins contained 6-histidine tag at their N-termini. To express mutant proteins E. coli XL1 Blue cells were transformed with the plasmids according to standard protocols and spread onto 3 --4 Petri dishes with LB agar media supplemented with ampicillin for selection. After overnight growth at 37C the plates were stored for 2 --5 days at room temperature or 4C to allow proteins to mature completely. Then, the plates were washed with PBS. Cells were disrupted by sonication, and soluble recombinant proteins were purified on the TALON metal-affinity resin (Clontech). Spectroscopy | Absorption spectra were recorded on a Beckman DU520 UV/VIS Spectrophotometer. A Cary Eclipse Fluorescence Spectrophotometer (Varian) was used for measuring excitation-emission spectra. For molar extinction coefficient determination, we relied on measuring mature chromophore concentration rather than total protein concentration. DsRed and its mutants were alkali-denatured with equal volume of 2 M NaOH. asCP and its mutants were acid-denatured with equal volume of 2 M HCl. Under these conditions, DsRed and asCP chromophores absorb at 452 and 430 nm, respectively . The amounts of chromophore (that correspond to amounts of matured protein) were equalized among samples, absorption spectra for the native proteins were collected. Absorbance intensities were compared to that of DsRed (extinction coefficient is 75,000 M-1cm-1) or asCP (extinction coefficient is 56,000 M-1cm-1), and molar extinction coefficient for each mutant was estimated. For quantum yield determination, the fluorescence of the mutants was compared to equally absorbing DsRed (quantum yield for DsRed was measured to be 0.70 ). PMID- 12019031 TI - Expression of monolysocardiolipin acyltransferase activity is regulated in concert with the level of cardiolipin and cardiolipin biosynthesis in the mammalian heart AB - Abstract | Background | Monolysocardiolipin acyltransferase (MLCL AT) catalyzes the acylation of monolysocardiolipin to cardiolipin in mammalian tissues. We previously reported that cardiac cardiolipin levels, MLCL AT and cardiolipin synthase activities were all elevated in rats made hyperthyroid by thyroxine treatment. In this study, we examined if cardiac mitochondrial MLCL AT activity was dependent upon the biosynthesis and level of cardiolipin in the heart. Rat heart mitochondrial MLCL AT activity was determined under conditions in which the levels of cardiac cardiolipin and cardiolipin synthase activity were either reduced or unaltered using four different disease models in the rat. In addition, these parameters were examined in a murine model of cardiac cell differentiation. Results | In rats made hypothyroid by treatment with 6-n-propyl-2-thiouracil in the drinking water for 34 days, cardiac cardiolipin content was decreased 29% (p < 0.025) and this was associated with a 32% decrease (p < 0.025) in cardiolipin synthase and a 35% reduction (p < 0.025) in MLCL AT activities. Streptozotocin-induced diabetes or hyperinsulinemia in rats did not affect cardiac cardiolipin content nor MLCL AT and cardiolipin synthase activities. Finally, cardiolipin content, MLCL AT and cardiolipin synthase activities were unaltered during murine P19 teratocarcinoma cell differentiation into cardiac myocytes. In all models, phospholipase A2 activities were unaltered compared with controls. Conclusion | We propose a general model in which the expression of MLCL AT activity is regulated in concert with the biosynthesis and level of cardiolipin in the heart. PMID- 12019031_Background TI - AB - Phospholipids are important structural and functional components of the biological membrane . Structurally, as major components of the biological membrane, they define compartmentalization of organelles and the protective barrier, the cell membrane, which surrounds cells. An important class of phospholipids are the polyglycerophospholipids. Cardiolipin (CL), the first polyglycerophospholipid discovered, was isolated from beef heart by Pangborn in 1942 . In the heart, CL represents approximately 12 --16% of the entire cardiac phospholipid mass and is found exclusively in mitochondria . In mammalian tissues CL is required for the reconstituted activity of a number of key mitochondrial enzymes involved in cellular energy metabolism including for example cytochrome c oxidase, carnitine palmitoyltransferase, creatine phosphokinase, pyruvate translocator, mono-, di- and tricarboxylate carriers, glycerol-3-phosphate dehydrogenase, phosphate transporter, ATP/ADP translocase and ATP synthase . Under experimental conditions in which CL was removed or digested away from these proteins with phospholipases, denaturation and complete loss in activity of many of these proteins was observed. CL interaction with these proteins was specific since substitution with other phospholipids did not fully reconstitute activity. The fatty acyl composition of CL also appeared to be important for this functional reconstitution. For example, the activity of delipidated rat liver cytochrome c oxidase was reconstituted by the addition of CL . The specific activity of reconstituted cytochrome c oxidase varied significantly with different fatty acyl compositions of CL. A strong positive correlation has been established between fatty acid unsaturation of CL and antioxidant production in cells . In staurosporine-treated granulosa cells undergoing apoptosis CL levels were reduced . Peroxidation of CL induced release of cytochrome c from mitochondria into the cytosol and this was associated with the induction of apoptosis . Suppression of CL peroxidation inhibited release of cytochrome c from mitochondria . Thus, the activities of the enzymes that synthesize and remodel CL play a pivotal role in maintaining the content and molecular composition of CL and hence may regulate a plethora of cellular processes from energy metabolism to apoptosis. In mammalian tissues CL is synthesized by condensation of phosphatidylglycerol with cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol catalyzed by CL synthase [for review see ]. Thyroxine treatment of rats stimulated the expression of rat liver and heart mitochondrial CL synthase activities . The increase in CL synthase activity accounted for the elevated levels of CL observed in these organs. We recently identified and characterized the activity of monolysocardiolipin acyltransferase (MLCL AT), the enzyme responsible for monolysocardiolipin (MLCL) acylation to CL in mammalian tissues . In another study, we showed that thyroxine treatment of rats elevated cardiac MLCL AT activity and postulated that MLCL AT may be a rate-limiting enzyme for the molecular remodeling of CL in the heart . The above studies prompted us to examine if cardiac MLCL AT activity was linked to CL biosynthesis and content in the heart. Our results, using four different disease models in the rat in which the level of cardiac CL is either reduced, elevated or unaltered, demonstrate that this is the case. We also observe this relationship in a model of murine cardiac cell differentiation. PMID- 12019031_Results TI - AB - Cardiac CL content, CL synthase and MLCL AT activities are reduced in hypothyroid rats | In previous studies we observed that cardiac MLCL AT activity was elevated when the cardiac CL content and CL synthase activity were elevated in hyperthyroid rats . We examined if cardiac CL content, CL synthase and MLCL AT activities were reduced in hypothyroid rats. Rats were made hypothyroid by the addition of 0.05% PTU to their drinking water for 34 days. This protocol was shown to produce decreased serum thyroid hormone levels and result in cardiac atrophy in the rat . As seen in Table , in rats that received PTU there was a 48% decrease (p < 0.025) in heart weight compared to controls. In addition, the heart to body weight ratio decreased, indicative of cardiac atrophy. Growth failure was demonstrated by the decreased body weights of the hypothyroid animals compared to controls. As a further control, the activity of an inner mitochondrial membrane marker, succinate dehydrogenase, was determined. Cardiac mitochondrial succinate dehydrogenase activity was reduced 23% (p < 0.025) from 30 +- 3 mumol/minmg to 23 +- 3 mumol/minmg protein in PTU-treated rats. These are documented characteristics of hypothyroidism . Table 1 | Body weight, heart weight, heart CL content, CL synthase and MLCL AT activities in normal and hypothyroid rats. Heart mitochondrial fractions were prepared from rats made hypothyroid by the addition of 0.05% PTU to their drinking water for 34 days and CL content, CL synthase and MLCL AT activities determined. We initially determined the activity of cardiac mitochondrial PA:CTP cytidylyltransferase, a rate-limiting enzyme of CL biosynthesis . PA:CTP cytidylyltransferase activity was 15.1 +- 1.2 pmol/min/mg protein and unaltered (14.7 +- 1.1 pmol/min/mg protein) in cardiac mitochondrial fractions prepared from hypothyroid rats. Hence, PA:CTP cytidylyltransferase served as a control for a mitochondrial enzyme not affected by hypothyroidism. When compared to controls, heart mitochondria prepared from hypothyroid rats exhibited a 29% decrease (p < 0.025) in CL content, a 32% decrease (p < 0.025) in CL synthase activity and a 35% decrease (p < 0.025) in MLCL AT activity . PLA2 activity was 4.2 +- 0.7 nmol/minmg protein and unaltered (4.0 +- 0.5 nmol/minmg protein) in cardiac mitochondria prepared from hypothyroid rats. Thus, cardiac mitochondrial CL content, CL synthase and MLCL AT activities were all reduced in hypothyroid rats. Cardiac CL content, CL synthase and MLCL AT activities are unaltered in streptozotocin-induced diabetic rats and in hyperinsulinemic rats | Previously we showed that cardiac phosphatidylglycerol levels were reduced in streptozotocin-induced diabetic rats but CL synthase activity and CL content were unaltered . We examined if streptozotocin-induced diabetes in rats altered MLCL AT activity or if hyperinsulinemia in rats altered CL synthase and MLCL AT activities in cardiac mitochondria. Rats were made diabetic by injection of steptozotocin or hyperinsulinemic by intraperitoneal addition of insulin. Subsequently, the hearts were removed and mitochondrial fractions prepared. Cardiac CL synthase activities were 3.0 +- 0.5 pmol/minmg protein in hyperinsulinemic rats and did not differ from control (3.1 +- 0.6 pmol/minmg protein) non-insulin injected animals. Cardiac CL content was 5.9 +- 0.5 nmol/mg heart and unaltered compared to controls (6.1 +- 0.1 nmol/mg heart). Cardiac MLCL AT activities were 38 +- 6 pmol/minmg protein in diabetic rats and 43 +- 9 pmol/minmg protein in hyperinsulinemic rats and did not differ from controls (40 +- 6 pmol/minmg protein saline injected and 41 +- 9 pmol/minmg protein non-insulin injected animals, respectively). Thus, in streptozotocin-induced diabetes and hyperinsulinemia, conditions in which the CL content and CL synthase activities were unaltered, MLCL AT activity was unaltered. CL content, CL synthase and MLCL AT activities are unaltered during cardiac cell differentiation | As a distinct model, we examined if CL synthase activity was altered in murine P19 teratocarcinoma cells induced to undergo differentiation into cardiac myocytes. We chose this model since differentiation of murine P19 cells into cardiac myocytes results in an increase in phosphatidylethanolamine biosynthesis, phosphatidylethanolamine mass and lysophosphatidylethanolamine acyltransferase activities . The cells were harvested at various times, 0 --8 days post DMSO addition, and MRNA analysis of markers of cardiac cell differentiation performed on cell lysates. GATA-4 is a member of the GATA family of zinc finger transcription factors and is an early marker of cardiac cell differentiation. As seen in Figure , GATA-4 was expressed at 4 days post DMSO addition relative to the constitutive expression of tubulin. As expected GATA-4 expression preceded the expression of other cardiac genes including B-natriuretic peptide (BNP), alpha myosin heavy chain (alphaMHC), beta myosin heavy chain (betaMHC) and Troponin C relative to the constitutive expression of tubulin. Thus, the P19 cells used in this study differentiated into cardiac myocytes. As previously shown CL content, MLCL AT and PLA2 activities were unaltered during P19 cell differentiation into cardiac myocytes . CL synthase activity was 2.7 +- 0.5 pmol/minmg protein in undifferentiated and unaltered (2.8 +- 0.3 pmol/minmg protein) in differentiated P19 cells. Together, the above five models using hyper- and hypothyroid, diabetic and hyperinsulinemic rats and murine P19 cell differentiation into cardiac myocytes all indicate that expression of mammalian cardiac mitochondrial MLCL AT activity appears to be regulated in concert with the biosynthesis and content of CL in the heart. Figure 1 | Expression of genes during differentiation of P19 cells into cardiac myocytes. Expression of genes during differentiation of P19 cells into cardiac myocytes. P19 cells were incubated with 1% DMSO for up to 8 days. At various times, 0 --8 days post DMSO addition, cells were harvested and mRNA levels of GATA-4, BNP, alpha MHC, beta MHC, troponin C and tubulin were determined by quantitative RT-PCR analysis. PMID- 12019031_Discussion TI - AB - Previous and current studies in the mammalian heart and liver support the hypothesis that CL content is regulated in concert with the level of CL synthase activity | In the CL biosynthetic pathway, PG is converted to CL by condensation with CDP-DG catalyzed by CL synthase . In vitro studies have indicated that alteration in cellular CL levels appears to have functional consequences. For example, reduction in the content of CL was shown to reduce oxygen consumption in mitochondria prepared from rat liver . Thus, maintenance of the appropriate content of CL in mammalian mitochondria is essential for proper mitochondrial function. Thyroxine treatment of rats was shown to stimulate the activity of rat liver mitochondrial CL synthase 2.5-fold . This elevation in rat liver mitochondrial CL synthase activity was suggested to account for the elevated levels of CL observed in livers prepared from hyperthyroid rats. In addition, CL synthase was shown to be elevated in heart mitochondria prepared from hyperthyroid rats and this was correlated with an increase in cardiac CL content . Prior to the current study, CL synthase activity had not been determined in any model of hypothyroidism. Hypothyroidism in the rat resulted in a 25% reduction in cardiac CL synthase activity. This reduction in CL synthase activity likely accounted for the reduced levels of cardiac CL observed in hearts prepared from hypothyroid animals. Previous studies in the rat have indicated that hypothyroidism also results in reduced CL levels in the liver [for review see ]. Thus, it is reasonable to assume that CL synthase activity would also be reduced in the liver of hypothyroid animals. In the current study, CL synthase activity was unaltered in diabetic and hyperinsulinemic rats and in a model of murine cardiac cell differentiation. In these models, the content of CL was unaltered. These data suggest that the level of CL produced in the mammalian heart is regulated in concert with the level of CL synthase activity. Previous and current studies in the heart support the hypothesis that cardiac MLCL AT activity may be regulated in concert with CL content and CL synthase activity | The data presented in this paper are entirely consistent with the conclusion that the expression of MLCL AT activity in the heart is regulated in concert with the biosynthesis and content of cardiac CL. Previously, we demonstrated that thyroxine-treatment of rats resulted in an increase in cardiac CL content, CL synthase and MLCL AT activities . In the present study rats made hypothyroid with PTU in the drinking water had reduced cardiac CL content, CL synthase and MLCL AT activities. In contrast, in streptozotocin-induced diabetes and hyperinsulinemia, pathological conditions in which cardiac mitochondial CL content and CL synthase were unaltered , MLCL AT activities were unaltered. In addition, CL content, CL synthase and MLCL AT activities were unaltered during cardiac cell differentiation. It is reasonable to propose that when the rate of synthesis and level of CL is either reduced or elevated expression of the activities of the enzymes that remodel CL should be correspondingly reduced or elevated. The activity of cardiac mitochondrial PLA2 was high (100-fold) relative to cardiac mitochondrial MLCL AT activity and was unaltered in all models examined . Since MLCL AT activity was either increased or decreased under conditions in which elevated or reduced CL remodelling was required, i.e. elevated or reduced CL synthesis, it is possible that MLCL AT may be rate-limiting for MLCL acylation to CL in the mammalian heart. However, it should be considered that other factors such as the intra-mitochondrial level of MLCL may be limiting for the acylation of MLCL to CL. PMID- 12019031_Conclusions TI - AB - A summary of our findings is presented in Table . In hyperthyroidism, when cardiac CL synthase activity and CL content are elevated an increase in MLCL AT activity is observed. In hypothyroidism, when cardiac CL synthase activity and CL content are reduced a decrease in MLCL AT activity is observed. Finally, when cardiac CL synthase activity and CL content are unaltered in streptozotocin-induced diabetes, hyperinsulinemia and murine P19 cell differentiation into cardiac myocytes, MLCL AT activity is unaltered. Thus, expression of MLCL AT activity is regulated in concert with the biosynthesis and content of cardiac CL. Table 2 | Summary of mammalian cardiac mitochondrial MLCL AT activities, CL synthase activities and CL content in various cardiac models. PMID- 12019031_Materials and Methods TI - AB - Male Sprague Dawley rats (125 --175 g) were used throughout the study and were housed in a temperature and light controlled room. They were maintained on Purina rat chow and tap water ad libitium. Treatment of animals conformed to the Guidelines of the Canadian Council on Animal Care. Rats were made hypothyroid by administration of (0.5% w/v) 6-n-propyl-2-thiouracil (PTU) in their drinking water for 34 days. Rats were made diabetic by injection of 60 mg/Kg steptozotocin. Hyperglycemia was confirmed 24 h later by urine and blood glucose analysis. Rats were made hyperinsulinemic by intraperitoneal addition of 3 units/day of insulin for 28 days. Murine P19 teratocarcinoma cells were kindly provided by Dr. Mona Nemer, Institute of Cardiovascular Research, University of Montreal, Montreal, Quebec, Canada. [1-14C]Linoleoyl-Coenzyme A was obtained from American Radiochemical Co., St. Louis MO. All other radiochemicals were obtained from Dupont, Winnipeg, Canada. MLCL was obtained from Avanti Polar Lipids, Alabaster, AL. Thin-layer chromatography (Silica gel 60, 0.25 mm thickness) plates were obtained from BDH, Toronto, Canada. Cell culture and reagents were products of Canadian Life Technologies (GIBCO) Burlington, Ontario, Canada. Ecolite scintillant was obtained from ICN Biochemicals, Costa Mesa, CA.. Lipids standards were obtained from Serdary Research Laboratories, Englewood Cliffs, NJ., USA. All other biochemicals were of analytical grade and obtained from either Fisher Scientific, Edmonton, Canada, Sigma Chemical Co., St. Louis, MO. or CanLab Division of Baxter Co. Winnipeg, Canada. The protocol for differentiation and culturing of murine P19 teratocarcinoma cells into cardiac myocytes was performed as described . P19 cells (5 x 105 cells/ml) were placed into 60 mm bacterial dishes, 1% dimethylsulfoxide (DMSO) was added and incubation continued for 48 h. The cells began to aggregate at this point. Cells were then transferred to a 100 mm bacterial dish and 1% DMSO was added for another 48 h. The cells in these 100 mm bacterial dishes differentiated into the cardiac cell lineage within eight days. At various days (0 --8) post DMSO addition cells were harvested and mRNA expression of GATA-4, BNP, alphaMHC, betaMHC, troponin C and tubulin were determined using quantitative RT-PCR analysis as described . A 10% homogenate from rat hearts or P19 cells was prepared in buffer (0.25 M sucrose, 0.145 M NaCl, 10 mM Tris-HCl, pH 7.4) and centrifuged for 10 min at 600 x g (Beckman J2-H with JA-20 rotor). The resulting pellet was washed once, resuspended in 5 ml buffer by 15 strokes of a hand-held Dounce (tight fitting) tissue grinder and designated the crude nuclear fraction. The supernatant from the first centrifugation was centrifuged at 10,000 x g for 10 min. The resulting pellet was resuspended in 1.5 ml buffer as described above and used as the source of mitochondrial fraction for enzyme assays. Protein in this fraction was determined by the method of Bradford . Phospholipase A2 (PLA2) was determined as described using phosphatidyl [14C]glycerol as substrate . CL synthase and phosphatidic acid (PA):CTP cytidylyltransferase activities were determined as described . MLCL AT activities were determined as described . Mitochondrial fractions (50 mug) were incubated for 30 min at 25C in 50 mM Tris-HCL, pH 8.0, 33 muM [1-14C]linoleoyl-Coenzyme A (68,700 dpm/nmol), 0.3 mM MLCL in a final volume of 0.35 ml. The reaction was initiated by the addition of [1-14C]linoleoyl-Coenzyme A substrate and terminated by addition of 3 ml of chloroform:methanol (2:1, by vol). 0.8 ml of KCL was added to facilitate phase separation. The aqueous phase was removed and the organic phase dried under nitrogen and resuspended in 25 mul of chloroform:methanol (2:1, by vol). A 20 mul aliquot was placed on a thin layer plate and CL was separated from other phospholipids in a solvent system containing chloroform:hexane:methanol:acetic acid (50:30:10:5, by vol). The silica gel corresponding to CL was removed and placed in plastic scintillation vials with 5 ml of aqueous counting scintillant. Radioactivity incorporated into CL was determined approximately 24 h later using a liquid scintillation counter. CL content was determined as described . Mitochondrial succinate dehydrogenase activity was determined as described . Students t-test was used for the determination of statistical significance. The level of significance was defined as p < 0.025. PMID- 12019031_List of abbreviations TI - AB - CL, Cardiolipin; MLCL AT, monolysocardiolipin acyltransferase; MLCL, monolysocardiolipin; PTU, 6-n-propyl-2-thiouracil; PLA2, phospholipase A2; PA, phosphatidic acid; DMSO, dimethylsulfoxide; CTP, cytidine-5'-triphosphate; ATP, adenosine-5'-triphosphate; ADP, adenosine-5'-diphosphate; BNP, B-natriuretic peptide; alphaMHC, alpha myosin heavy chain; betaMHC, beta myosin heavy chain; mRNA, messenger ribonucleic acid; STZ, Stre ptozotocin PMID- 12019031_Authors contribution TI - AB - Mr. William A. Taylor intitiated the experimental and edited the manuscript. Dr. Fred Y. Xu performed experimental studies related to the P19 cells and edited the manuscript. Mr. Brian J. Ma performed experimental studies related to the hyperinsulinemic rat model and edited the manuscript. Dr. Thomas C. Mutter performed experimental studies related to the hypothyroid rat model. Mr. Vernon W. Dolinsky initiated experimental studies related to the hypothyroid rat model and edited the manuscript. Prof. Grant M. Hatch conceived of the study, participated in its design and coordination, wrote and edited the manuscript. All authors have read and approved the final manuscript. PMID- 12052259 TI - The role of the Zn(II) binding domain in the mechanism of E. coli DNA topoisomerase I AB - Abstract | Background | Escherichia coli DNA topoisomerase I binds three Zn(II) with three tetracysteine motifs which, together with the 14 kDa C-terminal region, form a 30 kDa DNA binding domain (ZD domain). The 67 kDa N-terminal domain (Top67) has the active site tyrosine for DNA cleavage but cannot relax negatively supercoiled DNA. We analyzed the role of the ZD domain in the enzyme mechanism. Results | Addition of purified ZD domain to Top67 partially restored the relaxation activity, demonstrating that covalent linkage between the two domains is not necessary for removal of negative supercoils from DNA. The two domains had similar affinities to ssDNA. However, only Top67 could bind dsDNA with high affinity. DNA cleavage assays showed that the Top67 had the same sequence and structure selectivity for DNA cleavage as the intact enzyme. DNA rejoining also did not require the presence of the ZD domain. Conclusions | We propose that during relaxation of negatively supercoiled DNA, Top67 by itself can position the active site tyrosine near the junction of double-stranded and single-stranded DNA for cleavage. However, the interaction of the ZD domain with the passing single-strand of DNA, coupled with enzyme conformational change, is needed for removal of negative supercoils. PMID- 12052259_Background TI - AB - Escherichia coli DNA topoisomerase I is a representative example of type IA DNA topoisomerase (for reviews, see refs ). Its major biological role in the bacterial cell is the removal of excessive negative supercoils from DNA to maintain the DNA at optimal superhelical density along with DNA gyrase . The enzyme has a molecular weight of 97 kDa and the active site tyrosine responsible for DNA cleavage is found in the 67 kDa N-terminal transesterification domain. The structure of this 67 kDa domain has been determined by X-ray crystallography to be torus-like, indicating the need for protein conformational change for strand passage to take place after DNA cleavage . Relaxation activity requires the presence of the Zn(II) binding tetracysteine motifs found between the 67 kDa N-terminal domain (Top67) and the 14 kDa C-terminal single-stranded DNA binding domain . The three tetracysteine motifs do not form a stably folded structure on its own, but when combined with the 14 kDa C-terminal domain, forms a stably folded 268 amino acid DNA binding domain (ZD domain) that has higher affinity for single-stranded DNA than the 121 amino acid 14 kDa C-terminal region by itself . Recent sequence and structural analysis suggests that the 14 kDa domain is evolutionarily related to the three tetracysteine motifs and belongs to the zinc ribbon family . The ZD domain in E. coli topoisomerase I probably evolved from a domain that binds five Zn(II) originally. Figure 1 | Domain organization of E. coli DNA topoisomerase I. Domain organization of E. coli DNA topoisomerase I. Removal of negative supercoils from DNA by bacterial type IA topoisomerase involves the following steps: (1) binding of the enzyme to the junction of double-stranded and single-stranded DNA ; (2) cleavage of a single-strand of DNA near the junction with cleavage sequence preference of a cytosine in the -4 position to form the covalent intermediate ; (3) conformational change of the covalent enzyme-DNA complex to result in physical separation of the 5' phosphate covalently linked to the active tyrosine, and the 3' hydroxyl of the cleaved DNA; (4) passage of the complementary single strand through the break; (5) enzyme conformational change to bring the 5' phosphoryl end back into the proximity of the 3' hydroxyl group of the cleaved DNA; (6) religation of the phosphodiester bond. Although it is known that the ZD domain can function as a DNA binding domain, its exact role in these individual steps of removal of a negative superhelical turn from DNA by E. coli topoisomerase I remains to be defined. Using purified 67 kDa transesterification domain and 30 kDa ZD domain, results from experiments described here provide new insight into the action of these two individual domains in the enzyme mechanism. PMID- 12052259_Results TI - AB - Partial restoration of relaxation activity from mixing of Top67 and ZD domains | As reported previously , the N-terminal transesterification domain Top67 by itself did not exhibit any relaxation activity when assayed with negatively supercoiled plasmid DNA . The 30 kD C-terminal ZD domain also had no relaxation activity by itself, as expected. Partial relaxation of the input supercoiled DNA was detected when Top67 was mixed with the ZD domain prior to addition of DNA. A ratio of 2 ZD molecules added for each Top67 was found to be sufficient for maximum relaxation activity, with no increase in activity when higher ratio of ZD/Top67 was used (data not shown). The specific activity observed under this optimized condition was still about 10 fold lower than that of the intact enzyme. Analysis of the time course of relaxation with 6 pmoles of topoisomerase I or top67 reconstituted with ZD showed that negative supercoils were removed at a much slower rate by the reconstituted activity. Figure 2 | Partial restoration of relaxation activity by complementation of Top67 and ZD domains. Partial restoration of relaxation activity by complementation of Top67 and ZD domains. (a). Agarose gel electrophoresis was carried out to analyze the relaxation reaction products after 1 h of incubation. Lane 1: supercoiled plasmid DNA with no protein added; lane 2: Top67 alone; lane 3: ZD alone. Lanes 4 --7 (Top67 reconstituted with ZD) and lanes 8 --11 (topoisomerase I) have 6, 1.2, 0.24 and 0.05 pmoles of proteins added. (b). Time course of relaxation reaction catalyzed by 6 pmoles of topoisomerase I, or Top67 reconstituted with ZD Top67 and ZD domains have comparable binding affinities to single-stranded DNA but significantly different affinities for double-stranded DNA | The gel mobility shift assay was used to compare the binding affinities of Top67 and the ZD domain to a 5' end-labeled single-stranded oligonucleotide 35 base in length. As shown in Figure , these two domains had similar affinities for binding to the single-stranded substrate. The half maximal binding values based on the average of results from three different experiments were 0.02 muM for Top67 and 0.04 muM for the ZD domain. However, with the same oligonucleotide in a duplex form , Top67 exhibited much higher affinity (half maximal binding value = 0.07 muM) than the ZD domain (half maximal binding value > 5 muM). Figure 3 | Binding of Top67 and ZD to single-stranded and double-stranded DNA. Binding of Top67 and ZD to single-stranded and double-stranded DNA. The gel mobility shift assay was used to compare the binding affinities. The substrates used are (a): single-stranded 5'GAAAACTCACAGGAAGCGGCCGAAGCGATTCGTCC 3'; (b): the same labeled strand of hybridized to its complementary strand. Open circles: Top67; solid circles: ZD. Top67 can recognize cleavage sites preferred by E. coli DNA topoisomerase I | Previous studies have shown that E. coli DNA topoisomerase I cleavage of single-stranded DNA occurs with selectivity for sites with the C nucleotide base at the -- 4 position and that the enzyme preferentially cleaves at junctions of double-stranded and single-stranded DNA . Several different 5'-end labeled substrates were prepared and used in cleavage assays to compare the cleavage sites selected by Top67 versus topoisomerase I. The results showed that with single-stranded substrates, Top67 also preferred cleavage sites with a C nucleotide base at the -4 position as reported for most of the type IA topoisomerases . There were some differences from topoisomerase I in the relative distribution of cleavage products among the potential cleavage sites . Top67 appeared to be more non-discriminatory in selection of the possible cleavage sites with the C nucleotide in the -4 position. Addition of the ZD domain had no effect on the cleavage selectivity of Top67. A substrate with both single-stranded and double-stranded regions was constructed to mimic such junction in negatively supercoiled DNA. Top67 and topoisomerase I recognised the same cleavage site on this substrate . Maximal yield of cleavage products was obtained for both Top67 and topoisomerase I within seconds after mixing of the enzyme and DNA so any potential difference in cleavage rates between the Top67 and topoisomerase I is unlikely to account for the difference in relaxation efficiency. Figure 4 | Cleavage selectivity of topoisomerase I and Top67. Cleavage selectivity of topoisomerase I and Top67. This was analyzed using single-stranded 32mer (a), 31mer (b), or substrate with both single- and double-stranded regions (c). For (a) and (b), lane 1: no protein added, lane 2: topoisomerase I, lane 3: Top67, lane 4: Top67 mixed with ZD. For (a), lane 5: ZD alone. For (c), lane 1: topoiosmerase I, lane 2: Top67, lane 3: no protein added, lane 4: DNase I digestion pattern. Top67 cleavage sites are religated upon addition of high salt and Mg2+ | To test the religation capability of Top67, a 5'-end labeled oligonucleotide 61 base in length was first incubated with the enzyme in low ionic strength buffer to allow formation of the cleaved complex. Sodium chloride concentration was then increased to 1 M to induce reversal of cleavage and dissociation of the enzyme from the DNA. We observed that more complete and consistent reversal of cleavage was obtained with both topoisomerase I and Top67 if a low concentration of Mg2+ (4 mM) was also added with the NaCl. This is consistent with an early observation of dissociation of the enzyme-DNA complex in high salt upon addition of Mg2+. It has also been reported that addition of Mg2+ was apparently not required for observation of this reversal of cleavage. However, it is possible that some enzyme preparations may contain bound Mg2+ and the low concentration of bound Mg2+ might have been sufficient for reversal of cleavage, as postulated previously to explain the data . The results of this cleavage reversal experiment indicated that the ZD domain was not required for efficient reversal of cleavage and Top67 could carry out religation of cleaved DNA. Again the reversal of cleavage was complete for both Top67 and topoisomerase I within seconds after the addition of high salt and Mg2+ even when the reactions were carried out on ice (data not shown) so the lack of relaxation activity by Top67 is unlikely to be due to deficiency in religation. Figure 5 | Reversal of DNA cleavage by topoisomerase I and Top67. Reversal of DNA cleavage by topoisomerase I and Top67. A 5'-end labeled 61mer was used as substrate. C: no enzyme added. Lane 1: enzyme cleavage reaction stopped with SDS; Lane 2: enzyme cleavage reaction incubated with 1 M NaCl before SDS treatment; Lane 3: enzyme cleavage reaction incubated with 1 M NaCl and 4 mM MgCl2 before SDS treatment. The ZD domain is not required for catenation of double-stranded DNA circles | E. coli topoisomerase I can catalyze catenation of double-stranded DNA circles if the molecules contain single-strand scissions . To test if the Top67 can carry out double-stranded DNA passage at enzyme cleavage sites across from the DNA nicks, the yield of DNA catenanes were compared with that obtained with topoisomerase I. In contrast to the relaxation activity, the catenating activity of Top67 shown in figure was as efficient as that of full-length topoisomerase I, and the addition of the ZD domain had no effect . The rate of catenane formation for Top67 alone was similar to that of topoisomerase I . This catenation activity observed with topoisomerase I and Top67 was unlikely to be due to contaminating topoisomerase III activity since it was not observed with the ZD domain purified under almost identical procedures and a site-directed mutant with substitution of the active site Tyr319 by phenylalanine also did not exhibit this activity . Figure 6 | Catenation of nicked double-stranded DNA circles by topoisomerase I and Top67. Catenation of nicked double-stranded DNA circles by topoisomerase I and Top67. (a). Phage PM2 DNA circles with one or more single-strand scissions were incubated with 5 pmoles of proteins for 1 h. Lane 1: no enzyme added; Lane 2: topoisomerase I; Lane3: Top67; Lane 4: Top67 and ZD domain; Lane 5: ZD domain; Lane 6: mutant with Y319F substitution. (b). Aliquots of the reaction for topoisomerase I and Top67 were removed at different time points to analyse the time course of catenation. PMID- 12052259_Discussion TI - AB - There are two homologous type IA topoisomerases present in E. coli. Topoisomerase III has potent DNA decatenating activity for resolution of plasmid DNA replication intermediates, but much weaker relaxation activity than topoisomerase I . To exhibit maximal relaxation activity, topoisomerase III requires high temperature (52C) along with low magnesium and monovalent ion . In contrast, E. coli topoisomerase I was not active in the in vitro assay for resolution of plasmid DNA replication intermediates . Removal of the C-terminal 49 amino acids from the 653 amino acid topoisomerase III protein resulted in drastic reduction of catalytic activity . Fusion of the carboxyl-terminal 312 amino acid residues of E. coli topoisomerase I, which includes the entire ZD domain, onto the 605 N-terminal amino acids of topoisomerase III generated a hybrid topoisomerase that has relaxation activity resembling topoisomerase III along with weak decatenating activity . Although preferring single-stranded DNA as binding substrate, topoisomerase I had been shown to also bind double-stranded DNA , but there is no data available to indicate which domain in the enzyme is responsible for this interaction. The experiments described here measured directly the interaction of the ZD domain with both double-stranded and single-stranded DNA substrates. ZD domain was found to bind to single-stranded DNA, but not double-stranded DNA, with high affinity. This result indicated that with regard to the mechanism of E. coli topoisomerase I, the ZD domain was likely to function as a single-stranded DNA binding domain instead of having double-stranded DNA binding function as previously suggested . Even though Zn(II) binding transcription factors that recognise specific double-stranded DNA are well represented in eukaryotes , there are also numerous examples of Zn(II) coordination being required for interaction with single-stranded nucleic acid or damaged DNA with single-strand characteristics . The effect of removal of the ZD domain on the individual step of enzyme action was also investigated using Top67. The results indicated that Top67 was effective in binding to both double-stranded and single-stranded DNA. As a result, Top67 could position itself in the absence of ZD domain at the junction of double- and single-stranded DNA for subsequent DNA cleavage, as previously observed for intact topoisomerase I . Reversal of DNA cleavage also took place readily with Top67 upon addition of 1 M NaCl and 4 mM MgCl2. The ZD domain also was not required for selectivity of a cytosine in the -4 position relative to the cleavage sites. Despite its ability to recognise the DNA substrate and carry out DNA cleavage-religation, Top67 by itself cannot catalyze change of linking number in the relaxation of supercoiled DNA. The single-strand DNA substrate designated for the ZD domain in the catalytic mechanism of the enzyme may be the strand of DNA complementary to the strand first cleaved by the enzyme to form the covalent complex. This interaction with the passing strand of DNA would not be needed for the first two steps of enzyme mechanism up to the formation of the covalent complex. Our results showed that adding the purified ZD domain partially restored the relaxation activity. Therefore the ZD domain can supply the function that is missing in Top67 even when the two domains are not covalently linked. However, the resulting relaxation activity is much less efficient than that of the intact enzyme, suggesting that coordinated actions of the two domains are required for efficient removal of negative supercoils from DNA. The requirement of specific protein-protein interactions between the two domains could also account for the weak relaxation activity observed for the hybrid topoisomerase with ZD linked to topoisomerase III sequence . This proposed role for the ZD domain in interacting with the passing single-strand of DNA is also supported by the observation that there is no difference between Top67 and intact topoisomerase I in the formation of catenanes. This reaction involves passage of another double-stranded DNA circle, instead of the complementary DNA strand through the break generated by DNA cleavage so the ZD domain would not be expected to play any significant role. High concentration of DNA substrate is required to favor formation of catenanes catalyzed by topoisomerase I, and the enzyme also has to be present in higher concentration compared to the relaxation reaction. The double-stranded DNA-binding activity in E. coli topoisomerase III required for highly efficient decatenation activity is attributed to a 17-amino-acid residue with no counterpart in E. coli topoisomerase I . It may be required for interaction with the passing double-strand of DNA in the decatenation mechanism. The presence of this decatenation loop instead of the Zn(II) binding ZD domain in topoisomerase III may account for the dominance of the decatenation activity over the relaxation activity. Based on these results, we propose a model for the relaxation of supercoiled DNA by E. coli topoisomerase I modified from previous versions that have a number of common features but differ most significantly in the role of the Zn(II) binding domain . In this model, the subdomains in Top67 is responsible for interacting with the G-strand of DNA both upstream and downstream of the cleavage site. The ZD domain interacts with the passing single-strand DNA to be transported (T-strand). After cleavage of the DNA gate strand which becomes covalently linked to Tyr319 on Top67 (step 2), protein conformational change involving both Top67 and the ZD domain increases the distance between the covalently bound 5' phosphate and non-covalently bound 3' hydroxyl of the cleaved DNA gate strand while the passing DNA strand (T-strand) is guided through the "gate" via interaction with the ZD domain (step 3) to lead to change in linking number. A second enzyme conformational change positions the cleaved DNA ends for religation (step 4). The ZD domain can still interact with the T-strand of DNA even when not linked to Top67 in the same polypeptide, but efficiency of catalysis is reduced as a result, probably due to loss of coordinated action by the two domains. The presence of the ZD domain may enhance the transition of Top67 from a closed conformation to a more open conformation so that strand passage can take place through the "DNA gate". Previous data showed that although Zn(II) binding is not absolutely required for formation of the cleaved complex, it increased the amount of cleaved complex that can be isolated . When linked to Top67, the ZD domain also has some influence on the cleavage site selections. It has previously been observed that a mutation in the Zn(II) binding motif can affect the cleavage site selectivity of topoisomerase I even though Top67 by itself can recognize both the cytosine in the -4 position and the junction of single- and double-stranded DNA. To gain further details for this model of enzyme action, we are characterizing the protein-protein interactions between the Top67 transesterification domain and the ZD domain, as well as the protein conformational changes that can take place when the enzyme interacts with DNA substrate. Figure 7 | Model for removal of a negative supercoil by E. coli DNA topoisomerase I. Model for removal of a negative supercoil by E. coli DNA topoisomerase I. Subdomains I, II, III, IV found in the crystal structure of Top67 (4) are illustrated schematically along with the potential site for ZD domain (Z). G-strand: DNA strand cleaved to provide "DNA Gate". T-strand: DNA strand to be transported through the "DNA Gate". The hyperthermophilic topoisomerase I from Thermotoga maritima has been shown to coordinate one Zn(II) with a unique tetracysteine motif Cys-X-Cys-X16-Cys-X-Cys but Zn(II) binding is not required for relaxation activity . The sequence of this unique tetracysteine motifs is somewhat different from those present in other type IA topoisomerases in that the other tetracysteine motifs always had at least two amino acids separating the pairs of cysteines (Cys-X2-11-Cys), instead of just one amino acid (Cys-X-Cys) in T. maritima topoisomerase I . Therefore the structure and function of the single Zn(II) binding motif in T. maritima may differ from the multiple Zn(II) binding motifs in E. coli topoisomerase I. Direct interaction between DNA and the T. maritima Zn(II) binding motif has not been demonstrated. It has been suggested that the mechanisms of these two enzymes may be different . Direct interaction between the enzyme and the passing strand may not be necessary for the T. maritima topoisomerase I activity. The relaxation and decatenation activities of T. maritima topoisomerase I appear to be significantly more efficient than those of the E. coli topoisomerase I . Based on their primary sequences, a number of bacterial topoisomerase I enzymes do not appear to coordinate any Zn(II) with tetracysteines motifs while other type IA topoisomerase has up to 4 tetracysteine motifs . The topoisomerase I from Mycobacterium smegmatis has been demonstrated biochemically not to bind Zn(II) . In contrast, mutation disrupting the fourth Zn(II) motif of Helicobacter pylori topoisomerase I abolished enzyme function in vivo. Therefore there may be significant differences in the mechanisms of type IA topoisomerases from different organisms with respect to requirement of Zn(II) binding for relaxation activity. There is also another possible explanation for the varied number of tetracysteine motifs and requirement of Zn(II) for relaxation activity found in different type IA topoisomerases. The 14 kDa C-terminal region of E. coli topoisomerase I has been classified based on its structure to be in the Zn-ribbon superfamily [SCOP release 1.50, 7] even though it does not bind Zn(II). It also has high affinity for binding to single-stranded DNA on its own when separated from the three tetracysteine motifs . Based on the structural and DNA-binding properties of the E. coli topoisomerase I 14 kDa domain, one can conclude that it is possible for a subdomain in topoisomerase I to lose the Zn(II) binding cysteines during evolution and still maintains the Zn-ribbon structure and single-strand DNA binding properties . Finally, the in vivo catalytic activities of eukarytotic type IA topoisomerases, the topoisomerase III from various higher organisms may be related to their sequences. The transesterification domains of these eukaryotic enzymes have high degrees of identity to E. coli DNA topoisomerase III . However, the decatenation loop is not present in the eukaryotic topoisomerase III sequences and to date the decatenation activity has not been demonstrated for these enzymes. The number of potential Zn(II) binding cysteine motifs range from none in S. cerevisiae DNA topoisomerase III to four highly conserved tetracysteine motifs in the beta family of the topoisomerase III enzymes . The Zn(II) domain formed by these tetracysteine motifs may be required for interaction with single-strand DNA in removal of hypernegative supercoils or disruption of early recombination intermediates between inappropriately paired DNA molecules . PMID- 12052259_Conclusions TI - AB - We have shown that the ZD domain of E. coli DNA topoisomerase I is not required for the substrate recognition and DNA cleavage-religation action of the enzyme. We propose that the ZD domain interacts with the passing single-strand of DNA in the relaxation of negatively supercoiled DNA by this enzyme. PMID- 12052259_Materials and methods TI - AB - Enzyme and DNA | E. coli DNA topoisomerase I and the ZD domain were expressed and purified as described . To express the 67 kDa N-terminal transesterification domain (Top67), a stop codon at amino acid 598 was introduced into plasmid pJW312 used for topoisomerase I expression by site-directed mutagenesis employing the Chameleon-Mutagenesis kit from Stratagene. Top67 was expressed and purified with the same procedures as topoisomerase I. The oligonucleotides were custom synthesized by Genosys. The single-strand substrates and the top strand of the duplex substrates were labeled at the 5' termini with T4 polynucleotide kinase and gamma32P-ATP. The labeled oligonucleotides were purified by electrophoresis in a 12 or 15% sequencing gel. After elution from the gel slice, the labeled single-stranded oligonucleotides were desalted by centrifugation through a Sephadex G10 spin column. The duplex or heteroduplex substrates were prepared by mixing the labeled top strand with 4 fold excess of the unlabeled bottom strand, heating at 80C for three minutes, cooling to room temperature and purified by electrophoresis in a 20% non-denaturing polyacrylamide gel with TBE buffer. Plasmid pJW312 DNA used in relaxation assay was purified by CsCl centrifugation. Phage PM2 DNA was extracted from infected Pseudoalteromonas espejiana cells and PM2 DNA with one or more single-chain scissions used in the catenation assay was prepared as described . DNA relaxation assay | Top67 and the ZD domains at different concentrations were mixed and incubated at 37C for 10 min before addition to the 0.3 mug of supercoiled plasmid DNA in 20 mul of 10 mM Tris-HCl pH 8.0, 2 mM MgCl2, 0.1 mg/ml gelatin. After incubation at 37C for up to 1 h, the reaction was stopped by addition of 50 mM EDTA and electrophoresed in a 0.7% agarose gel and visualized by ethidium bromide staining as described . Gel mobility shift assay | The proteins were mixed with the 1 pmole of the labeled DNA substrates in 10 mul of 20 mM Tris-HCl pH 8.0, 100 mug/ml BSA, 12% glycerol and 0.5 mM EDTA. The samples were incubated at 37C for 5 min and then loaded onto a 6% polyacrylamide gel and electrophoresed with buffer of 45 mM Tris-borate pH 8.3, 1 mM EDTA. Electrophoresis was carried out at room temperature at 2 V/cm for 2 h. After drying of the gel, bands corresponding to the protein-bound oligonucleotides and unbound oligonucleotides were visualized by autoradiography, excised and counted in a Scintillation counter for quantitation. DNA cleavage assay | The cleavage assays were carried out with 1 pmole of 5' 32P-end labeled DNA substrate and 5 --10 pmoles of topoisomerase I or Top67 in 10 mul of the buffer used for the gel mobility shift assay. After incubation at 37C for up to 20 min, an equal volume of 90% formamide, 10 mM KOH, 0.25% bromophenol blue and 0.25% xylene cyanol was added to stop the reactions. The samples were analyzed by electrophoresis in a 12% sequencing gel followed by autoradiography. Salt and Mg2+ induced reversal of cleavage | The conditions were modified from those described previously . The cleavage reactions were incubated at 37C for 5 min and then divided into three aliquots. The cleavage products were trapped in one aliquot by the addition of SDS to 1%. NaCl (1 M) alone or NaCl with MgCl2 (4 mM) were added to the other aliquots followed by further incubation at 37C for up to 30 min before the addition of SDS. The products were analyzed as described for the cleavage reactions. Catenation of nicked DNA circles | The catenation reaction was carried out with 1.4 mug of nicked PM2 phage DNA in 20 mul of 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 10 mM KCl, 10 mM MgCl2. After incubation at 37C for up to1 h, the reactions were stopped with the addition of 1% SDS and 50 mM EDTA. The products were analyzed as described for the relaxation assay. PMID- 12052259_Authors' contributions TI - AB - Author 1 (A.A.) carried out all the experiments except the catenation assay. Author 2 (Y.T.) conceived of the study, participated in its design and coordination and carried out the catenation assay. All authors read and approved the final manuscript. PMID- 11996675 TI - Hisactophilin is involved in osmoprotection in Dictyostelium AB - Abstract | Background | Dictyostelium cells exhibit an unusual stress response as they protect themselves against hyperosmotic stress. Cytoskeletal proteins are recruited from the cytosolic pool to the cell cortex, thereby reinforcing it. In order to gain more insight into the osmoprotective mechanisms of this amoeba, we used 1-D and 2-D gel electrophoresis to identify new proteins that are translocated during osmotic shock. Results | We identified hisactophilin as one of the proteins that are enriched in the cytoskeletal fraction during osmotic shock. In mutants lacking hisactophilin, viability is reduced under hyperosmotic stress conditions. In wild type cells, serine phosphorylation of hisactophilin was specifically induced by hypertonicity, but not when other stress conditions were imposed on cells. The phosphorylation kinetics reveals a slow accumulation of phosphorylated hisactophilin from 20 --60 min after onset of the hyperosmotic shock condition. Conclusion | In the present study, we identified hisactophilin as an essential protein for the osmoprotection of Dictyostelium cells. The observed phosphorylation kinetics suggest that hisactophilin regulation is involved in long-term osmoprotection and that phosphorylation occurs in parallel with inactivation of the dynamic actin cytoskeleton. PMID- 11996675_Background TI - AB - Cells steadily face changes of the external osmolarity, to which they have to adapt. To withstand a steep increase in osmolarity, eukaryotic cells activate responses like "regulatory volume increase", accumulation of compatible osmolytes and stimulated expression of stress proteins . Recently, an exception from this scheme has been identified: Dictyostelium cells protect themselves against hyperosmolarity by largely rearranging cellular proteins, whereas no "regulatory volume increase", no accumulation of compatible osmolytes and no change of the expression pattern of the most abundant proteins were observed . Among the translocated proteins identified, cytoskeletal proteins appear to be predominant. In particular, the rearrangement of actin and myosin II to the cell cortex beneath the plasma membrane was shown to constitute a pivotal element of osmoprotection in Dictyostelium. These two proteins form the core of a rigid network resembling a shell-like structure . Conversely, DdLIM, a cytoskeletal protein involved in the formation of protrusions , is depleted from the cytoskeletal fraction under hypertonic conditions, which is in consistence with the rounding up and the retraction of protrusions of cells . 30 --60 min after onset of the shock condition, progressive actin phosphorylation is observed , which presumably leads to the inactivation of the dynamic actin system . The pivotal role of the cytoskeleton in hyperosmotic stress response is also reflected by the fact, that the cytoskeletal proteins beta-actinin and "120 kDa gelation factor" are essential to ensure survival under hyperosmotic conditions . However, the signalling pathways mediating protein translocation and actin phosphorylation largely remain to be elucidated. The best understood signalling pathway involves myosin II translocation: upon hyperosmotic stress an increase in cGMP concentration was observed, which is due to guanylyl cyclase activation. This in turn results in the activation of the kinase, which eventually phosphorylates myosin II heavy chain . Thereby, disassembly of myosin II filaments is induced as a prerequisite for their reassembly at the cell cortex , which is accomplished within 10 min after onset of hyperosmotic shock . In addition, DokA, a homologue of bacterial histidine kinases and cAMP are involved in hyperosmolar signal transduction , however, molecular targets of these signalling pathways during osmoregulation have not been identified yet. Hence, the translocation and phosphorylation of cytoskeletal proteins constitute pivotal osmo-responses in Dictyostelium. To further elucidate this unusual hyperosmolar stress response we attempted to identify additional proteins, which are translocated under hypertonic conditions and investigated, whether protein phosphorylation of a candidate protein is regulated in response to hyperosmolarity. We could identify hisactophilin as such a protein, which is translocated and phosphorylated during hyperosmotic shock. Hisactophilin is a 15 kDa protein in Dictyostelium, that consists of two highly identical isoforms Hisactophilin I and II, which are myristoylated at the N-terminus . The two isoforms exhibit 84% sequence identity and are both myristoylated and distributed between plasma membrane and cytoplasm . Due to this high degree similarity, both isoforms are in this manuscript referred to as hisactophilin, without distinguishing between them. The biochemical properties of hisactophilin, namely actin- and membrane binding have recently shown to be strongly pH-dependent, which is due to the high content of 26 --30% histidine residues . Therefore, a role also as pH-sensor was postulated for this protein . In addition to myristoylation, phosphorylation was shown to be a posttranslational modification of hisactophilin ; the physiological role of hisactophilin phosphorylation however is unknown. Hisactophilin appears to play an essential role in osmoprotection, as hisactophilin null cells are osmosensitive. PMID- 11996675_Results TI - AB - Hisactophilin is enriched in the cytoskeletal fraction of wild type cells exposed to hyperosmotic shock | To identify proteins, which are translocated to or depleted from the cytoskeleton upon hyperosmotic stress, we used 2-D electrophoresis as a differential method. Wild type cells were subject to hypertonic shock in liquid culture prior to cell lysis. The Triton X-100-insoluble cytoskeletal fraction was isolated and the proteins were separated by 2-D electrophoresis. Samples from three independently grown cell cultures were analyzed in parallel. As control, the same procedure was performed with wild type cells shaken in SPB buffer. Computer analysis of the silver-stained gels with the software package Melani II (Biorad) revealed, that a 15 kDa protein, consisting of several isoforms with a pI of 7.3 --7.5, is enriched in the cytoskeletal fraction of hyperosmotically shocked cells compared to control cells (a typical result is shown in Fig. , upper panels). Correspondingly, reduction of the protein amount in the cytosolic pool was observed under hypertonic conditions compared to the control (Fig. , lower panels), suggesting, that the protein is translocated from the cytosol to the cytoskeleton under hypertonic conditions. Search of protein databases for Dictyostelium proteins exhibiting a molecular weight of 15 kDa and a pI of 7.3 --7.5 resulted in hisactophilin as a candidate cytoskeletal protein, which consists of two isoforms, hisactophilin I and II . The identity of the characterized protein as hisactophilin was demonstrated by immunostaining with a monoclonal alpha-hisactophilin antibody . In addition, analysis of digested peptides of the protein eluted from 2-D gels by mass spectrometry demonstrated the identity of hisactophilin (data not shown). The enrichment of hisactophilin in the cytoskeletal fraction of hyperosmotically shocked cells was also confirmed by analyzing cytoskeletal fractions by 1-D SDS-PAGE followed by immunostaining with an alpha-hisactophilin antibody . As the actin-rich cytoskeleton is primarily found in the cell cortex of hypertonically shocked cells , we investigated, whether a hisactophilin II-GFP fusion protein expressed in wild type cells is translocated to the cell cortex after onset of hyperosmotic shock. In fact, the hisactophilin II-GFP fusion protein was found to form a thick layer beneath the plasma membrane of cells exposed to hypertonicity for 10 min, whereas only a weak staining of the plasma membrane was observed in control cells (data not shown). Figure 1 | Hisactophilin is enriched in the cytoskeletal fraction of hyperosmotically shocked cells. Hisactophilin is enriched in the cytoskeletal fraction of hyperosmotically shocked cells. (A) Hisactophilin is enriched in the cytoskeletal fraction of cells hyperosmotically shocked for 2 h with respect to control cells suspended in SPB buffer for 2 h. (upper frames). Correspondingly, less hisactophilin was found in the cytosol of cells exposed to hypertonicity when compared to cells suspended in SPB buffer (lower frames). Proteins from cytosolic and cytoskeletal fractions were separated by 2-D electrophoresis. The frames show a silver-stained region of the gel corresponding to a molecular weight of 15 kDa and to an IEP of 7.3 --7.5. The predominant spot of each panel is indicated with an arrow. (B) Identification of the protein in (A) as hisactophilin by Western blotting of a typical gel of the cytoskeletal fraction and immunostaining of with a alpha-hisactophilin antibody. (C) The enrichment of hisactophilin in the cytoskeletal fraction of cells exposed to hypertonicity was confirmed by 1-D SDS-PAGE. The cytoskeletal fraction was isolated from cells suspended in SPB buffer (-) or in SPB buffer/400 mM sorbitol (+) for 2 h. The proteins were separated by SDS-PAGE and blotted. Immunostaining was performed with an alpha-hisactophilin antibody (right panel). The membrane was subsequently coomassie-stained (left panel). Abb.: M= marker. Hisactophilin is phosphorylated upon hyperosmotic stress | As phosphorylation is an important regulatory modification of actin and myosin II under hypertonic conditions, we investigated, whether this also applies for hisactophilin. Wild type cells deprived of phosphate were suspended in Mes buffer and were metabolically labeled with 32P-orthophosphate. After 1 h incubation, hyperosmotic shock was initiated and samples were withdrawn at intervals. Hisactophilin was isolated after cell lysis by immunoprecipitation. The samples were analyzed by autoradiography and subsequently by immunostaining with an alpha-hisactophilin antibody. Hisactophilin, that was isolated from cells shocked for 2 h, was found to be phosphorylated, whereas no detectable hisactophilin phosphorylation was observed in control samples isolated from cells suspended in SPB buffer for 1 h or 3 h . Analysis of the kinetics of the phosphorylation reaction revealed, that the phosphorylation does not constitute an acute stress response . Phospho-hisactophilin progressively accumulated 20 --60 min after onset of the shock condition. The result of the densitometric analysis revealed that the kinetics of phospho-hisactophilin can be fitted by a quadratic equation . Figure 2 | Hisactophilin is phosphorylated in wild type cells exposed to hyperosmotic conditions Hisactophilin is phosphorylated in wild type cells exposed to hyperosmotic conditions (A). Cells were metabolically labeled with 32P-orthophosphate prior to hypertonic shock. Hisactophilin was immunoprecipitated from samples withdrawn at T = 0 and T = 2 h. The probe was separated by SDS-PAGE and blotted. Autoradiography (left panels A,B) was performed for 48 h and hisactophilin was subsequently identified by immunostaining (right panels A, B). (+) denotes samples from shocked cells, (-) denotes samples from control cells suspended in SPB buffer. The arrow indicates the position of 15 kDa proteins. (B) The amount of phospho-hisactophilin progressively increased in hyperosmotically shocked cells. The experiment was performed as described in (A). (C) Densitometric analysis of the autoradiography in (B) reveals an exponential increase in phospho-hisactophilin upon hyperosmotic stress: the kinetics can be fitted by a quadratic function displayed in the figure. The densities are given in relative arbitrary units, setting the background as 0. To test the specificity of hisactophilin phosphorylation as a hyperosmotic stress response, wild type cells were labeled with 32P-orthophosphate and were then exposed to various other stress conditions. Subsequently, samples were withdrawn and hisactophilin phosphorylation was detected as described above. The phosphorylation reaction was found to be specific for hypertonic conditions, as other stress conditions as heat stress, acid stress, oxidative stress and energy depletion did not result in a detectable hisactophilin phosphorylation . Figure 3 | Hisactophilin phosphorylation is not a general stress response. Hisactophilin phosphorylation is not a general stress response. Autoradiographies are shown on the left panels, the corresponding immunostainings are displayed on the right panels. (A) Wild type cells were metabolically labeled with 32P-orthophosphate and were then exposed to various stress conditions. Hisactophilin phosphorylation was detected as described in Fig. . : Control (cells shaken in SPB buffer). 2: Cellular Acidification. 3: Heat stress. 4: Energy depletion. 5: Oxidative stress. 6: Hyperosmotic stress. (B) Hisactophilin phosphorylation is dependent on osmolarity, but not on the osmolyte. Wild type cells were metabolically labeled with 32P-orthophosphate and were then exposed to either 200 mM NaCl, 350 mM glucose or 400 mM sorbitol for 1 h. As control, cells were shaken in SPB buffer (1). Hisactophilin phosphorylation was detected as described in Fig. 2. To prove that the phosphorylation of hisactophilin is dependent on osmolarity, but not on the osmolyte used, wild type cells were exposed to hypertonic stress conditions, using either NaCl, glucose or sorbitol as osmolyte. The concentrations of the osmolytes were adjusted to 400 mOsm in the case of sorbitol (400 mM) and NaCl (200 mM) and a slightly reduced osmolarity (350 mOsm) was chosen in the case of glucose. Analysis of hisactophilin phosphorylation in response to the hypertonic stress conditions revealed that phosphorylation occurred to the same extent in the presence of all three osmolytes, indicating that hisactophilin phosphorylation is a hyperosmolar stress response . Serine phosphorylation of Hisactophilin | Recently, it has been demonstrated in vitro using a crude kinase fraction, that hisactophilin is phosphorylated on threonine (95%) and on serine residue(s) (5%) . We addressed the question, whether the phosphorylated residues are identical under hyperosmolar conditions in vivo. We therefore labeled wild type cells with 32P-orthophosphate, exposed them to hypertonic stress and immunoprecipitated hisactophilin. The protein probe was hydrolyzed in the presence of constant boiling HCl and was then separated together with phosphoamino acid standards by 2-D thin layer electrophoresis. 32P-labeled phosphoamino acids originating from phospho-hisactophilin were detected by autoradiography. Identification of the phosphoamino acid was performed by staining the standards with Ninhydrin and subsequent comparison of the staining pattern with the autoradiography. As shown in Fig. , only phospho-serine could be detected under in vivo conditions, whereas no phospho-threonine or phospho-tyrosine was observed, indicating that serine phosphorylation is favored over threonine phosphorylation under hyperosmotic stress conditions in vivo. Figure 4 | Hisactophilin is phosphorylated on serine residue(s) in vivo. Hisactophilin is phosphorylated on serine residue(s) in vivo. Wild type cells were metabolically labeled with 32P-orthophosphate and were then exposed to 400 mM sorbitol for 2 h. Hisactophilin was isolated by immunoprecipitation and was analyzed by 2-D thin layer electrophoresis. Phosphoamino acid standards were detected by Ninhydrin staining (positions indicated by lines) and were compared to the autoradiography. Abbreviation: part. hydrol.=partially hydrolyzed Hisactophilin-deficient cells exhibit reduced viability under hyperosmotic stress conditions | To investigate whether hisactophilin plays a role in osmoregulation, we determined whether survival of his--cells was affected under hypertonic conditions. The viability of his--cells was found to be reduced by 75% 2 h after onset of the stress condition, whereas viability of the wild type cells was not significantly affected . Hence, the presence of hisactophilin is essential to ensure viability under hypertonic conditions. Figure 5 | Viability of his--cells is reduced under hypertonic conditions. Viability of his--cells is reduced under hypertonic conditions. his--cells and wild type cells were suspended in either 400 mM sorbitol or SPB buffer for 2 h. Samples were plated together with bacteria and plaques were counted after 2 --4 days incubation (n = 5). PMID- 11996675_Discussion TI - AB - Dictyostelium was shown to employ an unusual mechanism to cope with the effects of osmotic stress by rearranging its cellular cortex . As the cells round up, actin and myosin moieties are being redistributed underneath the plasma membrane. In this process the distribution of the actin-associated protein Hisactophilin was investigated in order to gain a better insight into osmo-protective mechanism of the cell. Hisactophilin was found to be enriched in the cytoskeletal fraction of wild type cells exposed to hyperosmotic stress . In addition, a thick layer of Hisactophilin II-GFP was formed at the cell cortex of cells, that were shocked for 10 minutes (data not shown), which correlates with the observation that actin and myosin II are translocated to the cell cortex within 10 minutes after onset of the stress condition . As hisactophilin was shown to enhance actin polymerization in vitro and as cells overexpressing hisactophilin II exhibit an increased F-actin content , this protein could concur in the formation of the rigid filamentous network of actin and myosin II filaments. The essential role of hisactophilin becomes evident when hisactophilin negative cells are investigated under osmotic stress conditions. They show a markedly reduced survival rate in a test were they are first exposed to high osmolarity for 2 hours and are then plated onto agar plates containing bacteria. This procedure involves a volume reduction and a subsequent volume increase caused by the re-dilution in the plating process. It therefore remains to be investigated whether hisactophilin deploys its function in the process of rearranging the cell's cortex upon volume reduction or is essential for reinforcing the cortex in its rounded up state. A role comparably to the family of MARCKS (myristoylated alanine-rich protein kinase C substrate) proteins in mammalian cells was postulated recently . These proteins are also myristoylated at the N-terminus and crosslink actin into a rigid meshwork at the membrane. Upon phosphorylation, MARCKS proteins are rejected from the membrane by electrostatic interactions, which results in the spatial separation of the cytoskeleton from the membrane . In fact, hisactophilin phosphorylation was found to be specifically induced under hypertonic conditions (Fig. , ). However, the phosphorylation reaction is too slow to account for the translocation of the protein within 10 minutes : phospho-hisactophilin progressively accumulated within 60 min with a kinetics strikingly similar to the kinetics of actin phosphorylation under hyperosmotic conditions . This points out the possibility, that actin and hisactophilin cooperate in two distinct phases of osmoregulation. During the first phase, the proteins are translocated within the first 10 minutes of stress which is accompanied by cortical reinforcement. The second phase is characterized by actin and hisactophilin phosphorylation reinforced after about 30 min of hypertonic shock. Actin phosphorylation was shown to correlate with an inactivation of the dynamic actin system . The identical kinetics suggests a similar role for hisactophilin during this process. In addition, the coinciding phosphorylation kinetics raises the possibility of a common regulation of hisactophilin and actin phosphorylation. The signalling pathways regulating actin and hisactophilin phosphorylation are unknown, however, the second messengers involved in osmoregulation identified so far, namely cGMP and cAMP could be excluded as modulators of hisactophilin phosphorylation, as an increase or decrease in these messengers did not affect hisactophilin phosphorylation (data not shown). This suggests the presence of an additional signalling pathway concomitantly regulating a serine kinase specific for hisactophilin and, due to the identical phosphorylation kinetics, a tyrosine kinase specific for actin . A conceivable signalling pathway regulating the hisactophilin translocation is a change in pH, as the biochemical properties of Hisactophilin are strongly pH-dependent: actin-binding is strongly increased in vitro when pH is lowered to = 7.0 . In fact, progressive cytosolic acification was observed during hyperosmotic stress, resulting in a pH drop from 7.5 to 6.8 15 min after onset of the stress condition . Hence, the translocation of hisactophilin to the actin-rich cytoskeleton can be attributed to the cytosolic acidification. Also, the enrichment of the elongation factor 1alpha in the cytoskeletal fraction of hyperosmotically shocked cells can be explained by cytosolic acidification, as enhanced binding of this protein to F-actin at pH 6.5 in vitro has been described recently . These examples are in accordance with the notion of pH changes as "synergistic messenger" . Changes in proton concentration affect the biochemical properties of proteins and can thereby act as signals. However, other signals have to concur to elicit the osmoprotective responses: the translocation of hisactophilin potentially caused by the drop in cytosolic pH is complemented by the relocation of myosin II mediated by cGMP and of actin by a yet unknown mechanism. Therefore, the role of hisactophilin in osmoprotection also exemplifies the numerous interactions of the signalling pathways that are necessary for osmoprotection in Dictyostelium. PMID- 11996675_Conclusions TI - AB - We could demonstrate that Hisactophilin is both translocated to the cytoskeleton and phosphorylated during hyperosmotic stress in Dicytostelium. The phosphorylation kinetics suggests a slow accumulation of phosphorylated hisactophilin resembling the phosphorylation kinetics for actin, which is known to eventual lead to cytoskeletal inactivation. Second messengers known to play an essential role during osmoprotection are not involved in hisactophilin phosphorylation suggesting a further signalling pathway being involved in the unusual osmoprotection mechanisms in Dictyostelium. PMID- 11996675_Materials and Methods TI - AB - Growth of Dictyostelium cells | D. discoideum strain AX2-214 (AX-2), referred to as wild type, was cultivated axenically in shaken suspension at 21C and harvested during exponential growth. Clones of HsII-GFP were cultivated as described for AX-2, but under the selection of 20 mug/ml Geneticin (G418). Clones of his--cells were cultivated as described for AX-2, but under the selection of 7.5 mug/ml Geneticin. Hyperosmotic shock in liquid culture | Hyperosmotic shock experiments were performed according to Schuster et al. (1996). Axenically grown Dictyostelium cells were harvested, washed twice in Soerensen Phosphate Buffer (SPB: 2.0 mM Na2HPO4, 14.6 mM KH2PO4, pH 6.0) and were then adjusted to 2*107 cells/ml with SPB buffer (osmolarity: 34 mOsm). Cell density was 1,6*107 cells/ml during the osmoshock, adjusted by the addition of SPB buffer/2 M sorbitol. To the control cells, SPB buffer was added until the same cell density was reached. The cell suspension was shaken for 1 h. SPB buffer/2 M sorbitol was added to a final concentration of 400 mM sorbitol (osmolarity of the buffer: 434 mOsm). The cell suspension was incubated up to 2 hours according to the experiment. Viability of Dictyostelium cells after exposure of the cells to hyperosmotic stress was determined as described . In short, his--cells were shaken in SPB buffer for 1 h prior to the hyperosmotic shock. Samples were withdrawn at intervals and cell suspension corresponding to 200 cells was plated on agar plates together with Klebsiella aerogenes bacteria. Plaques in the bacterial lawn were counted after 2 --4 days. Exposure to stress conditions | To acidify Dictyostelium cells, axenically grown wild type cells were washed twice in SPB buffer and were resuspended in SPB buffer/5 mM propionic acid, pH 6.0. To impose oxidative stress, the cells were incubated in SPB buffer/3 mM H2O2 for 60 min. To study heat stress, the cells were exposed to 30C for 30 min. Energy depletion was achieved by incubating the cells in SPB buffer/50 muM 2,4-dinitrophenol for 60 min. 2-D Gel electrophoresis | Preparation of the cytoskeletal fraction from hyperosmotically shocked cells and separation of the proteins by 2-D gel electrophoresis was performed as described recently . The cytoskeletal fraction was obtained by isolating the Triton X-100-insoluble fraction. The corresponding soluble fraction was defined as cytosolic fraction. 100 mug total protein was applied per gel. Metabolic labeling with 32P-orthophosphate | 50 ml cell suspension of axenically grown wild type cells were transferred to 300 ml phosphate-free medium buffered with 20 mM Mes, pH 6.0. The cells were shaken for 16 h and were subsequently washed twice in ice-cold 20 mM Mes, pH 6.0 (Mes buffer). The cells were adjusted to 2*107 cells/ml with Mes buffer. After incubating the cells for 30 min, 32P-orthophosphate (Amersham Pharmacia) was added to a final activity of 0.25 mCi/ml in the cell suspension. The cell suspension was shaken for 60 min, before the cells were exposed to hyperosmotic shock. Samples of 1 ml were withdrawn at intervals, the cells were washed twice with Mes buffer and were resuspended in 800 mul RIPA buffer (50 mM Tris/HCl, pH 7.5; 150 mM NaCl, 1% TritonX-100; 1% Natriumdeoxycholate; 0.1% SDS; 1 mM DTT; 1 mM EDTA; 100 muM Na-orthovanadate; Complete Protease Inhibitor Cocktail (Boehringer Mannheim)). The cell lysate was immediately frozen in liquid nitrogen. As control, samples were prepared from a cell suspension to which Mes buffer was added instead of Mes buffer/2 M sorbitol. Immunoprecipitation of hisactophilin | Thawed cell lysate from 32P-labeled cells was centrifuged for 20 min at 10000 g (4C). alpha-hisactophilin antibody 54-11-10 was added to the cleared lysate and was incubated for 3 h. Immunoprecipitation was performed by adding fixed Staphylococcus aureus cells (Pansorbin, Calbiochem) preincubated with rabbit-alpha-mouse IgG (Sigma). After 45 min incubation, the immunoprecipitate was washed twice with TBS and was analyzed by SDS-PAGE, Western blotting and autoradiography. Fluorescence microscopy | Axenically grown HisII-GFP cells were shaken in SPB buffer for 1 h at a cell density of 2*107 cells/ml. SPB buffer/2 M sorbitol was added to a final concentration of 400 mM sorbitol (cell density: 1,6*106 cells/ml). An aliquot of the living cells was allowed to adhere to glass cover slips for 10 min. Subsequently, fluorescence microscopic observations were carried out. As control, cells suspended in SPB buffer were analyzed. The equipment consisted in a Leitz Aristoplan fluorescence microscope, set at 450 --490 nm for excitation. Phosphoamino acid analysis | Phosphoamino acid analysis was performed as described . In short, hisactophilin was immunoprecipitated from 32P-labeled cells exposed to hyperosmotic stress for 60 min. Acid hydrolysis of the immunoprecipitate was performed, followed by the separation of the phosphoamino acids by electrophoresis in two dimensions on TLC plates together with phosphoamino acid standards. The plates were analyzed by autoradiography and by the detection of amino acids with Ninhydrin. Miscellaneous | Protein quantification, SDS-PAGE and Western blotting onto PVDF membrane were performed according to the published methods of Bradford , Laemmli and Towbin et al.. Western blots were treated with the mouse monoclonal alpha-hisactophilin antibody 54-11-10 . Antibodies were detected using peroxidase-coupled rabbit-alpha-mouse IgG and the Renaissance system (Du Pont). Prestained Seeblue marker (Novex) was used as a protein molecular weight standard. PMID- 12057022 TI - Training practitioners in preparing systematic reviews: a cross-sectional survey of participants in the Australasian Cochrane Centre training program AB - Abstract | Background | Although systematic reviews of health care interventions are an invaluable tool for health care providers and researchers, many potential authors never publish reviews. This study attempts to determine why some people with interest in performing systematic reviews do not subsequently publish a review; and what steps could possibly increase review completion. Methods | Cross-sectional survey by email and facsimile of the 179 participants in Australasian Cochrane Centre training events between 1998 and 2000. Results | Ninety-two participants responded to the survey (51 percent). Response rate of deliverable surveys was 82 percent (92/112). The remainder of the participants had invalid or no contact information on file. More than 75 percent of respondents felt that the current workshops met their needs for training. The most critical barriers to completion of a Cochrane review were: lack of time (80 percent), lack of financial support (36 percent), methodological problems (23 percent) and problems with group dynamics (10 percent). Conclusions | Strategies to protect reviewer time and increase the efficiency of the review process may increase the numbers of trained reviewers completing a systematic review. PMID- 12057022_Background TI - AB - Increasing emphasis is being placed on evidence-based medicine. The best evidence for treatment interventions comes from systematic reviews of randomised controlled trials, however, systematic reviews with meta-analysis of published and unpublished data are time- and labour-intensive. A major player in the evidence-based medicine movement has been the Cochrane Collaboration, an international organisation committed to 'preparing, maintaining and promoting the accessibility of systematic reviews of the effects of health care interventions.' The Australasian Cochrane Centre is one of the component centres of the Cochrane Collaboration, providing ongoing training and support to people in the Australasian region who prepare systematic reviews that are subsequently published on the Cochrane Library. The ultimate goal of the training program is to increase the number and quality of completed Cochrane reviews, and ensure that they are routinely updated. Currently, training occurs over two days, with the first day focusing on developing a protocol for a systematic review and the second, statistical analysis and interpretation of the review. Training sessions occur several times per year in locations throughout Australasia. In May 2001, the Australasian Cochrane Centre surveyed all of the participants of its training workshops during the years 1998 to 2000. We undertook this survey to: 1. determine why some people who attend Cochrane training workshop do not go on to publish a systematic review; and 2. what steps could increase systematic review completion. PMID- 12057022_Methods TI - AB - Survey Population and Contact Information | Our survey population included the 179 participants of the Australasian Cochrane Centre's training workshops from 1998 to 2000. Contact information for the year 2000 participants was drawn from the Australasian Cochrane Centre database (initiated in January 2000) of all workshop participants and current Cochrane reviewers in Australasia. Several partial data systems provided contact information for 1998 --1999 participants. Questionnaire | Participants were asked to respond to a cross-sectional survey of 21 'yes' or 'no' questions with a provision for open-ended comments. [See for the questionnaire used in this study.] The format of our internally developed questionnaire did not lend itself to statistical testing for internal consistency. No demographic details were collected, so that responses could be deidentified after receipt of the questionnaire. The initial questionnaire was sent via email or fax to all participants. A second email or fax was sent four weeks later, with a third survey sent via post to those participants who had not responded to the first two requests and had a postal address on file. Analysis | Both quantitative and qualitative methods were used in data analysis. The structured questions were analysed by SPSS for Windows software, release 10.0.5, and results were presented in the form of frequencies and percentages. Content analysis was used to analyse the qualitative responses. Publication status of workshop participants was determined by searching the Cochrane Library by each workshop participants' last name and first initial. PMID- 12057022_Results TI - AB - Response rate | Thirty-five questionnaires were returned as undeliverable (20 percent), with an additional 32 participants having no contact information on file with the Australasian Cochrane Centre (18 percent). [see Figure ] Of the 112 deliverable questionnaires (63 percent of participants), 54 participants (47 percent) responded after the first two mailings. With the third mailing, an additional 38 participants responded, creating an overall response rate for deliverable surveys of 82 percent. Eighteen people returned the survey without response, which provided us with 74 valid surveys (41 percent of participants) for analysis. Figure 1 | Survey Response of Workshop Participants Survey Response of Workshop Participants Publication status | Approximately 40 percent of these 1998 --2000 workshop participants have published a protocol or review on the Cochrane Library, as of 2001 issue 4. However, due to issues of anonymity of questionnaire response, we are unable to stratify the results based upon publication status. Findings from Questionnaire: Factors that Interfere with Systematic Review Completion | Lack of time emerged as the most critical barrier to completion of Cochrane reviews, with 80 percent of respondents citing this factor. Twenty respondents (36 percent) acknowledged lack of financial support, and twelve (21 percent), lack of institutional encouragement and support, as barriers to completion of their systematic review. Of the 31 people who offered qualitative comments, 11 suggested a need for dedicated time and support, with two needing better time management, and one wanting a grant to supply a research assistant. In a similar tone, seven people stated that other work or personal commitments take priority over Cochrane activities. [see Table ] Table 1 | Barriers for Training in, and Completion of, Systematic Review of the Literature Methodological barriers affected 23 percent of respondents. Three respondents specifically mentioned problems with translation of non-English papers, and two had no access to EMBASE (Excerpta Medica). In addition, one person was frustrated that a similar title for a Cochrane review had been registered, but not completed in a timely manner. Ten percent of respondents had problems with group dynamics, with two stating this factor as a significant delay to publication, and one complaining of contradictory editorial commentary. Thirteen respondents (22 percent) had yet to start a review. Nine respondents (16 percent) were working on a systematic review outside of the Cochrane Collaboration, with one qualitative commentator stating that the employer insisted on publication of the review prior to submission to Cochrane. A substantial minority (38 percent) of respondents felt that follow-up communication, especially via email, would help them complete their review. From the qualitative comments, this email could occur every one to six months, with 11 of the 12 respondents suggesting a time period of every three months or less. Findings from Questionnaire: Training Issues | Scheduling and content of the training workshops appeared to meet the needs of participants. Over 75 percent of respondents felt that the workshops were not too infrequent, too inconvenient, too far away, assumed too much knowledge, or were too long. Qualitative comments stressed the need for ongoing support after training. Three respondents requested a specific contact person, whereas another four asked for support to discuss general problems, statistical analysis issues, help selecting trials, and the creation of a bulletin board-style mentoring system. Three more respondents requested advanced training. In January 2000, the Australasian Cochrane Centre began collecting formal immediate evaluations from workshop participants. Analysis of these data indicates that the average participant self-ranks only slightly above an absolute beginner in systematic review methodology. Mean satisfaction with content, presentations, opportunity to ask questions, and overall satisfaction range between 4.5 to 4.7, on a scale from 1 = highly dissatisfied to 5 = highly satisfied. Finally, 83 percent of respondents stated that their Cochrane training had made them more likely to use the Cochrane Library to answer clinical questions. Only 45 percent, however, felt that their practice patterns had changed. PMID- 12057022_Discussion TI - AB - Lack of time is by far the most commonly reported barrier to completion of a systematic review. Financial, institutional, personal, and methodological issues are not problematic for the majority of respondents. These findings have important implications for both reviewers and entities commissioning systematic reviews. In the absence of funding for all or, at least, part of a systematic review, it will continue to be difficult for reviewers to do this research by taking more time out of their busy lives. The challenge becomes the ability to support researchers in minimising the time they spend on the process of the review, without affecting quality. Limitations in this study are several. First of all, the subjective nature of this study may have lead reviewers to state that lack of time was a barrier to completion of the project, when another reason, such as change in professional priorities was the 'true' barrier. Second, searching publications by last name and first initial may lead to either overstatement or understatement of publication status, as reviewers may have common last names, have changed their last names, or use a different first initial to the first name they use routinely. Finally, we had a large number of invalid addresses on file at the Australasian Cochrane Centre. The disruption of a move of the Australasian Cochrane Centre from Adelaide to Melbourne in 1999, with resultant staff turnover and loss of records, is responsible for much of our difficulty. However, we still had invalid contact information for almost one-quarter of the 2000 participants, whose training occurred less than 18 months prior to this survey. This lack of contact information is problematic as this survey suggests email communication could facilitate review completion. Strategies we plan to implement to improve the currency of our database include: 1. discussion at training events of the importance of informing the Australasian Cochrane Centre of change of address, 2. reminder notices on newsletters, and 3. liaison with other Cochrane entities. The year 2000 also saw a change in enrolment policy for the training workshops, allowing people to attend the course only if they had a title or protocol registered with the relevant Cochrane entity. Therefore, our results are likely to be more representative of recent participants of our program, committed to the Cochrane system. PMID- 12057022_Conclusions TI - AB - Lack of time, not skill, is the most common barrier to completion of a systematic review for authors who have attended such training given by the Australasian Cochrane Centre. One solution lies in advancements in methodology to make systematic reviews more efficient, such as automating aspects of the review or providing authors with specialised, centralised services in literature searches, project management, and the like. We expect that these advances in systematic review methodology, increased capacity for completing systematic reviews in Australasia, and realistic expectations regarding required investment of time for valid evidence will lead to an increased number and quality of systematic reviews published on the Cochrane Library. Ultimately, consumers and practitioners of health care around the globe should benefit from this increased knowledge. PMID- 12057022_Competing interests TI - AB - All authors were under the employ of the Australasian Cochrane Centre at the time of this survey. PMID- 12057022_Authors' contributions TI - AB - The authors contributed equally to the design and analysis of this project prior to Dr. Silagy's death in December 2001. Doctors Piehl and Green are responsible for all subsequent editorial changes. PMID- 12057022_Pre-publication history TI - AB - The pre-publication history for this paper can be accessed here: PMID- 12028592 TI - The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes AB - Abstract | Background | Activation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16 --24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using alphaCD3 antibody to stimulate the T cell receptor and alphaCD28 antibody to provide the co-stimulus. Results | Activation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with alphaCD3 and alphaCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF) 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF) 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK alpha/beta pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F. Conclusions | Both eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation. PMID- 12028592_Background TI - AB - The initiation of translation of mRNAs is an important control point in protein synthesis in eukaryotes and requires a set of initiation factors (eIFs). The cap-binding protein eIF4E recognises the 5'cap-structure of the mRNA, and is a component of the eIF4F complex consisting of eIF4E, eIF4G, a scaffolding protein , and eIF4A, an RNA helicase . Any secondary structure in the 5'untranslated region of the mRNA is thought to be unwound by eIF4A together with eIF4B or eIF4H . The 40S subunit of the ribosome binds to the eIF4F complex through an association between eIF4G and eIF3, which interacts directly with the 40S ribosomal subunit. The preinitiation complex, containing the 40S ribosomal subunit, eIF4F, eIF4B, and Met-tRNAi*eIF2*GTP, scans the 5'UTR until the AUG start codon is located. The subsequent hydrolysis of the GTP bound to eIF2 is promoted by eIF5, after which eIF2*GDP leaves the ribosome. The 60S ribosomal subunit can then join and the 80S complex is formed. eIF2 in the GDP-bound state is inactive and, in order to return to the active form again, the GDP is exchanged for GTP in a step promoted by the guanine nucleotide exchange factor eIF2B. The next stage in the translation process, the elongation step, can be regulated via changes in the activity of eEF2 . Phosphorylation of eEF2 at Thr56 results in its complete inactivation . Human primary T-cells are metabolically quiescent, with little ongoing DNA, RNA or protein synthesis . The low protein synthesis rate in quiescent T cells is associated with low levels of initiation factors in these cells. The rate of protein synthesis increase 2 --4 fold after 4 h of mitogenic stimulation , and it has been reported that the mRNA and protein levels for several translation initiation factors increased during T cell activation. The mRNA levels of eIF4A, eIF2alpha, and eIF4E increased rapidly after stimulation . However, the increase in the levels of the corresponding protein lagged significantly behind. It is therefore likely that increased levels of translation factors contribute to the pronounced stimulation of protein synthesis that occurs during T cell activation at later times, while modulation of the activity of several translation initiation factors e.g. by phosphorylation or association with binding proteins is important in the early phase of T cell activation . Increased phosphorylation of eIF4E in T lymphocytes has been reported under several conditions. Activation of quiescent mature porcine peripheral blood mononuclear cells with phorbol 12-myristate 13-acetate (PMA) or concanavalin A or stimulation of human primary T cells with PHA , PMA, or PMA plus ionomycin caused a rapid increase in the phosphorylation of eIF4E. Similarly, stimulation of the T cell receptor in the human leukaemic T cell line Jurkat with OKT-3, or treatment with PMA, increased eIF4E phosphorylation , and a significant increase in the amount of eIF4F complexes was also detected. The activity of eIF4E can also be modulated by its association with eIF4E-binding proteins, of which 4E-BP1 is the best-studied. Phosphorylation of 4E-BP1 leads to its dissociation from eIF4E, leaving eIF4E free to bind eIF4G and form eIF4F complexes . In a murine cytotoxic T cell line, interleukin-2 induced the phosphorylation of 4E-BP1 . 4E-BP1 is present in human primary T lymphocytes and becomes phosphorylated in response to PMA or PMA plus ionomycin . In several cell lines, an increase in eIF2B activity coincides with an increase in protein synthesis . One mechanism to regulate the activity of eIF2B is via phosphorylation of its epsilon-subunit (eIF2Bepsilon) by GSK-3, which causes a decrease in eIF2B activity . Stimulation of T cells with PMA plus ionomycin caused a rapid rise in eIF2B activity, which coincided with inactivation of GSK-3 , suggesting a role for dephosphorylation of eIF2Bepsilon. The activity of eIF2B can also be modulated by phosphorylation of the alpha-subunit of eIF2. eIF2 phosphorylated in its alpha-subunit acts as a competitive inhibitor of eIF2B . Stimulation of T cells with PHA did not cause significant changes in the phosphorylation state of eIF2alpha , excluding this mechanism of regulation under this condition. In this study we used the antibodies, alphaCD3 and alphaCD28, to activate resting human primary T lymphocytes. Engagement of alphaCD3 activates the T cell receptor, while cross-linking of alphaCD28 with the B7 receptor will supply a co-stimulatory signal, which is required for full activation of a resting T cell . We have studied the effects of T cell activation on protein synthesis and on the activities and/or phosphorylation states of several translation initiation factors. Furthermore, the signalling pathways involved in these changes have been investigated. PMID- 12028592_Results TI - AB - Activation of primary T cells with a physiological stimulus increased protein synthesis | We activated T cells with the antibodies, alphaCD3 and alphaCD28, for up to 24h and measured protein synthesis and the activation of several signalling pathways that are important for the regulation of translation factors and that have been shown to increase after activation of T cells . Figure 1 | Activation of T cells with alphaCD3 and alphaCD28. Activation of T cells with alphaCD3 and alphaCD28.A. Primary T lymphocytes were activated with both alphaCD3 and alphaCD28 for 4, 8, 16 or 24 h and for the last 45 min 10 muCi/ml of [35S]-labelled methionine was present. The experiment was performed in duplicate. Incorporation of [35S]-labelled methionine into equal amounts of protein was measured. Protein synthesis in control cells was set at 100%. Methionine incorporation ranged between 1000 and 2000 cpm per 50 mug of protein. (control cells at t = 0 h and t = 24 h are not significantly different, n = 3). B. T cells were activated by alphaCD3 and alphaCD28 or with PMA. Cells were activated for 30 min, harvested and 50 --80 mug of lysate was analyzed by SDS-PAGE and Western blotting. Antibodies that recognize the phosphorylated form of either ERK (pp42) or p38 MAPK (pp38) were used. Even loading of the gel was verified using anti-ERK2 (p42). Similar results were obtained in three sets of experiments. C. PKB activity was measured as described in Materials and Methods (p<0.05, n=4). The rate of protein synthesis in resting T cells is low, and activation of the cells with alphaCD3 and alphaCD28 led to a substantial increase in the incorporation of [35S]-methionine into protein. Within 24 h of activation, the rate of protein synthesis was increased 6-fold , depending on the blood donor. After 30 min of activation, phosphorylation of ERK and p38 MAPK, and the activity of PKB were measured. Phosphorylation of ERK2 and p38 MAPK increased already after 5 min and reached a maximum after 30 --60 min of treatment (data not shown). After 30 min of treatment, a clear phosphorylation of ERK and p38MAPK was detected . Treatment with PMA, a potent activator of PKC, was used as a positive control. PMA induced, in particular, a greater extent of phosphorylation of ERK than alphaCD3 plus alphaCD28. For p38 MAPK phosphorylation, the difference between these stimuli was less pronounced . An 1.5 fold increase in PKB activity was detected within 30 min of activation of T cells by alphaCD3 and alphaCD28. The increase in protein synthesis and the stimulation of various signalling pathways indicated that treatment of T cells with alphaCD3 and alphaCD28 led to activation of the cells. The cellular protein levels of eIF4E and eIF2Bepsilon do not change during the early phase of T cell activation | Previous studies using mitogenic stimuli showed that the levels of several initiation factor proteins increase later (>16 h) following T cell activation and probably contribute to the increase in protein synthesis . To examine whether activation of T cells with alphaCD3 and alphaCD28 also affected initiation factor levels, the amounts of the cap-binding protein, eIF4E, and of the catalytic subunit of the eIF2B complex, eIF2Bepsilon, were assessed at different time points . The amounts of eIF4E and eIF2Bepsilon protein each remained constant during the first 6 h. Figure 2 | The amount of eIF4E and eIF2Bepsilon protein did not change in the early phase of T cell activation. The amount of eIF4E and eIF2Bepsilon protein did not change in the early phase of T cell activation. T cells were activated with alphaCD3 and alphaCD28 for the indicated times. In each case, 600 mug of protein was used for an m7GTP Sepharose pull down to detect the amount of eIF4E or used in an immunoprecipitation reaction with alphaeIF2Bepsilon coupled to protein G to detect eIF2Bepsilon. The pull downs were analyzed by SDS-PAGE and Western blotting. The experiment was performed in duplicate. In this study, we have focused on the mechanisms underlying the initial response after activation of primary T cells by alphaCD3 and alphaCD28 and the concomitant increase in protein synthesis. Since the levels of initiation factor proteins did not change in this early phase of T cell activation, we considered the possibility that changes in the phosphorylation state and/or activities of several translation factors were involved in the initial activation of protein synthesis in T cells. Protein synthesis is regulated via multiple signalling pathways | Primary T lymphocytes were activated with alphaCD3 and alphaCD28, and after 1 h of activation, protein synthesis was increased 1.2 fold . To study the signalling events involved in this increase in protein synthesis, we performed the experiment in the presence of different specific signal transduction pathway inhibitors . The increase in overall protein synthesis was consistently blocked by each of the signal transduction inhibitors used, i.e. the PI 3-kinase inhibitor wortmannin, the mTOR inhibitor rapamycin, the p38 MAPKalpha/beta inhibitors SB203580 and SB202190, and the MEK inhibitor PD98059. It appears that the immediate activation of protein synthesis in T cells involves interplay between several signalling pathways. Figure 3 | The increase in protein synthesis requires signalling via several pathways. The increase in protein synthesis requires signalling via several pathways. T cells were preincubated with wortmannin (W, 100 nM), rapamycin (R, 100 nM), SB203580 (10 muM), SB202190 (10 muM) or PD98059 (PD, 50 muM) for 30 min before activation with both alphaCD3 and alphaCD28 for 1 h or the cells were treated with PMA (1 muM) for 1 h. For the last 30 min of activation 10 muCi/ml [35S]-labelled methionine was present. The experiment was performed in duplicate. Protein synthesis in control cells was set at 100%. Methionine incorporation ranged between 600 and 1500 cpm per 50 mug of protein. Incorporation of [35S]-labelled methionine into equal amounts of protein was measured using hot TCA precipitation (p<0.05 for the alphaCD3/28 and PMA treated samples; all other samples are not significantly different from the control, n=6). Stimulation of T cells with the more potent stimulus PMA for 1 h led to a substantially larger increase in protein synthesis (1.8 fold) compared to activation with alphaCD3 and alphaCD28 . Phosphorylation of eIF4E, eIF4F complex formation and 4E-BP1 phosphorylation remain unchanged after T cell activation | Phosphorylation of the cap-binding protein eIF4E can be regulated via the ERK and p38 MAPKalpha/betapathways , two pathways that appear to be important for the regulation of protein synthesis in T cells . Furthermore, the phosphorylation of eIF4E has been reported to be increased in response to several different treatments of primary T lymphocytes or the Jurkat T cell line . However, activation of T cells with alphaCD3 and alphaCD28 for up to 60 min did not cause a significant change in the phosphorylation state of eIF4E . Stimulation of T cells with either alphaCD3 or alphaCD28 alone was also insufficient to change the phosphorylation state of eIF4E (data not shown). We did detect a marked change in phosphorylation of eIF4E after 30 min of PMA treatment, indicating that the cells respond to this stimulus . In addition, treatment of the Jurkat T cell line with alphaCD3 and alphaCD28 caused phosphorylation of eIF4E that was already detectable after 30 min, demonstrating the effectiveness of the antibodies (alphaCD3 and alphaCD28) used . Figure 4 | Regulation of eIF4E phosphorylation and eIF4F formation. Regulation of eIF4E phosphorylation and eIF4F formation.A. Jurkat T cells were activated for 30 or 60 min by both alphaCD3 and alphaCD28. The primary T lymphocytes were activated for the indicated times with both alphaCD3 and alphaCD28 or with PMA. eIF4E was purified using m7GTP Sepharose, analyzed on a one-dimensional iso-electric focusing gel and detected by Western blotting. 4E and 4E-P indicate unphosphorylated and phosphorylated eIF4E respectively. B. 100 mug of total cell lysate from primary T cells treated for 1 h with alphaCD3 and alphaCD28 or PMA was analyzed by SDS-PAGE and Western blotting to detect 4E-BP1. (-) indicates untreated cells. The lane with 4E-BP1 from HeLa cell extract was obtained from a shorter exposure from the same blot. C. Formation of eIF4F was analyzed after 60 min activation of primary T cells with either alphaCD3, alphaCD28 or both. eIF4E was purified as described above and its association with eIF4G was analyzed by SDS-PAGE and Western blotting. An eIF4E blot was used to verify equal loading of all lanes. Similar results for eIF4E, eIF4G and 4E-BP1 were obtained in three independent experiments. An important way of regulating eIF4F assembly is through eIF4E-binding proteins such as 4E-BP1. Phosphorylation of 4E-BP1 leads to its release from eIF4E, allowing the latter protein to bind eIF4G . The phosphorylation of 4E-BP1 can be detected by virtue of a reduction in its mobility upon SDS-PAGE . As a control to demonstrate that different forms of human 4E-BP1 can be resolved on our gel system, we used HeLa cell extract and in this case three separate bands (alpha, beta and gamma) were indeed detected , indicative of differently phosphorylated forms. In resting T-cells, 4E-BP1 was mainly present in the unphosphorylated form (alpha-form) as reported before . We were unable to detect any changes in mobility of 4E-BP1, and therefore its phosphorylation after stimulation of the cells with alphaCD3 plus alphaCD28 or PMA . Formation of eIF4F complexes was studied by purification of eIF4E on m7GTP-Sepharose followed by a Western blot to detect associated eIF4G. In resting T cells, eIF4F complexes are already present, and after 1h of activation with alphaCD3, alphaCD28, or both antibodies, the amount of eIF4G bound to eIF4E remained unchanged . Similar results were obtained after 30 min of activation (data not shown). Surprisingly, no 4E-BP1 associated with eIF4E was detected, even though up to 2 mg of T cell extract was used in a m7GTP Sepharose pull down (data not shown). This could be due to low amounts of 4E-BP1 protein present in resting T cells. These data indicate that increased formation of eIF4F complexes is not required for the activation of protein synthesis in the early phase of T cell activation. Regulation of eIF2B activity after activation of T cells | In several cell types, an increase in overall protein synthesis coincides with an increase in eIF2B activity . We therefore examined the activity of eIF2B after activation of primary T cells . After 1 h of activation with alphaCD3 and alphaCD28, the activity of eIF2B increased 2.2 fold. The increase in eIF2B activity was not caused by a change in the phosphorylation state of the alpha-subunit of eIF2 or by an increase in the amount of eIF2Bepsilon protein (Figs. and and ). Immunoprecipitation of different amounts of T cell extracts showed that the eIF2Bepsilon antibody was able to detect different levels of protein in the immunoprecipitations within the same range used in Fig. (bottom panel). Taken these results together it suggested that eIF2B was regulated directly, e.g. via phosphorylation. To study which signal transduction pathways are involved in the regulation of the activity of eIF2B, the cells were activated in the presence of specific signal transduction pathway inhibitors and eIF2B activity was measured. The basal activity of eIF2B was slightly affected in the presence of the PI 3-kinase inhibitor wortmannin, the mTOR inhibitor rapamycin, and the p38 MAPKalpha/beta inhibitor SB203580, however the alphaCD3 plus alphaCD28-induced increase in eIF2B activity was completely blocked in the presence of each inhibitor. The MEK inhibitor PD98059 did not affect the basal eIF2B activity and was also able to inhibit the alphaCD3 and alphaCD28-induced increase in eIF2B activity, showing that all these signalling pathways are required to mediate the activation of eIF2B . Figure 5 | Regulation of eIF2B activity. Regulation of eIF2B activity.A. T cells were preincubated with wortmannin (W, 100 nM), rapamycin (R, 100 nM), SB203580 (SB, 10 muM), or PD98059 (PD, 50muM) for 30 min and left untreated (white bar) or the cells were activated with both alphaCD3 and alphaCD28 (black bar) for 1 h. Simultaneously cells were activated with PMA (hatched bar) for 1 h. An eIF2B assay was performed as described in Materials and Methods. Bars marked with * are significantly different from the untreated cells (p<0.05, n=5). B. Cell lysates from resting and stimulated cells (1 h alphaCD3 and alphaCD28) were analyzed by SDS-PAGE and Western blotting to detect phosphorylated eIF2alpha. eIF2B was immunoprecipitated from 400 mug of lysate using alphaeIF2Bepsilon and the amount of protein was analyzed by SDS-PAGE and Western blotting. Similar results were obtained in three experiments C. To test the sensitivity of the eIF2Bepsilon antibody different amounts of T cell extracts (as indicated) were immunoprecipitated with alphaeIF2Bepsilon and analyzed by SDS-PAGE and Western blotting. Similar results were obtained in two experiments. D. T cells were left untreated (white bar) or the cells were activated with both alphaCD3 and alphaCD28 (black bar) or PMA (grey bar) for 1 h. GSK-3alpha and beta were immunoprecipitated together from 400 mug of lysate and a kinase assay was performed as described in Materials and Methods (p<0.05, n=4). E. eIF2Bepsilon was immunoprecipitated from 400 mug of lysate from resting and stimulated cells (1 h alphaCD3 and alphaCD28) and the total amount of eIF2Bepsilon and the phosphorylation state of Ser540 were analyzed by SDS-PAGE and Western blotting. Similar results were obtained in two experiments. Activation of eIF2B after stimulation of the cells with PMA was about 2 fold higher than after stimulation with the antibodies. An increase in eIF2B activity after stimulation of primary T-cells with PMA/ionomycin has been reported before . It has been suggested that GSK-3 may be an important regulator of eIF2B activity, i.e. in response to insulin and during cell survival . Phosphorylation of eIF2Bepsilon by GSK-3 inhibits the activity of the eIF2B complex . GSK-3 activity is decreased only by a small extent (15%) after T cell activation with alphaCD3 and alphaCD28 . In contrast, PMA treatment reduced GSK-3 activity by about 50%, which is similar to previously reported data. GSK-3 phosphorylates eIF2Bepsilon on Ser540. Therefore, we analyzed the phosphorylation state of this site using a phospho-specific antibody. We were unable to detect any change in the phosphorylation of this site in response to alphaCD3 and alphaCD28, excluding a role for GSK-3 in the regulation of eIF2B activity in T cells under these conditions. Dephosphorylation of eEF2 | Elongation factor 2 (eEF2) plays an important role in the regulation of the rate of elongation, and therefore in the regulation of the rate of overall protein synthesis. Phosphorylation of eEF2 causes its inactivation . Phosphorylation of eEF2 was rapidly but only transiently decreased after activation of the primary T lymphocytes with alphaCD3 and alphaCD28 . Within 3 min, maximum dephosphorylation was reached and the phosphorylation level returned to a level similar to that of resting T cells by 10 min. Given the transient nature of these changes, it is unlikely that regulation of eEF2 plays a role in the sustained increase in the rate of protein synthesis after activation of T cells. However, dephosphorylation of eEF2 could play a role very early in T cell activation. Figure 6 | Dephosphorylation of eEF2 after T cell activation. Dephosphorylation of eEF2 after T cell activation. T cells were activated with alphaCD3 and alphaCD28 for the indicated times. 80 mug of protein was analyzed by SDS-PAGE and Western blotting. Phosphorylation of eEF2 was detected using a phospho-specific antibody (eEF2-P). ERK2 was detected as a loading control. Similar results were obtained in three experiments. Regulation of p70 S6 kinase upon T cell activation | Activation of p70 S6 kinase and phosphorylation of the ribosomal protein S6, an in vivo substrate of p70 S6 kinase, coincide with increased translation of specific mRNAs, namely the 5'TOP mRNAs . However, a recent report has questioned the role of p70 S6 kinase in 5'TOP messenger translation . We studied the effect of activation of T cells with alphaCD3 plus alphaCD28 on these proteins. The activity of p70 S6 kinase was increased about 1.5 fold after 60 min, and the increase in p70 S6 kinase activity was blocked by each of the signal transduction inhibitors used, i.e. wortmannin, PD98059, SB203580, and rapamycin . Similar results were obtained when the phosphorylation of the S6 protein was examined as a cellular read-out of p70 S6 kinase activity . Phosphorylation of S6 in response to PMA was greater than in response to alphaCD3 and alphaCD28, similarly to the situation for several translation factors, as described above. Figure 7 | Regulation of p70 S6 kinase. Regulation of p70 S6 kinase.A. Primary T lymphocytes were preincubated with wortmannin (W, 100 nM), rapamycin (R, 100 nM), SB203580 (SB, 10 muM), or PD98059 (PD, 50 muM) for 30 min before activation with both alphaCD3 and alphaCD28 (black bars) for 60 min. Bars marked with * are significantly different (p<0.01) from the activity in untreated cells (n=4). B. T cells were preincubated with wortmannin (W, 100 nM), rapamycin (R, 100 nM), SB203580 (SB, 10 muM), or PD98059 (PD, 50 muM) for 30 min before activation with both alphaCD3 and alphaCD28 for 60 min or the cells were stimulated with PMA for 60 min. Phosphorylation of the S6 protein was analyzed by SDS-PAGE and Western blotting using a phospho-specific S6 (Ser235) antibody. Similar results were obtained in four experiments. PMID- 12028592_Discussion TI - AB - The mechanisms underlying the regulation of protein synthesis following activation of resting primary T cells has not been widely studied. Early reports showed that stimulation of primary T cells with pharmacological stimuli, e.g. PHA or PMA, led to an increase in protein synthesis and protein levels of certain translation initiation factors within 16 h . More recently the regulation of protein synthesis and translation factors after a 6 h stimulation of primary T cells with PMA or PMA/ionomycin was described in detail . We investigated the regulation of protein synthesis and translation factors during the early phase of activation of resting T cells by alphaCD3 and alphaCD28, i.e. 1 h of activation. The protein synthesis rate in T cells rapidly increased after treatment , and several signalling pathways, i.e. ERK and p38 MAPK phosphorylation and PKB activation, were stimulated, showing the efficacy of the alphaCD3 and alphaCD28 antibodies in activating the cells. The increase in protein synthesis after 1 h was mediated via multiple signalling pathways, e.g. the MEK, p38 MAPKalpha/beta, PI 3-kinase and mTOR pathway, as indicated by the use of signal transduction inhibitors . In several cell types, the involvement of either MEK , PI 3-kinase or mTOR [,,-] in the activation of protein synthesis has been described. However, this is the first time that a role for the p38 MAPKalpha/betapathway in the regulation of overall protein synthesis has been described. This role is supported by the fact that two structurally unrelated p38 MAPKalpha/beta inhibitors, i.e., SB202190 and SB203580, were each able to block the increase in protein synthesis. The increase in protein synthesis after activation of T cells with alphaCD3 and alphaCD28 coincided with an increase in the activities of p70 S6 kinase and eIF2B and dephosphorylation of eEF2, which is indicative of an increase in its activity . The dephosphorylation of eEF2 was very transient, and therefore it is unlikely that eEF2 plays an important role in the sustained increase in protein synthesis after T cell activation. However, activation of eEF2 could be important for the initial increase in protein synthesis. The stimulation of p70 S6 kinase was mediated via similar signalling pathways (Fig. 7) to those underlying the activation of overall protein synthesis. Inhibition of PI 3-kinase, mTOR, p38 MAPKalpha/beta, or MEK during T cell activation by alphaCD3 and alphaCD28 prevented the activation of p70 S6 kinase, indicating that multiple signalling pathways are required for regulation of p70 S6 kinase activity. The effect of rapamycin on the activation of p70 S6 kinase in response to alphaCD3 and alphaCD28 has been reported previously . Inhibition of the activation of p70 S6 kinase by SB203580 has been described before in insulin-stimulated rat vascular smooth muscle cells [,-]. Furthermore, it has been reported that SB203580 (at the concentration used, 10 muM) can inhibit phosphorylation of PKB at Threonine308 and thus its activation . Since PKB is an upstream component of the signalling pathway towards p70 S6 kinase, this could provide a mechanism by which SB203580 blocks activation of p70 S6 kinase. Each of the other signalling pathways studied here has also been implicated in the regulation of p70 S6 kinase activity in a variety of cell types under a range of conditions [,-]. However, in human primary T cells all of them appear to be important for the regulation of p70 S6 kinase activity after stimulation with alphaCD3 and alphaCD28. The activity of the guanine nucleotide exchange factor, eIF2B, also was mediated via similar signalling pathways as the increase in protein synthesis . The increase in eIF2B activity after activation of T cells with alphaCD3 and alphaCD28, at the early times we examined, was not due to an increase in eIF2B protein level or to changes in eIF2alpha phosphorylation. Therefore, modification of the eIF2B protein complex probably caused the increase in eIF2B activity. The modulation of the activity of the eIF2B complex after activation of T cells with alphaCD3 and alphaCD28 required several different signalling pathways, e.g. MEK, p38 MAPKalpha/beta, mTOR and the PI 3-kinase pathway. These signalling pathways have been reported separately to be involved in the regulation of eIF2B activity . However, this is the first report where all these pathways are involved in the regulation of eIF2B in a single cell type. A small inactivation of GSK-3 was detected after activation of primary T cells with alphaCD3 and alphaCD28. However, no dephosphorylation of Ser540 in eIF2Bepsilon was detected , excluding a role for GSK-3 in regulating the activity of eIF2B under these conditions. In contrast, studies employing PMA/ionomycin-activated T cells , insulin treatment of various cell types , and cell survival have implied a role for GSK-3 in regulating eIF2B activity. In contrast to previously reported data using mitogenic stimuli to activate primary T cells or Jurkat T cells , eIF4E phosphorylation, association of eIF4G with eIF4E and 4E-BP1 phosphorylation remained unchanged after T cell activation using alphaCD3 and alphaCD28 as a stimulus . Signal transduction inhibitor studies showed that the MEK and p38 MAPKalpha/beta pathways are important for eIF4E phosphorylation in Jurkat T cells , and a role for MEK was demonstrated previously in primary T cells . The weaker activation of the ERK pathway in particular by alphaCD3 and alphaCD28 in primary T cells may well account for the absence of increased phosphorylation of eIF4E under these conditions. We did not observe increased phosphorylation of 4E-BP1 in response to alphaCD3 and alphaCD28, even though it has been reported to occur after cytokine stimulation of a murine cytotoxic T cell line or after 6 h of mitogenic stimulation of human primary T cells . However, Grolleau et al. showed that 4E-BP1 was present in primary T cells mainly in its dephosphorylated form, and no significant change was detected after PMA treatment. Our results are consistent with this last finding; 4E-BP1 is mainly present as one band, and no change in mobility is observed upon cell treatment. This is consistent with the observation that eIF4F complex formation did not alter. However, it remains surprising that eIF4F complexes are present when 4E-BP1 is completely dephosphorylated, and therefore presumably associated with eIF4E. We were unable to detect any 4E-BP1 associated with eIF4E, which is probably due to the low 4E-BP1 protein levels in resting T lymphocytes, thus explaining basal eIF4F formation. PMID- 12028592_Conclusions TI - AB - The treatment of primary T lymphocytes with alphaCD3 and alphaCD28 activates two key components of the translational machinery, p70 S6 kinase and eIF2B. The activities of these translation factors were regulated similarly to the activation of protein synthesis, consistent with an important role for the components in the activation of protein synthesis by alphaCD3 and alphaCD28. Interestingly, activation of protein synthesis, p70 S6 kinase, and eIF2B is inhibited by rapamycin, a compound that was first discovered as an immunosuppressant, suggesting that mTOR regulated translation is involved in the process of T cell proliferation. The activation of p70 S6 kinase is related to the regulation of translation of specific mRNAs, while the activation of eIF2B is likely to be required for stimulation of general protein synthesis . This suggests that increases in both specific and general protein synthesis are important in the early phase of T cell activation. PMID- 12028592_Materials and methods TI - AB - Primary T cell isolation and cell treatment | Buffy coats used for the isolation of T cells were prepared from freshly drawn blood from healthy human donors and were obtained from the Scottish National Blood Transfusion Service (Edinburgh, UK). Mononuclear leukocytes were isolated by Ficoll-Hypaque (Amersham-Pharmacia) gradient centrifugation. T cells were further enriched using nylon-wool columns. T cells were suspended in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated foetal calf serum, 1 mM glutamine and antibiotics/antimycotics (100 units/ml penicillin G sodium, 100 mug/ml streptomycin sulphate and 0.25 mug/ml amphotericin B). The cells were kept in 75 cm2 tissue culture flasks at a density of 4 x 106 cells/ml at 37C and 5% CO2. All tissue culture reagents were obtained from Gibco BRL. Measurement of protein synthesis rate | Cells were treated with alphaCD3 mouse IgG2a mAb (33/2A3) (1:1000 dilution from a hybridoma supernatant) and alphaCD28 mouse IgM mAb (CK243) (1:12 dilution from a hybridoma supernatant) for 1 h in the absence or presence of signal transduction inhibitors. For the last 30 min, 10 muCi/ml [35S]methionine was added to the cells. To harvest the cells, the cells were transferred to a microfuge tube and centrifuged at 6000 x g for 20 s. The cell pellet was lysed in 20 mM Hepes pH7.4, 50 mM beta-glycerophosphate, 0.2 mM EDTA, 1% Triton X-100, 10% (v/v) glycerol, 1 mug/ml leupeptin, 1 mug/ml pepstatin, 1 mug/ml antipain, 1 mM benzamidine, and 1 mM DTT. Part of the sample was used to measure the protein content with Protein Assay Reagent (Bio-Rad) and the rest was spotted in duplicate on to Whatman 3 MM paper and subjected to 'hot TCA precipitation'. Gel electrophoresis and Western blotting | T cells were activated with alphaCD3 and alphaCD28 for times indicated in the figure legends, harvested in Laemmli sample buffer and analyzed by SDS-PAGE and Western blotting. Phospho-p42/44(ERK) and phospho-p38 MAPK antibodies were obtained from New England Biolabs, the phospho-eIF2alphaantibody was a kind gift from Dr. Gary Krause (Detroit, USA), the phospho-S6 (Ser235) antibody was a kind gift from Dr. Dario Alessi (University of Dundee), the 4E-BP1 antibody was obtained from Santa Cruz (SC-6025), the eIF2Bepsilon antibody was raised in rabbit against the whole protein expressed in the baculovirus system , the phospho-specific antibody for Ser540 in eIF2Bepsilon was raised against the peptide SEEPDS(P)RGGC (S(P) indicates the phosphoserine) in sheep, and the phospho-eEF2 (Thr56) antibody was raised against the peptide GETRFT(P)DTRK (T(P) indicates phosphothreonine) . Kinase assays | For PKB assays, the cells were pelleted at 6000 xg for 20 s and harvested in 50 mM Tris-HCl pH 7.5, 1 mM sodium orthovanadate, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 muM microcystin LR, 1 mug/ml leupeptin, 1 mug/ml pepstatin, 1 mug/ml antipain, and 1 mM benzamidine-HCl. Antibodies directed against the three PKB isoforms (alpha, beta, and gamma) were simultaneously bound to protein G-Sepharose, and about 100 mug of protein was used in immunoprecipitation reactions. The immunoprecipitation and PKB assays were performed as described before . For p70 S6 kinase and GSK-3 assays, the cells were harvested in a buffer containing 50 mM Tris-HCl pH 7.5, 50 mM beta-glycerophosphate, 0.5 mM sodium vanadate, 1.5 mM EDTA, 1.5 mM EGTA, 0.5% Triton X-100, 1 mug/ml leupeptin, 1 mug/ml pepstatin, 1 mug/ml antipain, 1 mM benzamidine, and 1 mM DTT. A polyclonal antibody raised against a peptide sequence from p70 S6 kinase-1 was bound to protein G-Sepharose, and about 100 mug of extract was used in the immunoprecipitation reaction. Immunoprecipitation and the p70 S6 kinase assays (using a peptide substrate) were performed as described before . GSK-3alpha and beta were immunoprecipitated together from 150 mug of cell lysate and kinase assays were performed as described . Phosphorylation of eIF4E and eIF4F complex formation | Cells were pelleted at 6000 x g for 20 s and harvested in a buffer containing 20 mM Hepes pH7.4, 50 mM beta-glycerophosphate, 0.2 mM EDTA, 1% Triton X-100, 10% (v/v) glycerol, 1 mug/ml leupeptin, 1 mug/ml pepstatin, 1 mug/ml antipain, 1 mM benzamidine, and 1 mM DTT. Using m7 GTP Sepharose 4B (Amersham-Pharmacia; 15 mul of slurry diluted with 15 mul of Sepharose CL-4B), eIF4E was purified from approximately 2 mg of extract. For SDS-PAGE, Laemmli sample buffer was added and the samples were heated at 95C for 10 min. For one-dimensional iso-electric focusing analysis, the appropriate sample buffer was added . The samples were run on a 12.5% SDS-PA gel or on a one-dimensional iso-electric focusing gel, transferred to PVDF, and detected by Western analysis. eIF4E was detected with a polyclonal antibody raised in rabbit , and eIF4GI with a polyclonal antibody raised in sheep against the peptide CKKEAVGDLLDAFKEAN. Measurement of eIF2B activity | The cells were pelleted at 6000 x g for 20 s and lysed in a buffer containing 20 mM Tris-HCl pH 7.5, 50 mM beta-glycerophosphate, 100 mM KCl, 0.2 mM sodium orthovanadate, 0.2 mM EDTA, 0.2 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mug/ml leupeptin, 1 mug/ml pepstatin, 1 mug/ml antipain, 1 mM benzamidine, and 1 mM DTT. About 50 mug of cell lysate was used for the eIF2B assay, which was performed as described previously . PMID- 12028592_Authors' contributions TI - AB - Author 1 (MK) carried out all the experiments. Author 2 (CGP) participated in the design and coordination of this study. All authors read and approved the final manuscript. PMID- 12028592_Abbreviations TI - AB - eIF, eukaryotic initiation factor; eEF, eukaryotic elongation factor; ERK, extra-cellular regulated kinase; GSK-3, glycogen synthase kinase-3; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; m7GTP, 7-methyl guanosine triphosphate; mTOR, mammalian target of rapamycin; PI 3-kinase, phosphoinositide 3-kinase; PHA, phytohemagglutinin; PKB, protein kinase B; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; 4E-BP1, eIF4E-binding protein 1. PMID- 12006105 TI - Management of obstetric anal sphincter injury: a systematic review & national practice survey AB - Abstract | Background | We aim to establish the evidence base for the recognition and management of obstetric anal sphincter injury (OASI) and to compare this with current practice amongst UK obstetricians and coloproctologists. Methods | A systematic review of the literature and a postal questionnaire survey of consultant obstetricians, trainee obstetricians and consultant coloproctologists was carried out. Results | We found a wide variation in experience of repairing acute anal sphincter injury. The group with largest experience were consultant obstetricians (46.5% undertaking >= 5 repairs/year), whilst only 10% of responding colorectal surgeons had similar levels of experience (p < 0.001). There was extensive misunderstanding in terms of the definition of obstetric anal sphincter injuries. Overall, trainees had a greater knowledge of the correct classification (p < 0.01). Observational studies suggest that a new 'overlap' repair using PDS sutures with antibiotic cover gives better functional results. However, our literature search found only one randomised controlled trial (RCT) on the technique of repair of OASI, which showed no difference in incidence of anal incontinence at three months. Despite this, there was a wide variation in practice, with 337(50%) consultants, 82 (55%) trainees and 80 (89%) coloproctologists already using the 'overlap' method for repair of a torn EAS (p < 0.001). Although over 50% of colorectal surgeons would undertake long-term follow-up of their patients, this was the practice of less than 10% of obstetricians (p < 0.001). Whilst over 70% of coloproctologists would recommend an elective caesarean section in a subsequent pregnancy, only 22% of obstetric consultants and 14% of trainees (p < 0.001). Conclusion | An agreed classification of OASI, development of national guidelines, formalised training, multidisciplinary management and further definitive research is strongly recommended. PMID- 12006105_Background TI - AB - The importance of highlighting the problem of incontinence to professionals, and the need to focus on reducing underlying causes is emphasised in recent Department of Health documents . Anal incontinence may be defined as 'faecal or flatus incontinence which is a social or hygienic problem' . There is little doubt that vaginal delivery in general, and obstetric anal sphincter injury (OASI) in particular, are significant contributory factors in the development of anal incontinence . In the UK, anal incontinence in the year after birth is thought to affect nearly 40,000 mothers (1 in 20) annually . Post-partum anal incontinence may affect mothers psychologically as well as physically but many do not seek medical attention because of embarrassment or because they are easily discouraged from discussing it . In one study only one third of individuals with faecal incontinence had ever discussed the problem with a physician . In recent correspondence to the Continence Foundation, a woman describes 'the eternal shame of being with another person when the worst occurs' . The impact of this complication on the vulnerable postnatal mother and her baby is potentially catastrophic. Aside from the potential clinical and social implications there are important medico-legal issues . Furthermore, the treatment of postpartum anal incontinence itself is associated with very high cumulative costs . Perineal trauma after childbirth is of further importance because it lies behind the growing clamour for the right of a woman to choose whether to deliver by elective caesarean section . Obstetric anal sphincter injuries may be seen at the time of birth ('overt') or may be detected only after additional ultrasound investigation, after birth ('occult'). The incidence of 'overt' anal sphincter injury has previously been reported as being between 0.5 --3% of vaginal deliveries . Until recently, anal incontinence not due to 'overt' anal sphincter injury was attributed to pelvic neuropathy . The advent of anal endosonography altered this view by identifying further 'occult' obstetric trauma to the anal sphincter. This has been reported in 35% of primiparous women and a significant association has been demonstrated between these sonographic defects and anal incontinence. However, it has not been established whether these injuries were genuinely 'occult' or whether they had been missed by the doctor or midwife at delivery. There is evidence from one study that perineal anatomy is poorly understood by midwives and trainee doctors, who perform the bulk of deliveries in the UK. In this study, 41% of trainees and 16% of midwives incorrectly classified a partial or complete tear of the EAS as 'second degree'. Inconsistency in classification of tears would allow many injuries to pass, unrecognised. Clearly where an injury occurs, but is not detected, the incidence of anal incontinence may approach 100%. These women will frequently be referred to colorectal surgeons for further evaluation and possible 'secondary' repair. Even with recognition and 'primary' repair, the incidence of anal incontinence has been reported as over 50% and the actual incidence may be even higher . The reasons for the apparently poor outcome after primary repair are not clear, particularly as there is considerable controversy in the literature regarding the optimal obstetric management of OASI. Variation in outcome may be due to different methods and materials being used or to deficiencies in skill and training . Given the sub-optimal outcomes achieved when these injuries are repaired by obstetricians, it has been suggested that better results may be obtained if colorectal surgeons perform the primary surgery . The aims of this study were firstly, to establish the best available evidence for the management of OASI by conducting a systematic review of the literature. Secondly, we aimed to audit current practice amongst trainee and consultant obstetricians. Finally, we wished to explore the views of consultant colorectal surgeons with respect to optimal management of OASI. PMID- 12006105_Methods TI - AB - Literature review | The Cochrane Library and Cochrane Register of Controlled Trials were searched for relevant Randomised Controlled Trials (RCT), systematic reviews and meta-analysis. A search of MEDLINE and PUBMED (electronic databases) from 1966 up to April 2001 was also carried out. The databases were searched using the relevant MeSH terms, including all sub-headings and this strategy was combined with a Key-word search using -- Human; Female; Childbirth; Obstetric; Perineum; Anal sphincter; Tear; Injury; Rupture; Damage; Incontinence; Faecal; Anal; Repair; Surgery; Sutures; Randomised controlled trials; Meta-analysis. The bibliographies of retrieved articles were searched manually as well as conference proceedings and abstracts from obstetrics and gynaecology and coloproctology meetings. Survey participants | The study sample included all consultant obstetricians & gynaecologists in the Royal College of Obstetricians & Gynaecologists (RCOG) UK database. As a comprehensive national register of obstetric trainees is unavailable, we surveyed all trainee obstetricians in the two regions in which the authors are based (West Midlands and South West Thames regions). We also included all consultant members of the Association of Coloproctology of Great Britain and Ireland. Development of a questionnaire | The postal questionnaire that was sent to the consultant and trainee obstetricians enquired about those aspects of knowledge and practice that our preliminary literature review suggested would be linked to outcome. Questions relating to current experience, classification of perineal injury, acute management, follow-up, management of subsequent deliveries and training received were all included. A similar questionnaire inquiring about the management of primary OASI repair was sent to the consultant coloproctologists. Data processing and analysis | A Freepost envelope was included with the questionnaire. Non-responders received a second questionnaire. Data was entered onto an Access database. The software used for analysis was StatXact Turbo (CYTEL), Cambridge, Massachusetts. The Kruskal-Wallis test was used to compare ordinal data between the 3 groups and chi square tests were applied when the data were nominal. In order to highlight the differences between current evidence and current practice we have presented the results of our literature search in combination with the results of the survey. PMID- 12006105_Results TI - AB - Respondents | At the time of the survey, 1441 names appeared in the RCOG consultant database (UK) of whom 152 were non-practising obstetricians and 96 had retired. Of the 1193 consultants in active obstetric practice, 672 (56%) completed and returned the questionnaire. Of the 235 trainee obstetricians in the West Midlands and South West Thames regions, 148 (63%) completed and returned the questionnaire. Only 90 (23%) of the 385 members of the Association of Coloproctology replied, despite 2 mailings. The overall response rate was 50.2% (910/1813). The majority of responding consultant obstetricians, 438 (65%) and coloproctologists, 53 (59%) had been in post for more than 5 years. Of the trainee obstetricians-108 (73%) were post-MRCOG. The average numbers of acute OASI repairs (primary) performed annually by consultant obstetricians, obstetric trainees and colorectal surgeons are shown in Table . There were significant differences between the three groups. Table 1 | Number of acute OASI repairs performed per year Definition of OASI | The evidence | The literature review revealed a lack of consistency in the classification of OASI. One study, which examined all the obstetric texts (n= 65) in the RCOG library, found that 22% of authors classified anal sphincter injury as 'second degree' and a further 17% did not mention any classification . Obstetric anal sphincter injury (OASI) is classified as a '3rd degree' tear when there is any involvement of the external anal sphincter (EAS) but when the anal epithelium is involved it is '4th degree' and this was incorporated in the RCOG guidelines on the management of perineal trauma . The RCOG definitions of perineal injury, which are included in the Green Top Guidelines for the Management of Third and Fourth-Degree Perineal Tears Following Vaginal Delivery are shown in Table . A more descriptive classification suggested by Sultan was agreed at a recent consensus meeting on OASI where third degree tears were further classified into three subgroups according to the extent of damage to the external anal sphincter (EAS) and internal anal sphincter (IAS) . Table 2 | Classification of Injury The survey | Two hundred and twenty (33%) consultant obstetricians and 30 (22%) trainees considered a complete or partial external sphincter tear to be 'second degree' . There was widespread regional variation in the 'misclassification' of OASI as 'second degree'. This is emphasised by a ten-fold difference between some regions (higher in the northern regions) in rates of respondents considering a complete EAS tear to be 'second degree' . Table 3 | Classification of perineal tears Table 4 | Geographical variation in the definition of OASI Technique of repair following OASI | The evidence | The most common type of repair is an end-to-end repair, where either interrupted or figure-of-eight sutures are inserted into the sphincter muscle. There is little variation on the standard technique reported. One study described the end-to-end approximation of the anal sphincter, by suturing the outer fascial layer without inserting sutures in the muscle. However, this study has not been reproduced. When employed for secondary repair, mobilisation, scar excision and direct apposition lead to an incidence of sphincter repair disruption of 40% . A modification of an overlapping technique for sphincter repair, described by Parks for the secondary repair of OASI , was first described for acute OASI by Sultan in 1999 . The technique includes identifying the internal anal sphincter which, if torn, is repaired as a separate layer. Using this technique the authors found a significant reduction in anal incontinence (to 8%), which can be compared with 41%, seen in a previous study where the end-to-end technique was employed . There is only one published, prospective randomised study, comparing end-to-end and the overlap techniques . In this series of 112 primiparous women, no significant difference in continence was identified at 3 months' follow-up. The techniques used in this study were different to those described by Sultan . In particular, internal sphincter injury was not identified and repaired separately. There are two ongoing randomised controlled trials comparing the two methods (in Stoke and Liverpool) registered in the Cochrane clinical trials register . The survey | Coloproctologists favour the overlap technique for primary repair of OASI . This technique is reportedly being used by large proportions of obstetricians particularly trainees. Over 55% of responding consultant obstetricians (375/672) and coloproctologists (60/90) said that they would be interested in participating in a trial to compare the two methods of repair. Table 5 | Techniques of external anal sphincter repair Suture material | The evidence | No randomised controlled studies to assess the best suture material for sphincter repair were identified. Most texts that describe the repair still mention the use of chromic catgut . However, monofilament suture materials, such as Polydioxanone (PDS) or Polypropelene (Prolene) are thought to be better than catgut or Polyglactin (Vicryl) because of their longer half life. There is good evidence from randomised trials that synthetic materials such as Vicryl or Polyglycolic acid (Dexon) are preferable to catgut for repair of the perineum . Catgut sutures, made from bovine intestinal material, have recently been withdrawn from UK and European countries. The survey | Vicryl was the material most frequently used for sphincter repair (by 505 (75%) consultant obstetricians and 97 (66%) trainees). By contrast, only 22 (24%) coloproctologists used Vicryl, with greater numbers preferring PDS (41 (46%)). Obstetric experience with PDS was minimal (used by only 51 (8%) consultant obstetricians and 20 (14%) trainees). Antibiotic usage | The evidence | Intra-operative and post-operative broad spectrum antibiotics are recommended because the development of infection may be linked to a breakdown of the anal sphincter repair . Although data exists relating to antibiotic prophylaxis in colorectal surgery there are no randomised controlled studies which examine antibiotic use following OASI repair. The survey | Peri-operative antibiotics were recommended by 82 (91%) coloproctologists and by 528 (79%) consultant obstetricians and 130 (88%) obstetric trainees. Stool softeners | The evidence | A passage of a hard bolus of stool may disrupt repair and therefore most surgical textbooks and experts recommend use of laxatives. However the use of laxatives and stool softeners after OASI repair has not been evaluated in a randomised controlled trial. Indeed medical 'bowel confinement' practised by some colorectal surgeons following secondary anal sphincter repair, has been shown in a randomised trial to confer no benefit in terms of septic complications or functional outcomes . The survey | Stools softeners or laxatives were advised by 70 (78%) coloproctologists. Six hundred and eighteen (94%) consultants and 121 (82%) trainee obstetricians routinely prescribed laxatives following repair. Colostomy | The evidence | In a recent report comprising of 4 patients, temporary defunctioning colostomy has been described when failure to recognise and repair rectal mucosal injury lead to significant early perineal contamination . We could find no studies to support colostomy in the management of acute OASI. A small randomised trial showed no conclusive evidence that a defunctioning stoma confers any benefit for those patients undergoing secondary repair in terms of functional outcome and may be associated with higher morbidity and longer hospital stays related to the stoma closure . The survey | Twenty-seven (30%) coloproctologists recommend a covering colostomy for third and fourth degree tears (of whom 25 said 'only for 4th degree tears'). None of the obstetricians either performed or requested a colostomy for acute OASI. Follow-up | The evidence | There are no controlled trials comparing different protocols. We found two publications suggesting that all women with OASI should be followed-up by coloproctologists . At a recent consensus meeting on OASI follow up in a multidisciplinary clinic was recommended for women with persistent bowel symptoms after delivery. The survey | Coloproctologists suggest a follow-up period of more than 12 months, compared to the majority of obstetricians who follow-up patients for only six weeks . Table 6 | Duration of follow-up after OASI repair Management of subsequent delivery | The evidence | There were no data in the literature from controlled studies regarding the best mode of subsequent delivery following OASI. Evidence that women who experience even transient anal incontinence after vaginal delivery may be at increased risk of faecal incontinence after a further vaginal delivery has lead to calls that such women should be offered caesarean section. However, compared with vaginal delivery caesarean section carries a higher mortality and other forms of morbidity and should therefore not be offered routinely to all women . In the prospective study from Fynes and colleagues , women with transient anal incontinence or occult sphincter injury after first delivery were at high risk of faecal incontinence after a second vaginal delivery. Furthermore it is not clear whether or not pregnancy per se influences postpartum anal incontinence. Symptomatic anal incontinence has been reported after both elective and emergency caesarean deliveries. An alternative approach may be to routinely assess function by means of a continence questionnaire, anorectal physiology tests and endosonography and selectively offer caesarean section to those women with some degree of compromised function. Clearly those who have had successful continence surgery should be delivered by caesarean section . However it remains to be established whether caesarean section would be beneficial to pregnant women with severe incontinence who are due to have continence surgery some time after delivery. The survey | In the present survey most coloproctologists recommended caesarean section. By contrast most obstetricians allowed vaginal delivery after a previous OASI. Table 7 | Mode of subsequent delivery: Role of the coloproctologist in the management of OASI | The evidence | Although some authors suggest that coloproctologists are best trained to repair OASI, there are no data to support this statement and the debate continues . The survey | In the present survey only small numbers of consultants (103 (15%)), and fewer trainees (5 (3%)), called a coloproctologist or a general surgeon to assist during acute OASI repair, the indication in these cases being 'severe anatomical disruption'. Few of the respondents believed that a coloproctologist should be routinely involved in acute management of OASI; 17 (19%) coloproctologists, 169 (25%) consultants and 16 (11%) trainees). Training received in the management of OASI | The evidence | There are no research studies comparing different methods of training. There is only one study that has evaluated training in repair of OASI . Ninety four percent reported unsatisfactory training at the time of performing their first unsupervised OASI repair. The survey | 445 (64%) consultants and 184 (64%) trainees reported either 'a lack of' or 'unsatisfactory' training in the management of OASI. PMID- 12006105_Discussion TI - AB - Our literature review and national survey have shown that obstetric anal sphincter injury is an area of childbirth that has been largely ignored, in both research and educational terms. There is a poor evidence base for practice and there has been only one published randomised controlled trial in this area. Our survey had a good obstetric response rate but a poor response from the colorectal surgeons (this may be attributed to the fact that, only about one third of coloproctologists have a specialised interest in anorectal incontinence). Our survey shows wide variation within specialities, levels of seniority and different regions of the country. These findings were echoed at a national professional consensus which highlighted the importance of research into anal incontinence, including the problems of failed recognition and uncertainty about the best method of repair . There are two probable reasons for the underestimation of OASI. Firstly, as a result of lack of a consistent classification, OASI can be wrongly classified as a 2nd degree tear and therefore managed inappropriately. We found that 33% of consultants and 22% of trainees classified a partial or complete tear of the EAS as 'second degree' which echoes the previous smaller study by Sultan . One reason for misclassification may be a degree of confusion created by popular obstetric texts . Our analysis of the regional trends in classification indicates that more consultants in the Northern regions of the country prefer to classify OASI as 2nd degree. This may reflect the teaching in a popular obstetric textbook . To avoid confusion we support the unified and descriptive classification given by Sultan in which third degree tears are further sub-classified into 3a (less than 50% of thickness of external sphincter torn), 3b (more than 50% of thickness of external sphincter torn) and 3c (internal sphincter torn). Figure 1 | Regional variation of the definition of anal sphincter injury Regional variation of the definition of anal sphincter injury Secondly, underestimation may be due to lack of recognition of OASI because of lack of training. A recent study has shown that immediate assessment of all perineal tears following childbirth by a trained and experienced obstetrician can significantly increase the detection rate. In this study, rates of up to 15% were found, which might provide an explanation for the high incidence of 'occult' sphincter defects reported in previous prospective studies . We have recently presented national data showing nearly a hundred fold variation in detection rates of OASI . The reason for the poor outcome following repair can be attributed to inappropriate technique or to lack of operator expertise or to a combination of these factors. Despite a primary repair of acute obstetric anal sphincter injury, up to 59% of women suffer from persistent anal incontinence and persistent sonographic sphincter defects have been identified in over 80% . Anal incontinence may present for many years following OASI and can also deteriorate with time. Schofield and Grace have looked at the differential rates of the components of anal incontinence (faecal incontinence and flatus and urgency) in five studies which examined these outcomes after primary repair of third degree tears. The intervals of follow-up ranged from 6 weeks to 10 years. The overall mean rates were 25% (flatus +/- urgency) and 9% (faecal incontinence). The longer the follow-up, the higher the rate of incontinence. We have identified another 11 studies with long term follow-up (a mean of 41 months) after 3rd degree tears and 20 to 59% (mean 40%) reported anal incontinence symptoms . Responses to this national survey showed the principle area of disagreement to be 'the optimal method of repair'. However, the fact that half of all obstetricians now claim to be using the 'overlapping' method suggests that there is a change in practice taking place, despite the absence of good quality evidence to support this. Given that untutored use of the overlap method could potentially increase morbidity (as it requires more dissection and mobilisation prior to repair and could also result in a sphincter which is too tight), this may be considered an area of concern. In a recent 5 year follow-up study of incontinent women who had a secondary overlapping repair for obstetric trauma , although 50% improved only 4 of 38 patients were totally continent. The overall success of the overlap method 'seemed to deteriorate with time'. The authors wondered whether the technique itself contributed to this deterioration. Clearly there is an urgent need for further properly controlled trials of method of repair, with adequate long-term follow-up. As shown in our survey, there is a widespread interest in participating in a controlled trial in this area. If advantage is taken of this 'window of opportunity', considerable benefits for women should arise. The finding that only one third of UK consultant obstetricians reported that they were adequately trained to perform anal sphincter repairs requires further attention as this may well have serious clinical and medico-legal implications . We believe that obstetricians need more intensive and focused training in OASI and repair. A series of hands-on-workshops in the management of OASI, utilising a specifically designed model and animal models, has been initiated at St George's Hospital, London. Although it has been suggested that as colorectal surgeons are trained to perform a secondary sphincter repair they should be performing the primary repairs, there was little evidence that current systems could support such a significant change in organisation. Indeed, our survey showed that most coloproctologists have little or no experience of managing acute OASI. A lack of understanding of the circumstance of childbirth by coloproctologists may explain why 30% believed that a colostomy is appropriate management. Further interdisciplinary co-operation is clearly required. It is apparent from our survey that most obstetricians use Vicryl sutures and antibiotics, and prescribe stool softeners after repair. The ideal suture material for primary sphincter repair is not known although good results have been described using a delayed absorbable monofilament material such as PDS and some surgeons prefer prolene. Further research in this area is required. Given the large proportions of women who may suffer impaired anal continence even following repaired OASI, it is imperative to establish a system for follow-up. Further research into the most efficient and effective systems is required. Increasing awareness amongst women and community health professionals about the possible sequelae of OASI is important and easy access for appropriate follow-up and further investigation is essential. There is no evidence to indicate the ideal and safest mode of subsequent delivery. Until further research has been undertaken individual cases will need to be managed empirically. Where there has been secondary surgery or where symptoms have taken some time to improve the threshold for elective caesarean section will be lower. PMID- 12006105_Conclusions TI - AB - To encourage women to consider vaginal delivery positively, adverse outcomes need to be minimised. The results of our literature review and professional survey are informing the development of national guidelines based on current available best evidence . Support for research in this area has a broad mandate and reflects a need noted by both the research community and the research consumer . Randomised controlled trials of overlap versus end-to-end repair are currently underway . It is clearly of great importance that this research is supported and that further studies addressing the detail on training, prevention, recognition and management of OASI are commenced. PMID- 12006105_Competing interests TI - AB - None declared. PMID- 12006105_Appendix TI - AB - What is known about the subject / What the paper adds | Appendix see : Appendix Anal Sphincter Paper PMID- 12006105_Authors' contributions TI - AB - This study was conceived by RF, RJ, AS and SR. RF, RJ, AS, SR and PJ contributed to the design of the study. RF carried out the survey and data collection. RF and PJ analysed the data. The paper was written jointly by all authors. RF and RJ act as guarantors for this paper. PMID- 12006105_Pre-publication history TI - AB - The pre-publication history for this paper can be accessed here: PMID- 12052260 TI - Mutational analysis of human profilin I reveals a second PI(4,5)-P2 binding site neighbouring the poly(L-proline) binding site AB - Abstract | Background | Profilin is a small cytoskeletal protein which interacts with actin, proline-rich proteins and phosphatidylinositol 4,5-bisphosphate (PI(4,5)-P2). Crystallography, NMR and mutagenesis of vertebrate profilins have revealed the amino acid residues that are responsible for the interactions with actin and poly(L-proline) peptides. Although Arg88 of human profilin I was shown to be involved in PI(4,5)-P2-binding, it was suggested that carboxy terminal basic residues may be involved as well. Results | Using site directed mutagenesis we have refined the PI(4,5)-P2 binding site of human profilin I. For each mutant we assessed the stability and studied the interactions with actin, a proline-rich peptide and PI(4,5)-P2 micelles. We identified at least two PI(4,5)-P2-binding regions in human profilin I. As expected, one region comprises Arg88 and overlaps with the actin binding site. The second region involves Arg136 in the carboxy terminal helix and neighbours the poly(L-proline) binding site. In addition, we show that adding a small protein tag to the carboxy terminus of profilin strongly reduces binding to poly(L-proline), suggesting local conformational changes of the carboxy terminal alpha-helix may have dramatic effects on ligand binding. Conclusions | The involvement of the two terminal alpha-helices of profilin in ligand binding imposes important structural constraints upon the functions of this region. Our data suggest a model in which the competitive interactions between PI(4,5)-P2 and actin and PI(4,5)-P2 and poly(L-proline) regulate profilin functions. PMID- 12052260_Background TI - AB - The small actin binding protein profilin has multiple binding partners and is thought to play a key-role in the regulation of actin dynamics . Originally, profilin was identified as an actin sequestering protein but recently more complex effects on actin polymerization have been proposed because actin-profilin complexes can add to free barbed ends thereby stimulating actin polymerization . Profilins bind poly(L-proline) sequences and many proteins containing proline-rich stretches have been identified as profilin ligands. Of these the interaction with the enabled/vasodilator stimulated phosphoprotein (Ena/VASP) family is best documented . For several proline-rich proteins a direct link with signal transduction pathways has been described , thus positioning profilins at crossroads of multiple pathways that lead to actin remodeling . With the elucidation of the profilin-beta-actin crystal structure, the residues at the interface of both proteins were identified . Additionally, crystalographic, mutagenesis and spectroscopic studies have addressed the poly(L-proline) binding site and showed that a hydrophobic pocket between the amino and carboxy terminal alpha-helices forms the binding site for poly(L-proline) sequences [,-]. The interaction of profilin with phosphatidylinositol lipids has been functionally studied. In vitro, PI(4,5)-P2 dissociates actin:profilin complexes and these and other authors also demonstrated the specificity of the interaction between profilin I and PI(4,5)-P2 in both micellar form as well as in lipid vesicles . More recently it was shown that phosphatidylinositol (3,4)-bisphosphate and phosphatidylinositol (3,4,5)-triphosphate bind to profilin with even higher affinity than PI(4,5)P2 and that phosphatidylinositol (3,4,5)-triphosphate inhibits profilin sequestering activity much better than PI(4,5)P2. In addition, PI(4,5)-P2, bound to profilin, can only be hydrolyzed by phospholipase Cgamma1 (PLCgamma1), when this lipase is phosphorylated and activated, which occurs in response to transmembrane signaling . This leads to two, not mutually exclusive scenarios that profilins are involved in phosphoinositide metabolism or that PI(4,5)-P2 hydrolysis causes translocation of profilin from the membrane to the cytosol where it can interact with actin or other ligands. This suggests an important role for profilin-phosphoinositide interaction in vivo. The structural basis for this interaction is, however, only partly resolved (see below). The interaction of actin binding proteins with PI(4,5)-P2 is usually assigned to the binding of the negatively charged headgroup of the phoshoinositide to basic amino acids. In agreement with this is that the more positivily charged Acanthamoeba profilin II isoform has highest affinity for PI(4,5)-P2. Similarly, the more basic human profilin I isoform interacts better with PI(4,5)-P2 than does profilin IIa . The identity of the amino acids responsible for binding of profilins to PI(4,5)-P2 is a matter of debate, because there are discrepancies between studies on profilins from lower eukaryotes and from vertebrates . Based on comparison of the crystal structure of the two Acanthamoeba profilin isoforms, Fedorov and co-workers proposed that a surface with positive electrostatic potential, formed by residues 71, 80, 81 and 115 (corresponding to residues 74, 88, 90 and 125 in human profilin), was the main PI(4,5)-P2 binding site in Acanthamoeba profilin. This surface largely overlaps with the actin binding surface and hence this model explained the observed competition between actin and PI(4,5)-P2 for binding to profilin . Mutagenesis of the yeast homologue partially confirmed this model as residue 71, but not residue 80, is implicated in phosphoinositide binding . Based on the structural model, we previously suggested that Glu56 in mammalian profilin IIa would be responsible for the weaker interaction of this isoform because the negative charge of this residue reduces the large, positively charged surface around the hypothetical PI(4,5)-P2-binding site . In profilin I, which has a serine at position 56 this is less the case. In human profilin, however, only Arg88 and not Arg74, was argued to be involved in PI(4,5)-P2-binding since only the mutant in Arg88 showed decreased inhibition of PI(4,5)-P2 hydrolysis by PLCgamma . We and others have speculated that basic residues in the carboxy terminal alpha-helix of vertebrate profilins may be involved in PI(4,5)-P2-binding. First, Yu and coworkers postulated that the residues 126 to 136 (KCYEMSHLRR) of human profilin I are a modified version of the PI(4,5)-P2-binding motif in gelsolin (KSGLKYKK). Second, using photoactivatable homologues of PI(4,5)-P2, it was hypothesized that carboxy terminal basic residues in human profilin I are involved in contacting the negative headgroups of PI(4,5)-P2. Third, the observed competition between poly(L-proline) and PI(4,5)-P2 for binding to profilin is consistent with the proposal that the carboxy terminus of profilin is involved in PI(4,5)-P2-binding . Fourth, we have shown that mammalian profilins I and IIa have clearly different affinities for PI(4,5)-P2, even though their actin binding surface including Arg74 and Arg88, are well conserved. This suggests that still other residues must be involved in PI(4,5)-P2-binding. In this study we experimentally investigated this hypothesis using site directed mutagenesis of human profilin I. Our data clearly show that, in addition to Arg88, also Arg136 in the carboxy terminal helix has a major contribution to PI(4,5)-P2-binding. Given that mutant R136D, but not R88A, displays wild type actin binding activity, we propose that the PI(4,5)-P2 and actin binding sites only partly overlap. Our data also suggest a connection between PI(4,5)-P2-binding and the interaction with proline-rich ligands, since the profilin IIa mutant W3A, defective in poly(L-proline) binding shows increased PI(4,5)-P2-binding. Given the observed conformational changes upon poly(L-proline) and PI(4,5)-P2-binding we propose that correct orientation of the terminal alpha-helices is important for ligand binding. This is strengthened by the fact that the addition of a myc tag to the carboxy terminal helix of profilin IIa abolishes poly(L-proline) binding completely. PMID- 12052260_Results and discussion TI - AB - Mutational analysis of human profilin I | The goal of this study was to get a better insight into the structural basis of the interaction of vertebrate profilins with PI(4,5)-P2. To investigate the possible role of the above mentioned residues (see Background) in PI(4,5)-P2-binding and to obtain profilins that have reduced PI(4,5)-P2-binding capacity, we created a set of single and double mutants in the residues Ser56, Arg74, Arg88, Arg135 and Arg136 of human profilin I (Figure and Table ) and a mutant W3A defective in poly(L-proline) binding. Figure 1 | Three dimensional structure of human platelet profilin I (PDB entry, 1 fik). Three dimensional structure of human platelet profilin I (PDB entry, 1 fik). Helices are shown in red, beta-strands in blue, beta-turns in green and loops in grey. Residues mutated in this study are indicated with space filling: Trp3 in yellow (poly(L-proline) binding), Ser56 and Arg135 in pink, Arg 74 in green (actin binding), Arg88 and Arg136 in blue (PI(4,5)-P2 binding). Table 1 | Interaction of wild type and mutant profilins with a proline-rich peptide Wild type human profilin I as well as the mutants listed in Table were expressed in E. coli and all could be purified by poly(L-proline) affinity chromatography, except for the W3A mutant which does not bind poly(L-proline) (see below). We initially included the R88E mutant, but due to its instability, we were unable to purify this protein in sufficient amounts for biochemical analysis. Mutants have a similar fold and stability as wild type profilin I | We first probed whether the introduced mutations did not affect the conformation and stability by analyzing the conformational integrity of the mutants using circular dichroism (CD) spectra. We measured and compared spectra for wild type and mutant profilins between 184 and 260 nm . All mutants adopt a very similar fold as wild type profilin I. The wavelengths at which maximal and minimal peak values are observed do not or only slightly change. The small shoulders at lower wavelength, observed for the double mutants with R136D, suggests that mutation of this residue to aspartic acid affects in some way the stability or the position of the carboxy terminal alpha-helix. The differences are however too small to be interpreted quantitatively. Figure 2 | Circular dichroism spectra show that the mutants have a similar fold as wild type profilin I. Circular dichroism spectra show that the mutants have a similar fold as wild type profilin I. The molar ellipticity per residue weight is shown. The spectra of several single mutants (A) and of double mutants with altered PI(4,5)-P2 binding (B) are compared with that of wild type profilin I. To further test the stability of the mutants, especially the ones that show greatly altered binding to PI(4,5)-P2 (see below), we measured urea denaturation curves . For R136D and R88A/R136D we observed a very small shift of the transition to lower urea concentration when compared to wild type profilin I. On the contrary, R88E/R136D, which has the most pronounced phenotype (see below) displays a denaturation curve very similar to that of wild type profilin I. Together, these data show that the mutants are stable and correctly folded under the conditions used in the assays described below. Figure 3 | Urea denaturation curves for human profilin I and the three mutants that have strongly reduced PI(4,5)-P2 binding. Urea denaturation curves for human profilin I and the three mutants that have strongly reduced PI(4,5)-P2 binding. For each profilin the ratio of the intrinsic fluorescence (F) at two different wavelengths F(352 nm)/F(332 nm) is plotted versus the urea concentration. Wild type profilin I (closed squares), R136D (open circles), R88A/R136D (open triangles), R88E/R136D (closed circles). The inserted table lists the urea concentration at the midpoint of the fluorescence transition. These values are a measure for the stability of the proteins. Poly(L-proline) binding | To sensor more subtle effects on the poly(L-proline) binding of the mutants, we used surface plasmon resonance technology to monitor the binding of the mutants to the (GP5)3 peptide derived from VASP. The measured reasonance units (RU) for each mutant at three different concentrations are given in Table . Although it is not possible to calculate a Kd for profilin I by this method , from the obtained RU-values we can deduce relative affinities for the mutants as compared to wild type profilin I . The most severe effects are observed for R135D, R136D and double mutants containing one of these mutations. This is logical because Arg135 and Arg136 are located in the carboxy terminal alpha-helix, which is involved in poly(L-proline) binding. These residues do, however, not directly contact the proline-rich peptide nor do they stabilize any of the crucial poly(L-proline) binding residues . Instead they are oriented outward, away from the poly(L-proline) moiety in the co-crystal. Therefore, the mutations may induce a conformational change in the carboxy terminal helix, which distorts correct orientation of the poly(L-proline) binding residues. But as judged from the CD-spectra and modeling experiments, this structural change is probably very subtle . In addition, mutations may inhibit or facilitate the previously observed conformational changes that occur in profilin upon binding of poly(L-proline) . Even though mutations at positions 56, 74 or 88 and combinations thereof are distant from the poly(L-proline) binding site, they also result in lowered poly(L-proline) binding. Remarkably, mutations in this region in yeast profilin caused a similar phenotype . Apparently these mutations cause allosteric conformational changes, resulting in less efficient binding of the proline-rich peptide. Interaction of mutants with actin | We determined the dissociation constants of our mutants for alpha-skeletal muscle actin using capped filaments . Under these conditions, profilin displays only G-actin sequestering activity. In addition, we studied the effect of each mutant on non-steady state actin polymerization . To analyze the obtained curves we determined the amount of F-actin formed at a time point (indicated in Figure as T1/2) where the amount of F-actin in the absence of profilin is 50% of the amount formed after 1500 sec. In the presence of WT profilin I, only 12% of F-actin is formed at this time point. The values for the mutant profilins are given in Table . Figure 4 | Time course of alpha-actin polymerization in the absence or presence of several mutant profilins. Time course of alpha-actin polymerization in the absence or presence of several mutant profilins. 10 muM actin and 5 muM profilin are pre-incubated prior to addition of KCl and MgCl2 to a final concentration of 100 mM and 2 mM, respectively. Curves for actin alone (closed triangles), or in the presence of either wild type profilin I (closed circles), R74E (open squares), R136D (open circles) are shown. T1/2 is the time point where the F-actin amount in the actin alone sample reaches 50% of the total F-actin formed after 1500 sec. For each profilin I mutant the percentage of F-actin at T1/2 is determined and given in Table . Table 2 | The interaction of wild type and mutant profilins with alpha-actin. We could not calculate a Kd value for R74A, R74E, S56E/R74E, S56E/R74A and R74E/R88E because the concentration of the actin-profilin complex was nearly zero, leading to very high Kd estimates. This is consistent with the observation that these mutants have no activity in the time course polymerization assay. As determined from the crystal structure of the actin-profilin complex and mutagenesis studies , Arg74 is a crucial residue for actin binding, since it forms a salt bridge with the carboxyl group of Phe375. Consequently, changing the arginine to an alanine or glutamic acid abolishes this interaction completely. Arg88 is also part of the actin-profilin interface, but changing it to alanine decreases the affinity only three-fold, indicating that the binding is less stringent than for Arg74. Mutating Arg88 to leucine or to glutamic acid in combination with S56E (which on its own has no effect), however, abolished actin binding completely. Arg135 and Arg136 locate in the carboxy terminal helix on the opposite side of the molecule and do not participate in actin binding. As a consequence, mutations in these residues do not affect the affinity for actin to a significant extent (Figure and Table ). PI(4,5)-P2 binds to two distinct regions in human profilin I | We used microfiltration and gel filtration to assay the ability of the mutants to bind PI(4,5)-P2 (Figure , Table ). The results of both assays were comparable. Based on analogy with invertebrate profilins (see background) and combined with sequence comparison of profilin I and IIa, we expected S56E to contribute negatively to PI(4,5)-P2-binding. This is, however, not the case and thus this amino acid difference between profilin I and IIa cannot explain the different affinities of the two profilin isoforms for PI(4,5)-P2. A further difference with invertebrate profilins is the observation that mutating Arg74 to leucine, glutamic acid or alanine (this study and ) does not significantly affect PI(4,5)-P2-binding. Since substitution to an acidic residue at this position results in only a slight effect, we consider the contribution of Arg74 in PI(4,5)-P2-binding to be of minor importance. Consequently, also the double mutants S56E/R74A and S56E/R74E show nearly wild type PI(4,5)-P2-binding. Previously, it was shown that Arg88 is involved in PI(4,5)-P2-binding of human profilin I , in agreement with several crystal structures showing a phosphate or sulfate anion associated with Arg88 and surrounding residues . In our assays, R88A has a small effect on PI(4,5)-P2-binding . Unfortunately we were unable to purify mutant R88E for which we expected a more pronounced phenotype. The effect of the latter mutation can, however, be inferred from the double mutants R74E/R88E and S56E/R88E. Both mutants show reduced PI(4,5)-P2-binding, compared to S56E, R74E and S56E/R74E which display nearly wild type binding capacity . Figure 5 | PI(4,5)-P2-binding of profilin mutants. PI(4,5)-P2-binding of profilin mutants. A. Microfiltration of profilin-PI(4,5)-P2 complexes. 4 muM profilin is incubated with increasing concentrations of PI(4,5)-P2 as indicated and applied to a filter with MWCO of 30.000. Non-bound profilin passes through the filter upon centrifugation. The flowthrough is analyzed by SDS-PAGE and is shown here for wild type profilin, R135D, R136D and R135A/R136A. B. Examples of gel filtration experiments. Profilin (10 muM) was pre-incubated with increasing concentrations of PI(4,5)-P2 and run over a SMART Superdex75 gel filtration column. Free profilin elutes at 1.62 ml, while the profilin-PI(4,5)-P2 complex elutes in the void (0.96 ml). The profilin peak shifts to the void fraction upon binding to PI(4,5)-P2. Elution pattern of wild type profilin alone (black line), profilin with 40 muM PI(4,5)-P2 (dark grey line) and profilin with 150 muM PI(4,5)-P2 (light grey line) are shown. We calculated the peak surface of free profilin to determine the percentage of bound profilin for different PI(4,5)-P2 concentrations. These data were then plotted in curves as shown in C. C. Percentage of bound profilin in function of PI(4,5)-P2 concentration as determined from the gel filtration curves. Wild type profilin (closed circle), R136D (open circle), R88A (closed triangle), R88A/R136D (open triangle) and R88E/R136D (closed square) in the gel filtration experiment. The concentration of PI(4,5)-P2 where 50% of profilin is bound to the micelles was derived from these curves and is given in Table for the different mutants. Table 3 | PI(4,5)-P2-binding of mutants assayed by gel filtration Interestingly, mutant R136D has a more pronounced effect than R88A (Figure and Table ). In contrast, mutating the neighboring residue Arg135 has only a small effect on PI(4,5)-P2-binding. Combining mutations in Arg88 and Arg136 has an additive effect : R88A/R136D and R88E/R136D show a much larger reduction in PI(4,5)-P2-binding than the single mutants . This suggests that the reduced PI(4,5)-P2-binding seen for R136D is due to a direct loss of an interaction. Although we cannot exclude contribution from allosteric effects, modeling experiments substituting R136 with an aspartic acid (data not shown) show no significant change in position of the side-chain or of the carboxy terminal alpha-helix. We conducted gel filtration experiments at high profilin to PI(4,5)-P2 ratio's for wild type profilin I and the R136D mutant to assess if the mutation affects overall saturable binding ability. This seems, however, not to be the case (data not shown), since we found for both wild type and mutant a ratio of ten profilin molecules per PI(4,5)-P2 micelle, suggesting a stoichiometry of 1:8 profilin : PI(4,5)-P2 molecules, consistent with a previous report . Depending on the assay conditions used, variable values for the stoichiometry of the profilin : PI(4,5)-P2 complex were found, varying between 1:4 and 1:10 . Given this 1:8 stoichiometry, it is difficult to observe the loss of one interaction using PI(4,5)-P2 micelles. We note, however, that in case of the mutant higher concentrations of profilin and PI(4,5)-P2 than for wild type profilin were required to obtain saturation, in agreement with the lower affinity of this R136D mutant Lassing and Lindberg showed that the inhibition on actin polymerization of wild type human profilin I decreases in the presence of PI(4,5)-P2. If Arg136 is involved in PI(4,5)-P2-binding, then this mutant should be less affected in its inhibitory activity in the presence of PI(4,5)-P2. This is indeed what we observe . R136D behaves similar to wild type profilin I in the absence of PI(4,5)-P2 (Figure and ). In the presence of a 9-fold molar excess of PI(4,5)-P2 we observe, however, a significant difference. For R136D we measure only a small reduction in sequestering activity compared to an almost complete inhibition of the sequestering activity of wild type profilin I. In the presence of a 25-fold molar excess of PI(4,5)-P2, however, R136D loses its sequestering activity completely (data not shown), indicating that the mutation did not entirely abolish PI(4,5)-P2-binding. This is consistent with the results from the gel filtration experiment and implicates a role for other residues such as Arg88. Figure 6 | PI(4,5)-P2 inefficiently competes with actin for binding to R136D profilin I. PI(4,5)-P2 inefficiently competes with actin for binding to R136D profilin I. The curves shown are : 8 muM Mg2+-ATP-G-alpha-actin (5% pyrene labeled) alone (closed triangles) or with 4 muM wild type profilin I (closed circles), 4 muM R136D (open circles), 4 muM wild type profilin I and 36 muM PI(4,5)-P2 (closed squares), 4 muM R136D and 36 muM PI(4,5)-P2 (open squares). Recently we demonstrated that profilin IIa has a lower affinity for PI(4,5)-P2 than profilin I . This can be explained with the data presented in this paper. In profilin I, Arg136 is important for PI(4,5)-P2-binding. In profilin IIa, there is an aspartic acid at this position (Asp136) and the profilin I R136D mutant thus mimics the profilin IIa isoform with respect to PI(4,5)-P2-binding. An indirect role of tryptophan 3 in PI(4,5)-P2-binding | Based on experiments with photoactivatable PI(4,5)-P2 analogues, Chaudhary and coworkers (1998) suggested that hydrophobic residues in the amino terminal helix are involved in the interaction with PI(4,5)-P2. Trp3, the fluorescence of which is quenched in the presence of PI(4,5)-P2, is spatially close to Arg136 (see Figure ). Therefore we mutated the former residue to alanine, thereby reducing the hydrophobic moiety. Trp3 is a crucial residue for the interaction of profilin with poly(L-proline) [,-,] and as expected the W3A mutants of profilin I and IIa lack poly(L-proline) binding and were thus purified using alternative methods (see Materials and Methods). The dissociation constant for the actin-profilin I W3A-complex was similar to that of wild type profilin I . The profilin I W3A mutant did not show a significant decrease in PI(4,5)-P2-binding, suggesting this residue does not directly contribute to the interaction. Interestingly, the profilin IIa W3A mutant shows increased affinity for PI(4,5)-P2 and the affinity is comparable with that of wild type profilin I . Given the profilin I W3A data presented here and in view of the conformational changes observed upon ligand binding , we propose that mutating Trp3 in profilin IIa promotes/induces a conformation which is more competent for PI(4,5)-P2-binding (see below). Figure 7 | Profilin IIa W3A mutant has increased affinity for PI(4,5)-P2. Profilin IIa W3A mutant has increased affinity for PI(4,5)-P2. Percentage of bound profilin in function of PI(4,5)-P2 concentration as determined from gel filtration experiments described in Figure . The concentration of PI(4,5)-P2 where 50% of profilin is bound to the micelles was derived from these curves and is given in Table . Model for regulation of profilin-ligand interactions | The data presented here show that in addition to Arg88, Arg136 is involved in PI(4,5)-P2-binding of mammalian profilin I. Based on our quantitative gel filtration assay, the contribution of Arg136 is in fact more important than that of Arg88 and the double mutant hardly binds PI(4,5)-P2 micelles. We conclude that the PI(4,5)-P2 binding sites of profilin are located in two distinct regions of the molecule that are approximately 31 A apart (see Figure ). It is remarkable that there are no corresponding positively charged residue(s) in the carboxy terminus of yeast and Acanthamoeba profilins that could account for a similar interaction as found here for human profilin I. This may indicate that the structural basis for the interaction of PI(4,5)-P2 with profilins from lower and higher eukaryotes is partially different. We also note that Acanthamoeba profilin II has a ten fold lower affinity for PI(4,5)-P2 than human profilin I . Both PI(4,5)-P2-binding regions in vertebrate profilins are implicated in the interaction with another profilin ligand. Arg136 is close to several poly(L-proline) binding residues. Not surprisingly, mutations in Arg136 have also strongly decreased poly(L-proline) affinity, although Arg136 itself is not directly contacting proline-rich ligands. On the other hand, Arg88, involved in PI(4,5)-P2-binding is also part of the actin binding site . A partial overlap of actin- and PI(4,5)-P2-binding sites was also observed for actophorin and gelsolin , suggesting this is the basis for a general regulatory mechanism for several actin binding proteins, whose function is inhibited by PI(4,5)-P2. Our data thus offer an explanation for the previously observed competition between PI(4,5)-P2 and the two other profilin ligands : actin and poly(L-proline) . This offers a nice model for the regulation of profilin with its different ligands. Since PI(4,5)-P2 inhibits both actin and poly(L-proline) binding , it is conceivable that PI(4,5)-P2 may have a master regulatory function in the cell. When PI(4,5)-P2 is hydrolyzed after cell stimulation, profilin may be set free to interact both with proteins containing proline-rich regions and with actin to regulate actin dynamics. The concerted action in vivo of profilin-actin complexes with several proline-rich proteins such as Ena/VASP proteins, N-WASP and formins for the promotion of actin polymerization was suggested previously . Several of our mutants suggest allosteric communication within vertebrate profilins. Arg88 mutants have reduced poly(L-proline) binding, although this residue is not part of the poly(L-proline) binding pocket. Conversely, W3A (in profilin IIa) influences PI(4,5)-P2-binding, but appears not to be directly involved in PI(4,5)-P2-binding as suggested by the data on profilin I W3A, although a W3N mutation in profilin I results in a higher affinity for PI(4,5)-P2. These results suggest that the interaction of profilin with PI(4,5)-P2 and poly(L-proline) involve conformational changes, which have been experimentally observed before . The interaction of profilin with PI(4,5)-P2 induces an increase in alpha-helical content . We propose that the local structure of the neighboring binding sites may change upon binding of PI(4,5)-P2 and poly(L-proline). The fact that W3A of profilin I binds PI(4,5)-P2 similar to wild type, suggests that profilin I already has the correct conformation for optimal binding of PI(4,5)-P2 and that mutating Trp3 to alanine does not ameliorate this conformation further (see also below), while an asparagine at position 3 does have a positive effect . In contrast, the W3A mutation in profilin IIa increases the affinity for PI(4,5)-P2, suggesting that this mutation induces a conformational change which optimizes the interaction with PI(4,5)-P2 despite the presence of an aspartic acid at the nearby position 136. The profilin IIa structure is, however, optimal for strong poly(L-proline) binding. In modeled and energy minimized profilin IIa structures we observed that the terminal alpha-helices are further apart from each other suggesting better access to the poly(L-proline) binding cleft . From this point of view, it is logical to assume that changing the position of these terminal alpha-helices has dramatic effects on ligand binding. This idea is consistent with our observation that the addition of a myc-tag to the carboxy terminal end of profilin IIa results in the dramatic loss of poly(L-proline) binding despite the fact that all known proline interacting residues are present . The suggested conformational change in profilin IIa-myc does, however, not significantly influence the affinity for actin . Similarly, mouse profilin IIb, which has six additional amino acids at its carboxy terminus, does not bind poly(L-proline) . In addition, it has been reported that both amino- and carboxy terminal GFP fusion proteins of mammalian profilins display a dramatic loss in poly(Lproline) binding . Some fusion proteins even lack complete poly(L-proline) binding. Therefore we believe that the correct positioning of the terminal alpha-helices of profilin is a primary requirement for ligand interaction. It is clear that any distortion of the alpha-helices will reduce the interaction with poly(L-proline). Figure 8 | Addition of carboxy terminal myc-tag to profilin IIa dramatically reduces poly(L-proline) binding. Addition of carboxy terminal myc-tag to profilin IIa dramatically reduces poly(L-proline) binding. A. Biacore binding curves for 100 muM wild type profilin IIa (blue), 1 muM wild type profilin IIa (green), or 100 muM profilin IIa-myc (red) to the (GP5)3 peptide derived from VASP. Resonance units (R.U.) are a measure for the number of profilin molecules retained by the peptide on the sensor chip and this is also concentration dependent (see B.). Even at a 100 times higher concentration, profilin IIa-myc (100 muM) binds less efficient to the peptide than wild type profilin IIa (1 muM). B. R.U. values obtained with different concentrations of wild type profilin IIa and profilin IIa-myc. Note that the value for 100 muM wild type profilin IIa is different from the one in Table , due to a different amount of peptide coupled to the sensor chip. PMID- 12052260_Conclusions TI - AB - We have identified Arg136, besides the previously identified Arg88, of human profilin I as an important residue for the interaction with PI(4,5)-P2. Since Arg136 is part of the poly(L-proline) binding helix and Arg88 is located in the actin binding surface, we suggest that the interaction of profilin with its different ligands is regulated by competitive interactions, which may be partly allosteric. Our results also indicate that the position of the two large terminal alpha-helices is crucial for optimal ligand binding. The addition of (protein or peptide) tags to the carboxy terminus results in dramatic decreased affinity for poly(L-proline) ligands. Conceivably, this will result in altered interactions in cells and in vivo data obtained with tagged profilin isoforms should be carefully (re)interpreted. PMID- 12052260_Materials and methods TI - AB - Profilin mutagenesis and purification | The profilin I cDNA amplified by polymerase chain reaction from a human cDNA library was subcloned into pET11d . Site directed mutagenesis was performed by polymerase chain reaction with mutated oligonucleotide primers and pfu polymerase. Mutations were verified by sequencing. MC1061 E. coli harboring the pT7POL26 plasmid were used for expression of wild type and mutant profilin I, Proteins were subsequently purified by poly(L-proline) affinity chromatography . W3A mutants do not bind poly(L-proline), thus the flow-through of the poly(L-proline) column was loaded onto a DEAE column equilibrated in buffer A (20 mM Tris-HCl, pH 8.1, 1 mM EDTA, 1 mM DTT). The column was eluted with a 0 to 500 mM NaCl gradient in buffer A. Profilin eluted with 60 to 130 mM NaCl. The profilin containing fractions were pooled and loaded on a MonoQ column. The flowthrough of this column contained profilin and only very few other proteins. These contaminating proteins were then removed by gel filtration in buffer A. Other Protein preparations | We purified actin from rabbit skeletal muscle and isolated it as calcium G-actin by Sephadex G200 chromatography in G-buffer (5 mM Tris-HCl, pH 7.7, 0.1 mM CaCl2, 0.2 mM ATP, 0.2 mM dithiothreitol, 0.01% sodium azide) . Actin was pyrene labeled on cysteine 375 . Gelsolin was purified from human plasma . Circular dichroism | We performed CD measurements in the far UV region (184 --260 nm) for WT and mutant profilins at a concentration of 15 muM in 7 mM TRIS/HCl, pH 8 in a JASCO J-170 spectropolarimeter using a 1 cm pathway cell. The step resolution was 0.5 nm and the scan speed 20 nm/min. For each sample the average of 9 scans was obtained and spectra were normalized for concentrations. Denaturation curves | Profilin was diluted to 2 muM in increasing concentrations of urea (0 to 8 M) in 20 mM Tris-HCl pH 8.1, 1 mM EDTA, 1 mM DTT. The samples were incubated for 15 min. at room temperature and the intrinsic fluorescence change during a wavelength scan between 300 and 400 nm was measured in a Hitachi F4500 spectrophotometer with the excitation wavelength set at 295 nm. We recorded a shift of the emission peak from 332 nm to 352 nm upon denaturation with urea. For each sample we plotted the ratio F(352 nm)/F(332 nm) versus the concentration of urea in that sample (see Figure ) . Polyproline binding | A (GP5)3 peptide, derived from VASP, was used to compare the affinities of the profilin I mutants on a BiacoreX (Pharmacia). The amino terminally biotinylated peptide was coupled to a streptavidin coated Biacore biosensor chip (Pharmacia). The experiments were carried out and analyzed as described in . Actin binding assays | The affinity of the profilin mutants for alpha-actin was determined using gelsolin capped filaments as described in . To determine the effect on non-steady state actin polymerization we pre-incubated 10 muM actin (5 % pyrene labeled) with or without 5 muM profilin for 15 minutes at room temperature prior to the addition of a final concentration of 2 mM MgCl2 and 100 mM KCl. The fluorescence change was recorded using a Hitachi F4500 spectrophotometer. PI(4,5)-P2-binding | Microfiltration was performed as described using 4 muM profilin and different concentrations of PI(4,5)-P2 (Sigma) as indicated in Figure . For gel filtration experiments, 10 muM profilin was pre-incubated with PI(4,5)-P2 micelles for 30 min on ice prior to loading on a Superdex75 gel filtration column (SMART, Pharmacia). The peak surface of free profilin was determined and used to calculate the percentage of bound and free profilin in each sample. The competition experiment between actin and PI(4,5)-P2 was performed with 8 muM Mg2+-ATP-G-alpha-actin (5% pyrene labeled), 4 muM profilin and 36 muM PI(4,5)-P2 in 5 mM Tris-HCl, 0.2 mM ATP, 0.2 mM dithiothreitol, pH 7 in the absence of Ca2+ and Mg2+ to avoid precipitation of PI(4,5)-P2. Profilin and PI(4,5)-P2-micelles were incubated for 10 minutes on ice prior to addition of actin and subsequent incubation for 10 minutes at room temperature. Polymerization was started by adding KCl to a final concentration of 50 mM. PMID- 12052260_Abbreviations TI - AB - Circular dichroism : CD; enabled : Ena; phosphatidylinositol 4,5-bisphosphate : PI(4,5)-P2; phospholipase Cgamma1 : PLCgamma1; vasodilator stimulated phosphoprotein : VASP; reasonance units : RU. PMID- 12052260_Authors' contributions TI - AB - A.L. participated in design of the study, carried out the mutagenesis, the stability and CD experiments and the actin and PI(4,5)P2 binding studies and drafted the manuscript. V.J. purified the proteins and carried out the Biacore and microfiltration experiments. D.D. helped with the mutagenesis. J.V. participated in the design of the study. C.A. conceived the study, participated in the coordination and in the design of the study. PMID- 12069692 TI - Gene expression profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiation signals AB - Abstract | Background | Expression profiling holds great promise for rapid host genome functional analysis. It is plausible that host expression profiling in an infection could serve as a universal phenotype in virally infected cells. Here, we describe the effect of one of the most critical viral activators, Tat, in HIV-1 infected and Tat expressing cells. We utilized microarray analysis from uninfected, latently HIV-1 infected cells, as well as cells that express Tat, to decipher some of the cellular changes associated with this viral activator. Results | Utilizing uninfected, HIV-1 latently infected cells, and Tat expressing cells, we observed that most of the cellular host genes in Tat expressing cells were down-regulated. The down-regulation in Tat expressing cells is most apparent on cellular receptors that have intrinsic receptor tyrosine kinase (RTK) activity and signal transduction members that mediate RTK function, including Ras-Raf-MEK pathway. Co-activators of transcription, such as p300/CBP and SRC-1, which mediate gene expression related to hormone receptor genes, were also found to be down-regulated. Down-regulation of receptors may allow latent HIV-1 infected cells to either hide from the immune system or avoid extracellular differentiation signals. Some of the genes that were up-regulated included co-receptors for HIV-1 entry, translation machinery, and cell cycle regulatory proteins. Conclusions | We have demonstrated, through a microarray approach, that HIV-1 Tat is able to regulate many cellular genes that are involved in cell signaling, translation and ultimately control the host proliferative and differentiation signals. PMID- 12069692_Background TI - AB - Whole-genome expression profiling exemplified by the development of DNA microarrays represents a major advance in genome-wide functional analysis . In a single assay, the transcriptional response of each gene to a change in cellular state can be measured, whether it is a viral infection, host cell cycle changes, chemical treatment, or genetic perturbation. Specifically, systematic approaches for identifying the biological functions of cellular genes altered during these changes, such as HIV-1 infection, are needed to ensure rapid progress in defining significant host and viral genome sequences in directed experimentation and applications. Therefore, host cellular states can be inferred from the expression profiles, and the notion that the global transcriptional response constitutes a detailed molecular phenotype, such as class discovery, class prediction, drug target validation, and the classification of tumors by expression profiling has begun to receive considerable attention . Since its discovery, much of the mainstream human immunodeficiency virus type 1 (HIV-1) Tat research has focused on its ability to activate the HIV-1 LTR. However, to date, besides the transactivation activity on the HIV-1 promoter, few other effects exerted by HIV-1 Tat on cellular and viral genes has also been observed. The Tat protein has been shown to transcriptionally repress host cellular genes and be involved in the immunosuppression associated with viral infection. For instance, HIV-1 infection is able to down-regulate major histocompatibility complex type I (MHC-I) by various different viral proteins, including Tat which represses the transcription of MHC-I, Vpu which retains nascent MHC-I chains in the endoplasmic reticulum, and Nef which can mediate selective internalization of MHC-I molecules from the plasma membrane. MHC class I gene expression has also been shown to be reduced upon infection with the wild-type LAI virus or a Tat exon one recombinant virus . Tat has been shown to down-regulate mannose receptor, EDF-1, CD3-gamma, and TCR/CD3 surface receptor . Tat reduces mannose receptor levels and promoter activity in mature macrophages and dendritic cells by interfering with the host transcriptional machinery; resulting in decreased levels of surface mannose receptor needed for Ag (mannosylated albumin uptake) or pathogen capture (Pneumocystis carinii phagocytosis), and eventual delivery to MHC class II-containing intracellular compartments . EDF-1, a gene down-regulated when endothelial cells are induced to differentiate in vitro, was shown to be down-regulated by Tat at the transcriptional level, resulting in the inhibition of endothelial cell growth and in the transition from a nonpolar cobblestone phenotype to a polar fibroblast-like phenotype . When examining the in vivo effects of HIV-1 Tat protein in the Xenopus embryo, it was found that upon injection of synthetic Tat mRNA into zygotes, a marked delay in gastrulation occurred. This led to the altered specification of the anterior-posterior axis and partial loss of the anterior embryo structures. Mechanistically, HIV-1 Tat elicited a general suppression of gene expression, including that of Xbra and gsc, two early genes whose expression are required for proper gastrulation . In relation to the cell cycle, Tat has also been shown to bind to p53 and inhibit the transcription of p53 responsive elements, such as the p21/Waf1 gene promoter. Consequently, upon introduction of stress signals (e.g., gamma irradiation), HIV-1-infected cells lose their G1/S checkpoint, enter the S-phase inappropriately, and apoptose . Finally, the inhibition of Tat on translational machinery has also been noted. The potential translational inhibitory effects of the TAR RNA region is mediated by the activation of p68 (the interferon-induced 68-kilodalton protein kinase) kinase, which was down-regulated by Tat during productive HIV-1 infection . Although the mechanism of the host cellular down-regulation remains largely unknown, few reports have attempted to decipher the mechanism of the observed inhibition. For instance, the addition of Tat to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcriptional activity of the TH promoter, strongly suggesting ICER's involvement in Tat-mediated inhibition of gene expression . Aside from induction of ICER, Tat is capable of forming complex (es) with a component of TFIID, TAFII250 and Tip60 both of which contain histone acetyltransferase (HAT) activity. In these cases, Tat-TAFII250 and Tat-Tip60 do not affect the transcription from the HIV-1 LTR, but interfere with the transcription activity of cellular genes. It is postulated that different targets of HATs by Tat have different consequences. The interaction of Tat with p300/CBP and P/CAF stimulates its ability to transactivate LTR-dependent transcription, while Tat-TAFII250 or Tat-Tip60 interactions control the transcription of cellular genes. Here to better understand the host response to Tat, we have performed microarray experiments on HIV-1 infected cells expressing the Tat protein. To our surprise many host cellular genes were down-regulated when comparing HIV-1 infected latent cells to uninfected parental cells. Because most, if not all, latent infected cells available to date (e.g., ACH2, U1, J1.1, OM.10) have various expression levels of doubly spliced viral mRNAs, including Tat, Rev, Nef, Vpr, and other accessory proteins, we decided to perform the microarray in a system where Tat was constitutively expressed; asking whether Tat by itself, or in the absence of other accessory proteins, could still down-regulate host cellular genes. Consistent with latently infected cells, we found many cellular genes to be down-regulated in Tat expressing lymphocytes. The down-regulation is most apparent on cellular receptors that have intrinsic receptor tyrosine kinase (RTK) activity and signal transduction members that mediate RTK function; including the Ras-Raf-MEK pathway, and co-activators such as p300/CBP and SRC-1, which mediate gene expression related to hormone receptor genes. Interestingly, we also observed up-regulation of S-phase genes, as well as ribosomal genes involved in translation. Functionally, down-regulation of receptors may allow latent HIV-1 infected cells to either hide from the immune system or avoid extracellular differentiation signals normally regulated by receptors. Up-regulation of S-phase and translation genes may allow speeding of cells through the S-phase and subsequent accumulation at the G2 phase, where most of the cellular and viral translation may take place. Therefore, the presence of Tat may not only control activated transcription on HIV-1 LTR, but also aid in the subsequent translation of viral mRNA in the cytoplasm. PMID- 12069692_Results and discussion TI - AB - Host expression profiling in a sufficiently large and diverse set of profiles could allow additional hypotheses to be drawn regarding the function of genes based on the regulatory characteristics of their own transcripts . Here, we describe the effect of one of the most critical viral activators, Tat, involved in HIV-1 infection and pathogenesis. The rationale for these experiments came from the fact that many AIDS-infected patients who are either at stage III (non-progressors) or under highly active antiretroviral therapy (HAART) treatment show some level of doubly spliced viral messages in their infected cells. One of these messages, Tat, has been well studied and characterized, both from a viral activator standpoint, its effect on few host cellular genes, and its effect as an extracellular cytokine. However, to date there are no reported Tat gene expression analyses that detect more than a few cellular genes. We performed our microarray analysis first with a cDNA blot of 588 genes, which is known to contain various receptors, cytokines, transcription factors, DNA replication genes, and other additional well characterized genes. Figure shows the results of a microarray experiment from uninfected (CEM) and latently HIV-1 infected (ACH2) cells. By definition, latently HIV-1 infected cells contain integrated HIV-1 sequences in the host genome. To our surprise, we detected many cellular genes that were down-regulated in ACH2 cells as compared to CEM uninfected cells. ACH2 cells, similar to many other latent HIV-1 infected cells including U1, OM10.1, 8E5, and J1-1, express multiple doubly spliced messages including Tat, Rev, Nef, and Vpr . Therefore, it would be difficult to determine which one of these viral open reading frames was in fact controlling the observed changes in host gene expression. Figure 1 | Gene expression analysis of uninfected and HIV-1 infected cells. Gene expression analysis of uninfected and HIV-1 infected cells. A) Both CEM (uninfected) and ACH2 (latently HIV-1 infected) cells were grown to mid-log phase of growth and processed for RNA isolation. Total RNA was labeled with 32P-ATP and hybridized to human cDNA filters (Clontech, 588 genes). Blots were hybridized overnight, washed the next day, and exposed to a PhosphorImager cassette. B) Same as in panel A, except all the 588 genes were plotted as fold change vs. gene index (individual genes). Examples of three genes such as prothymosin-alpha, C-myc, and p21/Waf1, is shown on the diagram. C) Northern blot analysis of prothymosin-alpha, C-myc, p21/Waf1 and ubiquitin using 10 mug of total RNA, separated on 0.8% formaldehyde gel, and probed with 40 mer anti-sense oligos against respective genes. Bottom of panel C, last insert shows RNA ethidium bromide stain from CEM and ACH2 cells. Nonetheless, when mapping all the 588 genes, we found that 139 genes were activated above 1 fold and 449 genes were expressed below 1 fold . This is in sharp contrast to latent HTLV-1 infected Tax expressing cells, where more than two-thirds of the same set of genes were activated and scored above one . Some of the genes from HIV-1 latent cells were further processed as control experiments using Northern blot analysis. As can be seen in Figure , and consistent with previously published reports, p21/Waf1 was down-regulated and C-myc and pro-thymosin-alpha were up-regulated in HIV-1 latent cells. Collectively, these experiments point toward host cellular changes in the presence of doubly spliced HIV-1 RNA; however they do not explain whether Tat or other viral genes are responsible for the observed cellular changes. Therefore, we focused our attention on HIV-1 Tat protein, since Tat could readily be detected in immunoprecipitations from 35S labeled latent ACH2, U1, OM10.1, 8E5, and J1-1 cells (20, data not shown). We utilized a well-characterized system of H9 and H9/Tat cell lines for our next set of microarray experiments and increased our repertoire of the known cDNA genes from 588 to 2400. This was accomplished by using glass slides which were printed with cDNAs of 500 bases or higher, and could be used in hybridization with two different sets of RNAs labeled with either Tyramide linked Cy-3 or Cy-5. Results of such an experiment is shown in Figure , where H9 cytoplasmic Poly A+ selected RNA was labeled with Cy-5 and the H9/Tat RNA with Cy-3 prior to hybridization. When all the 2400 genes were plotted, some 695 genes were shown to be up-regulated above one fold and 1705 genes were down-regulated below one fold (right hand graphs). This was consistent with the results obtained from the ACH2 microarray experiment, where more than 2/3 of the cellular genes were down-regulated by one or more of the doubly spliced genes. We then arbitrarily chose a cut-off of three fold change for our next set of analyses. This was mainly because many of our in house microarray experiments with HIV-1, HTLV-1, and HHV-8 infection has shown a reproducible correlation between protein and mRNA levels when gene expression levels were up- or down-regulated by more than three fold (data not shown). A collection of all the genes above and below three fold are shown in Tables , , and . Based on existing literature, we categorized all of these genes into known pathways. For instance, genes in Table belong to receptors, co-receptors, and co-activators, genes in Table are all the translation related factors, and those in Table indicate genes that are involved in cytoskeleton, signal transduction, cell cycle, DNA repair, transcription, and chromatin remodeling processes. All genes have a number ratio of Cy3 to Cy5 (C3/C5) indicating a ratio of mRNA from H9/Tat over H9 cells. A brief name description and gene ID accession number is given to the right hand side of each ratio. All genes are divided into up (3 fold and higher) and down (3 fold and lower) regulated in Tat expressing cells. Below is a brief description of genes that we, along with the existing literature, were able to correlate with proliferative and/or differentiation signals. Figure 2 | Gene expression analysis from Tat expressing cells. Gene expression analysis from Tat expressing cells. Both H9 and H9/Tat cells were grown to mid-log phase of growth, processed for RNA preparation, and labeled with Tyramide linked Cy-5 (H9) or Cy-3 (H9/TAT). Labeled RNAs were hybridized simultaneously to a glass slide containing 2400 known cDNA genes (NEN Inc.). All genes were plotted similar to Figure and genes above & below 1 fold change were plotted (on the right hand side) to show activation and suppression of all genes. Table 1 | Receptors Table 2 | Translation Factors Table 3 | Receptor family members | It has long been known that infection by HIV-1 commonly leads to the down-regulation and the disappearance of CD4 receptors from the plasma membrane, a phenomenon referred to as receptor down-modulation. This, in turn, renders cells refractory to subsequent infection by the same or other viruses that use the CD4 receptor for entry; thus creating a state of super-infection immunity. Results in Table indicate that although few receptor genes were up-regulated, most of the cellular receptors in general, were down-regulated in the presence of the Tat protein. Most of these receptors or membranous proteins were initially discovered from immune or neuronal cells, hence they were given names related to the immune or nervous system. For instance, mRNA for the neuropeptide Y-like receptor (Acc# X71635), which was up-regulated in Tat expressing cells, was initially discovered as a G-protein coupled neuropeptide Y receptor, and later found to be homologous to the co-receptor CCR5 needed for HIV-1 infection of monocyte/macrophage cells. Therefore, most of the receptors listed in Table may in fact be expressed in various tissues and have multiple functions. Consistent with our microarray results on CCR5 up-regulation, experiments performed in peripheral blood mononuclear cells (PBMCs) with soluble Tat has shown selective entry and replication of CCR5 virus into cells . Up-regulation of HIV-1 coreceptor by Tat has also been reported, where a synthetic Tat protein that was immobilized on a solid substrate, up-regulated the surface expression of the chemokine receptors in purified populations of primary resting CD4+ T cells. Also, a similar result was seen from Tat protein actively released by HIV-1 infected cells, implying a potentially important role for extracellular Tat in rendering the bystander CD4+ T cells more susceptible to infection . We therefore tested whether H9/Tat cells, which showed an increase in CCR5 expression, could in fact allow better entry and infection of the CCR5 (R5) virus into cells. Figure shows the result of such an experiment, where H9/Tat cells allowed a better replication profile of the R5 than the CXCR4 (X4) virus. The increase in viral titer peaked after some 18 days of infection with the R5 virus, further implying that the CCR5 co-receptor allowed a better selection of R5 virus in Tat expressing cells. Figure 3 | Functional and physical confirmation of few genes from Tat expressing cells. Functional and physical confirmation of few genes from Tat expressing cells. A) Infection of mono- and T-tropic viruses into Tat expressing cells. Both HXB-2 and BaL strains of HIV-1 were infected into H9 and H9/TAT cells. Supernatants were collected every 3 days and further processed for p24 gag ELISA assays. B) Western blot analysis from H9 and H9/TAT expressing cells using co-activators (SRC-1), DNA damage (DNA-PK), activator (p300), and signal transduction (Ras, RAF, and MAPK) antibodies. TBP stands for TATA binding protein, which served as positive control in western blots. C) Western blot analysis from CEM (uninfected T-cell), ACH2 (infected T-cell), U937 (uninfected promonocytic), U1 (infected promonocytic), and PBMCs treated with purified Tat wild type or K41A mutant (100 ng/ml) proteins. Fifty microgram of whole cells lysates were processed for western blots with anti-DNA-PK, p300, RAF, and TBP antibodies. Another example of co-receptors with multiple functions is the leukotriene family member B4, which was down-regulated in Tat expressing cells (Acc# D89078, Table ). The cysteinyl leukotrienes (CysLT), LTC, LTD, and LTE, were first shown to be essential mediators in asthma . However, when the mouse leukotriene B4 receptor (m-BLTR) gene, was cloned it was shown to have significant sequence homology with chemokine receptors (CCR5 and CXCR4), co-receptors for many different HIV-1 clades . Along the same lines, when cells were infected with 10 primary clinical isolates of HIV-1, leukotriene B4 receptor was primarily utilized for efficient entry into cells which were mainly of the syncytium-inducing phenotype . Therefore, up-regulation of neuropeptide Y-like receptor and down-regulation of leukotriene B4 receptor in Tat expressing cells indicates a selective advantage of one class of virus (CCR5) over another (CXCR4). Other examples of consistency between our microarray results on receptors and the HIV-1 Tat literature, include the down-regulation of gene expression in uPAR (Acc# X74039), IP3 (Acc# D26070, D26351), Glu R flop (Acc# U10302), PPAR (Acc# L07592), alpha-2 macroglobulin receptor protein (Acc# M63959), and receptor tyrosine kinase (Acc# L36645, U66406) genes. The transmembranous urokinase-type plasminogen activator receptor (uPAR; CD87) focuses the formation of active plasmin at the cell surface, thus enhancing directional extracellular proteolysis. Interestingly, the promoter activity of the CD87 gene was shown to decline after infection , implying that post integration of HIV-1 may in fact down-regulate CD87 gene expression. Similarly, inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular calcium release channels involved in diverse signaling pathways and are required for the activation of T lymphocytes . Tat (also implicated as a neurotoxin) has been shown to release calcium from inositol 1,4, 5-trisphosphate (IP3) receptor-regulated stores in neurons and astrocytes causing premature apoptosis . Down-regulation of IP3 may therefore contribute to viral latency and maintenance of an anti-apoptotic state in cells. HIV-1 infection can cause extensive neuronal loss and clinically, a severe dementia. The cause of the neurotoxicity remains unclear as neurons are not infected, but the disturbance of glutamate-linked calcium entry has been implicated. It has been shown that HIV-infected brain has a decrease of mRNA and protein of the GluR-A flop subtype of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor in cerebellar Purkinje cells. The observed disturbance of AMPA receptors may contribute to the neurotoxic process in other vulnerable brain regions and clinically to the development of dementia . Interestingly, in a mouse model AMPA receptors in the cortex, striatum, hippocampus, and cerebellum declined by 29 --50% as early as 8 weeks post-retroviral inoculation. Thus, the reduction in AMPA receptor density may contribute to the development of the cognitive abnormalities associated with HIV-1 infection . Finally, patients with AIDS who are receiving therapy with HIV-1 protease inhibitors have been reported to be afflicted with a syndrome characterized by lipodystrophy (fat redistribution favoring the accumulation of abdominal and cervical adipose tissue), hyperlipidemia, and insulin resistance. Potential mechanisms for altered adipocyte function include, direct binding to PPARgamma or inhibition of transcription of PPARgamma promoter . The lipodystrophy syndrome may be a result of the inhibition of 2 proteins involved in lipid metabolism that have significant homology to the catalytic site of HIV proteases; namely cytoplasmic retinoic acid binding protein type 1 and low density lipoprotein-receptor-related protein . An additional mechanism of PPAR down-regulation may be related to Tat expression in latent cells. Translation associated factors | Viruses have evolved a remarkable variety of strategies to modulate the host cell translation apparatus with the aim of optimizing viral mRNA translation and replication. For instance, viruses including Herpes simplex virus type 1 (HSV-1) have been known to induce severe alterations of the host translational apparatus, including the up-regulation of ribosomal proteins and the progressive association of several nonribosomal proteins, such as VP19C, VP26, and the poly(A)-binding protein 1 (PAB1P) to ribosomes . In the case of HIV-1, approximately one infectious HIV-1 genome in an infected cell could be transcribed and translated into 50,000 to 100,000 physical particles . This poses an immense challenge for the virus to be able to transcribe, splice, transport, and translate its RNA into fully packaged virions in a timely fashion. Therefore, it would be advantageous for the virus to set the stage for each successive step necessary for viral progeny formation. One such event is Tat's ability to control genes that aid in translational machinery. As seen in Table , many of the critical components of a functional ribosome, including large subunits L 3, 6, 26, 31, 38, and 41, as well as S 6, 12, 20, and 24, and many of the translation initiation factors are up-regulated by Tat. This would imply that Tat up-regulates many ribosomal genes that may be necessary to produce functional ribosomes needed for viral mRNA translation. Therefore, interfering with translation could provide a new strategy for anti-HIV treatment. Along these lines, when the aminogylcosides (kanamycin, hygromycin B, paromycin and neomycin) due to their ability to inhibit protein synthesis by affecting ribosomal fidelity, or puromycin because of its competing ability with tRNAs for binding on the large ribosomal subunit, or cycloheximide which inhibit the large ribosomal subunit by preventing ribosomal movement along the mRNA, were used in active HIV-1 infection, it was found that both cycloheximide and puromycin produced the greatest decrease in HIV-1 inhibition, presumably by inhibiting the large subunit of the ribosome . Translation of HIV-1 RNAs pose a challenge since they all contain a TAR sequence at their 5' end. The Tat-responsive region (TAR) of HIV-1 exhibits a trans-inhibitory effect on translation by activating the interferon-induced 68-kilodalton protein kinase. Productive infection by HIV-1 has been shown to result in a significant decrease in the amount of cellular p68 kinase. The steady-state amount of p68 kinase was found to be reduced in cells stably expressing Tat. Thus, the potential translational inhibitory effects of the TAR RNA region, mediated by activation of p68 kinase, may be down-regulated by Tat during activation of the latent virus . Along these lines, a Tat peptide antagonist, which bound specifically to TAR RNA and competed with Tat for binding, reduced Tat-dependent translation . Finally, upregulation of translation genes in Tat expressing cells is specially intriguing in light of the recent discovery of internal ribosome entry sites (IRESs) in HIV-1 gag ORF . IRESs are thought to promote initiation of translation by directly binding to ribosomes, in a manner independent of the mRNA cap or of scanning through upstream sequences. Since, the TAR is located at the 5' end of all HIV-1 RNA transcripts and the presence of secondary structure at or near the 5' end of RNAs reduces the accessibility of the 5' cap to eIF4F, it is thought that this feature of HIV-1 mRNAs can inhibit their cap-dependent translation . Therefore, a possible function of the HIV-1 gag IRES might be to serve as a mechanism to bypass the structural barriers to cap-dependent translation by recruiting ribosomes easily and directly to the gag ORFs. IRES entirely contained within a translated ORF has been shown in the MMLV gag , and host mRNA encoding p110PITSLRE and p58PITSLRE. Along these lines, cap-dependent translation may be cell cycle regulated, especially when cells are arrested at the G2 phase of the cell cycle, where the cap-dependent translation of most cellular host cell mRNAs is inhibited . Modulation of signal transduction pathway | Results in Table indicate that many seemingly different pathways are being regulated by Tat. However, the signal transduction pathway, MAPK, has been shown to control and be upstream of DNA-replication, transcription, and cell cycle pathways . The mitogen-activated protein kinase (MAPK) pathway, consisting of the MAP kinase kinases (MKKs) 1 and 2, and extracellular signal-regulated kinases (ERKs) 1 and 2, which have been implicated in diverse cellular processes including proliferation, transformation, and cell differentiation . The MAP kinase (MAPK) pathway has emerged as a crucial route between membrane-bound Ras and the nucleus. This MAPK pathway encompasses a cascade of phosphorylation events involving three key kinases, namely Raf, MEK (MAP kinase kinase) and ERK (MAP kinase). The MAPK pathway controls ERKs 1 and 2, c-Jun N-terminal kinase (JNK), and p38. These signaling pathways in turn, activate a variety of transcription factors including NF-kappaB (p50/p65), AP-1 (c-Fos/c-Jun), and CREB phosphorylation, which in turn coordinate the induction of many genes encoding inflammatory mediators. Cytokine receptors such as IL-3, GM-CSF, and the interferons transmit their regulatory signals primarily by the receptor-associated Jak family of tyrosine kinases, and activate STAT transcription factors. Activated STAT5 proteins are detected in reduced levels in lymphocytes recovered from HIV-infected patients and immunocompromised mice. Both of these types of receptor signaling pathways have recently been shown to interact with serine/threonine kinases such as MAP kinases. A common intermediate pathway initiating from receptors to the nucleus is the Ras/Raf/MEK/ERK (MAPK) cascade, which can result in the phosphorylation and activation of additional downstream kinases and transcription factors such as p90Rsk, CREB, Elk, and Egr-1 . Therefore, it is intriguing that Tat expressing cells show down-regulation of MAPK components (Table , Figure and ), essential mediators between receptors and nuclear transcription factors. This would imply that latently infected cells that express Tat (doubly spliced RNA) and not the whole virus (all three classes of the RNA), can control signal transduction related to membrane and transcriptional signaling . Interestingly, Tat, through the RGD motif, which controls integrin-based cell signaling, has been reported to mediate the activity of phosphotyrosine phosphatase(s). This in turn which would lead to a decrease in the levels of phosphotyrosine-containing proteins such as ERK-2/p42MAPK kinases . Cysteine-rich and basic Tat peptides have been shown to inhibit VEGF-induced ERK activation and mitogenesis. These peptides also inhibited proliferation, angiogenesis, and ERK activation induced by basic fibroblast growth factor with similar potency and efficacy . Consistent with this model, it has been shown that treatment of neural cells with culture supernatants from HAART-treated subjects, which presumably contain extracellular Tat, resulted in down-regulation of the JNK, AKT, and ERK kinases . Finally, activation of MAPKs has been shown to activate the singly spliced and unspliced (genomic) latent HIV-1 virus. For instance, the signal transduction pathways that regulate the switch from latent to productive infection have been linked to MAPK. The induction of latent HIV-1 expression has been shown to be inhibited by PD98059 and U0126, specific inhibitors of MAPK activation. The MAPK acts by stimulating AP-1 and a subsequent physical and functional interaction of AP-1 with NF-kappaB, resulting in a complex that synergistically transactivates the HIV-1 . At the level of infection and entry, the activation of MAPK through the Ras/Raf/MEK (MAPK kinase) signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity can be enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK in the absence of extracellular stimulation . Also, following infection, efficient disengagement of the reverse transcription complex from the cell membrane and subsequent nuclear translocation, requires phosphorylation of the reverse transcription complex components by ERK/MAPK; demonstrating a critical regulation of an early step in HIV-1 infection by the host cell MAPK signal transduction pathway . Therefore, Tat down-regulation of the MAPK pathway in latent cells implies that much of the host signal transductions connected to activation are down-regulated, and at the same time, these cells may be refractory to subsequent infection by other viruses. Thymosin family members, and cell cycle | Prothymosin alpha (ProTalpha) belongs to the alpha-Thymosin family which comprises different polypeptides widely distributed within animal tissues. Although its role has remained controversial, it is involved in the increase of immediate early genes such as c-myc , which is upstream of cyclin D synthesis and necessary for cell division . In humans, ProTalpha is coded by a gene family of six members. One of them contains introns, exons and classic regulatory signals, while the remaining five are intronless genes located on chromosome 2 . There are two mRNA transcripts, which arise in a ratio of 9:1 (shorter/longer form), where only the long transcript is regulated by extracellular signals. It has been demonstrated that malignant tissues with accelerated cell cycle show higher levels of ProTalpha expression than normal or surrounding healthy tissues . ProTalpha was shown as a marker for breast cancer , hepatocarcinoma , and plasma levels of its derivative Talpha1 been proposed as a marker for the prognosis of lung cancer . In ligand blotting assays, ProTalpha bound only to chromatin pools and nuclear fractions where histone H1 was present . The analysis of the interaction of ProTalpha with H1-containing chromatin suggests a putative role for ProTalpha in the fine-tuning of the stoichiometry and/or mode of interaction of H1 with chromatin . Interestingly, HL-60 cells overexpressing ProTalpha show an enhancement of accessibility of micrococcal nuclease to chromatin, implying relaxed chromatin structure for enhanced cell cycle gene expression . A broad study using several mononuclear and fibroblastic cell lines has shown that ProTalpha mRNA accumulation is cell cycle phase-dependent. In the U937 monocytic cell line, ProTalpha mRNA peaked at the end of S/G2 phase and fell towards the entry into the new G1 phase. More prominent mRNA regulation was found in the fibroblastic cell lines CV1 and NIH3T3, with peak mRNA levels at the end of S-phase. In all cases the expression pattern coincided with that of cyclin B and Cdc2/cyclin B activation . It is interesting to note that Cdc2 (Acc# X05360), Cdc10 homolog (Acc# S72008), and Cdc37 (Acc# U43077) were all up-regulated in Tat expressing cells. Cdc2, a catalytic subunit of cyclin-dependent kinases, is required for both the G1-to-S and G2-to-M transitions. In the fission yeast Schizosaccharomyces pombe, the execution of Start requires the activity of the Cdc2 protein kinase and the Cdc10/Sct1 transcription complex. The loss of any of these genes leads to G1 arrest . Cdc37 encodes a 50-kDa protein that targets intrinsically unstable oncoprotein kinases including Cdk4, Raf-1, and v-src to the molecular chaperone Hsp90, an interaction that is thought to be important for the establishment of signaling pathways. Cdc37 expression may not only be required to support proliferation in cells that are developmentally programmed to proliferate, but may also be required in cells that are inappropriately induced to initiate proliferation by oncogenes. For instance, MMTV-Cdc37 transgenic mice develop mammary gland tumors at a rate comparable to that observed previously in MMTV-cyclin D1 mice, indicating that Cdc37 can function as an oncogene in mice and suggests that the establishment of protein kinase pathways mediated by Cdc37-Hsp90 can be a rate-limiting event in transformation . Also, analysis of proteins that co-immunoprecipitated with Cdk6 and Cdk4 has shown complexes containing both Hsp90 and Cdc37 . Cdc37 also promotes the production of Cak1. Cak1 in yeast is the human homolog of CAK trimeric enzyme containing CDK7, cyclin H, and MAT1. Both human and yeast Caks function as RNA polymerase II CTD kinase, Cdk activating kinase, and DNA damage/repair enzymes. Cdc37, like its higher eukaryotic homologs, promotes the physical integrity of multiple protein kinases, perhaps by virtue of a cotranslational role in protein folding . Finally, Hsp90/Cdc37 has recently been shown in the stabilization/folding of Cdk9 as well as the assembly of an active Cdk9/cyclin T1 complex responsible for P-TEFb-mediated Tat transactivation . Transcription and chromatin remodeling factors | A highly ordered chromatin structure presents a physical obstacle for gene transcription; presumably by limiting the access of transcription factors and RNA polymerase II core machinery to target DNA . In concert with the observation that corepressors are associated with HDAC activities , it appears that the transcriptional outcome of nuclear receptors is determined by the balance of histone acetylation and deacetylation activities, and that ligands serve as a switch to recruit HATs with the concomitant dismissal of HDACs. Signal transduction pathways add another layer of regulation to the functions of CBP/p300. In the case of the POU homeodomain factor Pit-1, transcriptional activity is potentiated by MAPK pathways . Therefore, down-regulation of MAPK pathway members in Tat expressing cells, as seen in Table , is consistent with decreased phosphorylation of DNA binding factors such as Pit-1, and overall lower DNA binding activity. Here, we describe the effect of coactivator proteins SRC-1 (Acc# AJ000882, U90661, Table ) and p300 (Acc# U01877, Table ), and their relation to differentiation genes such as retinoic acid receptor (RAR/PML, Acc#: X06614, Table ), and Leptin receptor variant (Acc#: U66496, Table ); all of which are down-regulated in Tat expressing cells . Figure 4 | Synthesis of IL-8 in Tat expressing cells. Synthesis of IL-8 in Tat expressing cells. Hela cells (pCEP4, and eTat) were either unblocked (unt), or blocked with hydroxyurea (Hu) (2 mM) for 14 h, released, washed twice with phosphate-buffered saline (PBS) and subsequent addition of complete medium . Supernatants were collected at 9 hrs after release for ELISA. All remaining suspension cells were treated with 1% serum for 48 hrs prior to addition of Hu. PHA-activated PBMCs were kept in culture for 2 days prior to addition of Tat protein. Approximately 5 x 106 PBMCs were used for treatment with either wild type or K41A Tat mutant (100 ng/ml) proteins. After an initial incubation for one hr with Tat proteins, cells were washed and cultured in complete media for 24 hrs, prior to IL-8 ELISA. Figure 5 | Predictive model for control of gene expression and signal transduction by constitutive Tat expressing cells. Predictive model for control of gene expression and signal transduction by constitutive Tat expressing cells. Down-regulation of SWI/SNF components such as BAF 170 and 60 along with coactivators CBP/p300 and SRC-1 may down-regulate a subset of cellular genes that depend on chromatin remodeling and/or co-activator function for their gene expression. Such genes depend on the presence of ligand receptors that require either both SRC-1 and p300 or individual co-activator for their activity. Over the past three decades a great deal of evidence has accumulated in favor of the hypothesis that steroid receptor hormones act via regulation of gene expression. The action is mediated by specific nuclear receptor proteins, which belong to a superfamily of ligand-modulated transcription factors that regulate homeostasis, reproduction, development, and differentiation . This family includes receptors for steroid hormones, thyroid hormones, hormonal forms of vitamin A and D, peroxisomal activators, and ecdysone . Nuclear hormone receptors are ligand-dependent transcription factors that regulate genes critical to such biological processes as development, reproduction, and homeostasis. Interestingly, these receptors can function as molecular switches, alternating between states of transcriptional repression and activation, depending on the absence or presence of a cognate hormone, respectively. In the absence of cognate hormone, several nuclear receptors actively repress transcription of target genes via interactions with the nuclear receptor corepressors SMRT and NCoR. Upon binding of the hormone, these corepressors dissociate from the DNA-bound receptor, which subsequently recruits a nuclear receptor coactivator (NCoA) complex. Prominent among these coactivators is the SRC (steroid receptor coactivator) family, which consists of SRC-1, TIF2/GRIP1, and RAC3/ACTR/pCIP/AIB-1. These cofactors interact with nuclear receptors in a ligand-dependent manner and enhance transcriptional activation via histone acetylation/methylation and recruitment of additional cofactors such as CBP/p300 . CBP/p300 has been implicated in the functions of a large number of regulated transcription factors based primarily on physical interaction and the ability to potentiate transcription when overexpressed . In the case of nuclear receptors, the interaction with CBP/p300 is ligand-dependent and relies on the conserved nuclear receptor functional domain, AF-2 (activation function 2). In vivo studies have supported the conclusion that CBP/p300 are components of the hormonal-regulation of transcription in fibroblasts isolated from a p300-/- mouse; and loss of the p300 gene severely affects retinoic acid (RA)-dependent transcription . In a separate study using hammerhead ribozymes that specifically cleave CBP or p300 mRNA, Kawasaki et al reported that reduced cellular CBP or p300 levels resulted in compromised expression of endogenous RA-inducible genes such as p21/Waf1 and p27 cdk inhibitors. Along this line, Tat expressing cells have lower levels of p21/Waf1 presumably due to inactivation of p53 and a lack of p300/RA- induced gene expression. Consistent with this interpretation, CBP and p300 harbor transcriptional activation of ligand-induced RA or ER function on a chromatinized template . The NcoA family members constitute SRC-1/NcoA-1 , TIF2/GRIP1/NcoA-2, and pCIP/ACTR/AIB1 proteins, which interact with liganded RA receptor (RAR), and CBP/p300. Overexpression of these NCoA factors enhances ligand-induced transactivation of several nuclear receptors . A weak intrinsic HAT activity has been reported in SRC-1/NCoA-1 and pCIP/ACTR/AIB1, suggesting that chromatin remodeling may also be a function of these NCoA factors ; although they do not appear to contain regions homologous to the HAT domains of CBP/p300 or p/CAF. Structure-function analysis of the NCoAs have revealed multiple copies of a signature motif, LXXLL, with conserved spacing that is required for interaction with nuclear receptors and CBP/p300 . Intriguingly, different LXXLL motifs are required for PPARgamma (Peroxisome Proliferator activated receptor gamma, a gene down-regulated in Tat expressing cells; Acc# L07592, Table ) function in response to different classes of ligands, suggesting distinct configuration of assembled complexes. Taken together, through the use of microarray technology, we have described one of the first observations about how Tat is able to control various host cellular machineries. Although our data is consistent with most of the cited literature on the effects of Tat in infected host and uninfected bystander cells, we caution that the transcriptional profiling in chronically infected cells such as ACH2 or H9/Tat cells may not necessarily be representative of the pattern of expression observed in most cells infected by other group M, N, or O HIV-1 isolates. We recently extended our observations by utilizing other HIV-1 infected cells which normally express Tat (U1), and addition of exogenous purified Tat to uninfected PBMCs. Preliminary results using western blots supports the idea that genes which were altered in H9/Tat system also showed a similar level of change in few of the tested genes . This notion of consistency was further confirmed using the IL-8 activation by Tat. Interleukin-8 (IL-8) belongs to the CXC chemokine family and is secreted by several different cell types, including monocytes, neutrophils, endothelial cells, fibroblasts, and T lymphocytes. IL-8 production (induced by several stimuli, including IL-1, TNF-, and phorbol myristate acetate) is primarily regulated at the transcriptional level. IL-8 is a potent chemotactic factor for granulocytes and T lymphocytes, and is found in HIV-infected individuals. The CXC chemokine IL-8 does not bind to CCR5. It has previously been shown that IL-8 mRNA induction was seen less then 1 h after Tat (72aa) stimulation, and levels remained elevated for up to 24 h, leading to IL-8 protein production . Along these lines, we have previously shown that the IL-8 gene is expressed in a cell cycle-dependent manner in cells that express the Tat protein, and the induction is during the S phase of the cell cycle and regulated by stable NF-kB binding to the IL-8 promoter . When looking for IL-8 at the G1/S border, we found that all Tat containing cells, including PBMCs that were treated with exogenous Tat showed an up-regulation of IL-8 in the supernatant , further implying that results obtained from the H9/Tat system may infact be of general physiological relevance in vivo. Figure 6 | Proposed model for changes in signal transduction. Proposed model for changes in signal transduction. A) Down-regulation of receptor tyrosine kinases (RTK) by Tat which modulates the phosphorylation and transcription of downstream effectors such as Ras, Raf, MEK, MAPK, and control transcription factor phosphorylation. B) Role of Tat in the increase of genes necessary for proliferation, such as Cdc2, Cdc37, and Prothymosin alpha, and down-regulation of differentiation genes, such as receptors, co-receptors, and signal transduction genes. Finally, throughout the current study we came across some technical findings that were critical in the confirmation of most of our results. For instance, few genes did not correlate in their activation or suppression levels when comparing fold changes between microarrays and protein levels using western blot analysis. We suspect this is because many genes that are transcribed may not necessarily be translated, due to their cell cycle stage, 5' stem and loop RNA structures, varying half-lives of proteins and mRNAs, and a host of other unknown variables. Also, specific changes that occur in a cell may not be required in redundant pathways that score for a specific function. This is commonly seen in the differences between HIV-1 infected or Tat expressing in vitro cell lines and AIDS patients PBMC samples. Therefore, other microarrays would have to be performed on purified infected PBMCs to confirm most of the changes observed in Tables , , and . Unfortunately, to date this particular issue is not feasibly addressable, since it is not possible to isolate a homogenous population of infected T- or Monocytic cells from AIDS patients. Also, confirmatory tests for protein expression would have to be done with both hydrophilic and hydrophobic extraction buffers. For instance, we have observed that PCNA protein, which is up-regulated in Tat expressing cells, extract best with hydrophobic buffers from the nucleus, presumably due to its binding to DNA replication machinery (data not shown). Future experiments will address issues related to differences between various HIV-1 Tat clades, host expression levels between T- and Monocytic cells, and its effect at various stages of the cell cycle. PMID- 12069692_Conclusions TI - AB - Expression profiling from HIV-1 or Tat expressing cells holds great promise for rapid functional analysis. Here, we have described the effect of Tat and its alterations with the host cellular gene expression. We observed that more than 2/3 of the cellular genes tested were down-regulated by Tat. These genes belong to receptor, co-receptor, and co-activator pathways that are part of serine/threonine receptor tyrosine kinase, Ras/Raf/MEK/ERK (MAPK) cascade, which control proliferative and/or differentiation signals. We also observed a great deal of increase in the host cell translation apparatus with the possible aim of optimizing viral mRNA translation prior to viral maturation and release. Therefore, HIV-1 accessory doubly spliced messages such as Tat, may control the host gene expression in latently infected cells, and determine not only viral transcription, but also the fate of post-transcriptional events. PMID- 12069692_Materials and method TI - AB - Cell culture | ACH2 cells are HIV-1 infected CD4 lymphocytic cells, with an integrated wild-type single-copy chromatinized DNA. The CEM T cell (12D7) is the parental cell for ACH2 cells. ACH2 cell lines has a single copy of LAI strain proviral sequence. The TAR has a point mutation at (C37 -> T), which no longer responds (efficiently) to Tat. However, the cell line is fully capable of making infectious virus in presence of TNF, PHA, PMA, and a host of other stimuli. H9 and H9/Tat cells are both CD4+ Lymphocytic cells, where H9 cells carry a control integrated vector without the Tat open reading frame, and H9/Tat cells carry integrated Tat expression vector. Both cell lines were a generous gift of George Pavlakis (NCI, NIH). U1 is a monocytic clone harboring two copies of the viral genome from parental U973 cells. All cells were cultured at 37C up to 105 cells per ml in RPMI-1640 media, containing 10% Fetal Bovine Serum (FBS) treated with a mixture of 1% streptomycin and penicillin antibiotics, and 1% L-glutamine (Gibco/BRL). Phytohemagglutinin-activated PBMC were kept in culture for 2 days prior to addition of Tat protein. Isolation and treatment of PBMC were performed by following the guidelines of the Centers for Disease Control. Approximately 5 x 106 PBMC were used for treatment of wild type and K41A Tat mutant (100 ng/ml) proteins. After an initial incubation for one hr with Tat proteins, cells were washed and cultured in complete media for 24 hrs, prior to western blots. pCEP4, eTat cells were HeLa cells stably transfected with either a backbone control plasmid (pCEP4; Invitrogen) or a plasmid expressing Tat (1 --86) with a C-terminal epitope tag (eTat) . HeLa cell lines containing either the control or eTat plasmid were selected by single-cell dilution. Both cell types were selected and maintained under 200 mug of hygromycin per ml. Verification of Tat transcriptional activity was achieved by electroporation of reporter plasmids as previously described . Cell cycle analysis | Hela cells were blocked with hydroxyurea (Hu) (2 mM) for 14 h. Following the block, cells were released by being washed twice with phosphate-buffered saline (PBS) and by the addition of complete medium. All suspension cells were treated with 1% serum for 48 hrs prior to addition of Hu. Supernatants were collected and analyzed by an IL-8 ELISA according to the manufacturer's instructions (Biosource International). For controls, each sample, approximately 1 x 106 cells was processed for cell sorting. Cells were washed with PBS and fixed by addition of 500 mul of 70% ethanol. For fluorescence-activated cell sorting (FACS) analysis, cells were stained with a cocktail of propidium iodide (PI) buffer (PBS with Ca2+ and Mg2+, RNase A [10 mug/ml], NP-40 [0.1%], and PI [50 mug/ml]) followed by cell-sorting analysis. FACS data acquired were analyzed by ModFit LT software (Verity Software House, Inc.). Cell extract preparation and immunoblotting | All cells were cultured to mid-log phase of growth, washed with PBS without Ca2+ and Mg2+, and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.2 mM Na3VO4, 1 mM DTT, 0.5% NP-40 and protease inhibitors (Protease inhibitor cocktail tablets, Boehringer Mannheim, one tablet per 50 ml). The lysate was incubated on ice for 15 min, and microcentrifuged at 4C for 10 min. Total cellular protein was separated on 4 --20% Tris-glycine gels (Novex, Inc.) and transferred to a polvinylidene difluoride (PVDF) membranes (Immobilon-P transfer membranes; Millipore Corp.) overnight at 0.08 A. Following the transfer, blots were blocked with 5% non-fat dry milk in 50 ml of TNE 50 (100 mM Tris-Cl [pH 8.0], 50 mM NaCl, 1 mM EDTA) plus 0.1% NP-40. Membranes were probed with a 1:200 --1:1000 dilution of antibodies at 4C overnight, followed by three washes with TNE 50 plus 0.1% NP-40. All antibodies used in this study were purchased from Santa Cruz Biotechnology. The next day, blots were incubated with 10 ml of 125I-protein G (Amersham, 50 mul/10 ml solution) in TNE 50 plus 0.1% NP-40 for 2 hrs at 4C. Finally, blots were washed twice in TNE 50 plus 0.1% NP-40 and placed on a PhosphorImager cassette for further analysis. Total RNA purification | Cells were grown to mid-log phase of growth (5.0 x 106), pelleted, and washed twice with cold D-PBS without Ca2+/Mg2+. Total RNA was extracted on ice using Trizol Reagent (Life Technologies, Inc.). Purified RNA was then analyzed on a 1% agarose gel for quality and quantity prior to each experiment. Glass slide microarray | Gene expression analysis was performed using MicromaxTM: Human cDNA Microarray System I (cat# MPS101, NEN Life Science Products). On a glass microarray slide, 2400 know human genes were arrayed into 4 separate grids (A, B, C, D), containing 600 genes each (gene description and location on microarrays available at NEN website: ). All human genes were ~2200 bp cDNAs, and were characterized from 50+ human cDNA libraries (AlphaGene, Inc., Woburn, MA). In addition to the human genes, three plant control genes were spotted on each grid and were utilized to balance the Cyanine-3 (Cy-3) and Cyanine-5 (Cy-5) fluorescence signals. A total of 8 mug each of H9 (control sample) and H9/Tat (test sample) mRNAs were reverse transcribed into Biotin and Dinitrophenyl (DNP) labeled cDNA, respectively. After cDNA quality and quantities were analyzed, both cDNAs were then pooled and simultaneously hybridized overnight at 65C onto the glass microarray. The next day, the microarray slide was serially washed in 0.5x SSC (Sodium Citrate-Sodium Chloride) + 0.01% SDS (Sodium Dodecyl Sulfate), 0.06x SSC + 0.01% SDS, and 0.06x SSC. Next, the Tyramide Signal Amplification (TSATM) was then used to amplify the Cy-3 and Cy-5 signals using antibody-enzyme conjugates, alpha-DNP-Horseradish peroxidase (HRP) and alpha-Streptavidin-HRP with Tyramide linked Cy-3 and Cy-5. Screening and data analysis was performed by NEN. cDNA filter hybridization | Gene expression of CEM and ACH2 were performed using Atlas Human cDNA Expression Array (Clontech Laboratories Inc., Palo Alto, CA) according to the manufacturer's directions. One mug of poly A+ RNA each was DNase I treated, purified using a CHROMA SPIN-200 column, and reverse transcribed into 32P-labeled cDNA. The CHROMA SPIN-200 column was used to purify the 32P-labeled cDNA from unincorporated 32P-labeled dNTPs and small (<0.1 kb) cDNA fragments. Each sample was then hybridized to a human cDNA expression array overnight with continuous agitation at 68C. The next day, the array was washed three times with gentle agitation, first wash with 2x SSC + 1% SDS and the last two washes with 0.1x SSC + 0.5% SDS at 37C. Array was exposed to a PhosphorImager Cassette and analyzed using ImageQuant software. Northern blots | Total cellular RNA was extracted using the RNAzol reagent (Gibco/BRL). Total RNA (20 mug) was isolated from various cells and ran on a 1% formaldehyde-agarose gel overnight at 75 V, transferred onto a 0.2 mum nitrocellulose membrane (Millipore Inc.), UV cross-linked, and hybridized overnight at 42C with 32P-end-labeled 40 mer oligo probes including p21/Waf1, C-myc, Pro-thymosin, Actin, Tat, and Ubiquitin (Loftstrand, Gaithersburg, Md.). Next day, membranes were washed two times for 15 min each, with 10 ml of 0.2% SDS-2XSSC at 37C, exposed, and counted on PhosphorImager Cassette. Viral infection and ELISA assay | Both H9 and H9/Tat cells were infected in the presence of 10 ug of polybrene. For PBMC infections, PHA activated PBMCs were kept in culture for 2 days prior to each infection. Isolation and treatment of PBMCs were performed by following guidelines from the CDC (Isolation, culture, and identification of HIV, Procedural Guide, July 1991, Atlanta, GA). Approximately 2 x 10 6 of H9 or H9/Tat cells, and 5 x 10 6 PBMC cells were infected with either an HXB-2 (CXCR4, T-tropic), or BaL (CCR5, Macrophage-tropic) at 5 ng of p24 gag antigen/ HIV-1 strain. Both viral isolates were obtained from the NIH AIDS research and reference reagent program. After 8 hrs of infection, cells were washed and fresh media was added. Samples were collected every 3rd day and stored at -20 C for p24 gag ELISA. Media from HIV-1 infected cells were centrifuged to pellet the cells and supernatants were collected, and diluted to 1:100 to 1:1000 in RPMI 1640 prior to ELISA. The p24 gag antigen level was analyzed by HIVAGTM-1 Monoclonal antibody Kit (Abbott Laboratories, Diagnostics Division). PMID- 12069692_Authors' Contributions TI - AB - CF, and FS carried out the ACH2 and H9/Tat microarrays. LD, CE, IZ, CGL, and KW aided in westerns, northerns, p24 and ELISA assays. AM, KK, SB, AP, and FK aided in data interpretation, Bioinformatics, literature searches and writing the manuscript. PMID- 12052258 TI - Hospital competition, resource allocation and quality of care AB - Abstract | Background | A variety of approaches have been used to contain escalating hospital costs. One approach is intensifying price competition. The increase in price based competition, which changes the incentives hospitals face, coupled with the fact that consumers can more easily evaluate the quality of hotel services compared with the quality of clinical care, may lead hospitals to allocate more resources into hotel rather than clinical services. Methods | To test this hypothesis we studied hospitals in California in 1982 and 1989, comparing resource allocations prior to and following selective contracting, a period during which the focus of competition changed from quality to price. We estimated the relationship between clinical outcomes, measured as risk-adjusted-mortality rates, and resources. Results | In 1989, higher competition was associated with lower clinical expenditures levels compared with 1982. The trend was stronger for non-profit hospitals. Lower clinical resource use was associated with worse risk adjusted mortality outcomes. Conclusions | This study raises concerns that cost reductions may be associated with increased mortality. PMID- 12052258_Introduction TI - AB - The last two decades brought about fundamental changes in the organization and delivery of medical services in the United States as payers seek to control the escalation in health care expenditures. Policies addressing these issues have been of two types. The first relies on containing costs through control of the prices paid to providers, beginning with the Prospective Payment System (PPS) for hospitals in 1983, Resource Based Relative Value Units (RBRVUs) payment for physicians in 1992 and the most recently implemented prospective payment for nursing homes. Such prospective payment systems provide the same incentives to cut costs to all providers, irrespective of the markets in which they are located and the competitiveness of their markets. The other relies on changing the focus of competition among health care providers, from quality based competition to price based competition . These policy changes were successful in bringing about a deceleration in hospital revenues and expenditures growth . Little is known, however, about what specific strategies hospitals adopted and the impact these strategies may have had on the quality of care patients receive. Previous studies found that hospitals increased efficiency in all clinical services following selective contracting in California. California hospitals also tended to specialize and differentiate themselves from similar hospitals in response to competitive pressures . A similar response, of increased specialization, was observed for a national sample of hospitals following implementation of PPS . In this paper we study another potential strategy that hospitals may adopt and which has not been addressed in the literature to-date. We investigate the hypothesis that hospitals in an increasingly price competitive environment, shift resources from activities related to clinical services, which are not easily observed and evaluated by patients, into hotel services which are easily observed. We study hospitals in California, comparing resource allocation during a regime dominated by quality competition and a regime dominated by price competition. We then examine the association between risk adjusted excess hospital mortality and resource use in clinical services, to investigate the potential impact on quality of medical care and health outcomes. Competition in California in the 1980s: a case study | The implementation of selective contracting in California in 1982 offers a unique natural experiment to study the response of hospitals to changes in the nature of competition. Unlike other health care markets, in which the change from quality to price based competition was gradual, driven by continuously increasing penetration of managed care, and often time confounded by other secular trends, the California legislation changed market conditions very rapidly for all hospitals in the state by permitting all health plans for the first time to contract with only a subset of hospitals. It thus allows a pre/post study design: hospitals resource allocation decisions during the quality competition regime (pre period) can be compared to decisions made during the price competition regime (post period). This natural experiment allows us to test the hypothesis that changes in the nature of competition are more likely to be associated with a shift of resources from clinical to hotel activities (and a concomitant deterioration in mortality outcomes) in more competitive hospital markets. As the level of hospital competition has not changed during the period (the Herfindahl-Hirschman Index (HHI) remained stable in all markets), this test is limited to the change in the nature of competition and is not confounded by the impact of changes in the level of competition on resource allocation decisions. During the same time period, California hospitals were also subject for the first time to price regulation, due to implementation of the Medicare PPS. While PPS also provided hospitals with incentives to lower their costs, it did so in a distinctively different manner than price competition. The PPS set the price per discharge hospitals were paid, thus creating incentives to lower costs, irrespective of market structure (7). The intensity of price based competition, on the other hand, is highly sensitive to the competitiveness of the hospital market. The analytical strategy of this paper is based on this distinction. Hospital competition, quality, resource allocation and health outcomes | Competition focused on prices, as is often the case in markets dominated by managed care, creates incentives to increase efficiency and possibly curtail resource use. With the exception of possible increases in administrative activities designed to contain costs in other areas (e.g. billing and utilization review) or to increase marketing efforts, such incentives to cut costs are likely to affect all aspects of hospital activities. Hospitals may also compete on quality, both quality of medical services and quality of hotel services and amenities. The importance of competition for quality is likely to be greater in markets in which hospitals compete for patients directly, as they do for all fee-for-service patients and for those enrolled in HMOs that offer a choice of hospitals within their market. Furthermore, to the extent that HMOs make their contracting decisions based on beneficiary hospital preferences, perceptions of quality are important competitive tools. Competition for quality , unlike competition for price , may lead to increased costs. Furthermore, it may affect clinical and hotel services differently. In markets where patients' choice of hospitals are increasingly important, hospitals are likely to compete more on quality attributes that patients observe and value. Given the difficulty that patients have in directly determining the quality of medical care they receive, and the relative ease with which they can evaluate the quality of hotel services (e.g. condition of the facility, quality of food) hospitals face incentives to shift resources from clinical activities to amenities. On the other hand, if patients rely on their physician's recommendations in choosing hospitals , and to the degree that physicians can assess clinical quality, albeit imperfectly, hospitals are faced with counter incentives, incentives that would promote resource use in clinical activities rather than hotel services. As a result, hospitals may face conflicting incentives: incentives to maintain or enhance the quality of hotel services on the one hand, and incentives to maintain activities that contribute to the quality of clinical care and health outcomes on the other. The actual choices that hospitals make about resource allocation depend on the relative strength of these opposing incentives. A model of changes in resource allocation | As the main hypothesis of interest is that the change in the nature of competition was associated with changes in hospital resource allocations, the model we hypothesize allows the marginal effects of market and hospital characteristics (Xt) on resource allocation (Yt), as measured by the coefficients in a regression model (betat), to vary over time: (1) Yt = alphat + betatXt + epsilont The models we estimated were difference models of the form (2) DeltaY = Deltaalpha + Deltabeta * X0 + betat * DeltaX + Deltaepsilon where Delta is the difference between year t and the base year, indicated by t = 0. From (2) it follows that betat, the vector of coefficients multiplying the change variables, measures the marginal effect of the variable in the end year, while Deltabeta, the vector of coefficients multiplying the level variables, measures the change in the marginal effect. The marginal relationship in year 0 is given by beta0 = betat - Deltabeta.. PMID- 12052258_Methods TI - AB - Sample | The initial sample included all 338 acute care hospitals in California that were in operation during both 1982 and 1989. Of those, 18 (5.3%) were excluded from the resource allocation analyses and 8 (2.4%) were excluded from the mortality analyses, because of incomplete data. Data sources | Financial, ownership and utilization data were obtained from the Hospital Annual Financial Disclosure Reports, filed annually by all California hospitals with the Office of Statewide Health Planning and Development (OSHPD). Risk adjusted mortality data were obtained from the Medicare Hospital Information Report published by the Health Care Financing Administration . Variable definitions | I. Resource allocation variables | Resource allocation was measured by expenditures per adjusted discharge. Adjusted discharges are a composite measure of input designed to account for both inpatient discharges and outpatient visits, using the methodology developed by the American Hospital Association. Expenditures per adjusted discharge were calculated separately for three categories: clinical, hotel and administrative services. Expenditures were aggregated by cost center, with each cost center assigned to one of the three services. The table in the lists all hospital cost centers and their assignment to hotel, clinical and administrative categories. The dependent variables in the resource allocation analyses were defined as the differences in expenditures per adjusted discharge, between 1989 and 1982, for each of the three categories. II. Quality variables | The dependent variables for the analyses of quality of clinical care were excess death rates from all causes and from 4 specific medical conditions that have relatively high death rates: acute myocardial infarction, congestive heart failure, pneumonia and stroke. We included in the analyses measures based on cause specific mortality in addition to overall mortality because prior studies have shown that these measures tend to be uncorrelated, and that hospitals performing well in one clinical area do not necessarily perform well in others. Measures based on overall mortality may therefore be biased towards zero, showing less variation compared with cause specific measures. Excess mortality was defined as the difference between the observed mortality rate for the hospital and a predicted, risk adjusted mortality rate. Observed and predicted mortality rates were obtained from the Medicare Hospital Reports . They are based on Medicare discharges and include all deaths within 30 days of admission, irrespective of the location of death. The risk adjustment methodology used by the Health Care Financing Administration, incorporates individual patients' age, gender, specific diagnoses and comorbidities, admission source, emergency or elective admission and the patient's risk group based on hospitalizations during the preceding 6 months . III. Independent variables | Competition was measured by the Herfindahl-Hirschman Index (HHI), defined as the sum of squared market shares of all hospitals competing in the same area. Hospital market areas and the HHI were calculated based on all payer zip code level patient flows, as described in Zwanziger et al. . To control for financial pressures hospitals may have been experiencing in addition to competition, we included variables measuring bad debt and charity as percent of total revenues and percent occupancy. To control for potential economies of scale the estimated models included total clinical standard units of measures reported in the California Financial Disclosure Reports. Ownership indicator variables included for-profit, not-for profit public, and not-for profit district ownership. The omitted category was private not-for profit hospitals. Teaching status was defined as hospitals with some residents. All payer DRG-based case mix index was included to account for differences in patients' severity. Median family income measured for the hospital's zip code area was included to capture demand effects and as a proxy for cross sectional wage variations. Analyses | We estimated regression models in which change in expenditures per adjusted discharge and excess mortality in 1989 were the dependent variables. Because all models were heteroskedastic, all reported tests of significance are based on White's robust standard errors . The Ramsey RESET test for specification errors was applied to all models to rule out the need for non-linear and interaction terms. The mortality models were weighted by the inverse of the standard error for the predicted mortality rate, to account for differences across hospitals in the accuracy of the excess mortality measures, which are due to differences in sample sizes . Since initial analyses indicated different associations (different betas) for for-profit and non-profit hospitals, we estimated fully interacted models, in which all variables were interacted with for-profit status. The hypotheses of significant marginal effect were therefore tested for the non-profit hospitals by a t test of the main effect and for the for-profit hospital by an F test of the linear restriction that the sum of the coefficients of the main and interaction effect are zero. PMID- 12052258_Results TI - AB - Description of sample hospitals | Table presents descriptive statistics for the hospitals included in the study. The majority of hospitals (52.6%) were private non-profit with the second largest group (26.0%) being for-profit institutions. Fifteen point four percent were teaching hospitals. The average hospital size did not change significantly over the 1982 --1989 period, remaining at 190 --200 beds. Occupancies declined significantly, from an average of 62.3% to 55.2%, and inpatient case mix increased significantly from 1.17 to 1.27, indicating that hospitals were treating sicker and more expensive patients at the end of the period. Both total expenditures and expenditures per adjusted discharge increased significantly. Table 1 | Descriptive Statistics The degree of competition among hospitals has not changed between 1982 and 1989. The HHI of around 0.3 suggests that competition was limited. (Markets with HHI values below 0.18 are considered moderately or very competitive ). The large variation in the HHI, however, indicates that many hospitals were located in competitive markets, with 25% of hospitals in markets with HHI below 0.17. Overall mortality rates averaged 10%. Average observed and predicted rates were very similar, but the variation in rates was higher for the observed rates compared with the predicted rates, suggesting substantial variations in excess mortality and quality across the sample. Resource allocation changes | Table reports the mean values for the dependent and independent variables included in the multivariate regressions. Table reports results by ownership -- for-profit and non-profit. These results are based on a fully interacted model estimated over pooled data by ownership. All models were highly significant (p < 0.01). The clinical services model explained 51% of the variation in expenditures per adjusted discharge, while the hotel and administrative services models explained 24% and 26% respectively. Table 2 | Means and standard deviations of variables included in the multivariate analyses Table 3 | Expenditures per adjusted Discharge -- Multivariate regression results Effect of competition | Table presents the marginal effect of competition, calculated from the regression results and using equations 2 and 3, for 1982 and 1989, as well as the change in these coefficients between the two years. (Note that the regression coefficients for the HHI variable were multiplied by -1 in table , such that a positive association means that expenditures per discharge increase with increased competition.) Table 5 | Marginal effect of competition on Expenditures per adjusted discharge (Positive values indicate increase with increased competition) Non-profit hospitals in more competitive areas had higher expenditures per adjusted discharge in all three categories in all years. The marginal effect was highest in clinical areas and lowest in administrative services. It declined significantly over time in both clinical and hotel services, but not in administrative services. The decline was almost three times as large in the clinical services compared with hotel services. By 1989, while the marginal effect of competition on expenditures in these services was still positive, it was no longer significantly different from zero. These findings are consistent with the hypothesis that the focus of competition on quality in 1982 has diminished significantly over the seven-year period we studied. The results for for-profit hospitals present a different picture. First, the association between expenditures per adjusted discharge and competition was statically significant only for administrative services. This association was also by far the strongest. It was negative, indicating that hospitals in more competitive markets spent less per discharge on administrative activities. The marginal effect was slightly smaller in 1989, suggesting that hospitals in more competitive areas may have reallocated resources into administrative services. They may have, for example, invested in better information and management systems that would allow them to better control costs. The association between competition and resource use was negative in clinical services and positive in hotel services. This is consistent with the hypothesis that for-profit hospitals compete on quality in those areas that can be easily observed by patients, namely hotel services, and cut back on resources in clinical services, where quality is more difficult for patients to evaluate directly. The change over the 1982 through 1989 period is also consistent with this hypothesis: the negative association between competition and clinical resources increased in 1989 as did the positive association between hotel resources and competition. The lack of significance of the associations may reflect the smaller number of for-profit hospitals in the sample and the resulting lower statistical power. (There were 83 for-profit hospitals and 13 independent variables, compared with 237 non-profit hospitals.) Other hospital and market characteristics | The strongest and most consistent relationship was between all payer case mix and expenditures per adjusted discharge in all categories (see table ). The association was substantially stronger for the clinical category and in non-profit compared with for-profit hospitals. Most other variables either exhibited no significant associations or no clear patterns. There were no significant differences in resource allocations by ownership. Teaching status was positively associated with clinical and hotel expenditures among the non-profit hospitals but exhibited a negative association among the for-profit hospitals. Percent bad debt and charity and percent occupancy had no significant relationship with expenditures among the non-profit but were associated with lower clinical and administrative expenditures among the for-profit hospitals. Median family income was associated with higher expenditure levels for all services among the non-profit hospital, but only with the clinical services among the for-profits. Risk adjusted excess mortality | Table reports the results of the regressions modeling the association between excess mortality (defined as the difference between observed and predicted mortality rate) and clinical expenditures per adjusted discharge, competition, ownership, and teaching status. The models explained between 1% and 5% of the variation in excess mortality. In all cases there was a negative association between clinical expenditure levels and excess mortality, implying that increased resources were associated with better mortality outcomes. This relationship was present for mortality from all causes as well as from the four specific causes, and was statistically significant at the 0.10 level or better. Table 4 | Risk adjusted excess mortality -- Multivariate Regression Results Table shows the increase in excess mortality that is associated with a decrease of 1 standard deviation (SD) in clinical expenditures per adjusted discharge, based on the estimated regression coefficients. For comparison, the table also provides the magnitude of 1 SD in excess mortality among the study hospitals. In all cases, the effect of a 1 SD in resources was less than a 1 SD in excess mortality. As expected due to the potential bias towards zero in the measures based on all causes, the associations were larger for the cause specific measures. Table 6 | Increase in excess mortality rates associated with 1 standard deviation decrease in clinical expenditures per adjusted discharge As discussed earlier, we hypothesized that competition may affect quality, including clinical quality, not only through its impact on resource use but also due to incentives to compete on quality. If indeed hospitals were competing on clinical quality, the association between the HHI and excess mortality, controlling for resource use, should have been positive and significant. In all cases, except for pneumonia, we did not find a significant relationship. In most cases there was also no significant relationship between ownership or teaching status and excess mortality. PMID- 12052258_Discussion TI - AB - In this paper we present a test of the hypothesis that changes in the nature of competition among California hospitals, resulting from selective contracting, were associated with changes in hospitals' resource allocation decisions. We find empirical evidence to suggest that resources have been shifted from clinical activities (which are not observed by patients) and into hotel services (which are more readily observable). These changes in resource allocation tended to be larger in hospitals located in more competitive areas. As the level of competition has not changed during the study period, the change in hospital behavior is likely to be a response to the change in the nature of competition. The relationship between resource allocation and competition differed by ownership. For-profit hospitals in more competitive areas had lower expenditures levels compared with those in less competitive areas. Among non-profit hospitals, we found the opposite -- clinical expenditures per adjusted discharge increased with competition. The trend over time, however, even though it was much stronger among the non-profit hospitals, was the same for both types. For both, the change in the marginal effect of competition (Deltabeta) was negative. As a result, the positive association between competition and clinical resource use among non-profit hospitals diminished and the negative association among the for-profit hospitals increased. The analyses of excess mortality demonstrate that clinical quality, at least as measured here, is positively associated with the amount of resources used in producing clinical services. Therefore, policies that create incentives for hospitals to limit resource use are likely to have an impact on health outcomes. Furthermore, as clinical quality is not easily observable by consumers, leading to disparity in incentives to provide hotel and clinical quality, more competitive areas are likely to experience a larger relative decline in resources allocated to clinical activities and hence worse mortality outcomes. The impact on health outcomes in non-profit hospitals may not be as large as it might have been because expenditure levels were curtailed not only in clinical areas, but in hotel services as well, although to a much lesser degree. This strategy spread the burden of cost containment efforts beyond clinical activities. If non-profit hospitals would have concentrated all their cost cutting efforts in clinical services, as did the for-profit hospitals, the impact on costs, and potentially on mortality outcomes, would have been 35% higher (see table ). The generalizability of the findings presented here is limited in several ways. First, quality was measured only in terms of excess mortality. While this is an important aspect of quality, it is likely to be an insensitive measure. Because measures based on mortality do not tend to be correlated with measures based on other outcomes , such as complications, one cannot deduce from this study that other aspects of clinical quality have been affected similarly by the changes in competition during the period. Furthermore, the mortality models we estimated, unlike the expenditures models, were cross sectional and are therefore subject to the usual concerns about potential bias due to omitted hospital specific effects. It should be noted, however, that the models did include the variables most likely to be associated with excess mortality -- patient level risks, expenditures, competition, ownership and teaching status. It should also be noted that while our findings with respect to changes in resource allocation are based on total expenditures, thus reflecting care for all patients, the mortality outcomes are based on the experience of Medicare patients only. Prior studies, however, suggest that patient care given by same provider does not vary by payer status . A more important generalizability question arises due to the sample selected for this study -- namely hospitals located only in California during the 1980s. Can the behavior of hospitals observed in this local and during this time period be assumed to generalize to other markets and other times? While the magnitude of the effects we measure in this study are clearly not generalizable, the directions of the effects are likely to transcend time and place, as they reflect basic behavioral responses to market incentives in accordance with theory. While this sample selection may be viewed as a limitation of this study, it is also its strength. By focusing on a period in which levels of competition were stable and where the change in the nature of competition can be traced to a specific legislative act, this study is able to test for the differential impact of price vs. quality competition, without confounding by other factors. PMID- 12052258_Conclusion TI - AB - In summary, the results of this study should be viewed as raising a cautionary question: are the hospital cost reductions that have been observed in California and nationally associated with increased mortality? PMID- 12052258_Authors' Contributions TI - AB - DM designed the study, performed the analyses, and wrote the manuscript. AB prepared the data set, and JZ participated in the design and analyses. PMID- 12052258_Competing interests TI - AB - None declared. PMID- 12052258_Pre-publication history TI - AB - The pre-publication history for this paper can be accessed here: PMID- 12079499 TI - Functional group interactions of a 5-HT3R antagonist AB - Abstract | Background | Lerisetron, a competitive serotonin type 3 receptor (5-HT3R) antagonist, contains five functional groups capable of interacting with amino acids in the 5-HT3R binding site. Site directed mutagenesis studies of the 5-HT3AR have revealed several amino acids that are thought to form part of the binding domain of this receptor. The specific functional groups on the ligand that interact with these amino acids are, however, unknown. Using synthetic analogs of lerisetron as molecular probes in combination with site directed mutagenesis, we have identified some of these interactions and have proposed a model of the lerisetron binding site. Results | Two analogs of lerisetron were synthesized to probe 5-HT3R functional group interactions with this compound. Analog 1 lacks the N1 benzyl group of lerisetron and analog 2 contains oxygen in place of the distal piperazine nitrogen. Both analogs show significantly decreased binding affinity to wildtype 5-HT3ASRs. Mutations at W89, R91, Y142 and Y152 produced significant decreases in binding compared to wildtype receptors. Binding affinities of analogs 1 and 2 were altered only by mutations at W89, and Y152. Conclusions | Based on the data obtained for lerisetron and analogs 1 and 2, we have proposed a tentative model of the lerisetron binding pocket of the 5-HT3ASR. According to this model, The N-benzyl group interacts in a weak interaction with R91 while the benzimidazole group interacts with W89. Our data support an interaction of the distal amino nitrogen with Y142 and Y152. PMID- 12079499_Background TI - AB - The cysteine-loop family of ligand gated ion channels (LGIC) is comprised of receptors with pentameric quaternary structure and at least two ligand binding sites present at the subunit interfaces . This receptor family is characterized by the presence of a critical disulfide loop structure within the binding site and an integral ion selective channel. LGIC receptors are found in both the peripheral and central nervous systems. Members of this family include the acetylcholine receptors , the gamma-amino butyric acid type A receptor (GABAAR) , and the glycine receptor (GlyR) . The first subunit of the 5-HT3R was cloned in 1991 . The sequence of this subunit was shown to be highly homologous to LGIC receptors and thus identified the 5-HT3R receptor as another member of this superfamily . Similar to other LGIC receptors, more than one subtype has been identified. Two splice variants of an A subunit (long and short forms), and a single B subunit have been cloned . Both the long and short forms of the A subunit are capable of forming functional homomeric receptors [5-HT3ALR and 5-HT3ASR] although some differences between an agonist and partial agonist activity have been observed . A third subtype is formed by a combination of the A and B subunits to produce a heteromeric receptor of unknown stoichiometry . Heteromeric receptors are pharmacologically and functionally distinct from the homomeric 5-HT3AL and 5-HT3AS receptors . 5-HT3Rs are distributed throughout the central and peripheral nervous system, playing a significant role in phenomenon such as anxiety, emesis and alcoholism. Antagonists to 5-HT3Rs are clinically efficacious in the treatment of chemotherapy-induced emesis and recent studies on human subjects have suggested their potential application in the treatment of early onset alcoholism . Hibert et al proposed an early model for the antagonist pharmacophore of the 5-HT3R . According to this model, all 5-HT3R antagonists contain an aromatic ring, a carbonyl oxygen or bioisosteric equivalent, and a basic nitrogen. According to Hibert's model, the basic nitrogen is located 5.2A from the centre of the aromatic ring and approximately 1.7A above plane of the ring. The carbonyl oxygen and the aromatic ring are coplanar and separated by a distance of 3.3A. Recent studies have expanded on this model to include another lipophilic region and a second hydrogen bonding interaction two atoms away from the first . A compound that contains all five pharmacophoric regions was synthesized by Orjales et al. This compound (1-(phenylmethyl)-2-piperizinyl benzimidazole or lerisetron) is shown in figure and is a potent 5-HT3R antagonist. Functional groups on this compound capable of forming interactions with the receptor are the distal amino group, a benzimidazole and a benzyl group in the N1 position of the benzimidazole. While Lerisetron contains no carbonyl group, the second nitrogen contained in the benzimidazole heterocycle could act as bioisostere of this functional group . Orjales demonstrated the importance of the N-benzyl group by synthesizing several N1 substituted analogs of Lerisetron. Removal of the N-benzyl group produced a 80-fold decrease in affinity, indicating a role for this group in interacting with the 5-HT3R. Other studies have supported this observation and suggest a more specific electrostatic interaction . Figure 1 | 5-HT3R antagonists. 5-HT3R antagonists. Granisetron and Lerisetron are shown along with the two analogs utilized to probe the functional group interactions of binding site amino acids. Analog 1 lacks the N-benzyl functional group. Analog 2 contains oxygen in place of the distal piperazine nitrogen. While structure-activity relationship studies and molecular modeling have led to the development of a detailed pharmacophore model, determining specific point interactions between 5-HT3 antagonists and binding site amino acids has proven difficult. Mutagenesis studies have identified the interaction of amino acids W89 and R91 in the binding of 5-HT3R ligands . Studies conducted in our laboratory have identified three additional putative binding site residues (Y140, Y142, and Y152) . W89 and R91 are present in a conserved region of LGIC receptors often referred to as loop D . Similarly, Y140, Y142 and Y152 are located in the region homologus to E loop region of nicotinic AchR. In this study, we have endeavoured to identify the amino acids interacting with the different functional groups present on the lerisetron molecule in order to develop a model for interaction of this compound with the 5-HT3R. Using site directed mutagenesis in combination with analogs of lerisetron, we have identified amino acids that appear to interact selectively with the terminal amino group, the N-benzyl group and the aromatic benzimidazole. PMID- 12079499_Results TI - AB - Functional activity of lerisetron | Whole cell patch-clamp experiments were performed to test the functional activity of lerisetron. No response was observed when lerisetron was applied alone (data not shown). When co-applied with 5-HT, lerisetron inhibited the absolute magnitude of the response with no apparent alteration of the response profile . The combination of several identical inhibition experiments produced a Ki value of 0.2 +- 0.03 nM for lerisetron inhibition of the 5-HT induced response. These data correspond well with previously reported data for this compound and verify the competitive antagonist action of lerisetron. Analogs of lerisetron have been shown to inhibit 5-HT3Rs in a similar manner . Figure 2 | Inhibition of 5-HT induced responses by Lerisetron. Inhibition of 5-HT induced responses by Lerisetron. tsA 201 cells transfected with 5-HT3ASR cDNA were clamped at a holding potential of -60 mV and perfused with 4 muM 5-HT (lower trace) or 4 muM 5-HT and 0.3 nM lerisetron (upper trace). Lerisetron reduced the peak 5-HT induced currents from 2.0 nA to 1.0 nA with no change in the response characteristics. An IC50 value was determined by a combination of similar experiments and determined to be 0.4 +- 0.03 nM. Importance of the N-benzyl and distal piperazine nitrogen to binding of lerisetron | The Ki value for lerisetron inhibition of [3H]-granisetron binding to wildtype receptors was determined to be 0.8 +- 0.19 nM (Figure and Table ). This value agrees with previously published data for this compound. The Ki values for analogs 1 and 2 under identical conditions are 25 +- 3.2 nM and 320 +- 82 nM respectively (Figure , Table ). The observed change in Ki represents the decreases in binding energy resulting from removal of the N-benzyl group (analog 1) and the distal piperazine nitrogen (analog 2). Figure 3 | Inhibition of [3H]-granisetron binding to mutant and wildtype receptors by lerisetron. Inhibition of [3H]-granisetron binding to mutant and wildtype receptors by lerisetron. Competition binding assays were performed as described in Materials and Methods. Each point represents a mean +- SE of n>4 experiments. Ki values calculated from this data and the relative magnitude of the shift in Ki compared to wildtype receptors are shown in Table . Y140A produced no significant change in Ki values. W89F, W89Y and R91A mutations produced moderate increases in Ki. Y142A and Y152A mutations resulted in increases in Ki of 160 and 190 fold respectively. Figure 4 | Inhibition of [3H]-granisetron binding to wildtype and mutant receptors by analog 1 and analog 2. Inhibition of [3H]-granisetron binding to wildtype and mutant receptors by analog 1 and analog 2. Competition binding assays were performed as described in Materials and Methods. Each point represents a mean +- SE of n> 4 experiments. IC50 values were determined from the data and a Ki value calculated using the Cheng-Prusoff equation. Ki values for inhibition on each mutant are shown in Table for all three compounds. A. Wildtype B. W89F C. W89Y D. R91A E. Y142A F. Y152A Table 1 | Summary of inhibition data obtained for lerisetron, analog 1 and analog 2 on mutant and wildtype receptors. Ki values were calculated from IC50 values as previously described. All data is the result of n > 4 experiments and is expressed as the mean +- SE. The relative change in Ki was calculated as the Ki (mutant) / Ki (wildtype) and indicates the increase in Ki relative to wildtype +- SE. * indicates significant difference in fold change value. Kd values for [3H]granisetron binding to wt and mutant receptors were reproduced from reference 25. Identification of amino acids interacting with Lerisetron | In order to determine the nature of the amino acids interacting with the distal amino and N-benzyl groups of lerisetron, we constructed 5-HT3ASRs containing mutations at W89, R91, Y140, Y142 and Y152. Figure shows inhibition of [3H]-granisetron binding by lerisetron on wildtype and mutant receptors. For most amino acids, an alanine substitution was constructed in order to effectively remove any amino acid interaction with the ligand. For W89, an alanine substitution has been shown to prevent binding of [3H]granisetron; therefore a less severe mutation was constructed. The W89F mutation produces a 18-fold change in Kd for [3H]-granisetron binding (18 +- 2 nM) and the W89Y mutation produces a 5.8-fold change in Kd (5.7 +- 0.7 nM). Mutation of amino acid R91 to alanine produced a 5-fold change in Kd for [3H]granisetron binding (4.9 +- 0.7 nM) . These data agree well with previously reported values . Alanine mutations at the tyrosine positions Y140, Y142 and Y152 also produced minor increases in Kd for [3H]granisetron binding (2.7 +- 0.19 nM, 4.5 +- 0.5 nM and 7.8 +- 1.1 nM respectively) . Only small changes in Ki for lerisetron were observed for the Y140A mutation while the Y142A and Y152A mutations produced large increases in the Ki (Table and Figure ). For W89F and W89Y, the changes in Ki observed for lerisetron were much smaller than for the alanine mutations at Y142A and Y152A, as would be expected for the less severe nature of these mutations. The changes were, however significant (p < 0.001 in both cases) and are similar to the changes in Kd reported for [3H]granisetron. The increase in Ki on the W89F mutant receptor was 4.8 +- 0.56 fold and the increase in Ki on the W89Y receptor was 3.6 +- 0.4 fold. The R91A mutant produced an increase in Ki of 7.6 +- 1.5 fold as compared to the wildtype receptor. These data indicated potential interactions of lerisetron with W89, R91, Y142 and Y152. Mutation of W89 | As mentioned above, the lack of [3H]-granisetron binding to W89A mutant receptors necessitated the use of W89F and W89Y mutations to analyze functional group interactions. The effects of these mutations on the Ki for analogs 1 and 2 are shown in figure , and Table . Analog 1 inhibited [3H]-granisetron binding to W89F receptors with a Ki of 170 +- 54 nM (7 +- 3.2 fold increase, p < 0.001) and W89Y receptors with a Ki of 81 +- 14 nM (3.2 +- 0.6 fold increase, p < 0.001). This reflects a significant increase in Ki and reflects a potential interaction of analog 1 with W89. The strength of this interaction is apparently similar to the strength of the interaction with [3H]-granisetron and lerisetron since the magnitude of the change is similar in both cases. Analog 2 also showed a significant increase in Ki as a result of the W89F and W89Y mutations. The magnitude of the change for W89F (5.1 +- 1.3 fold, p < 0.05) was similar to that observed for lerisetron and analog 1. The W89Y mutation produced a 6.8 +- 1.6 fold change in Ki (p < 0.05). Thus, all three compounds appear to form similar interactions with W89. Mutation of R91 | Mutation of R91 to alanine (R91A) resulted in a significant, but small increase in Ki for lerisetron of 7.6 +- 1.5 fold (p < 0.01). Figure shows the inhibition of [3H]-granisetron binding by analogs 1 and 2 at R91A mutant receptors. No significant change in Ki was observed on these receptors for either analog 1 (0.9 +- 0.28 fold) or analog 2 (0.56 +- 0.14 fold) as compared to the wildtype receptor. Mutation of Y142 | Mutation of Y142 to alanine produced one of the largest observed changes in Ki for lerisetron (Figure and Table ). The Ki obtained for lerisetron was 130 +- 28 nM, reflecting a change of 160 +- 37 fold compared to wildtype receptors. The Ki value for analog 1, in contrast, increased only 6.8 +- 2.3 fold (p < 0.01) as a result of this mutation (Figure and Table ). The Ki for analog 2 showed a similar change of 17 +- 0.77 fold (p < 0.01). While these Ki values are significantly different from wildtype values for each analog, the lack of larger effects suggests that neither analog 1 nor analog 2 bind as strongly as lerisetron to Y142. Mutation of Y152 | The Y152A mutation showed the most variability in its effects on Ki values for lerisetron, analog 1 and analog 2 (Figure , Figure and Table ). Lerisetron inhibited [3H]-granisetron binding with a Ki value of 150 +- 36; an increase of 190 +- 43 fold compared to wildtype values. The Ki value for analog 1 increased from 25 +- 3.2 nM (wildtype) to 2.5 +- 0.40 muM. This change of 100 +- 16 fold is slightly smaller, but not significantly different from the relative change observed for lerisetron. The Ki for analog 2 increased from 0.32 +- 0.08 muM on wildtype to 13 +- 4.2 muM on Y152A mutant receptors (40 +- 12 fold increase). The increase observed for analog 2 was significantly less than that observed for both lerisetron and analog 1. The smaller change in Ki for analog 2 suggests that analog 2 binds weakly to Y152 while lerisetron and analog 1 bind more tightly. PMID- 12079499_Discussion TI - AB - The purpose of this study was to ascertain the functional group interactions of lerisetron with specific amino acids present in the 5-HT3R binding site. To this end, we examined the effects of specific amino acid mutations on the binding of lerisetron and two analogs. These analogs, shown in Figure , are identical to lerisetron in structure; however, each analog lacks a single functional group found in lerisetron. By comparing the effects of a specific amino acid mutation on the Ki for inhibition of [3H]-granisetron binding, we can begin to identify the functional groups likely to participate in that interaction. Five different amino acids were tested: W89, R91, Y140, Y142 and Y152. Each of these amino acids has been shown in previous studies to alter the binding of 5-HT3R ligands . A summary of the changes in Ki is presented as a bar chart in Figure and . Figure 5 | Relative increases in Ki for lerisetron, analog 1 and analog 2 on mutant and wildtype receptors. Relative increases in Ki for lerisetron, analog 1 and analog 2 on mutant and wildtype receptors. The relative change in Ki was determined as Ki (mutant)/ Ki (wildtype) for each compound. A. W89Y, W89F, R91A B. Y140A, Y142A, Y152A. Lerisetron showed a significant increase in Ki for all mutants except Y140A. Increases in Ki were observed for analog 1 and 2 on W89 and Y152A. While lerisetron binds to wildtype receptors with high affinity, analog 1 binds with lower affinity (31 fold increase in Ki compared to lerisetron). Analog 2, which lacks the terminal nitrogen of lerisetron, binds with even lower affinity (a 400-fold fold increase in Ki compared to lerisetron -- Table ). These data demonstrate the relative importance of the N-benzyl and the terminal nitrogen moieties for lerisetron binding. Cation-pi interactions involving aromatic residues and a quaternary ammonium have been demonstrated to play a major role in binding of nicotinic ligands to nAchRs . Our data support a similar role for Y142 and Y152 in binding of lerisetron to the 5-HT3A receptor. Functional group interactions of W89 | The W89F mutation produced a significant increase in Ki for all three compounds. The magnitude of the change was similar in all cases. In addition, the increase in Ki was identical to the increase in Kd that has been observed for [3H]granisetron binding on this mutant . Alterations in Ki resulting from the W89Y mutation were slightly less, however the change was again the same for lerisetron, analog 1 and analog 2. These data suggest that all three compounds form binding site interactions with W89. The interaction between lerisetron and W89 is unlikely to be via the N-benzyl functional group since the Ki for analog 1 was also altered by this mutation. The same argument can be made for the distal piperazine nitrogen since the Ki for analog 2 also increased. The portion of the molecule common to all three compounds, the aromatic benzimidazole, is thus the most likely point of interaction for W89. Functional group interactions of R91 | The R91A mutation increased the Ki value for lerisetron inhibition of [3H]granisetron binding by 7.6 fold. This is a moderately small change for an alanine mutation, particularly considering that the smallest change in Ki for removal of a functional group on lerisetron (the N-benzyl group) was 31 fold. It is therefore likely that this interaction is either extremely weak or the change in Ki is the result of a structural change in the binding site. Previous studies concluded that R91 was an important interaction for the 5-HT3R agonist 5-hydroxytryptamine (5-HT), since the Ki for 5-HT inhibition increased over 3000 fold as a result of the R91A mutation . A change in Kd for [3H]granisetron binding to R91A was also observed. In order to determine whether the N-benzyl or distal piperazine nitrogen of lerisetron was involved in an interaction with R91, we tested both analog 1 and 2 on R91A mutant receptors. No change in Ki was observed for either compound. This result makes it much more difficult to assign the correct functional group to this amino acid since it suggests that one or both of the compounds is no longer binding the receptor in precisely the same manner as lerisetron. Considering the small change observed for lerisetron binding as a result of this mutation, even a slight reorientation of the molecule in the binding site could result in the loss of this interaction. Functional group interactions of Y142 | The Ki values for inhibition of [3H]granisetron binding by analogs 1 and 2 were altered only slightly by the Y142A mutation. The magnitude of the increase in Ki for lerisetron, however, was considerably larger (160 fold) and is indicative of an important interaction of the compound with Y142. The lack of a large change in Ki for both analogs makes it difficult to interpret this data since one or both of the compounds appears to be interacting differently with the binding site than lerisetron. Analogs 1 and 2 differ from each other both in the functional groups contained in the molecule and their structural similarity to lerisetron. Analog 2 is most similar in overall structure. The substitution of oxygen for the distal amino nitrogen alters the potential interactions formed at this position, but is likely to have a small effect on the overall size and shape of the molecule. Analog 1 is far less similar to lerisetron and more similar to the 5-HT3R antagonist granisetron. Previous studies have shown that the binding of granisetron is not affected by the Y142A mutation . Analog 1 may bind more similar to granisetron than lerisetron and thus would be unaffected by mutations at Y142. This is less likely to be the case with analog 2. The strength of the putative interaction at Y142 can be identified by examining the change in binding of lerisetron as a result of the Y142A mutation. The Y142A mutation produced a 160 fold change in Ki. This change reflects the binding energy lost as a result of the alanine substitution. The observed change in Ki on wt receptors is much larger than that observed for removal of the N-benzyl group (31 fold), but is similar to that observed for substitution of the distal amino nitrogen in analog 2 (400 fold). Taken together with the close structural similarity of analog 2 to lerisetron, it can be concluded that comparison of analog 2 and lerisetron should provide the best means of identifying the interaction at Y142. No change in Ki was observed for analog 2 as a result of the Y142A mutation indicating a lack of any significant interaction of this compound with Y142. These data support our hypothesis that Y142 interacts with the distal piperazine nitrogen of lerisetron. A second amino acid may also be involved since the change in Ki for lerisetron binding as a result of the Y142A mutation was smaller than the change produced by substitution of the piperazine nitrogen. As described below, one candidate for this second amino acid is Y152. Functional group interactions of Y152 | The Y152A mutation produced increases in Ki for all three compounds although the magnitude of the change differed. The increases in the Ki values were 190 fold for lerisetron, 98 fold for analog 1 and only 40 fold for analog 2. Thus, analog 1 retains much of its ability to interact with Y152 despite the absence of the N-benzyl group, while analog 2 interacts more weakly with this amino acid. Since the Ki for analog 1 is increased by the Y152A mutation, it is unlikely that the N-benzyl group interacts with Y152. The small change in Ki for analog 2 supports a partial interaction of Y152 with the distal piperazine nitrogen although some interaction with another group is also apparent. This other group would be expected to be in close proximity to the distal nitrogen. The most likely candidate is the other nitrogen of the piperazine ring. Thus Y152A may form a partial interaction with both piperazine nitrogens. PMID- 12079499_Conclusions TI - AB - Figure shows a hypothetical model of the lerisetron-binding site supported by our observations. The model illustrates the secondary structure of the region of the receptor from Y140 -- Y152 in a loop configuration. This structure is supported by site-directed mutagenesis data as well as structural predictions obtained from other LGIC receptors . The recent determination of the structure of a nicotinic acetylcholine binding protein that shares significant homology with the LGIC family also supports a loop structure in this part of the protein. The region from W89 through Y93 is shown as a beta-sheet as has been hypothesized based on site-directed mutagenesis studies of this strand of the 5-HT3R . Our data indicate the functional groups of lerisetron that may interact with W89, R91, Y142 and Y152. Figure 6 | Model of the 5-HT3R binding site. Model of the 5-HT3R binding site. The model illustrates the major points discussed in the text. Lerisetron is shown spanning the region between the D and E loops of the receptor. Both loops are illustrated as beta-sheets as described. The side chains are presumed to point in or out of the page depending on their position. W89, R91, Y140, Y142 and Y152 all presumably extend into the binding site. The terminal piperazine nitrogen is shown interacting primarily with Y142, but is also capable of forming an interaction with Y152. Y152 may also form an interaction with the second piperazine nitrogen. W89 is illustrated as forming an aromatic interaction with the benzimidazole. While R91 is shown in close contact to the N-benzyl group, the data support only a weak interaction at this point. W89 is shown interacting with the aromatic benzimidazole group of lerisetron although the precise position of W89 relative to this group is not known. The W89 interaction with this group is supported by the observed increase in Ki for lerisetron, analog 1 and analog 2. Since the benzimidazole group is common to all three compounds it is the most likely point of interaction. W89 also represents a common interaction in the binding site for both lerisetron and [3H]granisetron. Y142 is shown interacting with the distal piperazine nitrogen possibly through a cation-pi interaction. This orientation of an amino group interacting with an aromatic amino acid in a cation-pi interaction has been shown for the nicotinic acetylcholine receptor and has been hypothesized for many LGIC receptors [-,]. This conclusion is based on both the magnitude of the change observed on the wild type receptor for removal of the amino group (400 fold) compared to the effect of the Y142A mutation on lerisetron binding (160 fold) and the lack of any major change in Ki for analog 2 as a result of this mutation. Our data does not support an interaction of this amino acid with either the N-benzyl or benzimidazole portions of lerisetron. Y152 is shown positioned between the two piperazine nitrogens. This conclusion is supported by the smaller increase in Ki for analog 2 (40 fold) compared to that observed for lerisetron (190 fold). These results suggest a partial interaction of Y152 with the distal piperazine nitrogen. Since some change was observed, a second interaction is also likely. The functional group in closest proximity to the distal piperazine nitrogen is the other nitrogen on the piperazine ring. Another possibility would be the N-benzyl interaction, however, since the Y152A mutation also produced a large increase in Ki for analog 2, this conclusion is not supported by our data. R91 is shown as interacting with the N-benzyl group. This is a difficult conclusion to make considering the small effect of the R91A mutation on lerisetron binding. The interaction is included in the model based on structural information obtained from the crystal structure of AChBP . The region of this protein homologous to loop E and loop D of the 5-HT3A receptor suggests a loop structure from Y140 to Y152 and a 3-residue turn containing a glycine at position 147 and the beta-strand from W89 through Y93 oriented as shown in Figure . The orientation of lerisetron between W89 and Y142A as shown would enable the N-benzyl group to be positioned in close proximity to R91. If this is the case, then a small alteration in position of analog 1 or 2 in the binding site could result in the loss of this presumably weak interaction. The apparent alterations in the binding site location of analog 1 would be consistent with this hypothesis. An alternate hypothesis would place the N-benzyl group in a different position, interacting with another amino acid; either solely or in concert with R91. Our data support a binding site for lerisetron on the 5-HT3R that spans the D and E loop regions. Table shows the sequence alignment for the 5-HT3R, the alpha7 receptor and the AChBP for these loops. Sequence alignment of mouse 5-HT3 AR, alpha7 nAchR and AChBP result in alignment of the proposed D and E loop of the 5-HT3 AR with corresponding regions of the alpha7 nAchR and AChBP. The amino acids W89, R91 Y140, Y142 and Y152 of the 5HT3 A R can be aligned with W53, Q55, L102, R104, and M114 of the AChBP . These amino acids form a cluster in the proposed acetylcholine binding domain of AChBP similar to that proposed in our model. Both loops have been identified on the complementary face of the binding site of the nAChR. It is unknown if lerisetron utilizes amino acids on the principal face although none have been identified. The model for lerisetron binding will be further refined as its interactions with other binding site amino acids are investigated . Of particular interest would be potential interactions of the N-benzyl group that would account for the decrease in binding affinity of analog 2. Additional information gained from comparison of our model with the recent crystal structure of the AChBP demonstrates that lerisetron can be roughly 'fit' into the binding site such that all the residues line up as shown in our model. While this is not direct evidence that the model is correct, subsequent molecular modeling of the data presented in this paper may provide further support for our hypothesis. Our current model provides an initial working hypothesis that can form the basis of further investigation. Also, while it is unclear whether the information obtained in this study can be extended to other 5-HT3R ligands, a similar approach would be useful in identifying functional group interactions for mCPBG, 5-HT, dtC and granisetron. Table 2 | Sequence alignment of the mouse 5-HT3R with the mouse nicotinic alpha7 receptor and the AChBP. Two sequences are shown. The top sequence is the putative D loop containing W89 and R91 (homologous to W53 and Q55 of the AChBP). The lower sequence shows the putative E loop and contains Y140, Y142 and Y152 (homologous to L102, R104 and M114 of the AChBP). PMID- 12079499_Materials and Methods TI - AB - Mutagenesis | Wild type 5-HT3AS mouse receptor cDNA was derived from N1E-115 neuroblastoma cells as previously described . Mutant receptors were constructed using polymerase chain reaction (Quick change mutagenesis kit, Promega). All mutations were confirmed by DNA sequencing. Cell culture methods and transfections | tsA201 cells (a derivative of the HEK293 cell line) were grown in Dulbecco's modified Eagles medium (D-MEM) containing 10% FBS and 100-units/ml penicillin/streptomycin. Cultures were maintained in humidified atmosphere of 5% CO2 at 37C. For binding studies, tsA201 cells were plated at a density of 5 x 106 cells/75 cm2 and grown for 9 hours prior to transfection. Cells were transfected with 10 mug murine 5-HT3ASR cDNA using calcium phosphate co-precipitation (New Life Technologies, NY), then incubated 36 hours prior to harvesting. For whole cell patch clamp experiments, tsA201 cells were plated at a density of 0.25 x 106 cells/27 cm2 dish and grown 12 hours prior to transfection. Cells were washed with fresh culture medium then transfected with 10 mug 5-HT3ASR cDNA using Qiagen Superfect transfection reagent (Qiagen, CA). Transfected cells were incubated with this mixture for 2.5 hours, then divided into 35 mm culture dishes at a density of approximately 5 x 104 cells/dish and incubated for 24 hours at 37C before recording. Radioligand Binding Assay | Transfected cells were scraped from the dishes, washed twice with Dulbecco's PBS (New Life Technologies, NY), then resuspended in 1.0 ml PBS/100 mm dish. Cells were either used fresh or frozen at this step until needed. Immediately prior to use, cells were homogenized in PBS using a glass tissue homogenizer then centrifuged at 35 000 x g for 30 minutes in a Beckman JA20 rotor. Membranes were washed once more with PBS then resuspended in 1 ml PBS/100 mm dish. Protein content was determined using a Lowry assay (Sigma. Diagnostics, St. Louis, MO). Binding assays were performed in PBS. For Kd determinations, 100 mul of homogenate was incubated at 37C for 1 hour with varying concentrations of [3H] granisetron (NEN, MA). Specific binding of [3H] granisetron was determined as the bound [3H] granisetron not displaced by a saturating concentration of a competing ligand (100 muM mCPBG or 10 muM MDL-72222). Kd values were determined by fitting the binding data to the following equation using GraphPad PRISM (San Diego CA): B = Bmax [L] n / ([L] n + Kn), where theta is bound ligand, Bmax is the maximum binding at equilibrium L is the free ligand concentration and n is the Hill coefficient. For Ki determinations, 100 mul of homogenate was incubated at 37C for 2 hours with varying concentrations of inhibitor and [3H] granisetron (NEN, MA). Binding was terminated by rapid filtration onto a GF/B filters. The IC50 values were calculated by fitting the data to the following equation using GraphPad PRISM (San Diego CA): theta = 1/ (1+(L/IC50)), where theta is the fractional amount of [3H] granisetron bound in the presence of inhibitor at concentration L as compared to the amount of [3H] granisetron bound in the absence of inhibitor. IC50 is the concentration at which theta = 0.5. The Ki is calculated from the IC50 value using the Cheng-Prusoff equation. Electrophysiological Methods | Transfected tsA201 cells were transferred to a recording chamber and submerged in extracellular recording buffer containing 25 mM HEPES pH 7.4, 140 mM NaCl, 1.7 mM MgCl2, 5 mM KCl, 1.8 mM CaCl2. Patch electrodes (2 --2.5 MOmega) were filled with intracellular recording buffer containing 25 mM HEPES pH 7.4, 145 mM KCL, 2 mM MgCl2 and 1 mM EGTA. Cells were clamped in whole cell configuration at a holding potential of -60 mV. Currents elicited by agonist application were measured using an Axopatch 200B amplifier (Foster City, CA) under computer control (DataPac 2000, RUN Technologies). Agonists and antagonists were dissolved in extracellular solution and delivered to cells using a rapid perfusion system (Warner Instruments, Hamden, CT). For EC50 determinations, responses were normalized to the maximum response obtained from the full agonist 5-HT and fitted to the equation Psi= 1/1+(EC50/ [C] n), where Psi is the normalized current at 5-HT concentration [C], EC50 is the concentration of 5-HT needed to obtain half maximal activation and n is the apparent Hill coefficient. For inhibition experiments, cells were exposed to inhibitor alone for 30 s prior to co-exposure with 5-HT. Inhibited responses were calculated as a fraction of the response to 5-HT alone. Data were plotted as the fractional response versus the concentration of inhibitor and analysed using GraphPad software. The IC50 value was calculated as the concentration of antagonist inhibiting the 5-HT evoked response by 50%. A Ki value was calculated from the IC50 using the Cheng-Prusoff equation. Synthesis of Lerisetron and its analogs | All target molecules were prepared according to a general 2-step synthesis reported previously by Orjales et al. with only slight modification . Commercially available 2-chlorobenzimidazole, in dry DMF was treated with a slight excess of NaH, (1.1eq). After stirring for 1 hour at room temperature, one equivalent of the appropriate alkyl bromide was added slowly and the reaction mixture heated under reflux for > 5 hours (the reaction was monitored by TLC). Reaction product was partitioned between water and methylene chloride; organic layer was dried (Na2SO4) and concentrated in vacuum. The solid residue was purified by Flash chromatography, which afforded the corresponding N-substituted 2-Chlorobenzimidazole intermediates in good yield. The final step involved a nucleophilic substitution of the 2-chloro group by piperazine at high temperatures. The reaction was performed neat using 4 --10 fold excess piperazine and typically heated for a short period only, (30 --45 min). Similar work-up afforded a residue that was purified by either crystallization or chromatography. The yields ranged from 40 --95%. All compounds were characterized by NMR, MS, HRMS, and /or elemental analysis or were identical to literature reports. Materials | D-MEM, Penicillin-Streptomycin, fetal bovine serum, and Trypsin were obtained from New Life Technologies. 5-HT and MDL-72222 were obtained from RBI. [3H]-granisetron (84 Ci/mmol) was purchased from New England Nuclear. PMID- 12079498 TI - Low solubility of unconjugated bilirubin in dimethylsulfoxide -- water systems: implications for pKa determinations AB - Abstract | Background | Aqueous pKa values of unconjugated bilirubin are important determinants of its solubility and transport. Published pKa data on an analog, mesobilirubin-XIIIalpha, studied by 13C-NMR in buffered solutions containing 27 and 64 vol% (C2H3)2SO because of low aqueous solubility of mesobilirubin, were extrapolated to obtain pKa values in water of 4.2 and 4.9. Previous chloroform-water partition data on bilirubin diacid led to higher estimates of its pKa, 8.12 and 8.44, and its aqueous solubility. A thermodynamic analysis, using this solubility and a published solubility in DMSO, suggested that the systems used to measure 13C-NMR shifts were highly supersaturated. This expectation was assessed by measuring the residual concentrations of bilirubin in the supernatants of comparable DMSO-buffer systems, after mild centrifugation to remove microprecipitates. Results | Extensive sedimentation was observed from numerous systems, many of which appeared optically clear. The very low supernatant concentrations at the lowest pH values (4.1-5.9) were compatible with the above thermodynamic analysis. Extensive sedimentation and low supernatant concentrations occurred also at pH as high as 7.2. Conclusions | The present study strongly supports the validity of the aqueous solubility of bilirubin diacid derived from partition data, and, therefore, the corresponding high pKa values. Many of the mesobilirubin systems in the 13C-NMR studies were probably supersaturated, contained microsuspensions, and were not true solutions. This, and previously documented errors in pH determinations that caused serious errors in pKa values of the many soluble reference acids and mesobilirubin, raise doubts regarding the low pKa estimates for mesobilirubin from the 13C-NMR studies. PMID- 12079498_Background TI - AB - The saturation status of unconjugated bilirubin (UCB) is relevant to understanding the pathophysiology of jaundice and to interpreting experiments with UCB . UCB, in its diacid form (H2B), has a low solubility (So) of 51 nM in water . The total solubility (S) at any pH is determined by So, pKa values, and pH : S = [H2B] + [HB-] + [B=] = So(1 + Ka1/ [H+] + Ka1Ka2/ [H+]2) (Eq. 1) Self-association of UCB dianion, B=, can also increase S . Beginning in 1995, a series of papers reported the use of 13C-NMR to study the ionization of a close analogue of bilirubin-IXalpha, mesobilirubin-XIIIalpha (MBR), along with several soluble reference acids, in buffered mixtures of (C2H3)2SO and water . Due to the very low aqueous solubility of MBR, data were obtained only at two high concentrations of (C2H3)2SO (64 and 27 vol%). Its pKa values in "water" (actually in 1 vol% of ((C2H3)2SO), obtained by an extrapolation procedure based on the behavior of soluble reference acids, were 4.2 and 4.9, far lower than values of 8.12 and 8.44 obtained by solvent partition between chloroform and buffered aqueous solutions . These serious discrepancies led us to reexamine several experimental aspects of the 13C-NMR studies and the implications of their purported low pKa values for the solubility of UCB. Inaccuracies in the measurements of buffer pH and of the pKa values of the reference 13C-carboxylic acids, related to failure to correct for the strong effects of DMSO on the pKa values of weak acids, have been previously noted and acknowledged . In addition, as discussed later, a thermodynamic theory about solubility in mixed solvents, using the So values of 51 nM in water and 10 mM in pure DMSO , suggested that So of UCB would be only about 0.15 muM in 27 vol% DMSO and 2.2 muM in 64 vol% DMSO, which are well below concentrations used in the MBR studies. This implies serious supersaturation effects. In fact, turbidity was reported for some of the systems used in the 13C-NMR papers , indicating the formation of coarse suspensions. By definition, as stressed in the Conclusions section, pKa determinations are valid only for monomeric species and require that solutions are below saturation at all pH values studied. Even reversible aggregation of monomers in undersaturated solutions is known to affect pKa estimates . If supersaturation leads to the formation of fine sols or coarse suspensions, the data are unacceptable for determination of the pKa values of monomers. In the present paper, we assess whether some of the systems used in the 13C-NMR studies were supersaturated with MBR. Our experimental work on sedimentation was done with unconjugated bilirubin (UCB) and we used DMSO instead of its deuterated analogue, (C2H3)2SO. It is known that, when alkaline aqueous UCB is acidified to neutral or low pH's, "Usually a colloid suspension of bilirubin is formed and the solution remains clear, as observed by the naked eye, thus inviting an erroneous interpretation" . Such systems, which simulate true solutions, often show sedimentation on centrifugation . We here report that sedimentation of UCB from apparently clear solutions can likewise be extensive when UCB in DMSO is diluted with aqueous buffers to final mole fractions (N) of DMSO = 0.025, 0.086 and 0.31, corresponding to 9, 27, and 64 vol% of DMSO. A thermodynamic theory is used to examine the effect of added DMSO on So. Our findings, consistent with our partition-derived So, S and pKa values of UCB in aqueous buffers , indicate that the recently reported low pKa values of MBR in comparable (C2H3)2SO -water systems , were determined above saturation. PMID- 12079498_Results TI - AB - Residual UCB in the supernatants after centrifugation | Many systems initially appeared optically clear, but well over 90% of initial UCB sedimented on centrifugation of most samples below pH 7.2. Overall recoveries (UCB in supernatant + UCB in redissolved precipitate) were between 90 and 100%. Only residual [UCB] in supernatants are reported. Figs. plot the measured residual [UCB] in the supernatant vs. the initial [UCB] at NDMSO = 0.31, 0.086 and 0.025. Deviations of their ratio below unity (dotted lines) represent a decrease in [UCB], mainly from precipitation, but possibly also from limited degradation. At N = 0.31 , [UCB] after sedimentation varied from 2.8 muM at pH 5.86 to 166 muM at pH 8.38. At N = 0.086 , [UCB] ranged from 0.3 to 2.4 muM at pH 4.50 and from 1.4 to 6.4 muM at pH 7.05 and most of the UCB sedimented. By contrast, at pH 7.56 and 7.70 (phosphate), only minor precipitation was observed. At N = 0.025 ; [UCB] ranged from 0.1 to 2.7 muM at all three pH values (4.15 to 7.18). Figure 1 | A-C. Residual vs. initial UCB concentrations in buffered DMSO/water systems. A-C. Residual vs. initial UCB concentrations in buffered DMSO/water systems. Measured residual [UCB] in supernatants of buffered DMSO/water systems, after centrifugation for 5 min at 14,000 g, are plotted against the pipetted, initial UCB concentrations before centrifugation. Mole fractions (N) of DMSO were 0.310 (A), 0.086 (B) and 0.025 (C). Panels B and C are log-log plots. The dashed unity line indicates complete retention of UCB in supernatant. The buffers used and the corrected pH values of the DMSO-buffer systems were: Panel A -- (circle) Phosphate pH 8.4, (square) Tris pH 7.1, (diamond) Acetate pH 6.9, (triangle) Acetate pH 5.9; Panel B -- (circle) Phosphate pH 7.6 --7.7, (square) Tris pH 7.1, (triangle) Acetate pH 4.5; Panel C -- (circle) Phosphate pH 7.2, (square) Tris pH 7.0, (triangle) Acetate pH 4.15. [UCB] in each supernatant was determined from A at 458 nm, using the extinction coefficient of 0.0634 muM-1cm-1. Sedimentation of bilirubin-albumin complexes | The supernatant from the original supersaturated UCB-HSA system, when diluted with 1/8th vol. of buffer and centrifuged again, showed more sedimentation and a progressive rise in [UCB] as one moved down the column of fluid. By contrast, after dilution with 1/8th volume of DMSO to decrease UCB saturation, the supernatants produced no further sedimentation and there were no significant differences in [UCB] or protein concentrations along the axis of the fluid column. Thus, the UCB-HSA complex, which constitutes over 99.9% of the UCB in this system , did not sediment. PMID- 12079498_Discussion TI - AB - Findings and their relation to thermodynamic theory | Supersaturated aqueous systems of UCB, that are optically clear before centrifugation, may exhibit considerable variation in the extent of sedimentation [-,]. Although sedimentation is often extensive, it is generally incomplete and may not be observed at all. Our data in DMSO-water show the same features, which are expected from the complex kinetics of nucleation and growth of insoluble aggregates of UCB diacid (H2B), leading to the formation of a new solid phase . Our centrifugation, 5 min at 14,000 g, was quite mild, and the short 20-minute period between preparation of UCB-DMSO-water systems and centrifugation severely limited the time-dependent growth to large aggregates. Lack of sedimentation of the UCB-HSA complex (mol. wt. 68,000) indicates that fine colloids composed of 100 UCB molecules would be too small to sediment. Thus, supersaturated systems lacking coarse, insoluble aggregates may not show sedimentation, but any sedimentation observed indicates their presence. To evaluate the important effect of pH on sedimentation efficiency, we calculated S in water using chloroform-water partition data on UCB and the best measure of So in chloroform, 0.88 mM . S at any pH, e.g. 62 nM at pH 7.4 and 0.32 muM at pH 8.5, can be calculated from the fitted partition data, or, equivalently, from Eq. 1, using the partition-derived So in water of 51 nM and pKa values of 8.12 and 8.44 . In aqueous systems , the lowest [UCB] in water, below which no sedimentation was observed at 100,000 x g for a few hours, was 100 nM at pH 7.4, modestly higher than our partition-derived S of 62 nM . Even under such vigorous centrifugation, the lowest [UCB] increased rapidly with increasing pH, to 17 muM (150 times S) at pH 8.05 and 34 muM (230 times S) at pH 8.2 . This indicates increasing charge-stabilization of fine, non-sedimenting colloids of H2B by adsorbed UCB anions . In contrast, below pH 6.7, sedimentation of 10 muM UCB was nearly complete . This is compatible with a dearth of stabilizing UCB anions at this pH, as expected from the high pKa values of 8.12 and 8.44 . Our present data on residual [UCB] in DMSO-water systems likewise show decreased sedimentation with increasing pH . At each NDMSO, the lowest [UCB] were at the lowest pH values: 0.1 muM (N = 0.025, pH 4.15); 0.3 muM (N = 0.086, pH 4.5); and 2.8 μM (N = 0.31, pH 5.9). As in water, these are likely to be closest to the So values at each N. Indeed, they are only moderately higher than the corresponding So values of 0.07 muM, 0.15 muM and 2.2 muM, respectively, calculated from Equation 2 using So values of 51 nM in water and 10 mM in DMSO . log So,mixed = log So,water + (log So,DMSO - log So,water) x N (Eq. 2) Equation 2 is a thermodynamic relationship based on assumptions of complete ideality of mixing . In general, a roughly linear variation of log So with N at low N is expected. For example, data from 1-naphthoic acid in DMSO-water show that log So is a linear function of N up to N = 0.35. Such a relationship leads to a relatively small effect of low N values on So. Thus, according to Equation 2, So increases by a factor of only 1.4 at N = 0.025 and 2.9 at N = 0.086, but by a relatively larger factor of 44 at N = 0.31. This would markedly reduce the supersaturation factor ([UCB]/ So), which is a measure of the tendency of UCB to come out of solution at N = 0.31. This explains in part the relatively high [UCB] at high pH at N = 0.31 . The pH effects on [UCB] at each NDMSO are of interest also. The lowest [UCB] at each pH registered relatively small increases with significant increases in pH: for example from 0.1 muM (pH 4.14) to 0.2 muM (pH 7.0) at N = 0.025; from 0.3 muM (pH 4.5) to 1.4 muM (pH 7.1) at N = 0.086; and from 111 muM (pH 7.1) to 166 muM (pH 8.4) at N = 0.31. These increases are probably caused mainly by increasing charge-stabilization of colloidal aggregates, as in aqueous media . If, instead, the relatively small increases are ascribed entirely to increases in true solubility (S) at the high pH (Eq. 1), the required pKa values are about 7 at N = 0.025 and 0.086, and 8.5 at N = 0.31. The true pKas of UCB in DMSO-water are thus probably significantly higher. We note that some variability in sedimentation results from our short-term experiments, most evident at the low residual [UCB] in Figs. and , in part magnified by the log-log scale used. Some variability is expected, however, because of the complexity of the kinetic processes of nucleation, growth and flocculation that precede sedimentation. In Fig. , the difference between acetate and Tris buffers is quite small (note the linear scale), compatible with the 58% higher [H+] in the acetate buffer. In Fig. , the markedly lower sedimentation from phosphate buffers at pH 7.6 --7.7, as compared to Tris buffer at pH 7.1, can be ascribed mainly to the much higher pH values and ionic strength of the phosphate systems. Another significant factor may be the difference in charge between the buffer salts; phosphate is anionic whereas Tris is cationic and zwitterionic. The cationic species of Tris can, in principle, reduce the negative charges on the surface of the colloidal H2B sufficiently to facilitate the formation of coarser particles and, thus, increase sedimentation. Implications for pKa values of mesobilirubin-XIIIalpha (MBR) | In the recent 13C-NMR studies of the ionization of the 13C-COOH groups of MBR , it was assumed that the relevant physical properties of UCB and MBR, and of (CH3)2SO (DMSO) and (C2H3)2SO, are similar. Actually, as expected from the replacement of two vinyl groups in UCB with two ethyl groups in MBR, MBR is slightly more soluble in organic solvents and has a higher Rf on silica gel t.l.c. ; MBR is thus more hydrophobic and should be less soluble in water than is UCB. Our low [UCB] in DMSO-water systems at comparable N, therefore, indicate that many of the (C2H3)2SO/buffer systems used in the 13C-NMR studies were likely supersaturated with MBR. In those studies, the MBR concentrations used were stated to be 1 to 100 muM at N = 0.086 , compared to our lowest [UCB] of 0.3 muM at pH 4.5 and 1.4 muM at pH 7.05. At this N, 9 of 11 MBR data points were obtained at pH below 7.05 and 5 below pH 4.5 , so that even 1 muM MBR was likely to be supersaturated. At N = 0.31, our lowest [UCB], 2.8 muM at pH 5.9, was close to the lower limit of the 2 to 800 muM range of [MBR] used . Thus, many data points, obtained at pH values down to 2 , were probably from supersaturated systems, despite being optically clear. As noted here and elsewhere [-,], optical clarity gives no assurance of the absence of supersaturation. Actually, turbidity was reported in some of the 13C-NMR samples , indicating that coarse, insoluble aggregates of MBR were present. The claim that such turbidity did not affect 13C-NMR measurements contrasts with evidence that even small multimers can change NMR chemical shifts . It should be noted also that, at high concentrations of B=, extensive, reversible self-association of B= can lead to apparently stable supersaturation with no separation of an insoluble phase . For example, at pH 8.5 and a UCB concentration of 20 muM (63 times S), the weight-average aggregation number of UCB has been found to be 7.17 , corresponding to a molecular weight of 4,195. The aggregation number remained fairly high, 4.2, in 60% (w/v) ethanol . The successful application of equilibrium ultracentrifugation for that study suggests a complete absence of even small colloidal species of UCB. Self-association of MBR dianions in (C2H3)2SO-water mixtures cannot be ruled out on a priori grounds. It has been shown that neglect of self-association of B= leads to an artefactually low estimate of pKa values for UCB . In addition to the problems of insolubility, supersaturation and self-aggregation of the MBR systems in (C2H3)2SO-water , we had shown previously that inaccuracies in the pH measurements affected both the magnitude of DeltapKa (the change in pKa on adding (C2H3)2SO to water), as well as the degree of the variation of DeltapKa with N . This is important for extrapolating pKa values in (C2H3)2SO-water to pure water (N = 0). Indeed, remeasurement of one soluble acid raised its pKa by as much as 3 units at N = 0.31 . Thus, the inaccuracies in pH measurement produced serious errors in reported pKa values of more than fifteen soluble acids used as models for MBR, as well as for MBR itself . Many methods, using appropriate pH measurements, have been applied in the past to determine thermodynamic pKa values of soluble acids in non-aqueous or partially aqueous media, including DMSO-water systems . Many other relevant references were given in our prior paper . In that paper, our pKa measurements on acetic acid in DMSO-water systems were based on the potentiometric method, using properly calibrated glass electrodes, which determine the activity of H+, and on estimates of the activity coefficients of the acetate ion. This method, which is well established for aqueous solutions, yielded results in good agreement with data from the literature that was based on a very different method, measurements of electrical conductivity . In the 13C-NMR papers, therefore, it was not justified, to assume that pH values do not change on adding DMSO , or to use uncalibrated pH measurements for determination of the pKa values of soluble acids . Our sedimentation data and their interpretation indicate that significant additional uncertainties, not important for the soluble acids investigated, exist for the reported pKa values of the relatively insoluble MBR in (CD3)2SO-water (4.2 and 4.9 at N = 0.086 and 4.3 and 5.0 at N = 0.31), as well as their extrapolation to obtain pKa values of 4.2 and 4.9 in water . Indeed, if these low aqueous pKa values, along with the experimental S values at pH 8.5 of 0.32 muM , or 0.6 muM , are applied to Eq. 1, the calculated extremely low So values of UCB diacid of 4 or 8 x 10-15 M are seven orders of magnitude lower than the experimental So, 5.1 x 10 --8 M . Applying the So of 4 or 8 x 10-15 M to Eq. 2, moreover, would indicate massive supersaturation (up to 8 to 10 orders of magnitude) of MBR at the concentrations (1 --800 muM) used in the 13C-NMR studies . PMID- 12079498_Conclusions TI - AB - The present sedimentation data for UCB in DMSO-water demonstrate that the true solubilities of UCB, even at fairly high pH values, are low at DMSO mole fractions up to 0.31. The results and related considerations are compatible with similar results in purely aqueous solutions , and support both the estimated solubility (So) of 5.1 x 10 --8 M for uncharged UCB (H2B) in water, and the corresponding high aqueous pKa values of 8.12 and 8.44, derived from our partition studies . These were performed in undersaturated systems and took into account the self-association of B=. Our experimental data indicate problems of insolubility, supersaturation and self-aggregation of UCB in DMSO-water mixtures with compositions similar to the MBR systems in (C2H3)2SO -water . In (C2H3)2SO-water, DMSO-water or any other medium , properly determined pKa values for the dissociation equilibria of a diacid H2A (H2A <--> HA- + H+ and HA- <--> A= + H+) must pertain to monomeric H2A, HA- and A=, the solute species involved in the stated equilibria, and require unambiguous determination of [H+] or pH. Unless pKa values are determined for monomeric systems, relative concentrations of H2A, HA- and A= cannot be determined from the pKa values and the pH. The 13C-NMR data, suggesting low pKa values for MBR in (C2H3)2SO -water and water [-,], did not meet these essential requirements of proper pH measurements nor provide assurance that the MBR in every system was below saturation and not self-associated . The issues raised are not trivial, since the pKa and So values of UCB are clinically relevant to the effects of pH on the precipitation of calcium bilirubinates in pigment gallstones and the neurotoxicity caused by UCB diacid in severely jaundiced neonates . PMID- 12079498_Materials and Methods TI - AB - Materials | UCB (Calbiochem) was purified by alkaline extraction of a chloroform solution, recrystallized twice from chloroform-methanol , dried under Argon, stored invacuo in the dark and used within 6 weeks. DMSO was spectroscopic grade, 99.8% pure (UVASol, Merck). Human serum albumin (HSA, lot 903635) was from Calbiochem-Boehringer. All other chemicals were reagent grade (Merck). Water used was deionized and distilled. All flasks and tubes were Kimax glass, washed with 0.1 N HCl and rinsed 4X with water and then dried before use. Stock buffers, 1.0 M, were: Tris-HCl, pH 7.01; Na- phosphate, pH 6.85, or 6.99; and Na-acetate, pH 4.01. Stock UCB in DMSO (4 to 6 mM) and stock HSA, 613 muM in 0.1 M Tris-HCl buffer, pH 7.01, were prepared freshly for each experiment. Preparation of UCB-DMSO-buffer systems | Test systems (4.0 mL) of UCB were prepared in duplicate: to 0.4 mL of the stock buffer were added successively the appropriate volumes of water, DMSO and, finally, up to 150 muL of stock UCB/DMSO solution. To minimize UCB oxidation, all tubes and solutions were deoxygenated with Argon and kept in the dark . To determine the [UCB] in the UCB/DMSO stock, 6.0 muL, was added to 3.0 mL of the HSA stock. The absorbance, A, at 460 nm was read against a blank containing 2.0 mL of HSA stock plus 4.0 muL DMSO, and the [UCB] calculated using the extinction coefficient, epsilon, 47,000 M-1.cm-1. The pH measurement of each final DMSO/aqueous buffer system included an electrode calibration using the strong acid, HClO4. The 0.1 M phosphate buffer in N = 0.31 DMSO was centrifuged because of partial insolubility and only the supernatant was used. Centrifugation and analysis of residual UCB in supernatants | Fifteen min after mixing, samples were assessed visually for turbidity or precipitation. After Vortex-mixing, duplicate 1.8 mL aliquots were transferred to polypropylene tubes and centrifuged for 5 min at 25C and 14,000 g (Mikroliter centrifuge, Hettich, Tuttlingen, Germany). The supernatants were assayed spectrophotometrically within 20 min. The precipitates were washed once with 1.8 mL of water, again centrifuged, the water aspirated, and the packed precipitate dissolved in DMSO for spectrophotometry. Absorbance (A) was measured in triplicate at 458 nm on each sample, diluted, when necessary, with DMSO or DMSO/buffer mixture to A of 0.2 to 0.8; a comparable medium without UCB was used as a blank. In all systems, including the redissolved UCB sediments, we applied the extinction coefficient of 0.0634 muM-1cm-1 at the peak wavelength of 458 nm for UCB in pure DMSO . Preliminary calibration studies. of the effects of DMSO concentration and buffer composition on A had confirmed this value for pure UCB in pure DMSO and in DMSO/buffer systems containing 64 vol% DMSO. In the systems containing 27 and 9 vol% DMSO, the spectrum developed a plateau between 458 and 450 nm, but A at 458 nm remained within +- 10% of the value expected from applying the extinction coefficient of 0.0634 muM-1cm-1 to the measured quantity of UCB dissolved in each system. The variability is in part due to degradation, discussed above, and in part due to the low absorbances at [UCB] below the saturation limit in some buffer/DMSO systems. For these reasons, no corrections were made for these minor differences in A. Sedimentation of bilirubin-albumin complexes | To determine if UCB bound to HSA would sediment, we prepared a system containing HSA, 300 muM, and UCB 170 muM, in 0.1 M Tris-HCl buffer, pH 7.01. Microcentrifugation for 5 min yielded a small amount of precipitated UCB. Three aliquots of the clear supernatant were diluted with 1/8th volume of DMSO and a fourth aliquot diluted with 1/8th volume of buffer. The diluted samples (in duplicate) were then microcentrifuged for another 10 min and 25 muL samples taken from the top, middle and bottom of the fluid column in each tube, using a Hamilton syringe. Protein concentrations were determined with the Bio-Rad bicinchonic acid method, which is unaffected by bilirubin. After dilution with 1.8 mL DMSO, triplicate A readings were taken at 458 nm. PMID- 12079498_Abbreviations TI - AB - UCB, unconjugated bilirubin; H2B, UCB diacid; B=, UCB dianion; DMSO, dimethylsulfoxide; MBR, mesobilirubin XIIIalpha; N = mole fraction of DMSO in DMSO-aqueous buffer systems; NMR, nuclear magnetic resonance; S, solubility of UCB or MBR at a given pH; So, solubility of UCB diacid. PMID- 12079498_Authors' note TI - AB - An abstract of this work has been published (Gastroenterology 2000; 118:A1477) PMID- 12079498_Authors' contributions TI - AB - All three authors collaborated in the conception, design and writing of this study. The work was performed by JDO while he was a visiting professor at the Academic Medical Center in Amsterdam, the Netherlands. All authors read and approved the final manuscript. PMID- 12079500 TI - Identification of critical residues in loop E in the 5-HT3ASR binding site AB - Abstract | Background | The serotonin type 3 receptor (5-HT3R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family. Results | Thirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic alpha7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147) to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR. Conclusion | Our data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, mCPBG, d-tc and lerisetron. 5-HT and mCPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure. PMID- 12079500_Background TI - AB - Sequence homology between the serotonin type 3 receptor (5-HT3R), the nicotinic acetylcholine receptor (nAChR), the GABAA receptor and the glycine receptor suggests a large amount of structural similarity within this superfamily of ligand gated ion channels. This presumed structural homology can be used to guide site directed mutagenesis studies of particular receptor subtypes. On a gross level, all members of this superfamily assemble as pentameric receptors . In some cases, receptors can be assembled from a single subunit (5-HT3AR and nicotinic alpha7 receptors) [,-]. In other cases, at least two different subunits are required . The ligand binding site is thought to be formed at the junction between two subunits . Based on data obtained from affinity labeling and site directed mutagenesis studies, several regions of the nicotinic acetylcholine receptor have been implicated in ligand binding . Six essential loops appear to contribute to the binding site with the position of each loop differing slightly depending on whether the receptor is heteromeric or homomeric. For homomeric receptors such as the alpha7 nAChR, the loops are identified as A, B, C, D, E and F . The amino acids that interact with ligands presumably extend into the binding site from these loops. Subunits are composed of 2 faces (+ and -) with the + face of one subunit forming one side of the binding site and the -- face of another subunit forming the complimentary side. Some binding loops (A, B and C) are present on the -- face while the remaining loops (D and E) are present on the + face. . The individual amino acids that form binding interactions with the functional groups present on a ligand are likely to differ for each receptor subtype, reflecting the specificity of a particular binding site, however, the overall structure of the binding domain may be similar even for binding sites with different ligand specificities. Identification of ligand specificity requires identification of both the location or structure of individual binding site loops and the amino acids present in a particular receptor subtype. The purpose of this study is to extend the information available from other members of this receptor family to the 5-HT3R. Sequence homology and a presumed structural similarity to other ligand gated ion channels suggests that the E loop region of the 5-HT3AR forms part of the ligand binding domain for 5-HT3R ligands. This region extends from Y140 to K153 and is shown in Table . The homologous sequences of other representative members of this family are also shown. In the center of this region is a critical glycine residue that is thought to play a role in establishing a hairpin loop . Recent x-ray crystallographic data obtained from an ACh binding protein (AChBP) shows a loop structure in this region resulting from a 3 residue turn containing a glycine homologous to G147 of the 5-HT3R. On either side of this putative turn region are residues that have been identified as important to receptor binding . The formation of this loop structure brings amino acids on either side of glycine into close proximity and may form a binding pocket that will accommodate one or more functional groups. In order to identify the interaction of amino acids in this binding loop with 5-HT3R ligands, we have constructed alanine mutations of residues throughout this region and evaluated the alteration in binding affinity of 5 different classes of 5-HT3R ligands . Our data identifies 3 tyrosine residues that appear to interact selectively with each structural class and supports the existence of a loop structure in this region of the receptor. Figure 1 | 5-HT3R ligands. 5-HT3R ligands. Table 1 | Sequence Comparison of Putative Binding Loop Region. PMID- 12079500_Results TI - AB - All mutant receptors were tested for their ability to bind the 5-HT3R antagonist [3H]granisetron. Table shows the Kd values for wildtype mouse 5-HT3ASRs and the 13 alanine mutations we evaluated. [3H]granisetron is a potent antagonist of the wt 5-HT3R (Kd = 0.98 +- 0.12 nM). This value agrees with published data for this compound . Bmax values range from the 5.5 pmoles/mg protein observed for E148A to 0.30 pmoles/mg protein for the K153A mutation, indicating some variability in expression of the different receptors. In general, however, receptor expression was similar to that reported by other laboratories [,-]. No detectable binding was observed for G147A and V149A mutant receptors. For all other mutants, decreases in binding affinity (increased Kd) were observed although the magnitude of the change was less than 10 fold in all cases. A bar graph showing the change in Kd value resulting from each alanine mutation is shown in Figure . The largest decreases in binding affinity were observed for Y142A (4.6 fold, Figure ), E148A (5.3 fold) and Q150A -- K153A (6 -- 8 fold). Figure 2 | Changes in Ki or Kd (for binding of [3H]granisetron) as a result of alanine mutations. Changes in Ki or Kd (for binding of [3H]granisetron) as a result of alanine mutations. The values shown are the ratio of the Kd or Ki for wildtype and mutant receptors. The length of each bar illustrates the magnitude of change in either the Kd or Ki relative to wildtype while the direction indicates an increase (up) or decrease (down) in the Kd or Ki. A. Most alanine mutations produced little change in Kd. No binding was detected for the G147A and V149A receptors therefore no Kd could be determined. B. Changes in Ki were small for most receptors although large changes were observed for select ligands at Y140A, Y142A and Y152A receptors. The relative increases in Ki for these amino acids are as follows: 5-HT: Y140A 1.3 +- 0.30 fold, Y142A 110 +- 25 fold, Y152A 24 +- 5.6 fold mCPBG: Y140A 7.3 +- 1.7 fold, Y142A 160 +- 36 fold, Y152A 24 +- 5.6 fold d-tc: Y140A 50 +- 12 fold, Y142A 6.5 +- 1.5, Y152A 10.0 +- 2.3 fold lerisetron: Y140A 4.6 +- 1.1 fold, Y142A 160 +- 37 fold, Y152A 190 +- 43 fold Figure 3 | Saturation binding isotherms for binding of [3H]granisetron to wildtype, Y140A, Y142A and Y152A receptors. Saturation binding isotherms for binding of [3H]granisetron to wildtype, Y140A, Y142A and Y152A receptors. The data shown represent the specific binding of [3H]granisetron as determined from at least four identical experiments. Specific binding was determined as the fraction of total binding not inhibited by a saturating concentration of mCPBG or MDL-72222. Data were combined and fitted using non-linear curve fitting. Only small shifts in binding affinity to [3H]granisetron were observed on all three mutants. Kd values, Bmax and Hill coefficients are shown in Table . Table 2 | [3H]Granisetron binding to wildtype and mutant 5-HT3ASRs. Inhibition binding assays were also conducted. Four test compounds with structures representative of the major classes of 5-HT3R ligands were chosen: serotonin (5-HT, the endogenous agonist), m-chlorophenylbiguanide (mCPBG, agonist), d-tubocurarine (d-tc, antagonist) and lerisetron (antagonist). The Ki values for inhibition of [3H]granisetron binding by all four compounds are shown in Table . Little change in Ki value was observed for the majority of mutations. The values highlighted in bold in Table represent the Ki values for inhibition of [3H]granisetron binding on mutant receptors that increased over 10 fold compared to the Ki obtained for wildtype receptors. The bar chart in Figure illustrates the changes in Ki resulting from each mutation on the test compounds. Ratios of Ki are shown as positive for increases in Ki on mutant receptors versus wildtype and negative for decreases. A positive change thus corresponds to a decrease in binding affinity for the compound as a result of the mutation. Large decreases in binding affinity were observed for select compounds only on the Y140A, Y142A and Y152A mutations. Table 3 | Ki values for Inhibition of [3 H]granisetron binding to wildtype and mutant 5-HT3ASRs. A more detailed analysis of the competition binding data obtained for the Y140A, Y142A and Y152A mutations is shown in Figure . For 5-HT, the Y142A mutation produced a 110 fold increase in Ki and Y152A produced a 24 fold increase. No change in Ki was observed for the Y140A mutation . The 5-HT3R agonistmCPBG showed a similar profile for the changes in Ki values resulting from mutations of the three tyrosines . As was observed for 5-HT, the Y142A mutation produced a large increase in Ki (160 fold) while the Y14 0A and Y152A mutations produced only 7 and 24 fold changes respectively. Figure 4 | Inhibition of [3H]granisetron binding by 5-HT3R ligands. Inhibition of [3H]granisetron binding by 5-HT3R ligands. The data represent the combined results of at least four experiments. Assays were conducted as described in Materials and Methods. Fractional response is the fraction of [3H]granisetron binding obtained in the presence and absence of inhibitor. An IC50 value was determined from the data using non-linear curve fitting as described in the methods and the Ki value calculated using the Cheng-Prusoff equation, the Kd as determined in Figure and the concentration of ligand used to obtain the inhibition data. Kd values are reported in Table . A. 5-HT B. mCPBG C. dtC D. Lerisetron The Ki value for d-tc inhibition of [3H]granisetron binding was altered only slightly by the Y142A or Y152A mutations (6.5 fold and 10 fold changes respectively). The Y140A mutation, however, produced a 50 fold increase in the Ki compared to wildtype receptors . The Ki value for lerisetron inhibition of [3H]granisetron binding was increased 160 fold by the Y142A mutation and 190 fold by the Y152A mutation. Only a 4.6 fold change in Ki resulted from the Y140A mutation. Lerisetron was the only compound for which a large increase in the Ki was observed on the Y152A mutation . This mutation produced smaller changes in Ki for 5-HT and mCPBG (24 fold for both) and only a 10 fold change for d-tc. In whole cell patch clamp studies, 5-HT perfusion of cells transfected with Y140A and Y142A cDNA produced no responses at 5-HT concentrations of up to 1 mM although specific binding to these receptors was identified in receptor binding studies. Unlike Y140A and Y142A, Y152A receptors responded to application of 5-HT. Due to the low potency of 5-HT on these receptors, only a portion of the concentration response curve could be determined (up to 1 mM). The EC50 value was estimated as greater than 370 muM . This value shows a greater than 140 fold increase in EC50 compared to wildtype receptors; a larger change than was observed for the Ki (24 fold). The most dramatic change observed for whole cell currents was an alteration in the kinetics of the response elicited by application of 5-HT. Y152A mutant receptors displayed much slower rise times compared to wildtype receptors at all concentrations tested. Peak wt responses were typically obtained in less than 80 ms while Y152A responses required several seconds to plateau. Desensitization kinetics were also altered. While wt receptors desensitized rapidly, mutant receptors showed no desensitization during the 8 s perfusion time. Figure 5 | Whole cell patch clamp recording from tsA 201 cells transfected with wildtype and Y152A cDNA. Whole cell patch clamp recording from tsA 201 cells transfected with wildtype and Y152A cDNA. Data were obtained using whole cell patch clamp on tsA201 cells transfected with wt or Y152A mutant 5-HT3ASR cDNA. Cells were clamped at a holding potential of -60 mV and 5-HT applied with a rapid perfusion system. The application time for 5-HT is indicated by the bar above the curves. The responses indicated in A and B are representative examples of at least 4 sets of experiments. A. Wildtype 5-HT3ASR. 5-HT was applied as indicated for a 4 s duration. The concentrations of 5-HT used and the response amplitudes are: a. 0.3 muM (0.03 nA) b. 1.0 muM (0.96 nA) c. 3.0 muM (2.5 nA) d. 30.0 muM (4.4 nA), e. 100.0 muM (3.9 nA). B. Y152A mutant receptors. 5-HT was applied as indicated for an 8 s duration. The curves in B were offset vertically for clarity. The dashed line in each curve indicates the baseline level for that response. The concentrations of 5-HT used and the response amplitudes are: f. 3.0 muM (0.10 nA) g. 100 muM (0.14 nA) h. 300.0 muM (0.57 nA) i. 1.0 mM (1.0 nA). C. Concentration response curve for 5-HT activation of wt 5-HT3ASRs. Each data point represents the combined results of 4 experiments. Responses were normalized to the response obtained at 30 muM and the curves fit as described in the methods section. EC50 = 2.7 +- 0.25 muM, n = 1.6 +- 0.22. D. Concentration response curve for 5-HT activation of Y152A 5-HT3ASRs. Each data point represents the combined results of 4 experiments. The response did not plateau in the range of concentrations used and hence responses were normalized to the maximum concentration of 5-HT (1 mM). The EC50 was estimated by fitting the data to the following equation: I = Imax/(1 +(EC50/ [C])n), where is the normalized current at 5-HT concentration [C], EC50 is the concentration of 5-HT needed to obtain half maximal activation and n is the apparent Hill coefficient. Imax in this case represents the estimated maximum current relative to the response obtained at 1 mM. To obtain the curve shown in the figure, the Hill coefficient was fixed at the wt value of 1.6. The EC50 value obtained from this fit is 370 +- 27 muM. PMID- 12079500_Discussion TI - AB - The putative E-loop region of the LGIC family of receptors is homologous to residues Y140 through K153 in the 5-HT3R [,-]. Structure-function studies of this region have been conducted in several other members of this family of receptors including GABAA and nAChR subtypes. In each case, residues have been identified that alter either the binding of selective ligands or receptor function [,-]. In order to determine if this loop also contains residues critical to the structure or function of the 5-HT3R, we have constructed alanine mutations of amino acids throughout the homologous region of the mouse 5-HT3ASR and investigated the affects on binding of 5 different structural classes of 5-HT3R ligands. We have identified three tyrosine residues that appear to play a role in binding of selective ligands to this receptor. In addition, our data support the existence of a loop structure in this region as has been hypothesized for the nAChR and identified in a homologous AChBP . Representative members of 5 major structural classes of 5-HT3R ligands were tested on all mutants . These ligands include the antagonists [3H]granisetron, d-tc and lerisetron, and the agonists 5-HT and mCPBG. Most of the mutations tested produced only minor changes in binding affinity for these ligands. Large changes in binding are only apparent on Y140A, Y142A and Y152A receptors for select ligands. The resulting effects of individual mutations are specific to particular structural classes of ligands. The Y140A mutation altered the Ki for d-tc inhibition, but did not alter the Ki obtained for any other compound tested, while Y142A altered the Ki for mCPBG, 5-HT and lerisetron but had little if any effect on inhibition by d-tc. These data indicate the highly specific nature of the effects introduced by the alanine mutations and appear to reflect specific changes in ligand/receptor interaction. Binding of [3H]granisetron is altered only slightly by the alanine mutations introduced in this study (<10 fold). This result indicates the lack of involvement of amino acids in this binding loop in the binding of [3H]granisetron. It is apparent, however, that [3H]granisetron does occupy the same binding cleft as other 5-HT3R ligands as evidenced by the ability of 5-HT, mCPBG, d-tc and lerisetron to displace it from the binding site. The lack of any large change in binding of granisetron supports our contention that there is little global structural perturbation of the binding site resulting from the introduction of each individual alanine mutation. The Ki for inhibition of [3H]granisetron binding by 5-HT was increased 110 fold by the Y142A mutation. A similar increase was also observed for mCPBG (160 fold). In contrast, the Y140A and Y152A mutations produced relatively small changes in the Ki. Since both compounds are 5-HT3R agonists, they are likely to share the same binding interactions. One of these interactions appears to be with Y142. Other studies have also identified binding site interactions for agonists. R91, E106, F107, W183 and several residues adjacent to the M1 region have all been demonstrated to alter the action of 5-HT and/or mCPBG . Within the crystal structure of the AChBP, homologous residues are located in the apparent binding site . The effects of agonist on the 5-HT3R are mediated by their interaction with these binding site residues, resulting in stable receptor conformations, including the channel open state. Identification of interacting amino acids and their location in the tertiary structure of the receptor may provide clues to the mechanism of channel opening. For example, amino acids homologous to Y142 and W183 (R104 and W143 respectively) are in close proximity in the AChBP, although on complementary faces of the receptor subunits . Spier et. al. have suggested that W183 may be involved in a cation-pi interaction with the amino group of 5-HT and mCPBG . If W183 and Y142 are located near each other but on opposite faces of the binding site in the 5-HT3R, then they could potentially act in concert to help stabilize a conformation of the receptor leading to channel opening. d-Tubocurarine inhibition was uniquely altered by the Y140A mutation. An increase in Ki of 50 fold was observed for d-tc on this mutant. Little if any change resulted for any other mutation. Thus, while d-tc may form an interaction with the receptor at this binding loop, it appears to interact with Y140 rather than Y142 or Y152. This difference in binding site interactions of the antagonist d-tc and the agonists 5-HT and mCPBG could be the result of a slightly different positioning of d-tc in the binding site. The antagonists d-tc and [3H]granisetron also interact differently with this region since [3H]granisetron binding was not altered by mutations at any of the amino acids tested. While the binding of d-tc appears to involve some interaction with this binding loop, the binding of [3H]granisetron does not. Previous studies have indicated a point of overlap between [3H]granisetron and d-tc at W89 of the 5-HT3ASR. . These studies indicate that the binding regions for these antagonists are partially overlapping at W89 but not at Y140. In addition to Y140 and W89, d-tc has also been observed to interact with D206 and several other residues in loop C . The d-tc binding site appears to involve interaction with at least three different binding loops (A, E and C) although additional interacting amino acids may be found. Data obtained for d-tc is particularly valuable due to the rigid nature of this molecule. Determination of interacting functional groups and their relationship to individual amino acids could enable d-tc to be used as a molecular ruler to determine relative positions of these amino acids. Lerisetron inhibition of [3H]granisetron binding to mutant receptors was also investigated. Lerisetron is a potent 5-HT3R antagonist first synthesized by Orales et. al.. As was observed for 5-HT3R agonists, lerisetron binding was altered by the Y142A mutation. This mutation produced an increase in the Ki of 160 fold compared to wildtype receptors. This increase in Ki indicates an important interaction of lerisetron with Y142 and a similarity between the binding location of lerisetron, 5-HT and mCPBG. Lerisetron is the only antagonist tested that shares a binding site interaction in this region with agonists. In contrast to 5-HT and mCPBG, however, lerisetron also interacts with Y152 as indicated by the 190 fold increase in Ki on Y152A mutant receptors. Since neither d-tc nor [3H]granisetron interacts with Y142 or Y152, there appears to be a difference between the interactions formed by these antagonists compared to lerisetron. The binding sites of the three antagonists tested differ with respect to this binding loop. Since the role of a competitive antagonist is simply to block the binding of agonists and prevent channel opening, the specific amino acids that interact with the ligand can vary for different antagonists. This is less likely to be the case with agonists since they must produce a conformational change in the protein to exert their effects. Only two mutant receptors failed to bind [3H]granisetron; G147A and V149A. G147 is the conserved glycine in this putative binding loop. Chirara et. al. have suggested that the highly conserved nature of the glycine in this region may indicate the existence of a loop structure consisting of either gamma or a loose three residue-turn in the nAChR . Either of these turns would bring the two putative beta-strands together such that gammaL109, gammaY111 and gammaS115 and gammaY117 are all on the same side of an antiparallel beta-sheet. These residues have been identified by affinity labeling, site-directed mutagenesis or cysteine substitution to lie on the same surface. A classic 2-residue beta-turn would place these residues on opposite surfaces . Substitution of the conserved glycine by alanine may disrupt the structure of this region and prevent assembly or expression of the receptor. The recent determination of the crystal structure of an AChBP supports this hypothesis. The AChBP displays a large amount of homology to the amino terminal of LGIC receptors and thus may be similar in structure . The crystal structure of this protein reveals a loose 3 residue turn incorporating the conserved glycine residue . Homologous residues in other LGIC subunits have also been identified and are shown in Figure . The residues identified in this study as altering binding affinity of 5-HT3R ligands would also be present on the same surface if this structure is present in the 5-HT3R. While Y140 lies somewhat outside the region identified by Chiara in the nAChR (homologous to gammaN107), gammaL109 and gammaL119 are homologous to Y142 and Y152 of the 5-HT3ASR. The ability of lerisetron to interact with both Y142 and Y152 also supports the hypothesis that these two amino acids are present in a loop structure since the eight intervening residues would position Y142 and Y152 too far apart to permit them both to interact with a single ligand even if they were interacting with functional groups on opposite ends of the molecule. A loop structure would bring them into closer proximity and permit interaction with the small molecule lerisetron. All three tyrosine mutations were investigated using a whole cell patch clamp assay to determine if functional changes could be observed. Whole cell responses could not be obtained for Y140A or Y142A, although specific binding of [3H]granisetron was observed. These data suggest that, while the receptors do assemble and are capable of binding [3H]granisetron, they are either not transported to the cell surface or are non-responsive to 5-HT at concentrations of 1 mM or less. Y152A does produce functional channels however they display distinctly altered response kinetics when compared to wildtype receptors. Y152A responses do not show the rapid rise times observed in wt receptors. The extremely slow rise times observed for Y152A receptors may indicate a change in rate constants preceding channel opening. These changes in the rate constants for either agonist binding or channel opening also produce a 140 fold decrease in the observed EC50 for 5-HT activation. The slow rise is followed by a non-desensitizing phase of the response that is dramatically different from the fast desensitization observed for wt 5-HT3ASRs. Lack of desensitization could result from either a stabilization of the open state of the channel or a destabilization of the desensitized state. Mutations of homologous or nearby residues in both the nAChR and GABAA receptors have also been demonstrated to alter the agonist response. Mutation of the homologous residue in the GABAA receptor gamma-subunit (T142) to serine altered the efficacy of the agonist Flumazenil, converting it to a partial agonist . In the nAChR, mutation of mouse and rat epsilonP121 to leucine altered both the binding of acetylcholine and the stability of the open state of the channel. epsilonP121 is homologous to P154 in the 5-HT3R and is only two residues away from Y152. The authors of this study concluded that this portion of the acetylcholine binding site was closely linked to the channel opening region of the receptor . It is reasonable to conclude that the homologous region in the 5-HT3R may perform a similar function. The link between an agonist binding domain and a conformational change leading to channel opening is not unexpected since the two must obviously be linked. If binding to this region of the receptor is shown to be a critical step between the binding of agonists and the opening of the channel, further investigation of the amino acids in this loop may provide valuable clues to molecular basis of this process. PMID- 12079500_Conclusions TI - AB - Our data indicate an important role for this putative binding site loop in the interaction of the 5-HT3R with different ligands and illustrate the difference in binding of different structural classes of ligands. Each structural class shows different patterns of interaction with amino acids in this region of the receptor. [3H]granisetron does not appear to interact with any of the amino acids tested while d-tc interacts with only Y140, mCPBG and 5-HT with Y142 and lerisetron with both Y142 and Y152. Similar selective effects have been observed on other residues including W183, W89, F107 and E106 among others. Our data in conjunction with those of other laboratories indicates the differences in orientation of different ligands within the same binding cleft. These differences in orientation result in different amino acid/functional group interactions. As the structural detail of these interactions emerges, these differences could potentially be exploited to produce more potent and specific ligands. For example construction of a "hybrid" ligand that combines the interactions of [3H]granisetron with those of d-tc, 5-HT or mCPBG could produce an antagonist with increased affinity due to the additional binding energy of these interactions and a greater specificity since it would utilize more structural features of the binding site. The requirement of a glycine at position 147 and the ability of lerisetron to interact with both Y142 and Y152 also support the hypothesis that the secondary structure in this region of the receptor is formed by a loop structure. If the loose 3 residue turn proposed by Chiara et. al. and shown for the AChBP is present in the 5-HT3R, then Y140, Y142 and Y152 would be present on the same side of the sheet and all three would be capable of extending into the binding site. A similar observation has been made for the nAChR where gammaS111, gammaY117, gammaL119, deltaR113 and deltaT119 of the mouse nAChR receptor and gammaL109 and gammaY111 of the torpedo nAChR are all thought to be present in the binding site . PMID- 12079500_Materials and Methods TI - AB - Materials | [3H]granisetron was purchased from New England Nuclear, 5-HT from Spectrum, and mCPBG and d-tc from Research Biochemical International. Lerisetron was provided by Dr. Karen Kirschbaum at The University of Louisiana at Monroe, Monroe, LA. All other reagents were obtained from commercial sources. Site directed mutagenesis | Wild type 5-HT3AS mouse receptor cDNA was obtained from Dr. Michael White . Mutant receptors were constructed using either the Quick Change Mutagenesis kit (Stratagene) or the Altered Sites Mutagenesis kit (Promega). All mutations were confirmed by commercial DNA sequencing. Cell culture and transfection | tsA201 cells, a derivative of HEK293 cells, were seeded at a density of 5 x 106 cells/100 mm dish. Cells were grown in DMEM medium containing 10% FBS, 100 units/ml penicillin/streptomycin for nine hours in 5% CO2 and transfected with 10 mug mouse 5-HT3ASR cDNA per 100 mm dish using the calcium phosphate technique (New Life Technologies, NY). Media was changed 12 --14 hrs after transfection. The cells were allowed to grow for another 24 hours and then harvested. For whole cell patch clamp experiments, tsA201 cells were seeded to a density of 0.25 x 106 cells/60 mm dish. Cells were grown in DMEM culture medium containing 10% FBS and 100 units/ml penicillin/streptomycin for 12 hours prior to transfection. Transfections were performed using Superfect Transfection Reagent (Qiagen, CA). Ten mug of cDNA were mixed with DMEM medium containing no serum or antibiotics in a volume of 150 mul. Twenty mul of Superfect reagent were then added and the mixture incubated at room temperature for 15 min. The reaction was terminated by adding 1 ml of DMEM medium containing 10% FBS and 100 units/ml penicillin/streptomycin and the entire mixture added to cells in the 60 mm dish. Cells were exposed to Superfect Reagent for 3 hours. At that time, the reagent was replaced with DMEM medium containing 10% FBS and 100 units/ml penicillin/streptomycin and incubated for an additional 24 hours prior to use. Binding assays | Transfected cells were scraped from the dishes, washed twice with Dulbecco's PBS (New Life Technologies, NY), then resuspended in 1.0 ml ice cold PBS/100 mm dish. Cells were either used fresh or frozen at this step until needed. Immediately prior to use, cells were homogenized on ice in PBS using a glass tissue homogenizer then centrifuged at 35 000 x g for 30 minutes in a Beckman JA20 rotor (4C). Membranes were washed once more with PBS at 4C then resuspended in 1 ml PBS/100 mm dish. Protein content was determined using a Lowry assay (Sigma. Diagnostics, St. Louis, MO). Membranes were initially prepared and Bmax and Kd values determined in the presence and absence of a cocktail of protease inhibitors (Complete Protease Inhibitor Cocktail, Roche Diagnostics, Mannheim Germany). No change in Bmax was observed as a result of omitting the protease inhibitor (Bmax (+ protease inhibitor)= 3.1 +- 0.11 pmoles/mg protein vs Bmax (- protease inhibitor) = 3.2 +- 0.13 pmoles/mg protein.) hence all assays were performed in the absence of the cocktail. Binding assays were performed in PBS. For Kd determinations, 100 mul of homogenate was incubated at 37C for 1 hour with varying concentrations of [3H]granisetron (NEN, MA). Specific binding of [3H]granisetron was determined as the bound [3H]granisetron not displaced by a saturating concentration of a competing ligand. Kd values were determined by fitting the binding data to the following equation using Graphpad PRISM (San Diego CA): B = Bmax [L]n / ([L]n + Kn), where B is bound ligand, Bmax is the maximum binding at equilibrium L is the free ligand concentration and n is the Hill coefficient. Ki determinations, 100 mul of homogenate was incubated at 37C for 2 hours with varying concentrations of inhibitor and [3H]granisetron (NEN, MA). Binding was terminated by rapid filtration onto a GF/B filters. The IC50 values were calculated by fitting the data to the following equation using Graphpad PRISM (San Diego CA): theta = 1/ (1+(L/IC50)), where theta is the fractional amount of [3H]granisetron bound in the presence of inhibitor at concentration L as compared to the amount of [3H]granisetron bound in the absence of inhibitor. IC50 is the concentration at which theta = 0.5. The Ki is calculated from the IC50 value using the Cheng-Prusoff equation. Electrophysiological Recordings | Transfected cells were transferred to a recording chamber containing extracellular solution (140 mM NaCl, 1.7 mM MgCl2, 5 mM KCl, 1.8 mM CaCl2, 25 mM HEPES pH 7.4). Patch electrodes of resistance 2.5 --3.0 MOmega were filled with filtered intracellular solution containing 145 mM KCl, 2 mM MgCl2, 1 mM EGTA, 25 mM HEPES (pH 7.4). Cells were clamped in whole cell configuration at a holding potential of -60 mV. A continuous extracellular solution flow (0.8 ml/min) was maintained throughout the recording procedure. 5-HT was dissolved in extracellular solution and delivered to cells using a rapid perfusion system (Warner Instruments, Hamden, CT) at a rate matching the extracellular solution flow rate. The drug perfusions lasted for a period varying from 4 to 8 seconds. Currents elicited by agonist application were measured using an Axopatch 200 B amplifier (Foster City, CA). The data were plotted and analyzed by non-linear curve fitting (Graphpad PRISM, San Diego CA) according to the following equation: I = 1/(1 +(EC50/ [C])n), where is the normalized current at 5-HT concentration [C], EC50 is the concentration of 5-HT needed to obtain half maximal activation and n is the apparent Hill coefficient. PMID- 12086585 TI - Insertion of a small peptide of six amino acids into the beta7 --beta8 loop of the p51 subunit of HIV-1 reverse transcriptase perturbs the heterodimer and affects its activities AB - Abstract | Background | HIV-1 RT is a heterodimeric enzyme, comprising of the p66 and p51 subunits. Earlier, we have shown that the beta7-beta8 loop of p51 is a key structural element for RT dimerization (Pandey et al., Biochemistry 40: 9505, 2001). Deletion or alanine substitution of four amino acid residues of this loop in the p51 subunit severely impaired DNA binding and catalytic activities of the enzyme. To further examine the role of this loop in HIV-1 RT, we have increased its size such that the six amino acids loop sequences are repeated in tandem and examined its impact on the dimerization process and catalytic function of the enzyme. Results | The polymerase and the RNase H activities of HIV-1 RT carrying insertion in the beta7-beta8 loop of both the subunits (p66INS/p51INS) were severely impaired with substantial loss of DNA binding ability. These enzymatic activities were restored when the mutant p66INS subunit was dimerized with the wild type p51. Glycerol gradient sedimentation analysis revealed that the mutant p51INS subunit was unable to form stable dimer either with the wild type p66 or mutant p66INS. Furthermore, the p66INS/p66INS mutant sedimented as a monomeric species, suggesting its inability to form stable homodimer. Conclusion | The data presented herein indicates that any perturbation in the beta7-beta8 loop of the p51 subunit of HIV-1 RT affects the dimerization process resulting in substantial loss of DNA binding ability and catalytic function of the enzyme. PMID- 12086585_Background TI - AB - Human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) is a product of the gag-pol polyprotein precursor, which is subsequently cleaved by the pol-encoded protease to yield the active form of the enzyme . This multifunctional enzyme is responsible for copying the single stranded viral RNA genome into double stranded proviral DNA . HIV-1 RT is a heterodimer consisting of a 66 and 51 kDa polypeptide chain designated as p66 and p51, respectively. The p51 subunit is generated via endoproteolytic cleavage of the p66 subunit between Phe 440 and Tyr 441 . The larger subunit (p66) contains both polymerase and RNase H activities, while the smaller subunit (p51) lacks these functions, in context of the heterodimer . However, both the p66 and p51 monomers are functionally inactive when dissociated from each other . Several years have passed since it was first suggested that agents that could specifically disrupt the dimerization of HIV-1 RT might prove a worthwhile antiretroviral strategy , though such agents have yet to be developed. Despite the fact that p51 shares an identical amino acid sequence with the N-terminal portion of p66, the two subunits assume different global folding patterns in the formation of the asymmetric heterodimer . Structural determination through X-ray crystallography has revealed that the p66 subunit of HIV-1RT has its polymerase domain in an "open" conformation, with its subdomains forming a large cleft which accommodates DNA. In contrast, the p51 subunit assumes a compact folded conformation that causes the active site residues in this subunit to be buried and therefore, nonfunctional . It has been proposed that the open conformation of p66 is supported by interactions with a closed and compact p51 molecule . The two subunits interact mainly via their connection subdomains. Additional contacts, between the thumb subdomain of p51 and RNase H subdomain of p66 are also substantial . Although there have been conflicting reports regarding the DNA polymerase activity of recombinant preparations of the p51 homodimer , it has become clear that p51 mainly plays a supportive role in context of the p66/p51 heterodimer. Assembly of chimeric heterodimers formed by mixing subunits of HIV-1 RT and FIV-1 RT, has demonstrated that the p51 subunit of HIV-1 RT helps to preserve the functional integrity of the HIV-1 RT heterodimer . Despite the fact that several functions have been proposed for the p51 subunit, the mechanism whereby p51 performs these functions has remained largely undefined. Some of the proposed functions for p51 include: (i) stabilizing the t-RNA primer binding for the initiation of reverse transcription , (ii) enhancement of strand displacement DNA synthesis , and (iii) as a processivity factor in DNA synthesis . Cys->Ser mutation at position 280 in the p51 subunit has been shown to alter the RNase H activity of the heterodimeric enzyme, indicating that this residue in the thumb subdomain of p51 plays an important role in support of the RNase H activity of p66 . The emergence of a strain of HIV-1 resistant to the non-nucleoside RT inhibitor TSAO (Tertbutyldimethyl silyspiro amino oxathioledioside) displaying Glu->Lys mutation at position 138 in the p51 subunit of HIV-1 RT has also been reported , thus implicating p51 to play a more direct role in drug binding and/or the enzymatic activities of HIV-1RT. This report was initially surprising, since Glu138 of p51 was thought to be quite distant from the purported dNTP-binding pocket of HIV-1RT, as well as the NNRTI binding pocket. However, in light of our recent findings implicating this loop region of p51 as a critical structural element supporting the catalytic functions of p66, it seems feasible that mutation at position 138 in p51 effectively altered the binding of TSAO through its influence on the p66 catalytic subunit . Examination of the crystal structure of HIV-1 RT reveals the presence of a small groove like region on the floor of the polymerase cleft of p66 . The beta7-beta8 loop of p51, comprising of six amino acids denoted as SINNET appears to fit into this groove-like region and likely stabilizes the polymerase domain of p66. In an earlier communication, we have shown that the p51 subunit of HIV-1 RT is required to load the p66 subunit on to the template primer for DNA synthesis . Our recent studies indicate that the beta7-beta8 loop of the p51 subunit is essential for the catalytic function of the p66 subunit. Deletion of this loop or substitution of four amino acid residues with alanine within the beta7-beta8 loop of p51 severely impaired the DNA polymerase activity of the enzyme as a consequence of the inability of the enzyme to form stable dimers . These findings clearly establish the absolute requirement of the beta7-beta8 loop of p51 for RT dimerization. Nonetheless, the question regarding the optimal size and composition requirement of this loop for efficient dimerization remains unanswered. In the present article, we have addressed the impact of increasing the size of the beta7-beta8 loop on the dimerization process. As a preamble to these studies, we have increased the size of this loop by repeating its six amino acid sequence in tandem. The rationale for duplicating the loop sequence was to increase the size of this loop without significantly disrupting the interactions seen with the wild type beta7-beta8 loop. The resulting mutant derivatives of HIV-1 RT containing insertion of six amino acids in the beta7-beta8 loop in either or both the subunits were analyzed for their ability to form stable dimers and other biochemical characteristics. In this article, we present evidence that HIV-1 RT mutants, carrying insertion of six amino acids in the beta7-beta8 loop specifically in the second subunit, do not form stable dimers. This inability to dimerize substantially decreases the enzymes affinity for DNA consequently impairing its polymerase and RNase H activities. PMID- 12086585_Results TI - AB - Glycerol gradient ultra-centrifugation analysis | The ability of the HIV-1 RT mutants, carrying insertion of six residues in the beta7-beta8 loop in either one or both the subunits was analyzed by glycerol gradient sedimentation analysis. Fractions of 200 muL were collected from the bottom of the tube and aliquots of every third fraction were subjected to SDS PAGE. The results shown in Figure , indicate that the sedimentation peak for the wild type p66/p66 homodimer was between fractions 16 --19 of the gradient (Panel A), whereas the wild type p51 species sedimented between fractions 22 --28, as a monomeric protein (Panel C). The sedimentation pattern for the p66WT/p51INS mutant indicated two distinct peaks. While the p66WT subunit predominantly sedimented in between fractions 16 --19, at the predicted position of the homodimer, the p51INS subunit sedimented between fractions 22 --28, indicating a monomeric conformation (Panel D). This sedimentation profile indicates that the p66WT/p51INS mutant carrying the 6 amino acid insertion in the beta7-beta8 loop of its p51 subunit is unable to form stable heterodimer. Under these conditions, the p66WT subunit of the p66WT/p51INS mutant tends to homodimerize. The mutants, p66INS/p66INS (Panel B) and p66INS/p51INS (Panel F), exhibited a distinct sedimentation peak between fractions 22 --28 of the gradient, indicating that these enzymes are unable to form stable dimers. In contrast, the p66INS/p51WT mutant (Panel E), in which only the p66 subunit carried an insertion in the beta7-beta8 loop sedimented between fractions 16 --19 of the gradient, indicating that these two subunits can form stable heterodimers. These analyses imply that the proper size of the beta7-beta8 loop in the second subunit of HIV-1 RT is important for the formation of a stable dimeric enzyme. Figure 1 | (A) Sedimentation profile of the wild type and mutant HIV-1 RT and (B) Polymerase activity profile of the glycerol gradient fractions. (A) Sedimentation profile of the wild type and mutant HIV-1 RT. Mutant enzymes carrying 6 amino acids insertion in the beta7-beta8 loop of either or both the subunits were applied on 10 --30% linear glycerol gradient (5 mL) and centrifuged at 48,000 rpm in SW 50.1 rotor for 22 hours. Gradients were fractionated from the bottom and subjected to SDS polyacrylamide gel electrophoresis and Coomassie Blue staining. (B) Polymerase Activity profile of the glycerol gradient fractions. Every third fraction between 7 and 33 of the glycerol gradient was diluted 10-fold and analyzed for its polymerase activity on poly (rA)(dT)18 as described under Materials and Methods. The reaction products were resolved on an eight percent denaturing poly-acrylamide urea gel and subjected to PhosphorImaging. In order to correlate the sedimentation profile of these insertion mutants with their functional activity, we analyzed the polymerase activity in the various gradient fractions. These results are presented in Fig. . The polymerase activity profile of the gradient fractions of the wild-type p66/p66 and the p66INS/p51WT mutant revealed major polymerase activity peaks corresponding to fractions 16 --19 . This activity peak correlates with the protein band intensity seen in Fig. (panels A and E) and is also in agreement with the sedimentation pattern of these two enzymes. Interestingly, the activity profile of the p66WT/p51INS mutant also yielded a peak corresponding to gradient fractions 16 --19 , thus substantiating our contention that the p66WT subunit of the p66WT/p51INS mutant tends to self-dimerize and form the catalytically active p66 homodimer. The wild type p51 and the two mutants, p66INS/p66INS and p66INS/p51INS, the sedimentation profile of which indicated a monomeric conformation were conspicuously devoid of any polymerase activity . These results imply that the beta7-beta8 loop of the second subunit of HIV-1 RT is critical in forming functionally active dimeric enzyme. DNA polymerase activities of wild type HIV-1 RT and its insertion mutants | DNA polymerase activity of the wild type HIV-1 RT and its mutant derivatives were quantitatively determined on both heteropolymeric RNA (U5-PBS RNA) and DNA (49-mer) templates, primed with 32P labeled 17-mer PBS primer. As documented in Table , insertion of the six amino acid segment in both the subunits (p66INS/p66INS or p66INS/p51INS), resulted in significant loss of polymerase activity corresponding to greater than 90% reduction. Interestingly, when the p66INS mutant was dimerized with the wild type p51 (p66INS/p51WT), the polymerase activity was restored to wild type levels. The same result was obtained when p66INS was dimerized with p51 having Asp->Ala mutation at amino acid position 186 (one of the catalytically crucial carboxylate triad) but having an intact beta7-beta8 loop. The rationale for using p51D186A mutant was to ascertain that the wild type polymerase activity observed with p66INS/p51WT was not due to residual polymerase activity of p51. These results suggest that insertion of six residues in the beta7-beta8 loop of p51 but not in p66 is detrimental to the function of the heterodimeric enzyme. Table 1 | Effect of duplication of the beta7 --beta8 loop in either or both the subunits of HIV-1 RT on the polymerase activity of the enzyme As shown in figure , evaluation of the polymerase activity of the wild type HIV-1RT and its insertion mutants by primer extension assay generally mirrored the results observed in the TCA precipitation assay, summarized in Table . Insertion of six amino acid residues in the beta7-beta8 loops of both the subunits (p66INS/p66INS or p66INS/p51INS) resulted in significant impairment of polymerase activity of the mutant enzymes. On an RNA template, both these enzymes exhibited total lack of polymerase activity , though the p66INS/p51INS mutant exhibited residual primer extension capability on a DNA template , while the p66INSp/66INS mutant was inactive on this template. Consistent with the results of the TCA precipitation assay, both the mutants, p66INS/p51WT and p66INS/p51D186A, exhibited near wild type (p66WT/p51WT) polymerase activity on both RNA and DNA templates. Thus, the impairment of the polymerase activity seen with these mutants carrying insertion in either both the subunits or specifically in the second subunit may be related to the inability of these mutants to form stable dimers resulting in a closed polymerase cleft. Figure 2 | Analysis of polymerase products catalyzed by insertion mutants of HIV-1 RT. Analysis of polymerase products catalyzed by insertion mutants of HIV-1 RT. Primer extension reactions catalyzed by the wild type and mutant enzymes were on DNA (panel A) and RNA (panel B) templates, primed with 5'-32P labeled 17-mer primer. Each set of reactions was carried out for 30 seconds (lane 1) and 60 seconds (lane 2) at 37C and quenched by the addition of equal volume of Sanger's gel loading dye. The reaction products were resolved on an 8% polyacrylamide-7M urea gel and subjected to PhosphorImager analysis. Effect of insertion in either or both the subunits of HIV-1 RT on the DNA binding function of the enzyme | Earlier we have shown that substitution of four amino acids on the beta7-beta8 loop with alanine reduced the DNA binding ability of the enzyme. Similar results were obtained when four amino acids were deleted from the loop. We therefore, concluded that the effect was exerted via p51 since alanine substitution or deletion specifically in the p66 subunit had no effect on DNA binding and polymerase activity of the enzyme. We speculated that alanine substitution or deletion in the loop of the p51 subunit may shorten the interacting sphere of the loop which may not be able to induce opening of the polymerase cleft in p66, which is essential for DNA binding. Given the fact that shortening of this loop in p51 prevented DNA binding, we were interested in examining the effect of six amino acids insertion on DNA binding. Therefore, we determined the equilibrium dissociation constants (Kd) of E-TP binary complexes for the wild type enzyme and its mutant derivatives by gel mobility shift assay. For this purpose, we used a 33-mer heteropolymeric DNA template primed with 5' 32P-labeled 21-mer DNA. Results shown in figure and table indicate a 24-fold reduction in DNA binding affinity when both the subunits carried insertion (p66INS/p51INS) in their beta7-beta8 loops. Similar results were obtained with the p66INS/p66INS mutant. Interestingly, the DNA binding affinity was restored to the wild type levels, when the mutant p66 subunit was dimerized with the wild type p51 (p66INS/p51WT). We expected that the p66WT/p51INSmutant would also exhibit reduction in its affinity for DNA, since insertion of six residues in the p51 subunit was speculated to be detrimental for the dimerization process. However, the DNA binding affinity of the p66WT/p51INS mutant was similar to the wild type enzyme. Our glycerol gradient sedimentation analysis had revealed that the p66WT/p51INS mutant does not form stable dimers, rather the p66WT subunit tends to self dimerize. Thus, the wild type DNA binding affinity seen in case of the p66WT/p51INS mutant may be attributed to the presence of these p66WT/p66WT homodimeric species. Based on our observations from the sedimentation analysis, we propose that the loss of DNA binding function in case of the HIV-1RT insertion mutants results from a failure to form stable dimers. The wild type p51 alone exhibited very low DNA binding affinity with a Kd[DNA]1300-fold higher than the wild type heterodimeric enzyme. These results suggest that the optimal size of the beta7-beta8 loop in the second subunit of the HIV-1 RT dimer is crucial for opening the polymerase cleft of the p66 subunit. Figure 3 | DNA binding affinity of mutant HIV-1 RT carrying insertion in the beta7 --beta8 loop of either or both the subunits. DNA binding affinity of mutant HIV-1 RT carrying insertion in the beta7 --beta8 loop of either or both the subunits. The 49-mer DNA primed with 5'-32P labeled 21-mer DNA primer was incubated with varying concentrations of the individual enzyme at 4C for 10 min. The mixture was electrophoresed under non-denaturing conditions on a 6% (w/v) polyacrylamide gel and analyzed on phosphorImager (Left panel). The positions of the free template primer (TP) and enzyme bound template-primer (E-TP binary complex) are indicated for the wild type enzyme. The percent of E-TP complex formed as a function of enzyme concentration was plotted for determining the Kd values (Right panel). Table 2 | DNA and dNTP binding affinities of the mutant HIV-1 RT carrying insertion in the beta7 --beta8 loop of either or both the subunits Ternary complex formation by the wild type and mutant enzymes | In the crystal structures of the ternary complex of HIV-1 RT (E-DNA-dNTP) the finger subdomain moves by 20A towards the palm subdomain . In this finger closing conformation, the DNA is locked in a stable ternary complex poised for catalysis. An in vitro assay using dideoxy terminated primer annealed with the template which allows the next correct dNTP to bind in the ternary complex without actual DNA synthesis has recently been reported . Using this assay system, we have evaluated the ability of the insertion mutants to form the ternary complexes and the effect of DNA trap on such complexes. Since binding of dNTP to the enzyme is an ordered mechanism which occurs only after DNA binding, the extent of labeled TP remaining bound to the enzyme in the presence of dNTP and DNA trap represents the extent of ternary complex formed. The E-TP binary complex was formed at enzyme concentrations which binds 100% of the labeled template primer. The preformed E-TP complex was then incubated in the presence of next correct dNTP followed by addition of 300-fold molar excess of unlabeled TP as the DNA trap. We found that E-TP binary complex was completely competed out by the DNA trap (data not shown) while a significant amount of the E-TP binary complex converted to E-TP-dNTP ternary complex was resistant to competition with DNA trap suggesting the stability of the ternary complex. Table lists the apparent dNTP binding affinity for the WT enzyme and its insertion mutants determined from data shown in Fig. . It was observed that although the DNA binding affinity was severely affected in case of the p66INS/p51INS mutant, its apparent dNTP binding affinity in the ternary complex did not change with respect to the wild type enzyme. However, the p66INS/p66INS mutant was unable to form a ternary complex. These data suggest that these two mutants may have a different conformation and mode of interaction in the ternary complex. It is apparent that the p66INS/p66INS binds to TP in a nonproductive manner which may have a direct impact on dNTP binding in the ternary complex. Figure 4 | Apparent dNTP binding affinity of mutant HIV-1 RT carrying insertion in the beta7 --beta8 loop of either or both the subunits. Apparent dNTP binding affinity of mutant HIV-1 RT carrying insertion in the beta7 --beta8 loop of either or both the subunits. The 33-mer DNA primed with 5'-32P labeled dideoxy (ddC) terminated 21-mer DNA primer was incubated with the individual enzyme at 4C for 10 min. The binding affinity [Kd (dNTP)] of the wild type enzyme and its mutant derivatives in the ternary complex was determined by incubating the preformed E-TP binary complex of the individual enzyme species at different concentrations (0.26 --800 muM) of the next correct dNTP (dGTP). Following incubation with dGTP, 300 fold molar excess of the unlabeled TP was used as trap to remove the readily dissociable binary complexes from the ternary complex population. The extent of stable ternary complexes formed were resolved on 6% native polyacrylamide gel and analyzed by phosphorImaging. The [Kd [dNTP]], in the ternary complex for each enzyme was determined by quantifying the E-TP-dNTP ternary complexes as a function of increasing concentrations of dGTP and fitting the data to the equation for single-site ligand binding using Sigma-Plot. Lanes 1 --6 represent the ternary complex formed in the presence of dGTP at 0.2, 1.28, 6.4, 32, 160 and 800 muM, concentrations. Steady state kinetic analysis of HIV-1 RT and its insertion mutants | In order to determine whether alteration in DNA binding without any change in the apparent dNTP binding affinity of the insertion mutants is consistent with their kinetic parameters, we analyzed the steady-state kinetic parameters of these mutants. The results of this investigation are summarized in Table . On poly (rA).(dT)18, only the p66INS/p66INS mutant showed a significant increase in Km [dNTP]. This observation is in agreement with the apparent dNTP binding affinity data in the ternary complex, where p66INS/p66INS mutant was found to be defective in forming a productive ternary complex. This observation is also consistent with our suggestion that p66INS/p66INS binds nonproductively to TP that may influence the formation of ternary complexes. Interestingly, the p66INS/p51INSmutant carrying insertion in both the subunits did not display the same reduction in dTTP binding affinity. However, the p66INS/p66INS and p66INS/p51INS mutants displayed nearly 6,000-fold and 400-fold reduction in catalytic efficiency (kcat/Km) compared to their wild type counterparts, respectively, on this template primer. A 10-fold reduction in catalytic efficiency in case of the p66INS/p51WT was noted only on poly (rA). (dT)18 and may be template-primer specific. None of the enzymes displayed a significant reduction in Km [dNTP] when the heteropolymeric DNA\DNA template primer was used, although the p66INS/p66INS and p66INS/p51INS mutants exhibited drastic reduction in catalytic efficiency. The p66WT/p51INSdisplayed no change in either dNTP binding or catalytic efficiency. These results are in keeping with our analysis of the polymerase and DNA binding assay for this mutant. Once again, we believe that the wild type p66 subunit in this enzyme preparation tends to homodimerize since the p51INS fails to participate in stable dimer formation. This phenomenon masks the deleterious effect of the insertion mutation. Table 3 | Steady-State kinetic parameters of mutant HIV-1 RT carrying insertion in the beta7 --beta8 loop of either or both the subunits RNase H activity of the insertion mutants | Since the polymerase activity of the homo- and hetero-dimeric enzymes carrying insertion in the beta7-beta8 loop in both the subunits (p66INS/p66INS and p66INS/p51INS) was drastically impaired, it was of interest to examine how this insertion affects their RNase H activity. To evaluate this, we employed a 30-mer RNA-DNA hybrid, and examined the cleavage of the 5'-32P-RNA strand of the duplex by the wild type enzyme and its mutant derivatives. The result of this analysis is presented in Fig. . Similar to their polymerase activities, the RNase H activities of the p66INS/p66INS and p66INS/p51INS mutants were severely impaired. This is not surprising, since our analysis of the DNA binding function of these two mutants had indicated a substantial loss of DNA binding affinity, which in turn is expected to affect both the polymerase and RNase H functions. Dimerization of the p66INS subunit with the wild type p51 (p66INS/p51WT) carrying an intact beta7-beta8 loop resulted in substantial recovery of the RNase H activity. The RNase H activity seen in case of the p66WT/p51INS mutant was not surprising since the p66WT subunit of the p66WT/p51INSmutant tends to self-dimerize and form the catalytically active p66 homodimer. Figure 5 | RNase H activity of the wild-type HIV-1 RT and its mutant derivatives. RNase H activity of the wild-type HIV-1 RT and its mutant derivatives. The individual enzymes were incubated with 5'-32P-labeled 30-mer RNA annealed with 30-mer complementary DNA strand, at 37C for 30 sec and 1 min as described in Materials and Methods. The cleavage products were resolved on an 8% denaturing polyacrylamide-urea gel and analyzed on a phosphorImager. Lanes 1 and 2 represent reactions carried out for 30 sec and 1 min, respectively. PMID- 12086585_Discussion TI - AB - In an earlier investigation on the role of the p51 subunit of HIV-1 RT, we demonstrated that decrease in size of its beta7-beta8 loop impairs the catalytic function of the heterodimer . In the present studies, we demonstrate that maintaining the wild type size of this loop in the p51 subunit is critical for dimerization of the enzyme and its catalytic activity. Duplication of the beta7-beta8 loop sequence selectively in the p66 subunit did not affect the dimer formation, DNA binding or polymerase activity of the p66INS/p51WT mutant. However, insertion of the same amino acid residues in the beta7-beta8 loop of p51 prevented stable dimerization of the p51INS subunit with either p66INS or p66WT and adversely impacted the DNA binding, polymerase and RNase H activities. Earlier, we have shown that p51 facilitates the loading of the p66 subunit on to the template primer . Therefore, the impaired polymerase activity and template-primer binding affinity of HIV-1 RT mutants carrying insertion in p51 may be due to their inability to load the catalytic p66 (p66INS) on the template primer. These altered biophysical/enzymatic properties of these insertion mutants may be attributed to the reduced dimer stability. Crystal structures of HIV-1 RT show that p66 and p51 assume different folding patterns and tertiary structures . It has been proposed that p66 in a monomeric form exists in a closed conformation similar to p51 . Following dimerization with another molecule of p51 (or p66), it assumes an open conformation . The polymerase domain of p51 is buried within its core. This difference in tertiary structures between the two subunits makes the dimer asymmetric. The amino acid residues at the contact interface differ with respect to their position and location in the 3-D structure. The counterpart of amino acid residues of p66 located at the contact interface are buried in the p51 folded conformation, whereas those of p51 are scattered in the p66 'open' conformation . Figure 6 | Amino acid residues in the p51 subunit in contact with the p66 subunit. Amino acid residues in the p51 subunit in contact with the p66 subunit. The Calpha backbone of p51 and p66 is shown in orange and green, respectively. Amino acids residues of p51 making contact with the p66 subunit are represented as 'sticks' and are shown encircled. The location of analogous residues in p66 are also encircled. It may be noted that these residues in p66 are scattered. The residues circled within 'blue' are from fingers; those in light blue' are from 'connection' and those in pink are from 'thumb' subdomains. The template (blue) and primer (gray) are also displayed in this figure. The residues in blue circle are V21, K22, P25, P52, E53, N57, T131, N136, N137, E138, T139, in light blue circle are P392, I393, Q394, E396, T397, T400, W401, N418, P420, L422, and those in pink circle are N255, Q258, V261, N265, V276, L283, T286, L289. Of the several domain interactions between p66 and p51, the beta7-beta8 loop of p51 is strategically positioned to interact with the residues on the floor of the palm subdomain of p66. It has been suggested that the stability of the dimer is related to the buried surface area between the two subunits . In the nevirapine-bound HIV-1 RT crystal structure, the total contact surface area between the subunits is approximately ~4600 A2. The two major contact regions between the subunits which provide it stability are their connection subdomains and the thumb of p51 and RNase H domain of p66. These contacts account for approximately two third of the total buried surface area. Interestingly, the marginal decrease in the total surface area due to deletion of four residues in the beta7-beta8 loop does not account for the dimer instability, thus suggesting that polar interactions of residues in the beta7-beta8 loop of p51 with the palm subdomain of p66 may play a role in conferring stability to the heterodimer. The observation that a single point mutation at L289 of p66, a residue not in direct contact with p51, also destabilizes the dimer indicates that other factors may also contribute towards dimer stability. In order to analyze the impact of inserting the six amino acid peptide in the beta7-beta8 loop of p51, we used the molecular modeling approach. A search in the database of known protein structures employing the 'loop-search' algorithm of SYBYL yielded 100 loops, of which only five were sterically permissible. The loop exhibiting the best homology was incorporated in the modeled structure . This loop of 9 amino acids (RFNAHGDVN) from the protein S. lectin formed a short anti-parallel two strand beta-sheet. This inserted loop lies in the vicinity of the palm subdomain of p66 and exhibits additional hydrophobic and polar interactions with residues in the palm subdomain of p66, not seen in the wild type structure. These additional interactions are expected to enhance the stability of the dimer. However, sedimentation analysis indicates that the insertion mutants form unstable dimer. This implies that the insertion may have altered the relative position of the subdomains in the two subunits thereby perturbing the dimer stability. In summary, the beta7-beta8 loop of p51 is an important structural element involved in imparting stability to the heterodimer and in opening the polymerase cleft of p66 for catalysis. Figure 7 | Molecular model of the inserted peptide in the beta7 --beta8 loop of the p51 subunit. Molecular model of the inserted peptide in the beta7 --beta8 loop of the p51 subunit. Molecular modeling of the extended loop was performed by the 'Loop Search' option of SYBYL version 6.5 (Tripos Associates, St. Louis, MO). A total of 100 loops, matching the Calpha distances constrain between the end points of the loop residues, were obtained. The loops were examined for 'bumps' with the neighboring protein structure. Selection of the appropriate loop was based on the least root mean square deviation and maximum homology with the inserted loop sequence. A loop satisfying these criteria containing two small anti-parallel beta-strands is shown in cyan. The side chains of amino acid residues of p66 are shown in green and those of p51 (wild type) are shown in orange. The side chains of interacting residues in the inserted peptide are colored violet and are indicated by their position number in parentheses. Interactions seen in the wild type loop are shown in blue dotted line while additional interactions are shown in black dotted lines. PMID- 12086585_Materials and methods TI - AB - PfuTurbo DNA polymerase and PCR reagents were obtained from Stratagene, Inc. Restriction endonucleases, DNA modifying enzymes and HPLC-purified dNTPs were purchased from Roche Molecular Biochemicals. Fast flow chelating sepharose (iminodiacetic-Sepharose) for immobilized metal affinity chromatography (IMAC), Phosphocellulose and Q-sepharose was purchased from Amersham Pharmacia Biotech. The alpha-32P-dNTPs and gamma-32P-ATP were purchased from Perkin Elmer life sciences. The DNA oligomers were synthesized at the Molecular Resource Facility at UMDNJ. All other reagents and chemicals were of the highest available purity grade and purchased from Fisher, Millipore Corp., Roche Molecular and Bio-Rad. Plasmid and clones | The expression vector pET-28a and E. coli expression strain BL21 (DE3) were obtained from Novagen. The HIV-1RT expression clones (pKK223-3 RT66 and pET-28a-RT51) constructed in this laboratory were used for PCR amplification and construction of the insertion mutants in the p66 and p51 subunits of HIV-1 RT. An HIV-RNA expression clone pHIV-PBS was a generous gift from Dr. M. A. Wainberg . Insertion of 6 amino acid residues in the beta7 --beta8 loop | The pKK-RT66 clone containing two unique restriction sites, Hpa1 and Stu1, at codons 136 and 140 in the RT coding region was used for insertion of 6 amino-acid residues in the beta7-beta8 loop of the p66 and p51 subunit. The pKK-RT66 clone was digested with HpaI restriction enzyme to generate a blunt end at codon 136. For insertion, two complementary pre-kinased 18-mer synthetic DNA oligos having the following sequences: 5'-ATA AAC AAT GAG ACA ATA-3 (sense strand) and 3'-TAT TTG TTA CTC TGT TAT-5' (antisense strand) were hybridized. The 18-mer duplex DNA encoding the insertion peptide (Ile-Asn-Asn-Glu-Thr-Ile) was ligated with Hpa1 digested pKK-RT66 in between codon 135 and 136. The positive clones were screened in E. coli HB101 by the absence of an Hpa1 site and the correct orientation of the insertion was confirmed by DNA sequencing. This construct expresses the p66+6aa subunit without His tag sequences. A His-tag at the N-terminal of the p66+6aa subunit was introduced by sub cloning the Bal-I and Hind III fragment of pKKRT66+6aa into pET-28a-RT66 expression cassette. A unique Sac I site was also introduced in pKK-RT66 template at codon 440. The construction of P51+6aa was carried out by removal of the 360 bp fragment from pKK-RT66+6aa by restriction digestion with SacI followed by re-ligation of the vector ends. The insertion mutant in pET28a and pKK223-3 vectors were introduced into E. coli BL-21 (DE3) pLys S and E. coli JM109, respectively, for expression. Induction of the enzyme protein was carried out as described before for the wild type HIV-1RT . The enzyme with the hexahistidine-tag was purified from bacterial lysates by immobilized metal affinity chromatography , while non-hexahistidine-tagged enzyme was purified using the phosphocellulose and Q-Sepharose columns as described previously . Preparation of the heterodimeric enzyme with subunit specific insertion | The p51 subunit with a hexahistidine-tag and a non-tagged p66 were used to generate the heterodimers containing insertion in either or both of the subunits. For each set of heterodimers, 260 mug of p51 was mixed with 660 mug of p66 in the buffer containing 50 mM Tris HCl, pH 7.8, 60 mM KCl and 5 mM MgCl2. The rationale for using a 1:3 ratio of p51 to p66 was to saturate the His-tagged p51 with the non-tagged p66, ensuring heterodimer formation and eliminating excess p66 during IMAC purification. The mixture was incubated for 16 hours at 4C and applied to (0.5 mL) Ni2+ iminodiacetic-Sepharose (IDA-Sepharose) column, which was pre-equilibrated with the binding buffer (20 mM Tris HCl pH 7.8, 500 mM NaCl and 5 mM Imidazole). The column was washed with 15 mL of the same buffer to remove the excess of p66 that was not dimerized with p51 bound to the IDA-sepharose column. The heterodimeric RT was then eluted from the column with elution buffer (20 mM Tris HCl pH 7.8, 500 mM NaCl and 250 mM imidazole). Fractions of 0.5 mL were collected and an aliquot of each fraction was analyzed by SDS-PAGE using Coomassie Blue stain. The fractions containing approximately equal band intensity of p66 and p51 were dialyzed against a storage buffer (50 mM Tris HCl pH 7.0, 200 mM NaCl and 50% Glycerol) and this enzyme preparation was used in all experiments. Glycerol gradient ultra centrifugation | Fifty micrograms of the enzyme protein in 100 muL of buffer (50 mM Tris HCl, pH 7.8, 1 mM DTT and 400 mM NaCl) was carefully loaded onto 5 mL of 10 --30% glycerol gradients prepared in the same buffer. The gradients were centrifuged at 48,000 rpm in an SW48 rotor for 22 h at 4C. Fractions (200 muL) were collected from the bottom of the tube and aliquots of these fractions were electrophoresed using SDS PAGE and Coomassie Blue stain to identify the protein peak. The polymerase activity in the gradient fractions were analyzed by extension of the labeled (dT)18 annealed to poly (rA) template. Every third fraction between 7 and 33 of the glycerol gradient was diluted 10-fold and analyzed for its polymerase activity. Reactions were carried out at 37C for 2 min at 20 muM dTTP concentration and quenched with Sanger's gel loading dye . The reaction products were resolved by denaturing polyacrylamide-urea gel electrophoresis and analyzed on a PhosphorImager (Molecular Dynamics, Inc.). DNA polymerase assay | Polymerase activity of the HIV-1RT WT and insertion mutant enzymes was determined using two different template-primers: U-5PBS HIV-1 RNA and synthetic 49-mer U5-PBS DNA templates primed with the 17-mer PBS primer . Assays were carried out in a 50 muL volume containing 50 mM Tris HCl, pH 7.8, 100 mug/mL bovine serum albumin, 5 mM MgCl2, 1 mM dithiothreitol, 60 mM KCl, 100 nM template-primer, 50 muM of each of the four dNTPs with one of them being 32P-labeled (0.1 muCi/nmol dNTP) and 21 nM enzyme. Reactions were incubated at 37C for 3 min and terminated by the addition of ice-cold 5% trichloroacetic acid containing 5 mM inorganic pyrophosphate. Following termination, the reaction mixtures were filtered on Whatman GF/B filters. The filters were then dried, immersed in scintillation fluid and counted in a liquid scintillation counter. Gel analysis of RNA and DNA dependent polymerase activities | The U5-PBS HIV-1 RNA and heteropolymeric synthetic U5-PBS HIV-1 DNA templates primed with the 17-mer PBS DNA primer were used to assess the polymerase activities of the wild type and mutant heterodimeric enzymes. The primers were 5'-labeled using gamma-32P-ATP and T4 polynucleotide kinase according to the standard protocol . Polymerase reactions were carried out by incubating 2.5 nM template primer with 50 nM of the wild type HIV-1RT or its mutant derivative in a total reaction volume of 6 muL containing 25 mM Tris-HCl, pH 7.5, 10 mM dithiothreitol, 100 mug/mL bovine serum albumin, 5 mM MgCl2 and 50 muM of each dNTP. Reactions were initiated by the addition of enzyme and terminated by the addition of an equal volume (6 muL) of Sanger's gel loading dye . The reaction products were resolved by denaturing poly acrylamide-urea gel electrophoresis and analyzed on a PhosphorImager (Molecular Dynamics, Inc.). Template-Primer (TP) binding affinity of the wild type enzyme and its mutant derivatives | The dissociation constants (Kd) of the E-TP binary complexes of the wild type HIV-1 RT and its mutant derivatives were determined as described by Tong et al. . The heteropolymeric 33-mer DNA (0.4 nM) annealed to 5'-32P-labeled 21-mer primer (0.3 nM) was incubated with varying concentrations of the wild type enzyme and its mutant derivatives in a total volume of 10 muL containing 50 mM Tris-HCl, pH 7.8, 5 mM MgCl2 and 0.01 % BSA. Following incubation of the mixture for 10 min at 4C, equal volume of 2x gel-loading dye containing 0.25% bromophenol blue and 20% glycerol was added. The E-TP binary complexes formed were resolved at 4C on 6% native polyacrylamide gel using Tris-Borate buffer (85 mM Tris, 85 mM Boric acid, pH 8.0). The amounts of the TP in the binary complex (E-TP) and in free form with respect to the varying concentrations of the enzyme protein were determined by PhosphorImager (Molecular Dynamics, Pharmacia) analysis of the gel. The fraction of the bound DNA was plotted against enzyme concentration and the Kd [DNA] value was determined as the RT concentration at which 50% of DNA is bound. Ternary complex formation assay | The ternary complex (E-DNA-dNTP) formation was assessed by incubating the binary complexes of enzyme and dideoxy terminated template primer in the presence of next correct dNTP . The binary complexes were formed by incubating 10 --50 nM of the wild type enzyme or its mutant derivatives with 0.3 nM of 5'-32P-labeled dideoxy terminated 33-mer/21-mer template-primer as described above. The chosen concentration of enzyme was such that resulted in almost complete shift during E-TP complex formation. The E-TP-dNTP ternary complex formation was assessed by the addition of dNTP complementary to the next template base (in this case dGTP, 200 muM). Following incubation with dNTP at 4C for 10 min, 300 fold molar excess of a DNA trap was added to the incubation mixture to assess the stability of the binary and ternary complexes formed by the enzyme. The complexes were resolved on a 6% native polyacrylamide followed by phosphorImaging. The extent of E-TP-dNTP ternary complexes formed was quantified using ImageQuant software. RNase H activity assay | We used a 5'-32P labeled 30-mer synthetic U5-PBS RNA template annealed with a complementary 30-mer DNA to determine the RNase H activity of the enzymes . The reaction mixture contained labeled RNA-DNA hybrid (10 K Cerenkov cpm), 50 mM Tris-HCl pH 8.0, 60 mM KCl, 10 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 5 mM MgCl2, and 20 ng of enzyme in a final volume of 5 mul. Reactions were carried out at 37C for 30 sec and 1 min and terminated by the addition of equal volume of Sanger's gel loading dye . The cleavage products were analyzed on an 8% denaturing polyacrylamide-urea gel and scanned on a phosphorImager (Molecular Dynamics Inc.). PMID- 12097150 TI - Molecular cloning and tissue distribution of mammalian L-threonine 3-dehydrogenases AB - Abstract | Background | In mammals, L-threonine is an indispensable amino acid. The conversion of L-threonine to glycine occurs through a two-step biochemical pathway involving the enzymes L-threonine 3-dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase. The L-threonine 3-dehydrogenase enzyme has been purified and characterised, but the L-threonine 3-dehydrogenase gene has not previously been identified in mammals. Results | Transcripts for L-threonine 3-dehydrogenase from both the mouse and pig are reported. The ORFs of both L-threonine dehydrogenase cDNAs encode proteins of 373 residues (41.5 kDa) and they share 80% identity. The mouse gene is located on chromosome 14, band C. The amino-terminal regions of these proteins have characteristics of a mitochondrial targeting sequence and are related to the UDP-galactose 4-epimerases, with both enzyme families having an amino-terminal NAD+ binding domain. That these cDNAs encode threonine dehydrogenases was shown, previously, by tiling 13 tryptic peptide sequences, obtained from purified L-threonine dehydrogenase isolated from porcine liver mitochondria, on to the pig ORF. These eukaryotic L-threonine dehydrogenases also have significant similarity with the prokaryote L-threonine dehydrogenase amino-terminus peptide sequence of the bacterium, Clostridium sticklandii. In murine tissues, the expression of both L-threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase mRNAs were highest in the liver and were also present in brain, heart, kidney, liver, lung, skeletal muscle, spleen and testis. Conclusions | The first cloning of transcripts for L-threonine dehydrogenase from eukaryotic organisms are reported. However, they do not have any significant sequence homology to the well-characterised Escherichia coli L-threonine dehydrogenase. PMID- 12097150_Background TI - AB - In husbanded animals, dietary supplements of mixtures of indispensable amino acids regulate their growth and the activity of amino acid metabolising enzymes . The liver plays a critical role in regulating amino acid metabolism. Porcine-derived hepatocytes are being used in clinical studies of bioartificial liver organs . The regulation of amino acid supply to bioartificial organs and maintaining the activity of the amino acid-metabolising enzymes will be important in their development. From a metabolic perspective, threonine is one of three indispensable amino acids. In biological tissues, there are two major and one minor L-threonine degradation pathways. L-threonine is either catabolised by L-threonine 3-dehydrogenase (EC 1.1.1.103; gene abbreviation TDH) to 2-amino-3-ketobutyrate or by L-serine/threonine dehydratase (EC 4.2.1.16; L-threonine deaminase, gene abbreviation SDH) to NH4+ and 2-ketobutyrate in the major pathways and by threonine aldolase (EC 4.1.2.5; gene abbreviation GLY1) to yield glycine and acetaldehyde in the minor pathway . In both prokaryotic and eukaryotic cells, the conversion of L-threonine via 2-amino-3-ketobutyrate to glycine takes place in a two-step process . L-threonine dehydrogenase catalyses the reaction: L-threonine + NAD+ = 2-amino-3-ketobutyrate + NADH. The subsequent reaction between 2-amino-3-ketobutyrate and coenzyme A to form glycine and acetyl-CoA is catalysed by 2-amino-3-ketobutyrate coenzyme A ligase (EC 2.3.1.29; gene name KBL). If not processed by 2-amino-3-ketobutyrate coenzyme A ligase, the highly reactive intermediate, 2-amino-3-ketobutyrate, rapidly undergoes decarboxylation to form aminoacetone and CO2. L-threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase have been found to be associated physically on the inner membrane-matrix of mitochondria where the two enzymes form a complex with a stoichiometry of one threonine dehydrogenase tetramer to two 2-amino-3-ketobutyrate coenzyme A ligase dimers . The three-dimensional structure of Escherichia coli 2-amino-3-ketobutyrate coenzyme A ligase has been determined recently . Recently, we have cloned the murine and human 2-amino-3-ketobutyrate coenzyme A ligase cDNAs and wished to clone the first enzyme in this L-threonine catabolic pathway. The E. coli L-threonine dehydrogenase gene and its activity have been well characterised . I describe the first isolation of eukaryotic cDNA sequences encoding L-threonine dehydrogenases that have significant identity to 13 peptide sequences from the purified porcine L-threonine dehydrogenase enzyme , and the amino-terminus peptide of L-threonine dehydrogenase from the Gram+ Firmicutes bacteria, Clostridium sticklandii . However, they have no significant homology to the E. coli enzyme at the primary amino acid sequence level. PMID- 12097150_Results TI - AB - Previously we identified, by homology to the E. coli gene, both the mouse and human cDNAs for 2-amino-3-ketobutyrate coenzyme A ligase, the second enzyme in the biochemical pathway that converts L-threonine to glycine . In a search for the mouse cDNA of L-threonine dehydrogenase, which is the first enzyme in this pathway, I initially used the same approach. However, only expressed sequence tags belonging to sorbital dehydrogenase (and, with a much lower degree of homology, numerous isoforms of alcohol dehydrogenase) were identified. Nor were other candidate genes found in the human genomic sequence. Fortunately, Kao and Davis (1994) had previously purified and characterised the porcine L-threonine dehydrogenase protein, that they had isolated from liver mitochondria and partially peptide, sequenced. This peptide sequence was used to identify mouse ESTs with significant homology by a back translation to nucleotides search. The program ESTblast was used to construct a tentative mouse contiguous sequence from EST sequences. PCR primers were designed to match the 5' and 3' ends of the electronic contiguous sequence and used to amplify the gene from mouse liver and lung cDNA. After agarose gel electrophoresis, each primer set produced a single amplicon indicating that the gene is not alternatively spliced. The PCR products were cloned and sequenced. A blast search with the murine threonine dehydrogenase cDNA sequence of the pig EST database identified similar 5' and 3' ESTs (accession Nos. BE233801 and BI400146 respectively) and the sequence of these ESTs was utilised to design primers to amplify the pig L-threonine dehydrogenase from hepatocytes by RT-PCR. Analysis of murine and porcine L-threonine dehydrogenase cDNAs | The 1508 bp mouse sequence has an ORF which encodes a 373 residue protein and has a ATTAAA polyadenylation signal at 1460 --1465 (GenBank accession No. AY116662) . A second clone (accession No. AF134346) includes 63 bp of 5'UTR and utilises a more 5' ATTAAA polyadenylation signal at 1350 --1355. The predicted protein has a 41,461 Da molecular mass and an isoelectric point 8.45. The mouse genomic sequence for this cDNA is located on chromosome 14, band C (accession No. NW_000100, The Sanger Institute, UK). The gene spans 16.4 kb and consists of 9 exons. There is a 329 bp CpG island (64% CG) spanning the 5' untranslated exon . Figure 1 | The cDNA sequence and translation of murine L-threonine dehydrogenase. The cDNA sequence and translation of murine L-threonine dehydrogenase. There are two potential polyadenylation signals (attaaa at 1350 --1355 and 1460 --1465) shown in bold and underlined with the polyadenylation sites indicated by a. An * indicates the taa stop codon. The bold type and underlined nucleotide pairs indicate the positions of the exon/exon/ boundaries. Figure 2 | The gene structure of the murine TDH gene. The gene structure of the murine TDH gene. The gene spans 16.4 kb and consists of 8 translated exons and a 5' untranslated exon on chromosome 14, band C (supercontig accession No. NW_000100, The Sanger Institute, UK). There is a CpG island spanning the 5' untranslated exon. The ORF is indicated by closed boxes and the sizes, in bp, of the exons and introns are indicated. The pig sequence (GenBank accession No. AY095535) also has an ORF that encodes a 373-residue protein with a 41,432 Da molecular mass and an isoelectric point 7.67 . At the nucleotide level, the porcine and mouse ORFs have 78% identity and at the protein level have 81% identity and 94% similarity. The potential polyadenylation signal on the pig sequence is homologous to the most 5' signal on the mouse sequence. Figure 3 | The porcine L-threonine dehydrogenase cDNA sequence and translation. The porcine L-threonine dehydrogenase cDNA sequence and translation. The ORF (accession No. AY095535) is shown in bold and the 5' EST (accession No. BE233801) and the 3' EST (accession No. BI400146) are shown in lower case. The potential polyadenylation signal (attaaa) is shown in bold and underlined. Comparison of the porcine L-threonine dehydrogenase ORF with sequenced peptides from the porcine L-threonine dehydrogenase enzyme | Evidence that the porcine cDNA encodes for L-threonine dehydrogenase comes from the high degree of similarity to sequenced peptides from the purified and structurally characterised porcine L-threonine dehydrogenase protein isolated from liver mitochondria . The sequences of 13 porcine peptides have been aligned with the porcine ORF protein and have 98% identity over 212 residues . The 5 mismatched residues are probably due to errors in peptide sequencing since they are located towards the end of the sequences. Figure 4 | Comparison of the translation of the porcine L-threonine dehydrogenase cDNA and sequenced peptides from porcine L-threonine dehydrogenase. Comparison of the translation of the porcine L-threonine dehydrogenase cDNA and sequenced peptides from porcine L-threonine dehydrogenase. Porcine cDNA sequence, Ss cDNA; porcine sequenced peptides (Pig PEP). Individual pig peptides are alternatively, not underlined, then underlined and shown in bold. Conserved residues are shown by an (*) and strongly conserved residues by (:). Import into mitochondria | Mammalian L-threonine dehydrogenase is a nuclear encoded gene; the protein is synthesised in the cytoplasm and imported into mitochondria. The amino-terminal of the mature porcine L-threonine dehydrogenase protein isolated from mitochondria corresponds to amino acid residue 51 on the porcine L-threonine dehydrogenase ORF , which suggests that the pro-protein is cleaved to produce a 36 kDa mature enzyme. This value is close to that which would be expected since the mature porcine enzyme has a subunit molecular mass of 37 kDa on SDS-PAGE . The amino-terminal region of the mouse, fly and nematode L-threonine dehydrogenase proteins all have characteristics of mitochondrial targeting sequences , despite being the region of lowest similarity within the protein, having a high content of basic amino acids and few acidic amino acids . Figure 5 | Comparison of eukaryotic L-threonine dehydrogenase protein sequences. Comparison of eukaryotic L-threonine dehydrogenase protein sequences. The L-threonine dehydrogenase sequences are: mouse, MmTDH; pig, SsTDH; Drosophila melanogaster, DmCG5955; Caenorhabditis elegans, CeF3.4 (corrected as described in results). NAD, indicates those conserved residues likely to contact the nicotinamide cofactor by homology with the crystal structure of the UDP-galactose 4-epimerases. The locations of the exon/exon boundaries are shown on the translated protein as underlined residues. Sequence homology in other species | A database search revealed the presence of L-threonine dehydrogenase genes in the genome of other organisms. The fly, Drosophila melanogaster has a 6 exon gene located on chromosome 3L which translates into a cDNA of 1288 bp, encoding the 367 residue CG5955 protein (accession No. AAF51607) . The nematode, Caenorhabditis elegans has a 5-exon gene located on chromosome V (encompassing only the first 5 exons of the predicted 10 exons of the hypothetical gene product F08F3.4, accession No. AAB04871). By extending the fifth exon to the next polyadenylation site a 1217 bp cDNA is formed, encoding a 359-residue protein . The cDNA sequences of both these genes are supported by EST data. The fly and nematode proteins have over 52% identity and 88% similarity to the mammalian proteins in the 306-residue, central core of the enzyme. Four exon/exon boundaries are conserved in two or more of the genes . The search also revealed similar L-threonine dehydrogenase ESTs in amphibians, bony fishes, tunicates, flies, moths, mites, nematodes and trypanosomes, but not in higher plants and yeasts. Similarly, the gene for the second enzyme in this pathway, KBL, is also absent from the yeast, Saccharomyces cerevisiae , and no L-threonine dehydrogenase activity has been found in S. cerevisiae . That L-threonine dehydrogenase sequences have been evolutionarily conserved between the Gram+ bacteria and mammals is shown by the homology between mouse and the amino-terminus peptide sequence from the threonine dehydrogenase of the Gram+ Firmicutes bacteria, C. sticklandii which has 54% identity and 93% similarity over 28 residues . C. sticklandii is an amino acid fermenting anaerobic bacterium that can grow on threonine as a sole substrate. Together, the mouse and C. sticklandii sequences enabled the identification of putative L-threonine dehydrogenase genes in a number of bacterial species such as Thermoplasma acidophilum, T. volcanium and Staphylococcus epidermidis. An alignment with the putative L-threonine dehydrogenase sequence, the SAV0542 gene, from S. aureus that has 41% identity and 75% similarity to the mouse protein is shown in Fig. . Figure 6 | Comparison of murine and prokaryotic L-threonine dehydrogenase protein sequences. Comparison of murine and prokaryotic L-threonine dehydrogenase protein sequences. The L-threonine dehydrogenase sequences are: mouse, MmTDH; Staphylococcus aureus hypothetical protein from gene SAV0542 (accession No. BAB46113) , S. aureus; Clostridium sticklandii threonine dehydrogenase amino-terminal peptide, C.sticklandii NT . Mammalian threonine dehydrogenases have an NAD+ binding domain | A search of the protein structural database revealed that the closest matches with 19% identity were UDP-galactose 4-epimerases (GALE) from E. coli and Homo sapiens . GALE is a mixed alpha-helices/beta-sheet protein with a N-terminal NAD+ binding Rossmann-fold and belongs to the tyrosine-dependent oxidoreductase protein family (also known as short-chain dehydrogenases). The characteristic Tyr-X-X-X-Lys couple (residues 195 and 199) found in all family members are important for catalysis with the conserved tyrosine serving as the active-site base . By comparison with the crystal structures of the GALE proteins, two domains were identified and it is likely that the substrate, L-threonine, is located in the cleft between the two domains. The larger amino-terminus domain (residues 58 --231 on the mouse sequence) has a NAD+ binding motif. There are 12 conserved residues in the murine L-threonine dehydrogenase protein that are likely to contact the nicotinamide cofactor (Gly-62, Gly-65, Gly-68, Asp-88, Ile-107, His-127, Leu-131, Asn-147, Ser-169, Tyr-195, Lys-199 and Tyr-222) . The smaller carboxy-terminus domain (residues 232 --335) has little similarity to GALE and is likely to be involved in substrate binding. Expression of L-threonine dehydrogenase mRNA in mouse tissues | To identify which tissues are likely to contribute to L-threonine dehydrogenase activity in the mouse, reverse-transcriptase real time PCR was used to examine the tissue distribution of L-threonine dehydrogenase mRNA. By reverse-transcriptase real time PCR L-threonine dehydrogenase expression was found in all tissues examined, being highest in liver, high in testis and spleen and lowest in skeletal muscle, relative to the expression of beta-actin . Similar results were also obtained with another set of L-threonine dehydrogenase primers (located on exons 6 and 7) (data not shown). The expression of 2-amino-3-ketobutyrate coenzyme A ligase was also found in all tissues examined, being highest in liver and high in kidney. The expression level of the housekeeping gene beta-actin was similar in all tissues examined. Another housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was also used to standardise expression levels, but G3PDH expression showed greater variation between tissues, having higher expression in heart and skeletal muscle and lower expression in testis relative to beta-actin (data not shown). After 40 cycles of PCR amplification, the amplicons were specific as verified by melting curve analysis (data not shown) and agarose gel electrophoresis . Figure 7 | Expression of L-threonine dehydrogenase mRNA in tissues by semiquantitative PCR. Expression of L-threonine dehydrogenase mRNA in tissues by semiquantitative PCR. (A) The expression level in each tissue cDNA was normalised to the expression level of the housekeeping gene beta-actin. The ratio of L-threonine dehydrogenase mRNA to beta-actin mRNA (Y axis, arbitrary units) from each tissue was standardised to that of liver, which was taken as 100. (B) Ethidium bromide stained agarose gel of PCR products after 40 cycles of amplification. Threonine dehydrogenase, TDH; 2-amino-3-ketobutyrate coenzyme A ligase, KBL. The tissues examined were: brain, Br; Heart, He; kidney, Ki; liver, Li; lung, Lu; skeletal muscle, SM; spleen, Sp; testis, Te; and no cDNA control, -c; 100 bp ladder, m. PMID- 12097150_Discussion TI - AB - L-threonine dehydrogenase is the first enzyme in the biochemical pathway converting L-threonine, via 2-amino-3-ketobutyrate, to glycine. In a search for mammalian L-threonine dehydrogenase genes, clones were isolated from mouse and pig with ORFs very similar to sequenced peptides from the pig L-threonine dehydrogenase protein . The mouse and pig L-threonine dehydrogenase proteins also have significant homology to amino-terminus peptide sequences from chicken (Gallus gallus) and the bacteria, C. sticklandii . I conclude that the cloned cDNAs encode L-threonine dehydrogenase genes. The mammalian proteins belong to the tyrosine-dependent oxidoreductase protein family (also known as short-chain dehydrogenases). They do not have any significant homology to the well-characterised bacterial L-threonine dehydrogenase enzymes from E. coli and the Gram-negative plant pathogenic bacteria Xanthomonas campestris , which belong to the medium-chain, zinc-binding alcohol/polyol dehydrogenase family. However, they are distantly related with both short and long-chain dehydrogenases having characteristics of the NAD(P)-binding Rossmann-fold. Both short- and long-chain L-threonine dehydrogenases have no significant homology to the D-threonine dehydrogenase gene from Pseudomonas cruciviae that is a member of the 6-phosphogluconate dehydrogenase family. However, both types of L-threonine dehydrogenase utilise NAD+ as a co-factor, but the C. sticklandii enzyme only utilises NAD+ as an electron acceptor whereas the E. coli enzyme also catalyses the reduction of 3-acetylpyridine adenine dinucleotide and thionicotinamide adenine dinucleotide . In L-threonine dehydrogenases purified from vertebrate livers NAD+ is the lead substrate followed by L-threonine and the products are released in the order CO2, aminoacetone and NADH . All these reaction products inhibit threonine dehydrogenase activity. The short-chain and medium-chain threonine dehydrogenase also differ in their requirement for zinc ions with the E. coli enzyme requiring one ion per subunit and the C. sticklandii enzyme being inhibited by zinc ions . Their substrate specificity differs since the E. coli enzyme will also oxidizes L-serine in addition to L-threonine . In the mouse, expression of both threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase mRNAs were found in all tissues examined, both being highest in liver. In vertebrates, threonine dehydrogenase enzymatic activity is thought to be mostly confined to the liver when the mass of the organ is taken into consideration. In the rat, the activity in kidney and heart was 30% and in brain 10% of the hepatic activity: no activity was detected in other tissues . In the pig, activity in the pancreas was similar to that of liver, but no activity was found in other tissues . The mRNA data supports the suggestion that this pathway has a wider tissue distribution than had been thought previously. In vertebrates, L-threonine is degraded by two major enzymatic pathways. In normally-fed pigs and rats 80 to 87% of L-threonine catabolism occurs via threonine dehydrogenase . Probably, the 2 major pathways have different physiological roles with the cytosolic L-serine/threonine dehydratase enzyme being highly inducible by a high protein diet, starvation and cortisone, and thought to aid the homoeostatis of blood glucose by mobilising threonine and serine for hepatic gluconeogenesis. Whereas the mitochondrial threonine dehydrogenase enzyme is thought to act in the maintenance of free somatic threonine concentration derived from dietary threonine . With the identification of threonine dehydrogenase and L-serine/threonine dehydratase genes from rodents, the way is open to study their relative contribution to threonine catabolism under different physiological states. PMID- 12097150_Conclusions TI - AB - The mouse and pig L-threonine 3-dehydrogenase cDNAs have been cloned. They encode 373 residue proteins. The mouse gene is located on chromosome 14, band C. Mammalian L-threonine 3-dehydrogenases are mitochondrial enzymes that utilise NAD+. In agreement with this, the predicted proteins have mitochondrial targeting sequences in their amino-termini followed by an NAD+ binding domain. In murine tissues, the expression of L-threonine dehydrogenase mRNA was highest in the liver. PMID- 12097150_Materials and Methods TI - AB - Molecular cloning of murine and porcine L-threonine dehydrogenase cDNAs | Clones encoding the mouse L-threonine dehydrogenase cDNA sequence were obtained by touchdown PCR amplification from mouse liver and lung cDNAs using the Advantage cDNA polymerase mix (Clontech, UK) on a Perkin-Elmer 2400 thermocycler. The cycle conditions for the first 10 cycles were 94C for 5 sec, 72C less 0.4C per cycle for 3 min and for the next 20 cycles 94C for 5 sec, 68C for 10 sec, 72C for 3 min per cycle using primers (100 nM) derived from the sequence of the mouse ESTs D21787, forward 5'-CCGGCTCCCGCGTGGCGTTCTCAGCATCCA-3' and AV100443, first reverse 5' TTTTTTTTTTTTTTTTTGATACTTAAATTG-3' and second reverse 5'-TTTTTTTTTTTGCAAGCGATCGTT-3' (Amersham-Pharmacia Biotech, UK). The porcine (Sus scrofa) L-threonine dehydrogenase cDNA was cloned from primary hepatocytes (generously provided by Dr Leonard J. Nelson, Liver Cell Biology Laboratory, University of Edinburgh, UK). Total RNA was extracted using guanidine thiocyanate and treated with DNase-I to remove any contaminating genomic DNA (SV total RNA isolation system, Promega, UK). A comparison of the murine threonine dehydrogenase cDNA sequence with the pig EST database identified similar 5' and 3' ESTs (accession Nos. BE233801 and BI400146 respectively). From these EST sequences a gene-specific reverse transcriptase primer and PCR primers were designed to amplify a cDNA region encompassing the porcine L-threonine dehydrogenase ORF. Total RNA was reversed transcribed with AMV RNase H- reverse transcriptase (ThermoScript, Life Technologies, UK) at 50C using primer 5'-GTTTAATACAAATGTCACATACTTCCACAT-3'. The cDNA was amplified by touchdown PCR, as described above, with a final annealing temperature of 58C using the PCR primers 5'-GGAAGGCTGTTGGCAGAGACC-3' and 5'-CGCTTCCTCCGGCACTTCA-3'. PCR products were examined by agarose gel electrophoresis and stained with ethidium bromide. For cloning, PCR products were excised from low-melting point agarose gels and the agarose digested with agarase (Promega, U.K.). The PCR products were cloned into the T-A vector pCR-II-TOPO (Invitrogen, The Netherlands) and sequenced in both directions using the big dye terminator cycle sequencing ready reaction kit and AmpliTaq DNA polymerase FS and run on an ABI 373XL Stretch Sequencer (both from PE Applied Biosystems, UK). Tissue distribution of L-threonine dehydrogenase mRNA by semiquantitative PCR | Murine cDNA from 8 tissues (BD Clontech, UK) was analysed for the relative expression of L-threonine dehydrogenase and the housekeeping gene, beta-actin, by real time PCR. This was carried out on a GeneAmp 5700 Sequence Detection System using a SYBR Green I double-stranded DNA binding dye assay (both from AB Applied Biosystems). Approximately 0.4 mug of cDNA from each tissue was amplified by PCR using Taq Gold polymerase. Tissue master mixes were divided into gene specific mixes with the addition of PCR primers to a final concentration of 300 muM. The L-threonine dehydrogenase primers were; sense 5'-AAGCACGCGCCTGACTTC-3' and antisense 5'-CCGAGCATTGCTGTCATCTAGA-3' and produced a 99 bp amplicon. The 2-amino-3-ketobutyrate coenzyme A ligase primers were; sense 5'-TGCACGGTTGTCTTCTCAAATG-3' and antisense 5'-ACACAGCGGTCAATGTCTTCCT-3' and produced a 150 bp amplicon. The beta-actin primers were; sense 5'-CAGAAGGAGATTACTGCTCTGGCT-3' and antisense 5'-GGAGCCACCGATCCACACA-3' and produced a 93 bp amplicon. The PCR primers were designed (with the aid of the Primer Express program, AB Applied Biosystems) to amplify cDNA with both sense primers located on the penultimate exon and the antisense primers located on the last exon. The beta-actin primers were designed to amplify beta-actin specifically and not other actin isoforms or pseudogenes. The amplification conditions were; a 10 min hot start to activate the polymerase followed by 40 cycles of 95C for 15 sec and 60C for 1 min. The number of cycles required for the SYBR Green I dye fluorescence to become significantly higher than background fluorescence (termed cycle threshold [Ct]) was used as a measure of abundance. A comparative Ct method was used to determine L-threonine dehydrogenase gene expression. Expression levels of L-threonine dehydrogenase in each tissue cDNA sample was normalised to the expression levels of the housekeeping gene beta-actin (DeltaCt). The ratios of L-threonine dehydrogenase mRNA/beta-actin mRNA from each tissue were standardised to that of liver which was taken as 100% (DeltaDeltaCt). The formula 2-DeltaDeltaCt was used to calculate relative expression levels assuming a doubling of the DNA template per PCR cycle. To confirm amplification specificity, the PCR products from each sample were examined by melting curve analysis and subsequent agarose gel electrophoresis. PMID- 12084180 TI - A comparison of hospital readmission rates between two general physicians with different outpatient review practices AB - Abstract | Background | There has been a relentless increase in emergency medical admissions in the UK over recent years. Many of these patients suffer with chronic conditions requiring continuing medical attention. We wished to determine whether conventional outpatient clinic follow up after discharge has any impact on the rate of readmission to hospital. Methods | Two consultant general physicians with the same patient case-mix but markedly different outpatient follow-up practice were chosen. Of 1203 patients discharged, one consultant saw twice as many patients in the follow-up clinic than the other (Dr A 9.8% v Dr B 19.6%). The readmission rate in the twelve months following discharge was compared in a retrospective analysis of hospital activity data. Due to the specialisation of the admitting system, patients mainly had cardiovascular or cerebrovascular disease or had taken an overdose. Few had respiratory or infectious diseases. Outpatient follow-up was focussed on patients with cardiac disease. Results | Risk of readmission increased significantly with age and length of stay of the original episode and was less for digestive system and musculo-skeletal disorders. 28.7% of patients discharged by Dr A and 31.5 % of those discharged by Dr B were readmitted at least once. Relative readmission risk was not significantly different between the consultants and there was no difference in the length of stay of readmissions. Conclusions | Increasing the proportion of patients with this age- and case-mix who are followed up in a hospital general medical outpatient clinic is unlikely to reduce the demand for acute hospital beds. PMID- 12084180_Background TI - AB - The number of emergency general medical admissions continues to grow relentlessly, many patients having repeated admissions due to exacerbations of chronic disease. The rate of readmission is sometimes used as an indicator of discharge practice performance and targets to reduce this rate have been set in the UK National Health Service . In the United Kingdom National Health Service, there are three common patterns of follow up care following an emergency general medical admission: 1) in the community, by the patient's general practitioner (primary care physician), 2) at a hospital outpatient clinic provided by the general physician who supervised the inpatient episode and 3) at a hospital specialist clinic appropriate to the patient's condition. One potential method for reducing the rate of readmissions is more frequent outpatient clinic review of patients after discharge. However, there is limited evidence regarding the effectiveness of the current practice of outpatient review following discharge from hospital in reducing readmissions. We took the opportunity of studying two physician colleagues with similar clinical practice who differed in their outpatient follow up practices. Following discharge, one reviewed twice as many patients in his personal general medical outpatient clinic than the other. The aim of this study was to identify any difference in the rate of readmission and the length of stay of readmissions associated with these different review practices. PMID- 12084180_Methods TI - AB - The emergency take at Birmingham Heartlands Hospital is sub-specialised, patients with acute myocardial infarction, respiratory conditions and infectious diseases being admitted directly under the appropriate specialist team. All other patients come under the care of the general physician of the day who practices general medicine separately from his or her speciality interest. The two physicians were general physicians at Birmingham Heartlands Hospital and had been appointed in 1993 and 1995. Both had an equal share of the general medical admitting rota and so received an equivalent random sample of the total emergency medicine admissions over the course of a 12 month period. Both had a special interest in renal medicine but this study only relates to their general medical practice. Inpatient data was taken retrospectively by DC from the routinely collected hospital activity computer database, which records all inpatient episodes. Coding was performed by professional coders using diagnoses provided by clinicians at the time of discharge. All patients who had been admitted as an emergency to General Medicine, managed by Dr Rayner or Dr Temple and subsequently discharged by them between April 1997 and March 1998 were included in the analysis. Factors associated with readmission were explored using multiple logistic regression analysis with independent variables of age, sex, length of stay of original admission episode, HRG coding and discharging consultant. Length of stay was divided into three categories, <3 days, 3 --7 days and >7 days. Since the decision to review the patient in the clinic would be affected by the HRG code and length of stay of the original admission, as well as the consultant discharging the patient, whether the patient was reviewed in the clinic was not included as an independent variable. PMID- 12084180_Results TI - AB - A total of 1366 inpatient episodes were collected from 1289 patients, 70 patients having more than one episode. In 1280 discharge episodes (1203 patients) the patient left the hospital alive and so outpatient follow up was possible. This cohort was used for subsequent analysis. Because of the specialisation in the admitting arrangements relatively few patients were coded to respiratory system (HRG3 D) (table ). There was no significant interaction between consultant and HRG code in the logistic regression analysis, confirming that the case-mix was not significant different between the two consultants. There was similarly no difference in sex (Dr A = 51.7% male, Dr B = 51.2% male) or the age distribution . Figure 1 | Age distribution for patients discharged by each consultant Age distribution for patients discharged by each consultant Table 1 | HRG3 Chapter Codes for patients discharged by the two consultants. The number of patients offered and either attending or not attending a first outpatient appointment within 90 days of discharge is shown in Table . Dr B saw twice as many patients as Dr A, 124 (19.6% of discharges) versus 63 (9.8%). The timing of outpatient clinics of those patients reviewed following discharge is shown in Figure . Dr A reviewed patients slightly earlier than Dr B. The age and HRG distributions of patients seen were very similar, 66.3% being for the cardiac HRG chapter. Hence, Dr B saw twice as many patients coded in the cardiac group (HRG3 Chapter E) as Dr A, 84 patients (30% of possible follow ups) versus 40 (15.8%). Dr B also had a higher subsequent appointment rate (58.6% versus 32.1%), giving an average number of appointments per patient of 1.59 and 1.32 respectively. Figure 2 | Cumulative percentage of patients attending first general medical out-patient review clinic with time after discharge for each consultant. Cumulative percentage of patients attending first general medical out-patient review clinic with time after discharge for each consultant. Table 2 | Number patients attending and not attending (in brackets) the first follow-up appointment within 90 days of discharge. Following discharge, a number of patients were referred to other specialist clinics rather than being followed up in the general medical clinic of the admitting consultant. Similar numbers were referred to other clinics by each consultant, 177 (25%) by Dr A and 156 (23%) by Dr B, with a similar spread of specialities. There was no difference in the referral rate to cardiology clinics (33 patients from each consultant). Re-admission rates | From the cohort of 1203 patients discharged, 411 patients were readmitted to the Trust as an emergency under any speciality in the twelve months after discharge. 28.7% of patients discharged by Dr A and 31.5 % of those discharged by Dr B were readmitted at least once. Many patients had more than one re-admission and the total number of episodes generated by these 411 patients was 800 , Dr A's cohort generating 388 and Dr B's 412 episodes. Table 3 | Readmission rates following discharge episode. The age distribution for re-admissions was more skewed to the elderly than the initial cohort . The greatest rate of re-admission occurred in the first 6 weeks after discharge and continued steadily over the subsequent 11 months . Figure 3 | Age distribution for patients readmitted following discharge by each consultant. Age distribution for patients readmitted following discharge by each consultant. Figure 4 | Cumulative growth of readmissions during 12 month period after discharge by each consultant. Cumulative growth of readmissions during 12 month period after discharge by each consultant. The HRG coding for the re-admission episode closely matched that for the initial admission (see Additional file: ). Cardiac disorders (HRG3 Chapter E) was the dominant group. 76 (28.9%) of the 263 patients in this group discharged by Dr A (38% of the discharge cohort), were re-admitted at least once, generating 143 episodes (36.9% of readmission episodes). 64 (22.1%) of the 289 patients in this group discharged by Dr B (43% of the discharge cohort), were re-admitted at least once, generating 160 episodes (38.8% of re-admission episodes). Cardiac disorders (HRG3 Chapter E) made up 66% of patients followed up in outpatients A similar proportion of the patients who attended the follow-up general medical clinics for each consultant were readmitted within the 12 months following discharge, 6 of 63 for Dr A (10%) and 11 of 124 for Dr B (9%). Variables independently associated with readmission were increasing age (expressed in units of 10 years), particular HRG codes (both Digestive system, and Musculo-skeletal system, in comparison with all other HRG's -- see table ), and increasing length of stay (LoS) of the original inpatient episode. The discharging consultant was not a "risk factor" for readmission, and there was no interaction between consultant and HRG code. Table 5 | Variables independently associated with readmission from multiple logistic regression analysis. Length of stay for categories 3 --7 days and >7 days was compared to <3 days. Length of stay | Patients who were subsequently readmitted had a longer original length of stay than those not readmitted (mean 4.9 v 3.6 days, median 3 v 2 days). The length of the readmission episodes was much longer and did not differ between the consultants (mean 8.1 v 8.1 days, median 4 v 4 days). PMID- 12084180_Discussion TI - AB - This observational study compares two consultant physicians whose general medical practice, due to the specialist admissions arrangements for respiratory or infectious diseases, focussed on cardiovascular and cerebrovascular disease and deliberate self-harm. About one-third of patients were readmitted at least once over the twelve month period following discharge and the total number of readmission episodes equalled about half of the number of discharges. The risk of readmission was greater in older patients and in those with a longer initial inpatient stay. A longer length of stay may indicate more severe disease or less adequate social circumstances. The risk was less in those with digestive and musculo-skeletal conditions (HRG3 Chapters F and H), which would have included conditions that are less likely to recur, such as gastro-intestinal bleeding and musculoskeletal pain. For the middle length of stay group (3 --7 days) and digestive conditions, p-values are marginally non-significant; but the former is part of a clear trend with increasing length of stay, and the latter is so sharply distinguished from any other risks in specific HRG's that it was included. The rate of readmission for both physicians was high. However, similar rates of readmission have been reported from the USA and Australia in similar patient groups. The risk of readmission in these studies was also associated with patient characteristics such as age, co morbidity, depression and previous readmission events rather than the quality of patient care . As both consultants reviewed a proportion of patients in the outpatient clinic, it is not possible to exclude totally an effect of general medical clinic review on the rate of readmissions. Furthermore, only one outcome of treatment, readmission, has been looked at in this study and we have no way of comparing the quality of patient care or patient satisfaction. The rate of readmission was highest in the first six weeks after discharge. Approximately 50% of follow-up appointments were made for more than six weeks after discharge and hence these could not be expected to affect the early readmission rate. It would be interesting to study the impact of earlier outpatient review on readmission rates. This study compared two physicians with different thresholds for offering general medical outpatient clinic review following discharge. Although carried out by different consultants, the clinics were otherwise run in a similar and conventional way. It did not compare different types of outpatient clinic, which may be more effective than the conventional follow up clinic. Others have found similar difficulty in reducing the rate of readmission, either by involving a general practitioner in the discharge process or running a nurse-led heart failure clinic . More promising results have been reported in two studies of intensive intervention in the patients' home following admission for stroke , in which physician or physiotherapist visits reduced readmission rates, and for heart failure , in which visits by a cardiac nurse had a similar effect. PMID- 12084180_Conclusions TI - AB - One can conclude that a practice of following up a larger proportion of patients with this age- and case-mix in the general medical outpatient clinic does not reduce the requirements for inpatient acute medical beds. These results would not support an increase in the use of general medical review clinics, of the type described in this study, in an attempt to control the growth in emergency admissions. PMID- 12084180_Competing interests TI - AB - None declared. PMID- 12084180_Authors' contributions TI - AB - HCR conceived the study and wrote the manuscript. RMT participated in the design of the study and writing of the manuscript. TM performed the statistical analysis. DC performed the data extraction and analysis. All authors read and approved the final manuscript. PMID- 12084180_Pre-publication history TI - AB - The pre-publication history for this paper can be accessed here: PMID- 12110157 TI - Application of the development stages of a cluster randomized trial to a framework for evaluating complex health interventions AB - Abstract | Introduction | Trials of complex health interventions often pose difficult methodologic challenges. The objective of this paper is to assess the extent to which the various development steps of a cluster randomized trial to optimize antibiotic use in nursing homes are represented in a recently published framework for the design and evaluation of complex health interventions. In so doing, the utility of the framework for health services researchers is evaluated. Methods | Using the five phases of the framework (theoretical, identification of components of the intervention, definition of trial and intervention design, methodological issues for main trial, promoting effective implementation), corresponding stages in the development of the cluster randomized trial using diagnostic and treatment algorithms to optimize the use of antibiotics in nursing homes are identified and described. Results | Synthesis of evidence needed to construct the algorithms, survey and qualitative research used to define components of the algorithms, a pilot study to assess the feasibility of delivering the algorithms, methodological issues in the main trial including choice of design, allocation concealment, outcomes, sample size calculation, and analysis are adequately represented using the stages of the framework. Conclusions | The framework is a useful resource for researchers planning a randomized clinical trial of a complex intervention. PMID- 12110157_Background TI - AB - Antibiotic use in nursing home residents is an important public health issue given that 50% to 75% of residents are exposed to at least one course of antibiotics annually . Such overuse can potentially lead to the development of multi-drug antibiotic resistant bacteria, drug-related adverse effects, and harmful drug interactions . The appropriate use of antibiotics in this setting is challenging. The diagnosis of infection in residents of nursing homes is often difficult, since symptoms may be vague and signs difficult to elicit . The evaluation of residents is often made by nursing staff who have varying levels of expertise and knowledge about infectious diseases. Frequently, treatment decisions are made by busy clinicians who rely on nursing assessment as their primary source of information. Interventions to optimize antibiotic use in this setting therefore need to directly involve both nurses and physicians and have the potential for strong "buy-in" from these healthcare professionals. Such interventions are aimed at changing behavior and by necessity will involve multiple components. Recently, Campbell and colleagues published a framework for the design and evaluation of complex health interventions . Derived from a document drafted by members of the MRC Health Services and Public Health Research Board, the paper is based on the premise that a different construct is required for trials that evaluate complex interventions. Examples include interventions to improve delivery of health services, to change health professionals' behavior, or to promote health through strategies aimed at individuals, schools, healthcare facilities, or communities. It is apparent that such trials pose very different challenges when compared to clinical trials designed, for example, to assess the efficacy of pharmacologic therapy . Amongst these are the development, identification, documentation, and reproduction of the interventions. Although such challenges are well known , few published strategies exist for researchers about to embark on a clinical trial of these interventions. Campbell et al. propose that the development and evaluation of such complex interventions be considered in a series of phases, analogous to the sequential phases of drug development . The design and evaluation framework proposed by Campbell et al. appears to be well suited to interventions for optimizing antibiotic use in long-term care facilities. The objective of this paper is to evaluate the extent to which the various steps in the development of a cluster randomized trial to optimize antibiotic use in nursing homes are represented in the framework for the design and evaluation of complex health interventions . Often it is not possible to evaluate complex health interventions, such as strategies to improve antibiotic use in long-term care facilities, by randomizing individuals. Cluster randomized trials randomize groups of individuals, healthcare professionals, or institutions. Applying the development steps of this trial to the framework proposed by Campbell and colleagues allows for a formal evaluation of the framework's utility. PMID- 12110157_Methods TI - AB - The five phases of the framework of Campbell et al include 1) a pre-clinical or theoretical phase, 2) Phase I, identification of the components of the intervention, 3) Phase II, definition of the trial and intervention design, 4) Phase III, methodological issues for the main trial, and 5) Phase IV, promoting effective implementation of the intervention. Using the framework, the corresponding stages of the nursing home trial development are identified and described. PMID- 12110157_Results TI - AB - Preclinical or theoretical phase | Overview | The purpose of this phase is to determine if the intervention has the potential to have the desired effect. This step can take into account previous studies demonstrating empirical evidence for the intervention as well as the theoretical basis for the intervention. Review of the theoretical basis for an intervention may lead to an altered hypothesis and improved specification of "potentially active ingredients" . The first step in the development of the algorithms was to systematically review the literature for data that could lead to strategies for reducing antibiotic use. Lower respiratory infections, urinary infections, and skin and soft tissue infections account for the majority of bacterial infections in residents of long-term care facilities . No data to support reducing antibiotics for lower respiratory or skin infections was located. In contrast, data to support reducing antibiotic use for urinary indications was found. Asymptomatic bacteriuria, the presence of bacteria in the urine in the absence of urinary symptoms, occurs in up to 50% of older institutionalized women and 35% of institutionalized older men . There are four randomized controlled trials that demonstrate no benefit in treating asymptomatic bacteriuria in residents of long-term care facilities . Despite the evidence, one third of all prescriptions for urinary indications in residents of nursing homes are for asymptomatic bacteriuria . Accordingly, the algorithms indicate that urine should not be cultured and antibiotics should not be prescribed in the absence of clinical features of urinary infection (Figures and ). The algorithms also consider the increased risk of urinary infection with indwelling urinary catheters and with specific urinary symptoms . Figure 1 | Diagnostic algorithm. Diagnostic algorithm. This algorithm guides physicians and nurses in the ordering of urine cultures for nursing home residents with suspected infections. Figure 2 | Treatment algorithm. Treatment algorithm. This algorithm allows physicians and nurses to optimize antibiotic use in residents with suspected infections. Phase I: defining components of the intervention | Overview | This phase, often inter-related to the previous phase, emphasizes modeling or simulation studies to improve understanding of the components of an intervention. Use of qualitative research and descriptive studies may help define components of the intervention. Furthermore, in this phase potential barriers in trials that seek to change behavior may be elicited . In order to assess the potential effectiveness of the two algorithms, we applied them to data from a study assessing antibiotic use in Ontario chronic care facilities . In this modeling exercise, we found that the algorithms could reduce antibiotic use for urinary indications by 80%. Previously, we had conducted focus groups among physicians and nurses who provide care to nursing home residents. Our objective was to explore the perceptions, attitudes, and opinions of those involved in the process of prescribing antibiotics for asymptomatic bacteriuria . Twenty-two physicians and 16 nurses, each in two focus groups, participated. Both physicians and nurses emphasized that there was a wide range of indicators, more subtle than the classic symptoms of urinary tract infection, that influence the ordering of cultures and the prescribing of antibiotics. Because residents in long-term care facilities frequently have cognitive impairment and are unable to articulate their symptoms, health care providers rely instead on signs of more subtle functional and behavioural changes. Misconceptions about the definition of symptoms of urinary tract infection were also discussed. Although the nurses participating in the focus groups had learned that foul-smelling urine alone was not sufficient reason to order a culture, a number of physicians still felt that this was one of the main reasons why cultures are ordered. Other key findings included the influence nurses have over physician prescribing through their description of the clinical situation, often conveyed over the telephone. Physicians attributed their use of antibiotics to treat bacteriuria in residents with no urinary symptoms to relatively vague non-urinary symptoms (e.g. lethargy, change in functional status). To these physicians, this meant that such residents were not in fact "asymptomatic", despite the fact that absence of urinary symptoms alone was used to define asymptomatic bacteriuria in the clinical trials . This was an important barrier to evidence-based care that would not have been discovered without using a qualitative approach. To assess the factors leading to the ordering of urine cultures, we conducted a prospective survey in four nursing homes. Nursing staff were asked to complete a brief questionnaire every time a request for a urine culture was made. They were asked to specify the signs and symptoms present at the time the culture was ordered and to specify who initiated the request for the culture. Physicians, after they received the results of the urine culture, were asked to indicate the clinical reasons for their therapeutic decision. Forty-eight percent of the 545 urine cultures obtained over six months were ordered by nursing staff and 52% by physicians. The urine culture was suggested by nursing staff 56% of the time. For 67% of urine culture orders, there were no urinary symptoms present at the time the culture was requested. One in every three urine cultures ordered resulted in an antibiotic prescription. Of these prescriptions, one third were for asymptomatic bacteriuria. Phase II: defining trial and intervention design | Overview | In this phase, the information gathered in phase I is used to develop the optimum intervention and study design. The feasibility of delivering the intervention is tested and the acceptability to healthcare providers and patients is assessed . The diagnostic and treatment algorithms were tested and further refined in a three-month pilot study conducted in four nursing homes. Administrators and directors of nursing of the homes felt that the algorithms should be introduced to the nursing staff by each facility's infection control practitioner. To ensure that the infection control practitioners themselves understood the algorithms, they were asked to decide whether urine cultures should be obtained and antibiotics ordered for six case-scenarios using the algorithms. Using this "train the trainer" approach, the practitioners then conducted brief seminars with registered nursing staff to introduce the algorithms in their facilities. This approach was unsuccessful however. Semi-structured interviews with staff revealed that in two of the homes there were nurses who were completely unfamiliar with the algorithms two-thirds of the way into the pilot study. This led to the development of a standardized 30-minute presentation of six case-scenarios facilitated by one of two study investigators. Participation was active, staff were asked to decide whether to order antibiotics and urine cultures and to justify their answers using the algorithms. In addition, a video-tape replicating the seminar material was made and distributed to the nursing homes. The use of logs to document appropriate adherence to the algorithms was also piloted during the three-month pilot. Office visits to introduce the algorithms to nursing home physicians were conducted using the six case-scenarios presented to nursing staff. Feedback about the algorithms from both physicians and nurses was positive, both groups felt that the algorithms were user friendly and feasible in the long-term care setting. Additionally, the initial poor adherence to the algorithms indicated that regularly scheduled on-site visits should be another component of the intervention, serving as an important reminder or cue to the participants. Meetings with the local champion for the study (usually the director of nursing) to discuss barriers or obstacles to completing the study were therefore held during the pilot. Feedback about adherence to the protocol was given. Such follow up visits were acceptable to staff and administrators as they were not overly time consuming. Phase III: methodological issues for the main trial | Overview | Campbell and colleagues state that this step addresses issues normally posed by randomized trials, such as sample size, inclusion and exclusion criteria, methods of randomization, as well as the challenges of complex interventions . The main antibiotic trial uses a randomized matched pair design where one of 11 pairs of nursing homes (matched by size and case-mix), has been randomized to the clinical algorithms and the other to usual management of presumptive urinary tract infections. Only free-standing, community-based nursing homes are eligible i.e. those representative of the majority of nursing homes in Canada and the United States. To be eligible, nursing homes must also: 1) have 100 or more residents (since the required sample size of antibiotics courses will be achieved sooner); 2) have no stated policy for diagnosis or treatment of urinary tract infections; 3) agree to refrain from introducing new management strategies for antibiotic utilization or urinary tract infection during the study. The primary outcome of this trial is the total number of antibiotic courses prescribed. Other outcomes include 1) the proportion of antibiotic courses prescribed for urinary indications, 2) rates of urine cultures ordered, 3) hospitalization rates for urinary tract infections, and 4) mortality rates. The last two outcomes are being measured to ensure that the algorithms can safely be applied. Within a nursing home, randomization of individual healthcare providers to the algorithm likely would introduce bias due to contamination. Therefore, for the quantitative component of this study, the nursing home will serve as the unit of allocation and analysis. To conceal allocation, one nursing home in each pair was arbitrarily assigned an even number (and the other odd) by a hospital administrative assistant not associated with the study or any of the investigators and who was blinded to the nursing homes' identity. A statistician who is not associated with the study and who was unaware of the nursing home pairs, generated a list of 11 random numbers using a random numbers table, one for each pair of nursing homes. The corresponding odd or even numbered nursing home in each pair was assigned the intervention. An absolute reduction in 20% of all antibiotic use, which corresponds to a reduction in the proportion of antibiotic prescriptions for urinary indications from 30% to 10%, was considered the minimal clinically important effect worth detecting. To detect this difference, for an alpha of 0.05 and 80% power, adjusting for the effect of within cluster dependency, 4513 prescriptions need to be collected in total. This means that 20 or 10 pairs of nursing homes will need to be followed for 10 months. Given the potential for withdrawals of nursing homes, 22 homes will be recruited. A paired t-test will be used to analyse the within-pair differences between the proportions of antibiotics prescribed for urinary indications in matched pairs of nursing homes. This way, the fact that the denominator of the proportions is actually an outcome is taken into consideration. Differences in rates of overall antibiotic use (antibiotic courses per 1000 resident days) will be compared using a paired t-test. Rates of antibiotic use for urinary indications (antibiotic courses per 1000 resident days and defined daily dosages/1000 resident days), rates of urine cultures obtained (urine cultures per 1000 resident days), rates of hospitalization (per 1000 resident days), and overall mortality rates will be compared using paired t-tests and Wilcoxon signed rank test. Logistic regression analysis that adjusts for the effect of clustering will be performed to adjust for potentially important co-variates such as nursing home size, proportion of residents bed/wheelchair bound, and pharmacy automatic stop dates. Phase IV: promoting effective stability of the intervention, assessing for the existence of adverse effects | Qualitative studies to assess the attitudes and perceptions of both nurses and physicians will help us understand the key elements to maintaining sustainability should the intervention prove to be successful in reducing antibiotic use. We plan to conduct focus group studies for nursing staff and semi-structured interviews amongst physicians and directors of nursing care. If the intervention is not successful, this qualitative approach may help explain the barriers leading to failure of the intervention. Although the trial itself is meant to examine the effectiveness of the intervention, including adherence and adverse effects, if the results demonstrate that the intervention can reduce overall antibiotic use and is safe, one possibility is to conduct a cohort study of the algorithms in nursing homes over a large geographic area will be made. PMID- 12110157_Discussion TI - AB - The application of the trial to improve antibiotic use in nursing home residents to the framework demonstrates that the framework is a useful resource for health services researchers. Not surprisingly, the stages of the framework have been used in previous studies of complex interventions, even before the framework was published. One recent example is the Rapid Early Action for Coronary Treatment (REACT), a multi-component, community-based educational intervention of community development, public education, provider education, and patient education (31). The description of the trial described in this paper however allows for a focused assessment of the components of this framework. The pre-clinical phase of the antibiotic trial involved a summary of the evidence needed to construct the algorithms. This phase should include a systematic review of the literature with respect to the intervention , a point omitted in the pre-clinical phase description of the design paper but discussed in the Medical Research Council paper described in the text . It was also evident that the preclinical and first phase cannot be clearly demarcated, given that it was the modeling study in phase I which actually determined if the intervention had the potential to have the desired effect, the stated aim of the preclinical phase . The use of qualitative research to help define the intervention was extremely valuable. Focus group studies helped to accurately identify and anticipate barriers to the intervention, even prior to the pilot study. Determining physicians and nurses attitudes, beliefs, and behaviors toward antibiotic use for urinary infections highlighted the importance of nonspecific, non-urinary symptoms and the key role of nurses in the process of antibiotic treatment of asymptomatic bacteriuria in institutionalized older adults. As specified in the framework, these findings illustrate the utility of using qualitative methods early in the development of complex interventions. Focus groups and semi-structured interviews will also be conducted at the end of the main trial. We anticipate that such qualitative data will help us identify strategies to sustain the algorithms should the algorithms prove to be effective in reducing antibiotic use . Results of the questionnaires administered to nurses and physicians used in developing the algorithms provided convincing evidence about the need to target both ordering of urine cultures and antibiotic prescribing in order to reduce overall antibiotic use. The particular importance of introducing the algorithms to nurses in order to culturing of urine was emphasized. This confirmed the qualitative findings. It is notable that although the phases of the framework are described as being sequential , components of the frameworks might vary temporally, as exemplified by the qualitative and questionnaire data described in phase I being collected prior to the actual development of the algorithms. The pilot study was instrumental in refining and introducing the algorithms. One of the most valuable aspects of the pilot study was finding the optimal way to introduce the algorithms to nurses and physicians. Perhaps more emphasis could have been placed on strategies to introduce the complex health interventions in the design framework . An important methodological issue for randomized trials of complex health interventions is allocation concealment. This was a key consideration in the design and implementation of the antibiotic trial. Empiric evidence exists to suggest that failure to conceal allocation can result in larger biased estimates of effect than failure to blind . When a limited number of clusters are randomized, such as in this trial, there is extra concern . Although it is often impossible to blind patients, practitioners, and researchers to the intervention, it is always possible to conceal allocation . Such issues are well described in the Medical Research Council paper described in the text . Sample size calculation is an important challenge in cluster-randomized trials. Cluster randomization reduces power and the sample size generally needs to be substantially increased. Further elaboration on this topic in the Phase III of the framework would be of benefit to researchers. PMID- 12110157_Conclusion TI - AB - The application of a randomized trial to optimize antibiotic use in nursing homes to the framework reported by Campbell and colleagues demonstrated that the framework is a useful resource for researchers embarking on a clinical trial of a complex health intervention. PMID- 12110157_Competing interests TI - AB - None declared. PMID- 12110157_Pre-publication history TI - AB - The pre-publication history for this paper can be accessed here: PMID- 12121577 TI - Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases AB - Abstract | Background | The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised. Results | We have characterised two novel human gene products, hAps1, encoded by the NUDT11 gene, and hAps2, encoded by the NUDT10 gene. These cytoplasmic proteins are members of the DIPP subfamily of Nudix hydrolases, and differ from each other by a single amino acid. Both metabolise diadenosine-polyphosphates and, weakly, diphosphoinositol polyphosphates. An apparent polymorphism of hAps1 has also been identified, which leads to the point mutation S39N. This has also been characterised. The favoured nucleotides were diadenosine 5',5"'-pentaphosphate (kcat/Km = 11, 8 and 16 x 103M-1s-1 respectively for hAps1, hAps1-39N and hAps2) and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 x 103M-1s-1 respectively for hAps1, hAps1-39N and hAps2). Both hAps1 and hAps2 had pH optima of 8.5 and an absolute requirement for divalent cations, with manganese (II) being favoured. Magnesium was not able to activate the enzymes. Therefore, these enzymes could be acutely regulated by manganese fluxes within the cell. Conclusions | Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10. We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers. PMID- 12121577_Background TI - AB - In addition to the canonical ribonucleoside and deoxyribonucleoside phosphates and cofactors, cells contain a large number of minor nucleotides. Among these are the diadenosine polyphosphates (ApnA, where n = 2 --7 ). Ap3A and Ap4A are the most intensively studied of these and are generally present in the soluble fraction of eukaryotic and prokaryotic cells at concentrations between 10 nM and 5 muM . Platelet dense granules, adrenal chromaffin granules and certain synaptic vesicles have been reported to contain high concentrations of Ap5A and Ap6A in addition to Ap3A and Ap4A, all of which can be exocytosed following appropriate stimuli and bind to target cell purinoceptors causing a variety of physiological responses in the cardiovascular and central and peripheral nervous systems [,-]. However, although Ap6A has been detected in erythrocytes , there are no substantiated measurements of Ap5A and Ap6A in the soluble fraction of nucleated cells, and it is likely that they are typically present at concentrations much lower than those of Ap3A and Ap4A. The estimated 7 muM Ap5A reported in cardiac muscle may also be confined to granules. Whilst the extracellular diadenosine polyphosphates have partially characterised signalling properties, the possible functions of the soluble, intracellular compounds remain unclear. On the one hand, they may simply be by-products of several enzymic reactions (e.g. those catalysed by aminoacyl-tRNA synthetases and other ligases ). On the other, they may have one or more of a number of important regulatory roles, including involvements in DNA replication/repair, metabolic stress responses, the determination of cellular fate, and the regulation of enzyme activities and ion channels (see ). For example, Ap5A is a potent inhibitor of adenylate kinase . It is clear that cells have a variety of relatively specific enzymes able to degrade these compounds. These include symmetrically-cleaving Ap4A hydrolases (in prokaryotes), members of the histidine triad protein family such as the FHIT tumour suppresser protein and (in lower eukaryotes) the related Ap4A phosphorylases, and most widespread of all, members of the Nudix hydrolase family (see for reviews). Nudix hydrolases cleave predominantly the diphosphate linkage in compounds of general structure: nucleoside diphosphate linked to another moiety, X. Some family members have a relatively broad substrate specificity, while others have a much more restricted range. They all possess the Nudix sequence signature motif Gx5Ex5 [UA]xREx2EexGU (where U is an aliphatic hydrophobic amino acid), which represents the catalytic site of the enzyme. Animal and plant Ap4A hydrolases (EC 3.1.6.17) degrade Ap4A, Ap5A and Ap6A, always producing ATP as one of the products. They are most active towards Ap4A; however, the related enzymes from Escherichia coli and Rickettsia prowazekii show a marked preference for Ap5A. More recently, distinct members of the Nudix family that prefer Ap6A have been purified and characterised. The three human enzymes termed diphosphoinositol polyphosphate phosphohydrolases 1 , 2alpha and 2beta (hDIPP-1, -2alpha and -2beta) as well as Schizosaccharomyces pombe Aps1 and Saccharomyces cerevisiae Ddp1p all favour Ap6A over Ap5A. Activity with Ap4A is lower, and is in fact lacking in the case of Ddp1p. Remarkably these Ap6A hydrolases are also able to utilise the structurally unrelated non-nucleotide substrates, diphosphoinositol pentakisphosphate (PP-InsP5) and bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4), hence they are commonly referred to as the DIPP subfamily. These substrates are generally favoured in vitro, with kcat/Km ratios being 50 --500-fold higher for PP-InsP5 compared to Ap6A, except for DIPP-2beta, which has a significantly reduced relative activity with PP-InsP5 compared to the others. The intracellular levels of diphosphoinositol polyphosphates appear to be lower than those of the nucleotide substrates. PP-InsP5 levels are often between 1 and 3% of the levels of InsP6, which has been estimated as being between 15 and 100 muM (see ) and [PP]2-InsP4 levels are an order of magnitude lower , and as such are virtually undetectable in many cell types. PP-InsP5 turnover is regulated by the tumour promoter thapsigargin , while [PP]2-InsP4 turnover is regulated by cyclic AMP- and cyclic GMP-dependent processes via an undefined mechanism . However, as for the ApnAs, no clear functions have yet been found for the diphosphoinositol polyphosphates, although they have been implicated in vesicle trafficking , apoptosis and DNA repair . We describe here two further members of the human DIPP subfamily with novel properties. These proteins, hAps1 and hAps2, products of the NUDT11 and NUDT10 genes respectively, closely resemble the other DIPPs in primary structure, but show a selectivity towards Ap5A and Ap6A and reduced activity towards PP-InsP5 and [PP]2-InsP4. They also display a novel pattern of tissue-specific expression. PMID- 12121577_Results TI - AB - Sequence alignment | An initial BLAST search of the GenBank expressed sequence tag (EST) database with the hDIPP-1 sequence identified two closely related but previously uncharacterised predicted proteins, which we have called hAps1 and hAps2 (human ApsixA hydrolases 1 and 2). Alignment of the cDNA sequences with the human genome indicated that the genomic sequences encoding these proteins, FLJ10628 and LOC139770, lie about 150 kb apart on the X chromosome at Xp11.23 and are transcribed in opposite directions . In accordance with the guidelines for the Nudix protein family, the genes for hAps1 and hAps2 have been designated NUDT11 and NUDT10 respectively by the HUGO Gene Nomenclature Committee (see ). These proteins are each 164 amino acids long and are most similar to hDIPP-2beta, hAps1 showing 76, 90 and 91% identity at the amino acid level to hDIPP-1, 2alpha and 2beta, respectively. In particular, they both possess the additional Gln residue (Q86 in DIPP-2beta) that distinguishes hDIPP-2beta from hDIPP-2alpha and hDIPP-1 and which is responsible for the reduced activity of hDIPP-2beta towards diphosphoinositol polyphosphates compared to the other DIPPs. hAps1 and hAps2 are also identical to the hDIPP-2 enzymes throughout the Nudix motif . These observations led us to anticipate that hAps1 and hAps2 would be potent dinucleoside polyphosphate hydrolases with reduced activity towards diphosphoinositol polyphosphates. The existence of two such similar expressed gene products was surprising. Comparison of the open reading frames (ORFs) of hAps1 and hAps2 revealed 6 differences at the nucleotide level, leading to five silent changes (T8, E28, E69, D88 and stop), and an Arg-Pro substitution at position 89. A comparison of the cDNAs for the two gene products showed further divergence in the 3' untranslated region. It appears that the evolution of these closely related genes occurred recently, as hAps1 and hAps2 show greater similarity with each other than with the Aps-like sequences from mouse (of which two genes, XM_135784 and XM_135786 appear to yield the same protein product) or cow . Further examination of all available hAps1 ESTs revealed a sequence divergence, leading to a point mutation S39N, in the coding region. This is present in two ESTs and was detected in 10% (3 from 30) of polymerase chain reaction (PCR) products generated from a range of normal human cDNA samples from diverse sources. In all other respects investigated, including the 3' untranslated region, it appeared to be identical to hAps1. This would suggest that this minor variant, hAps1-39N, represents a polymorphism present in the human population. Its properties were also examined in this study. Figure 1 | The NUDT11/NUDT10 region of chromosome Xp11. The NUDT11/NUDT10 region of chromosome Xp11.23. The two known exons of NUDT11, encoding hAps1, and NUDT10, encoding hAps2 are shown represented as boxes. Introns are represented by angled connecting lines. The coding region of each gene is shaded. Solid arrows indicate the direction of transcription of each gene. Figure 2 | Sequence alignment of hAps1, hAps2, hDIPP-1 and hDIPP-2 (alpha and beta). Sequence alignment of hAps1, hAps2, hDIPP-1 and hDIPP-2 (alpha and beta). The figure shows an alignment of the amino acid sequences of hAps1, hAps2, hDIPP-1 and hDIPP-2 (alpha and beta), generated by the MAP algorithm. Black shading denotes amino acids in at least two of the proteins were identical. Physicochemical similarity is denoted by grey shading. Figure 3 | Comparison of hAps1 with translated EST sequences from mouse and cow. Comparison of hAps1 with translated EST sequences from mouse and cow. No full-length bovine EST for the hAps1 homologue is available, hence the composite of two separate sequences is shown. A predicted rat protein from genomic sequencing is also shown. Table 1 | Tissue sources of known ESTs for hAps1 and hAps2 Purification and properties of hAps1 and hAps2 | The ORFs for hAps1, hAps1-39N and hAps2 were cloned into the pGEX6P-1 expression vector and expressed in Escherichia coli (E. coli). After incubation with isopropyl beta-D-thiogalactoside (IPTG) (100 muM) at 26C for 8 hours, a major protein of the expected size was induced and expressed in a soluble form in each case. The glutathione S-transferase (GST)-tagged recombinant proteins were purified by chromatography on Glutathione Sepharose 4 Fast Flow resin . Figure 4 | SDS-PAGE of purified hAps1, hAps1-39N and hAps2. SDS-PAGE of purified hAps1, hAps1-39N and hAps2. GST-tagged hAps proteins were prepared as described under "Materials and methods". Approximately 10 mug of (a) hAps1, (b) hAps1-39N and (c) hAps2 were analysed by SDS-PAGE. Purity was determined by Coomassie-blue staining a 4 --12% Bis-Tris NuPage gel (Novex). Molecular weight standards (MultiMark multi-colored standards) were from Novex. Initially, the ability of hAps1 and hAps2 to metabolise Ap5A was determined at 37C in the presence of 50 mM HEPES, pH 7.6 and 100 muM substrate. A divalent cation was essential for activity, with Mn2+ by far the most effective between 2 and 6 mM . Cu2+ supported less than 30% and Zn2+ and Co2+ each less than 3% of the maximum activity. Ni2+, Ca2+ and, surprisingly, Mg2+ were unable to activate hAps1 or hAps2. When assayed in the presence of 1 mM MnCl2, hAps1 and hAps2 showed alkaline pH optima of pH 8.5 , a feature common among Nudix hydrolases. Activity increased from undetectable at pH 6 to over 70% maximum between 7.5 and 9. Previous characterisation of hDIPP-1, 2alpha and 2beta, as well as S. pombe Aps1 was performed in a buffer containing 1 mM MnCl2 at pH 7.6. In order to compare results for hAps1 and hAps2 directly with hDIPP-1, hDIPP-2 and S. pombe Aps1, we have performed assays using identical conditions. Figure 5 | Properties of hAps1 and hAps2. Properties of hAps1 and hAps2. (a) Mn2+-dependency of hAps1 (filled circles) and hAps2 (open circles) activity towards Ap5A (100 muM). (b) pH dependency of hAps1 (filled circles) and hAps2 (open circles) activity towards Ap5A (100 muM). When hAps1, hAps1-39N and hAps2 were assayed with a wide range of potential substrates all three proteins were found, as expected, to metabolise Ap5A and Ap6A. Of the other substrates tested, only p4A and p4G showed significant rates of hydrolysis by all three, while Ap4A and Gp4G were also effective substrates for hAps2. All three enzymes showed a marked preference for adenine over guanine nucleotides. Table shows rates of metabolism and products formed where identified. Km and kcat values for each enzyme with Ap5A and Ap6A are shown in Table . The most rapidly metabolised substrate appeared to be Ap5A, although Ap6A was bound with higher affinity. Table 2 | Nucleotide substrate utilisation by hAps1, hAps1-39N and hAps2 Table 3 | Kinetic parameters of hAps1, hAps1-39N and hAps2 Prolonged treatment of Ap6A and Ap5A identified the major products as adenosine 5'-monophosphate (AMP) plus adenosine 5'-pentaphosphate (p5A) and AMP plus adenosine 5'-tetraphosphate (p4A) respectively. In neither case did p5A or p4A accumulate as these are metabolised almost as rapidly as they are formed, p5A being hydrolysed sequentially to p4A then ATP. In this respect, hAps1 and hAps2 show similar modes of action to hDIPP-1 and hDIPP-2 (S.T. Safrany, data not shown). Nudix hydrolases have been found to be sensitive to inhibition by fluoride. Hydrolysis of Ap5A by hAps1 and hAps2 was found to be inhibited non-competitively with Ki values of 13.0 +- 0.5 and 30 +- 1 muM respectively (data not shown). Despite the overall high degree of sequence similarity between hAps1, hAps2 and the DIPPs, hAps1 and hAps2 were found to have little activity towards PP-InsP5 or [PP]2-InsP4. hAps1 and hAps1-39N showed first order rate constants (k-1) with PP-InsP5 of 19 +- 4 and 7 +- 5 mug-1min-1 respectively. Activity of hAps2 was similar to that of hAps1, with a k-1 = 13 +- 6 mug-1min-1. These compare with k-1 values of 2200, 2000 and 220 mug-1min-1 for hDIPP-1, -2alpha and -2beta respectively . Km and kcat values were determined for each of the proteins and these suggested that the reduction in activity towards PP-InsP5 compared with hDIPP-1 and hDIPP-2alpha was primarily due to a reduced affinity for this substrate. Activity towards [PP]2-InsP4 was even weaker. hAps1 and hAps2 gave k-1 = 1.0 +- 0.2 and 0.9 +- 0.2 mug-1min-1 respectively. hAps1-39N showed no activity towards [PP]2-InsP4 under the conditions tested. In contrast, hDIPP-1, -2alpha and -2beta gave k-1 values of 320, 90 and 32 mug-1min-1 respectively, whereas S. pombe Aps1 and S. cerevisiae Ddp1p showed similar activity with k-1 = 8.3 and 1.7 mug-1min-1 respectively . Tissue distribution | Analysis of the tissue distribution of hAps1 and hAps2 gene expression was performed using PCR and human tissue cDNA samples. Using primers selective for hAps1, PCR products were readily visible from brain, pancreas and testis and less so from lung, thymus, prostate, ovary, small intestine and heart (the least) . Selective primers for hAps2 produced strong signals from brain and liver, but also from heart, placenta, lung, kidney, pancreas, spleen, prostate, testis and ovary . No signal for hAps1 was detected in placenta, liver, skeletal muscle, kidney, spleen, colon or peripheral blood leukocytes. Likewise, no signal for hAps2 was found in skeletal muscle, thymus, small intestine, colon or peripheral blood leukocytes. A number of tumour cell line cDNA samples were also tested; hAps1 was present in prostate PC3, and ovary GI-102 cells. The prostate PC3 cell line was also found to express hAps2. Tumour cell lines from breast GI-101, lung LX-1, colon CX-1, lung GI-117, colon GI-112 and pancreas GI-103 were found to express neither hAps1 nor hAps2. These results are in broad agreement with the sources of ESTs for these two protein families . This list is for comparison and is not exhaustive. In contrast, hDIPP-1 was found in all the tissues and cell types tested above (S.T. Safrany, unpublished data). Figure 6 | Tissue distribution of hAps1, hAps2 and beta-actin. Tissue distribution of hAps1, hAps2 and beta-actin. PCR using human tissue first-strand cDNA as template was performed with primers specific for beta-actin and for hAps1 or hAps2. 35 cycles of amplification were used for beta-actin, 40 cycles for hAsp1 and hAps2. Whilst an exhaustive analysis has not been performed for hDIPP-2, it was found to be highly expressed in heart, skeletal muscle, kidney and pancreas, and weakly expressed in brain, placenta lung and liver . Subcellular localisation | The subcellular localisation of hAps1, hAps1-39N and hAps2 was investigated by individual expression of the three proteins tagged at the N-terminus with EGFP in HEK293 and PC12 cells, and visualisation of the expressed fusion proteins by deconvolved fluorescence microscopy. These results show that in both cell types EGFP-tagged proteins showed a cytosolic location indistinguishable from EGFP-hDIPP-2alpha, EGFP-hDIPP-2beta and EGFP alone (Fig. and ). Similar data were obtained with proteins tagged at the C-terminus with EGFP (data not shown). This is in agreement with localisation predicted using PSORTII. Figure 7 | Subcellular localisation of EGFP, EGFP-hDIPP-2 (alpha and beta) and hAps1 and hAps2. Subcellular localisation of EGFP, EGFP-hDIPP-2 (alpha and beta) and hAps1 and hAps2. Proteins fused to the C-terminus of EGFP were expressed in (a) HEK293 or (b) PC12 cells for 24 h and visualised by fluorescence microscopy. PMID- 12121577_Discussion TI - AB - The human genome contains at least 18 Nudix genes, several of which encode multiple products, and 9 Nudix hydrolases have been characterised so far to varying degrees (see ). These include hDIPP-1, hDIPP-2alpha and hDIPP-2beta, encoded by the NUDT3 (hDIPP-1) and NUDT4 (hDIPP-2alpha and hDIPP-2beta) genes, all of which hydrolyse both the higher order diadenosine polyphosphates and diphosphoinositol polyphosphates. In this report, we describe the cloning and functional characterisation of hAps1 and hAps2, two new members of the DIPP subfamily with a novel pattern of expression and substrate specificity. A polymorphism of hAps1 (hAps1-39N) has also been found, its activity and localisation appearing broadly similar to those of hAps1. Unlike the other subfamily members, hAps1 and hAps2 show little activity towards diphosphoinositol polyphosphates, both gene products favouring diadenosine polyphosphates, ApnA, where n = 5 or 6. In each case, Ap6A binds with higher affinity than Ap5A, whereas the kcat for the latter is greater. Given that no other PP-InsP5 hydrolase activity was observed during the purification of rat hepatic DIPP (most similar to hDIPP-1) and assuming that tissue expression and physiology are broadly conserved between human and rat, these results would suggest that Aps-like proteins do not contribute significantly to inositol phosphate metabolism. Both hAps1 and hAps2 show typical Nudix hydrolase requirements, namely an alkaline pH optimum in vitro and a requirement for divalent cations . An unusual feature of hAps1 and hAps2 is that they both require Mn2+ for activation, and that Mg2+ is without effect. Optimal [Mn2+] is approximately 4 mM, which is in contrast to that estimated for free intracellular [Mn2+] of below 0.5 muM . Free [Mn2+] in our in vitro assays was not determined. It is well acknowledged that Mn2+ binds serum albumin very tightly, but despite this we would anticipate free [Mn2+] in the assays to be far in excess of intracellular levels. It is known that the activity of metal ion-requiring enzymes in different cellular compartments can be regulated through the controlled trafficking of these ions and therefore through the controlled access of these enzymes to their required ions. This principle has been most thoroughly studied in the case of Ca2+, although analogous observations have been made with Cu2+ and Mn2+. Since the [Mn2+] throughout the cell is known to be non-uniform, the unusual requirement of the hAps enzymes suggests a possible mechanism of regulation through access to this ion. Several biochemically distinct Ap5/6A hydrolases have been described previously. S. pombe Aps1, S. cerevisiae Ddp1p and hDIPP-1 and hDIPP-2 have all been shown to metabolise Ap5A and Ap6A. The activities of hAps1 and hAps2 towards Ap5A and Ap6A are comparable to hDIPP-1, but their activities towards the diphosphoinositol polyphosphates PP-InsP5 and [PP]2-InsP4 are greatly reduced, thus making them some 100 --300-fold more selective towards Ap5A and Ap6A than hDIPP-1 or hDIPP-2. S. pombe Aps1 and S. cerevisiae Ddp1p, the sole representatives of the DIPP subfamily in the two yeast genomes, appear to favour the diphosphoinositol polyphosphates. Their modes of action have been shown to differ from their mammalian counterparts. The major products of Ap6A hydrolysis by S. pombe Aps1 and S. cerevisiae Ddp1p are ADP and p4A . As with hDIPP-1 and hDIPP-2 (Safrany, data not shown), hAps1 and hAps2 produced AMP and p5A as major products. With Ap5A as substrate, S. pombe Aps1 favoured the production of ADP plus ATP, whereas S. cerevisiae Ddp1p favoured production of AMP and p4A. In this respect hAps1 and hAps2 as well as hDIPP-1 and hDIPP-2 resemble Ddp1p. Due to the chromosomal proximity of Aps1 and Aps2, and the greater degree of sequence identity with each other than with the Aps-like genes from mouse, rat or cow, it seems likely that this gene pair has arisen as the result of a recent gene duplication event on the short arm of the X chromosome . A popular view of the evolution of catalytic motifs envisages the duplication of relatively non-specific progenitors followed by adaptation to perform special tasks, and the Nudix hydrolase motif appears to be a good illustration of this process. The existence of multiple forms of these enzymes would suggest that the diadenosine polyphosphates have significant biological functions, with the specialised task of hAps1 and/or hAps2 being the regulation of Ap5A and Ap6A levels. PMID- 12121577_Conclusions TI - AB - Previously, the human DIPP family comprised hDIPP-1, a ubiquitous enzyme able to hydrolyse both inositol phosphates and diadenosine polyphosphates, and hDIPP-2alpha and -2beta, which are predominantly diphosphoinositol polyphosphate phosphohydrolases highly expressed in heart, skeletal muscle, kidney and pancreas. We have now identified two further members, hAps1 and hAps2, which have high selectivity towards the diadenosine polyphosphates. The distinction between DIPP and ApnA hydrolase activities reinforces the importance of Q86 in DIPP-2beta and the role of Q85 in hAps1 and hAps2. The addition of this amino acid in hDIPP-2 has been previously shown to have a substantial negative effect upon its activity towards diphosphoinositol polyphosphates, whereas Ap5A hydrolase activity was increased. The expression of hAps1 and hAps2 is also restricted. Of the tissues tested, only pancreas showed high levels of hDIPP-2 and hAps1, and only heart showed high levels of hDIPP-2 and hAps2, although ESTs were found suggesting that hDIPP-2 is expressed in testis, ovary and prostate. The presence of hAps1 and hAps2 in such a variety of tissues suggests that Ap5A and Ap6A may have roles other than as secreted molecules. We find that hAps1 is present in the pancreas, but there is no suggestion that it is secreted. For this reason, hAps1 and hAps2 are expected to control intracellular Ap5A and Ap6A levels. In conclusion, we have identified two human Nudix hydrolases that share a novel substrate specificity, metabolising predominantly Ap5A and Ap6A. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept of Ap5A and Ap6A acting as intracellular messengers. PMID- 12121577_Authors' contributions TI - AB - NRL performed the cloning and subcloning, determined subcellular localisation in mammalian cells, participated in sequence analysis and obtained the genomic alignment. AGMcL assisted in the writing of the manuscript. STS conceived of this study, prepared recombinant enzymes, performed enzyme assays, determined the tissue distribution, participated in sequence analysis and drafted the manuscript. All authors read and approved the final manuscript. PMID- 12121577_Materials and Methods TI - AB - Materials | Ap4G, Ap5G and p4A were purchased from Jena Bioscience, Jena, Germany. Other nucleotides were purchased from Sigma. Non-radioactive PP-InsP5 was a kind gift from J.R. Falck (University of Texas Southwestern Medical Center, Dallas, TX, USA). [3H]-PP-InsP5 and [3H]-[PP]2-InsP4 were prepared as previously described . Cloning and plasmid construction | PCR products corresponding to the hAps1 sequence were amplified from a human testis library (Clontech), using the primers SSD019 (5'-ggcaggatccaagtgcaaacccaaccagacg-3') and SSD020 (5'-ggcaggatccttagggatcgctatctggcg-3') (BamHI sites are underlined). PCR was carried out using a HiFidelity Expand Kit (Roche), and the products cloned into the vector pCR2.1TOPO (Invitrogen) and sequenced. Surprisingly, three variant sequences were found (termed hAps1, hAps1-39N and hAps2). These were subcloned as BamHI fragments into the BamHI site of pGEX-6P1 (Amersham Pharmacia Biotech) (starting at K2, to compare with K3 of hDIPP-1 and hDIPP-2) and fully re-sequenced. The 3' nucleotide of SSD019 introduced a silent mutation into hAps2, and was allowed to remain. Likewise, SSD020 also introduced a silent mutation into the stop codon of hAps2. This was also allowed to remain. Vectors for the expression of Nudix hydrolase proteins fused to the C-terminus of Enhanced Green Fluorescent Protein (EGFP) were produced by ligating a cDNA encoding each protein as a BamHI digestion fragment from the GST-fusion vectors described above, into pEGFP-C1 (Clontech) previously digested with BglII and BamHI. Bioinformatics | Multiple sequence alignments were performed using MAP and BoxShade, both run by EMBnet, Lausanne, Switzerland. Blast searches of EST and genomic databases were performed using the facilities provided by NCBI, Bethesda, MD, USA. Expression and purification of hAps1, hAps1-39N and hAps2 | Full-length hAps1, hAps1-39N and hAps2 were expressed and purified from E. coli as GST-fusion proteins. Expression plasmids based on pGEX-6P1 were transformed into E. coli strain BL21, and induced at 26C overnight with 100 muM IPTG. Cells were harvested in buffer A (20 mM Tris, 150 mM NaCl, 2 mM DTT, 0.1 mM EGTA, pH 7.5), supplemented with 5 mug/ml leupeptin and 1 mug/ml aprotinin, and disrupted by sonication (3 x 15s). Particulate matter was removed by centrifugation. The supernatant was applied to (and subsequently eluted from) a 5 mm x 5 cm Glutathione Sepharose 4 Fast Flow column (Amersham Pharmacia Biotech) at a flow rate of 1 ml/min. The column was washed for 5 min with buffer A. Bound protein was eluted with a gradient generated by mixing buffer A with buffer B (buffer A plus 25 mM glutathione, pH 7.5) as follows: 0-10 min, 0-50% buffer B; 10-13 min, 100% buffer B. Fractions containing pure hAps1, hAps1-39N or hAps2 were supplemented with glycerol (10% v/v final), and stored at -80C. Tissue distribution | The tissue distribution of hAps1 and hAps2 expression was determined by PCR from human tissue cDNA samples (human cDNA panels, Clontech) and human cDNA libraries (testis from Clontech, brain from Dr Peter Cheung of the MRC Protein Phosphorylation Unit, Wellcome Trust Biocentre, Dundee, Scotland, UK). beta-Actin-specific and hAps1 and hAps2-specific primers were used. The hAps1 primers were SSD049 (5'-cgtcttcgaacagaaccaggatcg-3'), and SSD045 (5'-caaaagccacacacatggtgcc-3') and the hAps2 primers were SSD050 (5'-cgtcttcgaacagaaccaggaccc-3') and SSD046 (5'-gtgcaacaacctggagaatagtcattgta-3'). Primers SSD045 and SSD046 were selective for the 3' untranslated regions of hAsp1 and hAsp2 respectively. beta-Actin cDNA was amplified using beta AS (5'-acactgtgcccatctacgaggg-3') and beta AA (5'ccttctgcatcctgtcagcaatg-3'). PCR was carried out using a HiFidelity Expand Kit (Roche), and cDNA samples from heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas (human MTC panel I #K1420-1, Clontech), spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes (human MTC Panel II #K1421-1, Clontech). The following tumour cell lines were also tested: breast GI-101, lung LX-1, colon CX-1, lung GI-117, prostate PC3, colon GI-112, ovary GI-102 and pancreas GI-103 (human MTC Tumor Panel #K1422-1, Clontech). Subcellular localisation | The subcellular localisation of hAps1, hAps1-39N and hAps2 proteins fused to EGFP was investigated using transfected HEK 293 epithelial cells and PC12 pheochromocytoma cells. PC12 cells were grown in DMEM/5% foetal bovine serum/5% horse serum. HEK293 were grown in DMEM/10% foetal bovine serum. Both cell types were grown on coverslips and transfected using Fugene-6 (Roche) with expression vectors for each Nudix hydrolase fused to the C-terminus of EGFP. 24 hours after transfection, cells were observed using a Leica inverted stage fluorescence microscope and a Hamamatsu Orca charge-coupled-device camera. Images were analysed using Improvision OpenLab deconvolution software. Enzyme assays | For the analysis of nucleotide metabolism, assays were performed as previously described , unless otherwise stated. The UV-absorbing (260 nm) nucleotide reaction products were analysed using a 4.6 x 125 mm Partisphere 5 mum SAX HPLC column. Substrates and products were eluted at 1 ml/min by the following gradient generated by mixing water with buffer C (1.3 M (NH4)2HPO4, pH 4.8 with H3PO4): 0-5 min, 0% buffer C; 5-55 min, 0-50% buffer C; 56-70 min, 0% buffer C. pH optima of recombinant enzymes were determined in a series of buffers comprising 1 mM MnCl2, 100 mug/ml BSA and 15 mM MES, 15 mM HEPES or 15 mM Tris (pH 6-9 with HCl or NaOH). Divalent ion-dependency was determined in a buffer comprising 50 mM HEPES, 100 mug/ml BSA, pH 7.6, and 50 muM-10mM (where possible) divalent metal as the chloride salt. Inositol phosphate metabolism was determined exactly as described previously . Km and kcat values were determined by hyperbolic regression analysis. PMID- 12121577_Authors' note TI - AB - During the reviewing of this manuscript a paper was submitted and accepted in the Journal of Biological Chemistry (K Hidaka, JJ Caffrey, L Hua, T Zhang, JR Falck, GC Nickel, L Carrel, LD Barnes, SB Shears: An adjacent pair of human NudT genes on chromosome X are preferentially expressed in testis and encode two new isoforms of diphosphoinositol polyphosphate phosphohydrolase. J Biol Chem in press). DIPP-3beta corresponds to hAps1, whereas DIPP-3alpha corresponds to hAps2. PMID- 12149129 TI - Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine AB - Abstract | Background | Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein. Results | The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature. It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent. Conclusion | It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain. PMID- 12149129_Background TI - AB - Acetylcholinesterase (AChE, EC 3.1.1.7) is a serine hydrolase, which catalyzes the hydrolysis of acetylcholine. This enzyme is the target of organophosphate and carbamate insecticides which phosphorylate or carbamoylate the serine of the active site blocking the hydrolysis of the neurotransmitter acetylcholine. The post-synaptic membrane then remains depolarised and synaptic transmission cannot take place so the insect dies. These compounds are used to control various agricultural pests: insects, acari and nematodes. One of the consequences is that pesticide residues remain in the environment and are potentially toxic for all animals including humans since cholinergic transmission is well conserved. Insecticide residues can be detected with biosensors using AChE as biological element. Such a method has been described to detect low levels of contaminants in crops, soil, water or food samples using various kinds of biosensors . Drosophila AChE was found to be the most sensitive enzyme when compared to enzymes of non-insect origin and in-vitro-mutagenesis was used to select enzymes up to 300-fold more sensitive . But like most enzymes from mesophilic organisms, Drosophila AChE is not stable, and this instability precludes its utilization in biosensors. It can be stabilized by additives: protein such as bovine serum albumin, reversible inhibitor or polyethylene glycol. Alternatively, stabilization can also be achieved by encapsulation in liposomes . Another way to stabilize the enzyme is to use in vitro mutagenesis to modify the primary structure of the protein. This method has the advantage of stabilizing the enzyme during its synthesis leading to higher production and higher purification yields. Several kinds of mutation may stabilize a protein, among them there is elimination of free sulfhydryl groups. Wang et al. suggested that removal of nonessential free cysteines by serine could be a general method for preparing muteins with higher stability, specific activity and homogeneity. Subsequent assays have shown that mutation of free cysteines has been found to increase the half-life of proteins . However, some exemples have been reported where elimination of a free cysteine did not improve the stability of protein and even decreased it . There are eight cysteines in the wild type cholinesterase , six are involved in intrachain disulfide bonds, one is involved in an interchain disulfide bond and one, at position 290 (328 with the precursor numbering) remains free . This free cysteine is half buried in the protein, located in a loop near the disulfide bond formed by cysteines 292 and 307 (Fig. and ) . When comparing the primary sequence of cholinesterases, it appears that this cysteine is not conserved and thus is not essential for the function of the enzyme. In the present study, we used mutagenesis to change this free cysteine for a hydrophobic residue with a similar size, a valine to enhance the stability of the enzyme. Figure 1 | Position of cystein 290 near the disulfide bond formed by cysteines 292 and 307. Position of cystein 290 near the disulfide bond formed by cysteines 292 and 307. Figure 2 | Orientation of cysteine 290 towards the center of the protein. Orientation of cysteine 290 towards the center of the protein. PMID- 12149129_Results and discussion TI - AB - Thermodynamic and thermal (also called long-term) stabilities are usually closely related since thermal denaturation is considered to be a two-step process involving a reversibly unfolded intermediate . But the two stabilities do not necessarily correlate due to independent unfolding of microdomains . Therefore, several denaturation methods were applied. In all situations analyzed, denaturation of AChE was irreversible and followed apparent first order kinetics. Stability was then characterized by the half-life (t50), the time at which 50% of an initial enzymatic activity is preserved. The effect of freezing was measured from the recovery after one freeze-thaw cycle . Table 1 | Stability of wild type and mutated AChE (mean +- SEM) and an index of the increase in stability i.e. the ratio between the measurement made for the mutant and the wild type enzymes. We first analyzed denaturation with the most common methods used to study protein denaturation: incubation at high temperature or in the presence of urea. Incubation of the wild type enzyme at 45C resulted in the progressive irreversible denaturation of the enzyme with a 17 min half-life. Mutation of cysteine 290 to valine increased this half-life to 24 min. It also provided resistance to urea denaturation: the mutated enzyme was 2.7-fold more stable than the wild type if we consider the ratio of half-life time in 4 M urea . Lyophilisation is the most efficient method to store Drosophila AChE, however freezing denatures the enzyme at dilutions below the concentration of 1 mmole per liter . We thus checked if the C290V mutation protects the enzyme at the concentration of 10 nM. Following one freezing to -20C, 75% of the wild type enzyme was denatured while only 50% of the mutated enzyme activity was lost. The utilization of AChE in a biosensor can last several days in some continuous flow devices. In fact, as the inhibition is irreversible, the longer AChE is incubated with the sample, the lower the detection limit of the biosensor. However, the enzyme activity has to decrease from inhibition and not from spontaneous denaturation of the protein. We thus incubated the wild type and the mutated enzyme at 20C. Half the enzyme was denatured in 1.5 days; the mutation doubled this half life. Detection of insecticides in food requires their extraction with organic solvent. Although the solvent should be eliminated before the assay, low amounts may remain in solution and inactivate the enzyme. We used acetonitrile as model because it is soluble in water. This organic solvent strongly denatured the protein which had a half-life in 20% acetonitrile of 1.3 minutes. This half-life was slightly increased to 1.4 minutes by the mutation. As some proteases may be present in the sample to be assayed for pesticide residues, we tested the effect of the mutation on the proteolytic sensitivity of the enzyme. Actually, it has been reported that a protein's resistance to proteolysis increases with its conformational stability due to the fact that the susceptibily to proteolysis reflects the rate of local unfolding . Our data support this relation between stability and proteolytic susceptibility since the mutated enzyme was 1.3 more resistant to proteolytic attack than the wild type enzyme in the experimental conditions used. Several hypotheses can be put forward to explain this increased stability. A first hypothesis involves the deletion of the highly reactive sulfhydryl group which could interact with the neighboring disulfide bond. Thiol-disulfide interchanges have been reported in proteins including acetylcholinesterase and changes involving a free sulfhydryl group and a disulfide bond have been found to result in the irreversible denaturation of proteins . As the disulfide exchange reaction originates from nucleophilic attack on a sulfur atom of the disulfide, the proximity of the disulfide bond formed by cysteines 292 and 307 enforces this hypothesis. The inter Cbeta distance between Cys 290 and Cys 292 or Cys 307 are 5 A or 7.7 A respectively . Actually, mutation of cysteine 292 to valine resulted in an inactive protein suggesting that disruption of the bond between cysteines 292 and 307 results in misfolding since the loop is not involved in the structure of the active site. A free sulfhydryl group may also mediate the formation of dimer and oligomer. Although oligomerization usually has a stabilizing effect , it can sometimes lead to loss of biological activity . As the side chain of cysteine 290 points towards the interior of the protein and is inaccessible to the solvent, it is unlikely that this cysteine is responsible for the formation of tetramers. Air oxidation of thiols is an important source of thermal inactivation of lysozyme . To test the relevance of this phenomenon for stability, we used the "foot printing system" of Tullius and Dombrowski using the EDTA complex of iron(II) to generate hydroxyl radical form hydrogen peroxide as previously performed with AChE . The C290V mutation did not increase the half life of the protein. Thus, stabilization does not seem to originate from the elimination of an oxidation site. The contribution of individual amino acids to the stability of a specific protein has often been found to be difficult to assess since the elimination of one type of interaction often affects other types of interactions. This can happen in our mutation: by replacing a cysteine by a valine, we not only eliminated a reactive thiol, we also increased the hydrophobicity of the side chain which is oriented towards the heart of the protein . As increasing the hydrophobicity of buried residues usually increases the stabilityof proteins , we may hypothesize that this rule applied here. Thus, removal of the free cysteine 290 most probably stabilized Drosophila AChE by two mechanisms: inhibition of disulfide exchange and increase of the hydrophobicity of the side chain. PMID- 12149129_Methods TI - AB - Protein production and purification | cDNA encoding Drosophila melanogaster AChE and mutant were expressed with the baculovirus system . We expressed a soluble dimeric form deleted from a hydrophobic peptide at the C-terminal end with a 3 x histidine tag replacing the loop from amino-acids 103 to 136. This external loop is at the other end of the molecule with respect to the active and its deletion does not affect either the activity or the stability of the enzyme. Secreted AChE was purified to homogeneity using the following steps, ammonium sulfate precipitation, ultrafiltration with a 50 kDa cut off membrane, affinity chromatography with procainamide as ligand, NTA-nickel chromatography and anion exchange chromatography . Activity was recorded at 25C in 25 mM phosphate buffer pH 7, with 1 mM acetylthiocholine iodide as substrate using the method of Ellman et al. . Residue numbering followed that of the mature protein. Denaturation | All denaturation experiments were performed with 10 picomoles enzyme in 1 ml 25 mM phosphate buffer pH7 at 25C. AChE was incubated in denaturing conditions and the remaining activity was measured by taking aliquots at regular times since residual enzymatic activity is dependent on the time of incubation. To analyze thermosensitivity, enzymes were incubated at 45C instead of 25C and 1 mg/ml Bovine Serum Albumin was added to the buffer. Aliquots were mixed with cold buffer chilled on ice and the solution was incubated at 25C for ten minutes before recording the remaining activity. For urea denaturation, unfolding of AChE was induced by adding 4 M urea to the incubation buffer. Aliquots were diluted 10-fold in 25 mM phosphate buffer pH7 before measuring the remaining activity. The effect of freezing was estimated by measuring the remaining activity after one day at -20C. Stability at room temperature was measured by recording the activity after incubation at 20C. The effect of organic solvent was followed by incubation of the enzyme in 20% acetonitrile. The effect of protease sensitivity was determined by incubation of AChE with 0.1 mg/ml pronase. To follow the effect of oxygen radical on denaturation 10 mM H2O2, 1 mM FeCl3, 2 mM EDTA and 40 mM ascorbic acid were added to the incubation medium following the procedure of Weiner et al. . PMID- 12149129_Authors' contributions TI - AB - A.B-L performed the in vitro mutagenesis experiment, I.F. and M.A. produced and purified the proteins. S.M. and C.L. carried out the stability measurements, D.F. designed the study. All authors read and approved the final manuscript. PMID- 12126482 TI - Priority setting for new technologies in medicine: A transdisciplinary study AB - Abstract | Background | Decision makers in health care organizations struggle with how to set priorities for new technologies in medicine. Traditional approaches to priority setting for new technologies in medicine are insufficient and there is no widely accepted model that can guide decision makers. Discussion | Daniels and Sabin have developed an ethically based account about how priority setting decisions should be made. We have developed an empirically based account of how priority setting decisions are made. In this paper, we integrate these two accounts into a transdisciplinary model of priority setting for new technologies in medicine that is both ethically and empirically based. Summary | We have developed a transdisciplinary model of priority setting that provides guidance to decision makers that they can operationalize to help address priority setting problems in their institution. PMID- 12126482_Background TI - AB - Priority setting is a challenge for every health care system in the world because demand for health care outweighs the supply of resources allocated to finance it. Which programs should a regional health authority fund? Which drugs should be placed on the drug benefit formulary? Which patients should be admitted to a hospital's critical care unit? Which priorities should a research granting body support? There are no widely accepted models for legitimate and fair priority setting in health care to help address these questions. Traditionally, approaches from health economics are promoted as the solution to the problem of priority setting. Health economics provides necessary but insufficient tools (e.g. cost-effectiveness analysis, program budgeting and marginal analysis ) to aid priority setting decision makers. Studies of actual priority setting show that these tools have only limited influence on decision making and the analyses are often unavailable when needed . Priority setting for new technologies, for example, is frequently conducted under conditions of varying degrees of evidence about the safety, effectiveness, and appropriateness of particular interventions . Moreover, there is no consensus regarding the values these approaches emphasize (i.e. efficiency) . The Institute of Medicine Panel on Cost-Effectiveness in Health and Medicine argued that, "... CEA [should] be used as an aid to decision makers who must weigh the information it provides in the context of ... other values ." Other values important to priority setting include equity, the health of individuals as against communities, the "rule of rescue," and democratic decision making . Unpacking these values helps to clarify choices, but reasonable people, having diverse moral views, disagree about what constitutes a fair allocation of resources to meet competing health care needs. In the absence of consensus on guiding principles, the problem of priority setting becomes one of procedural justice -- legitimate institutions using fair processes . Discipline-specific ethical approaches to priority setting (e.g. from philosophy, law, political science, medicine, and health economics) are insufficient because they are not grounded in actual experiences of priority setting in health care institutions and because the values that they contribute to priority setting conflict. Empirical descriptions of priority setting that focus, for example, on considerations of safety, effectiveness, and appropriateness of particular preventive, diagnostic or therapeutic interventions are also insufficient. This is because, though grounded in actual experiences of priority setting, they cannot alone provide normative guidance about what should be done. What is needed, therefore, is a synthesis that incorporates both ethical and empirical considerations. A transdisciplinary model, based in empirical realities of decision making and justified by ethical values, can provide such a synthesis. In this paper, we develop a transdisciplinary model of health care priority setting. Transdisciplinary research -- "researchers work [ing] jointly using shared conceptual framework drawing together disciplinary specific theories, concepts and approaches to address common problems" -- can be contrasted with interdisciplinary research -- "researchers work [ing] jointly but still from a disciplinary-specific basis on [a] common problem" -- and multidisciplinary research -- "researchers work [ing] in parallel from their respective disciplinary bases to address a common problem" . By integrating an ethically based account of health care priority setting (accountability for reasonableness) with an empirically based one (diamond model) as described below, we have engaged in a transdisciplinary effort. The result is a model because it consists of plausible relationships proposed among concepts and sets of concepts . However, the innovation here is not the resulting model, though the transdisciplinary model is an advance on its parent-models, but rather the transdisciplinary approach to the problem of priority setting. PMID- 12126482_Discussion TI - AB - Accountability for reasonableness: an ethically-based account of priority setting | Daniels and Sabin identify two key problems at the heart of health care priority setting: legitimacy and fairness . The legitimacy problem poses the question: under what conditions should authority over priority setting decisions be placed in the hands of a particular organization, group or person? The fairness problem poses the question: when does a patient or clinician have sufficient reason to accept priority setting decisions as fair? An institution's priority setting decisions may be considered legitimate and fair if they satisfy four conditions of accountability for reasonableness: relevance, publicity, appeals, and enforcement . Table 1 | The four conditions of accountability for reasonableness Although developed in the empirical context of US managed care organizations, the strength of accountability for reasonableness lies in its ethical roots found in theories of democratic deliberation, grounded in the disciplines of philosophical ethics and political philosophy. These roots connect the priority setting process in accountability for reasonableness with broader democratic processes. The limitation of accountability for reasonableness is that it does not sufficiently explain how an institution might go about operationalizing the model. For example, how should institutional administrators go about putting the relevance condition into practice? Diamond model of health care priority setting: An empirically-based account | Our case study of priority setting for new technologies in medicine in two health care institutions provides a descriptive account of how priority setting decisions are actually made . In the case study, we analyzed documents, interviewed people and observed meetings of groups making priority setting decisions. Based on these data, we developed the diamond model of health care priority setting . Figure 1 | The diamond model of priority setting The diamond model of priority setting The six elements are described briefly below: * Institutions | Priority setting occurs in an institution with a mandate to set priorities; variations between institutional cultures affect priority setting. * People | Groups of people contributing a spectrum of factors and arguments are involved in priority setting. * Factors | Factors considered important by priority setting decision makers include benefit, evidence, cost, cost-effectiveness, and equity. * Reasons | Priority-setting decisions involve clusters of factors that vary according to the decision. The previous conception of priority setting as trade-off (eg, equity vs efficiency) was too simplistic and abstract to describe actual priority setting reasoning. Reasons are assembled by combining these factor-clusters in support of a particular decision. Finally, each decision and rationale is compared with previous decisions and rationales in a casuistry or "case law" that helps to ensure consistency. * Process | Priority setting includes certain procedural safeguards such as transparency, disclosure of conflicts of interest, fair access to decision makers, and fair chairing and leadership of the priority setting group. * Appeals | Decisions are open to review based on new information or arguments. The diamond model reflects the fact that legitimacy and fairness are not all or none phenomena. Instead, they lie along a spectrum and priority setting can be more or less legitimate and fair. Much as a gem's flawlessness depends on the flawlessness of its facets, legitimate and fair priority setting depends on the legitimacy and fairness of each of six elements identified in our study. The strength of the diamond model lies in its empirical roots, grounded in the discipline(s) of the social sciences. Because it reflects the realities of actual decision making, it can more easily be put into practice than an ethically based account. The major limitation of the model is that simply because a group makes priority setting decisions in a particular way does not make these decisions "right". An ideal model of health care priority setting will need to specify what should be done (i.e. justified ethically) and how it can be done (i.e. based in empirical reality). A transdisciplinary model of priority setting in health care institutions | By combining accountability for reasonableness with the diamond model, we developed a transdisciplinary model of priority setting that has a solid ethical justification and deep empirical roots so it is more easily put into practice in a particular context (see Figure ). Figure 2 | A model of accountability for priority setting in health care institutions A model of accountability for priority setting in health care institutions We integrated these two accounts as follows . First, because reasons are central to priority setting, we collapsed together the elements of "reasons" and "factors" in our diamond model into the concept of "rationales" and positioned this as the central concept of the transdisciplinary model. Second, we arranged three elements of the diamond model -- processes, people, and appeals -- around the central concept of rationales (see Table , left hand column), because we discovered that conditions of accountability for reasonableness -- relevance, publicity, and appeals (Table , middle column) -- bound these elements to rationales. Third, we replaced these conditions with operational goals: reasonableness, transparency, responsiveness, and accountability (Table , right hand column). Finally, we situated the model in the "institutions" element of the diamond model, which accords with the enforcement condition of accountability for reasonableness, and supports an operational goal of accountability. Table 2 | Relationships among diamond model elements, accountability for reasonableness conditions, and transdisciplinary model operational goals This new model is a more practical and user-friendly version of accountability for reasonableness that can guide health care decision makers in actual priority setting. There would be constraints in each domain from accountability for reasonableness. Institutions would have to be duly delegated to make these decisions; people would have to represent an appropriate array of perspectives; the people in the context would have to determine what factors should be included in the rationales; the process would have to make reasons transparent not only to participants but to people affected by the decisions; and appeals would have to be responsive to the rationales. Implications for decision makers | Imagine that a health care institution such as a hospital, managed care organization, or regional health authority is charged with priority setting for new technologies in medicine. The following steps, based on the transdisciplinary model, offer guidance to the institution to ensure that their priority setting is legitimate and fair: Step 1 | Bring together a committee of fair-minded people to make priority setting decisions. The group should include a broad spectrum of individuals from within the organization, as well as patients and members of the public. They should focus on the reasonableness of the rationales for each decision. Step 2 | Put into place processes to ensure the transparency of the decision making activities (e.g. public notices and town hall meetings) and to ensure that the rationales are widely available. Step 3 | Design methods to hear appeals regarding the rationales for decisions and to respond to further evidence or arguments. Step 4 | Develop mechanisms of institutional accountability to ensure that the first three steps are followed. Comparison with current practice | Although studies of actual priority setting practices are rare, they often resemble aspects of the transdisciplinary model. In the US, accountability for reasonableness was itself developed in the context of private sector managed care institutions and is being applied to pharmaceutical benefit management . In Canada, the diamond model was developed in the context of public sector institutions making decisions about new technologies in cancer and cardiac care. In the UK, Oxfordshire Health Authority has developed a "Priorities Forum" that illustrates many aspects of the transdisciplinary model . There may be differences in how some public or private institutions might go about implementing the transdisciplinary model. Many public institutions are already obligated by law to disclose their decision making practices and outcomes and to include a broad representation of public stakeholders. Adoption of the transdisciplinary model would therefore entail a less radical transformation of their institutional culture and practices than will be the case for many private institutions that have, up until this point, been under no such obligations . Both accountability for reasonableness and the diamond model -- and therefore the transdisciplinary model -- were developed in the context of priority setting for new technologies. It would be reasonable to expect that the transdisciplinary approach might be more broadly applicable, for example, to decision making about bed closures or space allocation or staffing, or around strategic, capital, and operational planning both at the hospital level and at the clinical level. If this exercise were repeated in different contexts, the resulting transdisciplinary models may very well look quite different. This transdisciplinary model may not be fully generalizable to all contexts, though we expect that it will offer some guidance to decision makers across many priority setting contexts. PMID- 12126482_Summary TI - AB - Priority setting decision makers have had little guidance to help them develop their processes. Traditional discipline-specific approaches are insufficient because they cannot tell decision makers what to do in specific contexts, and empirically derived models of actual decision making are insufficient because they cannot provide normative guidance. We have developed a transdisciplinary model of priority setting that provides guidance to decision makers that they can operationalize to help address priority setting problems in their institution. PMID- 12126482_Funding TI - AB - The project was funded by a grant from the Medical Research Council of Canada/Canadian Institutes of Health Research and the Physicians Services Incorporated Foundation. Dr. Gibson is supported by a Canadian Health Services Research Foundation post-doctoral award. Dr. Singer is supported by a Canadian Institutes of Health Research Investigator award. PMID- 12126482_Competing interests TI - AB - None declared PMID- 12126482_Pre-publication history TI - AB - The pre-publication history for this paper can be accessed here: PMID- 12204944 TI - Peripheral Blood Lymphocyte Subsets in Adolescents: a Longitudinal Analysis from the REACH Project AB - | Flow cytometry analysis of lymphocyte subset markers was performed for a group of sexually active, human immunodeficiency virus (HIV)-negative adolescents over a 2-year period to establish normative data. Data were collected in the REACH Project (Reaching for Excellence in Adolescent Care and Health), a multicenter, longitudinal study of HIV-positive and high-risk HIV-negative adolescents. Two- and three-color flow cytometry data were collected every 6 months for these subjects. We determined the effects of gender, race, and age on the following lymphocyte subset markers: total CD4+ cells, CD4+ naive cells, CD4+ memory cells, all CD8+ cells, CD8+ naive cells, CD8+ memory cells, CD16+ natural killer cells, and CD19+ B cells. Gender was the demographic characteristic most frequently associated with differences in lymphocyte subset measures. Females had higher total CD4+ cell and CD4+ memory cells counts and lower CD16+ cell counts than males. Age was associated with higher CD4+ memory cell counts as well as higher CD8+ memory cell counts. For CD19+ cells, there was an interaction between age and gender, with males having significantly lower CD19+ cell counts with increasing age, whereas there was no age effect for females. Race and/or ethnicity was associated with differences in total CD8+ cell counts and CD8+ memory cell counts, although both of these associations involved an interaction with gender. PMID- 12204944_Introduction TI - AB - The interaction between infection with human immunodeficiency virus (HIV) and immunologic responses to infection has led to the development of new and more detailed laboratory methods. There are limited normative data available for immunologic assays and flow cytometry measures, especially for specific age populations, such as adolescents. Adolescence is a unique period of development, characterized by profound physiologic and psychosocial changes . Gender and age differences in immunologic cell numbers and function have been observed for both healthy and diseased subjects. Studies of adolescents and adults have demonstrated gender differences as well as some age differences in immune cell counts as characterized by flow cytometry studies and limited number of functional assays . Age may be an important factor influencing immunologic responses to infection, especially in younger children .Other factors, including race and genetic characteristics, may also influence immunologic cell numbers. Understanding how these factors influence the immune system in healthy individuals is key to beginning to understand age, gender, and race differences in immune system-based diseases and the adolescent's immunologic response to infection with HIV and other infectious agents. HIV infection is a major issue confronting adolescents . Recent data show marked increases in the number of HIV infections in adolescents and young adults, especially in the number of infections due to heterosexual transmission in young women and in the number due to male-to-male transmission in young men . The REACH (Reaching for Excellence in Adolescent Care and Health) Project of AMHARN (The Adolescent Medicine HIV/AIDS Research Network) recruited and longitudinally followed a cohort of high-risk youths not infected with HIV to establish normative data for this population . The study set out to establish gender, age, and racial differences in a set of immunologic markers in a longitudinal analysis. Before comparisons can be made for HIV-infected adolescents, normative data must be established for groups related to age, race and/or ethnicity, and gender. We present data for a group of phenotypic markers in an HIV-negative adolescent cohort. PMID- 12204944_MATERIALS AND METHODS TI - AB - The REACH Project recruited HIV-positive and high-risk HIV-negative adolescents (age range, >=13 to <19 years) into a study of biomedical and behavioral features of HIV infection as observed while under medical care for HIV infection and adolescent health. The HIV-negative subjects served to establish adolescent normative data for a number of biological measures. HIV-negative subjects were recruited from adolescent clinics serving high-risk adolescents based on the high seroprevalence rates in the geographic areas and on sexual risk or needle-using risk of the adolescents. The characteristics of the cohort, recruitment and eligibility criteria, and study design have been reported elsewhere . The HIV-negative subjects were determined to be so on the basis of results from an enzyme-linked immunosorbent assay (ELISA) performed within 30 days of enrollment into the REACH study. The HIV ELISA was repeated annually to confirm HIV-negative status. The HIV-negative youth, to qualify for enrollment into the study, had a history of either sexual intercourse or injection drug use. Blood samples for HIV-negative subjects were collected at 15 clinical sites every 6 months (see Appendix). The following flow markers were analyzed, along with an automated differential count, at a local AIDS Clinical Trials Group-certified laboratory: CD3+/CD4+ (helper T cell), CD3+/CD8+ (suppressor and/or cytotoxic T cell), CD3-/CD56+/CD16+ (natural killer cell), and CD19+ (total B cell). Additional flow markers were analyzed centrally at the Immunology Core Laboratory at The Children's Hospital of Philadelphia, as previously reported . These markers included the following cell subsets: CD4+/CD45RO-/CD45RA+ (naive helper cell), CD4+/CD45RO+/CD45RA- (memory helper cell), CD8+/CD45RO-/CD45RA+ (naive suppressor cell), and CD8+/CD45RO+/CD45RA- (memory suppressor cell). A cross-sectional analysis of 192 subjects by two-sided tests with a type I error of 5% and a power of 80% can identify a standardized effect of 0.20, considered small, or a correlation of 0.20. Repeated analyses, with subject's age at the data collection visit as the time variable, were performed using Generalized Estimating Equations (GEE) software (SAS software, version 8.1; [SAS Institute]). P values of 0.05 or less were considered significant for two-sided tests. Multiple comparison corrections were not used. Summary statistics for cell counts are presented as means and standard deviations, which include only one observation per subject in each age category, although individual subjects may have contributed to more than one age category. For analysis, counts were log-transformed to make skewed distributions more nearly normal, as assessed by the Shapiro-Wilks test. Separately for each lymphocyte subset, GEE models were used to regress log counts on gender, subject's present age, and race and/or ethnicity of these HIV-negative subjects. The three two-way interactions of the demographic variables were also examined; models with interactions also retained main effects. Because multiple visits in the same age category were included, these GEE models used more data than appear in the summary of means and standard deviations. PMID- 12204944_RESULTS TI - AB - At the time of this analysis, 198 HIV-negative subjects were enrolled in the REACH cohort. Two HIV-negative transgender males were excluded and 4 subjects did not have a complete lymphocyte subset panel evaluation performed, leaving 192 subjects eligible for analysis. The demographic characteristics of the eligible subjects are shown in Table . For each eligible subject, all study visits at which a complete lymphocyte subset panel evaluation had been performed were included in the analysis. The total number of study visits included was 779, with one to nine visits per subject. The distribution of ages at the times the visits were made is also shown in Table . The means and standard deviations by gender and age for each of the eight subsets are provided in Table . Figures to are a graphical display of means by gender and age. The significant statistical associations, by cell type, are shown in Table . There was a significant association with one or more of the demographic characteristics for 6 of the 8 lymphocyte subsets that were examined. No association with the three demographic characteristics was found for CD4+ naive cells or for CD8+ naive cells. Gender was the demographic characteristic most frequently associated with differences in lymphocyte subset counts. Gender had a significant main effect or interaction for 6 of the 8 subsets: total CD4+ cells, CD4+ memory cells, total CD8+ cells, CD8+ memory cells, CD16+ cells, and CD19+ cells. Females had higher total CD4+ cell counts, higher CD4+ memory cell counts, and lower CD16+ cell counts. For total CD8+ cells and CD8+ memory cells, there was an interaction between gender and race. African-American males had significantly lower total CD8+ counts than did other males; there was not a significant difference in total CD8+ counts by race for females, although African-American females had nearly significantly higher total CD8+ counts. For CD8+ memory cells, African-American and Hispanic males had higher mean counts than males classified as Caucasian or other, while African-American and Hispanic females had lower mean counts than females classified as Caucasian or other. None of these differences were statistically significant when data for males and females were modeled separately. For CD19+ cells, there was an interaction between gender and age. Males had significantly lower CD19+ cells counts with increasing age, whereas there was no age effect for females. Age was associated with differences in three of the eight subsets: CD4+ memory cells, CD8+ memory cells, and CD19+ cells. Older subjects had higher CD4+ memory cell counts and higher CD8+ memory cell counts. The gender by age interaction for CD19+ cells is described in the preceding paragraph. Race and/or ethnicity was associated with differences in only two of the eight subsets: total CD8+ cells and CD8+ memory cells. Both of these associations involved an interaction with gender, as was discussed above. FIG.1. | Graphical representation of peripheral blood lymphocyte subsets (CD4 cells) observed in adolescents. Graphical representation of peripheral blood lymphocyte subsets (CD4 cells) observed in adolescents. FIG. 3. | Graphical representation of peripheral blood lymphocyte subsets (CD16 and CD19 cells) observed in adolescents. Graphical representation of peripheral blood lymphocyte subsets (CD16 and CD19 cells) observed in adolescents. TABLE 1 | Characteristics of the cohort TABLE 2 | Mean counts (and standard deviations) of lymphocyte subsets by gender and age TABLE 3 | Significant associations between demographic characteristics and lymphocyte subsets, calculated by multivariate linear regression PMID- 12204944_DISCUSSION TI - AB - We have investigated the impact of gender, race, and age on T-lymphocyte subset populations as measured by flow cytometry in adolescents identified as at high risk for HIV infection in a national, longitudinal study. Gender was the demographic factor most frequently associated with differences in lymphocyte subsets. In females, we found higher CD 4+ T-lymphocyte counts and higher CD4+ memory T-lymphocyte counts, and lower CD 16+ T-lymphocyte counts than in males. Our study demonstrated some limited gender differences in CD8+ cells and CD19+ cells counts, but only with the influence of either race or age. Since this was a longitudinal study in a predominantly African-American and Hispanic group of adolescents, it serves as an important reference when studying disease processes that affect the immune system in the adolescent population. Our data for CD4+ T lymphocyte differences by gender are consistent with those previously reported for minority and Caucasian adolescents. Bartlett and colleagues studied a group of minority youth and found higher levels (both totals and percentages) of CD4+ T lymphocytes in females than in males and lower levels of B cells in females than in males . In another study of primarily Caucasian adolescents from 12 to 19 years, Tollerud and colleagues found higher levels of CD4+ T lymphocytes in females than in males among older adolescents . Our studies demonstrate that much of this gender difference is mainly in the memory CD4+ T-cell subset. We did not find significantly higher B-cell levels in males than in females, as was found by Bartlett. There is no direct explanation for this; however, we had fewer adolescents in the younger age ranges, which may explain some of this difference. A number of studies have reported increases in CD4+ T cells in females . Studies of adults have identified some similar gender differences. Higher mean total CD3+ T-cell and mean total CD4+ T-cell counts have been observed in females than in males; in addition, lower CD16+ cells have also been noted in adult females than in males . Differences in immune cell numbers may be secondary to hormonal differences (androgens or estrogen or both). Gender may affect the immune system at many levels. Differences in sex steroid regulation and production are present in males and females early in development, with the most profound differences noted during adolescence with the onset of puberty. Pubertal hormonal changes occur under the control of the hypothalamic-pituitary-gonadal axis, which leads to increases in sex hormone levels . Gender has an effect on both B- and T-cell responses and the secretion of immune modulators, including cytokines and chemokines . Cell proliferation occurs under the influence of cytokines, and some differences in T-cell numbers between males and females may be related to differences in cytokine regulation . Whether the increased levels of circulating CD4+ T cells found in females in this study is secondary to the influence of specific cytokines requires further investigation. Sex hormones may influence the immune cell populations through several indirect pathways. Sex hormones may impact immune cell numbers through thymic pathways. Studies of castrated male mice demonstrate increased number of both CD4+ T cells and CD8+ T cells . In addition, evidence suggests that thymic involution is modulated by androgens . Sex hormones may also influence T cells directly through cell receptors for sex steroids, as has been found for CD8+ T cells . During adolescence, the hormonal influence on immune cell numbers may be most apparent; however, the impact of sex hormones on immunity may begin much earlier. There are detectable estrogen receptors in fetal thymus as well as fetal lymphoblast cells, suggesting that sex hormones may influence lymphocyte maturation in the thymus . Whether the interactions between lymphocyte receptors and sex hormones lead to differences in immune cells numbers later in life is not clear. Gender differences in circulating T-cell subsets most likely reflect a complex regulatory mechanism, some of which may be under the influence of gender differences in sex steroids. The impact of gender on the immune system is evident from gender differences in immune system diseases particularly autoimmune disorders. Systemic lupus erythematosis, rheumatoid arthritis, myasthenia gravis, and Hashimoto's disease all have a higher prevalence among women . We found no independent influences of race and/or ethnicity on any of the immunologic measures in our study. We are limited, however, by the relatively small numbers of Caucasion subjects in our cohort. Rates of new HIV infections in adolescents appear to be greatest in minority groups . Thus, racial or ethnic differences in immune responses would be important to determine in a more heterogeneous population. We also found limited influence of age on the T-cells subsets studied with the only independent impact of age on memory CD4+ T cells and CD8+ T cells. Again, our study is limited by small numbers of younger adolescent subjects. Studies in younger children show a significant impact of age on T-cell populations, with signficantly higher numbers of CD4+ T lymphocytes in young infants . Differences between adolescents and adults have been reported: adolescent females begin to demonstrate CD4+ T-cell levels that are increased compared to those observed in males, and females also begin to demonstrate CD8+ T cell levels that are lower (such levels in adults have also been reported) . Our numbers may be too small to demonstrate subtle differences in the transition from adolescence to adulthood. Our study is unique as a longitudinal analysis of gender, age, and race and/or ethnicity differences in specific immunologic cells in high-risk youth. Although we found some significant differences, our study has several limitations. First, as most of our youth were sexually mature at the time of entry into the study, we were unable to analyze the data by Tanner-stage subgroups. Second, the mechanisms leading to gender differences were beyond the scope of this study. Finally, our population was primarily minority; thus, the findings may not apply to all populations. However, understanding the impact of gender, race, and age on immune cell numbers in healthy individuals is essential for subsequent evaluation in disease states. PMID- 12204944_APPENDIX TI - AB - The following investigators, listed in order by the numbers of subjects enrolled at their respective clinics, are participating in this study: L. Friedman, L. Pall, D. Maturo, A. Pasquale (University of Miami); D. Futterman, D. Monte, M. Alovera-DeBellis, N. Hoffman, and S. Jackson (Montefiore Medical Center); D. Schwarz and coauthor B. Rudy (University of Pennsylvania and the Children's Hospital of Philadelphia); M. Tanney and A. Feldman (Children's Hospital of Philadelphia); M. Belzer, D. Tucker, C. Hosmer, K. Chung (Children's Hospital of Los Angeles); S. E. Abdalian, L. Green, C. McKendall, and L. Wenthold (Tulane Medical Center); L. J. D'Angelo, C. Trexler, C. Townsend-Akpan, R. Hagler, and J. A. Morrissy (Children's National Medical Center); L. Peralta, C. Ryder, S. Miller, and S. Calianno (University of Maryland); L. Henry-Reid and R. Camacho (Cook County Hospital/University of Chicago); M. Sturdevant, A. Howell, and J. E. Johnson (Children's Hospital, Birmingham); A. Puga, D. Cruz, and P. McLendon (Children's Diagnostic and Treatment Center); M. Sawyer, J. Tigner, and A. Simmonds (Emory University); P. Flynn, K. Lett, J. Dewey, S. Discenza (St. Jude Children's Research Hospital); L. Levin and M. Geiger (Mt. Sinai Medical Center); P. Stanford and F. Briggs (University of Medicine and Dentistry of New Jersey); and J. Birnbaum, M. Ramnarine, and V. Guarino (SUNY Health Science Center at Brooklyn). The following investigators have been responsible for the basic science agenda: C. Holland (Center for Virology, Immunology, and Infectious Disease, Children's Research Institute, Children's National Medical Center), A. B. Moscicki (University of California at San Francisco), D. A. Murphy (University of California at Los Angeles); S. H. Vermund (University of Alabama at Birmingham); P. Crowley-Nowick (The Fearing Laboratory, Brigham and Women's Hospital, and Harvard Medical School, Boston, Mass.), coauthor S. D. Douglas (University of Pennsylvania and the Children's Hospital of Philadelphia). Staff from sponsoring agencies include A. Rogers and A. Willoughby, (NICHD), K. Davenny and V. Smeriglio (NIDA), E. Matzen, (NIAID), B. Vitiello (NIMH). PMID- 12204952 TI - Comparison between the Skin Snip Test and Simple Dot Blot Assay as Potential Rapid Assessment Tools for Onchocerciasis in the Postcontrol Era in Ghana AB - | Successful control of onchocerciasis through mass distribution of ivermectin needs to be coupled with reliable, sensitive, specific, yet affordable diagnostic methods to monitor and ensure the efficacy of such measures. The effort put into the development of diagnostic methods for onchocerciasis that can substitute for or work in combination with the present "gold standard," the skin snip test, has resulted in the discovery of a number of immunogenic proteins with potential use as diagnostic tools in the postcontrol era. Most of these proteins have now been produced through recombinant DNA techniques. However, when costs are not a trivial issue, none of them have yet found their way into the areas where the disease still exists. In the present study, we have evaluated the performance of a simple dot blot assay which uses a mixture of native proteins designated PakF as a serious contender in the quest for a less invasive and more sensitive method to detect Onchocerca volvulus infection in areas with diverse endemicities. Our results indicate that the assay we propose is more sensitive than the skin snip test and shows high specificity, both characteristics required for a suitable tool for the monitoring of onchocerciasis in the postcontrol era. PMID- 12204952_Introduction TI - AB - Onchocerciasis (river blindness) occurs in 34 countries in Africa, Latin America, and the Arabian Peninsula. The vast majority of the estimated 18 million persons infected with the causative agent, the parasite Onchocerca volvulus, live in Africa . One of the goals of the World Health Organization is to eliminate onchocerciasis as a public health problem. The classical method to detect O. volvulus infection consists of examining small skin biopsy specimens (skin snips) by microscopy for the presence of O. volvulus microfilariae (mf). However, this very specific method is invasive. The process may be uncomfortable, which may result in poor compliance from individuals, especially in communities subjected to repeated tests . Much effort has been put into the development of less invasive serodiagnostic methods to support the initiatives aimed at elimination of the disease. The program in West Africa, known as the Onchocerciasis Control Program (OCP), has been successful in meeting its goal of eliminating onchocerciasis as a public health problem in the 11 countries where the program has been active . As a result of the OCP's success, the efforts of the program are now turning to surveillance, with the goal of rapidly detecting and controlling outbreaks of infection in the onchocerciasis-free zones . In the postivermectin era, however, there is still no diagnostic test available for large-scale use in onchocerciasis-free zones or areas where the disease is still endemic, although with very low mf density. We have previously described a simple dot blot assay (DBA) using a native low-molecular-weight antigen fraction of female O. volvulus parasites, designated PakF , which is a useful diagnostic test in settings where the disease is nonendemic , and with its high specificity and sensitivity, it could have potential as a screening tool for onchocerciasis under certain circumstances. The DBA using this antigenic fraction (PakF-DBA) showed very high sensitivity and specificity when tested in Guatemala (9a), a country where onchocerciasis is endemic but where no other human filariasis has been reported . In this study, we describe the evaluation of the PakF-DBA for sera from individuals from localities with diverse endemicities in the Volta region of Ghana and compare the results with those obtained using the skin snip test. This region of Ghana has pockets of high and low transmission of onchocerciasis, and mass treatment has been practiced in some of these areas. Screening for onchocerciasis in these areas has always been done using the skin snip test; however, repeated ivermectin treatment results in decreased mf load, making the skin snip test less sensitive and reliable. We also evaluate the potential cross-reactivity of the assay with other filarial infections and show that sera from patients with circulating Wuchereria bancrofti do not cross-react in this assay. We discuss the potential use of the PakF-DBA as a less expensive and therefore affordable method for rapid screening and monitoring of areas where control measures have been established. (G. Guzman conducted this research in partial fulfillment of the requirements for his Ph.D. thesis at Karolinska Institute, Stockholm, Sweden, 2002.) PMID- 12204952_MATERIALS AND METHODS TI - AB - Study population. | The study was carried out in five localities of the district of Ho in the Volta region of Ghana with diverse endemicities (hypo- and hyperendemicity). Thirty individuals, who volunteered to participate, were selected from each locality, half of whom were within the age range of 5 to 17 years. Informed consent was obtained from all the participants in the study or their parents or guardians. The localities were as follows: (i) Aflapke (LOC1; male-to-female ratio, 0.9; child-to-adult ratio, 1), (ii) Klave (LOC2; male-to-female ratio, 0.9; child-to-adult ratio, 1), (iii) Aboete, Anorme, Asifiafe, and Luvudo (LOC3; male-to-female ratio, 2.3; child-to-adult ratio, 1), (iv) Honuta Gborgame (LOC4; male-to-female ratio, 0.8; child-to-adult ratio, 1), and Kpedze Anoe (LOC5; male-to-female ratio, 1.0; child-to-adult ratio, 1). Study samples. | Capillary blood was obtained by pricking the middle finger with a disposable hemolancet. Five circles of blood were blotted onto previously labeled 90-mm-diameter round, fast-drying filter paper (no. 597; Schleicher & Schuell, Dassel, Germany). When the blood spots were dry, filters were placed inside labeled plastic zip-lock bags and stored in the freezer compartment of a refrigerator (approximate temperature, -20C). Blood collection was done according to the ethical and safety policies in Ghana. Pools of sera from mf+ and nonexposed Guatemalan individuals from a previous study were used as positive and negative controls, respectively. A panel of Indian sera from areas where W. bancrofti is endemic, including 9 microfilaremic patients (W. bancrofti mf+; age range, 10 to 40 years) and 16 patients with chronic infection but no circulating mf in the blood (chronic patients [CP]; age range, 20 to 50 years), were used as other filaria controls. In addition, 15 individuals living in the same area with no evidence of filariasis (endemic controls [EN]) were included. These sera were from an area where onchocerciasis is not reported. Diagnosis of onchocerciasis. | Parasitological examination for O. volvulus was done by four-site skin snips and microscopic examination for the presence of mf following 24-h incubation in saline at room temperature (approximately 20C). The presence of mf of O. volvulus in any of the skin snips confirmed the diagnosis. Skilled technicians could distinguish O. volvulus from other filaria, such as Streptocerca, which is prevalent in the study area. The microfilarial density (MFD) was determined by dividing the average number of mf by the average weight of the skin snips in milligrams. Thus, an mf+ diagnosis corresponded to an individual with an MFD of >0. Antigen preparation (TSF). | Intact noncalcified nodules obtained in a previous study were digested by the collagenase method of Schulz-Key et al. . Female worms were washed and frozen at -70C in RPMI 1640 medium containing gentamicin (0.2 mg/ml). Tris-soluble antigen fractions (TSF) were prepared as previously described and stored at -20C . The total protein concentration was estimated by the Bradford assay . IEC. | PakF fractions were prepared separately from TSF by ion-exchange chromatography (IEC) as described previously, except that neither a fast protein liquid chromatography system nor a peristaltic pump was used . We simplified the IEC preparation by using a handheld ion-exchange column as described elsewhere (unpublished data). One hundred and twelve micrograms of TSF (1 to 1.3 mug/mul) was diluted to 0.5 ml in equilibration buffer (50 mM Tris-HCl, pH 9.0, containing 0.05% Tween 20). This was passed manually through a 1-ml HiTrap-Q ionic column (Pharmacia, Uppsala, Sweden) at a flow rate of 0.5 ml/min using a 1-ml syringe as a reservoir. The sample was eluted with equilibration buffer at 0.5 ml/min, and the eluate was collected in eight 350-mul aliquots before the buffer was changed to equilibration buffer containing 0.5 M NaCl. Two final 350-mul aliquots were collected using this buffer. Each fraction was tested for antigenicity and polypeptide composition using a DBA (see below) and one-dimensional gel electrophoresis as previously described . Serum elution from blood spots. | Blood was extracted as previously described . Briefly, one disk, 6 mm in diameter, was cut out from a blood blot on each filter and extracted for 2 h in 2 ml of phosphate-buffered saline (PBS; pH 7.4) containing 0.05% Tween 20 (PBST). After slow vortexing, the disk was removed and the eluted serum was collected. In some instances, eluted sera were tested before and after heat treatment at 56C for 30 min. DBA. | The DBA was performed as described previously with some modifications . Briefly, nitrocellulose strips, 4 mm wide and 3 cm long, were placed in mini-incubation trays (Bio-Rad Laboratories, Richmond, Calif.), and droplets (0.5 mul) of unconcentrated and undiluted PakF (approximately 5 ng) and TSF (64 ng; positive control) were adsorbed on the strips. The strips were allowed to dry at room temperature and were then blocked with 1% nonfat milk powder in PBST for 1 h under gentle agitation on a rocking shaker. The strips were incubated for 1 h with 1 ml of eluted serum. The strips were washed three times for 5 min each time with PBST and then incubated for 1 h with alkaline phosphatase-labeled goat anti-human immunoglobulin G (Calbiochem, La Jolla, Calif.) diluted 1:2,000 in PBST. The strips were washed three times as before with PBST and twice with PBS only before development of the immunoreactive spots with BCIP (5-bromo-4-chloro-3-indolylphosphate)-nitroblue tetrazolium (Sigma Chemical Co., St. Louis, Mo.). The color reaction was allowed to proceed for 10 min and then was stopped with several washes with distilled water. The strips were air dried in the dark. The DBA was performed blind, and thus the results from the skin snips were not made available to the person performing the assay until after all the analyses had been done. Control sera were tested first, diluted 1:4,000 in PBST. Evaluation of the dot blots. | Nitrocellulose strips were evaluated by visual examination and, for purposes of quantification, also by densitometry. Densitometric values were obtained as the integrated intensity of all the pixels in a spot excluding the background and were expressed as arbitrary units (Bio Image, Ann Arbor, Mich.). A visually positive sample was determined by identifying two spots of reactivity where PakF was applied to the nitrocellulose. The absence of reactivity in those spots was assessed as a negative result . Analysis of results. | Prevalence was defined as the number of skin snip-positive (MFD > 0) and DBA-positive (by visual examination) individuals in each locality studied. The sensitivity and positive predictive values of the PakF-DBA were calculated by comparison to the skin snip as a "gold standard." Sensitivity was calculated as the proportion of positive individuals detected by both methods to those detected by the skin snip test. Similarly, the positive and negative predictive values were calculated as the proportions of positive and negative individuals by both methods to those positive or negative by DBA . Statistics. | Pearson's correlation coefficients (Pcc) were used to compare the prevalences of the skin snip test and PakF-DBA, and the significance was tested using the Fisher z transformation. FIG. 1. | (A) Reproducibility in the production of PakF. (A) Reproducibility in the production of PakF. Lanes 1 to 4 show the electrophoretic patterns of PakF fractions obtained at different times, showing the mixture of at least five distinct bands that can be detected by silver staining. (B) Clear-cut difference between positive (+) and negative (-) samples in the PakF-DBA test. In a typical onchocerciasis-positive sample, the two spots where PakF had been adsorbed, as well as the procedural control (TSF), are recognized by the serum sample. Negative samples do not show reactivity against PakF, although they may still react against some proteins present in the whole soluble extract, TSF. (C) Graphic representation of the densitometric data obtained by measuring the densities of the spots in all the individual DBA strips representing the five localities under study. A line at 0.1 U of integrated intensity indicates the cutoff value for visually negative samples. PMID- 12204952_RESULTS TI - AB - Skin snip test results. | Of the 150 individuals tested, 99 were skin snip positive and 51 were skin snip negative. A cluster of 40 negative individuals was found in the age range 5 to 20 years, and the remaining 11 were distributed between 21- and 60-year-olds . No Streptocerca mf were detected in the skin snips of any of the participants in the study. The prevalence of individuals with mf+ skin snips varied from 43.3 to 76.7% in the five localities . A closer evaluation of the <18-year-old age group shows that only one of the 15 individuals in LOC2 had a positive skin test, resulting in a prevalence of 6.7% . In this age group, the highest prevalence was in locality LOC3. DBA results. | Figure shows the reproducibilities in the production of PakF at different times. Figure shows the result of a positive DBA test. For a sample to be DBA positive, the two dots where PakF has been adsorbed need to show reactivity. TSF, included as a procedural control, may or not be reactive depending on the degree of cross-reactivity of the proteins in the whole soluble extract. In the case of onchocerciasis, infected individuals normally recognize TSF even if they do not recognize PakF, as they may react to other proteins present in the whole extract. In order to represent these results in a more graphic manner, densitometric values were generated for each sample, obtaining the mean of the integrated intensity of each pair of spots on each DBA strip. Using this system, we were able to assign a numeric value of intensity to spots which were visually weak or very weak. Those visually negative fell in the intensity range of 0 to 0.1, and those visually positive had an integrated intensity above 0.1. This allowed us to generate a graph of integrated intensities for the entire study population . Figures and show the results obtained by DBA in all the areas under study. While the individual intensities of the spots varied, there was a clear-cut difference between positive and negative controls. In total, 125 samples were positive by DBA and 25 were negative, with a cluster of 22 DBA-negative samples within the age range 5 to 17 years. Individual analysis of each locality showed that the majority of positive individuals in the age range 5 to 17 years were inhabitants of the villages grouped as LOC3 and those from LOC5 . The MFD in relation to age followed a distribution pattern very similar to that of the DBA versus age (not shown). Comparison of DBA and skin snip results. | Tables and show the comparisons of the DBA and the skin snip test results, using visual assessment of the strips, for the whole study population and those aged 5 to 17 years, respectively. Stratified by age , the results clearly show that the DBA results closely followed the skin snip results in all age groups. However, the DBA consistently showed more positive individuals in all groups except the older age groups, with the biggest discrepancy between the DBA and skin snip test seen in the younger age groups. A closer look at the 5-to-17-year-old age group in the individual localities showed a similar trend between the DBA and skin snip test, with LOC2 having the lowest DBA positivity but LOC5 showing DBA prevalence akin to that of LOC3. The PakF-DBA detected more positive individuals than the skin snip test. Moreover, 98 out of the 99 skin snip-positive individuals were detected by DBA, for a sensitivity of 99% and a predictive value of 78%. A positive correlation (Pcc = 0.899; P < 0.05) between the prevalences estimated by the skin snip test and PakF-DBA was found when the whole study population was analyzed, which did not vary appreciably when the 5-to-17-year-old population was analyzed separately (Pcc = 0.893; P > 0.05). There was no significant correlation (r = 0.102; P > 0.05) between the prevalences estimated by the two methods among those older than 18 years. Cross-reactivity of PakF-DBA with other filarial sera. | The specificity of the PakF-DBA was assessed using sera from Indian individuals with bancroftian infection with apparently no exposure to O. volvulus. None of the W. bancrofti mf+ sera or the control sera were positive by DBA. However, two CP and two EN sera reacted positively on the DBA . Pooled Guatemalan mf+ and nonexposed sera, used as positive and negative controls, were positive and negative, respectively, when tested at the same dilution. In total, 4 out of 40 Indian sera were positive by DBA, resulting in a 90% specificity of the assay. FIG. 2. | DBA results obtained from serum eluted from finger prick blood, using PakF as an antigen. DBA results obtained from serum eluted from finger prick blood, using PakF as an antigen. Each strip represents an individual serum. The picture shows the immune reactivity of each individual to duplicate spots of PakF (top) and the unfractionated TSF (bottom). The samples are from LOC1, but the last two strips correspond to a pool of mf+ (GUmf+) and nonexposed (N-GU) controls, respectively. The strips were visually scored as positive (+) or negative (-) as indicated in the legend to Fig. . FIG. 3. | DBA results from LOC2, LOC3, LOC4, and LOC5. DBA results from LOC2, LOC3, LOC4, and LOC5. Visual assessment of the DBA strips was performed as indicated in the legend to Fig. . FIG. 4. | Specificity of PakF-DBA. Specificity of PakF-DBA. The graphic representation of densitometric data shows that the PakF-DBA is highly specific when sera from individuals with other filarial infections, other than onchocerciasis, are analyzed. Two W. bancrofti CP and two EN were positive by PakF-DBA, whereas none of the microfilaremic (MF) individuals reacted to PakF. Pools of Guatemalan O. volvulus-infected (GUmf+) and nonexposed (N-GU) samples were used as positive and negative controls, respectively. Visual assessment: samples classed as negative had an integrated intensity of <0.1 U, whereas those classed as positive had an integrated intensity of >0.1 U. TABLE 1 | Comparison of PakF-DBA and skin snip test in diagnosis of onchocerciasis in different age ranges TABLE 2 | Performance of PakF-DBA compared to skin snip test for diagnosis of onchocerciasis in the whole study population (5 to 78 years old) TABLE 3 | Performance of PakF-DBA compared to skin snip test for diagnosis of onchocerciasis in the age range 5 to 17 years PMID- 12204952_DISCUSSION TI - AB - Since the advent of successful control of onchocerciasis through measures including mass distribution of ivermectin, the need for developing surveillance programs to detect recrudescence of infection in the OCP area, as well as safe, inexpensive, and rapid diagnostic tests for O. volvulus infection, has been highlighted. In the present study, we have evaluated the performance of a simple DBA (PakF-DBA) as a serious contender in the quest for a less invasive and more sensitive method to detect O. volvulus infection in areas with diverse endemicities. With the PakF-DBA, we were able to detect a trend similar to that of the skin snip test, but the PakF-DBA detected a higher proportion of infected individuals than the skin snip test. These included 98 out of 99 detected by the more invasive gold standard test. This could indicate that the sensitivity of the DBA described here is higher than that achieved with the skin snip test. Although we have used analytical densitometry to generate comprehensive graphs, we have shown that the results obtained by visual examination of the DBA strips correspond well with densitometric data (Guzman et al., submitted). The clear-cut difference between positive and negative samples allows for visual assessment instead of the use of a sophisticated apparatus such as a densitometer. This is of particular interest in areas where the use of delicate or sophisticated readout devices is not practical. Therefore, the visual results of the DBA strips were enough to establish a correlation between the prevalences estimated by the skin snip test and those estimated by DBA in all age groups. The fact that the DBA results were comparable to those obtained by skin snip tests indicates the possibility that the overall estimates of prevalence generated by DBA from a sentinel population could replace those generated by skin snips. The major difference in the number of positive individuals detected by DBA and skin snip test was observed in the groups aged 5 to 17 years . When analyzed separately, a positive and significant correlation between the prevalences estimated with the PakF-DBA and the skin snip test was found for the population aged 5 to 17 years. However, no such correlation was observed in the group of adults studied (>17 years old). Due to the long life span of the adult worms, the continuous exposure in areas with ongoing transmission of onchocerciasis, and the unknown duration of circulation of antibodies against the proteins contained in PakF, it is not likely that an antibody-based assay like the PakF-DBA would show a better performance than the skin snip test when an age group of >20 years is selected as a sentinel population to assess the efficacy of control measures. Our results are in accordance with those of other studies that have shown that the most useful indicator group for detecting recrudescence, as well as for determining the intensity of infection in a community, is the population 5 to 15 years of age . The PakF-DBA may be positive in more specimens due to a number of reasons, including higher sensitivity, lower specificity, or the capacity to detect even early (prepatent) infection. The possibility of detecting prepatent disease can only be evaluated in long-term longitudinal studies. Our results suggest that the PakF-DBA has a higher sensitivity coupled with high specificity. There is an emphasis in the World Health Organization strategy on the need for surveillance methods to be highly specific even at the cost of low sensitivity. The problem of specificity is that of the quality of the gold standard. If the gold standard has low sensitivity, then assays with higher sensitivities can be viewed as having low specificities. This is the problem with the highly specific but low-sensitivity skin snip test. However, we have previously established the specificity of the PakF-DBA assay to be 100% using a panel of sera from noninfected individuals from areas where no other filaria is present (Guatemala, in Central America) (Guzman et al., submitted). In the later studies, we used sera from individuals infected with other coendemic parasites in the area, mainly Ascaris lumbricoides, in order to rule out any cross-reactivity due to the carbohydrate-containing antigens widely shared among nematodes, primarily phosphocholine-containing antigens . In addition, we ruled out cross-reactivity with a series of sera from filaria-infected patients. In the present study, we have also approached this question by extending the pool of test samples with well-characterized sera from individuals with other filarial infections from areas where onchocerciasis is not endemic. The sensitivity of the PakF-DBA using these sera was 90%. Of importance, however, is the fact that those positive in the PakF-DBA were not the individuals with circulating bancroftian mf, for whom nine of nine sera were negative by the PakF-DBA test. Combined analysis using the present data and the additional panels of sera from areas where other filarias, but not O. volvulus, are endemic resulted in 96% specificity (Guzman et al., submitted). These results also suggest that the PakF-DBA could be useful in screening children and adolescents born after control measures have been established, since they form a desirable sentinel population to detect recrudescence of infection in controlled areas . In the areas of endemicity, the need for assays that provide rapid results is advocated. The time needed to obtain qualitative results from the PakF-DBA was comparable to that for the rapid version of the skin snip test, i.e., 3 h. Thus, although a quantification of the level of infection is beyond the capabilities of the DBA, in terms of a rapid assessment for surveillance purposes, it represents an affordable alternative that offers more sensitivity than the skin snip test. Moreover, the test can be performed using only a few drops of finger prick blood, and this represents an advantage in terms of a higher compliance from the individuals in areas under constant screening to evaluate the impact of control measures. Several promising diagnostic methods, based on detection of specific antibodies, parasite DNA, or parasite antigens in clinical specimens, have been developed for onchocerciasis in recent years . However, none of these have been implemented under field conditions in the areas of endemicity. Antibody tests using recombinant antigens have been based on standard, indirect enzyme-linked immunosorbent assays, which can be performed in a simple laboratory. The antigen and DNA detection methods are performed over 2 or 3 days, and they require more, in terms of laboratory infrastructure, equipment, and expensive reagents, than the enzyme-linked immunosorbent assay . In either case, the costs involved in both the acquisition of instrumentation and the transfer of the technology are not trivial, thus making the development of less expensive, stable, and reliable methods a necessity in low-income countries where onchocerciasis is being controlled or still exists. Simple yet reliable approaches like the use of a less expensive diagnostic technique that could be implemented under field conditions are of particular importance, especially when the successor to OCP, the African Program of Onchocerciasis Control, is facing funding difficulties which could threaten its future success . The PakF-DBA described here, unlike antigen and DNA detection methods, does not require pretreatment of samples or expensive supplies and equipment (e.g., PCR). We have evaluated the PakF-DBA in heat-inactivated eluted sera (56C; 30 min) and found no difference in intensity between heated and unheated sera (data not shown). In addition, we have observed that PakF is highly stable at working temperatures normally found in the areas of endemicity, and it does not require the use of anti-immunoglobulin G4 conjugates to reduce the background, since the molecules comprising PakF do not seem to be recognized by such an isotype (Guzman et al., submitted). A possible disadvantage, compared to antigen or DNA detection methods, is that the PakF-DBA does not distinguish between past and present infections, but this is a problem of all antibody-based assays. Nevertheless, the PakF-DBA may still be a suitable tool for following changes in transmission when used in younger age groups. Furthermore, antibody testing still holds great promise as a means for monitoring changes in the transmission of O. volvulus after mass treatment of populations . Criticisms of the use of native human-derived material in the PakF-DBA have previously been made in terms of reproducibility of material and safety. We have made many preparations of PakF, and the reproducibility in performance in the DBA has been high. A reliable, sensitive, and specific recombinant protein would be valuable, but in its absence, native proteins still have a role. Where the question of possible exposure to contaminant viruses is concerned, PakF, which is a protein fraction stable at high temperatures, may be heat treated before use (Guzman et al., submitted). In addition, the PakF-DBA can be used with heat-inactivated sera. Concern about the availability of parasite material to prepare PakF is justified. However, in areas where no control measures have been established, the relative abundance of nodules from which the worms are freed makes it possible to obtain large amounts of PakF to be used in rapid screening of sentinel populations. In our hands, the amount of TSF obtained from a batch of one to three worms is enough to run a HiTrap-Q column up to 10 times. Each purification round produces 350 mul of PakF that can be applied to nitrocellulose strips without further treatment. The yield after 10 purification rounds (3.5 ml) allows the analysis of at least 3,000 samples, using the format of duplicate PakF spots per strip. Even if the format is changed to use 1 mul per spot, the number of samples that could be tested is large enough, considering that the material comes from only one to three worms. In the areas of endemicity, where nodulectomy is a common practice not only for control but also for aesthetic purposes, the availability of worms for PakF production would not be an insurmountable difficulty. We and others have shown variations in the protein compositions of O. volvulus parasites from different geographic areas . However, we have prepared PakF from worms isolated in both Ghana and Guatemala, used it in crossed analysis with sera from the two countries, and obtained similar results regardless of the origin of the parasite material (Guzman et al., submitted). Therefore, geographical variations would not be of concern in screening a particular area with parasite material obtained from worms from another region. In conclusion, the present study provides evidence that it is still possible to implement simple techniques for the monitoring of onchocerciasis which offer high sensitivity and specificity while being affordable in financially challenged areas. Where costs are an issue, and in the absence of more elaborate diagnostic tests in the areas of endemicity, simple tests like the PakF-DBA could still deliver reliable information that can be used to monitor changes in the transmission of onchocerciasis. PMID- 12204955 TI - Specificities and Opsonophagocytic Activities of Antibodies to Pneumococcal Capsular Polysaccharides in Sera of Unimmunized Young Children AB - | An enzyme immunoassay (EIA) for antibodies to pneumococcal capsular polysaccharides (Pnc PSs) detects in some cases antibodies that are cross-reactive within different Pnc PSs. Recently, it has been suggested that for detection of only serotype-specific antibodies, EIA can be modified by removing cross-reactive antibodies by absorption with an irrelevant PS, e.g., the type 22F PS. The opsonophagocytosis assay measures the functional activities of antibodies in vitro, and the results of that assay correlate with in vivo protection better than measurement of the antibody concentration by EIA. We compared these different methods for measuring antibodies to type 1, 6B, 11A, 14, 19F, and 23F Pnc PSs in the sera of unimmunized young children who had been monitored for pneumococcal carriage, acute otitis media, and acquisition of antibodies to Pnc PSs from 2 to 24 months of age. Serum samples with antibody increases after contact with a pneumococcus of a homologous serotype contained specific antibodies and often had opsonophagocytic activity (OPA) (20 of 46). In samples with antibody increases from children who had not had contact with a pneumococcus of a homologous serotype, the antibodies found to be type specific by conventional EIA were usually cross-reactive and infrequently had OPA (10 of 68). When type 22F PS absorption was used in the EIA, most of the false antibody increases were eliminated, but most of the true antibody increases were still detected and the association between the antibody concentration detected by EIA and OPA was improved. However, there were serotype-dependent differences in the frequency of OPA. Use of absorption with a heterologous PS in EIA should be encouraged, and both the specificity of EIA and the sensitivity of opsonophagocytic assays should be further evaluated and improved. PMID- 12204955_Introduction TI - AB - Immunity against Streptococcus pneumoniae (pneumococcus) is mediated by phagocytosis in the presence of complement and antibodies to pneumococcal capsular polysaccharides (Pnc PSs) . The in vitro opsonophagocytic activities (OPAs) of serum antibodies are believed to represent the functional activities of the antibodies in vivo and thus to correlate with protective immunity . Enzyme immunoassay (EIA) for the measurement of the concentrations of antibodies to Pnc PSs has been widely used to measure immunity to pneumococci and the immunogenicities of pneumococcal vaccines. However, for the estimation of immunity, a good correlation between the concentration of immunoglobulin G (IgG) measured by EIA and the OPAs of antibodies is needed. The correlation between the two methods has been reasonably good with postimmunization serum samples from infants and adults . However, sera from unimmunized individuals may have lower OPAs than expected on the basis of the antibody concentration obtained by EIA . The Pnc PS preparations used in the present EIAs are contaminated with a common cell wall PS (CPS) , and antibodies to CPS should be absorbed to improve the specificity of the EIA . Recently, several investigators have reported that despite absorption with CPS, antibodies cross-reactive with several types of Pnc PSs are still measured by EIA . The reason for this cross-reactivity has not been confirmed. It has been suggested that the Pnc PS preparations used as EIA antigens contain impurities or cross-reactive epitopes common to many serotypes . Removal of the cross-reactive antibodies by absorption with an irrelevant heterologous PS, e.g., the type 22F PS, improves the correlation between the antibody concentration obtained by EIA and the OPA . Thus, type 22F PS absorption has been suggested as an additional step in EIAs for antibodies to Pnc PSs. Cross-reactive antibodies are found more often in the sera of unimmunized infants and adults than in the sera of infants and adults immunized with pneumococcal vaccines, suggesting that the majority of the antibodies induced by vaccination are Pnc PS type or group specific . The origin and development of the cross-reactive antibodies by age has not been studied. We have previously described the natural development of antibodies to Pnc PSs, as detected by EIA, during the first 2 years of life , and antibody responses in children with pneumococcal acute otitis media (AOM) in a Finnish Otitis Media (FinOM) Cohort Study . Pneumococcal carriage and AOM induced antibodies to the homologous Pnc PS, but there were serotype-specific differences . Low concentrations of antibodies were also produced after contact with pneumococci with heterologous serotypes and even with no detectable contact with pneumococci . In this study, we evaluated the specificities and OPAs of antibodies in selected serum samples from our previous study . We used a set of samples that had twofold or greater increases in antibody concentrations compared to the concentration in a sample taken 6 months earlier and with approximately 1 mug or more of anti-Pnc PS antibodies per ml, as measured by the conventional EIA. Such samples were selected from children with and without a previous contact with pneumococci of the homologous serotype. The effect of the type 22F PS absorption step in the EIA was also evaluated. PMID- 12204955_MATERIALS AND METHODS TI - AB - Study population and sera. | Serum samples for this study were selected from among the samples from participants in the FinOM Cohort Study used in our previous study . The study population consisted of 329 healthy Finnish children who were monitored prospectively in a special study clinic from 2 to 24 months of age . During scheduled visits at 2, 3, 4, 5, 6, 9, 12, 15, 18, and 24 months of age, interview data and nasopharyngeal (NP) swab specimens for detection of pneumococcal carriage were obtained. In addition, NP aspirates (NPAs) and middle ear fluid (MEF) samples were obtained from patients with respiratory infection and from patients with a diagnosis of AOM, respectively. Serum samples (5 ml of venous blood) were obtained from the children at scheduled visits at 6 (+-14 days), 12 (+-14 days), 18 (+-28 days), and 24 (+-28 days) months of age. The sera were stored at -20C. The NP swabs, NPAs, and MEF samples were collected and cultured as described previously for detection of pneumococcal carriage or the etiology of AOM. Pneumococci were identified and serotyped by standard methods . The development of antibodies by age has been reported earlier . Serum samples used in this study. | Selected serum samples taken at the scheduled visits at 18 or 24 months of age were used in this study. The following criteria were used for selection: a twofold or greater increase in the concentration of antibody to PS of type 1, 6B, 11A, 14, 19F, or 23F compared to the concentration in the sample taken during the previous scheduled visit (i.e., at 12 or 18 months, respectively) and the presence of an antibody concentration of approximately 1 mug/ml or greater. An antibody concentration of at least 1 mug/ml is needed to achieve detectable OPA in infant sera taken after vaccination . The serum samples were further grouped according to pneumococcal culture findings . Sera in the Pnc PS contact-positive (Pnc 6B+, Pnc 11A+, Pnc 14+, Pnc 19F+, and Pnc 23F+) groups were from children who had had at least one culture-confirmed contact with a pneumococcus of the indicated serotype (pneumococci of that serotype were cultured from NP swabs, NPAs, and/or MEF samples) between the two scheduled visits. Accordingly, sera in the Pnc PS contact-negative (Pnc 1-, Pnc 6B-, Pnc 11A-, Pnc 14-, Pnc 19F-, and Pnc 23F-) groups were from children who had not had any detectable contact with the respective serotype by the indicated age, despite the increase in antibody concentrations. These children may have had contacts with other pneumococcal serotypes or no contacts with pneumococci at all. Data were derived from a total of 82 serum samples from 75 children. Some samples were included more than once. PSs. | Capsular PSs of S. pneumoniae serotypes 1 (lot 1211368), 11A (lot 963596), 14 (lot 2020510), 19F (lot 2033178), 22F (lots 1450702 and 2045906), and 23F (lot 1417200) were obtained from American Type Culture Collection (ATCC; Manassas, Va.). Capsular PS of serotype 6B was received via collaboration with the National Institute of Public Health and the Environment (Bilthoven, The Netherlands). The CPS used for absorption of anti-CPS antibodies from the sera was from the Statens Serum Institut (Copenhagen, Denmark). EIA. | The conventional EIA was performed as described earlier by Kayhty et al. . The results are expressed as micrograms per milliliter, calculated on the basis of the officially assigned IgG concentrations in reference serum sample 89-SF . In addition to the conventional EIA, antibody concentrations were determined by EIA with an absorption step with type 22F PS; the lowest serum dilution was incubated with 10 mug of CPS per ml and 30 mug of type 22F PS per ml for 1 h at room temperature before the EIA. The interassay coefficient of variance for both EIA methods was <15%. Inhibition EIA. | Serum samples were diluted 1:100 in 10% fetal bovine serum (Life Technologies, Ltd., Paisley, Scotland) in phosphate-buffered saline containing 10 mug of CPS per ml and aliquoted and placed into three separate tubes. Either homologous (type 1, 6B, 11A, 14, 19F, or 23F) or heterologous (type 22F) Pnc PS (30 mug/ml) was added, and the tubes were incubated for 1 h at room temperature. A control tube containing only CPS was included. The concentration of 30 mug/ml was found to be optimal for absorption for the PS types studied . After absorption, EIA was performed as described above. The percent inhibition was calculated from the optical density values. Inhibition of antibody binding by <=20% was considered low, and inhibition of antibody binding by >=80% was considered effective. Opsonophagocytosis assay. | The OPAs of the antibodies were analyzed by measuring the killing of live pneumococci by differentiated HL-60 cells in the presence of serum antibody and complement. A modification of the method described by Romero-Steiner et al. was used. S. pneumoniae serotypes 1, 6B, 14, 19F, and 23F (reference strains received from the Centers for Disease Control and Prevention, Atlanta, Ga.) and serotype 11A (reference strain obtained from the World Health Organization Collaborating Center for Reference and Research on Streptococci, Prague, Czech Republic) were grown in Todd-Hewitt broth supplemented with 0.5% yeast extract and kept frozen (-70C) in aliquots in Todd-Hewitt broth with 15% glycerol. OPA is expressed as a titer that is the reciprocal of the serum dilution with 50% killing compared with the bacterial growth in the controls without serum. A titer of 4 was given to sera with undetectable OPAs. To demonstrate the specificities of opsonic antibodies, serum samples were preincubated with either CPS or homologous or heterologous Pnc PS (30 min at room temperature with 1 mg of PS or CPS in 1 ml of undiluted sera) prior to the assay for OPA. OPA was totally removed only from samples preincubated with the homologous Pnc PS. Statistical analyses. | The average antibody concentrations were expressed as geometric mean concentrations (GMCs) and the average OPAs were expressed as geometric mean OPAs (GMOPAs). TABLE 1 | Selection of serum samples from among the FinOM Cohort Study samples for the different groups PMID- 12204955_RESULTS TI - AB - In the whole FinOM Cohort Study population, the number of serum samples with twofold or greater increases in antibody concentrations that yielded an approximately 1 mug/ml or higher concentration in the sample obtained at 18 or 24 months of age depended on the serotype . Among the serum samples from children without a previous contact with the homologous serotype, increases in the concentrations of antibodies to type 1, 6B, 11A, 14, and 23F PSs were detected in 4 to 9% of serum samples and increases in the concentrations of antibodies to type 19F PS were detected in 20% of the serum samples . Among the children with antibody increases but without contact with homologous type 1, 6B, 11A, 14, 19F, or 23F pneumococci, 8 of 13, 7 of 8, 16 of 20, 7 of 10, 20 of 34, and 5 of 12, respectively, had had contact with other serotypes of pneumococci. Antibody increases occurred more frequently (11 to 70%) in children with contact with a homologous serotype . Specificities of antibodies and effect of type 22F PS absorption on the antibody concentration measured by EIA. | The specificities of antibodies to serotypes 1, 6B, 11A, 14, 19F, and 23F measured by EIA were analyzed by inhibition of antibody binding with the homologous PS (type 1, 6B, 11A, 14, 19F, or 23F) or a heterologous PS (type 22F). Serotype-specific antibodies were considered those that were not inhibited (<20%) by the heterologous PS but that were effectively inhibited (>80%) by the homologous PS. Cross-reactive, nonspecific antibodies were inhibited (>20%) by heterologous PS. The antibodies to serotypes 6B and 14 in the serum samples from the Pnc 6B+ and Pnc 14+ groups were highly specific. In most cases the level of inhibition by heterologous PSs was <20%, and it was never >80% . Furthermore, they were mainly inhibited only by the homologous PSs: >80% inhibition by homologous PSs was seen in three of four and five of six serum samples, respectively . However, poorer inhibition by homologous PSs was found in the serum samples from the Pnc 6B- and Pnc 14- groups. It should be noted that three of the eight serum samples in the Pnc 6B- group were from children who had had contact with serotype 6A, and in two of the three serum samples the antibodies to PS type 6B were specific. Consequently, in all groups the concentrations of antibodies to type 6B and type 14 PSs obtained either by the conventional EIA or by EIA with the type 22F PS absorption step were equal; in addition, neither the GMCs nor the antibody concentrations in the individual serum samples (data not shown) were affected by the use of type 22F PS absorption. Antibodies to serotype 11A were highly specific in the Pnc 11A+ group: heterologous inhibition of <20% was detected in all 16 serum samples and homologous inhibition of >80% was detected in 15 of the 16 serum samples . In the Pnc 11A- group, however, heterologous inhibition of >20% was detected in 9 of 17 serum samples. Consequently, the EIA with type 22F PS absorption gave lower antibody concentrations than the conventional EIA for the Pnc 11A- group but not the Pnc 11A+ group . Very similar observations were made for serotype 23F: antibodies were specific in the Pnc 23F+ group but mostly cross-reactive in the Pnc 23F- group. The type 22F PS absorption step in EIA markedly reduced the concentrations of antibodies to type 23F PS in the Pnc 23F- group but not the Pnc 23F+ group . Antibodies to type 19F PS had a complex pattern. The specificities of the antibodies were somewhat better in the Pnc 19F+ group than in the Pnc 19F- group; 7 of 13 and 4 of 13 serum samples, respectively, were specific and were not inhibited (>20%) by the heterologous PS . However, many serum samples showed antibody binding that could not be effectively (>80%) inhibited by either type 19F or type 22F PS. All the serum samples from the Pnc 19F- group had antibodies with poor specificities; 5 were effectively (>80%) inhibited by heterologous PS, in addition to the type 19F PS, and in 5 additional serum samples the level of inhibition by either the homologous PS or the heterologous PS was low. The EIA with the type 22F PS absorption step gave lower concentrations than the conventional EIA for both the Pnc 19F- and Pnc 19F+ groups. However, the difference was more remarkable in the Pnc 19F- group . Serotype 1 was not cultured from any of the samples during the study. In spite of this, 13 children had increased levels of antibodies to the type 1 Pnc PS, as measured by conventional EIA . The antibodies to serotype 1 were not specific: inhibition by the homologous serotype as well as inhibition by the heterologous serotype was >80% for all serum samples . The EIA with type 22F PS absorption detected practically no antibodies to the type 1 Pnc PS . OPAs of antibodies. | Higher OPAs against pneumococci of serotypes 6B, 11A, 14, and 23F were measured in the sera of children taken after contact with a homologous serotype than in the sera of children with no contact with a homologous serotype . Even though the concentrations of specific antibodies (determined by EIA with type 22F PS absorption) to type 6B and type 14 PSs were equal, the OPAs were higher and were found more frequently in the Pnc 6B+ and Pnc 14+ groups than in the Pnc 6B- and Pnc 14- groups . Although most (16 of 26) of the serum samples in the Pnc 19F+ and 19F- groups had concentrations of antibodies to type 19F PS of >=1 mug/ml, as measured by EIA with type 22F absorption, none of the samples had OPAs against the type 19F strain . Antibodies to the type 1 PS were removed from all serum samples by type 22F PS absorption. Accordingly, none of the serum samples had OPAs against the type 1 strain . Overall, the sera of children with contact with pneumococci of a homologous serotype had OPA (titers, >=8) more often than the sera of children without homologous contact (20 of 46 versus 10 of 68 for the pneumococcal serotype contact-positive versus the pneumococcal serotype contact-negative groups combined; Table ). Furthermore, the antibodies were more often specific in the samples of children with contact with pneumococci of the homologous serotype than in the samples of children without contact with pneumococci of the homologous serotype . Only 3 of the 45 samples with cross-reactive antibodies had OPAs, whereas 27 of the 69 samples with specific antibodies had OPAs . Association between antibody concentration and OPA. | The concentrations of antibodies to type 6B, 11A, 14, and 23F PSs in serum measured by the conventional EIA and the EIA with type 22F PS absorption were compared to the OPAs . For these analyses, serum samples in the pneumococcal serotype contact-positive group and the pneumococcal serotype contact-negative group were combined. Serotypes 1 and 19F were not included because no OPAs against these serotypes were detected . For serotypes 6B and 14, the two EIAs gave very similar antibody concentrations , and thus, the type of EIA did not influence the association of concentration and OPA . For serotypes 11A and 23F the cross-reactive nonfunctional antibodies were markedly inhibited by type 22F PS and the association between the concentrations and the OPAs of antibodies was improved when EIA with the type 22F PS absorption step was used to determine the antibody concentration . FIG. 1. | Association between IgG concentration (in micrograms per milliliter), as measured by conventional EIA or by EIA with type 22F PS absorption, and OPAs (as titers) of antibodies to type 6B, 11A, 14, and 23F Pnc PSs. Association between IgG concentration (in micrograms per milliliter), as measured by conventional EIA or by EIA with type 22F PS absorption, and OPAs (as titers) of antibodies to type 6B, 11A, 14, and 23F Pnc PSs. Serum samples were taken from unimmunized children at 18 or 24 months of age. Horizontal lines, a titer of 4 was given to sera with undetectable OPA; vertical lines, a concentration of 1.0 mug/ml was used as a cutoff for the EIAs. TABLE 2 | Number of serum samples with levels of inhibition of <20% and >80% with the indicated homologous and heterologous (type 22F) Pnc PSs, GMCs of antibody measured by conventional EIA or EIA with type 22F PS absorption, and GMOPAs of antibodies to Pnc PSs 1, 6B, 11A,14, 19F, and 23F in sera of unimmunized children in the different groups TABLE 3 | Numbers of serum samples taken at 18 and 24 months of age from unimmunized children in the different groups containing either specific or cross-reactive antibodies to type 1, 6B, 11A, 14, 19F, and 23F Pnc PSs and OPAs of the samples PMID- 12204955_DISCUSSION TI - AB - We have previously studied the natural development of antibodies to Pnc PSs and its association with previous contacts with pneumococci, nasopharyngeal carriage, or AOM in the FinOM Cohort Study population . Previous contacts were associated with increased concentrations of antibodies to the homologous serotype, but there were serotype-specific differences. However, the children developed low concentrations of antibodies to all the Pnc PS types studied even without contact with the homologous serotype . The reason seems to be the fact that the detection of antibodies to Pnc PSs is affected by the cross-reactivity of the Pnc PS antigens used in the present EIA . We studied here the specificities and functional activities, as measured by opsonophagocytosis assay, of these antibodies and correlated these characteristics with culture-confirmed contacts with pneumococci. We also evaluated the possibility of using the type 22F PS absorption step in EIA to improve the specificity. For the whole study population, antibody increases yielding >=1 mug of antibodies per ml in the children without contact with the homologous serotype were rare compared to the antibody increases in children with contact with the homologous serotype: 20 versus 29% for serotype 19F and 4 to 9% versus 11 to 70% for the other five serotypes studied. We included in this study serum samples taken at either 18 or 24 months of age; these ages of sample collection were chosen to exclude maternal antibodies. Sera were grouped according to the pneumococcal contacts that the children had had by the indicated age. In general, serum samples from children with contact with a homologous serotype contained serotype-specific antibodies. Moreover, these samples often had OPAs, although there were clear serotype-specific differences. In contrast, antibodies in serum samples from children without contacts with a homologous serotype were mostly cross-reactive and infrequently had OPAs. Previous studies by us and other investigators with sera from adults have shown that detection of antibody to serotype 14 by EIA is serotype specific, whereas detection of antibody to the other serotypes is hampered by cross-reactivity . The present data with sera from a pediatric population confirm that antibodies to type 14 PS are highly specific. All serum samples in the Pnc 14+ group also had OPA against type 14. In contrast, only a few serum samples in the Pnc 14- group had OPA against type 14. This is interesting since this difference in OPA between the Pnc 14+ and the Pnc 14- groups cannot be explained by differences in antibody concentrations or antibody specificity. The origins of the antibodies in samples in the Pnc 14- group are not known; the antibodies were not absorbed by type 22F PS, and in a few cases they also had OPA. It is possible that some contacts with pneumococci remained undetected or that the children had had contacts with bacteria with PSs similar to type 14, e.g., the type III PS from group B streptococci . In the case of serotype 14, the increase in antibody levels or the concentration alone was not as good a marker of previous contact with type 14 as OPA was. The results of the present study confirm that the Pnc PS type 6B preparation used here and in our previous studies (-) is specific. However, although the specificities of antibodies to PS 6B were good in both the Pnc 6B+ and the Pnc 6B- groups, only a few serum samples had OPA. This may be due to the overall low antibody concentrations that were insufficient for detection of OPA by the present opsonophagocytosis assay. A previous contact with serotype 6A, which is cross-reactive with 6B, explained the increases in the levels of antibodies to type 6B PS in three of the eight serum samples in the Pnc 6B- group, and in two of the three samples the antibodies specificities were high. However, the antibodies in these three samples had no OPA against type 6B, despite the presence of anti-type 6B antibody concentrations equal to those in the samples from the Pnc 6B+ group showing OPA. This is in accordance with data showing that antibodies to type 6A elicited by vaccines containing type 6B may not be functional against type 6A strains or that more antibodies to type 6B PS are needed for killing of a type 6A strain than for killing of a type 6B strain . Altogether 26 serum samples with >=1 mug of IgG against type 19F PS per ml, as detected by the conventional EIA, were analyzed, and none of these serum samples had OPA against type 19F. The specificities of the antibodies in samples from children without previous contact with type 19F were poor. However, despite the higher concentrations and better specificities of antibodies in sera from children with cultures positive for type 19F, the sera showed no OPA. The specificities of antibodies to type 19F, as determined by EIA, in general, were confusing; in some cases antibody binding could not be inhibited by either the homologous PS or the heterologous PS. Furthermore, high concentrations of antibodies to type 19F PS even in the sera of vaccinated children do not seem to offer protection against AOM . Also, higher anti-type 19F antibody concentrations than anti-type 6B antibody concentrations are needed in the sera of vaccinated children for OPA . We have previously described that antibodies to type 1 PS can be detected by conventional EIA in serum samples of children participating in the FinOM Cohort Study, even though type 1 pneumococcus was not detected in any of the NP, NPA, or MEF samples during the study . We have now shown that these antibodies are not specific for type 1 PS and therefore are probably produced by a non-type-specific antigenic stimulus. These antibodies possessed no OPA against type 1 pneumococci. Use of the type 22F PS absorption step in the EIA removed all antibodies to type 1 PS detected by the conventional EIA in all samples. The data obtained in this study highlight the need for heterologous type PS absorption in EIA, especially when samples from an unimmunized population are studied. Type 22F PS absorption did not have a notable effect on the concentrations of antibodies to type 6B and type 14 Pnc PSs. However, the type 6B antigen was especially chosen for this study , and the commercially available preparations seem to be more cross-reactive , indicating that, in general, the type 22F PS absorption step is also needed for the anti-type 6B EIA. Because the nature of the cross-reactivity is unknown , the specificity and amount of antibodies inhibited by type 22F PS cannot be determined. It is, however, probable that the antibodies removed are not serotype specific and, in principle, should not be detected. Data from us and others show that the type 22F PS absorption step increases the association between the antibody concentration obtained by EIA and OPA. Furthermore, the results of the present study suggest that if antibody detection by EIA is used to study the seroepidemiology of pneumococcal infections, an EIA with type 22F PS absorption instead of the conventional EIA is highly recommended. The present opsonophagocytosis assay requires approximately 1 mug of antibody per ml for detection of OPA in the sera of immunized infants . As shown here, the concentration of specific antibodies needed may be even higher in sera of unimmunized children and may be different for the different serotypes. In the present study, high concentrations of specific antibodies to type 11A and 19F PSs were insufficient for OPA, while lower concentrations were associated with OPA against type 14 and 23F strains. It is possible that in addition to the antibody concentration and specificity, other qualitative characteristics of the antibodies, e.g., antibody avidity, affect OPA . Contacts with pneumococci seem to elicit the development of specific and functional antibodies, and thus, detection of OPA in sera could be an indicator of a serotype-specific pneumococcal contact. However, the sensitivity of the assay for OPA described here should be improved to detect activity in the sera of unimmunized individuals. PMID- 12204964 TI - Affordable CD4+-T-Cell Counting by Flow Cytometry: CD45 Gating for Volumetric Analysis AB - | The flow cytometers that are currently supported by industry provide accurate CD4+-T-cell counts for monitoring human immunodeficiency virus disease but remain unaffordable for routine service work under resource-poor conditions. We therefore combined volumetric flow cytometry (measuring absolute lymphocyte counts in unit volumes of blood) and simpler protocols with generic monoclonal antibodies (MAbs) to increase cost efficiency. Volumetric absolute counts were generated using CD45/CD4 and CD45/CD8 MAb combinations in two parallel tubes. The percentage values for the various subsets were also determined within the leukocyte and lymphocyte populations utilizing a fully automated protocol. The levels of agreement between the newly developed method and the present industry standards, including both volumetric and bead-based systems using a full MAb panel for subset analysis, were tested by Bland-Altman analyses. The limits of agreement for CD4 counts generated by the volumetric methods using either CD45/CD4 (in a single tube) or the full Trio MAb panel (in three tubes) on the CytoronAbsolute flow cytometer were between -29 and +46 cells/mm3 with very little bias for CD4 counts (in favor of the Trio method: +8 CD4+ lymphocytes/mm3; 0.38% of lymphocytes). The limits of agreement for absolute CD4 counts yielded by the volumetric CD45/CD4 method and the bead-based method were between -118 and +98 cells/mm3, again with a negligible bias (-10 CD4+ lymphocytes/mm3). In the volumetric method using CD45/CD8, the strongly CD8+ cells were gated and the levels of agreement with the full Trio showed a minor bias (in favor of the Trio; +40 CD8+ cells/mm3; 5.2% of lymphocytes) without a significant influence on CD4/CD8 ratios. One trained flow cytometrist was able to process 300 to 400 stained tubes per day. This workload extrapolates to a throughput of >30,000 samples per year if both CD45/CD4 and CD45/CD8 stainings are performed for each patient or a throughput of >60,000 samples if only CD45/CD4 counts are tested in a single tube. Thus, on the basis of the high efficiency and excellent agreement with the present industry standards, volumetric flow cytometers with automated gating protocols and autobiosamplers, complemented by generic CD45, CD4, and CD8 MAbs used in two-color immunofluorescence, represent the most suitable arrangements for large regional laboratories in resource-poor settings. PMID- 12204964_Introduction TI - AB - Dedicated flow cytometers are designed to enumerate the absolute numbers and percentages of lymphocyte populations, such as subsets of T cells, B cells, and NK cells. In the clinical service for monitoring human immunodeficiency virus (HIV) disease, the primary aim is to deliver absolute CD4+-T-cell counts, and this is achieved with a remarkably high level of precision . Nevertheless, the various cytometric systems differ in complexity . It has recently been documented that routine CD4-T-cell enumeration can be simplified without compromising quality , leading to cost-effective services for patients who receive generic antiretroviral drugs in resource-poor settings . Among the flow cytometers, dedicated instruments operating as "single platforms" are preferred due to their convenience and accuracy . These single platforms are based either on a volumetric principle by counting CD4+ T cells in a unit volume of blood or on the concept of adding known numbers of fluorospheres, or "microbeads," to each sample . These beads are, however, precision products that can increase running costs. Consequently, services handling large numbers of samples had to revert to "double platforms" operating a panleucogating strategy with CD45 monoclonal antibody (MAb) to secure a much less expensive but still accurate mode of operation . Indeed, the CD45-based gating, an example of the "heterogeneous" gating strategy, is a more reliable protocol when used with autogating in aging samples than the conventional gating strategies that utilize morphological scatter gates . Despite the present interest in improving the efficacy of routine flow cytometry , the performance of volumetric flow cytometric systems operating with CD45-based gating and generic MAbs has not yet been assessed. We have therefore investigated the following topics: (i) the agreement between the results of CD45/CD4 staining using simple panleucogating on volumetric single platforms and those obtained on the full volumetric and bead-based systems during CD4+-T-cell enumeration, including both absolute counts and CD4 percentage values (among leukocytes and lymphocytes); (ii) the increased sample throughput using CD45/CD4 staining; (iii) the extension of this protocol to include a second tube for CD45/CD8 staining in order to obtain CD4-plus-CD8 counts and CD4/CD8 ratios; and finally, (iv) the use of volumetric CD45 staining for generating absolute and differential counts for leukocyte subsets . Our study reveals the practical advantages of volumetric two-color flow cytometry with CD45/CD4 and CD45/CD8 staining using generic MAbs. Volumetric cytometers, equipped with biosamplers of high capacity, Microsoft Windows-based autogating software, and reporting systems, efficiently handle 300 to 400 samples during a working day. As many as 15 parameters, including CD4 and CD8 analysis together with hematological leukocyte differentials, can be generated for cost-efficient monitoring of HIV-infected patients in large regional laboratories. (Part of this research was presented at Monitoring and Diagnostic Tools for the Management of Antiretroviral Therapy in Resource-Poor Settings, a workshop held in Bethesda, Md., 11 to 13 November 2001, and arranged by Virology Education, B.V., Utrecht, The Netherlands.) FIG. 1. | Recent events leading to affordable CD4-T-cell enumeration by flow cytometry. Recent events leading to affordable CD4-T-cell enumeration by flow cytometry. NIBSC, National Institute for Biological Standards and Control; NEQAS, UK National External Quality Assessment Service, Sheffield, United Kingdom; QASI, Quality Assessment & Standardization for Immunology, Ottawa, Canada. PMID- 12204964_MATERIALS AND METHODS TI - AB - Clinical samples. | Samples (n = 93) were received for routine immunological diagnosis at an HIV-immunology laboratory and included patients at various stages of HIV infection as part of the routine diagnostic and quality assurance activity at the Royal Free Hospital. No extra specimens from HIV-seropositive patients were required. Twelve additional samples were taken from healthy volunteers 21 to 59 years of age as approved by the Institutional Ethics Committee . These whole-blood samples were collected in EDTA and analyzed within 24 h using a "lyse-no-wash" procedure . Briefly, in each tube, 10 or 20 mul of a diluted mixture of antibodies was admixed with 50 or 100 mul of whole blood, respectively. After 15 min of incubation at room temperature, 2.0 ml of lysing solution (0.17 M NH4Cl) was added. The samples were counted after a final 15-min incubation . Instrumentation. | Absolute lymphocyte subset counting was performed on three systems: (i) a CytoronAbsolute (Ortho Diagnostic Systems Inc., Raritan, N.J.) operating with Ortho Trio reagents and Immunocount II software to provide absolute counts by a volumetric method ; (ii) a FACSCalibur (Becton Dickinson Immunocytometry Systems, Oxford, United Kingdom) operating with TruCOUNT tubes preloaded with a known number of beads (46,295 beads per tube); cell concentrations were calculated with the formula (number of events in the region containing the cell population/number of events in the region containing beads) x (46,295/test volume [i.e., 50 mul]); and (iii) a CytoronAbsolute system operating with Immunocount II software using panleucogating in order to record three parameters: side scatter (SSc), green fluorescence (CD45-fluorescein isothiocyanate [FITC]), and orange fluorescence (CD4-phycoerythrin [PE] or CD8-PE). The volumetric absolute counting properties of the Cytoron were utilized with both systems i and iii. . The capacities of the automatic biosampler devices used were 100 (systems i and iii) and 40 (system ii) tubes. Total and differential white blood cell (WBC) counting was performed on a Bayer 120 hematology analyzer in the hospital's hematology laboratory as part of the routine service. Reagents. | During the volumetric-control procedure on the Ortho CytoronAbsolute, Ortho Trio MAbs were used . These included three tubes comprising in tube 1 isotype controls (immunoglobulin G1 [IgG1] plus IgG2a-FITC-IgG1 plus IgG2a-PE-IgG2a-PECy5), in tube 2 CD4(OKT4)-FITC-CD8(OKT8)-PE-CD3(OKT3)-PECy5, and in tube 3 CD16(3G8)-FITC-CD19(OKB9)-PE-CD3(OKT3)-PECy5 (original clone designations are shown in italics). During the bead-based control procedure on the FACSCalibur, TruCOUNT tubes were used in combination with MultiTEST reagents (Becton Dickinson Immunocytometry Systems) to obtain absolute CD4 counts . These included CD3(SK7)-FITC-CD8(SK1)-PE-CD45(2D1)-PerCP-CD4(SK3)-APC. The new CD45-based protocol was also based on the volumetric procedure performed on the Ortho Cytoron. Two tubes containing two-color immunofluorescence (IF) reagents were each tested. Tube 1 contained CD45(2D1)-FITC-CD4(RFT4)-PE, and tube 2 comprised CD45(2D1)-FITC-CD8(RFT8)-PE. These generic reagents are available in unconjugated form from the National Institute for Biological Standards and Control (Potters Bar, United Kingdom). Gating strategies for CD4 and CD8 enumeration. | On the Ortho Cytoron, we employed the Trio reagents and obtained absolute counts for the following cell types: T cells (CD3+; low side scatter), CD4+ T lymphocytes (CD3+ CD4+ CD8-), CD8+ T lymphocytes (CD3+ CD8+ CD4-), B cells (CD19+; low side scatter), NK cells (CD3- CD16+), and total lymphocytes (CD3+ T plus CD19+ B plus CD16+ NK cells referred to as Immunosum) . Percentage values for CD4+ T lymphocytes (CD4%) were derived as the number of CD4+ T cells divided by the total number of lymphocytes based on the criteria of CD4+, CD3+, and CD8- cells/Immunosum . The CD4/CD8 ratios were calculated as (CD3+ CD4+)/(CD3+ CD8+) values. The internal quality control for pipetting errors was based on CD3 replicates using Immunocount II software : samples for which the CD3 replicates differed from the average absolute CD3 count by >5% were automatically flagged for further inspection. The event threshold was set to operate on forward scatter. On the FACSCalibur, the gating strategy recommended by the manufacturer was used, with the threshold set for red fluorescence (CD45) in a single tube . For the new protocol, the gating strategy was based on CD45 panleucogating . A threshold was first set for green (CD45) fluorescence, and all WBCs were identified using a heterogeneous CD45/SSc dual-parameter histogram (CD45+ to CD45+++ in gate A). All WBC events in gate A were then sent to a CD4/SSc histogram, where CD4 T cells were counted (CD4++/SSc+ in gate E ). The same gating strategy was applied for CD8 counting in a second tube. Here, only lymphoid cells with bright CD8 expression were counted as CD8 T cells (CD8++/SSc+ in gate F . The CD4/CD8 ratios were calculated as (CD4++ SSc+)/(CD8++ SSc+) values. All these gating strategies were set to operate automatically and printed with all details . The internal quality control for pipetting errors was based on CD45 WBC replicates using the Immunocount II program. If the CD45 total WBC replicates differed from the average absolute CD45 count by >5%, the samples were flagged. All flagged samples or those where the operator had detected gating irregularities were subsequently reanalyzed. In the second stage of the analysis, the different CD45 staining intensities among the leukocyte populations were used to identify lymphocytes (CD45+++/SSc+ in gate B), monocytes (CD45++/SSc++ in gate C), and granulocytes (CD45+/SSc+++ in gate D). Using absolute counting and a two-color IF panel in two parallel tubes, the following 15 parameters were distinguished and stored: (i) total WBC counts, (ii) absolute CD4-T-cell counts, (iii) CD4-T-cell percentage among WBC, (iv) CD4-T-cell percentage among lymphocytes, (v) absolute CD8-T-cell counts, (vi) CD8-T-cell percentage among WBC, (vii) CD8-T-cell percentage among lymphocytes, (viii) absolute CD4- plus CD8-T-cell counts, (ix) CD4/CD8 ratio, (x) absolute lymphocyte counts, (xi) absolute monocyte counts, (xii) absolute granulocyte counts, (xiii) lymphocyte percentage among WBC, (xiv) monocyte percentage among WBC, and (xv) granulocyte percentage among WBC. In samples where a single tube was analyzed with CD45/CD4, 10 parameters (i to iv and x to xv) were recorded. Data handling and statistical analysis. | All results have been recorded in Microsoft Access-based spreadsheets. Following consultations with clinicians, forms were created for reports. Depending on the requests, these could include the single parameter of absolute CD4 count or all 10 to 15 parameters recorded above. After we tested whether the differences between the methods were normally distributed , Bland-Altman plots were used to investigate the agreement between the results obtained in two different systems, such as the panleucogating analysis on a volumetric flow cytometer versus a conventional "industry-standard" method. The standard techniques included the volumetric flow cytometer, CytoronAbsolute, using the full Trio panel, as well as the Becton Dickinson FACSCalibur running the TruCOUNT bead-based system. The absolute CD4+- and CD8+-lymphocyte counts and the percentages of these subsets among leukocytes and lymphocytes were studied. Bland-Altman (or bias) plots examine whether two methods have sufficient agreement to be used interchangeably. The average of values obtained by the two methods is plotted on the x axis, and the difference between the methods is plotted on the y axis. The average difference between the methods (bias), its 95% confidence intervals, and the limits of agreement (bias +- 2 standard deviations) are shown on the plots. The Pollock modification is identical to the Bland-Altman analysis except that the percentage difference is expressed between the compared methods, which is best suited to illustrate a systematic bias across a wide range of absolute counts. FIG. 2. | CD45/CD4 double staining on blood using panleucogating with volumetric absolute counting. CD45/CD4 double staining on blood using panleucogating with volumetric absolute counting. The graph report form is printed to document the autogating procedure. First, the CD45 side scatter histogram and gate A (all leukocytes) are established (left). All events of gate A are sent to the second display of CD4 side scatter (right). The CD4+ and lymphoid cells (in E) are automatically gated to provide the absolute CD4-T-cell count. E/A x 100 is the value of the CD4% among all leukocytes. The number of events in gate B (absolute lymphocyte count), gate C (absolute monocyte count), and gate D (absolute granulocyte count) are also defined. E/B x 100 is the CD4% value among lymphocytes. A parallel tube for CD45/CD8 double staining can also be run to provide the total of 15 parameters listed in Materials and Methods. QA, quality assurance; NEQAS, UK National External Quality Assessment Service. TABLE 1 | Age, sex, and HIV status of patient population studied PMID- 12204964_RESULTS TI - AB - CD4+-T-cell enumeration using primary CD45 and CD4 gating. | Total lymphocytes were identified by volumetric counting (i) as the sum of T cells, B cells, and NK cells (Immunosum) using Trio MAbs (referred to as a full Trio panel) and (ii) as cells with a bright CD45 expression and lymphoid side scatter in the CD45-based protocol. No significant systematic bias was observed between the two methods (bias = -8 lymphocytes/mm3 ). Next, CD4+-T-lymphocyte counts were determined by the full Trio panel and the CD45/CD4-based protocol, i.e., in the presence and absence of a CD3 reagent, respectively. The CD4% values among lymphocytes generated by the two methods showed a minimal bias of +0.38% (in favor of the Trio MAbs ). The absolute CD4-T-cell counts yielded by the two methods also showed excellent agreement (bias = +8 CD4+ cells/mm3; limits of agreement, between -29 and +46 CD4 cells/mm3 ). The agreement between the volumetric absolute CD4-T-cell counts using CD45/CD4 gating and the bead-based TruCOUNT tube was also determined. An average bias of -10 CD4 cells/mm3 was observed with widened limits of agreement (-118 and +98 CD4 cells/mm3), similar to the values previously observed between the standard volumetric and bead-based single-platform technologies . Efficiency of the CD45/CD4 gating protocol on a volumetric system. | After having documented CD4 enumeration using a CD45/CD4-based protocol by volumetric flow cytometer, we assessed the sample throughput of the system. A technician with a month of experience in flow cytometry processed 100 clinical samples using the autobiosampler. The steps of the procedure were monitored for time. A batch of 100 samples was processed in 95 min. The automated acquisition on the flow cytometer lasted for 120 min , allowing the operator a 25-min break before starting to prepare the following batch. Three batches of 100 samples could be prepared within the normal 8-h working day. An additional batch was also prepared at the end of the day for unattended acquisition during the late hours, to be ready for inspection by the next morning. In total, 300 to 400 clinical samples could be processed each day. CD8+-T-cell enumeration using primary CD45 and CD8 gating. | We next evaluated the agreement for CD8 enumeration between the CD45-based protocol and the full Trio panel. With the latter protocol, similar to the other currently recommended protocols, CD8+ T cells are counted when they coexpress both CD3 and CD8 molecules. Using the CD45-based protocol in the absence of a CD3 reagent, a tight gate was placed around lymphoid cells with bright CD8 expression (median, 130 x 103 antibody binding capacity per cell), excluding most NK cells (range, 10 x 103 to 110 x 103 antibody binding capacity per cell; median, 24 x 103 ABC per cell) from the CD8 gate. The correlation was excellent throughout the whole CD8 range , but a systematic bias was observed (+40 CD8 T cells/mm3; 95% confidence interval, +32 to +48) , representing +5.2% of CD8+ lymphocytes . The CD4/CD8 ratios were also evaluated in samples by the volumetric method, using the CD45/CD4 and CD45/CD8 protocols in two parallel tubes, and compared to the CD4/CD8 ratios observed with the full Trio panel . The agreements were good with virtually no bias (-0.05 CD4/CD8; limits of agreement, between -0.21 and +0.12 CD4/CD8) . We extended our study to investigate the agreement between the sum of the CD4+ and CD8+ T cells generated with the volumetric CD45 protocol and the T cells observed using CD3 staining in the full Trio protocol. The CD45-based protocol failed to identify CD3+ lymphoid cells doubly negative for CD4 and CD8 antigens (CD3+ CD4- CD8-). There was a bias of +125 T cells/mm3 in favor of the CD3+-T-cell counts recorded on the full Trio panel. These CD3+ CD4- CD8- T cells represented a 10.1% bias throughout the whole range of the T-cell counts . WBC subset enumeration using CD45-based protocols on a volumetric flow cytometer. | The expression of CD45 antigen is the common feature of all WBCs, and the CD45 staining intensity plus SSc distinguishes lymphocytes, monocytes, and granulocytes . We investigated the agreement between counts generated by this method and those yielded by a hematology analyzer in the routine hematology laboratory of our institution. The agreements for total WBCs, lymphocytes, and granulocytes were good, with a minimal bias of -136 WBCs/mm3, -70 lymphocytes/mm3, and +78 granulocytes/mm3 . However, the agreement for monocyte enumeration was poor. The hematology analyzer underestimated the monocyte counts with a bias of -179 monocytes/mm3. This is a large value, representing 35 to 40% of the total monocyte counts, as already reported for several hematology analyzers . FIG. 3. | Bland-Altman plots to establish the agreements between the volumetric CD45/CD4 protocol, single tube, and the "state-of-the-art" single-platform technology. Bland-Altman plots to establish the agreements between the volumetric CD45/CD4 protocol, single tube, and the "state-of-the-art" single-platform technology. The parameters studied were the total absolute lymphocyte counts (a), CD4-T-cell percentage values among lymphocytes (b), and absolute CD4-T-cell counts (c and d). The standard technologies used were the full lymphocyte subset panel (three tubes) tested with the Ortho Trio panel on the CytoronAbsolute (a, b, and c) and the TruCOUNT bead-based method (one tube) performed on a FACSCalibur (d). FIG. 4. | Evaluation of the agreement between the volumetric absolute CD8-T-cell counts generated with the CD45/CD8 protocol (CD8++ SSc+) and the full Trio panel (CD3+ CD8+). Evaluation of the agreement between the volumetric absolute CD8-T-cell counts generated with the CD45/CD8 protocol (CD8++ SSc+) and the full Trio panel (CD3+ CD8+). The results using linear regression (a) and the Bland-Altman plot (b) and its Pollock modification (c) are shown. In the Pollock modification, the differences between the two methods of counting CD8 T cells were expressed as a percentage of the total CD8 counts to illustrate the systematic bias at a 5% level. FIG. 5. | Bland-Altman plots (a and b) and the Pollock modification (c) to establish agreements on the Cytoron between the volumetric CD45/CD4-plus-CD45/CD8 two-tube protocol and the standard volumetric method using Trio reagents. Bland-Altman plots (a and b) and the Pollock modification (c) to establish agreements on the Cytoron between the volumetric CD45/CD4-plus-CD45/CD8 two-tube protocol and the standard volumetric method using Trio reagents. The parameters studied were the CD4/CD8 ratios (a) and the sum of the absolute CD4- plus CD8-T-cell counts versus the CD3+-T-cell counts (b and c, respectively). In the Pollock modification (c), the differences in total T-cell counts were expressed as percentages of T-cell counts to illustrate the regular underestimation of total CD3+-T-cell counts, at a 10% level, by the CD45 protocol. This bias is due to the existence of CD3+ CD4- CD8- T lymphoid cells. TABLE 2 | Timetable of routine operation using a biosampler with a 100-tube capacity PMID- 12204964_DISCUSSION TI - AB - The need to improve laboratory services for regions of the world where the HIV epidemic threatens to destroy the fabric of life has revitalized efforts to identify the most efficient techniques for monitoring HIV disease. The present changes relate to the common areas of routine immunology and hematology, such as quality assurance, sample processing, and transportation, as well as to the challenges of how to optimally count blood cells . Immunological methods, based on the specificities and discriminating capacities of MAbs, have recently made an impact by recognizing even minor subsets of functionally divergent blood cells . By using flow cytometry, the true power of directly identifying cells by antibodies, as opposed to first investigating merely their morphological features, is now documented, and the strategy of primary immunological gating is widely used . The relevant examples include CD45 for leukocytes and their subpopulations , CD3 for T cells , CD4 for the major T-cell subset and monocytes , and CD8 for the minor T-cell subset and some NK cells . Reliable total lymphocyte counts have been achieved by the Immunosum technique , providing the sum of the immunogated CD3+ (T), CD19+ (B), and CD16+ (NK) cells instead of using only the lymphocytic scatter appearance. The commonly used display on the cytometers is referred to as a heterogeneous, or morphospectral, protocol to show the IF of cells stained with MAb (on one axis) and the side scatter profile of cells (on the other axis) . In our study, we combined immunological CD45 gating with volumetric absolute counting on single platforms in order to introduce a robust method for WBC counting and for enumerating CD4+ and CD8+ T cells. Our four main findings are as follows. First, this study confirms our previous work, also performed on volumetric flow cytometers , as to the good agreement between the absolute CD4+-T-cells counts obtained by direct CD4 gating and by CD4+ CD3+ coexpression . Importantly, however, when we previously used CD4 MAb on its own without CD45, reliable CD4%-per-lymphocyte values could be obtained only with the constant vigilance of an experienced operator, who frequently had to manually modify the lymphocyte gates, a time-wasting procedure . We have now added CD45 gating to the protocol and report the excellent agreement between lymphocyte counts determined by CD45-side scatter and by the Immunosum method using the full Trio panel . Thus, CD45 staining improves the efficiency of the new autogated protocol , saving effort and technicians' time. Similarly, this gating strategy shows no bias compared to the bead-based CD4 counts but reveals occasional differences leading to a wider spread . This discrepancy might be a bead-related phenomenon, because similar results are seen when CD4 counts obtained by panleucogating on a double platform are compared to counts obtained by the bead-based method . The second conclusion is that the CD45/CD4 protocol on a volumetric cytometer provides an efficient system in which a trained flow cytometrist can run large numbers of tubes per day (>300 samples using CD45/CD4 alone ). If two parallel tubes are used with CD45/CD4- and CD45/CD8-double-stained cells, >150 blood samples can be studied. Obviously, such intensive diagnostic activity needs to be supplemented by clerical help and supervisory capacity. Nevertheless, this capacity illustrates the high efficiency of flow cytometry compared to that of manual methods such as the Dynabeads system , where a single assistant can manually handle only 15 to 20 samples per day. A hugely increased workload for CD4-T-cell enumeration is in line with the expected demand generated by the arrival of generic drugs for antiretroviral therapy. The larger regional centers dedicated to nationwide support with organized sample transportation using TransFix blood stabilizers will require this increased service capacity. The technical efficiency of this technology is directly related to three factors: (i) the fluent operation with a robust autogating process, where only <2 to 4% of samples need attention for regating (see above) , (ii) the use of an efficient autobiosampler , and (iii) a convenient system using a Windows environment and a Microsoft Access database for feedback to the clinicians. Flexible reporting, based on consultation with clinicians, may include only CD4 counts or any of the 15 parameters listed in Materials and Methods. The third finding of our study is related to the use of CD3, the specific T-cell marker. Arguably, CD3 is not required to identify CD4+ T cells . However, the CD8+-lymphocyte populations are more complex and display CD8 antigen over a wide range (15 x 103 to 140 x 103 CD8 molecules/cell ). The CD8+ cells include 80 to 92% proper CD8+ CD3+ T cells that display CD8 at a high level (CD8++; 80 x 103 to 140 x 103/cell) and 8 to 20% CD8+ CD3- NK cells that express CD8 at a lower level (CD8+; <80 x 103/cell). It is therefore logical to place a tight gate around the CD8++ population and compare these results with those obtained by counting CD3+-gated CD8 T cells . The results described above show that the CD8++ gate underestimates CD3+ CD8+ counts by 5.2% . This bias is apparently too modest to influence the CD4/CD8 ratios (bias, -0.05 ). An extra advantage of running both CD45/CD4 and CD45/CD8 tubes is the availability of CD4- plus CD8-T-cell counts that disregard the CD3+ CD4- CD8- T cells. We have argued elsewhere that these double-negative T cells represent a functionally different, mostly T-cell receptor alphabeta-negative subset that should not be included in the total T-cell counts . Finally, Loken et al. have documented the differential expression of CD45 antigen on lymphocytes, granulocytes, and CD14+ monocytes. In our study, the CD45 analysis is combined with volumetric counting in order to generate absolute leukocyte differential counts. These parameters, when defined on hematological counters, can be error prone , and the monocyte counts are frequently underestimated . On the other hand, the monocyte counts obtained by CD45 gating and carefully confirmed by the CD14 monocytic marker expression are more accurate. Consequently, the methods described above, in combination with the use of stabilized blood preparations with long shelf lives , will assist the establishment of long-awaited quality assurance schemes for leukocyte differentials and absolute counts, which are required to coordinate the performance of the wide variety of different hematology analyzers. In conclusion, the present volumetric CD45/CD4 flow cytometry, assisted by more affordable sources of MAbs, has wide applicability in the routine laboratories operating in economy-conscious environments. The specification required for the two-color IF plus side scatter used in this study is within the reach of the newly designed, battery-operated, smaller-volumetric-flow cytometers that carry red diodes or other small light sources as the sole source of light excitation and are also capable of performing bead-based enzyme-linked immunosorbent assays with the multiplexing technology in the area of the differential diagnosis of infectious diseases . TABLE 3 | Comparative performances of a hematology analyzer (Bayer 120) and the simplified CD45-based protocol on the CytoronAbsolute for absolute WBC enumeration PMID- 12204943 TI - Laboratory Diagnosis of Visceral Leishmaniasis AB - PMID- 12204943_ TI - AB - The group of diseases known as the leishmaniases are caused by obligate intracellular protozoa of the genus Leishmania . Natural transmission of leishmania is carried out by a certain species of sandfly of the genus Phlebotomus (Old World) or Lutzomyia (New World). These are present in three different forms: (i) visceral leishmaniasis (VL), (ii) cutaneous leishmaniasis, and (iii) mucocutaneous leishmaniasis. The visceral form, also known as black sickness or kala-azar in Asia, is characterized by prolonged fever, splenomegaly, hepatomegaly, substantial weight loss, progressive anemia, pancytopenia, and hypergammaglobulinemia and is complicated by serious infections. It is the most severe form of the disease and, left untreated, is usually fatal. Although confirmed cases of VL have been reported from 66 countries, 90% of the world's VL burden occurs on the Indian subcontinent and in Sudan . After recovery, some patients (50% in Sudan and 1 to 3% in India) develop post-kala-azar dermal leishmaniasis (PKDL), which requires prolonged and expensive treatment . PKDL patients also play an important role in VL transmission . VL is typically caused by the Leishmania donovani complex, which includes three species: L. donovani, Leishmania infantum, and Leishmania chagasi. The clinical features of VL caused by different species are different, and each parasite has a unique epidemiological pattern. On the Indian subcontinent, the disease is almost exclusively caused by L. donovani. The initial report of Leishmania tropica causing VL in India was refuted by us and others . L. infantum is responsible for VL in children in the Mediterranean basin. However, due to increasing prevalence of human immunodeficiency virus (HIV) infection in this region, HIV-VL coinfection in the adult population is being reported frequently. L. chagasi causes VL in children in Latin America, where lymphadenopathy is a dominant clinical feature. L. tropica, the causative organism of Old World cutaneous leishmaniasis, is reported to produce visceral disease in nonimmune persons . Similarly, visceralization by Leishmania amazonensis, has also been reported . Clinical manifestations of all forms of VL change from time to time, and this is the case more so in AIDS patients . PMID- 12204943_EPIDEMIOLOGY TI - AB - Leishmania infections are worldwide in distribution: they are found in five continents. The disease is endemic in the tropical and subtropical regions of 88 countries. There are an estimated 12 million cases worldwide; 1.5 to 2 million new cases occur every year. Cutaneous forms are most common (1 to 1.5 million cases per year), representing 50 to 75% of all new cases, and 500,000 cases of VL occur every year . The geographical distribution of leishmaniasis is limited to the areas of natural distribution of the sandfly, the vector for the disease. Economic development, including widespread urbanization, deforestation, and development of newer settlements, besides migration from rural to urban areas, is responsible for the spread of the sandfly as well the reservoir system of leishmania . Moreover, the number of new host populations, i.e., populations of immunodeficient HIV-infected patients, is increasing, especially in southern Europe and Africa . Leishmania-HIV coinfection is regarded as an emerging disease especially in southern Europe, where 25 to 70% of adults with VL have AIDS as well; leishmaniasis behaves as an opportunistic infection, and it has been proposed that it be included as an AIDS-defining illness. Moreover, the presence of the leishmania parasite outside the reticuloendothelial system, e.g., in the peripheral blood, in HIV-infected patients makes these patients a reservoir and source of infection for the vectors. The parasite load in peripheral blood is generally so high that transmission among intravenous drug users by use of shared syringes has also been demonstrated . The resurgence of leishmaniasis, its emergence in newer geographical areas and in newer hosts, besides changing the clinical profile of infected patients, has put forward newer challenges in the areas of diagnosis, treatment, and disease control. PMID- 12204943_PRINCIPLES FOR DIAGNOSIS OF LEISHMANIASIS TI - AB - The diagnosis of VL is complex because its clinical features are shared by a host of other commonly occurring diseases, such as malaria, typhoid, and tuberculosis; many of these diseases can be present along with VL (in cases of coinfection); sequestration of the parasite in the spleen, bone marrow, or lymph nodes further complicates this issue. Laboratory diagnosis of leishmaniasis can be made by the following: (i) demonstration of parasite in tissues of relevance by light microscopic examination of the stained specimen, in vitro culture, or animal inoculation; (ii) detection of parasite DNA in tissue samples; or (iii) immunodiagnosis by detection of parasite antigen in tissue, blood, or urine samples, by detection of nonspecific or specific antileishmanial antibodies (immunoglobulin), or by assay for leishmania-specific cell-mediated immunity. Demonstration and isolation of parasite. | The commonly used method for diagnosing VL has been the demonstration of parasites in splenic or bone marrow aspirate. The presence of the parasite in lymph nodes, liver biopsy, or aspirate specimens or the buffy coat of peripheral blood can also be demonstrated. Amastigotes appear as round or oval bodies measuring 2 to 3 mum in length and are found intracellularly in monocytes and macrophages. In preparations stained with Giemsa or Leishman stain, the cytoplasm appears pale blue, with a relatively large nucleus that stains red. In the same plane as the nucleus, but at a right angle to it, is a deep red or violet rod-like body called a kinetoplast . After identification, parasite density can be scored microscopically by means of a logarithmic scale ranging from 0 (no parasite per 1,000 oil immersion fields) to +6 (>100 parasites per field) . The sensitivity of the bone marrow smear is about 60 to 85%. Splenic aspirate, though associated with risk of fatal hemorrhage in inexperienced hands, is one of the most valuable methods for diagnosis of kala-azar, with a sensitivity exceeding 95%. It requires no special equipment, from the patient's standpoint is generally preferable to the more painful bone marrow aspirate, and has proven to be safe and relatively easy to perform in experienced hands. For patients suspected to have VL, splenic aspirate can be performed even when spleen is not palpable, after demarcating the area of splenic dullness by percussion. The only risk of splenic puncture is bleeding from a soft and enlarged spleen. At our treatment center, fatal bleeding has occurred only twice in 9,612 splenic aspirate procedures performed over the last 10 years. To avoid the risk of excessive blood loss, splenic puncture should be avoided in patients with a platelet count of less than 40,000 platelets/mul and a prothrombin time of more than 5 s over the control. A tissue specimen, e.g., a spleen, liver, or lymph node tissue specimen, may be subjected to imprint cytology by the repeated pressing of its cut flat surface on microscopic slides. The smear is fixed with absolute alcohol and stained with Giemsa stain. In imprint cytology, a monolayer of cells is formed and amastigotes are easily identifiable. The results are expressed as the number of leishmania per 100 host cell nuclei. Tissue specimens can also be subjected to histology, and the presence of parasites can be demonstrated by standard hematoxylin and eosin stain. Tissue specimens are usually uneven in thickness; consequently the amastigotes are unevenly distributed. Long searches may be required to demonstrate the parasite. The sensitivity of the test can be increased by staining the specimen with fluorescent dye-tagged antibodies to the surface receptors of the parasite. Fluorescein isothiocyanate isomer- or rhodamide B isothiocyanate-conjugated antiserum is usually used for this purpose. Fluorescent dye-conjugated monoclonal antibodies are also used for speciation of the parasite. Culture of parasite can improve the sensitivity of detection of parasite, but leishmania culture is rarely needed in routine clinical practice. However, cultures are required for (i) obtaining a sufficient number of organisms to use an antigen for immunologic diagnosis and speciation, (ii) obtaining parasites to be used in inoculating susceptible experimental animals, (iii) in vitro screening of drugs, and (iv) accurate diagnosis of the infection with the organism (as a supplement to other methods or to provide a diagnosis when routine methods have failed). Leishmania strains can be maintained as promastigotes in artificial culture medium. The culture media used may be monophasic (Schneider's insect medium, M199, or Grace's medium) or diphasic (Novy-McNeal Nicolle medium and Tobies medium). We prefer diphasic medium containing modified diphasic rabbit blood agar overlaid with RPMI 1640 (Gibco BRL, Grand Island, N.Y.) for primary isolation, and we prefer M199 medium containing 20% fetal calf serum to amplify parasite numbers . Hockmeyer's medium, which is Schneider's commercially prepared culture medium supplemented with 30% heat-inactivated fetal calf serum with 100 IU of penicillin and 100 mug of streptomycin, is simple to use and satisfactory for diagnosis of VL, but it is expensive . Culture tubes are inoculated with 1 to 2 drops of bone marrow or splenic aspirate and incubated at a temperature between 22 and 28C. The tubes are examined weekly for the presence of promastigotes by phase-contrast microscopy or by wet mount of culture fluid for 4 weeks before being discarded as negative. If promastigotes are present, they are maintained by weekly passage to fresh medium. Blood can also be used to isolate the parasite, but the method is slow and takes longer. Aseptically collected blood (1 to 2 ml) is diluted with 10 ml of citrated saline, and the cellular deposit obtained after centrifugation is inoculated in culture media. Contamination of the culture media by bacteria or yeast species or other fungi usually complicates the culture but can be avoided by use of good sterile techniques and by the addition of penicillin (200 IU/ml) and streptomycin (200 mug/ml) to the medium (for bacteria), as well as 5-flucytosine (500 mug/ml) (as an antimycotic agent) . In vitro culture of the amastigotes is done for chemotherapeutic studies and to study the interrelationship of the amastigotes and macrophages. The amastigotes are grown in tissue or macrophage culture. These cell lines are produced from (i) human peripheral blood monocytes, after these are set apart by density sedimentation with lymphocyte separation medium (LSM; Organon-Teknika, Durham, N.C.), in which case a new batch of macrophages must be produced anew ; (ii) macrophage cell lines, e.g., P388D and J774G8 lines from mice; and (iii) dog sarcoma and hamster peritoneal exudates of cell lines, in which case continuous culture can be achieved . The parasite can also be demonstrated after inoculation of laboratory animals (such as hamsters, mice or guinea pigs) with infected specimen . Animal inoculation is not usually employed as a diagnostic test, since several months may be required to obtain a positive result. Golden hamster is the animal of choice for maintaining L. donovani complex . It can be infected via many routes, including across mucous membranes, but intraperitoneal and intrasplenic routes are preferred. Both amastigotes and promastigotes can infect the animal. After inoculation, the animal is examined weekly for signs of infection, such as cutaneous lesions, hepatosplenomegaly, or metastatic lesions. Amastigotes can be harvested by biopsy from the spleen and the liver of an animal that is under anesthesia and that is allowed to survive following the procedure as a source of infective parasite. In the absence of signs of obvious infection, the animal is generally sacrificed after 4 months, at which point liver and spleen samples are examined for the presence of the parasite. In areas of endemicity, recognition of species of leishmania is rarely required. However, identification of an organism to the species level is helpful epidemiologically and is also important for the treatment of and prognosis determination for global travelers who are not immune to the parasite and tend to develop unusual manifestations of the disease . Identification of species of the L. donovani complex is particularly difficult, because morphologically the species are almost indistinguishable from each other. For species-level identification, a large amount of promastigotes is obtained by culture of the organism and the species-specific isoenzyme pattern is analyzed by cellulose acetate electrophoresis . Typing of washed live promastigotes by direct agglutination test with species-specific monoclonal antibodies is another highly sensitive taxonomic tool frequently utilized for this purpose . Species-level identification can also be done by analysis of amplified minicircle kinetoplast DNA (KDNA), by choosing primers from conserved regions of different leishmania species KDNA minicircles . Yet another method used for identification of species of leishmania is the analysis of the in vitro promastigotes' released antigenic factors, which are different for different leishmanial species . Although demonstration of even a single amastigote upon microscopic examination of tissue smears or multiple promastigotes in cultures is considered sufficient for positive diagnosis of the disease, the sensitivity of the tissue examination, except in the case of splenic aspirate, is low. Moreover, the procedure(s) for obtaining tissue specimen(s) is traumatic and associated with considerable risk. Identification of amastigotes requires considerable expertise and training and is subject to the ability of the observer. Besides, culturing parasites is expensive and time consuming and requires expertise and costly equipment, severely restricting its use in routine clinical practice. DNA detection method. | Due to the limitations inherent in techniques used for detection of parasites, new approaches to the detection of parasites, such as DNA hybridization, have been attempted since the early 1980s. Although these methods had considerable sensitivity (detecting as few as 50 to 100 parasites) , their potential use in routine diagnosis is hampered by the complex procedure of hybridization. The development of PCR has provided a powerful approach to the application of molecular biology techniques to the diagnosis of leishmaniasis. Primers designed to amplify conserved sequences found in minicircles of KDNA of leishmanias of different species were tested in various tissues of relevance. Such a target was eminently suitable because the kinetoplast is known to possess thousands of copies of minicircle DNA. In recent years, PCR-based diagnostic methods with a wide range of sensitivities and specificities have been described . In a study reported from Sudan, PCR was found to be more sensitive than microscopy for the detection of Leishmania parasites in lymph node and bone marrow aspirations. However, its sensitivity for the detection of Leishmania DNA in the blood of parasitologically proven VL cases was only 70% . In another study reported from India, in which a species-specific primer for L. donovani (LDI primer) was used, the sensitivity of PCR with whole blood from VL patients was 96% and Leishmania DNA was detected in skin specimens from 45 of 48 patients with PKDL (sensitivity, 93.8%) . A PCR-enzyme-linked immunosorbent assay (ELISA) technique using a primer that was able to identify 33 L. infantum strains from 19 different zymodemes has been developed. It has a sensitivity higher than that of other diagnostic techniques, e.g., indirect fluorescent-antibody (IFA) test, parasite culture, or microscopy, and was able to detect a minimum of 0.1 promastigote or 1 fg of genomic material. This PCR-ELISA technique can potentially be used for diagnosis of VL from peripheral blood samples . PCR done from blood spots on filter paper can also be used as a screening test to identify Leishmania infection in immunocompromised patients with high parasite loads in peripheral blood. The sensitivity of this technique for detecting leishmania (75%) was considerably higher than the respective sensitivities of microscopy (26.3%) and blood culture (42.3%) (17). However, PCR assay with buffy coat preparations to detect Leishmania was 10 times more sensitive than that with whole-blood preparations, and particularly good results were obtained when proteinase K-based methods were used. Proteinase K-based PCR was able to detect 10 parasites/ml . A fluorescent DNA probe specific for a conserved region of the small subunit rRNA gene of Leishmania and a pair of flanking primers, when used for DNA amplification in one assay, proved to be a highly specific and rapid diagnostic modality to detect infection with Leishmania . Using this rapid fluorogenic PCR technique, DNA could be amplified from 27 strains of cultured Leishmania, and the turnaround time from fresh human tissue biopsy to test result was found to be less than 24 h . Besides being a highly sensitive and specific tool for diagnosis of both VL and PKDL and a useful method for species identification , PCR can also be used to distinguish between relapse and reinfection in treated VL patients. Restriction fragment length polymorphism analysis of the PCR-amplified minicircle of leishmanial DNA can be utilized for this purpose . PCR could also prove to be an important tool in assessing the success of VL treatment: of patients treated for VL who tested negative by PCR with lymph node tissue, none relapsed or developed PKDL, while more than half of patients who tested positive by PCR with lymph node tissue either relapsed or developed PKDL after apparent cure of disease following supervised treatment . On the other hand, a substantial number of the patients who tested positive by PCR, after apparent cure, did not relapse or develop PKDL, a result that suggests the limitation of PCR in deciding the end point of treatment. The PCR positivity observed in these patients may be due to nonviable parasite. Similarly, PCR results for healthy endemic controls may be positive , which may lead to the erroneous conclusion that they suffer from VL. In these healthy endemic controls, a combination of direct agglutination test (DAT) (which shows low titers in healthy endemic controls) and PCR may be helpful in defining the status of these patients. Immunodiagnosis. (i) Antigen detection. | Antigen detection is more specific than antibody-based immunodiagnostic tests . This method is also useful in the diagnosis of disease in cases where there is deficient antibody production (as in AIDS patients). De Colmenares et al. (20) from Spain have reported two polypeptide fractions of 72-75 kDa and 123 kDa in the urine of kala-azar patients. The sensitivities of the 72-75-kDa fractions were 96%, and the specificities were 100%. Besides, these antigens were not detectable within 3 weeks of anti-kala-azar treatment, suggesting that the test has a very good prognostic value . A new latex agglutination test (KATEX) for detecting leishmanial antigen in urine of patients with VL has showed sensitivities between 68 and 100% and a specificity of 100% in preliminary trials. The antigen is detected quite early during the infection and the results of animal experiments suggest that the amount of detectable antigen tends to decline rapidly following chemotherapy. The test performed better than any of the serological tests when compared to microscopy. Large field trials are under way to evaluate its utility for the diagnosis and prognosis of VL . (ii) Antibody detection. | For several decades, nonspecific methods, which depend upon raised globulin levels, have been used in the diagnosis of VL. Some of the tests used for detecting these nonspecific immunoglobulins are Napier's formol gel or aldehyde test and the Chopra antimony test. Since these tests depend upon raised globulin levels, results can be positive in a host of conditions . Lack of specificity, as well as varying sensitivities, renders them highly unreliable. Several immunodiagnostic methods which are more sensitive and specific have been developed. They are useful in identifying specific cases and can be used for community surveillance. The human body makes an attempt to fight against VL by producing some of the highest levels of antibodies found in response to any disease, all to no avail. This is due to polyclonal activation of the B cells, resulting in marked elevation of levels (in serum) of immunoglobulin G (IgG) and IgM against various nonspecific proteins and haptens . The consistent presence of high levels of antibodies against parasite antigens can simplify diagnosis of VL. Several serological techniques are based on detection of these antibodies. The specificity of the antibody depends upon the antigen or epitope used in the test, as the parasite stimulates production of a wide array of antibodies, including group-, genus-, and species-specific antibodies. Therefore, the sensitivity may depend upon the test and its methodology, but the specificity will depend on the antigen rather than the serological procedure used. In most serological tests, the sensitivity and specificity data are compared against demonstration of parasites in various tissues. Conventional methods for antibody detection included gel diffusion, complement fixation test, indirect hemagglutination test, IFA test, and countercurrent immunoelectrophoresis . However, aside from practical difficulties at peripheral laboratories, the sensitivities and specificities of most of the above tests have been the limiting factors. Except for the IFA test, which is used on a limited scale, these tests are rarely used for routine diagnosis of VL. In 1988, a modified DAT was reported to be useful in kala-azar and is being used in several countries of endemicity . In this test, the trypsinized whole promastigotes are formalin fixed and stained with Coomasie brilliant blue; serum from the patient is then incubated with the antigen, and agglutination is observed the next day. Use of an 0.8% concentration of 0.1 M 2-mercaptoethanol in the sample diluent further improves its performance . DAT in various studies has shown to be 91 to 100% sensitive and 72 to 100% specific . In Sudan, in specially set up field laboratories, Medecins Sans Frontieres uses DAT for diagnosis of VL; patients with high titers receive treatment, and a confirmatory parasitic diagnosis is done in those with low titers . From India, several laboratories reported satisfactory sensitivity and specificity levels for this test . Although DAT showed a high degree of repeatability within the centers, its reproducibility across the centers was quite weak . Moreover, difficult field conditions, the fragility of aqueous antigen, the lack of cold chain, and batch-to-batch variations in the antigen, along with the nonstandardization of test readings, have severely limited its widespread applicability in regions of endemicity. Freeze-dried antigens developed in Belgian and Dutch laboratories are likely to overcome some of these handicaps . Unless this improved antigen is produced indigenously to make it affordable and DAT is made user friendly with one-step dilution and reduced incubation time, its field use is unlikely in countries of endemicity like India. Like most antibody-based tests, DAT may yield positive results for a long time after complete cure and thus has not proved to be of much prognostic value . ELISA has been used as a potential serodiagnostic tool for almost all infectious diseases, including leishmaniasis. The technique is highly sensitive, but its specificity depends upon the antigen used. Several antigens have been tried. The commonly used antigen is a crude soluble antigen (CSA). It is prepared by repeated freezing and thawing (four to six cycles) of a suspension of promastigotes in phosphate-buffered saline, followed by cold centrifugation at 10,000 to 20,000 x g. The supernatant is used as soluble antigen and is used to coat ELISA plates after estimation of protein content (100 to 5,000 ng/ml). The sensitivity of ELISA using these concentrations of CSA is reported to range from 80 to 100%, but cross-reactions with sera from patients with trypanosomiasis, tuberculosis, and toxoplasmosis have been recorded . On the other hand, when various selective antigenic masses (116 kDa, 72 kDa, and 66 kDa) were used, a specificity of 100% could be achieved, but only at the cost of sensitivity, which went down to as low as 37.5% . Palatnik-de-Souza et al. described the use of fucose-mannose ligand as the antigenic molecule. It is a 36-kDa glycoprotein present throughout the life cycle of leishmania (amastigote and promastigote stages). Its use in ELISA has been found to result in 100% sensitivity and 96% specificity . In a recent study, it was found that the sensitivity and specificity of ELISA in diagnosing VL could also be increased by the use of soluble antigens derived from promastigotes cultivated in a protein-free medium. One study, done with 129 VL and 143 cutaneous leishmaniasis patients, showed a sensitivity of 95% . A recombinant antigen, rK39, has been shown to be specific for antibodies in patients with VL caused by members of the L. donovani complex . This antigen, which is conserved in the kinesin region, is highly sensitive and predictive of the onset of acute disease. The antigen is derived from L. chagasi, which in the United States is used for veterinary purposes, though it is not approved for human use. High antibody titers in immunocompetent patients with VL have been demonstrated. This antigen has been reported to be 100% sensitive and 100% specific in the diagnosis of VL and PKDL by ELISA . Another important facet of anti-rK39 antibody is that the titer correlates directly with the disease activity, indicating its potential for use in predicting response to chemotherapy. It was previously shown that anti-rK39 antibody titers were 59-fold higher than those of antibody against CSA at the time of diagnosis, and with successful therapy, it fell sharply at the end of treatment and fell further during follow-up monitoring. In patients who experience disease relapse, the titer rose steeply again . The diagnostic and prognostic utility of rK39 for HIV-infected patients has also been demonstrated . Because of the conditions prevailing in areas of endemicity, any sophisticated method cannot be employed on a wider scale. There is a need for a simple rapid and accurate test with good sensitivity and specificity, which can be used without any specific expertise. A promising ready-to-use immunochromatographic strip test based on rK39 antigen has been developed as a rapid test for use in difficult field conditions. The recombinant antigen is immobilized on a small rectangular piece of nitrocellulose membrane in a band form, and goat anti-protein A is attached to the membrane above the antigen band. After the finger is pricked, half a drop of blood is smeared at the tip of the strip, and the lower end of the strip is allowed to soak in 4 to 5 drops of phosphate-buffered saline, placed on a clean glass slide or tube. If the antibody is present, it will react with the conjugate (protein A colloidal gold) that is predried on the assay strip. The mixture moves along the strip by capillary action and reacts with rK39 antigen on the strip, yielding a pink band. In the strip of patients who are infected, two pinkish lines appear in the middle of the nitrocellulose membrane (the upper pinkish band serves as a procedural control). In the first extensive field trial in 323 patients, we found the strip test to be 100% sensitive (confidence interval, 98 to 100%) and 98% specific (confidence interval, 95 to 100%) . Several studies from the Indian subcontinent reported the test to be 100% sensitive . However, when evaluated in Sudan, the sensitivity of the test was only 67%. In the Sudan study, all the parasitologically confirmed VL patients who tested negative by the rK39 strip test showed IgG against rK39 by micro-ELISA (though at lower titers) . In a study done in southern Europe, the rK39 strip test results were positive in only 71.4% of the cases of VL . These differences in sensitivity may be due to differences in the antibody responses observed in different ethnic groups . When tested for PKDL, the test had a 91% sensitivity . High levels of specificity (97 to 100%) have been reported uniformly for this test; however, with a later version of the rK39-treated strips, some (12.5%) healthy endemic control subjects also tested positive . While such reactions might be considered to be false positive, these probably represent subclinical infections: PCR assay for L. donovani was positive in a few of these cases . Anti-rK39 IgG may be present in serum for an extended period after successful treatment for VL; thus, patients with suspected relapse of VL with a past history of infection would not be candidates for diagnosis by strip testing. Another drawback of this format is that an individual with a positive rK39 strip test result may suffer from an illness(es) (malaria, typhoid fever, or tuberculosis) with clinical features similar to those of VL yet be misdiagnosed as suffering from VL. Notwithstanding these limitations, the rK39 immunochromatographic strip test has proved to be versatile in predicting acute infection, and it is the only available format for diagnosis of VL with acceptable sensitivity and specificity levels which is also inexpensive (similar1 to 1.5 U.S. dollars) and simple and can be performed even by paramedics in prevailing difficult field conditions. Specific antibodies can also be detected by Western blotting. For this type of testing, promastigotes of L. donovani are grown to log phase and lysed and the soluble protein is run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The separated proteins are electroblotted onto a nitrocellulose membrane and probed with serum from the patient. The sensitivity of this technique can be enhanced using the chemiluminescent antibody probes. Using Western blotting, one can find even minor antigenic differences among various organisms and thus detect cross-reactive antigens. However, the process is time consuming, technically cumbersome, and expensive . (iii) Skin testing. | Delayed type hypersensitivity (DTH) or T-cell-mediated immunity is a group-specific immune response. The Montenegro skin test (leishmanin skin test) is a test for DTH specific to leishmaniasis, but its role is limited . In this method, 0.5 ml of phenol-killed whole parasites (5 x 107 promastigotes) is injected on the volar aspect of the forearm of the patient. After 48 to 72 h, the size of induration is measured and compared with the size of induration produced by injection of a phenol-saline control in the other forearm. Presently, there is no available standardized leishmanin reagent. All leishmanins are said to be alike and nonspecific. The test is negative in acute cases of VL due to the absence of DTH and is positive only in cases where kala-azar has been cured . FIG. 1. | Microphotograph showing intracellular and extracellular L. donovani Microphotograph showing intracellular and extracellular L. donovani bodies in splenic aspirate from a patient with visceral leishmaniasis. PMID- 12204943_HIV-LEISHMANIA COINFECTION TI - AB - Atypical clinical presentations of VL in HIV-infected patients pose a considerable diagnostic challenge. In fact, the clinical triad of fever, splenomegaly, and hepatomegaly is found in less than half of such patients, though more so in patients with low CD4 counts (<50 CD4 cells/mm3) . In these patients, leishmaniasis can present with gastrointestinal involvement (stomach, duodenum, or colon); ascites; pleural or pericardial effusion; involvement of lungs, tonsils, and skin; and even as widely disseminated disease . The diagnostic principles remain essentially the same as those for non-HIV-infected patients. The presence of amastigotes may be demonstrated in buffy coat preparation. Sometimes the presence of amastigotes in unusual sites may be demonstrated (e.g., amastigotes may be present in specimens from bronchoalveolar lavage, pleural fluid, or biopsy specimens from the gastrointestinal tract). For HIV patients, the sensitivity of antibody-based immunologic tests like the IFA test and ELISA is low . Since the parasite load is quite heavy in these patients, the presence of leishmania amastigotes in the bone marrow can often be demonstrated, but there are well-described instances in the literature where amastigotes were not demonstrable on bone marrow, though they were found at unexpected locations like the stomach, the colon, or the lungs. PCR analysis of the whole blood or its buffy coat preparation may prove a useful screening test for these patients, obviating the need for traumatic procedures. PMID- 12204943_CONCLUSIONS TI - AB - Various noninvasive tests, with various specificities and sensitivities, are available for the diagnosis of leishmaniasis ; however, none have become popular in areas of endemicity. Very few are commercially available; generally speaking, they also are expensive, require skilled personnel, expensive equipment, and electricity, and are technically demanding. Parasite diagnosis by splenic, marrow, or skin lesion remains the "gold standard," with its usual limitations. DAT can be performed only in a few centralized laboratories that are equipped for the purpose (and have trained personnel); cost, multiple steps, incubation, and antigenic variations are limiting factors. The rK39 strip test has the potential to be used for diagnosis of VL under field conditions. Other tests, which are likely candidates for diagnosis and prognosis of leishmaniasis in the future, are KATEX and a field-adaptable version of PCR, which would be simple, inexpensive, and easily available. TABLE 1 | Sensitivities and specificities of various methods used for diagnosis of visceral leishmaniasis PMID- 12204970 TI - Performance of Two Commercial Glycoprotein G-Based Enzyme Immunoassays for Detecting Antibodies to Herpes Simplex Viruses 1 and 2 in Children and Young Adolescents AB - | In 61 patients 1 to 14 years of age, the Gull/Meridian enzyme-linked immunosorbent assay (ELISA) had a sensitivity of 100% for herpes simplex virus type 1 (HSV-1) and specificities of 74% for HSV-1 and 48% for HSV-2. In 128 similarly aged patients, the HerpeSelect ELISA (Focus Technologies) showed sensitivities of 80% for HSV-1 and 88% for HSV-2, and specificities of 97% for HSV-1 and 100% for HSV-2. PMID- 12204970_Introduction TI - AB - Herpes simplex virus type 1 (HSV-1) infection is common in childhood and may be indistinguishable from viral or bacterial stomatitis, otitis media, and upper respiratory tract infections (, , 14). HSV-2 infections are unusual after the neonatal period and before sexual debut; seroprevalence begins to rise in late adolescence . Virologic methods for diagnosing HSV infection in children are limited by the need to collect samples early in the clinical course and by the need to perform the vigorous swabbing that is necessary to obtain infected cells from mucosal surfaces or from lesions. Serologic tests to detect HSV antibodies are available commercially. Some tests can distinguish HSV-1 from HSV-2 antibodies on the basis of type-specific antigens of glycoprotein G-1 (gG-1) and gG-2, respectively . The first such test to be approved by the Food and Drug Administration was an enzyme-linked immunosorbent assay (ELISA) from Gull Laboratories, Salt Lake City, Utah that was sold under the Premier brand by Meridian Diagnostics (Cincinnati, Ohio) (termed the Gull/Meridian ELISA). This ELISA was both sensitive and specific in a premarket evaluation of adult sera . To assess the accuracy of the Gull/Meridian ELISAs for children and adolescents, we tested blood samples from healthy children from southern Texas (n = 61; mean age, 7.4 years; range, 1 to 13 years) with kits purchased from Gull Laboratories and compared these results to those obtained by Western blotting (WB), a well-validated "gold standard" . Later, when the Gull/Meridian ELISAs were withdrawn from the market, we extended this study to evaluate the HerpeSelect HSV-1 and HSV-2 ELISAs from Focus Technologies (formerly MRL) on pediatric sera (n = 128; mean age, 5.7 years; range, 1 to 13 years) that had been sent to the University of Washington Virology Laboratory for HSV type-specific serology. Our comparison studies revealed substantial differences among the performances of these ELISAs. The seroprevalence of HSV-1 determined by WB in Texas-based patients was 49% (30 of 61 positive); no patient was positive for HSV-2. Of the sera from Seattle, 46 of 128 (36%) were seropositive for HSV-1 by WB and 8 of 125 (6%; ages 11 days to 14 years) were positive for HSV-2. All eight samples with discordant results for HSV-1 by the Gull/Meridian ELISA and WB were false positive by the ELISA. The sensitivity for the HSV-1 Gull/Meridian ELISA was 100%, with a negative predictive value (NPV) of 100% . The specificity for HSV-1 was 74%, with a positive predictive value (PPV) of 79%. The sensitivity of the Gull/Meridian HSV-2 ELISA could not be evaluated (none were HSV-2 WB positive). Thirty-two of 61 samples (52%) were positive by the Gull/Meridian HSV-2 ELISA, giving a specificity of 48% and a PPV of 0%. TABLE 1 | Results of WB and two ELISA for HSV-1 and HSV-2 antibodies in patients aged 1 to 14 years from Texas and Washington The results for 5 of 128 pediatric sera tested by the HerpeSelect HSV-1 ELISA were equivocal, and the sera could not be classified as negative or positive for comparison. For the remaining 123 sera, the HerpeSelect HSV-1 ELISA had a sensitivity of 80% and a specificity of 97%, with a PPV and an NPV of 95 and 89%, respectively . Two sera had equivocal results with the HerpeSelect HSV-2 ELISA. Neither was positive by WB for HSV-2; one was positive for HSV-1 antibody by both WB and the HerpeSelect HSV-1 ELISA. In addition, three sera had atypical HSV-2 results by WB and could not be scored by that test as either negative or positive. Of the 123 evaluable result sets for HSV-2 antibody, HerpeSelect HSV-2 ELISA had a sensitivity of 88%, a specificity of 100%, a PPV of 100%, and an NPV of 99% . Thus, two commercial ELISAs had very different performance characteristics with pediatric sera. The Gull/Meridian ELISA (based on immunoaffinity-purified gG-1 and gG-2) suffered from very low specificity and unacceptably low PPVs, especially for HSV-2. At least one widely used reference test, the gG-1 and gG-2 immunodot enzyme assay, is based on antigens similar to those in the Gull/Meridian ELISAs . The HerpeSelect ELISAs had high specificity for both HSV-1 and HSV-2 but surprisingly low sensitivity for HSV-1 (80%). Our study raises important issues about the use of these gG-based HSV type-specific serologic tests in children. The very low specificities of the Gull/Meridian ELISAs were surprising in light of previous data for adults . Although these tests could be more sensitive than WB, this higher sensitivity (if true) appears to apply only to children . Alternatively, some young people may have circulating factors that nonspecifically bind glycoprotein G in this particular ELISA. A previous report may have given an early, unrecognized warning of a unique problem with Gull/Meridian ELISAs for pediatric sera . It is unclear whether the specificity problem with Gull/Meridian ELISAs for pediatric sera is restricted to the Gull antigen. Studies using immunoaffinity-purified HSV-2 antigens in gG-2-based tests other than the Gull/Meridian ELISA suggest a reasonably low HSV-2 prevalence rate in children ; these studies give no insight into ELISA performance for antibodies to HSV-1 in pediatric sera. The HerpeSelect HSV-1 and HSV-2 ELISAs are based on baculovirus recombinant gG-1 and gG-2 . Unlike the Gull/Meridian ELISAs, these tests with pediatric sera gave no indication of excessive false-positive HSV-1 results (two of 77 sera) or HSV-2 results (0 of 116 sera) . However, the sensitivity for HSV-1 was only 80%. Two of nine false-negative sera were from infants under 1 year of age and could have represented low-titer maternal antibodies. The other seven sera were from children with a median age of 4.5 years (range, 3 to 12) who might have been in the process of seroconverting, based on the appearance of the WB profiles (data not shown). The accuracy of HSV serologic testing for children appears questionable with current ELISAs. False-positive HSV-1 results may lead to inappropriate treatment or to unnecessary antiviral prophylaxis in immunosuppressed patients. Positive HSV-2 tests for children suggest the occurrence of sexual abuse. Our limited testing of the HerpeSelect ELISAs provides cautious optimism that these tests are reasonably accurate for children. However, prospective studies using virologic diagnosis of infection as the gold standard are needed. A serologic diagnosis of HSV infection in children should be made with caution. A negative test should be followed by testing in 6 to 8 weeks to detect seroconversion. Positive results for HSV-2 antibodies should be confirmed by a second type-specific test, such as WB or the Focus HerpeSelect immunoblot . PMID- 12204953 TI - Calcium Phosphate Nanoparticles Induce Mucosal Immunity and Protection against Herpes Simplex Virus Type 2 AB - | Previously we reported that calcium phosphate nanoparticles (CAP) represented a superior alternative to alum adjuvants in mice immunized with viral protein. Additionally, we showed that CAP was safe and elicited no detectable immunoglobulin E (IgE) response. In this study, we demonstrated that following mucosal delivery of herpes simplex virus type 2 (HSV-2) antigen with CAP, CAP adjuvant enhanced protective systemic and mucosal immunity versus live virus. Mice were immunized intravaginally and intranasally with HSV-2 protein plus CAP adjuvant (HSV-2+CAP), CAP alone, phosphate-buffered saline, or HSV-2 alone. HSV-2+CAP induced HSV-specific mucosal IgA and IgG and concurrently enhanced systemic IgG responses. Our results demonstrate the potency of CAP as a mucosal adjuvant. Furthermore, we show that systemic immunity could be induced via the mucosal route following inoculation with CAP-based vaccine. Moreover, neutralizing antibodies were found in the sera of mice immunized intranasally or intravaginally with HSV-2+CAP. Also, the results of our in vivo experiments indicated that mice vaccinated with HSV-2+CAP were protected against live HSV-2 infection. In conclusion, these preclinical data support the hypothesis that CAP may be an effective mucosal adjuvant that protects against viral infection. PMID- 12204953_Introduction TI - AB - Since mucosal surfaces act as the primary point of entry for most pathogens and the first line of defense against them, vaccines inducing effective mucosal immunity may reduce rates of infection and decrease the morbidity and mortality of infectious diseases. Currently, no safe and effective mucosal vaccine adjuvants are approved for human use. Mucosal vaccine delivery is a promising strategy. Mucosal vaccines administered in one part of the body can elicit an antibody response in mucosal tissues remote from the site of initial antigen exposure. This effect occurs because of the common mucosal immune system . A major obstacle to developing a mucosal vaccine in humans is finding a safe and effective adjuvant. Experimental mucosal adjuvants include cholera toxin, heat-labile enterotoxin, mutant toxins (LTK63 and LTR72), CpG oligodeoxynucleotide, polymerized liposomes, microparticles, and interleukins or immune modulators. None of these adjuvants is approved for use in humans . Biodegradable calcium phosphate particles have been investigated as an alternative to aluminum adjuvants for parenteral vaccines. Clinical studies conducted in France described the use of a calcium phosphate adjuvant for secondary or booster immunizations against diphtheria and tetanus . Calcium phosphate has also been used for allergen desensitization . Early studies indicated that calcium phosphate particles produce strong adjuvant effects, induced less immunoglobulin E (IgE) than aluminum adjuvants, and elicited only minimal local irritation in animal experiments and human clinical trials . Here, we describe a unique formulation of calcium phosphate nanoparticles (CAP) which is distinct from the formulations of calcium phosphate described by European scientists and demonstrate its use as an effective mucosal adjuvant. Our results indicate that following viral challenge, mice immunized with CAP-based formulations of herpes simplex virus type 2 (HSV-2) glycoprotein exhibited significantly increased survival rates and less severe clinical infection than controls. These findings demonstrate that CAP delivered as a mucosal adjuvant confers protective antiviral immunity. PMID- 12204953_MATERIALS AND METHODS TI - AB - Formulation of subunit vaccine. | The preparation of partially purified HSV-2 glycoproteins has been described previously . Briefly, infected cells were collected and sonicated. The viral suspension was centrifuged at 5,500 x g for 15 min. Supernatant was collected and treated with 1% IGEPAL (Sigma Chemical Co., St. Louis, Mo.) lysis buffer for 30 min on ice. The solution was centrifuged at 18,500 x g for 2 h. The supernatant was dialyzed against phosphate-buffered saline (PBS) at 4C and stored at -80C. Then 1 mg of HSV-2 protein was added to 7.5 ml of 12.5 mM calcium chloride, followed by the addition of 7.5 ml of 12.5 mM dibasic sodium phosphate and 1.5 ml of 15.6 mM sodium citrate. The solution was stirred until the final average particle size was less than 1.2 mum, as determined with a Coulter N4Plus Submicron particle sizer, and was treated with 129 mM cellobiose overnight. The total protein inside CAP was 123 mug. The particle containing HSV-2 protein was coated again with 3.877 mg of HSV-2 proteins by coincubation for 1 h at 4C. The final concentration of CAP plus HSV solution was 2 mg of HSV/ml and 10 mg of CAP/ml. The control vaccines were PBS, CAP alone, and HSV-2 protein alone. Animals. | Female BALB/c mice, 6 to 8 weeks old and weighing 25 g, were obtained from Charles River Laboratories. The mice were maintained in standard housing with a normal diet of Purina rodent chow 5001. Immunization and sample collection. | Eight groups of five female BALB/c mice were inoculated intravaginally or intranasally with HSV-2+CAP (20 mug of viral protein plus 100 mug of CAP per dose per mouse), HSV-2 alone (20 mug per dose per mouse), or CAP alone (100 mug per dose per mouse) in a total volume of 50 mul (intravaginally) or 10 mul (intranasally). The mice received two inoculations, on days 0 and 7. Samples were collected 7, 14, and 38 days after the last immunization. Blood was obtained from the orbital sinus, and the serum samples were stored at -20C. Mucosal samples were collected 14 days after the last immunization by vaginal lavage with 100 mul of PBS. The sediments were removed by centrifugation, and samples were pooled and stored at -20C. ELISA. | HSV-specific antibodies were detected by an end-point dilution enzyme-linked immunosorbent assay (ELISA) as described previously . Titers for IgG in plasma samples were expressed as group mean +- standard error of the mean of values for individual animals. Titers for IgA and IgG in mucosal samples were expressed as the mean of triplicate assays from pooled mucosal samples. HSV-2 challenge experiment. | Using methods reported previously , mice were injected subcutaneously with DepoProvera (Upjohn, Kalamazoo, Mich.) at a concentration of 2 mg/mouse in 50 mul of distilled water on the 45th day following primary immunization. Five days later, the mice were challenged intravaginally with 106 PFU of HSV-2. Mice were examined daily for genital pathology, and the clinical scoring was performed by an investigator blinded to the animal's immunization status. Clinical pathology was scored on a 5-point scale: 0, no apparent infection; 1, slight redness of external vagina; 2, severe redness and swelling of external vagina; 3, genital ulceration with severe redness, swelling, and hair loss of genital and surrounding tissue; 4, severe ulceration of genital and surrounding tissue and paralysis; and 5, death. Neutralization assay. | As reported previously , Vero cells were propagated in culture plates. Pooled mouse serum samples from day 38 after the last immunization were incubated with HSV-2 and assessed for the presence of HSV-2-specific neutralizing antibodies by plaque assay. The titer is the reciprocal of the serum dilution required to inhibit the cytolysis of a confluent monolayer of Vero cells by 50%. Statistical analysis. | Pathological data were analyzed by analysis of variance to determine the difference between groups. PMID- 12204953_RESULTS TI - AB - As indicated in Fig. , both the intranasal and intravaginal HSV-2+CAP-vaccinated mice showed a high titer of HSV-specific mucosal IgA and IgG in vaginal lavage fluid at 14 days after the last immunization. Serological IgG and IgG2a titers determined on day 38 after the last immunization showed a systemic response in the mice after intranasal or intravaginal immunization with HSV-2+CAP compared to PBS, CAP alone, or HSV-2 alone . The neutralization assay was performed at day 38 following secondary immunization. Neutralizing antibodies were found in both the intranasally and intravaginally HSV-2+CAP-immunized mice at titers of 1:40 and 1:80, respectively. Notably, neutralizing antibodies were absent in the mice inoculated with PBS alone, CAP alone, or HSV-2 alone. Resistance to HSV-2 infection was evaluated by monitoring clinical pathology. On days 6, 8, and 10, the reduced clinical severity in mice intravaginally immunized with HSV-2+CAP achieved statistical significance (P < 0.05) compared to mice immunized with PBS, CAP alone, or HSV-2 alone (Fig. , right panel). One of five mice intravaginally inoculated with HSV-2+CAP died from HSV-2 infection, whereas all of the mice intravaginally vaccinated with PBS, HSV-2 alone, and CAP alone developed severe disease and died by day 8 or 10. Similarly, the mice vaccinated intranasally with HSV-2+CAP showed reduced clinical severity compared with mice immunized with PBS, CAP alone, or HSV-2 alone at days 8 and 10 (Fig. , left panel). Two of five mice intranasally vaccinated with CAP+HSV-2 died, compared with the controls (i.e., recipients of PBS, CAP alone, and HSV-2 only), all of which died eventually. All surviving mice were kept for 2 more weeks and recovered gradually. FIG. 1. | Groups of five female BALB/c mice were immunized on days 0 and 7 by intranasal or intravaginal delivery of PBS (vertically striped bars), CAP alone (open bars), HSV-2 alone (horizontally striped bars), or HSV-2+CAP (solid bars). Groups of five female BALB/c mice were immunized on days 0 and 7 by intranasal or intravaginal delivery of PBS (vertically striped bars), CAP alone (open bars), HSV-2 alone (horizontally striped bars), or HSV-2+CAP (solid bars). The antigen concentration and vaginal lavage fluid dilution used in the ELISA were 100 mug/ml and 1:1, respectively. FIG. 2. | The antigen concentration and antibody dilution used in the IgG ELISA were 6 mug/ml and 1:200, respectively. The antigen concentration and antibody dilution used in the IgG ELISA were 6 mug/ml and 1:200, respectively. The antigen concentration and antibody dilution used in the IgG2a ELISA were 100 mug/ml and 1:50, respectively. Each bar represents the group mean antibody level for mice immunized intranasally or intravaginally with PBS (vertically striped bars), CAP alone (open bars), HSV-2 alone (horizontally striped bars), or HSV-2+CAP (solid bars). FIG. 3. | Five BALB/c mice per group were immunized intranasally or intravaginally with PBS (vertically striped bars), CAP alone (open bars), HSV-2 alone (horizontally striped bars), or HSV-2+CAP (solid bars) and challenged intravaginally with 106 PFU of HSV-2 at 43 days after the last immunization. Five BALB/c mice per group were immunized intranasally or intravaginally with PBS (vertically striped bars), CAP alone (open bars), HSV-2 alone (horizontally striped bars), or HSV-2+CAP (solid bars) and challenged intravaginally with 106 PFU of HSV-2 at 43 days after the last immunization. Clinical pathology was scored as described in the text. The stars indicate P values of <0.05 for HSV-2+CAP versus PBS, CAP alone, and HSV-2 alone. PMID- 12204953_DISCUSSION TI - AB - The mucosal tissues are the primary routes of entry into the body for microbial pathogens. Vaccines inducing mucosal immunity prevent the transmission of infection via mucosal surfaces. However, no mucosal vaccine adjuvant is currently approved for human use. Because of the weak inherent immunogenicity of some antigens targeted for vaccine development, such as epitope subunits and recombinant peptides, there is a great need for safe and efficient mucosal adjuvants. The only adjuvants used in licensed vaccines in the United States are aluminum compounds, which effectively enhance immune responses . However, human studies have shown them to be weak adjuvants for inducing cell and humoral immunity to some virus protein subunits (S. J. D. Bell, personal observation). Additionally, alum can elicit an IgE antibody response that increases the risk of allergic reactions. We have reported previously that CAP delivered intraperitoneally with HSV-2 and Epstein-Barr virus proteins induced high titers of IgG2a antibody and neutralizing antibody and facilitated a high degree of protection against viral infection in a murine model . In this study, using HSV-2 protein as a model antigen, we evaluated the immunity and efficacy of an HSV-2+CAP experimental vaccine. Our results indicated that mice vaccinated either intravaginally or intranasally with HSV-2+CAP had high antibody levels at mucosal surfaces and effective neutralizing antibody titers and were protected against virus infection. We assumed that the neutralizing antibody prevented the attachment of pathogens to the epithelial surfaces and conferred protection against subsequent viral infection. Our findings also confirmed the previous studies showing that antibodies can efficiently neutralize virus in mucosal areas. The immune system within the female lower genital tract is the initial defense against sexually transmitted diseases. Our study suggested that intravaginal immunization induced relatively higher levels of mucosal IgG and IgA than intranasal immunization, providing optimal protection against HSV-2 infection. This observation supports the findings of others and suggests that genital local immunity and Th1 response in association with other protecting factors, such as local production of antibodies and viral clearance from the vaginal mucosa, play a major role in HSV-2 infection in mice. Our next step is to prove that CD4+ T cells secreting gamma interferon and B cells or natural antibodies are critical for immune protection against lethal genital HSV-2 reinfection. The exact mechanism of the adjuvant action of CAP is not fully understood. M cells in the mucosal tissues are known to reside exclusively in the epithelium and deliver foreign material by transepithelial transport from the lumen to the underlying mucosa-associated lymphoid tissue. Particulate antigens are desirable because they permit M cells to translocate across the tight epithelial barrier to mucosal dendritic cells. Therefore, the particulate mucosal vaccine created from the combination of soluble antigens formulated within CAP provides the desirable size and functional attributes to induce effective mucosal immunity. Recent comparative studies have indicated that microparticles are potent adjuvants for mucosal delivery . However, microparticles are not an ideal size for inducing cellular immunity because they tend to be too large, and it is believed that M cells, dendritic cells, macrophages, and local lymph nodes are more effective at taking up smaller particles. Advantageously, CAP are generally in the preferred size range (i.e., less than 1.2 mum, versus 1 mum-sized polymers) and also stimulate cellular immunity and cytotoxic T lymphocyte responses (unpublished data). Based on these results, we conclude that (i) the CAP-based HSV-2 subunit vaccine appears to concurrently induce both systemic and mucosal immunity and (ii) CAP shows great potential as a safe and effective mucosal vaccine adjuvant for humans, given its relative absence of side effects and lack of IgE antibody induction. PMID- 12204973 TI - Determination of the Nucleotide Sequences of Heat Shock Operon groESL and the Citrate Synthase Gene (gltA) of Anaplasma (Ehrlichia) platys for Phylogenetic and Diagnostic Studies AB - | The 1,670-bp nucleotide sequence of the heat shock operon groESL and the 1,236-bp sequence of the citrate synthase gene (gltA) of Anaplasma (Ehrlichia) platys were determined. The topology of the groEL- and gltA-based phylogenetic tree was similar to that derived from 16S rRNA gene analyses with distances. Both groESL- and gltA-based PCRs specific to A. platys were also developed based upon the alignment data. PMID- 12204973_Introduction TI - AB - Anaplasma (Ehrlichia) platys is a bacterial parasite of dog platelets that causes infectious cyclic thrombocytopenia . A. platys has been shown to be closely related to Anaplasma marginale, Anaplasma centrale, and Anaplasma phagocytophila, including the former human granulocytic ehrlichia (HGE) agents Ehrlichia equi and Ehrlichia phagocytophila, based on 16S rRNA gene sequences ; however, little information is available regarding the natural history of the pathogen. The 16S rRNA gene had been the only known gene sequence of A. platys before the heat shock protein gene (groEL) was sequenced recently . A groEL sequence analysis supported the phylogenetic relationship between A. platys and related species. The groESL operon contains a spacer region between groES and groEL which is thought to be more divergent than the coding regions . However, the nucleotide sequence of the spacer region of A. platys has not been studied yet. Thus, the nucleotide sequences of groES and the spacer region between groES and groEL were analyzed for additional phylogenetic characterization of A. platys. More recently, we sequenced the citrate synthase gene (gltA) of 13 species, including Ehrlichia, Anaplasma, and Neorickettsia, for phylogenetic analyses and found higher variation than for the 16S rRNA gene . The topology of the gltA-based phylogenetic tree confirmed the reorganization of genera in the families Rickettsiaceae and Anaplsmataceae reported recently . However, the gltA sequence of A. platys has yet to be determined. Thus, the nucleotide sequences of A. platys gltA were also analyzed to support the phylogenetic relationship of A. platys among related species. We also propose to use sequence data from groESL and gltA with greater differences among species to develop an A. platys-specific PCR method. New PCR primers to specifically detect A. platys fragments were developed based on the alignment data of these two genes for a diagnostic assay. The A. platys DNA analyzed in this study was supplied from a dog infected in Somieres, France . The dog had a history of a clotting disorder. At the time of bleeding, the platelet count was 256,000/mul, which was in the normal range, and A. platys was observed within 58% of platelets on a Giemsa-stained peripheral blood smear. To evaluate the species-specific PCR designed in this study, DNA from other strains of A. platys were used. DNA from a dog infected with A. platys in Venezuela was kindly provided by E. B. Breitschwerdt, North Carolina State University . DNA from a dog infected with A. platys in Okinawa, Japan, was also used . For amplification of the groESL operon of A. platys, EEgro1F and EEgro2R were used to amplify an approximately 1,700-bp fragment with an annealing temperature of 55C . The amplification products were purified using the QIAquick PCR purification kit (Qiagen GmbH, Hilden, Germany) and sequenced. Five primers shown in Table were used to complete the sequence of groESL. Fluorescence-labeled dideoxynucleotide technology was used for DNA sequencing reactions (Perkin-Elmer, Applied Biosystems Division, Foster City, Calif.). Samples were then sequenced using a Perkin-Elmer ABI Prism 377 automated DNA sequencer at the DNA Core Facility of the Center for Gene Research, Yamaguchi University. TABLE 1 | Oligonucleotide sequences of primers used in this study The strategy for determining the gltA sequence was similar to that used in our previous report . A partial sequence of A. platys gltA was first determined using two sets of degenerate primers, F1b and EHR-778R, and F1b and HG1085R . These primers were designed based upon the sequence of gltA of A. phagocytophila, A. marginale, and A. centrale. The amplification conditions were the same as in the previous study , with an annealing temperature of 53C. The amplification products were purified and sequenced as described above. After a partial determination of the sequence, the unknown areas of the 3' and 5' ends of the gene were determined using the Universal Genome Walker kit (Clontech Laboratories, Palo Alto, Calif.). Briefly, genomic DNA was digested with EcoRV, DraI, PvuII, StuI, and ScaI. DNA fragments were ligated with a Genome Walker adaptor, which had one blunt end and one end with a 5' overhang. A ligation mixture of the adaptor and ehrlichial genomic DNA fragments was used as a template for PCR. This PCR was performed using an adaptor primer supplied by the manufacturer and A. platys gltA-specific primers to walk downstream on the DNA sequence . For the amplification, the conditions were as in our previous report . The sequences of A. platys and the registered sequences of other related species deposited in GenBank were analyzed for phylogenetic relationships. Multiple alignment analysis, the calculation of distance matrices, and the construction of phylogenetic trees were performed with the ClustalW program version 1.8 in the DNA Data Bank of Japan (Mishima, Japan; ). The distance matrices for the aligned sequences with all gaps ignored were calculated using the Kimura two-parameter method , and the neighbor-joining method was used for constructing a phylogenetic tree . The stability of the tree obtained was estimated by bootstrap analysis for 100 replications using the same program. Tree figures were generated using the TreeView program, version 1.61 . A primer set, forward primer PLA-HS475F and reverse primer PLA-HS1198R, was designed based upon the alignment data to specifically amplify an A. platys groESL fragment. Another set of primers, PLA-CSM136F and PLA-CS1359R, was also designed based upon the alignment data of gltA. PCR conditions were the same as described above but with an annealing temperature of 58C and the use of 40 cycles. The specificity of the reaction was tested with DNA extracted from the three strains of A. platys and related species, including A. phagocytophila (formerly the HGE strain Webster) (J. S. Dumler), E. equi strain California (J. E. Madigan), E. phagocytophila strain 1602 (A. Garcia-Perez), A. marginale strain Florida (G. H. Palmer), E. canis strain Oklahoma (J. Dawson), Wolbachia pipientis (M. Taylor), and Neorickettsia helminthoeca (Y. Rikihisa). The sensitivities of both PCR systems were also examined using DNA from A. platys strain France. The DNA was diluted 10-fold from 1:1 to 1:10,000 with distilled water. A 1,670-bp groESL fragment of A. platys was determined to contain 41 bp of the partial groES, 51 bp of the spacer region, and 1,577 bp of the groEL coding region. The groESL operon was compared with that of other Anaplasma and Ehrlichia bacteria reported previously and was found to be closely related to the operon of A. phagocytophila and A. marginale, with 81.4 and 78.8% identity, respectively. The level of similarity among groESL sequences was much lower than that for the 16S rRNA gene sequence in the same species (98.6% with A. phagocytophila and 96.1% with A. marginale). The spacer length of 51 nucleotides was similar to that for related Anaplasma species: 52 bp for A. phagocytophila and 47 bp for A. marginale. The percent identities of the nucleotide sequence in the spacer region of A. platys compared to that of A. phagocytophila and to that of A. marginale were 74.5 and 72.3%, respectively, revealing a greater degree of divergence than for the entire groEL coding region. After the initial identification of the 955-bp partial sequence of A. platys gltA using the degenerate PCR strategy, the full-length open reading frame extending from the ATG start codon to the TAA stop codon was determined using the Genome Walker PCR method. The length of the gltA open reading frame was 1,236 bp and encoded a protein of 411 amino acids. The complete gltA sequence was compared with that of other Anaplasma and Ehrlichia bacteria reported previously and found to be closely related to those of A. phagocytophila and A. marginale, with 62.7 and 63.2% identity, respectively. The level of similarity among ehrlichial gltA was much lower than that for the 16S rRNA gene sequence in the same species. Thus, the sequence of gltA is much more variable among these species than is groESL. The length of the gltA sequence of A. platys (1,236 bp) is the same as that of A. phagocytophila but slightly shorter than that of A. marginale and A. centrale (1,254 bp). In topology, the gltA-based phylogenetic tree was very similar to the tree derived from analysis of the 16S rRNA gene analyses and the groEL-based tree reported previously . However, the trees constructed from gltA and groESL nucleotide sequences showed more distance than the 16S rRNA-based trees. These findings also support the use of gltA-based and groESL-based comparisons in determining the phylogeny of Anaplasma, Ehrlichia, and Neorickettsia agents and strengthen the 16S rRNA- and groESL-based phylogeny reported recently . There have been reports of A. platys-like organisms identified based on 16S rRNA analysis, including bacteria from white-tailed deer in North America and from a cow in South Africa . It would be interest to determine the phylogenetic position of these agents by analyzing the gltA and groESL gene sequences. FIG. 1. | Phylogenetic relationship of various Anaplasma, Ehrlichia, and Neorickettsia spp. Phylogenetic relationship of various Anaplasma, Ehrlichia, and Neorickettsia spp. based on the nucleotide sequences of gltA (a) and groESL (b) genes. The neighbor-joining method was used to construct the phylogenetic tree with the ClustalW program. The scale bar represents 10% divergence. The numbers at nodes are the proportions of 100 bootstrap resamplings that support the topology shown. The GenBank accession numbers of the groESL sequences used to construct the phylogenetic tree and aligned data are as follows: A. phagocytophila (strain HGE agent), ; E. equi, ; E. phagocytophila, ; A. marginale, ; E. chaffeensis, ; E. canis, ; E. muris, ; Ehrlichia sp. detected from I. ovatus, ; Ehrlichia (Cowdria) ruminantium, ; Neorickettsia (Ehrlichia) risticii, ; Neorickettsia (Ehrlichia) sennetsu, ; Rickettsia prowazekii, ; Bartonella henselae, . The GenBank accession numbers of the gltA sequences used for comparative analysis are as follows: A. phagocytophila (strain HGE agent), ; E. equi, ; E. phagocytophila, ; A. marginale, ; A. centrale, ; E. chaffeensis, ; E. canis, ; E. muris, ; Ehrlichia sp. detected from I. ovatus, ; E. (C.) ruminantium, ; N. (E.) risticii, ; N. (E.) sennetsu, : N. helminthoeca, ; R. prowazekii, ; B. henselae, . PCR is a powerful tool for epidemiological or diagnostic purposes because of its high sensitivity and specificity; however, there are few molecular tools available for A. platys. All PCR assays reported previously were developed based upon 16S rRNA gene sequences . In the present study, new PCR primers were designed to amplify A. platys DNA specifically and were based upon the alignment data of groESL and gltA of A. platys and closely related Anaplasma and Ehrlichia species. The PCR produced a fragment of 724 bp from groESL and 1,459 bp from gltA with the DNA from A. platys strain France . The sensitivities of the PCR systems were examined by using diluted DNA. Both groESL- and gltA-based PCRs detected DNA diluted 1:100 , similar to the 16S rRNA-based method . As 5 mul of the original DNA solution contained genomic DNA from approximately 375 platelets infected with A. platys, both PCR systems can detect DNA from 3.75 infected platelets in a reaction mixture. The specificity of both systems was also examined using DNA of three A. platys strains from different geographic locations, France, Japan, and Venezuela, and using DNA from related species, including A. phagocytophila and A. marginale. Figure shows that both PCR systems were specific for A. platys. Furthermore, the gltA-based PCR amplifies the whole gltA sequence of A. platys, which contains both start and stop codons. As the sequences of gltA and groESL have greater variation than the sequence of 16S rRNA, the sequence analysis of PCR products may supply useful information for phylogenic studies of the agents. Our findings suggest that both groESL- and gltA-based PCRs are useful for the specific detection of A. platys DNA, and they would be additional molecular tools for both phylogenetic study and diagnosis in veterinary medicine. FIG. 2. | The sensitivities of the A. platys The sensitivities of the A. platys-specific PCR based upon the groESL (lanes 1 to 6) and gltA (lanes 7 to 12) genes were evaluated. DNA equivalent to that from 375, 37.5, 3.75, 0.375, and 0.0375 infected platelets was used as a template for the amplicons demonstrated in lanes 1 and 7, 2 and 8, 3 and 9, 4 and 10, and 5 and 11, respectively. Distilled water was used in lanes 6 and 12 as a negative control. Lane M, 100-bp DNA ladder. FIG. 3. | A. platys A. platys-specific PCRs based upon the groESL (A) and gltA (B) genes were evaluated for their specificity with DNA from A. platys strains from Somieres, France (lane 1), Okinawa, Japan (lane 2), and Venezuela (lane 3), and from A. marginale (lane 4), A. centrale (lane 5), A. phagocytophila strain HGE agent (lane 6), E. equi (lane 7), E. phagocytophila (lane 8), W. pipientis (lane 9), E. canis (lane 10), and N. helminthoeca (lane 11), with distilled water as a negative control (lane N). Positive bands (arrows) were observed only with DNA of the three strains of A. platys in both groESL- and gltA-based PCRs. PMID- 12204973_Nucleotide sequence accession numbers. TI - AB - The nucleotide sequences of groESL and gltA of A. platys strain France determined herein have been deposited in the GenBank database under the accession numbers and , respectively. PMID- 12204966 TI - Suppression of Growth by All-trans Retinoic Acid Requires Prolonged Induction of Interferon Regulatory Factor 1 in Cervical Squamous Carcinoma (SiHa) Cells AB - | All-trans retinoic acid (ATRA) suppresses growth of cervical dysplasias in vivo, although the sensitivity to retinoids is frequently lost during cervical carcinogenesis. It has been suggested that prolonged treatment or use of higher doses of retinoids might offer favorable response rates. We found SiHa cervical squamous carcinoma cells that were virtually resistant to ATRA-induced growth-inhibitory effects at physiological doses (10-7 to 10-6 M) to be more responsive at pharmacological doses (10-5 to 10-4 M). The growth inhibition by high-dose (10-4 M) ATRA was associated with a sustained activation of interferon regulatory factor 1 (IRF-1), while a low dose (10-6 M) of ATRA activated IRF-1 only transiently. Antisense IRF-1 inhibited the high-dose (10-4 M), ATRA-mediated growth arrest; forced expression of IRF-1 caused a significant reduction in cell growth. High-dose (10-4 M) ATRA increased binding of NF-kappaB and STAT1 proteins to sequences that originated from the IRF-1 promoter region, while low-dose (10-6 M) ATRA induced only NF-kappaB binding. A delayed tyrosine phosphorylation of the signal transducer and activator of transcription-1 (STAT1) was observed after high-dose (10-4 M) but not low-dose (10-6 M) ATRA treatment. In agreement with this, induction of IRF-1 mRNA by ATRA was only modest and transient in a STAT1 knockout cell line, suggesting the importance of STAT1 in sustained IRF-1 expression. Our data showed that ATRA is capable of inducing dose-dependent cellular changes, which might be appropriate to overcome resistance to retinoids that frequently develops during cervical carcinogenesis. PMID- 12204966_Introduction TI - AB - Vitamin A and its natural or synthetic derivatives (collectively known as retinoids) are potent regulators of growth of various malignancies, including cervical cancer . In clinical trials, ATRA could reverse or suppress low-grade or moderate- to high-grade cervical dysplasias . However, retinoids were not effective in patients with more advanced dysplasias ; this finding is similar to the resistance to ATRA observed with HPV16-transformed cervical keratinocytes in vitro . It has been suggested that prolonged treatment or use of higher doses of retinoids might offer a favorable response rate . Retinoids are potent modulators of cellular proliferation and differentiation. In cervical carcinoma cells, retinoic acid induces interferon regulatory factor (IRF-1) , which is responsible for growth arrest . In growth-arrested cells, IRF-1 mRNA expression is markedly elevated, but its expression declines prior to and during DNA synthesis: in this context, IRF-1 is a tumor suppressor . IRF-1 expression can be stimulated by ATRA at the level of transcription through a gamma interferon-activated site (GAS) , via an NF-kappaB site , or directly via a retinoid-responsive element found in the promoter of the IRF-1 gene. Accordingly, our aim was to determine the effects of different doses of ATRA on the expression and regulation of IRF-1 and on the subsequent inhibition of growth in cervical squamous carcinoma (SiHa) cells. PMID- 12204966_MATERIALS AND METHODS TI - AB - Cell lines. | The HPV16-positive cervical squamous carcinoma cell line (SiHa) was purchased from ATCC and maintained at 37C in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum in a 5% CO2 atmosphere. 2fTGH and U3A fibroblast cell lines were gifts from George Stark (The Cleveland Clinic Foundation Research Center, Cleveland, Ohio), and they were maintained at 37C in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum plus 250 mug of hygromycin/ml in a 5% CO2 atmosphere. Plasmids. | The expression vector that contains IRF-1 cDNA (pHuIRF3-1) was kindly provided by Tadatsugu Taniguchi (Department of Immunology, Graduate School of Medicine and Faculty of Medicine, University of Tokyo, Tokyo, Japan). The IRF-1 cDNA was PCR amplified and subcloned into the pCR3.1 bidirectional eukaryotic TA cloning vector (Invitrogen, Carlsbad, Calif.) in both sense and antisense orientations, according to the manufacturer's instructions. Antibodies. | Anti-STAT1 and anti-phospho-STAT1 antibodies were purchased from Upstate Biochemical, Inc. (Lake Placid, N.Y.). The anti-IRF-1 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). MTT assay. | The growth rates of cells were measured with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 2 x 103 cells in 0.2 ml of culture medium were plated in each well of a 96-well culture plate. A total of 10 wells per time point were used for treatment. Cells were analyzed at regular intervals of 2, 4, 6, 8, and 10 days by the addition of 40 mul of MTT, 1.25 mug of MTT/ml of phosphate-buffered saline (PBS), to each well of the plate. The cells were incubated at 37C for 2.5 h, the medium was aspirated, and the cells were then lysed in 100 mul of dimethyl sulfoxide. Conversion of MTT to formazan by metabolically viable cells was monitored at 570 nm in an enzyme-linked immunosorbent assay reader, and the results were analyzed by regression analysis from triplicate experiments. Western blot analysis. | Cells were washed, scraped in PBS, and then centrifuged. The pellet was resuspended in a RIPA buffer containing protease inhibitors and kept on ice for 60 min. After centrifugation the protein content of the supernatant (whole-cell extract) was determined by a Bio-Rad DC (Hercules, Calif.) method. Fifty micrograms of whole-cell extract was analyzed by standard methods on a sodium dodecyl sulfate-polyacrylamide gel as described earlier . Proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad) and incubated with the primary antibody at 4C overnight. Protein bands were detected by an enhanced chemiluminescent method (ECL; Amersham Pharmacia, Piscataway, N.J.). Bands were analyzed on an AlphaImager system (Alpha Innotech, San Leandro, Calif.). Blots were stripped and reprobed several times. Equal protein loads were checked by rehybridizing the blots with a glyceraldehyde-3-phosphate dehydrogenase (G3PDH) antibody (Advanced Immunochemical, Long Beach, Calif.) that served as a constitutively expressed internal control. RNA isolation and semiquantitative reverse transcription-PCR. | Cells were washed with PBS and directly lysed in TriReagent-LS (Molecular Research Center, Inc., Cincinnati, Ohio) and precipitated according to the manufacturer's recommendations. One microgram of RNA was reverse transcribed (SuperScript II; Gibco/BRL, Grand Island, N.Y.) and subjected to PCR amplification as described earlier . Primers for IRF-1 were custom designed and synthesized (Genosys, The Woodlands, Tex.). Primers for G3PDH were purchased from Clontech (Palo Alto, Calif.). PCR fragments were resolved by agarose gel electrophoresis and transferred to a nylon membrane (Amersham Pharmacia, Piscataway, N.J.) and hybridized with end-labeled oligonucleotide probes. Oligonucleotide probes were custom designed and synthesized. Autoradiograms were analyzed by densitometry (AlphaImager; Alpha Innotech, San Leandro, Calif.). Electrophoretic mobility shift assay (EMSA). | Nuclear extracts were prepared by lysing cells in a buffer containing 10 mM HEPES (pH 7.9), 60 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 0.5% NP-40, and 1 mM phenylmethylsulfonyl difluoride. Five to ten micrograms of nuclear extracts were incubated in a binding buffer (10 mM Tris-HCl [pH 7.6], 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 6% glycerol, 1 mM dithiothreitol, 1 mug of polydIC) in the presence or absence of a 100-fold molar excess of unlabeled competitor DNA and/or appropriate antibodies on ice for 10 min. Following incubation, 10,000 cpm of 32P-end-labeled oligonucleotide probe was added and the reaction was incubated at room temperature for an additional 30 min. The DNA-protein complexes were separated from free probe by electrophoresis on a 4 to 6% polyacrylamide gel. The gel was dried and subjected to autoradiography. Oligonucleotides were custom designed and synthesized (Genosys, Inc.). Annealing of individual oligonucleotides was done according to standard protocols. EMSA oligonucleotides. | For gel shift competitions and experiments, the following double-stranded oligonucleotides were used. (The sense strand is shown. The oligonucleotides were synthesized by Genosys, Inc., and annealed according to a standard protocol.) IRF1-NF-kappaB , TTAGCGGGATTCCCCAGCCCT; IRF1-GAS , AGCCTGATTTCCCCGAAATGAC. Plasmid transfection. Cells were transfected with the recombinant IRF-1 plasmid using a GenePORTER 2 transfection reagent (GTS Inc., San Diego, Calif.). Cells were plated at 5 x 105 cells/60-mm dish density in 1 ml of culture medium 24 h before the transfection so that they will be 60 to 90% confluent on the day of transfection. Then, 4 mug of plasmid DNA was diluted in 80 mul of serum-free medium plus 20 mul of GenePorter 2, which gives a final volume of 100 mul. This mixture was incubated at room temperature for 10 min and then was added to the cells. After 24 h, fresh medium was added. Transfections were made in triplicate. Controls such as mock-transfected and vector-transfected cells were included. PMID- 12204966_RESULTS TI - AB - ATRA inhibits growth of SiHa cells in a dose-dependent fashion. | SiHa cells were grown in the presence or absence of ATRA for various time points . Cell growth was determined by an MTT assay. Apparently, physiological (low) doses (10-7 and 10-6 M) of ATRA did not inhibit cell growth significantly, while administration with pharmacological (high) doses (10-5 and 10-4 M) of ATRA caused significant growth inhibition. IRF-1 participates in ATRA-induced growth inhibition. | IRF-1 is an important regulator of cell proliferation , so its role in ATRA-induced growth inhibition was investigated. Accordingly, protein lysates from SiHa cells treated with low-dose (10-6 M) or high-dose (10-4 M) ATRA were evaluated by Western immunoblotting. ATRA at a concentration of 10-6 M increased protein levels of IRF-1 at a peak of fourfold 6 h after treatment , while induction of IRF-1 protein by 10-4 M ATRA was sustained. We also determined IRF-1 protein levels 24 h after treatment with various concentrations of ATRA . Apparently, induction of IRF-1 at 24 h was dose dependent; significant induction occurred at high doses. In another set of experiments, SiHa cells were transiently transfected with an IRF-1 expression vector. Using a trypan blue exclusion test, we determined the cell number 48 h posttransfection. The results (representing the mean of three independent measurements in which the standard deviation did not exceed 4%) showed that IRF-1 transfection significantly reduced cell numbers, to 65% of the level of untreated control cells. Also, transient transfection of an antisense IRF-1 construct abrogated ATRA-induced growth arrest. Prolonged induction of IRF-1 requires STAT1. | IRF-1 mRNA levels were increased only moderately and transiently after treatment with ATRA in a STAT1 knockout cell line (U3A), while treatment of the parental (STAT1 wild-type) line (2fTGH) with ATRA increased it significantly and for a longer time . Only high-dose ATRA induces phosphorylation of STAT1. | Considering the role of STAT1 in regulation of IRF-1 expression, we determined the phosphorylation status of STAT1 after ATRA treatment. Treatment with 10-6 M ATRA did not affect STAT1 tyrosine phosphorylation . In contrast, 10-4 M ATRA treatment significantly elevated STAT1 tyrosine phosphorylation between 3 to 12 h posttreatment. STAT1 levels, however, remained unchanged during ATRA treatment. NF-kappaB binding to an oligonucleotide from the IRF-1 promoter is independent of ATRA concentrations. | IRF-1 transcription is regulated via NF-kappaB sites in the IRF-1 gene promoter . Accordingly, we tested NF-kappaB binding to oligonucleotides corresponding to NF-kappaB-binding sites in the IRF-1 promoter by a gel-shift assay. As shown, NF-kappaB (mostly the p65-p50 complex) binding to the IRF-1 promoter was increased after 30 min by both low- and high-dose ATRA treatment (Fig. and B). STAT1 binding to an oligonucleotide from the IRF-1 promoter depends on ATRA concentration. | IRF-1 transcription could be also regulated via GAS/SIE sites in the IRF-1 gene promoter . Accordingly, we tested STAT1-binding oligonucleotides corresponding to a GAS site in the IRF-1 promoter by a gel-shift assay. In the extracts of ATRA-treated cells, binding of STAT1 to a GAS site was observed only after high-dose, but not low-dose, ATRA treatment . FIG. 1. | Effects of ATRA on cell growth. Effects of ATRA on cell growth. SiHa cells were seeded in a multiwell plate and treated with either low-dose (10-7 and 10-6 M) or high-dose (10-5 and 10-4 M) ATRA for the times indicated. An MTT assay was employed to determine the amounts of viable cells. Results are shown as means +- standard deviations (n = 3). FIG. 2. | Effects of ATRA on IRF-1 protein levels in SiHa cells. Effects of ATRA on IRF-1 protein levels in SiHa cells. Confluent cultures of SiHa cells were treated with either low-dose (10-6 M) or high-dose (10-4 M) ATRA for the times indicated. Top, Western immunoblotting was performed on the total cell lysate as described in Materials and Methods. Bottom, Densitometric results are expressed as fold change to the untreated controls. Values are shown as means +- standard deviations (n = 3). FIG. 3. | Dose-dependent induction of IRF-1 by ATRA. Dose-dependent induction of IRF-1 by ATRA. Confluent cultures of SiHa cells were treated for 24 h with various doses of ATRA as indicated. Western immunoblotting was performed on the total cell lysate as described in Materials and Methods. Densitometric results are expressed as fold change to the untreated controls. Values are shown as means +- standard deviations (n = 3). FIG. 4. | Prolonged induction of IRF-1 requires STAT1. Prolonged induction of IRF-1 requires STAT1. STAT1 knockout (U3A) or STAT1 wild-type (2fTGH) cells were treated with ATRA (10-6 M) for different time points. IRF-1 mRNA levels were determined by reverse transcription-PCR together with the constitutively expressed G3PDH. IRF-1 levels are given as ratios of IRF-1/G3PDH. Values are shown as means +- standard deviations (n = 3). FIG. 5. | Effects of different doses of ATRA on tyrosine phosphorylation of STAT1 in SiHa cells. Effects of different doses of ATRA on tyrosine phosphorylation of STAT1 in SiHa cells. Confluent cultures of SiHa cells were treated with low-dose (10-6 M) or high-dose (10-4 M) ATRA for the times indicated. Western immunoblotting was performed using anti-phosphoSTAT1 (STAT1p) as well as anti-STAT1 antibodies. Experimental data shown are representative of three independent experiments. FIG. 6. | Effects of different doses of ATRA treatment on binding of cellular proteins to oligonucleotides representing NF-kappaB or GAS sites of the IRF-1 promoter. Effects of different doses of ATRA treatment on binding of cellular proteins to oligonucleotides representing NF-kappaB or GAS sites of the IRF-1 promoter. Nuclear extracts from SiHa cells treated with low-dose (A and C) or high-dose (B and D) ATRA were incubated with labeled oligonucleotides representing the NF-kappaB (A and B) or GAS (C and D) binding sites of the IRF-1 promoter, and an EMSA was performed as described in Materials and Methods. In panels A and B we showed only the 30-min time point that represented the first appearance of NF-kappaB binding. STAT1 binding to the GAS oligomer (C and D) appeared after 6 h of treatment. Next, 100x cold ligand was used to demonstrate specificity of both probes (data not shown). Tetradecanoyl phorbol acetate (TPA)- or gamma interferon (IFNgamma)- treated cell extracts were used as positive controls. Preincubations with specific anti-NF-kappaB p65 and p50 or STAT1 antibodies were performed to demonstrate the presence of these proteins in the observed complexes (data not shown). PMID- 12204966_DISCUSSION TI - AB - Induction of IRF-1 mRNA and the consequent growth inhibition in cervical squamous carcinoma cells by ATRA have been demonstrated . Clinically, retinoids reversed or suppressed low-grade or moderate- to high-grade cervical dysplasias but were not effective in patients with more advanced dysplasias . Although immortalized keratinocytes are more sensitive to growth control by retinoids than their normal counterparts , at later stages transformed keratinocytes frequently become resistant to retinoids . SiHa cervical squamous carcinoma cells have been reported to be unresponsive to retinoids . In our experiments, SiHa cells exhibited resistance to 10-7 to 10-6 M ATRA-induced growth inhibition, but 10-5 M and especially 10-4 M ATRA significantly inhibited the growth of SiHa cells . A hallmark of growth inhibition is the high expression of the IRF-1 gene . Ultimately, this induction of IRF-1 is responsible for the observed growth inhibition of target cells. Introduction of an IRF-1 expression vector into SiHa cells caused growth arrest(see above). Similarly, in the presence of an antisense IRF-1 expression plasmid, high-dose (10-4 M) ATRA did not inhibit cell growth (viable cells were at 95% of control level, compared to 60% with ATRA but without the IRF-1 expression plasmid). IRF-1 expression was elevated in ATRA-treated cells in a dose-dependent fashion: low-dose (10-6 M) ATRA increased it temporarily, while high-dose (10-4M) ATRA caused a sustained expression of IRF-1 (Fig. and ). However, only high-dose (10-5 to 10-4 M) but not low-dose (10-7 to 10-6 M) ATRA could significantly inhibit cell growth in vitro. These data strongly suggested that duration of IRF-1 expression is critical for cell growth inhibition in this system. Our next set of experiments aimed to determine the molecular events behind this dose-dependent induction of IRF-1 by ATRA. In STAT1 knockout cells , ATRA moderately and temporarily induced IRF-1 mRNA , while IRF-1 induction in the parental cell line (2fTGH) was long lasting under the same conditions. Activation (tyrosine phosphorylation) of STAT1 occurred only after high-dose (10-4 M), but not low-dose (10-6 M), ATRA treatment in SiHa , suggesting that the capability of high-dose ATRA to activate STAT1 was responsible for the long-lasting activation of IRF-1 expression and consequent inhibition of cell growth. Interestingly, the kinetics of this tyrosine phosphorylation was delayed (3 to 13 h) compared to the immediate-early (5 to 15 min) phosphorylation by cytokines . Similarly, binding of the activated STAT1 to the GAS site was a late event (6 h). We observed that ATRA treatment ---at both 10-6 and 10-4 M concentrations ---induced binding of NF-kappaB to a double-stranded oligonucleotide originating from the IRF-1 promoter , similar to that described by others . However, binding of STAT1 to its cognate GAS elements in the IRF-1 promoter was observed only after high-dose (10-4 M) ATRA treatment , suggesting that STAT1 activation was responsible for the sustained IRF-1 expression. Phosphorylation of STAT1 and its binding to the IRF-1 promoter occurred with delayed kinetics compared to IRF-1 activation via NF-kappaB binding. The effects of GAS and NF-kappaB sites on the activity of the IRF-1 promoter might be synergistic and could be accountable for the long-lasting activation of IRF-1 expression. It has been postulated that retinoids directly increase the expression of transcription factors (STAT1 or IRF-1) that play key roles in JAK-STAT signaling, thereby restoring interferon (IFN) sensitivity . Our data suggest that ATRA could restore sensitivity to itself by a dose-dependent induction of STAT1, resulting in a sustained activation of the IRF-1 signal that might be critical in determining downstream cellular responses. The downstream targets might include the activation of apoptotic events associated with the IRF-1/CAS pathway or NF-kB induction or both. The role of these pathways in high-dose ATRA-induced inhibition of cell growth, however, needs further evaluation. PMID- 12204956 TI - In Vitro Whole-Blood Analysis of Cellular Immunity in Patients with Active Coccidioidomycosis by Using the Antigen Preparation T27K AB - | Measurement of cellular immunity in human coccidioidomycosis has important diagnostic and prognostic implications. The coccidioidin skin test has been the standard for the measurement of this, but it is not available in the United States. We examined the utility of measuring surface expression of CD69 on T lymphocytes in whole blood incubated with the coccidioidal antigen preparation T27K as an alternative to the skin test. Seventy donors with active coccidioidomycosis were studied. The mean fluorescent intensity (MFI) of CD69 expression on CD3 lymphocytes in response to T27K was 28.61 +- 1.77, significantly greater than the control response of 11.45 +- 0.78 (P < 0.001). The MFI CD69 response to T27K above that for the control (MFI CD69 above control) was 6.35 +- 2.18 for seven subjects with disseminated coccidioidomycosis who were studied within 5 months of diagnosis. This was significantly below the value of 20.17 +- 3.17 for 18 subjects with pulmonary coccidioidomycosis studied within 5 months of diagnosis and the value of 19.58 +- 2.91 for 27 subjects with disseminated coccidioidomycosis studied after 5 months of diagnosis (for both, P < 0.05). There was an inverse correlation between coccidioidal clinical score and MFI CD69 above control for all 34 subjects with disseminated coccidioidomycosis (r = 0.362; P = 0.036) but not for the 36 subjects with pulmonary disease (r < 0.001; P = 0.993). Among 30 subjects for whom data were available, there was a highly significant association between the MFI CD69 above control and the supernatant concentrations of gamma interferon, interleukin-2 (IL-2), and tumor necrosis factor alpha (for all, P < 0.001), but not for IL-4, IL-5, or IL-10. These data indicate that in vitro assessment of CD69 expression on T lymphocytes by using T27K may be a useful measure of cellular immune response among subjects with active coccidioidomycosis. PMID- 12204956_Introduction TI - AB - The association between expression of delayed-type hypersensitivity and the severity of illness in human coccidioidomycosis has been recognized for more than 50 years . Specifically, expression of delayed-type hypersensitivity is predominantly absent often among individuals who have severe, extrathoracic dissemination. On the other hand, delayed-type hypersensitivity is usually present among those with mild, self-limited illness . Moreover, the failure to express coccidioidal delayed-type hypersensitivity appears to predict subsequent relapse after therapy for patients with active coccidioidomycosis is halted . Immunologically, delayed-type hypersensitivity is an expression of cell-mediated immunity. While measurement of induration at the site of intradermal injection of antigen has been the traditional way to measure cell-mediated immunity, in vitro laboratory techniques also are capable of assessing cellular immunity in coccidioidomycosis . In vitro methods are advantageous in that they do not require exposure of the subject to foreign antigen or its preservative and results are not dependent on injection technique, time of reading, and measurement of induration size. In coccidioidomycosis, the use of in vitro methods to measure cell-mediated immunity has taken on further importance because no skin-test reagent has been available in the United States for the past several years. In the present study, we assessed in vitro expression of cellular immunity in coccidioidomycosis by measuring the T-lymphocyte surface expression of CD69, a glycoprotein that is expressed by activated lymphocytes and NK cells , after incubation of whole blood with the antigen preparation T27K. In the present work, we compared CD69 expression with simultaneous cytokine production among donors with various forms of active coccidioidomycosis. We have previously shown that in vitro assessment of cellular immunity by flow cytometry with the antigen T27K distinguishes immune and nonimmune healthy donors on the basis of coccidioidal skin testing when the antigen was still available . PMID- 12204956_MATERIALS AND METHODS TI - AB - Human subjects. | Seventy patients with active coccidioidomycosis were sequentially recruited and tested as they entered the Valley Fever Clinic of the Southern Arizona Veterans Affairs Health Care System. In this medical center, all those with a diagnosis of coccidioidomycosis are referred to the Valley Fever Clinic. Donors known to be infected with human immunodeficiency virus or known to have undergone allogeneic transplantation were excluded from the study. A variety of clinical data were collected at the time of study, and a clinical score of severity of coccidioidomycosis was determined. This score is based on clinical symptoms, the severity and number of clinical sites of disease, and the immune diffusion complement-fixing coccidioidal antibody titer. In this system, a higher score is associated with more severe coccidioidal illness . The study was approved by the Human Subjects Committee of the University of Arizona. In addition to these patients, 20 healthy donors with known reactivity to coccidioidin and without active coccidioidomycosis and 11 healthy donors without known coccidioidal immunity were also studied. Whole-blood preparation and incubation. | Approximately 5 ml of blood was drawn by venipuncture from each donor and placed into tubes containing lithium heparin. Aliquots of 0.5 ml were added to 15-ml polypropylene conical centrifuge tubes (Corning, Corning, N.Y.) to which 10 or 20 mug of T27K per ml was added. Previous studies did not reveal differences between the two concentrations. Nothing was added to control tubes. T27K was prepared as a solution as described previously . Samples were incubated for 18 to 24 h at 37C in 95% air-5% CO2, with the caps of the tubes kept loose and without rocking. At the end of this time, 50 mul of 20 mM EDTA (GIBCO, Grand Island, N.Y.) was added to each sample for 15 min at room temperature. Subsequently, 5.0 ml of FACSlyse (Becton Dickinson Immuno-cytometry Systems, [BDIS], San Jose, Calif.) was added to each tube to lyse red blood cells. The tubes were incubated for 10 min at room temperature, and then the contents were mixed. Following this, the samples were centrifuged, resuspended in 5.0 ml of wash buffer (phosphate-buffered saline with 1% bovine serum albumin and 0.1% NaN3), and recentrifuged; and the pellet was resuspended in 1.0 ml of wash buffer. To 200 mul of each sample was added 20 mul of phycoerythrin (PE)-labeled anti-CD69 and 20 mul of peridinin chlorophyll protein-labeled anti-CD3 (BDIS), and the mixture was incubated for 30 min at room temperature in the dark. Then, 2.0 ml of wash buffer was added to each sample, the samples were centrifuged again, and the pellet was resuspended in 500 mul of phosphate-buffered saline. After this step, the samples were immediately used for flow cytometry. Flow cytometry. | Cells were analyzed with a single argon-ion laser flow cytometer (488 nm; FACScan; Becton Dickinson, Mountain View, Calif.) with Cell Quest software (Becton Dickinson). Prior to each assay, general calibration and compensation were established by using two-color CaliBRITE beads (Becton Dickinson), prepared according to the directions of the manufacturer, with the results analyzed by using FACSComp software (version 4.1; Becton Dickinson). For every use, a printout was generated detailing the photomultiplier tube (PMT) voltages, laser current, and power. Over the 6-month period encompassing this study, the FLI PMT voltage varied by 14 V (620 to 634 V), the FL2 PMT voltage varied by 26 V (664 to 680 V), and the FL3 PMT voltage varied by 32 V (704 to 736 V). In addition, the laser current varied between 5.18 and 5.22 A, and the laser power varied between 15.00 and 15.05 mW. In addition to these general steps, a sample was prepared from T27K-stimulated cells for each experiment containing fluorescein isothiocyanate-labeled immunoglobulin G2a, PE-labeled immunoglobulin G1, and PerCP-labeled anti-CD3. From this, a CD3+-lymphocyte gate was established and the PMT voltage for each channel was set. Compensation was then optimized by analyzing samples containing fluorescein isothiocyanate-, PE-, and PerCP-labeled anti-CD8 within the CD3+-lymphocyte gate. Compensation was considered optimized when the positive populations were aligned over the negative populations. The surface expression of CD69 was then assessed among CD3 lymphocytes after counting of 10,000 of these cells for each sample and was recorded as the mean fluorescent intensity (MFI). Cytokine bead assay. | The plasma supernatant was drawn off some of the samples prior to preparation for flow cytometry and was frozen at -70C. Subsequently, these samples were thawed and the concentrations of the cytokines gamma interferon (IFN-gamma), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-alpha), IL-4, IL-5, and IL-10 were measured by a flow cytometric bead assay (BDIS). Samples with levels >5,000 pg/ml, the upper limit of the assay, were not rerun at dilution but were instead listed as 5,000 pg/ml. Statistics. | Statistical analysis of continuous variables between two groups was performed by the Student t test, and the paired t test was used for comparison of data within subjects. Linear regression was used to determine the association of continuous variables. A contingency table was used to assess differences in frequencies between groups by the Fisher exact test being used for two-by-two tables. In all cases, a P value of <0.050 was considered statistically significant. PMID- 12204956_RESULTS TI - AB - Description of patients. | The 70 patients consisted of 34 individuals with disseminated coccidioidomycosis and 36 patients with pulmonary disease. Reflecting the population of veterans from which the study population was derived, 67 of the subjects in the study were male, with a median age of 66.9 years (age range, 26.8 to 85.4 years). In addition, 21 were African American, 67 were non-Hispanic, and 5 were active cigarette smokers. The median time since the diagnosis of coccidioidomycosis upon entry into the study was 8.9 months (range, 0.2 to 289.4 months). Forty-seven patients were receiving some type of antifungal therapy at the time of the study, while 23 were not. Thirty-four of the patients had underlying diseases. Of these, diabetes was the most common and occurred in 16 patients. Expression of CD69 in response to T27K. | Among all 70 patients, the mean +- standard error of the mean MFI CD69 in response to incubation with T27K was 28.61 +- 1.77, significantly greater than the response of the control, whose samples were not incubated with T27K, of 11.45 +- 0.78 (P < 0.001). In order to account for the low but persistent expression of CD69 by CD3 lymphocytes without antigen stimulation, subsequent data are expressed as the difference, for each donor, in MFI CD69 between the sample incubated with T27K and the control sample not incubated with T27K (MFI CD69 above control). Hence, the results for each donor were normalized by subtracting the results for the sample containing T27K from the results for the control sample that did not contain T27K. The mean +- standard error of the mean MFI CD69 above control in response to T27K for all 70 patients was 17.16 +- 1.66 and ranged from -1.45 to 53.7. The MFI CD69 above control for the 34 patients with disseminated coccidioidomycosis was 16.86 +- 2.52 and was not significantly different from value for the 36 patients with pulmonary disease, 17.44 +- 2.22 (P = 0.861). The results for the patients with active coccidioidomycosis were not significantly different from the value of 15.21 +- 1.58 obtained for the 20 healthy immune donors with known coccidioidal immunity (P = 0.549). On the other hand, the MFI CD69 above control for 11 healthy nonimmune donors was only 1.58 +- 0.69, significantly less than the values for the patients with active coccidioidomycosis and the healthy, immune donors (for all, P < 0.001). There were no differences in the MFI CD69 above control response to T27K of CD3 cells on the basis of underlying disease, including diabetes, age of 60 years or more, or race (for all, P > 0.050) (data not shown). Because antifungal therapy may have changed the initial coccidioidal cellular immune response over time, particularly among those with disseminated disease, data were next analyzed by comparing subjects who were studied within 5 months of diagnosis to those who were studied after 5 months of diagnosis. These data are displayed in Fig. . Among the seven subjects with disseminated coccidioidomycosis studied within 5 months of diagnosis, the MFI CD69 above control was 6.35 +- 2.18, significantly less than the value of 20.17 +- 3.17 for the 18 subjects with pulmonary coccidioidomycosis studied within 5 months of diagnosis (P = 0.016) and the value of 19.58 +- 2.91 for the 27 patients with disseminated coccidioidomycosis studied more than 5 months after diagnosis (P = 0.031). This difference was not due to a difference in background expression of CD69. The expression of CD69 in control, unstimulated samples from those with a diagnosis of pulmonary coccidioidomycosis of less than 5 months' duration was 8.45 +- 1.38, not different from the value of 10.83 +- 3.30 for those with disseminated disease of less than 5 months' duration (P = 0.434). Association of CD69 expression in response to T27K and clinical coccidioidomycosis score. | As shown in Fig. , among the 34 subjects with disseminated coccidioidomycosis, the MFI CD69 above control in response to T27K was found to be significantly and inversely associated with the clinical coccidioidomycosis score (r = 0.362; P = 0.036). On the other hand, among the 36 donors with pulmonary coccidioidomycosis, there was no correlation between MFI CD69 above control in response to T27K and the clinical coccidioidomycosis score (r < 0.001; P = 0.993). Association of CD69 response with cytokine production. | For 30 subjects, the plasma supernatant was saved and subsequently tested to determine the relationship between CD69 expression and cytokine production after incubation with 10 mug of T27K per ml. Fifteen of these donors had disseminated coccidioidomycosis and 15 had pulmonary disease. As depicted in Fig. , there was a highly significant and positive association between MFI CD69 above control and the concentrations of IFN-gamma, IL-2, and TNF-alpha in the plasma supernatant (for all three, P < 0.001). The levels of IL-4, IL-5, and IL-10 were low and not significantly associated with MFI CD69 above control. The relation between CD69 expression and cytokine production was not different among the donors with disseminated or pulmonary coccidioidomycosis (data not shown). FIG. 1. | MFI CD69 above control for CD3 lymphocytes in response to T27K among 34 donors with disseminated coccidioidomycosis (black boxes) and 36 donors with pulmonary coccidioidomycosis (open boxes). MFI CD69 above control for CD3 lymphocytes in response to T27K among 34 donors with disseminated coccidioidomycosis (black boxes) and 36 donors with pulmonary coccidioidomycosis (open boxes). Patients studied within 5 months of diagnosis were compared to those studied more than 5 months after diagnosis. *, P < 0.05 when donors with disseminated coccidioidomycosis studied within 5 months of diagnosis are compared to donors with pulmonary disease studied within 5 months of diagnosis and donors with disseminated disease studied after 5 months of diagnosis. Values in parentheses indicate the number of subjects in each group. FIG. 2. | Correlation between clinical coccidioidomycosis score and MFI CD69 above control in response to T27K among 34 subjects with disseminated coccidioidomycosis and 36 subjects with pulmonary coccidioidomycosis. Correlation between clinical coccidioidomycosis score and MFI CD69 above control in response to T27K among 34 subjects with disseminated coccidioidomycosis and 36 subjects with pulmonary coccidioidomycosis. FIG. 3. | Comparison of MFI CD69 above control for CD3 lymphocytes after incubation with 10 mug of T27K per ml and the concentrations of IFN-gamma, IL-2, TNF-alpha, IL-4, IL-5, and IL-10 in plasma supernatants among 30 subjects with coccidioidomycosis. Comparison of MFI CD69 above control for CD3 lymphocytes after incubation with 10 mug of T27K per ml and the concentrations of IFN-gamma, IL-2, TNF-alpha, IL-4, IL-5, and IL-10 in plasma supernatants among 30 subjects with coccidioidomycosis. PMID- 12204956_DISCUSSION TI - AB - Measurement of delayed-type hypersensitivity by skin testing has been the standard technique by which cell-mediated immunity has been determined among individuals with coccidioidomycosis . This test is not available in the United States, and alternatives are required to assess coccidioidal cellular immunity. Assessment of cellular immunity in coccidioidomycosis serves several purposes. First, it may be used to establish the prevalence of coccidioidal infection in a population within a geographic region . Second, it may be used to determine new infection, if prior assessment of immunity has been performed and has been found to be lacking. Finally, expression of cell-mediated immunity appears to reflect a protective host response and may auger an improved clinical outcome among individuals with symptomatic coccidioidomycosis . In the present study, the surface expression of CD69 on CD3 lymphocytes after incubation with the coccidioidal antigen preparation T27K was used to ascertain the cellular immune status of donors with active coccidioidomycosis. The response to T27K of samples from all donors was significantly above that for unstimulated control samples and above the values obtained for a group of healthy, nonimmune donors. When the MFI CD69 above control in response to T27K was examined, there was no overall difference in response between donors with disseminated coccidioidomycosis and those with pulmonary disease, nor were there differences in response on the basis of underlying disease, age, or race. The average age of the patients with active coccidioidomycosis was nearly 67 years. While this appears old, recent data indicate that symptomatic coccidioidomycosis is far more prevalent among those aged 60 years and over than among younger subjects . Because all patients with active coccidioidomycosis at the medical center were referred to the coccidioidomycosis clinic and were eligible for study, the older age most likely reflects the predisposition for symptomatic illness among an older age group rather than any other factor. However, there were significant differences on the basis of the length of time between diagnosis and study. When studied early in the course of their illness, those with disseminated disease had a decreased response to T27K compared to the response of either those studied later or those with pulmonary disease. These data suggest that antifungal therapy may ameliorate the depression in cellular immune response that occurs in disseminated coccidioidomycosis that has been observed previously . Barbee and Hicks have demonstrated such a return in cellular immune response with therapy in a small cohort of patients using an in vitro lymphocyte transformation assay. We are prospectively examining the cellular immune response over time in patients with various forms of coccidioidomycosis on antifungal therapy to more fully explore this question. The coccidioidal antigen preparation T27K used in these experiments is the soluble, aqueous supernatant obtained after mechanically disrupted thimerosal-preserved spherules are centrifuged at 27,000 x g. Previous work with formalin as the preservative has shown that it protects mice from experimental coccidioidal infection when combined with alum . To date, the components of T27K have not been fully defined, but several coccidioidal antigens have been identified in the preparation, including chitinase, chitobiase, aspartyl protease, Ag2/proline-rich antigen, alkaline phosphatase, serine protease, and tube precipitin. Some of these antigens have been identified in both coccidioidin and spherulin, but neither of the latter two preparations has ever been fully characterized. The use of T27K in an in vitro assay is in contrast to earlier studies, in which either coccidioidin or spherulin was used as the skin-test reagent. While it would be of great interest to compare these older antigen preparations to T27K, they are, unfortunately, no longer available. Because of the laboratory and bioterrorism risk, preparation of either requires a licensed biosafety level 3 facility. Moreover, significant lot-to-lot variability occurs with these preparations, making any comparisons difficult. In previous work, we have found that the in vitro response to T27K among healthy donors corresponds closely with skin-test positivity to spherulin . The clinical score is a measure of coccidioidal severity and is increased in patients with severe disease and declines as disease improves with antifungal therapy. There was a significant inverse association between the score of coccidioidal illness among donors with disseminated coccidioidomycosis and the expression of CD69. However, no such association was seen among those with active pulmonary disease. This observation is consistent with observations first made by Smith and Beard with the use of coccidioidin skin testing and with the findings of other investigators who used both skin testing and in vitro assays of cell-mediated immunity . CD69 expression on CD3 lymphocytes in response to T27K among donors with coccidioidomycosis was directly and positively associated with production of the cytokines IFN-gamma and IL-2 but not with the production of the T-helper type 2 cytokines IL-4, IL-5, and IL-10. This suggests that CD69 expression is associated with a T-helper type 1 cellular immune response in patients with coccidioidomycosis. These data and our previous work demonstrating clear distinctions between healthy immune and nonimmune donors suggest that T27K is a biologically useful antigen in determining the cellular immune response to coccidioidomycosis. The close association between the production of T-helper type 1 cytokines, particularly IFN-gamma, and CD69 expression in response to T27K suggests that assessment of these cytokines alone or in association with CD69 expression might be useful in measuring the coccidioidal cellular immune response. At this time, we do not have enough data to determine whether this is the case. Studies to examine this are planned for the future. Expression of CD69 on lymphocytes has been shown to be associated with cellular immune function in patients with a variety of other conditions . In addition, other rapid assays have been found to be useful in determining the cellular immune response in patients with other granulomatous conditions. In particular, the measurement of IFN-gamma in whole blood incubated with tuberculin antigen has been found to compare favorably with purified protein derivative skin testing in patients with a variety of forms of tuberculosis . In the present study, expression of CD69 on CD3 lymphocytes in response to the coccidioidal antigen preparation T27K was a rapid, easily performed assay that required less blood than many standard clinical laboratory tests and provided useful information regarding coccidioidomycosis-specific cellular immunity. Therefore, even if the reagents for performing coccidioidal skin testing were still available, there would be several advantages to using an in vitro system, such as measurement of CD69 expression. The present study, while demonstrating clinical associations with the in vitro assay, did not clearly show a direct relationship between the assay result and clinical disease or outcome. It is imperative that further studies examining the utility of in vitro measurement of coccidioidal cellular immunity be performed to further assess this possibility. PMID- 12204957 TI - Immunoglobulin G Antibody against Helicobacter pylori: Clinical Implications of Levels Found in Serum AB - | The clinical significance of high levels of antibody against Helicobacter pylori is still unclear. We sought to evaluate whether the serum antibody levels could predict the presence of macroscopic gastroduodenal disease, to identify factors that correlate with antibody levels in a multivariate context, and to determine the predictive value of antibody levels for diagnosing H. pylori infection. The grades of gastritis and density of H. pylori colonization were scored separately using the updated Sydney system for antral and body mucosa. An enzyme-linked immunosorbent assay (ELISA) for the quantitative detection in serum of IgG antibodies to H. pylori was performed. Of the 170 dyspeptic patients, 105 (62%) had H. pylori infection. There was no difference in antibody levels among endoscopic findings of normal mucosa, chronic gastritis, and duodenal ulcer. On multivariate linear regression analysis, the status of H. pylori infection, mononuclear cell infiltration of body mucosa, and age correlated with antibody levels. The negative predictive value for antibody levels of <30 U/ml is 94%, and the positive predictive value of antibody levels of >70 U/ml is 98%. We conclude that serum antibody levels do not predict the severity of gastroduodenal diseases or the density of H. pylori colonization in H. pylori-infected dyspeptic patients. Higher levels are associated with the presence of H. pylori infection, the chronic gastritis score of the corpus, and older age. Setting a gray zone is necessary for ELISA, since the accuracy in this zone does not allow a precise determination of H. pylori status. PMID- 12204957_Introduction TI - AB - Helicobacter pylori has been established as an important etiological factor for chronic gastritis and duodenal ulcer . It is also associated with gastric ulcer and gastric cancer . As the test-and-treat policy for H. pylori infection is gradually being accepted by general practitioners, a simple, reliable, and noninvasive diagnostic test for H. pylori has become essential in clinical practice . An easier and cheaper way to diagnose H. pylori infection noninvasively is to test for antibodies to the infection. Enzyme-linked immunosorbent assay (ELISA) has been the most commonly used serological test because it is suited for screening large populations . Clinically, some patients are very concerned about contracting H. pylori infection when they are told they have high antibody levels. However, the clinical significance of the levels is still unclear. The correlation between anti-H. pylori antibody levels and the severity of histological gastritis or H. pylori density has been studied with conflicting results . This discrepancy in results may arise from differences in classification and grading of gastritis, the numbers of subjects, consideration of confounding factors, and statistical methods. One study reported that immunoglobulin G (IgG) antibody levels correlated with the grades of antral polymorphonuclear leukocyte infiltration and antral bacterial density , while another study demonstrated that having higher levels implies advanced antral gastritis with either acute or chronic inflammation . Both studies concluded that levels in themselves do not provide a method for predicting the presence of an ulcer. However, the sample size was small in one study, and no body mucosa was evaluated in either study. Therefore, we conducted this study to identify which factors correlate with antibody levels in a multivariate context and to determine if antibody levels could predict the presence of macroscopic gastroduodenal disease. The predictive value of antibody levels for diagnosing H. pylori infection was also evaluated. PMID- 12204957_MATERIALS AND METHODS TI - AB - Dyspeptic patients who were scheduled for upper gastrointestinal endoscopy were recruited into the study. Patients with any of the following conditions were excluded: (i) ulcer complications, such as bleeding, stenosis, or perforation; (ii) previous stomach surgery; (iii) intake of any substitute for benzimidazoles or preparations containing bismuth within 1 month prior to administration of the test; or (iv) having been treated with or currently on anti-H. pylori therapy. For serology studies, blood was drawn immediately after endoscopy, and sera were collected and stored at -70C until they were assayed. Histology. | During endoscopy, two sets of biopsy specimens from the antrum and the greater curvature of the midbody were obtained for rapid urease testing (CLO Test; Delta West, Bentley, Australia) and histology. Hematoxylin and eosin stains were used to grade the gastritis. The grades of gastritis were assessed for infiltration of mononuclear cells or neutrophils, mucosal atrophy, and intestinal metaplasia using visual analogue scales described in the updated Sydney system on a four-point scale (0, normal; 1, mild; 2, moderate; and 3, marked). A modified Giemsa stain was used to assess the density of H. pylori . The number of lymphoid follicles was recorded. All histological sections were evaluated by the same pathologist, who was blinded to the patients' clinical conditions. Quantitative ELISA. | Serum specimens were tested for the presence of IgG antibodies against H. pylori using a quantitative ELISA (HEL-pTEST II; AMRAD, Kew, Australia). Reference standards were used to produce a standard curve to quantitate H. pylori antibody levels in patient samples. The results were expressed in arbitrary units per milliliter. The antigen was an inactivated native antigen of H. pylori. On the day of testing, we added 100 mul of diluted specimens, diluted positive and negative controls, and duplicates of reference standards 1 to 4 to the appropriate wells of the microtiter plate. The plate was incubated for 15 min at room temperature and then washed six times with a wash buffer. After the washing, 100 mul of sheep anti-human IgG conjugated to horseradish peroxidase was added to each well. After a further 15 min of incubation, 100 mul of substrate reagent was added to each well and incubated in the dark for 15 min at room temperature. Then, stopping solution (100 mul of H2SO4) was added to each well to terminate the enzymatic reaction. The absorbance was read within 30 min using a 450-nm-pore-size filter with a 620-nm-pore-size filter as a reference. H. pylori status. | A patient was classified as H. pylori infected if both the CLO and histological tests were positive. A patient was classified as non-H. pylori-infected if both methods were negative. Patients who had only one positive CLO or histology test were considered to be of indeterminate status. Statistical analysis. | Continuous data were analyzed by the two-tailed Student's t test or analysis of variance (ANOVA). When ANOVA of multiple groups revealed a difference at the 5% level, the post hoc multiple-comparison technique was used to determine which pairs of groups were responsible for the overall difference. Spearman's rank correlation was used to examine the relationship between antibody levels and grades of gastritis. Multiple stepwise linear regression was also used to identify which factors were related to the antibody levels. The data were processed using SPSS software. Positive and negative predictive values were calculated for the ELISA (HEL-pTEST II), using gastric biopsy results as the "gold standard." PMID- 12204957_RESULTS TI - AB - One hundred and seventy patients (88 men and 82 women; mean age +- standard deviation [SD], 43.7 +- 13.5) were included in the study. There were 34 patients with normal endoscopic findings, 62 patients with gastritis, 57 patients with duodenal ulcers, 5 patients with gastric ulcers, 2 patients with combined gastric and duodenal ulcers, and 10 patients with other findings. Based on the proposed gold standard, 105 patients were infected, 64 were not infected, and 1 was of indeterminate status. Anti-H. pylori antibody levels did not show a normal distribution (skewness, 1.888; kurtosis, 2.839), but its logarithm did (skewness, 0.033; kurtosis, -0.967), so logarithmic transformation was performed in the assessment of anti-H. pylori antibody levels during the two-tailed Student's t-test or ANOVA by Scheffe's method or a linear regression. There was no difference in antibody levels between men and women or smokers and nonsmokers or among different blood groups. The influence of drinking habits on antibody levels was not evaluated because only six patients were regular drinkers. For dyspeptic patients with H. pylori infection, there was no difference in antibody levels among the endoscopic findings for the normal mucosa (n = 12), chronic gastritis (n = 36), and duodenal ulcers (n = 46) (223 +- 220, 227 +- 223, and 228 +- 196 U/ml [mean +- SD], respectively; P = 0.99). The mean serum anti-H. pylori antibody levels in relation to the grading of the four histological features of the antral mucosa and body mucosa are illustrated in Fig. and , respectively. We see a trend for the mean antibody levels to rise as the grades of gastritis and H. pylori density increase. Significant correlations were found between the antibody levels and the grades of gastritis in the antral mucosa (Spearman's rank correlation: r = 0.586 and P < 0.001 for neutrophil infiltration, r = 0.620 and P < 0.001 for mononuclear cell infiltration, r = 0.429 and P < 0.001 for atrophy, r = 0.630 and P < 0.001 for H. pylori density, and r = 0.438 and P < 0.001 for lymphoid follicles) and in the body mucosa (Spearman's rank correlation: r = 0.598 and P < 0.001 for neutrophil infiltration, r = 0.618 and P < 0.001 for mononuclear cell infiltration, r = 0.481 and P < 0.001 for atrophy, r = 0.680 and P < 0.001 for H. pylori density, and r = 0.190 and P < 0.05 for lymphoid follicles). However, if the calculation was restricted to H. pylori-infected patients, only the grades of neutrophil infiltration and mononuclear cell infiltration, atrophy, and the number of lymphoid follicles in the body mucosa retained significant correlation with antibody levels (r = 0.248 and P < 0.05, r = 0.292 and P < 0.005, r = 0.218 and P < 0.05, and r = 0.230 and P < 0.05, respectively). This is also noted in estimating correlation between antibody levels and age. Significant correlations were found when all subjects were considered (Spearman's rank correlation: r = 0.200; P < 0.01), but there was no significant correlation in H. pylori-infected patients (Spearman's rank correlation: r = 0.154; P = 0.117). However, if age is divided into older (>=45 years) and younger (<45 years) groups, the difference in antibody levels between the two groups in H. pylori-positive patients approached significance (median, 164, and range, 38 to 823 versus 124 and 21 to 804; P = 0.09). Intestinal metaplasia in antral specimens was observed in 18 patients (15 with score 2, 2 with score 3, and 1 with score 4), so scores 2, 3, and 4 were grouped together. There was no difference in the mean antibody levels between patients with and without intestinal metaplasia (P = 0.984). No intestinal metaplasia was observed in the body specimens. A multiple stepwise linear regression was performed to analyze the relationship between antibody levels and the following predictors: sex, age, smoking, blood groups, grades of antral and body gastritis, number of lymphoid follicles, H. pylori density, and status of H. pylori infection. Table shows that the status of H. pylori infection, grade of mononuclear cell infiltration of body mucosa, and age were three independent factors correlated with antibody levels (R = 0.780; R2 = 0.608). Figure shows the distribution of anti-H. pylori antibody (IgG) levels in H. pylori-infected and uninfected patients (n = 169). The negative predictive value of antibody levels of <20 U/ml is 100%, and that for levels of <30 is 94%. The positive predictive value of antibody levels of >70 U/ml is 98%. There were 27 patients (16%) whose antibody levels fell between 30 and 70 U/ml. Among them, 14 patients were H. pylori infected and the rest were non-H. pylori infected, judged by the reference standards. This suggests that little more than a guess is involved when making a positive-negative decision in this gray zone. FIG. 1. | Comparison of serum anti-H. pylori Comparison of serum anti-H. pylori antibody (IgG) levels among groups based on grading scores of four histological features of antral mucosa. Scores: 0, normal; 1, mild; 2, moderate; and 3, marked. Each column represents the mean +- standard error of the mean. **, P < 0.01 compared to a score of 0; ++, P < 0.01 compared to a score of 1. FIG. 2. | Comparison of serum anti-H. pylori Comparison of serum anti-H. pylori antibody (IgG) levels among groups based on grading scores of four histological features of body mucosa. Scores: 0, normal; 1, mild; 2, moderate; and 3, marked. Each column represents the mean +- standard error of the mean. **, P < 0.01 compared to a score of 0; ++, P < 0.01 compared to a score of 1. FIG. 3. | Distribution of anti-H. pylori Distribution of anti-H. pylori antibody (IgG) levels in H. pylori-infected and uninfected patients (n = 169). TABLE 1 | Multiple stepwise linear regression analysis of factors correlated with antibody levels PMID- 12204957_DISCUSSION TI - AB - In our present study, there was no difference in antibody levels of the normal mucosa, gastritis, and duodenal ulcers in H. pylori-infected patients. This is in accordance with the study by Sharma et al., who reported that H. pylori serology cannot be used to predict the presence or absence of gastroduodenal ulcer disease . Similarly, Sheu et al. reported no difference in antibody levels between nonulcer and ulcer subgroups , and Hsu et al. reported that levels of anti-H. pylori in serum are not predictive of the severity of macroscopic gastrointestinal damage. The correlation between anti-H. pylori antibody levels and the severity of histological gastritis or H. pylori density has been studied with conflicting results . Significant correlation was reported in studies in which non-H. pylori-infected patients were included in the analysis . On the other hand, no such correlation was found in one report in which only H. pylori-infected patients were analyzed . More than 90% of H. pylori-infected patients have detectable serum IgG antibodies, and the vast majority of H. pylori-infected patients have chronic active gastritis in the antral biopsy specimens. Therefore, the status of H. pylori infection is a crucial confounding factor in analyzing the relationship between antibody levels and histological gastritis. If non-H. pylori-infected patients are included in the analysis, the correlation between antibody levels and gastritis scores or H. pylori density may be misleading due to their common association with the status of H. pylori. Our study also supports these observations. In this study, grades of active and chronic inflammation, atrophy, lymphoid follicles, and H. pylori density were correlated with antibody levels in the antral mucosa but were not correlated if only H. pylori-infected patients were analyzed. Our results had some differences from the reports of Hsu et al. and Sheu et al. . In the study by Hsu et al., antibody levels did not correlate with grades of antral mononuclear cell infiltration, mucosal atrophy, or intestinal metaplasia but did correlate with grades of antral neutrophil infiltration and antral bacterial density in H. pylori-infected patients. The ELISA kit they used was the same as ours. Since anti-H. pylori antibody levels do not show a normal distribution, logarithmic transformation is more appropriate when performing a parametric test. Therefore, the variance may result from the small number of cases in their study (n = 36) and different statistical methods. In the study by Sheu et al., antibody levels correlated with the severity of acute or chronic inflammation. However, the rate of absence of neutrophil infiltration (score, 0) in H. pylori-infected patients in their study was 63%. This is contrary to the well-established concept that the majority of H. pylori-infected patients have chronic active gastritis in the antral biopsy specimens. In addition, the ANOVA model with Duncan's test is not an appropriate statistical method for data with unequal numbers in the groups. In this study, grades of active and chronic inflammation, atrophy, and lymphoid follicles in the body mucosa remained significantly correlated with antibody levels in H. pylori-infected patients. The correlation coefficients, however, were reduced to half of those when non-H. pylori-infected patients were included. This indicates that the status of H. pylori infection is an important contributing factor, which has been demonstrated in the multivariate linear regression model. The data in this study were obtained from dyspeptic patients, so they may not generalize to asymptomatic subjects. There has been a report showing that diffuse antral gastritis with prominent lymphoid follicles has the highest antibody levels . However, in that study, no statistical data were presented, so we calculated the difference between diffuse antral gastritis and chronic atrophic gastritis from the data presented in the table and found no statistical difference (0.274 +- 0.025 versus 0.266 +- 0.013 [mean +- SD]; P = 0.757). In our study, antibody levels of <30 or >70 U/ml had good predictive value for the absence or presence, respectively, of H. pylori infection. For individuals having a value between 30 and 70 U/ml, the so-called gray zone, a different diagnostic method is recommended to assess H. pylori status more accurately. When antibody levels are >70 U/ml, higher levels do not improve the positive predictive value. However, our previous study of the rapid serological test has demonstrated that antibody levels in ELISA are correlated with the density of the reaction band in the rapid serological test . The higher the antibody levels, the more clearly the band is noted. The lower the antibody levels, the more likely that it will be regarded as negative in the rapid serological test due to a very weak color change of the reaction band. Thus, higher antibody levels have a favorable effect on the diagnosis of H. pylori infection in rapid serological tests. There were several studies demonstrating that the sensitivity of serological diagnosis of H. pylori infection was higher in the older group (>=45 years) than in the younger group (<45 years) . Our study showing higher antibody levels in the older group in H. pylori-infected patients could explain this phenomenon, because the higher the antibody levels, the fewer false negatives there will be. In conclusion, levels of IgG anti-H. pylori antibody in the serum do not predict the presence of macroscopic gastroduodenal diseases or the density of H. pylori colonization in H. pylori-infected dyspeptic patients. The presence of H. pylori infection, higher updated Sydney system chronic gastritis scores of the corpus, and older age are associated with higher antibody levels. Setting a gray zone is necessary for ELISA, since the accuracy in this zone does not allow a precise determination of H. pylori status. PMID- 12204962 TI - Clinical and Epidemiological Relevance of Quantitating Hepatitis E Virus-Specific Immunoglobulin M AB - | Diagnosis of acute hepatitis E by detection of hepatitis E virus (HEV)-specific immunoglobulin M (IgM) is an established procedure. We investigated whether quantitation of HEV IgM and its ratio to HEV total Ig furnished more information than conventional IgM tests that are interpreted as positive or negative. A previously described indirect immunoassay for total Ig against a baculovirus-expressed HEV capsid protein was modified to quantitate HEV-specific IgM in Walter Reed (WR) antibody units by using a reference antiserum and the four-parameter logistic model. A receiver-operating characteristics curve derived from 197 true-positive specimens and 449 true-negative specimens identified 30 WR units/ml as an optimum cut point. The median HEV IgM level in 36 patients with acute hepatitis E fell from 3,000 to 100 WR units/ml over 6 months, suggesting that 100 WR units/ml would be a more appropriate cut point for distinguishing recent from remote IgM responses. Among three hepatitis E case series, determination of the HEV IgM-to-total-Ig ratio in acute-phase serum revealed that most patients had high ratios consistent with primary infections whereas a few had low ratios, suggesting that they had sustained reinfections that elicited anamnestic antibody responses. The diagnostic utility of the new IgM test was similar to that of a commercially available test that uses different HEV antigens. In conclusion, we found that HEV IgM can be detected specifically in >95% of acute hepatitis E cases defined by detection of the virus genome in serum and that quantitation of HEV IgM and its ratio to total Ig provides insight into infection timing and prior immunity. PMID- 12204962_Introduction TI - AB - Hepatitis E is acute, self-limited hepatitis caused by a virus of the same name (hepatitis E virus [HEV]) that is excreted in feces and transmitted orally. In large parts of Asia and Africa, this disease is common, causing sporadic and epidemic illness . Diagnosis of acute hepatitis E is based on detection of the HEV genome in serum or feces by reverse transcription-PCR (RT-PCR) or detection of newly elicited antibodies to HEV, in particular HEV-specific immunoglobulin M (IgM) . An IgM test is marketed in Asia ; this test uses recombinant HEV antigens derived from the carboxyl terminus of the capsid protein (ORF-2) and ORF-3. The good diagnostic utility of the marketed test has been characterized . Moreover, several research laboratories have developed IgM tests based on alternative recombinant HEV (rHEV) antigens expressed in bacteria or by use of the baculovirus system . Recently, we reported an indirect enzyme immunoassay (EIA) for total Ig against a baculovirus-expressed HEV capsid protein that quantitated antibodies to HEV in Walter Reed (WR) antibody units by using a reference antiserum and the four-parameter logistic model . We modified this test to detect HEV-specific IgM and employed the IgM and total-Ig tests together to characterize serum specimens from patients with suspected acute hepatitis E. We investigated whether quantitation of HEV IgM and its ratio to HEV total Ig furnished more diagnostic or epidemiological information than conventional IgM tests that are interpreted as positive or negative. Here we report the development of an HEV IgM quantitation standard, the protocol for the IgM test, the kinetics of HEV IgM and total-Ig responses over 6 months in a case series of patients with hepatitis E, an extensive characterization of the test's sensitivity and specificity, the use of the IgM-to-total-Ig ratio to identify rare cases of clinically overt reinfection, and our test's good concordance with the marketed IgM test. We found that quantitation of IgM and total Ig together furnished novel insight into infection timing and prior immunity. PMID- 12204962_MATERIALS AND METHODS TI - AB - RT-PCR. | Serum specimens were tested for the HEV genome, indicating viremia during acute infection, by use of previously published protocols that detect either a conserved region of ORF1 or ORF2 . The previously unpublished HEV ORF2 nested PCR primers, designated set 3, are listed in Table . Reference human antibodies. | Equal aliquots of acute-phase serum from 20 hepatitis E patients from Nepal were pooled; each case was diagnosed by detection of HEV viremia by RT-PCR. Pool 6, created by diluting the acute-phase serum pool with approximately 3 volumes of serum with HEV-specific total-Ig levels of <0.1 WR unit/ml, was designated the HEV IgM quantitation standard. Pool 7, created by diluting pool 6 with approximately 3 more volumes of the same negative serum, was designated the IgM positive control. Relative potency. | The relative potencies of reference antisera and working antigen lots were determined by parallel line assay and calculation of a common slope, as previously described . rHEV antigens. | The antigen for all assays was a 56-kDa recombinant capsid protein truncated at the amino and carboxyl ends to comprise amino acids 112 to 607 of the 660-amino-acid protein. The protein, made in Spodoptera frugiperda cells by using a baculovirus expression vector, was prepared by Novavax as previously described . All tests used 33 WR antigen units/ml; antigens were from one of the lots previously characterized . EIA protocols. | The IgM assay protocol was identical to the total-Ig protocol except that the goat anti-human Ig-horseradish peroxidase (HRP) conjugate was replaced with goat anti-human IgM-HRP (Kirkegaard and Perry). The optimal 1:4,000 dilution of anti-IgM conjugate was determined by testing twofold dilutions to find the highest signal-to-noise ratio. Serum specimens. | Serum specimens stripped of personal identifiers were from archives at the Walter Reed Army Institute of Research (WRAIR, Silver Spring, Md.) and the Armed Forces Research Institute of Medical Sciences (Bangkok, Thailand). All were from volunteers enrolled in research protocols approved by local institutional review boards and the Human Subjects Research Review Board of the U.S. Army Surgeon General. The majority of hepatitis E serum specimens came from three consecutive case series in Nepal: pregnant women enrolled in an observational cohort study, a cross-sectional study of intrafamilial HEV transmission that identified hepatitis E patients and subclinical infections in persons domiciled with them, and surveillance of hepatitis E among soldiers. IgM EIA control parameters. | Control parameters were developed to ensure accuracy and consistency. Eighteen wells on each 96-well plate were utilized for the following duplicate controls: six half-log dilutions of the HEV IgM quantitation standard (pool 6), the positive control (pool 7), the same negative control as for the total-Ig test, and a no-serum control. Thirty consecutive technically adequate runs were used to calculate limits for control parameters as the mean +- 1.96 (standard deviation) of log-transformed values (expressed as optical density [OD] or WR units per milliliter, as appropriate). Thereafter, assays were accepted according to these limits. Quantitation. | In using the four-parameter logistic model for quantitation, accuracy is greatest at the midpoint of the standard curve and least at the lower and upper limits. We used the procedures for quantitation developed for the total-Ig test (testing in duplicate, OD limits prompting sample dilution and retesting) to ensure consistency . Comparison of antibody potency by WRAIR and Genelabs Technologies IgM EIAs. | To characterize the relationship between antibody potency determined by the WRAIR IgM EIA and that determined by a widely used commercial test (HEV IgM enzyme-linked immunosorbent assay; Genelabs Technologies, Singapore), we tested dilutions of three specimens in both tests. The commercial test employs a mixture of recombinant HEV ORF2 and ORF3 polypeptides expressed in Escherichia coli which are absent from the WRAIR test's 56-kDa rHEV capsid antigen and the ORF2 polypeptide SG3 (amino acids 334 to 660), also expressed in E. coli, which overlaps 53% of the 56-kDa rHEV capsid antigen. The commercial-test results were expressed in OD units, whereas WRAIR EIA results were expressed in WR units per milliliter. Three regression lines were derived, and the portions of each titration curve above each assay's cut point were compared. Additionally, serial serum specimens from six patients with hepatitis E confirmed by detection of HEV viremia were tested in both assays, and their results were compared. TABLE 1 | HEV ORF2 "set 3" nested PCR primers PMID- 12204962_RESULTS TI - AB - Potency of reference antibodies. | As previously described , we quantified HEV-specific total Ig by using arbitrary WR units per milliliter defined by a convalescent-phase reference serum (pool 4). We chose to quantify HEV IgM also by using WR units per milliliter, in this case by an IgM quantitation standard called pool 6, arbitrarily defined to contain 860 WR units of HEV IgM/ml. The relationship between a unit of HEV IgM and a unit of HEV total Ig is undefined, but these units are roughly equivalent, as pool 6, defined to contain 860 WR units of HEV IgM/ml, was determined to contain 820 WR units of HEV total Ig/ml by parallel line assay against pool 4. Dilutions of pool 6 were used on each assay plate to establish an IgM standard curve. Pool 7 also was used on every assay plate as an IgM positive control. EIA control parameters and assay stability. | To ensure EIA accuracy and consistency, we empirically set control parameters and used individual plate standards and controls . These control parameters were derived from 30 consecutive technically adequate assays. The limited variation of these parameters over those 30 assays is evidence that a skilled serologist can achieve consistent assay performance . Kinetics of IgM and total-Ig responses in patients with hepatitis E. | Consecutive patients with suspected acute viral hepatitis hospitalized at one center in Nepal were evaluated for hepatitis E by an RT-PCR test for viremia. Thirty-six cases of hepatitis E were identified, and HEV IgM and total-Ig levels were determined for four serial serum specimens from each of these patients collected over approximately 6 months . The initial geometric mean IgM level was 3,000 WR units/ml at a median of 8 days after illness onset; the level declined slightly over the next 2 weeks and then exponentially over the next 5 months, nevertheless remaining easily detectable at 100 WR units/ml at a median of 190 days after illness onset. Geometric mean total Ig against HEV was greater than IgM at every time point analyzed, as was expected, but the decline in total Ig mirrored that in IgM. IgM test sensitivity, specificity, and cut point. | After determining the range of IgM levels likely to occur among patients, we analyzed the performance of the IgM test in order to identify a cut point. Specificity was assessed with serum specimens from 449 persons at low risk for acute HEV infection ; these were considered true-negative specimens. Approximately two-thirds were from healthy persons of all ages, including some residing in Nepal, a country where hepatitis E is endemic. Specimens from Nepal residents were collected several months before the annual epidemic, when the monthly infection rate is <2 per 10,000 (M. P. Shrestha and R. M. Scott, unpublished data). Other healthy donors were from the United States or Thailand, where hepatitis E is not endemic. Additionally, about one-third of the true-negative specimens were from patients in Thailand recently infected with bacteria or viruses other than HEV that cause hepatitis. The distribution of HEV IgM levels detected in this negative specimen set is shown in the upper histogram of Fig. . Most values were between 1 and 16 WR units/ml. Sensitivity was assessed with serum specimens from 197 persons with acute hepatitis E infection diagnosed by detection of HEV viremia. Of these, 94% had clinically overt hepatitis E, while 6% had had subclinical infections. These patients were predominantly from Nepal; a few were from Indonesia or Bangladesh; all had their specimens submitted to our laboratories for serologic diagnosis. The distribution of HEV IgM levels detected in this positive specimen set is shown in the lower histogram of Fig. . Most values were between 200 and 20,000 WR units/ml. A receiver-operating characteristics curve was constructed from the test results described above by using cut points of 10, 20, 30, 40, 60, and 100 WR units/ml . A cut point of 30 WR units/ml combines maximal sensitivity and specificity. Nevertheless, because elevated HEV IgM levels appear to persist for 6 months after illness onset, a cut point of 100 WR units/ml seems more appropriate for distinguishing recent from remote infection. Among the 36 cases for which data are summarized in Fig. , the proportion with HEV IgM levels above the cut point of 100 WR units/ml fell from 92% within 3 weeks of illness onset to 83% at 8 weeks after onset and 53% at 6 months after illness onset. Ratio of HEV IgM to total Ig. | We determined the ratio of HEV IgM to total Ig for all 197 serum specimens in the true-positive specimen set. The histogram of those results shows that most values were between 0.1 and 10 and were distributed symmetrically around a median value of 1.0. Nevertheless, <5% of specimens (n = 8) had distinctly low ratios, such that these specimens appeared to constitute a separate population. Serology data for acute-phase and early-convalescent-phase serum specimens from cases representing the 25th, 50th, and 75th percentiles were compared to data from the eight outlying cases with markedly low HEV IgM-to-total-Ig ratios . All patients had HEV viremia demonstrated in the first specimens. Of the eight outlying cases, seven represented patients with clinically overt hepatitis E whereas one case was an inapparent infection detected in a family member of a patient with hepatitis E. The timing of the first specimen collection after illness onset for these eight cases was similar to, or even earlier than, that for the 25th-, 50th-, and 75th-percentile cases, excluding late collection as an explanation for low IgM levels. Nevertheless, there are striking contrasts: among the eight cases with low IgM-to-Ig ratios, the levels of IgM are low and the levels of total Ig are extremely high. These findings suggest that typical cases of hepatitis E represent primary infections with a typical evolution of Ig isotypes from IgM to IgG, whereas a minority of cases represent reinfection (secondary infections), with a typical anamnestic Ig response characterized by low levels of IgM and extremely high levels of IgG. Comparison of WRAIR and Genelabs Technologies IgM EIAs. | We found a consistent and highly correlated relationship between antibody binding assessment by the WRAIR IgM EIA and that by a commercial test widely available in Asia, for pool 6 and for acute-phase serum specimens from two typical hepatitis E cases in Nepal . Nevertheless, a proportion of each titration curve was to the right of the WRAIR cut point (30 WR units/ml) (i.e., positive) but below the Genelabs cut point (i.e., negative). The greater sensitivity of the WRAIR test for low levels of antibody may confer some advantage on that test. The relative utility of the two tests was compared directly in a sample of 19 specimens collected at varying intervals after disease onset (median of 40 days of follow-up after disease onset) from six hepatitis E patients diagnosed by detection of viremia. All specimens were positive in both tests. A line plot of results for each case, determined by the WRAIR test (Fig. , upper graph) and the Genelabs test (Fig. , lower graph), shows that the kinetic responses were similar in four cases and different in two (cases 7 and 10). Overall, the performance of the two tests appeared similar. FIG. 1. | Plots of the HRP conjugate and of positive (pool 7), negative, and no-serum (antigen) controls over 30 technically adequate assays. Plots of the HRP conjugate and of positive (pool 7), negative, and no-serum (antigen) controls over 30 technically adequate assays. FIG. 2. | Levels of HEV IgM (solid line) and total Ig (dashed line) among 36 patients with hepatitis E proven by detection of HEV viremia. Levels of HEV IgM (solid line) and total Ig (dashed line) among 36 patients with hepatitis E proven by detection of HEV viremia. Each patient had four consecutive serum specimens collected. Data are grouped by median day of specimen collection; these values plus the associated interquartile ranges in parentheses are given below the plot of HEV IgM. Error bars represent the 95% confidence interval for the geometric mean. Horizontal dotted reference lines are drawn at 30 and 100 WR units/ml. The table below the line plot gives the proportions of patients with HEV IgM detected at four median time points for assay cut points of 100 and 30 WR units/ml. FIG. 3. | (Top) Histogram of HEV IgM levels determined in a true-negative specimen set (n = 449). (Top) Histogram of HEV IgM levels determined in a true-negative specimen set (n = 449). The interval scale is logarithmic; the mode is 6 WR units/ml. Vertical dotted reference lines are drawn at 30 and 100 WR units/ml. (Bottom) Histogram of HEV IgM levels determined in a true-positive specimen set (n = 197). The interval scale is logarithmic; the mode is 2,512 WR units/ml. Vertical dotted reference lines are drawn at 30 and 100 WR units/ml. FIG. 4. | Receiver-operating characteristic plot for IgM EIA based on true-negative and true-positive specimen sets. Receiver-operating characteristic plot for IgM EIA based on true-negative and true-positive specimen sets. FIG. 5. | Histogram of HEV IgM-to-total-Ig ratios for the true-positive specimen set (n = 197). Histogram of HEV IgM-to-total-Ig ratios for the true-positive specimen set (n = 197). The interval scale is logarithmic; the mode is 1.6. Vertical reference lines mark cumulative distribution percentiles, as follows: dotted lines, 5 and 95%; solid lines, 25 and 75%; dashed-and-dotted line, 50%. Cases to the right of the 5% reference line (n = 189) appear to represent primary infections, whereas cases to the left of the 5% reference line (n = 8) appear to represent secondary infections with anamnestic antibody responses. FIG. 6. | Comparison of IgM quantitation by WRAIR and Genelabs Technologies EIAs. Comparison of IgM quantitation by WRAIR and Genelabs Technologies EIAs. Titration of three acute-phase serum specimens from hepatitis E patients is represented. Horizontal dotted reference line, cut point for the Genelabs test; vertical dotted reference line, cut point (30 WR units/ml) for the WRAIR test. FIG. 7. | Serial determinations of HEV IgM levels for six patients with acute hepatitis E confirmed by RT-PCR detection of HEV viremia in the initial specimen. Serial determinations of HEV IgM levels for six patients with acute hepatitis E confirmed by RT-PCR detection of HEV viremia in the initial specimen. The upper graph shows results obtained by using the WRAIR IgM test; the bottom graph shows results obtained by using the Genelabs Diagnostics test. All 19 specimens were positive in each test. The cut point for each test is shown as a dashed line. TABLE 2 | IgM EIA control parameters defined by their variation over 30 consecutive assays TABLE 3 | Composition of the true-negative specimen set (n = 449) TABLE 4 | Serology data for all secondary cases and representative primary cases of hepatitis E in a sample of 197 patients from areas where hepatitis E is endemic PMID- 12204962_DISCUSSION TI - AB - We have adapted our previously reported indirect EIA, which is able to quantitate HEV total Ig accurately and reproducibly, to quantitate HEV IgM. This is the third report describing an IgM EIA that uses a recombinant HEV capsid protein expressed in the baculovirus system, but the first HEV IgM test to offer quantitation or to be so exhaustively characterized. The performance of the IgM test was extensively evaluated, and assay specificity, sensitivity, and consistency were carefully documented. We used the IgM and previously described total-Ig tests to determine HEV IgM levels, total-Ig levels, and their ratio in acute-phase serum specimens from almost 200 cases of hepatitis E occurring in countries where hepatitis E is endemic. This dual testing allowed us to make the novel observation that a small proportion of disease is characterized by an anamnestic antibody response, suggesting that these cases result from secondary infection of a person who had previously recovered from a primary infection. We chose an indirect EIA format for detecting and quantitating HEV IgM despite its inferior specificity compared to an IgM isotype-capture EIA. We rejected developing an IgM isotype-capture EIA after initial experiments demonstrated poor sensitivity despite use of substantially greater amounts of the rHEV antigen. We inferred from this observation that the rHEV antigen, in the physical form used in this assay (thawed from -70C storage one to three times before use) and at concentrations that were economically feasible, was inefficient at bridging layers of specific Ig. This behavior of our rHEV antigen limited the assay format to an indirect test. One of the weaknesses of an indirect test is that IgM-rheumatoid factor in a test serum, which has activity against the Fc portion of IgG directed against HEV antigen, may elicit a false-positive result . We substantially reduced the risk of such nonspecific reactions by testing serum initially diluted 1:1,000. The other potential weakness of an indirect EIA format is reduced sensitivity due to competition between virus-specific IgM and IgG for antigen binding sites. The test sensitivity of 92 to 97%, depending on the cut point, suggests that IgG competition is not a limitation of this particular assay. A necessary step in developing the quantitative IgM EIA was the creation of reference antibody pools of HEV IgM by using human serum from Nepal, where hepatitis E is endemic. By trial and error, we set the potency of the IgM quantitation standard so that a WR unit of IgM and a WR unit of total Ig (M plus G plus A isotypes) were comparable. There is no international HEV IgM reference standard, as the available World Health Organization HEV antibody standard contains only low levels of HEV-specific IgM (105.6 WR units/ml). We can provide samples of our IgM and total-Ig reference standards (available from the Department of Virus Diseases, WRAIR, upon request) to interested laboratories who wish to prepare their own in-house standards. Of the several approaches for EIA quantitation of an unknown by using a standard curve, we chose the four-parameter logistic model, which is generally considered the most accurate and reproducible . We retained all procedures from our total-Ig test, previously shown to be reproducibly accurate to below the cut point (7% median error in quantitation of the mid-range standard). To achieve accurate and reproducible quantitation, an operator must perform this test with great care, using well-controlled reagents. This is more likely to be possible when the test is performed routinely, as might be expected in a research serology laboratory or a regional or national public health laboratory. We had access to serial serum specimens collected over 6 months from 36 hepatitis E patients. These specimens demonstrated that IgM antibody levels were very high soon after illness onset, declined little over several weeks, and then declined rapidly to low levels over the next 4 to 6 months. This is typical of IgM responses to other acute, self-limited, systemic viral infections . The weeks-long duration of markedly elevated IgM levels after disease onset means that diagnosis using even relatively insensitive IgM detection methods should be successful, even if patients come to medical attention late. Moreover, the months-long duration of IgM responses to HEV may be a boon to hepatitis E outbreak investigations, since these typically commence months after the index case occurs. A sensitive IgM test should be able to identify most disease cases from late-convalescent-phase serum specimens; the WRAIR test meets this criterion by detecting HEV IgM above a cut point of 30 WR units/ml in 92% of specimens collected a median of 2 months after disease onset and in 83% of specimens collected 6 months after disease onset. Additionally, since many HEV infections are known to be subclinical , sensitive tests for IgM may enable identification of all infected persons rather than those with disease only. The preceding paragraph illustrates some of the ways in which an HEV IgM test might be used. For different uses, different assay cut points may be appropriate. The receiver-operating characteristics curve identified 30 WR units/ml as the cut point suitable for outbreak investigations, in which it is necessary to find remotely infected persons, whereas it identified 100 WR units/ml as a marginally less sensitive cut point that could distinguish a recent infection from a remote one. We began this study anticipating that an HEV IgM test would improve serological diagnosis of hepatitis E, then based on detection of HEV-specific total Ig or IgG. We confirmed that detection of HEV IgM is the best serological test for diagnosis of hepatitis E. Yet the most interesting aspect of our work was the observation that in some cases of hepatitis E, there was a weak or absent IgM response. By combining HEV IgM and total-Ig tests, we identified primary and anamnestic HEV immune responses among adolescent and adult hepatitis E patients in areas of HEV endemicity, distinguished by widely divergent HEV IgM-to-total-Ig ratios. We inferred that anamnestic responses resulted from reinfection (secondary infection) of a person having an immunologic memory of prior HEV infection. In our experience, secondary infections are uncommon, comprising <5% of overt hepatitis E cases. This is an important observation because previously, some authorities have speculated that waning immunity explained why most cases of hepatitis E occurred among adults. The fact that more than 90% of hepatitis E cases in areas of HEV endemicity occur in patients who have a primary antibody response refutes this speculation of waning immunity, since previously exposed persons should mount an anamnestic response upon reexposure. On the other hand, we do speculate that asymptomatic secondary infections may be more common than symptomatic ones. Ultimately, the true prevalence of secondary infection and disease must be assessed prospectively. Such studies also may identify the risk factors that are associated with partial failure of immunity and determine whether clinical outcomes differ between primary and secondary disease. If secondary disease is associated with high-dose HEV exposure or waning immunity, hepatitis E vaccines now under development may have public health utility even among previously exposed persons, if they can be demonstrated to boost protective immunity. Finally, we wanted to evaluate the WRAIR IgM test against the test marketed widely in Asia by Genelabs Diagnostics. The comparison was initiated by testing in parallel serial dilutions of three acute-phase specimens using the WRAIR and Genelabs Diagnostics tests. We found that the tests performed similarly across a range of dilutions, suggesting that these tests were comparable for serological diagnosis of acute disease. On the other hand, the results suggested that the WRAIR test would be more versatile in outbreak investigations based on its apparent superiority at detecting low levels of IgM reflecting remote infection. The comparison was completed by testing a separate panel of 19 specimens from 6 hepatitis E patients with viremia by both tests. In this limited assessment, both tests sensitively detected true acute HEV disease over a median of 40 days of follow-up. This result is consistent with an earlier report by Ghabrah and others , who found 96% concordance (kappa, 0.87) between an IgM test using an antigen similar to that used in the WRAIR test and the Genelabs IgM test. In conclusion, the test method described here enables accurate quantitation of HEV IgM, including low levels associated with remote infection. Detection of HEV IgM is the method of choice for laboratory diagnosis of hepatitis E. By combining quantitation of HEV IgM and total-Ig levels, a new class of secondary infections can be detected. The epidemiological implications of secondary infections are clear, but their clinical implications must be assessed. PMID- 12204960 TI - Use of Egg Yolk-Derived Immunoglobulin as an Alternative to Antibiotic Treatment for Control of Helicobacter pylori Infection AB - | The present study evaluated the potential use of immunoglobulin prepared from the egg yolk of hens immunized with Helicobacter pylori (immunoglobulin Y [IgY]-Hp) in the treatment of H. pylori infections. The purity of our purified IgY-Hp was 91.3%, with a yield of 9.4 mg of IgY per ml of egg yolk. The titer for IgY-Hp was 16 times higher than that for IgY in egg yolk from nonimmunized hens, and IgY-Hp significantly inhibited the growth and urease activity of H. pylori in vitro. Bacterial adhesion on AGS cells was definitely reduced by preincubation of both H. pylori (108 CFU/ml) and 10 mg of IgY-Hp/ml. In Mongolian gerbil models, IgY-Hp decreased H. pylori-induced gastric mucosal injury as determined by the degree of lymphocyte and neutrophil infiltration. Therefore, in this experimental model, H. pylori-associated gastritis could be successfully treated by orally administered IgY-Hp. The immunological activity of IgY-Hp stayed active at 60C for 10 min, suggesting that pasteurization can be applied to sterilize the product. Fortification of food products with this immunoglobulin would significantly decrease the H. pylori infection. In conclusion, the IgY-Hp obtained from hens immunized by H. pylori could provide a novel alternative approach to treatment of H. pylori infection. PMID- 12204960_Introduction TI - AB - Helicobacter pylori is the most common cause of gastritis and gastric ulcers and plays a pivotal role in the development of gastric carcinomas . Often a significant portion of those infected do not show symptoms, although chronic infection increases the risk of the development of H. pylori-associated gastric disease. There have been tremendous efforts to evaluate numerous therapeutic regimens for eradication of H. pylori infections. Successful treatment of H. pylori infections most often employs antibiotic therapy, consisting of some combination of metronidazole, amoxicillin, clarithromycin, and either bismuth or a proton pump inhibitor . However, antibiotic therapy fails in 10 to 15% of cases due to the development of antibiotic resistance . Increasing occurrence of antibiotic resistance would further complicate the treatment of H. pylori infections. Consequently, it is important to seek new therapies for a wider means of treating, suppressing, or preventing H. pylori infection without drug resistance problems. The concept of protecting a host with passively derived antibodies is not new. It has been shown that oral administration of antibacterial or antiviral immunoglobulins, through infant formulae or other diet, is effective in preventing intestinal infection . However, oral administration of antibodies is prohibitively expensive when large amounts of antibodies are required . Recently, chicken egg yolk was recognized as an inexpensive, alternative antibody source, and the usefulness of egg yolk immunoglobulin Y (IgY) has been assessed for therapeutic application by passive immunization therapy through oral ingestion of IgY, as in fortified food products for prevention or control of intestinal infections, such as those caused by enterotoxigenic Escherichia coli , Salmonella enterica serovar Typhimurium , and rotavirus . These studies, taken together, provide the potential advantage of using IgY with specificity to H. pylori (IgY-Hp) for controlling H. pylori-associated gastric disease and subsequently prevent disease resulting from chronic infection. Nevertheless, there has been no report so far on the use of IgY in the prevention and treatment of H. pylori infections. Furthermore, for the practical application of IgY-Hp, together with food or pharmaceutical materials to prevent H. pylori-associated disease, the stability of IgY toward heat, acid, and pepsin should be ensured. The aim of this study was to evaluate the potential use of IgY-Hp in the prevention and treatment of H. pylori infections. To achieve this objective, we studied, in vitro and in vivo, the activity of IgY-Hp against H. pylori. The effect of IgY-Hp on gastric mucosal injury induced by H. pylori was evaluated in vivo using Mongolian gerbils. The stability of IgY-Hp was also investigated. PMID- 12204960_MATERIALS AND METHODS TI - AB - H. pylori preparation and immunization. | H. pylori (ATCC 43504) was cultured in brucella broth (Difco Laboratories, Detroit, Mich.), supplemented with 5% (vol/vol) bovine calf serum (PAA Laboratories Inc., Parker Ford, Pa.) and antibiotics (amphotericin B, 2.5 mug/ml; vancomycin, 10 mug/ml; trimethoprim, 5 mug/ml; and polymyxin B, 2.5 IU/ml; all were from Sigma Chemical Co. [St. Louis, Mo.]) at 37C under 10% CO2 at 200 rpm. The H. pylori was harvested by centrifugation at 12,000 x g for 10 min and disrupted by sonication. Cellular material was removed by centrifugation, and the supernatant was collected (H. pylori whole-cell lysate). The protein concentrations were determined by bicinchoninic acid methods (Pierce, Rockford, Ill.). Brown Leghorn hens (25 weeks old; n = 15) were immunized intramuscularly with H. pylori whole-cell lysate (200 mug/ml, protein) using an equal volume of Freund's complete adjuvant (Difco Laboratories). Each hen was injected at four different sites (250 mul per site) of the leg muscle. Three booster injections, with Freund's incomplete adjuvant, were given at 2-week intervals following the first injection. One month after immunization, the eggs laid were collected daily for 1 month and stored at 4C. The egg yolk was separated, pooled, and frozen prior to purification of IgY. Isolation and purification of IgY-Hp. | Isolation of IgY-Hp was carried out by the method described by Akita and Nakai with modification. Egg yolk was separated from the white, and the yolk preparation was mixed with an equal volume of distilled water for 30 min, followed by the addition of 0.15% (wt/vol) lambda-carrageenan (Wako Pure Chemical Laboratory, Osaka, Japan). After centrifugation at 10,000 x g at 20C for 30 min, the water-soluble fraction (WSF) was collected and filtered through a Whatman filter paper (no. 1) to remove solid lipid materials. The resulting IgY-containing filtrate was further purified by salt precipitation (19% [wt/vol] sodium sulfate) and ultrafiltration (UF) using a UF membrane (Millipore Corp., Bedford, Mass.) cartridge with a molecular weight cutoff of 100 kDa. Purity and yield of IgY were monitored at various stages by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The IgY content was measured by its absorbance at 280 nm. SDS-PAGE. | According to the method of Laemmli , 10% PAGE was done with a Mini-PROTEAN II Cell (Bio-Rad Laboratories, Hercules, Calif.). Under nonreducing conditions, samples were diluted 1:4 with sample buffer (62.6 mM Tris-HCl, pH 6.8, 25% [vol/vol] glycerol, and 2% SDS [vol/vol]). Under reducing conditions, samples were diluted with sample buffer containing 5% (vol/vol) beta-mercaptoethanol and heated for 5 min at 100C. Fifteen microliters of the samples was loaded into each well (3 mug of protein per well). Prestained SDS-PAGE standards (Bio-Rad Laboratories) and standard chicken IgY (Promega, Madison, Wis.) were used as molecular weight markers. ELISA. | To assess the antibody activity of IgY-Hp to H. pylori, we performed the enzyme-linked immunosorbent assay (ELISA) by Akita et al. with modification. Ninety-six-well plates were coated with H. pylori whole-cell lysate (500 ng/well). After blocking with 1% (wt/vol) bovine serum albumin, 100 mul of IgY-Hp (1 mg/ml) was added using a twofold serial dilution. The plates were then washed with phosphate-buffered saline (PBS)-Tween (0.05% Tween 20 in PBS [pH 7.2]) and incubated for 1 h after the addition of alkaline phosphatase-conjugated goat anti-chicken IgY (Promega). The plates were washed with PBS-Tween, and disodium p-nitrophenyl phosphate (Sigma) was added as substrate to each well. After incubation for 10 min, the reaction was stopped by addition of 3 M NaOH. The absorbance was measured at 405 nm using a microplate reader (Multiskan MS; Thermo Labsystems, Helsinki, Finland). Heat, acid, and pepsin stability of IgY-Hp. | IgY-Hp solutions were incubated at 0, 4, 10, 25, 35, 60, 70, 80, and 90C for 10 min. The heat-treated IgY-Hp was cooled in an ice bath. For the pH stability test, the pHs of IgY-Hp solutions were adjusted to the desired pH (pH 2 to 8) with NaOH or HCl, the solutions were incubated at 37C for 4 h, and then each IgY-Hp solution was neutralized. For the pepsin stability tests, the pHs of the IgY-Hp solutions were adjusted to pH 2, pH 4, and pH 6, respectively. IgY-Hp solution of each pH were incubated with 15 mug of pepsin (Sigma)/ml at 37C for 0.5, 1, 2, and 4 h. After incubation, each IgY-Hp solution was neutralized to inactivate the pepsin. The remaining antibody activity was measured by ELISA, following the heat, pH, and pepsin treatments. Antibody activity was represented as a percentage of control. Colony counting. | H. pylori (108 CFU/ml) was incubated with IgY-Hp (1 and 10 mg/ml) for 6 h at 37oC under 10% CO2 at 50 rpm. After incubation, H. pylori was diluted with brucella broth via a 10-fold series dilution. Each 100 mul was inoculated onto brucella agar containing 5% (vol/vol) bovine calf serum, and the plate contents were incubated at 37C under 10% CO2 for 10 days. The colonies were identified as H. pylori by Gram staining and urease, oxidase, and catalase activities. The growth rate (percentage of control) was calculated by colony counting compared to results for the control. Urease activities. | Both IgY-Hp (1 and 10 mg/ml) and H. pylori (108 CFU/ml) were incubated at 37C under 10% CO2 for 6 h at 50 rpm, and then 50 mul of urea base (2% urea and 0.03% phenol red) was added and allowed to react for 30 min. Urease activity was quantified by measuring the optical density at 550 nm, using a modification to the method of Fauchere and Blaser , and was represented as % of control. H. pylori adhesion on AGS cells. | The human gastric carcinoma cell line AGS was obtained from the Korean Cell Line Bank (Seoul, Korea). Cells (105 cells/ml) were cultured in RPMI 1640 medium (Gibco BRL, Grand Island, N.Y.), supplemented with 10% (vol/vol) fetal bovine serum (HyClone Laboratories, Logan, Utah) and antibiotics (100 U of penicillin/ml and 100 mug of streptomycin/ml; HyClone) at 37C under 5% CO2 for 24 h. AGS cells were checked for their H. pylori colonization level in the presence or absence of IgY-Hp using scanning electron microscopy (SEM). SEM. | For electron microscopy investigation of bacterial adhesion, 300 mul of preincubated IgY-Hp (10 mg/ml) and H. pylori (108 CFU/ml) was added to the AGS cells on the chamber slide (Nalge Nunc International, Naperville, Ill.). After incubation for 4 h, the chamber slide was washed with PBS and fixed with 2.5% (vol/vol) glutaraldehyde for 24 h. After washing with PBS, the material was postfixed with 1% OSO4 for 60 min and then washed twice with PBS. Fixed material was dehydrated through a graded ethanol series from 50 to 95%, followed by two washes with absolute ethanol for 15 min. Dehydrated material, in absolute ethanol, was critical point dried in a critical point drier (HCP-2; Hitachi, Tokyo, Japan). Critical point dried material was mounted on an aluminum stub and coated with gold. Adherence of H. pylori to AGS cells was observed by SEM (S2500; Hitachi). Passive immunization with IgY-Hp against H. pylori infection in Mongolian gerbils. | Mongolian gerbils (6-week-old males) were purchased from Bio Animal 21 (Sung-Nam, Korea). The gerbils were infected with H. pylori (0.5 ml, 108 CFU/ml, orally) three times at 12-h intervals. Two weeks after inoculation, immune response to infection was monitored by ELISA. H. pylori-infected gerbils were randomly divided into three groups as follows: (i) H. pylori infection only (n = 10), (ii) treatment with 1 mg of IgY-Hp treatment/ml (n = 10), and (iii) treatment with 10 mg of IgY-Hp/ml (n = 10). Negative control groups were administered IgY-Hp (1 and 10 mg/ml) alone (n = 10). IgY-Hp (1 ml) was administered daily, using an oral feeding needle, for 4 weeks. After the gerbils were fasted for 24 h, the antral portion of stomach was quickly removed and used for histological examinations. Formalin-fixed tissue was processed routinely in paraffin and stained with hematoxylin and eosin for examination by light microscopy. Gastric mucosal injury was classified and scored on a scale of 0, 1, 2, or 3, according to the updated Sydney system . Statistical analysis. | All data were expressed as the means +- standard deviations (SD). The statistical significance was evaluated by Student's t tests. P values of <0.05 were considered statistically significant. PMID- 12204960_RESULTS TI - AB - Purification and characterization of IgY-Hp from egg yolk. | Isolation of egg yolk IgY, using the lambda-carrageenan method followed by a UF system, was effective. The purity of the IgY obtained was 91.3%, with a yield of 9.4 mg of IgY per ml of egg yolk . The egg yolk proteins obtained during purification of IgY were analyzed using SDS-PAGE. As shown in Fig. , the IgY finally purified, using the UF system, was pure and dissociated into heavy and light chains of 64 and 25 kDa, respectively. The electrophoretic patterns were in perfect accordance with commercially available IgY (Promega) (Fig. , lane 2). After treating the egg yolk with lambda-carrageenan, the number of proteins in the WSF were significantly decreased. These contaminating proteins were removed by salting out with 19% sodium sulfate. The IgY preparation was concentrated and desalted using the UF system. The IgY and IgY-Hp obtained from hens without and with H. pylori immunization, respectively, were examined for immunological properties by ELISA. The ELISA titers were 640 and 10,240 for IgY and IgY-Hp, respectively. These results indicate that IgY-Hp is highly specific to H. pylori . The heat, pH, and pepsin stabilities of IgY-Hp were evaluated by ELISA . The IgY-Hp was stable at 40C. However, at 60C for 10 min, IgY-Hp lost approximately 20% of its antibody activity. The antibody activity of IgY-Hp significantly decreased at 80C and lost up to 90% of its activity. IgY-Hp showed broad stability between the pHs 4 and 8. However, IgY-Hp lost 80 and 70% of its initial activity at pH 2 and 3, respectively. The antibody activity of IgY-Hp, as determined by ELISA, was almost lost when incubated with pepsin at pH 2, while about 92 and 99% of the activity remained at pH 4 and 6, respectively. Effects of IgY-Hp on growth and urease activity of H. pylori in vitro. | To determine the effect of IgY-Hp on the growth and urease activity of H. pylori, we compared growth rates and urease activity with those of the control. The growth rates for H. pylori were 60.0% +- 9.5% and 10.0% +- 10.8% compared with those found for the control, after incubation with 1 mg and 10 mg of IgY-Hp/ml, respectively . When H. pylori (108 CFU/ml) was incubated with IgY-Hp (1 and 10 mg/ml) for 6 h, the urease activity of H. pylori was 75.4% +- 8.5% and 15.5% +- 7.4% compared with that of the control . Effect of IgY-Hp on H. pylori-induced gastritis in the Mongolian gerbils model. | Mongolian gerbils were inoculated with H. pylori. Two weeks after the inoculation, the status of infection was confirmed by measuring the serum antibody (IgG) against H. pylori. The H. pylori-infected gerbils were determined by high IgG titer (data not shown). Oral administration of 10 mg of IgY-Hp/ml for 4 weeks to the H. pylori-infected gerbils improved lymphocyte infiltration compared to that found for the H. pylori infection group with no IgY-Hp (1.2 +- 0.8 versus 2.5 +- 0.5; P < 0.01) . However, the group that was administered 1 mg of IgY-Hp/ml showed no statistically significant difference with the H. pylori infection group (2.4 +- 1.1 versus 2.5 +- 0.5) . The group treated with 10 mg of IgY/ml significantly improved neutrophil infiltration (0.9 +- 0.7 versus 2.3 +- 0.8; P < 0.01) . There was no statistically significant difference between the group treated with 1 mg of IgY-Hp/ml and the H. pylori infection group (2.2 +- 0.5 versus 2.3 +- 0.8) . A high dose of IgY-Hp treatment decreased H. pylori-induced lymphocyte and neutrophil infiltration in gastric mucosa. However, a low dose of IgY failed to protect gerbils from H. pylori-induced gastric mucosal injury. Effect of IgY-Hp on H. pylori attachment on AGS cells. | Figure shows SEM of AGS cells and those infected with H. pylori (108 CFU/ml) in the presence and absence of IgY-Hp (10 mg/ml). Numerous rod-shaped H. pylori organisms, attached to the AGS cells' surfaces, were observed when cells were cultured with H. pylori . On the other hand, the number of adhering H. pylori organisms dramatically decreased when cells were pretreated with IgY-Hp . FIG. 1. | SDS-PAGE patterns of IgY purified from egg yolk. SDS-PAGE patterns of IgY purified from egg yolk. (A) Coomassie-stained SDS-10% PAGE under nonreducing conditions. (B) Reducing conditions of SDS-10% PAGE. Lanes: 1, molecular size marker, 2, chicken IgY, standard immunoglobulin (Promega), 3, egg yolk, 4, WSF after lambda-carrageenan treatment, 5, IgY obtained after salting out, and 6, final IgY obtained by UF. FIG. 2. | The reaction between IgY-Hp and H. pylori The reaction between IgY-Hp and H. pylori. One hundred microliters of IgY-Hp (1 mg/ml) was added with a twofold serial dilution in 96-well plates coated with H. pylori whole-cell lysate (500 ng/well), and the titers were measured using ELISA. IgY was isolated from the egg yolk of nonimmunized hens, and IgY-Hp was obtained from H. pylori-immunized hens. FIG. 3. | Heat, pH, and pepsin stability of IgY-Hp. Heat, pH, and pepsin stability of IgY-Hp. IgY-Hp was treated at various temperatures for 10 min (A), at various pHs for 4 h (B), and with pepsin (15 mul/ml) (C) at pH 2, 4, and 6 for 0.5, 1, 2, and 4 h. Remaining activities after the treatments were measured using ELISA and are expressed as a percentage of the initial activity. FIG. 4. | Effect of IgY-Hp on the growth and urease activity of H. pylori. H. pylori Effect of IgY-Hp on the growth and urease activity of H. pylori. H. pylori (108 CFU/ml) was incubated with of IgY-Hp (1 and 10 mg/ml) for 6 h at 37C under 10% CO2 at 50 rpm. After incubation, bacteria were inoculated on agar plates. The growth rate (percentage of control) was calculated by colony counting compared to the control. Both IgY-Hp (1 and 10 mg/ml) and H. pylori (108 CFU/ml) were incubated at 37oC under 10% CO2 for 6 h at 50 rpm and then 50 mul of urea base (2% urea-0.03% phenol red) was added and reacted for 30 min. Urease activity was measured by a spectrophotometer at 550 nm and is represented as a percentage of the control. The results are shown as the mean +- SD. * and **, statistically significant differences from the values for the control (P < 0.05 and P < 0.01, respectively). FIG. 5. | Effect of IgY-Hp on H. pylori Effect of IgY-Hp on H. pylori-induced gastric mucosal injury. IgY-Hp (1 and 10 mg/ml) was orally administered to Mongolian gerbils for 4 weeks. The lymphocytes and neutrophils were measured by the degree of lymphocyte and neutrophil infiltration, respectively. The results are shown as the mean +- SD. *, statistically significant differences from the values for the H. pylori infection group (P < 0.01). FIG. 6. | SEM findings for H. pylori SEM findings for H. pylori-infected AGS cells in the presence or absence of IgY-Hp. (A) AGS cells. (B) AGS cells infected with H. pylori (108 CFU/ml). (C) AGS cells infected with H. pylori (108 CFU/ml) pretreated with IgY-Hp (10 mg/ml). TABLE 1 | Summary of yield and purity of IgY from egg yolk PMID- 12204960_DISCUSSION TI - AB - The present study demonstrates that IgY-Hp prepared from the egg yolk of hens immunized with H. pylori is effective in the treatment of H. pylori infections. H. pylori infections are widespread in humans, and, although they can be cured using antimicrobial therapy, this large-scale use of antibiotics leads to the emergence of antibiotic-resistant strains . Moreover, antimicrobial therapeutic cures of H. pylori infections do not lead to immunity from reinfection , and the emergence of antibiotic-resistant strains can increase failure-of-therapy and relapse rates . Consequently, there have been tremendous efforts to seek alternatives to antibiotic-based therapies for a more widely available means of treating, suppressing, or preventing H. pylori infections without drug resistance problems. Recent work in several laboratories, using several animal models, has shown that immunization with defined native and recombinant antigens of H. pylori can protect against H. pylori infections . The mechanisms of protection are still poorly understood. Although prophylactic and therapeutic immunization has been successful in animal models, efficacy data for humans are still lacking. On the other hand, treatment of H. pylori infection by oral administration of active antibodies specific to H. pylori may have merit, due to the antibody being recognized by H. pylori, thus inhibiting adhesion of the bacterium to human epithelial cells more efficiently. Many studies have shown that egg yolk from an immunized hen has an antibody capable of specific recognition in an abundant quantity and is therefore economical . We estimated that 1 ml of the egg yolk (15 ml per egg) contains about 9.4 mg of IgY. A hen lays about 250 eggs (about 4,000 ml of egg yolk) in a year; thus, the eggs laid by an immunized hen in a year would yield 40 g of IgY. Oral administration of IgY from chicken egg yolk has been used successfully by many researchers in preventing many intestinal diseases, such as those caused by enterotoxigenic E. coli and human rotavirus . Sugita-Konishi et al. reported that IgY, obtained from hens immunized with a mixture of formalin-treated pathogenic bacteria, inhibited the growth of Pseudomonas aeruginosa. The production of Staphylococcus aureus enterotoxin A and adhesion of Salmonella typhimurium serovar Enteritidis to cultured human intestinal cells were also inhibited. IgY antibodies inhibited the colonization of teeth by Streptococcus mutans, thus preventing plaque formation in humans . However, there has been no report so far on the use of IgY in the prevention and treatment of H. pylori infections. In this study, IgY-Hp, obtained from hens immunized with H. pylori whole-cell lysate, dramatically inhibited the growth of H. pylori in vitro. If the antibody IgY had no specific effect, no inhibition of bacterial growth would occur. It seems clear from our observations, using gerbil models, that IgY-Hp decreased H. pylori-induced gastric mucosal injury, as determined by the degree of lymphocyte and neutrophil infiltration. Therefore, the therapeutic value of orally administered IgY-Hp, against the experimental model in gerbils, lies in its ability to inhibit the bacterial organism. More convincing evidence in support of the specificity of these antibodies comes from inhibition of H. pylori attachment to AGS cells, as confirmed by SEM. Although the mechanisms by which IgY-Hp prevented H. pylori colonization are yet not elucidated, our results suggest that IgY-Hp would inhibit H. pylori adherence properties. Of interest, the IgY-Hp used in this study significantly inhibited urease activity. The relationship between inhibitions of urease activity and adhesion properties needs to be clarified. In this study, IgY-Hp seems to have two properties: inhibiting adhesion of the bacterium to gastric epithelial cells and demonstrating powerful urease-inhibiting activity. For practical application of IgY-Hp together with food or pharmaceutical materials to treat H. pylori infection, the stability of IgY-Hp toward heat, acid, or pepsin was studied by measuring the remaining activity by ELISA. Immunologically, IgY-Hp stayed active at 60C for 10 min, suggesting that pasteurization can be applied to sterilize the product. The fortification of food products with this immunoglobulin, together with its higher productivity and effectiveness, would significantly decrease the activities of H. pylori infections. In conclusion, the encouraging results of this study indicate that the IgY-Hp obtained from hens immunized by H. pylori may provide a novel approach to the management of H. pylori infections in humans. However, many problems remain unsolved for the clinical application, such as the effect of IgY-Hp in humans, the persistence of the effect of IgY-Hp after cessation of the application, and the potential for eradicating an established infection. PMID- 12204959 TI - Bacillus Species Are Present in Chewing Tobacco Sold in the United States and Evoke Plasma Exudation from the Oral Mucosa AB - | Five Bacillus species, predominantly Bacillus megaterium and Bacillus pumilus, were isolated from two popular brands of commercially available chewing tobacco [(5.0 +- 1) x 106 CFU/ml of supernatant; results for four experiments]. Moreover, the supernatant of the Bacillus culture evoked plasma exudation from postcapillary venules in the intact hamster cheek pouch, exudation that was mediated by the kallikrein/kinin metabolic pathway. Taken together, these data indicate that Bacillus species contaminate chewing tobacco commercially available in the United States and elaborate a potent exogenous virulence factor(s) that injures the oral mucosa. PMID- 12204959_Introduction TI - AB - Habitual use of chewing tobacco is on the rise in the United States . This practice may be associated with oral mucosa inflammation and injury in susceptible individuals and may predispose chewing tobacco users to oral epithelial cell dysplasia and cancer . Previous studies from our laboratory showed that chewing tobacco elicits plasma exudation from the oral mucosa by activating oral keratinocytes and the kallikrein/kinin metabolic pathway . However, the toxic constituent(s) of chewing tobacco that accounts for these responses has not been characterized . A physiological disorder of the tobacco plant, termed "frenching," has been known to the tobacco industry since colonial times . Although the etiology of this condition is uncertain, so-called organic toxins produced by Bacillus species and found in the soil where tobacco plants are grown have been implicated . It is conceivable that Bacillus spores could contaminate chewing tobacco processed for human use and germinate when placed onto the oral mucosa. These bacteria could then elaborate potent virulence factors, such as proteinases, that activate oral keratinocytes and the kallikrein/kinin metabolic pathway in the oral mucosa, leading to plasma exudation and tissue injury . The purpose of this study was to begin to address this issue by determining whether Bacillus species contaminate commercially available chewing tobacco and, if so, whether they evoke plasma exudation from the intact oral mucosa. PMID- 12204959_MATERIALS AND METHODS TI - AB - Culture of chewing tobacco. | One box each of two popular chewing tobacco brands (moist snuff; Skoal Cherry and Skoal Spearmint [U.S. Tobacco Co., Richmond, Va.]) were purchased at a local grocery store in Chicago, Ill. One-half gram of chewing tobacco from each box was mixed with 50 ml of sterile, double-distilled water for 5 min. Then samples of the mixture were transferred onto blood, mannitol salt, and MacConkey agar plates by sterile cotton swabs and streaked for isolated colonies . The blood and mannitol salt agar plates were incubated under aerobic, anaerobic, and 10% CO2 conditions. MacConkey agar plates were incubated aerobically at 37C. After 48 h, the cultures were examined and a Gram stain of the isolated colonies was performed . After an additional 48 h of incubation 13 biochemical tests were conducted to identify the isolated gram-positive, spore-forming Bacillus species . A viable count was done to determine the number of organisms present in each sample. The most frequently isolated Bacillus species was inoculated into 7 ml of Trypticase soy broth and incubated for 72 h . Thereafter, the culture was centrifuged (15,000 x g), and the supernatant was filtered through a 0.22-mum-pore-size filter, snap-frozen in liquid nitrogen, and stored at -70C until used (see below). Determination of macromolecular efflux from the oral mucosa. | To determine the effects of the Bacillus supernatant on plasma exudation from the intact oral mucosa, we used the in situ microcirculation of the hamster cheek pouch as previously described in our laboratory and by other investigators (, , , -, ). Briefly, we used adult, male golden Syrian hamsters (mean body weight, 130 +- 1 g) that had been anesthetized with pentobarbital sodium and on which tracheostomics had been performed. The cheek pouch microcirculation was visualized with a fluorescence microscope (magnification, x40). Macromolecular leakage was determined by extravasation of fluorescein isothiocyanate-labeled dextran (FITC-dextran; molecular mass, 70 kDa), the intravascular tracer, which appeared as fluorescent spots or leaky sites around postcapillary venules. The number of leaky sites in three random microscopic fields was counted, averaged, and expressed as the number of leaky sites per 0.11 cm2 of cheek pouch, which corresponds to the area of one microscopic field (, , , -, ). With a spectrophotofluorometer, the concentration of FITC-dextran in the plasma and suffusate was determined based on a standard curve of FITC-dextran concentration versus percent emission. The clearance of FITC-dextran was determined by calculating the ratio of the concentration of FITC-dextran in suffusate (nanograms per milliliter) to that in plasma (milligrams per milliliter) and multiplying this ratio by the suffusate flow rate (2 ml/min). The experimental design has been used previously in our laboratory (, , -, ). After the buffer was suffused for 30 min (the equilibration period), FITC-dextran was injected intravenously and the number of leaky sites and the clearance of FITC-dextran were determined for 30 min. Next, the Bacillus supernatant (1:1,000 dilution in bicarbonate buffer, pH 7.4) was suffused over the cheek pouch for 20 min. The number of leaky sites was determined every minute for 20 min and at 5-min intervals for 60 min thereafter. The clearance of FITC-dextran was determined before the application of the supernatant and every 5 min after for 60 min. In another group of animals, 1 muM Hoe140 or 1 muM NPC 17647, both structurally distinct selective bradykinin B2 receptor antagonists (, , -), was suffused over the check pouch for 30 min before the check pouch was suffused with the Bacillus supernatant (1:1,000 dilution in bicarbonate buffer, pH 7.4) for 20 min. The observer of the cheek pouch microcirculation was unaware which hamster belonged in which treatment group. In preliminary studies, we determined that suffusion of bicarbonate buffer (vehicle) or of a 1:1,000 dilution of the bacteriological medium in bicarbonate buffer (pH 7.4) for the entire duration of the experiment or for 20 min, respectively, was associated with no visible leaky-site formation and no significant increase in the clearance of FITC-dextran (four animals; P > 0.5). The concentration of the Bacillus supernatant used in these experiments was based on preliminary experiments. The concentrations of Hoe140 and NPC 17647 used in these experiments were based on previous studies in our laboratory . Chemicals and drugs. | FITC-dextran was obtained from Sigma Chemical Co. Hoe140 and NPC 17647 were gifts from Aventis Pharmaceuticals and Nova Pharmaceutical Corporation, respectively. All drugs were dissolved in saline. Drugs were prepared fresh before each experiment and were diluted in saline to the desired concentrations. Data and statistical analyses. | When a test compound was suffused over the cheek pouch, we determined the maximal change in the number of leaky sites and the clearance of FITC-dextran and noted it as the response to that compound. Data are expressed as means +- standard errors of the means, except for body weight, which is expressed as mean +- standard deviation. Statistical analysis was performed using two-way analysis of variance and the Newman-Keuls test for multiple comparisons (StatView; SAS Institute Inc., Cary, N.C.) on a personal computer. A P of <0.05 was considered statistically significant. PMID- 12204959_RESULTS TI - AB - Culture of chewing tobacco. | Gram stain of cultures of both brands of chewing tobacco revealed large gram-positive, spore-forming bacilli in all samples (four separate experiments were conducted in duplicate). Biochemical analysis of these isolates revealed the presence of five distinct species of Bacillus, i.e., eight isolates of Bacillus megaterium, six of Bacillus pumilus, five of Bacillus brevis, two of Bacillus licheniformis, and one of Bacillus subtilis in the four experiments. The mean colony count was (5.0 +- 1.0) x 106 CFU/ml of supernatant (7.0 x 106 CFU/ml for experiments 1 and 2, 3.0 x 106 CFU/ml for experiment 3, and 2.1 x 106 CFU/ml for experiment 4). No other bacteria were isolated from either brand of chewing tobacco. Determination of macromolecular efflux from the oral mucosa. | Suffusion of saline (vehicle) alone for the entire duration of the experiment evoked no visible leaky-site formation and no significant increase from baseline in the clearance of FITC-dextran (four animals; P > 0.5). Repeated suffusions of B. megaterium supernatant (1:1,000 dilution in bicarbonate buffer, pH 7.4) for 20 min, each with 30-min suffusions of saline in between, were associated with significant and reproducible leaky-site formation [(5.4 +- 0.9)/0.11 cm2 and (6.0 +- 1.1)/0.11 cm2; each group contained four animals; P < 0.05 in comparison to results with saline, and P > 0.5 for within-group comparisons] and an increase in the clearance of FITC-dextran [(25.2 +- 7.5 ml)/min x 10-6 and (26.9 +- 5.9) ml/min x 10-6; each group contained four animals, P < 0.05 in comparison to results with saline, and P > 0.5 for within-group comparisons). Leaky-site formation was visible within 7 to 8 min after the start of suffusion and was maximal 4 to 5 min after the suffusion of B. megaterium supernatant was stopped. The number of leaky sites and the clearance of FITC-dextran returned to baseline 20 min after the suffusion of B. megaterium supernatant was stopped. Repeated suffusions of 1 muM Hoe140 or 1 muM NPC 17647 alone for 30 min, like suffusion of saline alone, evoked no visible leaky-site formation and no significant increase from baseline in the clearance of FITC-dextran (each group contained four animals; P > 0.5). Suffusion of Hoe140 and NPC 17647 (1 muM each) for 30 min significantly attenuated B. megaterium supernatant-induced leaky-site formation and the increase in clearance of FITC-dextran from the cheek pouch (each group contained four animals; P < 0.05 in comparison to results for B. megaterium supernatant alone). FIG. 1. | Effects of B. megaterium Effects of B. megaterium supernatant (1:1,000 dilution in bicarbonate buffer, pH 7.4; CT Bacillus), suffused for 20 min onto the intact hamster cheek pouch, on leaky-site formation (A) and the clearance of FITC-dextran (molecular mass, 70 kDa) (B) in the absence and presence of Hoe140 and NPC 17647 (1 muM each), two structurally distinct selective bradykinin B2 receptor antagonists. Data are given as means +- standard errors of the means and represent maximal values observed 5 min after the suffusion of the supernatant was stopped. Each group contained four animals. *, P of <0.05 in comparison to results with saline; +, P of <0.05 in comparison to results with B. megaterium supernatant alone. PMID- 12204959_DISCUSSION TI - AB - This study produced two new findings. First, we found that five distinct Bacillus species, predominantly B. megaterium and B. pumilus, contaminate, in relatively large numbers, two popular brands of chewing tobacco commercially available in the United States. Second, suffusion of a diluted B. megaterium supernatant onto an intact hamster cheek pouch, an established animal model for studying the mechanisms underlying the deleterious effects of smokeless tobacco in the oral mucosa , significantly increases macromolecular efflux from the cheek pouch. This response was mediated by local elaboration of bradykinin, because Hoe140 and NPC 17647, two structurally distinct selective bradykinin B2 receptor antagonists (, , -), significantly attenuated B. megaterium supernatant-induced responses. Taken together, these data indicate that Bacillus species contaminate commercially available chewing tobacco and elaborate a potent exogenous virulence factor(s) that activates the kallikrein/kinin metabolic pathway in the intact oral mucosa. This, in turn, evokes plasma exudation from postcapillary venules, which leads to interstitial edema and tissue dysfunction. Whether Bacillus species contaminate other brands of commercially available chewing tobacco remains to be determined. The mechanisms underlying bradykinin production by B. megaterium supernatant in the intact hamster cheek pouch were not elucidated. Nonetheless, the results of this study indicate that a potent bacterium-derived exogenous virulence factor(s) activates the kallikrein/kinin metabolic pathway in the oral mucosa to elaborate bradykinin, a potent edemagenic mediator . Previous studies have implicated proteinases released by oral bacteria, such as subtilisin and gingipain RgpA, in bradykinin-induced oral mucosa injury and inflammation in vivo (, , -). It remains to be determined whether Bacillus species contaminating commercially available chewing tobacco elaborate proteinases that directly activate the kallikrein/kinin metabolic pathway in the oral mucosa or whether a virulence factor(s) released by these bacteria stimulates oral keratinocytes, the first cells in the oral mucosa exposed to chewing tobacco and Bacillus species, to elaborate proteinases that activate this metabolic pathway (, , -). Likewise, the role of various chemical constituents of chewing tobacco in modulating the germination and expression of virulence factors by Bacillus species in the oral mucosa should be addressed . Notwithstanding the findings described above, the relevance of the present acute experimental study of the intact hamster cheek pouch to oral mucosa inflammation and injury observed in susceptible habitual chewing tobacco users is uncertain; the results of this study do not include the number of bacteria required to elaborate sufficient quantities of soluble virulence factors to injure the oral mucosa or whether Bacillus species spores and bacteria adhere to oral keratinocytes (, , , -, , ). However, habitual users consume relatively large quantities of chewing tobacco continually and spit it out rather than rinsing their mouths thoroughly after each application . This practice creates a local environment in the oral mucosa that is conducive to the germination of Bacillus species spores and to the elaboration of soluble virulence factors . Certain Bacillus species, including B. licheniformis and B. pumilus, isolated from chewing tobacco in the present study, have been shown to cause opportunistic infections and pulmonary inflammation in humans . The results of the present study extend these observations by showing that Bacillus species contaminating commercially available chewing tobacco elicit oral mucosa inflammation in experimental animals. Clearly, additional experimental and clinical studies are warranted to address these issues. In summary, we found that five distinct Bacillus species, predominantly B. megaterium and B. pumilus, contaminate in relatively large numbers chewing tobacco commercially available in the United States. Once germinating, these bacteria elaborate a potent exogenous virulence factor(s) that activates the kallikrein/kinin metabolic pathway in the intact oral mucosa, leading to plasma exudation and tissue dysfunction. PMID- 12204954 TI - Serodiagnosis of Mice Minute Virus and Mouse Parvovirus Infections in Mice by Enzyme-Linked Immunosorbent Assay with Baculovirus-Expressed Recombinant VP2 Proteins AB - | Mice minute virus (MMV) and mouse parvovirus (MPV) type 1 are the two parvoviruses known to naturally infect laboratory mice and are among the most prevalent infectious agents found in contemporary laboratory mouse colonies. Serologic assays are commonly used to diagnose MMV and MPV infections in laboratory mice; however, highly accurate, high-throughput serologic assays for the detection of MMV- and MPV-infected mice are needed. To this end, the major capsid viral protein (VP2) genes of MMV and MPV were cloned and MMV recombinant VP2 (rVP2) and MPV rVP2 proteins were expressed by using a baculovirus system. MMV rVP2 and MPV rVP2 spontaneously formed virus-like particles that were morphologically similar to empty parvovirus capsids. These proteins were used as antigens in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MMV or anti-MPV antibodies in the sera of infected mice. Sera from mice experimentally infected with MMV (n = 43) or MPV (n = 35) and sera from uninfected mice (n = 30) were used to evaluate the ELISAs. The MMV ELISA was 100% sensitive and 100% specific in detecting MMV-infected mice, and the MPV ELISA was 100% sensitive and 98.6% specific in detecting MPV-infected mice. Both assays outperformed a parvovirus ELISA that uses a recombinant nonstructural protein (NS1) of MMV as antigen. The MMV rVP2 and MPV rVP2 proteins provide a ready source of easily produced antigen, and the ELISAs developed provide highly accurate, high-throughput assays for the serodiagnosis of MMV and MPV infections in laboratory mice. PMID- 12204954_Introduction TI - AB - Mice minute virus (MMV) (formerly minute virus of mice) and mouse parvovirus (MPV) type 1 are among the most prevalent infectious agents found in contemporary laboratory mouse colonies . Although the immunosuppressive strain of MMV (MMVi) can induce a potentially lethal renal hemorrhagic disease when experimentally inoculated into neonatal mice , clinical disease and histologic lesions have not been reported for mice naturally infected with MMV. Similarly, clinical disease and histologic lesions have not been observed in mice naturally or experimentally infected with MPV . Despite the absence of clinical disease and histopathology, murine parvoviruses can have significant deleterious effects on research due to their immunomodulatory effects both in vivo and in vitro (, , -). In addition, there is significant potential for MMV and MPV to be transmitted among animals within a facility due to the high degree of environmental stability of these agents . Therefore, identification of infected laboratory mice is critical to minimize the impact of murine parvovirus infections on research. Serologic evaluation for the presence of antiparvovirus antibodies has typically been used to diagnose MMV and MPV infections in mice , although the type of immunoassay and the source of diagnostic antigen vary significantly among rodent diagnostic laboratories. The most common methods used for the diagnosis parvovirus infections in mice include the enzyme-linked immunosorbent assay (ELISA), the indirect fluorescent-antibody assay (IFA), and the hemagglutination inhibition (HAI) assay, with the ELISA being preferred due to its high-throughput capability. More important than methodology is the choice of parvovirus antigen. MMV antigens have been generated from cell lines experimentally infected with the prototype strain of MMV (MMVp), which grows very well in vitro, yielding highly concentrated preparations of MMV antigen. This antigen works well for the detection of MMV infections in mice by the ELISA, IFA, and HAI assay formats , but MMVp antigen also displays some cross-reactivity with antibodies directed against MPV . This cross-reactivity is primarily the result of antibodies generated to the nonstructural (NS) proteins of MPV binding to the NS proteins of MMVp in the antigen preparation, reflecting the high degree of homology among the NS proteins of the rodent parvoviruses . Cell culture-propagated MPV can be used as antigen to detect anti-MPV antibodies . However, high-titer stocks of MPV are extremely difficult and expensive to obtain via cell culture propagation, and this has largely precluded the use of MPV antigens in ELISAs. For the past few years, a recombinant nonstructural protein of MMV (rNS1), which is highly conserved among rodent parvoviruses, has been used as antigen in serologic ELISAs to detect MMV and MPV infections . However, recent findings indicate that this assay lacks sensitivity in detecting infections, particularly MPV infections . Thus, serodiagnostic assays for MMV and MPV that use capsid proteins as antigens are needed. The goals of this study were to develop sensitive and specific recombinant antigen-based ELISAs to detect parvovirus-infected mice. Here we report on the cloning of the genes that encode the major capsid proteins (VP2) of MMV and MPV, the expression of recombinant VP2 (rVP2) of MMV and MPV with baculovirus vectors, and the use of these recombinant proteins as antigens in ELISAs to detect anti-MMV or anti-MPV antibodies in sera from experimentally infected mice. Our results show that the newly developed ELISAs are highly sensitive and specific for detecting parvovirus infections in mice. PMID- 12204954_MATERIALS AND METHODS TI - AB - Viruses. | MPV-1b was propagated in CTLL-2 murine cytotoxic T cells as described previously . MMVi was a kind gift from Dave Pintel of the University of Missouri. Construction of recombinant baculoviruses. | Standard molecular biology techniques were performed as described previously . The MMV VP2 and MPV VP2 genes were amplified by PCR. The MMV VP2 gene was amplified with platinum Pfx DNA polymerase (Invitrogen, San Diego, Calif.) according to the protocol of the manufacturer. Each 50-mul reaction mixture contained 1.25 U of polymerase, 290 ng of MMVp pML-n plasmid (an MMVp clone that was a kind gift from Dave Pintel, University of Missouri) as template, and primers MMV VP2 2664-2682 forward (5'-gcggatccgtcgacATGAGTGATGGCACCAGCC-3') and MMV VP2 4436-4413 reverse (5'-gcggtaccgcggccgcTTAGTAAGTATTTCTAGCAACAGG-3'). The uppercase letters of the primers correspond to MMV genome sequences, while the lowercase letters of the forward primer represent engineered restriction endonuclease sites for BamHI and SalI and the lowercase letters of the reverse primer represent engineered restriction endonuclease sites for KpnI and NotI. Thermocycling parameters consisted of an initial denaturation step (94C, 2 min), followed by 35 cycles of denaturation (94C, 15 s), annealing (55C, 30 s), and extension (68C, 2 min). The MPV VP2 gene was amplified by a nested PCR strategy. A region of the MPV genome was amplified with platinum Pfx DNA polymerase (Invitrogen) according to the protocol of the manufacturer with the primers MPV 2634-2653 forward (5'-GCACAGCAAAGAACTCAGAC-3') and MPV 4440-4419 reverse (5'-CAGAAAGAAAGAACATGGTTGG-3') and an annealing temperature of 55C. Each 50-mul reaction mixture contained 1.25 U of polymerase and 370 ng of template DNA from MPV-infected CTLL-2 murine cytotoxic T cells that was purified by the DNeasy (Qiagen Inc., Valencia, Calif.) protocol. An aliquot (0.5 mul) of the amplification product from the first PCR was used as template to amplify the MPV VP2 gene by using the Expand High Fidelity PCR system (Roche, Indianapolis, Ind.) and internal primers MPV VP2 2664-2682 forward (5'-gcgaattcggatccgcATGAGTGATGGCGCCGAGC-3') and MMV VP2 4436-4413 reverse (see above) with an annealing temperature of 55C. The uppercase letters of the primers correspond to regions of the MPV genome, and the lowercase letters of the forward primer represent engineered restriction endonuclease sites for EcoRI and BamHI. The amplified MMV VP2 and MPV VP2 genes were electrophoresed for 1 h at 80 V on a 1% SeaPlaque GTG agarose gel (FMC BioProducts, Rockland, Maine) that contained 0.5% ethidium bromide. Amplicons were visualized under UV light, excised from the gel, and purified with the Zymoclean Gel DNA recovery kit (Zymo Research, Orange, Calif.); and DNA concentrations were determined by measuring the A260 with a spectrophotometer (Gene Quant; Amersham Biosciences Corp., Piscataway, N.J.). The purified MMV and MPV VP2 PCR products were digested with BamHI and NotI. The MMV VP2 gene was directionally cloned, in frame, into the His-tagged fusion vector pFastBac HTb (Invitrogen) to create the plasmid MMV-VP2-pFastBac HTb. The MPV VP2 PCR product was directionally cloned, in frame, into the His-tagged fusion vector pFastBac HTc (Invitrogen) to create the plasmid MPV-VP2-pFastBac HTc. Plasmid MMV-VP2-pFastBac HTb or MPV-VP2-pFastBac HTc was used to construct independent recombinant baculovirus expression vectors MMV-VP2 Bac and MPV-VP2 Bac, respectively, by using the Bac-to-Bac Baculovirus expression system (Invitrogen). High-titer stocks of recombinant baculoviruses were generated in Spodoptera frugiperda (Sf-9) insect cells (Invitrogen), and viral titers were determined by plaque assay . Production of recombinant proteins and purification of ELISA antigens. | Standard insect cell and baculovirus propagation protocols were followed . Pilot experiments were performed to determine the kinetics for maximal production of each recombinant protein . To generate large quantities of MMV rVP2 or MPV rVP2 protein for use as antigens, 1-liter pendulum spinner flasks (Wheaton Science Products, Millville, N.J.) were seeded with 600 ml of High Five insect cells (Invitrogen) at a density of 9 x 105 cells per ml in complete insect cell medium (Insecta-FIVE medium; Mediatech, Inc., Herndon, Va.) supplemented with 1.36 mg of l-glutamine per ml and 5% fetal bovine serum. Cells were infected with recombinant baculovirus at a multiplicity of infection of 1.0 for MMV-VP2 Bac or 0.1 for MPV-VP2 Bac. At 72 h postinoculation the cells were pelleted by centrifugation at 500 x g for 15 min at 4C, resuspended in 50 ml of phosphate-buffered saline (PBS), and disrupted by six 30-s cycles of sonication (Sonifer Cell Disrupter 185; Branson Ultrasonics Corp., Danbury, Conn.) with a cup sonicator (Heat Systems Ultrasonics, Farmingdale, N.Y.) on ice. Insoluble material was pelleted by centrifugation at 20,000 x g for 1 h at 4C. The clarified supernatant of the MMV rVP2 preparation (semipurified protein) was harvested and used as antigen in the MMV rVP2 ELISA. Uninfected High Five insect cells cultivated and processed in the same manner were used as controls in the MMV ELISA. To further purify the MPV rVP2 for use as antigen in the MPV ELISA and to purify the MMV rVP2 and MPV rVP2 proteins for electron microscopy, clarified supernatants of vector-infected insect cells were loaded onto 10 to 40% cesium chloride gradients and centrifuged at 26,500 rpm in an SW48 rotor (Beckman Coulter, Inc., Fullerton, Calif.) for 16 h at 4C in an ultracentrifuge (Beckman Coulter, Inc.). Following centrifugation, the area of the gradient containing the rVP2 proteins was visualized as a blue band at a density of 1.31 g/cm3. This fraction was harvested, and the recovered proteins were dialyzed overnight at 4C against PBS through a 10,000-Da-cutoff membrane (Pierce Endogen, Rockford, Ill.). The concentrations of the recovered proteins were determined by a bicinchoninic acid procedure , and the proteins were stored at -80C until use. Western blot analysis. | Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P transfer membranes (Millipore, Bedford, Mass.) . Molecular weight markers were included on each gel to facilitate the calculation of the sizes of the protein bands. Membranes were blocked with 5% nonfat dry milk in PBS (1.4 mM NaH2PO4, 10.8 mM Na2HPO4, 137.9 mM NaCl [pH 7.4]) and probed with diluted sera (1:200) from mice experimentally infected with MMV, mice naturally infected with MPV, or parvovirus-free mice. The membranes were incubated sequentially in solutions of biotinylated goat anti-mouse antibody diluted 1:1,000 (Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.) and avidin DH (Vector Laboratories, Inc.) and were developed with peroxidase substrate solution (Kirkegaard & Perry, Gaithersburg, Md.). Parvovirus serology. | For the MMV ELISA, semipurified MMV rVP2 protein was diluted to 1.0 mug/ml in coating buffer (0.1 M NaHCO3 [pH 9.6]) and insect cell control protein was diluted to 0.5 mug/ml in coating buffer; 200 mul of each was coated onto separate wells of an Immulon 2 96-well plate (Dynex Technologies, Inc., Chantilly, Va). For the MPV ELISA, CsCl-purified MPV rVP2 protein was diluted to 0.1 mug/ml in coating buffer, and 200 mul was loaded into test wells of an Immulon 2 96-well plate. Control wells were uncoated. Proteins were allowed to bind to the plates for 48 h at 4C. The wells were emptied and washed three times in PBS with 0.05% Tween 20 (PBS-Tween). Blocking buffer (PBS with 0.5% nonfat dry milk and 0.05% Tween 20) (300 mul) was added to each well, the mixture was incubated for 30 min at room temperature, and the buffer was discarded; 200 mul of serum diluted 1:100 in blocking buffer was added to experimental and control wells. After incubation at 37C for 2 h, the plates were washed three times in PBS-Tween. Two hundred microliters of alkaline phosphatase-labeled secondary antibody [F(ab')2 fragment goat anti-mouse immunoglobulin G (heavy and light chains); Jackson Immunoresearch Laboratories, Inc.]) diluted 1:7,000 in PBS-Tween was added to each well, and the plate was incubated at 37C for 2 h. The plates were washed five times in PBS-Tween. One hundred microliters of phosphatase substrate solution (1 mg of Sigma 104 phosphatase substrate [Sigma Chemical Company, St. Louis, Mo.] per 1 ml of substrate buffer [2 mM MgCl2, 27.5 mM NaCO3, 22.5 mM NaHCO3 {pH 9.8}]) was added to each well, and the plate was incubated in the dark at room temperature for 45 min. The optical density (OD) of each well was measured (Kinetic Reader EL312E; Bio-Tek Instruments, Inc., Winooski, Vt.) and automatically calculated as the OD at 405 nm (OD405) minus the OD490 to correct for optical interference. For each serum sample, the final OD was calculated as the OD of the test well minus the OD of the control well. The cutoff OD value for each ELISA was set at 3 standard deviations above the mean for 30 serum samples from mice negative for parvovirus infections. The MMV rVP2 ELISA and the MPV rVP2 ELISA were compared to a parvovirus ELISA that uses a recombinant nonstructural protein (rNS1) of MMV as antigen. The rNS1 ELISA was performed as described previously . Performance of ELISAs. | The following were calculated for each ELISA: sensitivity = TP/(TP + FN) x 100, specificity = TN/(TN + FP) x 100, positive predictive value = TP/(TP + FP) x 100, negative predictive value = TN/(TN + FN) x 100, and accuracy = (TP + TN)/(TP + FP + TN + FN), where TP is the number of samples with true-positive results, TN is the number of samples with true-negative results, FP is the number of samples with false-positive results, and FN is the number of samples with false-negative results. For each ELISA, a Kruskal-Wallis one-way analysis of variance on ranks was performed to test for differences between groups of mice. Statistical significance was set at a P value of <=0.01. Mice, experimental infections, and sera. | Four-, 8-, and 12-week-old male Hsd:ICR(CD-1) mice and 12-week-old male BALB/cAnNHsd, C3H/HeNHsd, C57BL/6NHsd, and DBA/2NHsd mice were obtained (Harlan Sprague-Dawley, Inc., Indianapolis, Ind.). The mice were separated into experimental and control groups of three to five animals each according to strain, age, and dose of viral inoculum. Mice were inoculated with 5 x 104 to 1 x 106 50% tissue culture infective doses of MPV-1b diluted in TE (50 mM Tris base, 10 mM EDTA [pH 8.7]) or with TE by intragastric gavage under isofluorane anesthesia. At 4 weeks postinoculation, the mice were euthanized with an inhaled overdose of carbon dioxide, blood was collected by cardiocentesis, and the sera were collected. For the MMV experimental infections, pregnant female C3H/HeNHsd mice were obtained (Harlan Sprague-Dawley), and neonatal male and female pups were inoculated oronasally with 5 x 104 50% tissue culture infective doses of MMVi. Mice were euthanized with an inhaled overdose of carbon dioxide at 8 weeks postinoculation, blood was collected by cardiocentesis, and the sera were collected. Sera from all mice were diluted 1:5 in PBS, heat inactivated at 56C for 30 min, and stored at -20C until use. The source colonies for all mice were documented to be free of known mouse pathogens, including MPV and MMV. Each experimental and control group was housed separately in microisolation cages, and all animal manipulations were performed in a class II biological safety cabinet by standard microisolation technique. All animal studies were approved by the University of Missouri or the University of Arizona Institutional Animal Care and Use Committee. Sera from MPV-inoculated mice that were positive for anti-MPV antibodies by an MPV IFA, sera from MMV-inoculated mice that were positive for anti-MMV antibodies by an MMV IFA, and sera from uninfected mice were used in the ELISAs. The MMV and MPV IFAs were performed as described previously . In Western blot experiments, sera from MMV-inoculated mice that were positive for anti-MMV antibodies by IFA were used as MMV-positive sera, sera from FVB/NCr mice that were naturally infected with MPV were used as MPV-positive sera, and sera from uninfected mice were used as negative sera. The mice naturally infected with MPV were positive for anti-MPV antibodies by MPV IFA, positive for MPV DNA by MPV PCR of mesenteric lymph nodes, negative for anti-MMV antibodies by MMV IFA, and negative for MMV DNA by PCR of mesenteric lymph nodes. Electron microscopy. | The MPV rVP2 and MMV rVP2 proteins were expressed and purified by CsCl gradient centrifugation as described above, with the exception that the recovered fractions were diluted in PBS and centrifuged at 41,000 rpm for 5 h in an SW 41 rotor (Beckman) at 4C to pellet the proteins. The pellets of each protein were resuspended in 1% ammonium acetate. A small droplet of each sample in 4% phosphotungstate was deposited on a carbon-coated grid and examined with a Hitachi H-6700 transmission electron microscope. PMID- 12204954_RESULTS TI - AB - MMV rVP2 and MPV rVP2 self-assemble into VLPs. | The VP2 genes of MMV and MPV were cloned to create the baculovirus vectors MMV-rVP2 Bac and MPV-rVP2 Bac. These vectors were used to express the recombinant capsid proteins MMV rVP2 and MPV rVP2 in insect cells. It is known that many parvovirus recombinant VP2 proteins, including MMV rVP2, assemble into virus-like particles (VLPs) when expressed in baculovirus expression systems . To determine if baculovirus-expressed MMV rVP2 or MPV rVP2 proteins self-assembled into VLPs, we subjected clarified supernatants of insect cells infected with the MMV-rVP2 Bac vector or the MPV-rVP2 Bac vector to CsCl gradient ultracentrifugation. Each gradient yielded a visible blue band at a density of 1.31 g/cm3 of CsCl, which is slightly less dense than previously reported densities of MMV VLPs and empty parvovirus capsids (1.32 g/cm3 of CsCl) . Transmission electron microscopic examination of negative stained preparations of the collected bands revealed the presence of VLPs morphologically similar to empty parvovirus capsids . The MMV VLPs and MPV VLPs were isometric and approximately 20 nm in diameter. Western blots of MMV rVP2 and MPV rVP2. | To determine if MMV rVP2 and MPV rVP2 reacted with natural antibodies to MMV or MPV and if the recombinant proteins shared cross-reactive epitopes, CsCl-purified MMV rVP2 and MPV rVP2 were subjected to SDS-PAGE and Western blot analysis. The Western blots were probed with MMV-positive sera, MPV-positive sera, or parvovirus-negative sera. MMV rVP2 was visualized as a 68-kDa immunoreactive protein band, and MPV rVP2 was visualized as a 69-kDa immunoreactive protein band . The molecular masses of the recombinant proteins represent the molecular mass of the VP2 protein plus the molecular mass of the leader peptide of the fusion protein. The MMV rVP2 protein reacted strongly with four of five MMV-positive serum samples, reacted weakly with one of five MMV-positive serum samples, and displayed cross-reactivity with two of five MPV-positive serum samples. The MPV rVP2 protein reacted strongly with all five MPV-positive serum samples and cross-reacted weakly with two of five MMV-positive serum samples. The MMV rVP2 and MPV rVP2 proteins did not react with sera from uninfected mice. In an ELISA format, MMV-positive sera reacted only with MMV rVP2, MPV-positive sera reacted only with MPV rVP2, and parvovirus-negative sera did not react with either antigen. MMV- and MPV-specific ELISAs. | The baculovirus-expressed MMV rVP2 and MPV rVP2 proteins yielded large amounts of readily produced antigens to develop the MMV- and MPV-specific ELISAs. From 600-ml suspension cultures of vector-infected insect cells, 250 mg of semipurified MMV rVP2 antigen or 1.2 mg of CsCl gradient-purified MPV rVP2 was produced. The semipurified preparation of MMV rVP2 was used as antigen for the MMV ELISA, and the CsCl-purified MPV rVP2 was used as antigen for the MPV ELISA. These antigen preparations were chosen on the basis of preliminary ELISA experiments that showed the marked immunoreactivity of semipurified MMV antigen with MMV-positive sera and the poor immunoreactivity of semipurified MPV antigen with MPV-positive sera. Strong immunoreactivity of MPV rVP2 in an ELISA was obtained only when CsCl-purified MPV rVP2 was used as antigen (data not shown). The baseline cutoff OD405 values used to designate positive sera were calculated to be 0.160 for the MMV ELISA and 0.093 for the MPV ELISA. To compare the performances of the MMV ELISA and the MPV ELISA to that of a previously described generic parvovirus ELISA that detects anti-NS1 antibodies, sera from mice experimentally infected with MMV or MPV and sera from uninfected mice were tested by the MMV ELISA, the MPV ELISA, and the rNS1 ELISA . The MMV ELISA was 100% sensitive and 100% specific in detecting MMV-infected mice and had a 100% positive predictive value, a 100% negative predictive value, and an accuracy of 100%. The MPV ELISA was 100% sensitive and 98.6% specific in detecting MPV-infected mice and had a 97.2% positive predictive value, a 100% negative predictive value, and an accuracy of 99%. The rNS1 ELISA was 21% sensitive and 100% specific in detecting MPV-infected mice, was 0% sensitive and 100% specific in detecting MMV-infected mice, and had a 100% positive predictive value, a 28% negative predictive value, and an accuracy of 76% for detecting mice infected with MMV or MPV. These results demonstrate that the MMV ELISA and the MPV ELISA are sensitive and specific and markedly outperform the rNS1 ELISA for detecting antibodies to MMV or MPV. The performances of the MMV ELISA and the MPV ELISA were further characterized by examining the intensities of the OD values generated when sera from MMV-infected mice, MPV-infected mice, and parvovirus-negative mice were evaluated . For the MMV ELISA, the median absorbance value for sera from MMV-infected mice was statistically higher than the median absorbance values for sera from mice infected with MPV or sera from parvovirus-free mice (P < 0.01). For the MPV ELISA, the median absorbance value for sera from MPV-infected mice was significantly higher than the median absorbance values for sera from mice infected with MMV or sera from parvovirus-free mice (P < 0.01). Prevalence of MMV and MPV infections in research mice. | In addition to evaluating sera from experimentally infected mice, we also evaluated all mouse serum samples that were submitted for routine serologic evaluation over a 4-week-period to the University of Missouri Research Animal Diagnostic Laboratory (Columbia, Mo.). These sera were from 83 research institutions throughout the United States and Canada and were obtained from a variety of immunocompetent mouse strains and stocks. Of 2,473 mouse serum samples tested, 205 serum samples (8.3%) were positive for MPV by ELISA, 38 serum samples (1.5%) were positive for MMV by ELISA, and 32 serum samples (1.3%) were positive for both MMV and MPV by ELISA. Seventeen institutions had sera positive for MPV, three institutions had sera positive for MMV, and five institutions had sera positive for both agents. A group of 22 mice that were serologically positive for MMV and MPV by ELISA were also evaluated for the presence of MMV and MPV DNA by PCR analysis of the mesenteric lymph nodes. Of these 22 mice, 19 were positive for MMV and MPV by PCR. Collectively, these data suggest that MPV and MMV infections are present in research mouse colonies, MPV infections are more prevalent than MMV infections, and dual infections with MPV and MMV occur in some mice. FIG. 1. | Transmission electron micrographs of negative stained preparations of MPV VLPs produced from baculovirus-expressed MPV rVP2 protein (A) and MMV VLPs produced from baculovirus-expressed MMV rVP2 protein (B). Transmission electron micrographs of negative stained preparations of MPV VLPs produced from baculovirus-expressed MPV rVP2 protein (A) and MMV VLPs produced from baculovirus-expressed MMV rVP2 protein (B). The VLPs were purified by CsCl gradient ultracentrifugation. FIG. 2. | Western blot analysis of MPV rVP2 VLPs or MMV rVP2 VLPs separated by SDS-PAGE. Western blot analysis of MPV rVP2 VLPs or MMV rVP2 VLPs separated by SDS-PAGE. The blot was probed with sera from MPV-infected mice (lanes 1 to 5), MMV-infected mice (lanes 6 to 10), and uninfected mice (lanes 11 and 12). Each lane contains 5 mug of protein; test sera were run at a 1:200 dilution. The positions of the MPV rVP2 and the MMV rVP2 are indicated by arrows on the left and right, respectively. The positions of the molecular mass standards are indicated on the left. FIG. 3. | Serum antibody responses of mice experimentally infected with MMV or MPV or of uninfected mice tested by MMV ELISA (A) or MPV ELISA (B). Serum antibody responses of mice experimentally infected with MMV or MPV or of uninfected mice tested by MMV ELISA (A) or MPV ELISA (B). The datum points are presented as box plots; for each experimental group the lower boundary of the box indicates the 25th percentile, the upper boundary of the box indicates the 75th percentile, and the line within the box marks the median value. The line below the box marks the 10th percentile, the line above the box marks the 90th percentile, and datum points outside this range are indicated (*). *, P < 0.01 compared to each other experimental group. TABLE 1 | Comparison of serologic test results for uninfected mice and mice experimentally infected with MPV or MMV PMID- 12204954_DISCUSSION TI - AB - In this report, we describe the development of ELISAs that detect anti-MMV or anti-MPV antibodies in the sera of parvovirus-infected mice. The MMV ELISA and the MPV ELISA displayed high sensitivities and high specificities for the detection of species-specific parvovirus antibodies in MMV- and MPV-infected mice, and each ELISA had high positive and negative predictive values. These assays are ideally suited for use as primary serologic tests to screen mouse colonies for MMV and MPV infections because the assays were highly accurate and because ELISAs are readily automated, making it possible to test large numbers of samples quickly and at relatively little expense. In addition, large quantities of the MMV rVP2 and the MPV rVP2 antigens used in these assays were readily produced with the baculovirus expression system, providing ample antigens for high-volume testing. This is particularly significant for MPV because it is difficult and expensive to produce large quantities of MPV-specific ELISA antigens in vitro from MPV-infected cells. The MMV and MPV ELISAs performed with high sensitivities in detecting MMV and MPV infections in mice, respectively, and these two assays were much more sensitive in detecting parvovirus infections in mice than a generic parvovirus ELISA that uses a recombinant nonstructural protein (rNS1) of MMV as antigen. This is consistent with previous work that shows that mice experimentally inoculated with MPV, especially mice infected after 12 weeks of age, develop anticapsid antibodies more so than anti-NS1 antibodies . The VP2 protein is the major capsid protein of the parvovirus virion and can be presented to the immune system upon exposure to the virus or during viral replication. However, nonstructural proteins, including NS1, are not present in the parvovirus virion and are produced more transiently upon infection. Thus, an undetectable anti-NS1 humoral immune response could result from a nonproductive parvovirus infection or from a productive infection that produces an insufficient amount of NS1 to induce an immune response . The MMV and MPV ELISAs also performed with high specificities in detecting MMV and MPV infections in mice, respectively. Previously described ELISAs that use whole-virus preparations of MMV or rNS1 proteins as antigens are not specific for the identification of MMV- and MPV-infected mice and require the use of secondary testing with MMV- or MPV-specific HAI assays to elucidate the true infection status of the animal. Thus, the high specificities of the newly developed MMV and MPV ELISAs eliminate the need for secondary testing by the HAI assay. An interesting feature of the baculovirus-expressed MMV and MPV rVP2 proteins was that they self-assembled into VLPs that were morphologically similar to empty parvovirus capsids. Self-assembly of baculovirus-expressed recombinant parvovirus VP2 proteins has been reported for many parvoviruses including MMV ; however, this is the first report of self-assembly of MPV rVP2 into VLPs. MMV rVP2 and MPV rVP2 were expressed as fusion proteins that contained an N-terminal polyhistidine tag with the intent of purifying these proteins by metal-affinity chromatography. In preliminary experiments, MMV rVP2 and MPV rVP2 could not be successfully purified by affinity chromatography (data not shown). However, this proved to be unnecessary because the semipurified preparation of MMV rVP2 was highly immunoreactive and therefore required no further purification. While the semipurified preparation of MPV was weakly immunoreactive, highly immunoreactive MPV rVP2 in the form of MPV VLPs was easily purified by CsCl ultracentrifugation. The N-terminal polyhistidine tag and leader sequences added an estimated 3.6 kDa to each rVP2. While the leader peptides did not alter the ability of the rVP2 proteins to assemble into VLPs, they may have caused a slight decrease in the densities of the VLPs (1.31 g/cm3 of CsCl) compared to previously reported densities of MMV VLPs and empty parvovirus capsids (1.32 g/cm3 of CsCl) . The formation of VLPs by the MMV and MPV rVP2 proteins may have contributed to the high sensitivities of the MMV and MPV ELISAs. Interestingly, the MMV rVP2 protein that was subjected to Western blotting reacted strongly with only four of five MMV-positive serum samples. The one MMV-positive serum sample that reacted weakly with MMV rVP2 by Western blotting was strongly reactive with MMV rVP2 by ELISA. It is possible that conformational epitopes present on the exterior of the MMV VLPs were not present when MMV rVP2 was disrupted or denatured for Western blot analysis. Our data also suggest that formation of MMV and MPV VLPs likely enhanced the specificities of the MMV and MPV ELISAs over those which would be attained with unassembled MMV rVP2 or MPV rVP2 proteins as antigens. Sera from two MPV-infected mice strongly cross-reacted with MMV rVP2 proteins on Western blots. These sera did not cross-react with MMV rVP2 in an ELISA format. In addition, two serum samples from MMV-infected mice displayed weak cross-reactivity with MPV rVP2. These two serum samples were not cross-reactive with the MPV rVP2 protein in an ELISA format. A possible explanation for these observations is that in the ELISA format, the MMV rVP2 and MPV rVP2 VLPs behave antigenically like whole virions and anticapsid antibodies are able to associate only with antigenic epitopes present on the exterior of the VLPs. However, when the VLPs are subjected to SDS-PAGE and Western blotting, the VLPs are disrupted and the proteins are denatured, possibly exposing cross-reactive epitopes. The MMV and MPV ELISAs were used to survey sera obtained from numerous mouse colonies from geographically disparate locations in North America. The prevalence of sera positive for a parvovirus was nearly 10%, with the majority of these sera being positive for MPV. This is consistent with a recent survey that showed that in the United States the prevalence of parvovirus infections among laboratory mouse colonies is high . Given the potential for parvovirus infections to alter or invalidate studies that use infected mice (, , -), there is a need for highly accurate, high-throughput assays to detect parvovirus infections in mice. Baculovirus-expressed MMV rVP2 and MPV rVP2 proved to be excellent antigens for the development of such assays. The MMV and MPV ELISAs performed with high sensitivities and specificities and markedly outperformed another widely used parvovirus ELISA that detects antibodies to NS1 proteins in infected mice. The two newly developed serologic assays offer rapid, inexpensive, and accurate methods for the screening of research mouse colonies for MMV and MPV infections. PMID- 12204972 TI - CD4+ CD25+ T-Cell Production in Healthy Humans and in Patients with Thymic Hypoplasia AB - | Regulatory T cells are found primarily in the CD4+ CD25+ fraction of T cells and play an important role in the prevention of autoimmunity. We examined CD4+ CD25+ T cells in 33 healthy children and adults and compared them to a population with an inherited form of thymic hypoplasia and a predisposition to autoimmune disease. Absolute numbers of CD4+ CD25+ T cells were markedly higher in healthy infants than in infants with chromosome 22q11.2 deletion syndrome. PMID- 12204972_Introduction TI - AB - Regulatory T cells constitute an important contributor to the state of tolerance . Little is known about their function and production in humans, and nothing is known about their development in humans . Murine studies first defined these cells as a component of CD4+ CD25+ (CD4 CD25) T cells. Several important features of these murine cells have been described. They exit the thymus from day 4 of life onwards, and they appear to require a T-cell receptor with high affinity . They are antigen specific in their activation but are non-antigen specific as effectors. As effectors, they are naturally nonproliferative, and their function is generally detected by their ability to inhibit proliferation of other cells or other effector functions, such as cytolytic activity. Although their exact mechanism of action is controversial, there is agreement that cell contact is required. These cells have been demonstrated to be responsible for the prevention of organ-specific autoimmunity in several murine models. Mice which have undergone a thymectomy between days 1 and 3 of life have a delay in the appearance of CD25+ T cells. These mice develop organ-specific autoimmune disease; the specific disease is dependent on the strain of mice used . Infusion of CD25+ T cells can abolish the organ-specific autoimmune disease. In thymectomy models, the ratio of CD25+ to CD25- T cells is normal. The CD25+ T cells appear late and in lower numbers but retain their normal proportion in the T-cell compartment. This has suggested that adequate quantitative production of regulatory T cells early in life may be a critical part of the development of tolerance. Human cells with regulatory properties express very high levels of CD25 and have suppressive effects on effector functions similar to that seen in mice . They are present in adults at levels similar to those seen in mice, i.e., approximately 6 to 10% of CD4 T cells. The studies described above documented the important role of the thymus in the production of CD4 CD25 T cells and the role of CD4 CD25 T cells in the prevention of autoimmunity. In this study, we examined a population of children who have thymic hypoplasia as a result of a heterozygous deletion of chromosome 22q11.2. This deletion results in a phenotype which has also been called DiGeorge syndrome, velocardiofacial syndrome, or conotruncal anomaly face syndrome. Approximately 80% of patients with the deletion will have an immunodeficiency as a consequence of the thymic hypoplasia . The immunodeficiency is typically a pure defect in T-cell production, and patients have on average 50 to 70% of the normal numbers of T cells . Autoimmune disease develops in approximately 10% of patients, although the pathophysiologic mechanism for this is not understood. This study examined the developmental appearance of CD4 CD25 T cells in the peripheral blood of 33 children and adults with chromosome 22q11.2 deletion and in 33 healthy age-matched controls in an effort to determine whether thymic hypoplasia could be associated with diminished numbers of this important T-cell subset, as is seen in mice with neonatal thymectomies. Unselected patients with hemizygous deletions of chromosome 22q11.2 as detected by the Oncor/Vysis N25 probe were included as "patients" in this analysis. Control samples were selected from clinics performing well-child care or allergy evaluations (children with negative RAST tests). Institutional review board approval was obtained for this study, and consent was obtained from each participating patient or parent of the patient. Three-color flow cytometry was performed to define CD3, CD4, and CD25. Only CD25hi CD4 T cells were included in the CD25-positive gate, in accordance with what is known regarding the expression of CD25 on human regulatory T cells . The fraction of CD25 T cells within the CD3 population and the frequency of CD25 T cells within the CD4 population did not vary with age and showed no difference between controls and patients. In both patients and controls, the CD25 fraction of CD4 T cells was typically 6 to 15%. In both patients and controls, the CD25 fraction of CD3 T cells was typically 2 to 10%. Differences were readily apparent between patients and controls when the CD4 CD25 proportion of total lymphocytes was examined and the CD4 CD25 absolute number was determined . There is a distinct age-related decline in the CD4 CD25 fraction and number of cells from birth through approximately 36 months of age in the controls. The differences between the CD4 CD25 T-cell numbers in children less than 3 years of age and those over 9 years of age are significant, with P being 0.001. In contrast, the fraction and number of CD4 CD25 T cells were similar throughout all ages examined in the chromosome 22q11.2 deletion population. As a result, the fraction and the absolute number are markedly lower in the patients than controls in infancy. The mean CD4 CD25 T-cell count in patients under 3 years of age was 7 cells/mm3, while it was 75 cells/mm3 in the controls (P = 0.001). The mean CD4 CD25 T-cell count in patients 3 to 9 years of age was 3 cells/mm3, while it was 14 cells/mm3 in controls (P = 0.005). The mean CD4 CD25 T-cell count in patients over the age of 9 years was 3 cells/mm3, while it was 10 cells/mm3 in controls (P = 0.002). Six patients had autoimmune disease. The mean CD4 CD25 T-cell count in these six patients was no different than the mean in the non-autoimmune disease patients. FIG. 1. | (Top) Fractions of CD4 CD25 T cells within the total lymphocyte gate; (bottom) absolute numbers of CD4 CD25 T cells. (Top) Fractions of CD4 CD25 T cells within the total lymphocyte gate; (bottom) absolute numbers of CD4 CD25 T cells. Gray diamonds represent patients with autoimmune disease. m, months. This is the first study to examine peripheral blood CD4 CD25 T cells in human children. We have shown that newborns generally have high numbers that decline rapidly over the first 2 to 3 years of life. This is dependent on T-cell production, because the fraction of CD4 CD25 cells within the T-cell subset remains relatively constant throughout childhood. Patients with chromosome 22q11.2 deletion with developmental thymic hypoplasia have markedly fewer CD4 CD25 T cells in infancy. The significance of these findings is twofold. Patients with chromosome 22q11.2 deletion syndrome have a relatively pure quantitative defect in T-cell production. Thus, this study suggests that regulation of CD4 CD25 T-cell production early in life in humans is directly related to thymic capacity. CD4 CD25 T cells are produced in similar proportions with other T cells postnatally. Secondly, this study suggests that one contributor to the increased prevalence of autoimmune disease could be diminished CD4 CD25 T cells. This is comparable to what was seen in the murine neonatal thymectomy model. The CD4 CD25 T cells appear late, and the absolute numbers remain diminished. In this murine model, approximately 20% of the mice developed autoimmune gastritis by 1.5 to 3 months of age after neonatal thymectomy . Extrapolating from the murine model, our finding of markedly diminished CD4 CD25 T-cell numbers early in life suggests that this phenomenon could play a role in the predisposition to autoimmune disease in patients with chromosome 22q11.2 deletion syndrome. Other factors are required to define the type of autoimmune disease and to further determine susceptibility. PMID- 12204961 TI - Immunoglobulin G Antibody Response to Infection with Coccoid Forms of Helicobacter pylori AB - | An increasing number of studies support a potential role for coccoid forms in Helicobacter pylori infection. Evidence for this was obtained through scanning microscopy, genetic analysis for virulence traits, examination of the presence and activity of key enzymes, and other methods. We studied the serum immunoglobulin G responses to coccoid H. pylori forms by enzyme-linked immunosorbent assay (ELISA) and immunoblotting and compared them with those of bacillary cells. Sera from a total of 295 infected individuals were studied; these included sera from 100 patients with duodenal ulcers, 98 patients with nonulcer dyspepsia, 11 patients with gastroduodenal cancer, and 86 asymptomatic individuals. Initially, we characterized and selected coccoid and bacillary antigenic preparations by one-dimensional (1-D) and 2-D gel electrophoresis and immunoblotting. Data showed that coccoid and bacillary preparations with comparable protein contents have similar patterns in 1-D and 2-D electrophoresis gels and antigenic recognition at blotting. These results revealed that coccoid and spiral antigens in ELISA can equally recognize specific antibodies to H. pylori in sera from infected individuals. The analysis of the spiral and coccoid preparations by Western blotting showed no major differences in antigen recognition. No specific bands or profiles associated with a single gastric condition were identified. PMID- 12204961_Introduction TI - AB - Helicobacter pylori spiral-coccoid dimorphism is observed both in vivo and in vitro. It is generally accepted that the coccoid forms arise as a response to stress conditions, e.g., in vitro aerobiosis , temperature changes , extended incubation , and in vivo antibiotic treatment . Since they were first described, coccoid H. pylori forms were considered an irreversible phase that leads to cell death . Present knowledge suggests that coccoid cells are not dead but actually dormant . Coccoid forms may therefore play a role in the survival, and eventually in the transmission, of this microorganism. A number of reports suggest that coccoid forms maintain cell structures, metabolism, DNA indemnity , and gene expression . There are also reports indicating that H. pylori cells are able to survive for prolonged periods in the environment, especially in water and under conditions of starvation . This would not be surprising if we took into account that coccoid forms are biologically important for other pathogenic bacteria, such as Vibrio vulnificus or Campylobacter jejuni . This study of coccoid forms may help us to better understand the natural history of H. pylori infection. H. pylori infection induces a strong local inflammatory response which often is insufficient to eradicate the pathogen, and this failure may be responsible for the chronicity that these gastric diseases often demonstrate. It is not fully understood how the immune system is involved in clinical outcomes. One point upon which investigators agree is that the presence of specific antibodies can be used as an epidemiological indicator of infection . Some studies suggest that noninvasive serologic tests may be of value to confirm treatment success . Although many studies have focused on the impact of bacillary H. pylori cells on immune status , there is no information on the potential role of coccoid forms. The aim of this work was to study the immunoglobulin G (IgG) immune response of colonized individuals against coccoid H. pylori forms and compare it with that elicited by its spiral counterpart. PMID- 12204961_MATERIALS AND METHODS TI - AB - Strains. | We studied 21 strains of H. pylori isolated in our laboratory from gastric biopsy samples of Chilean adults. The isolates were confirmed through microscopy, culture, and rapid urease testing. Antigen preparation. | All strains were grown under microaerophilic conditions at 37C on chocolate agar and a Skirrow antibiotic pool. Spiral cells were collected after 3 days in phosphate-buffered saline (PBS). The coccoid cells were harvested after 30 days at room temperature under aerobic conditions. Coccoid morphology was confirmed by Gram stain (100 fields) and by the strains' inability to grow in appropriate conditions. The coccoid and bacillary antigens were prepared by the acid glycine extraction method , standardized in their protein content (Bio-Rad Labs, Hercules, Calif.), and maintained frozen (-20C) until analysis. SDS-PAGE antigen characterization. | H. pylori proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with 4 and 7% stacking and running gels, respectively. The bands were visualized with silver stain, and the gels were analyzed by Quantity One software (Bio-Rad). 2-D electrophoretic antigen characterization. | The H. pylori preparations were first separated by isoelectric focusing according to a procedure described by Celis et al. . Antigens (200 mug/capillary) were incubated at room temperature with 40 mul of lysis solution (9.8 M urea, 2% NP-40, 2% ampholyte 3/10, 100 mM dithiothreitol) for 15 min. Preparations were loaded into the capillaries and covered with 20 mul of overlay solution (8 M urea, 1% ampholyte 7/9, 5% NP-40, 100 mM dithiothreitol). Gels were run at 200 V for 2 h, 500 V for 2 h, and 800 V for 16 h in two-dimensional (2-D) electrophoresis equipment (Protean II; Bio-Rad). After an electrophoretic run under similar conditions, the protein spots were visualized by silver staining and analyzed by 2-D Bio-Rad software. Western blot antigen analysis. | The H. pylori coccoid and bacillary antigens were evaluated by Western blot analysis . In brief, strips were blocked with skimmed milk, confronted with 1:150 serum dilutions, and maintained overnight at room temperature. Membranes were then incubated with an anti-human IgG alkaline phosphatase conjugate (Sigma). Reaction was revealed with 5-bromo-4-chloroindolylphosphate, nitroblue tetrazolium (GIBCO), and MgCl2 (Merck). The molecular masses of the proteins were calculated by interpolating the proteins in a curve constructed with reference markers (Bio-Rad). Serum panel. | The sera obtained previously were from 295 colonized individuals: 100 duodenal ulcer (DU) patients, 98 nonulcer dyspepsia (NUD) patients, 11 gastric cancer patients, and 86 asymptomatic individuals. Colonization of asymptomatic subjects was confirmed by the presence of IgG antibodies to H. pylori. ELISA for IgG antibodies to spiral and coccoid preparations. | Enzyme-linked immunosorbent assay (ELISA) was previously standardized in our laboratory ; the conditions were optimized by check board analysis. In brief, flat-bottom plates (Maxisorp; Nunc, Roskilde, Denmark) were covered with either bacillary or coccoid H. pylori surface antigen preparation (0.125 mug/well). After blocking with PBS containing 10% skim milk, serum diluted 1:600 was added, the plates were incubated for 2 h at 37C, and their reactivity was revealed with anti-human IgG alkaline phosphatase conjugate diluted 1:1,000 (Sigma). Wells were washed, and the substrate was added (p-nitrophenyl phosphate; Sigma) and maintained for 30 min at 37C. The reaction was stopped with 3 M NaOH. Results were expressed as optical density (OD) at 405 nm. All serum samples were evaluated in duplicate. Adsorption assay. | Four sera with high ELISA levels were tested to evaluate the presence of common antigens between both preparations. Serum samples were assayed before and after adsorption with bacillary and coccoid antigens separately. Briefly, a total of 25 mug of antigen was incubated with 400 mul of diluted sera (in PBS diluted 1/20) under gentle agitation for 1 h at 37C and overnight at 4C. Adsorbed sera were centrifuged at 6,000 x g, and the supernatant was saved and kept frozen at -20C until ELISA was performed. Statistics. | Comparisons between values obtained by ELISA based on coccoid and bacillary forms were performed through the Student's t test (STATISTICA software). PMID- 12204961_RESULTS TI - AB - H. pylori antigens. | The coccoid and spiral preparations revealed numerous electrophoretic bands ranging from 10 to 29 and 11 to 26 bands, respectively . The molecular weights were calculated to be between 6.5 and 169 for the bacillary form and 6.5 and 200 for the coccoid form. Based on the frequency of band occurrence (50% or higher), a common profile of the following approximate molecular weights of 18 bands was drawn: 6.5, 7.5, 9, 12, 14.5, 21, 27, 30, 35, 53.5, 57, 61.5, 68, 72, 78, 84, 94, and 104. Occasionally, the coccoid or spiral preparation showed a single band (200 or 66) not visible in the other form. 2-D electrophoretic analysis. | 2-D electrophoretic analysis revealed that coccoid preparations produced fewer spots than their spiral counterparts (135 and 163 spots, respectively). The spiral preparations showed spots in a molecular weight range of 22 to 219, and the coccoid forms showed spots in a range of 25 to 245. The pI range of the protein spots was similar for both preparations, ranging from 4.5 to 8.5. Only a few strains (n = 3) produced some spots observed exclusively in the coccoid forms (molecular weights of 245, 149, 136, 71, and 42 with pIs of 6.5, 7.1, 7, 4.4, and 4.5, respectively). In another three strains, the intensity of the spots was higher in the coccoid form . Western blot analysis. | All 21 H. pylori strains were challenged with homologous sera. We found variability in the number and intensity of bands recognized by the homologous sera. The number of reactive bands for the different strains ranged from 9 to 20 for the coccoid antigens and from 9 to 19 for the bacillary form. ELISA antigen selection. | Twelve out of 21 strains were selected due to the higher number of bands detected on the blot , and a higher protein content was revealed by 1- and 2-D electrophoresis. Strains with exclusive spots or higher intensity in the coccoid forms were also included . ELISA for IgG antibodies to spiral and coccoid preparations. | The IgG responses to H. pylori against coccoid and bacillary preparations are shown in Table . As observed, all the serum panels tested were reactive with both the coccoid and bacillary H. pylori preparations. The OD reading values showed high variability, which was reflected in elevated standard deviation values. The average ELISA values for patients with different gastric conditions were similar whether coccoid or spiral capturer antigens were used. The only exception was the cancer group, which showed lower values against both coccoid- and spiral-based antigens by ELISA. When the values were compared within each patient group, results showed significantly higher OD readings for the coccoid capturer antigen (P < 0.05). The only exception was the NUD patients, who had similar values for both antigens (P = 0.6551) . Adsorption assay. | The results of this assay showed that all the OD values decreased similarly (more than 90%) when sera were adsorbed with either coccoid or bacillary antigens. Western blot analysis with the antigen preparation used in ELISA. | A representative subset of serum samples tested (22 DU patients, 14 NUD patients, 11 cancer patients, and 21 asymptomatic individuals) was blotted against the coccoid and spiral capturer antigens used by ELISA. The results showed that sera from infected individuals recognized H. pylori epitopes with a wide individual variability. Accordingly, we could not find a common recognizable profile from patients with similar gastric conditions. However, some bands appeared with a higher frequency in most sera (molecular weights of ca. 104, 87, 66, 63, 61, 35, and 28) . Some bands were recognized only among the antigens presented by the bacillary form (molecular weights of 49, 32, 30, and 19) in serum samples from ulcer patients. Other bands were recognized only in the coccoid antigen by serum samples from asymptomatic individuals (molecular weights of 171, 156, 135, 116, 104, and 32), and yet other bands were recognized by sera from the ulcer patients (molecular weights of 144, 135, and 98). Serum samples from many asymptomatic subjects had antibodies to a 77-molecular-weight antigen; this epitope was not recognized by serum samples from symptomatic patients . To get a general picture of the epitopes recognized by blotting, we tested the antigens applied during ELISA against four pools of six sera representing each patient group. The results showed that the four groups of sera recognized the same bands in coccoid and bacillary antigens, with some minor differences in band intensity. The common profile recognized by the four sera groups is shown in Fig. . FIG. 1. | SDS-PAGE of bacillary (lanes 1 through 6) and coccoid (lanes 7 through 12) antigens of six Helicobacter pylori strains included for antigen characterization. SDS-PAGE of bacillary (lanes 1 through 6) and coccoid (lanes 7 through 12) antigens of six Helicobacter pylori strains included for antigen characterization. MW, molecular weight standard. FIG. 2. | Comparative 2-D gel electrophoresis of bacillary (a) and coccoid (b) protein preparations from a single strain. Comparative 2-D gel electrophoresis of bacillary (a) and coccoid (b) protein preparations from a single strain. Arrows show two spots found only in the coccoid form. The ovals show the spots that became more intense in the coccoid preparation. MW, molecular weight standard. FIG. 3. | Immunoblot of bacillary (even lanes) and coccoid (pair lanes) preparations from the 12 selected H. pylori Immunoblot of bacillary (even lanes) and coccoid (pair lanes) preparations from the 12 selected H. pylori strains for ELISA. Each antigenic preparation was tested against homologous serum. Lanes: MW, molecular weight standard; 1 and 2, strain 1; 3 and 4, strain 2; 5 and 6, strain 3; 7 and 8, strain 4; 9 and 10, strain 5; 11 and 12, strain 6; 13 and 14, strain 7; 15 and 16, strain 8; 17 and 18, strain 9; 19 and 20, strain 10, 21 and 22, strain 11; 23 and 24, strain 12. FIG. 4. | Antigenic protein bands of bacillary (lanes 1, 3, 5, and 7) and coccoid (lanes 2, 4, 6, and 8) pools used in ELISA recognized by pooling of sera from patients with different gastroduodenal conditions. Antigenic protein bands of bacillary (lanes 1, 3, 5, and 7) and coccoid (lanes 2, 4, 6, and 8) pools used in ELISA recognized by pooling of sera from patients with different gastroduodenal conditions. Lanes: 1 and 2, sera from gastroduodenal ulcer patients; 3 and 4, sera from NUD patients; 5 and 6, sera from cancer patients; 7 and 8, sera from asymptomatic individuals. TABLE 1 | IgG responses in OD units to bacillary and coccoid antigens in different groups of patients TABLE 2 | Frequency of band recognition of bacillary and coccoid antigen pools by sera from patients with different gastroduodenal conditions PMID- 12204961_DISCUSSION TI - AB - The amount of scientific information gained in the last 2 decades on H. pylori is impressive. Genetic and biochemical characteristics, and many aspects of virulence, have already been well described . The mechanism of transmission of H. pylori, however, is less well understood. Much research confirms that the oral-oral route is how one becomes infected with H. pylori . This does not, however, fully explain how H. pylori-related infections are so widespread the world over and why the prevalence is significantly higher in less-developed areas. One can postulate that the fecal-oral route, via contaminated water and/or foodstuffs, could be the missing link that explains the high prevalence of H. pylori infection in areas with poor sanitation . In order to satisfy this theory, one must explain how spiral cells are able to survive the hostile environmental factors that do not support them. Because coccoid cells are normally present both in vivo and in vitro, these forms could explain the persistence of H. pylori under hostile environmental conditions. To explore this issue, we attempted to determine whether the coccoid forms have any impact on the immune response to H. pylori in colonized individuals. We established a specific ELISA aimed at evaluating and comparing the immune response to coccoid and spiral forms against a panel of sera from symptomatic and asymptomatic infected individuals. The results revealed that all infected individuals either with or without symptoms were highly reactive to both forms. However, the coccoid antigen preparations usually gave significantly higher OD readings than their bacillary counterparts. The biologic significance of these differences is probably not relevant. It is tempting to speculate that the higher values found with coccoid antigen in ELISA may be due to the overexpression of one or more major epitopes in the coccoid morphology. However, the results obtained in the blotting assays make this explanation less probable. This is because we did not find any major differences in band recognition when sera were assayed against coccoid or bacillary antigens. The data from 68 serum samples analyzed indicated that the IgG response to the coccoid antigen was as strong as that to the spiral one . Most of the bands recognized by blotting were similar to those described by Nilsson et al. . We found no specific bands or profiles that could be associated with a particular morphological state or specific gastric condition. Kimmel et al. obtained similar results: when comparing antigenic patterns recognized in sera from different patients, they found no association of specific H. pylori antigens with antibodies in patients with specific gastroduodenal pathologies. The results presented here indicate that the coccoid forms of H. pylori are able to induce a humoral immune response. They also show that their immune response is similar to that induced by the bacillary forms. The analysis of the antigenic preparations applied in this study by 1-D and 2-D electrophoresis and immunoblotting showed that the total protein content of the coccoid forms is very similar to that of the spiral ones. Furthermore, it established that the cell proteins remain intact, as demonstrated by their immunogenic potential. The results of ELISA with adsorbed sera support this idea. These assays using cross-adsorbed sera confirm the presence of common antigens in coccoid and bacillary preparations. An important subject that remains to be elucidated is whether coccoid forms also elicit a cellular immune response or, in particular, whether there are differences in Th1 or Th2 involvement when coccoid antigens are presented. Future studies in our laboratory will focus on some of these issues. PMID- 12204951 TI - Antibody Responses of Cattle with Respiratory Coronavirus Infections during Pathogenesis of Shipping Fever Pneumonia Are Lower with Antigens of Enteric Strains than with Those of a Respiratory Strain AB - | The serum antibody responses of cattle with respiratory coronavirus infections during the pathogenesis of shipping fever pneumonia were analyzed with different bovine coronavirus antigens, including those from a wild-type respiratory bovine coronavirus (RBCV) strain (97TXSF-Lu 15-2) directly isolated from lung tissue from a fatally infected bovine, a wild-type enteropathogenic bovine coronavirus (EBCV) strain (Ly 138-3), and the highly cell culture-adapted, enteric prototype strain (EBCV L9-81). Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) activities were tested. Sequential serum samples, collected during the onset of the respiratory coronavirus infection and at weekly intervals for 5 weeks thereafter, had significantly higher IN and HAI titers for antigens of RBCV strain 97TXSF-Lu15-2 than for the wild-type and the highly cell culture-adapted EBCV strains, with P values ranging from <0.0001 to 0.0483. The IN and HAI antibody responses against the two EBCV strains did not differ significantly, but the lowest titers were detected with EBCV strain L9-81. PMID- 12204951_Introduction TI - AB - The role of coronaviruses as respiratory pathogens in cattle was recently recognized when numerous coronavirus strains were isolated from the nasal secretions and lung tissues from cattle with fatal cases of shipping fever pneumonia (SFP). These isolates are referred to as respiratory bovine coronaviruses (RBCV) . The enteropathogenic potential of coronaviruses was identified previously when these viruses were isolated from diarrhea fluid or intestinal samples from neonatal calves with severe diarrhea . In addition, coronaviruses were implicated in winter dysentery of adult cattle . These coronaviruses are referred to as enteropathogenic bovine coronaviruses (EBCV). Significant phenotypic, antigenic, and genetic differences separate the newly recognized RBCV from EBCV . Furthermore, infectivity-neutralizing (IN) monoclonal antibodies (MAb) distinguished between the EBCV wild-type strain LY138-3 and the highly cell culture-adapted EBCV strain L9-81, as well as the Norden vaccine strain . Bovine coronaviruses are enveloped, have a positive single-stranded RNA genome of about 31 kb, and belong to the Coronaviridae family of the order Nidovirales . The spike (S) and the hemagglutinin esterase (HE) glycoproteins of the five structural proteins function in the infectious process through adsorption and coadsorption to the receptors on susceptible host cells . The S and HE glycoproteins are both capable of hemagglutinating rodent and chicken red blood cells (RBC). The HE but not S glycoprotein facilitates the release of viruses adsorbed to RBC in the receptor-destroying enzyme (RDE) test mediated by an acetylesterase (AE) component of the HE (-, , ). These two glycoproteins induce IN and hemagglutinin-inhibiting (HAI) antibodies. The objectives of the present investigations were to assess the antibody responses of cattle during a naturally occurring and experimentally monitored infection that had induced severe pneumonic signs, including SFP. The kinetics of IN and HAI antibody responses of these cattle were analyzed with antigens of an endogenous RBCV strain, which were isolated from lung tissue from a fatally infected bovine from this SFP epizootic, and with antigens of the wild-type and the highly cell culture-adapted EBCV strains. The analysis was conducted with sera sequentially collected from cattle which had primary and secondary immune responses to the RBCV infection. PMID- 12204951_MATERIALS AND METHODS TI - AB - Experimental design. | One hundred five 6- to 8-month-old cattle were involved in this specific epizootic and were subjected to nasal swab and blood sampling and testing at the time of assembly at the order-buyer's barn (day 0), after transport on day 7, and thereafter on days 14, 21, 28, and 35, during the pathogenesis of and recovery from SFP . Nasal swab samples were taken for virological and bacteriological studies, while blood samples for serum harvest were collected for immunological investigations. The cattle were separated into five response groups based on clinical signs of respiratory tract diseases and results of RBCV isolations . Response groups 1 and 5 were utilized in the present analysis. Response group 1 included 72 cattle that exhibited clinical signs of respiratory tract disease and that nasally shed RBCV on day 0, day 7, or both. Seven randomly selected cattle of the group shedding RBCV on day 7 were investigated in detail. Seven calves in response group 5 were also included in these serological investigations because they remained clinically healthy and RBCV isolation negative during the epizootic and the 5-week follow-up period. To analyze sequential serum samples from the 14 cattle selected for their IN and HAI antibodies, we used antigens of three specific strains of bovine coronaviruses. Cell line, virus isolates, and virus purification. | Clone G of human rectal tumor (HRT-18) cells was used at the 24th passage level for RBCV and EBCV propagation . The wild-type RBCV strain 97TXSF-Lu15-2 was used at its second passage in clone G cells after initial isolation from lung tissue from a calf that died on day 8 of the epizootic . The highly cell culture-adapted EBCV strain L9-81 was originally isolated from bovine fetal kidney cell cultures as the EBCV Mebus strain . The EBCV L9-81 strain was propagated in 75 sequential passages in different bovine cell cultures, and the final six passages were done in clone G cells. The virulent wild-type EBCV strain LY138-3 from intestinal samples of a diarrheic calf was initially maintained by oral inoculation of conventional calves with induction of enteritis . It was used as an antigen in its third clone G passage. Partially purified preparations of RBCV strain 97TXSF-Lu15-2 and EBCV strains L9-81 and LY138-3 were employed for serological tests . IN assay. | Serum samples were prepared in quadruplicate and tested for IN activities according to a previously reported method . Serum 1745 with an IN titer of 128 and BCV antibody-free serum from a normal calf with an IN titer of <8 were included as positive and negative controls, respectively . The serum IN titers were expressed as the reciprocal of the highest serum dilution that completely inhibited cytopathic changes in 50% of the wells. Assays for HA and RDE. | The assays for hemagglutinin (HA) and RDE were performed as reported previously with washed rat RBC prepared as 0.5% suspensions with phosphate-buffered saline (pH 7.4) containing 0.05% bovine serum albumin. HAI test. | The HAI titers of serum samples were determined with partially purified virus stocks, which were diluted to contain 8 to 16 U of both HA and RDE as antigens . Again, serum 1745 with an HAI titer of 128 was included as a positive control, while BCV antibody-free serum from a normal calf with an HAI titer of <8 was used as a negative control . The serum HAI titers were expressed as the reciprocal of the highest dilution of serum sample that completely inhibited the aggregation of rat RBC. Data analysis and statistical methods. | The serum IN and HAI titers were transformed to base 2 logarithms for statistical analysis. All data were presented as means +- standard errors of the means. The IN and HAI activities of the two response groups were compared by an analysis of variance of repeated measures designed with a split-plot arrangement of treatments. Pairwise comparisons of treatment and day differences were assessed with Scheffe's test. Interaction effects were examined with pairwise t tests of least-squares means for preplanned comparisons of treatments at specific day levels. All tests were considered significant at a probability of <0.05. PMID- 12204951_RESULTS TI - AB - Comparison of IN activities of serial serum samples with antigens of RBCV strain 97TXSF-Lu15-2 and EBCV strains L9-81 and LY138-3. | The antibody responses with serum samples collected from cattle in response groups 1 and 5 had significantly higher overall kinetics of IN activities against RBCV strain 97TXSF-Lu15-2 than against EBCV strain L9-81, with P values of 0.0001 and 0.0001, and EBCV strain LY138-3, with P values of 0.0001 and 0.0025, respectively. Significant differences in IN activities against RBCV strain 97TXSF-Lu15-2 and EBCV strain L9-81 were observed in serum samples collected from cattle in response group 1, with P values of 0.0439, 0.001, <0.0001, <0.0001, and <0.0001 for results on days 7, 14, 21, 28, and 35, respectively. Similarly, the cattle in response group 5 had significantly lower levels on days 7, 14, and 28, with P values of 0.0095, 0.0164, and 0.0164, respectively. Significant differences between serum IN activities against RBCV strain 97TXSF-Lu15-2 and these against the EBCV strain LY138-3 antigen were also detected for cattle in response group 1, with P values of 0.0029, <0.0001, 0.001, and <0.0001 for results for days 14, 21, 28, and 35, respectively, and for cattle of response group 5 on days 7, 14, and 28 (P values of 0.0446, 0.0399, and 0.0335, respectively). Interestingly, IN antibody titers against EBCV strains L9-81 and LY138-3 in cattle of response groups 1 and 5 did not differ significantly during this 5-week investigation. Comparison of HAI activities of serial serum samples with antigens of RBCV strain 97TXSF-Lu15-2 and EBCV strains L9-81 and LY138-3. | The kinetics of the responses in HAI antibodies against RBCV strain 97TXSF-Lu15-2 were significantly greater than those against EBCV strain L9-81, with P values of 0.0001 and 0.0001 for the results for cattle of response groups 1 and 5, respectively. The differences between RBCV strain 97TXSF-Lu15-2 and EBCV strain LY138 were significant at a P value of 0.0001 for both groups 1 and 5 . The HAI activities against RBCV strain 97TXSF-Lu15-2 and EBCV strain L9-81 were remarkably different in serum samples collected from cattle in response group 1 for days 7, 14, 21, 28, and 35, respectively, with P values of 0.0483, 0.0004, 0.0035, <0.0001, <0.0001, and <0.0001. The HAI activities showed similar differences among cattle in response group 5 from day 0 onward with a P value of <0.0001. Levels of HAI antibodies against RBCV strain 97TXSF-Lu15-2 and EBCV strain LY138-3 were also substantially different for the cattle in response group 1 for days 7, 14, 21, 28, and 35 (P values of 0.0035, 0.0483, <0.0001, 0.0001, and <0.0001, respectively), and for the cattle in response group 5 for days 0, 7, 14, 21, 28, and 35 (P values of 0.0024, <0.0001, <0.0001, <0.0001, <0.0001, and <0.0001, respectively). Significant differences between serum HAI antibody kinetics against EBCV strain L9-81 and EBCV strain LY138-3 were not observed in cattle of either response group. FIG. 1. | IN (A and B) and HAI (C and D) antibody responses against RBCV strain 97TXSF-Lu15-2 (circles), EBCV strain L9-81 (triangles), and EBCV strain LY138-3 (squares) in serum samples from cattle. IN (A and B) and HAI (C and D) antibody responses against RBCV strain 97TXSF-Lu15-2 (circles), EBCV strain L9-81 (triangles), and EBCV strain LY138-3 (squares) in serum samples from cattle. Cattle in response group 1 (A and C) shed RBCV on day 7 and had clinical signs of respiratory tract disease, while cattle in response group 5 (B and D) did not develop adverse clinical signs and remained virus isolation negative, despite exposure to the animals that were shedding virus (response group 1). Data are means +- standard errors of the means (error bars). Results are shown for seven cattle (A and C) and for seven cattle (B and D). PMID- 12204951_DISCUSSION TI - AB - Prospectively designed and experimentally controlled sampling and testing of cattle during a naturally occurring SFP epizootic created fortuitous circumstances for analyzing the kinetics of antibody responses of cattle to a native, wild-type RBCV infection . This experiment also provided an excellent opportunity to compare antibody reactivities with both the wild-type RBCV and the highly cell culture-adapted and wild-type EBCV strains in serum samples collected from cattle involved in a naturally occurring RBCV infection. These conditions differ from experimentally induced infections that routinely involve giving virus inocula adapted to, and propagated in, cell cultures. Importantly, the IN and HAI antibody responses against RBCV were significantly greater than those detected with the wild-type EBCV strain LY138-3 and the highly cell culture-adapted EBCV. The EBCV strain L9-81 is virtually identical to the Norden vaccine strain that is used in attempts to protect against intestinal coronavirus infections of neonatal calves. Antigens of the highly cell culture-adapted EBCV strains reacted minimally with antibodies induced by naturally occurring RBCV infections. These findings call for analysis and a search for appropriate antigens in efforts to immunize cattle against these currently prevailing RBCV infections. Bovine coronaviruses contain a large, single-stranded RNA genome and may mutate in the natural host to generate quasi-species differences . Phenotypic changes may result from adaptation to, and propagation, of the virus in cell cultures, effecting the selection of host cell virus mutants. Differences in infectivity between the highly cell culture-adapted EBCV strain L9-81 and the wild-type strains of RBCV and EBCV consisted of a greatly expanded range of permissive host cells. This EBCV strain replicates readily, particularly in the presence of trypsin and in cultured bovine fetal kidney, spleen, thyroid, or adrenal cells, as well as in Vero cells . In contrast, the wild-type RBCV strains had restricted host cell ranges, because only clone G cells were permissive for initial isolation from clinical samples at low passage levels . Differences between the RBCV and EBCV strains also were observed in the HA patterns, cell fusion, and AE activities . Antigenic differences between RBCV and EBCV could not be detected by tests such as immunodiffusion immunofluorescence, immunoblotting, or enzyme-linked immunosorbent assays with antigens that include all the structural proteins of these coronaviruses . MAb specific for S glycoprotein with IN activity distinguished between EBCV strain L9-81 and several wild-type strains of EBCV . Differences between EBCV strain L9-81 and the wild type strain LY138-3 of EBCV in inhibition of AE activity with HE-specific MAb and in HA (4) reactivities were also detected. Genotypic differences consisted of changes in the regions of the polymerase and associated genes, in the genes of S and HE glycoproteins, and in other nonstructural genes . Nucleotide and deduced amino acid mutations were within the A and B immunoreactive domains of the S-I subunit of the spike protein of the RBCV strains but not the EBCV strains, which could be the basis for the observed diversity in antigenic stimulation during these naturally occurring infections . Comparatively, the differences in the kinetics of the primary bovine antibody responses to naturally occurring and experimentally monitored RBCV infections with the antigens of the wild-type RBCV and EBCV strains as well as the highly cell culture-adapted EBCV appear to be similar to responses of chickens to infections with the coronavirus of infectious bronchitis. Early antibodies generated during a primary immune response distinguished several serotypes of infectious bronchitis in both IN and HAI tests . PMID- 12204950 TI - Comparison of In-House and Commercial Slides for Detection by Immunofluorescence of Immunoglobulins G and M against Bartonella henselae and Bartonella quintana AB - | We compared the sensitivities and specificities of indirect fluorescent antibody tests developed in our laboratory and commercially available from Focus Technologies (FT; formerly MRL Diagnostic) for detection of serum antibodies to Bartonella spp. Serum samples tested were from patients with culture- or PCR-confirmed Bartonella quintana or B. henselae infections causing cat scratch disease (CSD), chronic bacteremia, or endocarditis. At a cutoff titer of 64, the FT test had higher sensitivity than our in-house test in detecting anti-B. henselae immunoglobulin G (IgG) antibodies in CSD patients (91.2 versus 52.9%; P < 0.001). The specificity in serum samples from 85 control patients was, however, lower with the FT test (87%) than with the in-house test (98.8%) (P = 0.002). A cutoff titer of 128 improves the specificity for the FT test but lowers the sensitivity to 85%. For patients infected with B. henselae, our in-house test, but not the FT test, enabled endocarditis to be detected more reliably. With the in-house test, titers of IgG against B. henselae of >=1,024 were found only in endocarditis patients and not in CSD patients. With the FT test, 19.1% of CSD patients had titers of IgG against B. henselae of >=1,024 (P < 0.001). Our in-house technique also improved detection of anti-B. quintana antibodies in homeless patients with endocarditis. IgG titers of >=1,024 were present in 75% of serum samples, but only in 16.7% of serum samples with the FT test (P = 0.004). Since each test has advantages over the other, the serological diagnosis of Bartonella infections would benefit if both tests were used concurrently. PMID- 12204950_Introduction TI - AB - There are now 18 species within the genus Bartonella. Three are well-known human pathogens: Bartonella bacilliformis , B. quintana , and B. henselae . Others have been isolated from patients on single occasions only: B. elizabethae (one endocarditis case) , B. vinsonii subsp. berkhoffii (one endocarditis case) , and B. vinsonii subsp. arupensis (one case of bacteremia) . B. clarridgeiae , and B. washoensis have been tentatively associated with cat scratch disease (CSD) and myocarditis, respectively. B. quintana and B. henselae are species of worldwide distribution . B. quintana was first described as the agent of trench fever in 1918 and is now known to be responsible for louse-borne bacteremia and endocarditis in homeless people and bacillary angiomatosis in AIDS patients . Humans are the only known reservoir of B. quintana, and transmission among people occurs via the body louse . B. henselae, a species first recognized in 1990 , is the main etiological agent of CSD and is also responsible for bacillary angiomatosis and peliosis hepatitis in immunocompromised (mainly AIDS) patients , as well as bacteremia and endocarditis . Cats are the main reservoir of B. henselae, and people become infected following cat scratches or bites. A role for the cat flea (Ctenocephalides felis) as a vector for human transmission has been proposed . B. henselae comprises two distinct genotypes, B. henselae Houston and B. henselae Marseille . Diagnostic techniques for infections with Bartonella spp. include culture of the pathogen , molecular biology techniques, especially PCR, to amplify Bartonella sp. genes , and serology . The usefulness of these various techniques may vary with the disease present. Culture of Bartonella spp. may be successful with blood from bacteremic patients , from cardiac valve specimens from endocarditis patients , and from skin, lymph node, or other organ biopsy samples from patients with bacillary angiomatosis . By contrast, B. henselae is only rarely isolated from patients with CSD . Amplification of Bartonella sp. DNA in tissue biopsy samples is an invasive technique and is mainly useful for patients with CSD (lymph node specimen) , bacillary angiomatosis (angiomatous lesions of skin or other organs) , or endocarditis (cardiac valve when removed) . Serology is the only noninvasive diagnostic technique, and it has been evaluated in the diagnosis of CSD , bacteremia , and endocarditis . The indirect fluorescent antibody (IFA) test was first described for CSD by Regnery et al. , who used B. henselae grown in Vero cells as antigens. It remains the most frequently used technique, and many laboratories have performed Bartonella serology using tests developed in-house, with reported sensitivities varying considerably, from nearly 100% to less than 30% . Commercially prepared antigen slides are now available for B. henselae and B. quintana serology , and in this report we compare the sensitivity and specificity of one of these tests with our in-house IFA test, which has been used by our laboratory for 10 years. We compared the abilities of the two tests to detect immunoglobulin G (IgG) and IgM antibodies in serum samples from patients known to have CSD (B. henselae), chronic bacteremia (B. quintana), or endocarditis (B. henselae and B. quintana). PMID- 12204950_MATERIALS AND METHODS TI - AB - Patients and serum samples. | Serum samples from 104 patients infected with Bartonella spp., comprising CSD patients (n = 68), patients with endocarditis (n =17; 5 patients with B. henselae endocarditis and 12 with B. quintana endocarditis), and homeless people (n = 19) with chronic B. quintana bacteremia but without endocarditis, were used in the study. Diagnoses of CSD were based on PCR amplification of B. henselae DNA from lymph node samples of patients with regional lymphadenopathy and close contact with cats . Bartonella endocarditis was diagnosed by positive blood cultures or PCR amplification of DNA of the organism from valve samples . Diagnoses of B. quintana bacteremia for the 19 homeless patients were based on blood culture results . Controls included 40 apparently healthy blood donors (group A), 10 patients initially thought to have CSD because of lymphadenopathy but later, by culture, found to have tuberculosis (group B), and 35 patients who were seropositive for bacterial or viral diseases not related to Bartonella species and for whom stored serum samples were available (group C). Group C included patients seropositive for cytomegalovirus , Epstein-Barr virus , hepatitis B virus , human immunodeficiency virus , acute Q fever , chronic Q fever , or Mediterranean spotted fever caused by Rickettsia conorii . Immunofluorescence assays. | All serum samples were examined by IFA for the presence of antibodies to B. quintana and B. henselae. For antigens we used either our slides prepared in-house or commercial slides from Focus Technologies (FT), Cypress, Calif. (distributed in France by Eurobio, Paris, France). We prepared in-house slides by using B. henselae strain Houston-1 or B. quintana strain Oklahoma as the antigen, as previously described . Briefly, bacteria were grown in the human endothelial cell line ECV 304 for 2 weeks before infected cells were recovered and lysed by sonication. Cell suspensions were centrifuged at 1,500 rpm (700 x g) for 10 min to remove cell debris, and bacteria were purified by using a sucrose gradient (phosphate-buffered saline [PBS] with 25% sucrose; 6,000 x g for 30 min). The resulting pellet was washed in PBS twice (6,000 x g for 10 min), and purified bacteria were pelleted by centrifugation (10,000 x g for 10 min), resuspended in 1 ml of 0.1% paraformaldehyde, and incubated at 4C for 24 h for fixation. The bacterial suspension was repelleted by centrifugation (10,000 x g for 10 min), resuspended in sterile distilled water, and stored at -80C before use. This whole-cell antigenic preparation was plated onto Dynatech slides and used for detection of either IgM or IgG antibodies by IFA. FT uses the same B. henselae Houston-1 and B. quintana Oklahoma strains for preparation of its commercial slides. Bacteria are grown in Vero cells for preparation of slides used to detect IgG and on blood agar before being harvested and diluted in yolk sac fluid for slides used to detect IgM. For both the FT and in-house tests, IgG titers of >=64 and IgM titers of >=20 were used as cutoff titers with B. henselae or B. quintana antigens. Statistical analysis. | Differences in antibody titers between groups and/or between IFA techniques were analyzed by using the chi2 test for categorical variables or Student's t test for continuous variables. A two-sided P value of <0.05 was considered significant. PMID- 12204950_RESULTS TI - AB - With serum samples from CSD patients, the sensitivities of the in-house and FT tests using a cutoff titer of 64 for detection of anti-B. henselae IgG antibodies were 52.9 and 91.2%, respectively (P < 0.001). Median IgG titers were 64 with the in-house test (range, <64 to 256) and 256 with the FT test (range, <64 to 1,024) . IgM antibodies were detected at a low titer (i.e., 20) for only two patients with the in-house test and one patient with the FT test. IgG antibodies cross-reacting with B. quintana were detected in most of the serum samples (35 of 36 [97.2%]; median titer, 64) from CSD patients with the in-house test, but in only one serum sample with the FT test (P < 0.001). All endocarditis patients had IgG titers of >=64 against both B. quintana and B. henselae antigens. However, significant differences in antibody titers were found between the in-house and FT tests. With the in-house test, 9 of 12 (75%) serum samples from patients with B. quintana endocarditis had homologous IgG titers of >=1,024, whereas such high titers were present in only 2 of 12 (16.7%) patients with the FT test (P = 0.004). Also, median homologous IgG titers in serum samples from B. quintana endocarditis patients were 1,024 with the in-house test but only 256 with the FT test (P < 0.01) . Anti-B. henselae cross-reacting antibodies were found in all B. quintana endocarditis patients (median IgG titers of 1,024 and 256 with the in-house and FT tests, respectively). All five B. henselae endocarditis patients had homologous IgG titers of >=1,024 with both tests (range, 1,024 to 4,096; median titers of 2,048 and 1,024 with the in-house and FT tests, respectively). Most of them had IgG antibodies which cross-reacted with the B. quintana antigen, albeit at lower titers (median titers of 512 and 256 with the in-house and FT tests, respectively). No antibodies to B. quintana were found in 2 of 19 (10.5%) and 3 of 19 (15.8%) serum samples from homeless patients with B. quintana bacteremia with the in-house and FT tests, respectively. For the remaining patients, anti-B. quintana IgG antibodies were found at low titers (range, 64 to 512; median, 128 with both tests) . Cross-reacting anti-B. henselae IgG antibodies were found for most of these patients, at similar titers. The two bacteremic patients with a homologous IgG titer of 512 had no echocardiographic evidence of endocarditis. None of the 40 serum samples from blood donors (group A) were found to contain antibodies to B. quintana or B. henselae by either of the two tests. Among the group B controls, only one had an IgG titer (of 64) detectable by the in-house test, while with the FT test, serum samples from this and two additional controls had IgG titers of 64. No reactive antibodies were found for the 35 group C controls when the in-house test was used. In contrast, serum samples from 8 of 35 (22.8%) group C controls had IgG titers (of 64 to 256) to Bartonella spp. in the FT test. Cross-reacting IgG antibodies were found in serum samples from three cytomegalovirus patients, two chronic Q fever patients, and three patients with Mediterranean spotted fever. Thus, by using a cutoff titer of >=64, the specificities of the in-house and FT tests for reactive IgG were both 100% for blood donors (group A); 90 and 70%, respectively, for group B; and 100 and 90%, respectively, for group C (P = 0.002). For all 85 controls taken together, the specificities of the FT and in-house tests were 87 and 98.8%, respectively (P = 0.002). To ensure a specificity of >=95% with the FT test, a titer of >=128 rather than >=64 should have been considered the cutoff. With this cutoff titer, the sensitivity would be lower (85.3%) for CSD patients, but this would still be superior to that of the in-house test. FIG. 1. | Anti-B. henselae Anti-B. henselae IgG titers in CSD patients. FIG. 2. | Homologous IgG titers in patients with B. quintana Homologous IgG titers in patients with B. quintana endocarditis. FIG. 3. | Anti-B. quintana Anti-B. quintana IgG titers in homeless patients with chronic B. quintana bacteremia. PMID- 12204950_DISCUSSION TI - AB - Bartonella serology has been performed in our laboratory for several years now, by using B. quintana or B. henselae grown in cell culture as the antigen. The aim of our study was to compare the sensitivity and specificity of our in-house test to that of a recently available commercial test from FT. With CSD patients, our results confirm the previously reported high sensitivity of the FT serological test , whereas a lower sensitivity and significantly lower median IgG titers were found with our in-house test. Reactive IgM antibodies were rarely detected for CSD patients by either test, and these antibodies would thus appear to be of little use diagnostically, as previously mentioned . The sensitivity of the B. henselae IFA test first described by Regnery et al. for detection of CSD has been reported from different laboratories as 32% to 100% . Enzyme immunoassay (, ,, , , ) and Western blotting techniques have not been consistently superior to IFA for the diagnosis of CSD. Several factors may influence the apparent sensitivity of a serological test. First, sensitivity may change according to disease case definition. Historically, a CSD case has been defined as the combination of regional lymphadenopathy with characteristic histopathological features (granuloma), the presence of an inoculation skin lesion (e.g., cat scratch), a history of cat contact, a positive result on a skin test using an antigen prepared from a CSD lymph node exudate, and negative laboratory results for other causes of lymphadenopathy . More recently, less restrictive case definitions have been used (e.g., regional lymphadenopathy and a recent contact with a cat ), since the skin test antigen is no longer available and lymph node biopsies are performed infrequently. However, a number of patients (e.g., 14% in the study of Flexman et al. ) with elevated anti-B. henselae antibody levels report no previous contact with a cat, and many patients present with clinically atypical cases of CSD . Use of a restrictive case definition for CSD may erroneously increase the sensitivity of a serological test because only patients with evident epidemiological and clinical findings suggestive of CSD will be considered. Thus, in a series of 354 suspected cases of CSD where serum samples were examined for anti-B. henselae antibodies, Flexman et al. reported a sensitivity of 18% (with a cutoff titer of >=1:64). However, when only the 98 patients who met the clinical case definition of CSD (lymphadenopathy and recent contact with a cat) were considered, the sensitivity was 54%. Accurate determination of the sensitivity and specificity of a serological test requires well-defined patient groups (infected and uninfected), identified by objective diagnostic procedures rather than clinical and epidemiological data. In a few recent studies, B. henselae DNA amplification from lymph node biopsy samples was used for confirmation of the diagnosis of CSD in a portion of cases . Giladi et al. recently evaluated an enzyme immunoassay for detection of anti-B. henselae antibodies in PCR-confirmed cases of CSD. We report the first evaluation of IFA tests for Bartonella-infected patients for whom diagnoses were unambiguously established by either culture or DNA amplification using PCR. Diagnoses of diseases not related to Bartonella spp. in control patients were also carefully documented The apparent sensitivity of a serological test may also be greatly influenced by the cutoff values used. Usually, cutoff titers are chosen to obtain a specificity of at least 95% in control patients . The control groups are usually composed of a mixture of apparently healthy blood donors and patients with a disease distinct from the disease being tested. Using an IFA test for detection of anti-B. henselae IgG antibodies at a cutoff titer of 64 in CSD patients, Regnery et al. , and Zangwill et al. in the United States, reported sensitivities of 88 and 84%, respectively, but the specificity was only similar90%. In each study, specificity was high at similar99% when a titer of 512 was considered the cutoff, but sensitivities decreased to only 67 and 64%, respectively. Bergmans et al. in The Netherlands reported a sensitivity as low as 31.8% by using a cutoff titer of 512, which was needed to obtain a specificity of >=95% in blood donors. The cutoff titer may depend on the seroprevalence of antibodies to Bartonella spp. in the population, and this may vary with geographical region . We found a specificity of 98.8% with the in-house test but only 87% with the FT test for the 85 controls we studied. With the FT test, the specificity was only 70% for the 10 patients with tuberculous lymphadenopathy. In our laboratory, cultures of about 5% of lymph node biopsy samples submitted yield Mycobacterium tuberculosis, while Bartonella spp. are demonstrated in approximately 30% of samples by culture or PCR. A highly specific test for CSD is critical in order to accurately rule out other, more serious diseases which may present as chronic lymphadenopathy, including tuberculosis or lymphoma. To ensure a specificity of >=95% with the FT test, a cutoff titer of >=128 should be used instead of >=64. It is becoming clear that some patients with typical CSD have no detectable antibodies to B. henselae, as shown in our study. Demers et al. reported a seronegative patient diagnosed by culture of B. henselae from a lymph node. Serology may be negative early in the course of the disease . Also, the antigenic preparation used for serology may greatly influence the results. Low antibody titers are usually found when B. henselae grown on agar is used as the antigen rather than cell culture-grown organisms . Poor antibody responses to B. henselae antigen have also been ascribed to the antigenic variability between the two B. henselae serotypes that have been described . Both the in-house and FT tests use B. henselae strain Houston-1 grown in cell culture as the antigen. Discrepancies between the two techniques with CSD and endocarditis patients may be explained by the mode of antigen preparation and conservation, as this may greatly influence antigenic-epitope recognition by serum antibodies . Finally, other Bartonella species (including B. clarridgeiae) may cause CSD , and antibodies to these organisms do not react with B. henselae. This was not the case in our study, since our PCR results showed that all CSD patients were infected with B. henselae, and the lack of detectable antibodies to B. henselae in some patients may then truly represent a lack of sensitivity in the serological tests. For homeless patients with chronic B. quintana bacteremia, both the FT and in-house tests detected only low levels of homologous IgG antibodies, and both failed to detect any reactive antibodies in many patients with culture-positive B. quintana bacteremia. There are few serological data on B. quintana infections. Although two large epidemics of trench fever occurred during the first and second World Wars , B. quintana had not yet been cultured and serology was unavailable . More recently, data from homeless people with B. quintana infections indicate that most suffer from chronic, intermittent bacteremia and that many do not have detectable antibodies to B. quintana when infections are diagnosed . The in-house and FT tests both detected homologous antibodies in patients with B. henselae endocarditis. However, only IgG titers obtained with the in-house technique allowed efficient discrimination between endocarditis patients and those with CSD. All B. henselae endocarditis patients presented with homologous IgG titers of >=1,024, whatever the technique used. In contrast, none of the CSD patients presented with IgG titers of >=1,024 with the in-house technique, whereas 13 of 68 (19.1%) had IgG titers of >=1,024 with the FT technique (P < 0.001). Thus, our in-house technique, but not the FT technique, may help define patients with a high likelihood of Bartonella-related endocarditis. In our experience, this may be critical, because Bartonella sp. endocarditis is a chronic, clinically unspecific disease. In this study, an anti-B. henselae IgG titer of >=1,024 with the in-house technique was 100% predictive of endocarditis , which is in accordance with previous reports from our laboratory . The in-house technique was more effective than the FT technique in detecting homologous IgG antibodies in serum samples from homeless patients with B. quintana endocarditis. The strain of B. quintana and the number of passages in culture have previously been reported to influence seroreactivity . The FT and in-house techniques use the same B. quintana Oklahoma strain, grown in cell cultures. The use of a cell culture- rather than an agar-grown antigen may favor the detection of specific antibodies, as previously described for B. henselae . With the in-house test, a homologous IgG titer of >=1,024 was 100% predictive of B. quintana endocarditis. Many B. quintana endocarditis patients, however, presented with lower IgG titers, and titers of 256 or 512 were found in 5 of 12 (41.6%) endocarditis patients. Such titers were also found in 7 of 19 (36.8%) homeless people with chronic bacteremia but with no endocarditis. These two groups of patients, then, could not be reliably differentiated by using IgG titers to B. quintana. Cross-reactions have been described between various Bartonella spp. and with Chlamydia trachomatis , Coxiella burnetii , and many other species, including Rickettsia rickettsii, Ehrlichia chaffeensis, Treponema pallidum, Francisella tularensis, and Mycoplasma pneumoniae . Cross-reactivity of serum samples containing IgM antibodies directed at Epstein-Barr virus capsid antigen has also been reported . The cross-reactivity between Bartonella sp. and Chlamydia sp. antigens has been particularly troublesome, and endocarditis supposedly due to a Chlamydia sp. on the basis of serology has later been shown to be due to a Bartonella sp. . The possibility of cross-reactions interfering with the serological diagnosis of Bartonella endocarditis is removed if all suspect serum samples are also tested for antibodies to C. burnetii and Chlamydia spp. In conclusion, our study has shown the high sensitivity of the FT test in detecting IgG against B. henselae in CSD patients. It should, however, be used with a cutoff titer of 128 to raise the specificity of the test to >=95%. Highly specific tests are essential for CSD, because a misdiagnosis may delay the detection of more serious conditions (e.g., lymphoma). Our in-house test had an advantage over the FT test in that it allowed CSD patients to be reliably differentiated from those with B. henselae endocarditis. An IgG titer of >=1,024 was 100% predictive of B. henselae endocarditis. Our in-house test was also more effective than the FT test at detecting antibodies to B. quintana in homeless patients, especially those with endocarditis. An anti-B. quintana IgG titer of >=1,024 was 100% predictive of endocarditis, although many patients with endocarditis presented with lower antibody levels. Since both tests have advantages, concurrent use of both tests should be considered in order to improve the diagnosis of B. henselae and B. quintana infections. PMID- 12204967 TI - Double-Blind Study To Evaluate Flow Cytometry Analysis of Anti-Live Trypomastigote Antibodies for Monitoring Treatment Efficacy in Cases of Human Chagas' Disease AB - | The validation of flow cytometry analysis of anti-live trypomastigote antibodies (FC-ALTA) to monitor cure after treatment of Chagas' disease was evaluated with serum samples from treated and nontreated chagasic patients. After optimization of the original technique, toward better sensitivity and applicability to field surveys, we design a double blind study of 94 coded samples classified into the following categories: patients not treated (NT) and patients treated but not cured (TNC), both presenting positive conventional serology and xenodiagnosis; patients treated and cured (TC), showing negative serology and xenodiagnosis; and patients treated under evaluation (TUE), who presented positive or oscillating conventional serology (CSA) but negative xenodiagnosis. Coded samples, diluted 1:256, were assayed by incubation with live cell culture trypomastigotes, which were subsequently stained with fluorescein isothiocyanate-conjugated anti-human immunoglobulin G, with prior fixation and analysis by flow cytometry. The results were expressed as the percentages of positive fluorescent parasites (PPFP) for each individual sample, establishing 20% PPFP as the cutoff between negative and positive results. Our data demonstrated that all NT and TNC presented positive results while all but one TC had a PPFP lower than 20%. Analysis of TUE demonstrated a wide degree of reactivity, with PPFP values that were negative (PPFP <= 20%), low positive (20% < PPFP <= 50%), and high positive (PPFP > 50%). As TUE with negative PPFP presented negative xenodiagnosis and positive or oscillating CSA, they were classified as dissociated according to the criteria of Krettli and Brener (J. Immunol. 128:2009-2012, 1982) and could indeed be considered cured after chemotherapy. This study demonstrates and validates the use of FC-ALTA to easily identify anti-live trypomastigote membrane-bound antibodies, offering another approach for investigating and monitoring the efficacy of specific chemotherapy in cases of human Chagas' disease. PMID- 12204967_Introduction TI - AB - Chagas' disease is a parasitic infection caused by the protozoan Trypanosoma cruzi, which is widespread in South and Central America, and transmitted mainly by an invertebrate vector, triatomine . Although autochthonous cases are rare in other localities, transmission of Chagas' disease by blood transfusion may represent an increasing public health problem in countries in which Chagas' disease is not endemic . Chemotherapy with nitroheterocyclic compounds has been indicated for the treatment of acute cases . In patients with chronic disease, however, the value of chemotherapy is still controversial. So far, it has been recommended for the treatment of patients with recent chronic disease who have not yet developed clinical symptoms . One of the major challenges regarding the evaluation of treatment effectiveness is the lack of truthful laboratorial approaches for use as tools for cure criteria. Two categories of tests are available, including serological and parasitological methods. Parasitological tests are based on parasite demonstration by hemoculture, xenodiagnosis, or parasitological molecular test (PCR), whereas serological methods evaluate the presence of specific antibodies by immunological methods, such as indirect hemagglutination (IHA), indirect immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). Using serological approaches, Krettli and Brener proposed that sera from chronic chagasic patients present two types of anti-parasite antibodies with different functional activities named lytic antibodies (LA) and conventional serology antibodies (CSA). LA are associated with resistance in active ongoing infection and can be detected by complement-mediated lysis (CoML) and indirect immunofluorescence, all with live trypomastigotes . On the other hand, CSA are neither associated with resistance nor able to bind to live parasites, but they do react to soluble or fixed epimastigote antigens and are detected by different immunological methods . Considering these findings, clinical trials have demonstrated that LA gradually become negative after treatment, whereas CSA can remain positive over decades. So it has been proposed that the cure criterion for Chagas' disease should be based on either negative parasitological tests or the absence of anti-live trypomastigote antibodies (ALTA) . A flow cytometric method to detect ALTA (FC-ALTA) has been previously described as a new approach for monitoring the efficacy of treatment in cases of human Chagas' disease. The performance of FC-ALTA has been demonstrated to be comparable to that of CoML, offering an alternative method for easily identifying anti-live T. cruzi membrane-bound antibodies . In this study, we describe an optimization of FC-ALTA analysis and evaluate its performance for clinical studies. A double-blind study, with serum samples from treated and nontreated chagasic patients, was conducted to validate the method, reemphasizing its applicability for monitoring cure after treatment of Chagas' disease. Moreover, the data presented here offer an optimization of the original technique, making our method more sensitive and applicable to field studies. PMID- 12204967_MATERIALS AND METHODS TI - AB - Patients. | The inclusion of all subjects in our investigation had the approval of the FIOCRUZ Ethical Committee (Brazilian Health Ministry). In this study, we analyzed 94 patients (53 females and 41 males) ranging in age from 6 months to 68 years assisted by one of us at the Faculdade de Medicina, Universidade Federal de Goias, GoiAnia, Goias, Brazil. Chagas' disease diagnosis was established in all patients by positive xenodiagnosis and three positive serological tests, including IHA, IFA, and ELISA. All patients in the acute (n = 13) and subacute (n = 3) phases were treated with benznidazole (BZ) (60 mg/kg of body weight/day for 60 days). Patients in the chronic phase (n = 78) were treated with BZ (n = 44), allopurinol (n = 3), nifurtimox (n = 3), or BW349C59 (n = 6) or not treated (n = 22). In this study, we did not focus on the efficacy of the different therapeutic schemes used since it was not the major goal of our present investigation. After clinical, parasitological, and serological follow-up studies, ranging from 3 to 26 years, patients were classified into three different categories: not treated (NT) (n = 22), all patients with chronic disease; treated but not cured (TNC) (n = 19), 4 patients with acute- and 15 patients with chronic-phase disease; treated and cured (TC) (n = 25), 8 patients with acute-, 3 patients with subacute-, and 14 patients with chronic-phase disease; and treated under evaluation (TUE) (n = 28), 1 patient with acute- and 27 patients with chronic-phase disease . NT and TNC patients had positive results on both serological and parasitological tests. Patients were considered TC only when both tests, serological and parasitological, were consistently and repeatedly (at least eight times) negative. Patients presenting with positive (n = 17) or oscillating (n = 11) serology with negative parasitological tests were considered TUE. Samples were coded and stored pure at -20C or in 50% glycerol at room temperature. Conventional serological tests. | Three kinds of serological tests were performed as recommended by the World Health Organization : IHA (Imunoserum, Sao Paulo, Brazil), IFA (Biolab, Rio de Janeiro, Brazil), and ELISA (Abbott). At the beginning of the study, we used the complement fixation reaction, which was available at that time, to screen some patients. Xenodiagnosis. | Xenodiagnosis was performed on all patients before and after treatment as described by Cerisola et al. by using triatomine from Dipetalogaster maximus and Triatoma infestans according to availability. Forty triatomines, after a 15-day fast, were allowed to feed on each patient for 30 min. Microscopic examination of intestinal contents was carried out 30 and 60 days after feeding. The number of xenodiagnoses per patient ranged from 8 (320 triatomines) to 114 (4,500 triatomines). Cell line and parasites. | NTCT clone 929 cells (L929, ATCC CCL 1) were maintained in our laboratory by serial passages and frozen in liquid nitrogen. For the assays, 5 x 105 L929 cells were seeded in tissue culture flasks (25 or 75 cm2, Falcon; Becton Dickinson, San Jose, Calif.) with 10 ml of RPMI medium (GIBCO, Grand Island, N.Y.) containing 10% fetal bovine serum (FBS) and incubated at 37C in humidified air containing 5% CO2. After 2 or 3 days, the monolayer was infected with 3 x 106 to 7 x 106 trypomastigotes of T. cruzi strain CL . Trypomastigotes were obtained initially from infected mice and subsequently from the supernatant of another infected monolayer. Infected cells were washed daily with RPMI-5% FBS. The cultures were maintained in RPMI and 10% FBS at 33C, 5% CO2, and 95% humidity . After 5 to 7 days of incubation, the trypomastigotes started being released into the supernatant. The cell culture was transferred to a 50-ml tube and centrifuged at low speed (100 x g for 10 min at room temperature) to remove cell debris and some contaminating amastigotes. Trypomastigotes recovered from the supernatant were washed three times (1,000 x g for 15 min at 4C) and resuspended in phosphate-buffered saline (PBS) and 10% FBS at a concentration of 1 x 106 parasites/ml. A 50-mul sample was used for FC-ALTA. FC-ALTA. | The immunofluorescence reaction was performed as described by Martins-Filho et al. , modified as follows. Live trypomastigotes (500,000) were incubated at 37C for 30 min in the presence of a 1:256 final dilution of serum (whole or diluted 1:1 in glycerol), using a coded serum sample. After incubation with sera, parasites were washed once with PBS containing 10% FBS. Parasite suspensions were reincubated at 4C for 1 h or 37C for 30 min in the dark in the presence of fluorescein isothiocyanate (FITC)-conjugated anti-human immunoglobulin G (IgG) antibody (anti-whole molecule) (Sigma, St. Louis, Mo.) diluted 1:400 in PBS-10% FBS. The use of different temperatures and times of incubation with FITC-conjugated anti-human IgG antibody represented the major change from the original method described by Martins-Filho et al. , where incubation was performed only at 4C for 60 min. Each assay included an internal control of nonspecific binding as well as positive and negative controls. To monitor unspecific binding, parasites not exposed to human serum were incubated with FITC-conjugated anti-human IgG. Positive controls included sera from NT chagasic patients, whereas samples from uninfected individuals were included as negative controls. After being stained, labeled parasites were washed with PBS-10% FBS and fixed on ice for 30 min with a fix solution (10 g of paraformaldehyde per liter, 1% sodium cacodylate, 6.65 g of sodium chloride per liter, and 0.01% sodium azide, pH 7.2). Flow cytometry analysis was performed up to 24 h after parasite fixation. FACScan data storage and analysis. | Flow-cytometric measurements were performed on a Becton Dickinson FACScan interfaced to an Apple Quadra FACStation. CellQuest software was used for both data storage and analysis. Trypomastigotes were identified based on their specific forward (FSC) and side (SSC) laser-scattering properties. Following FSC and SSC adjustments, parasites were localized on FSC (size) by SSC (internal complexity-granularity) dot plot distribution . Parasites were then selected by gating on the FSC by SSC dot plot distribution. An average of 8,000 to 9,000 gated trypomastigotes was analyzed for relative fluorescence intensity by using single FITC histograms for each individual sample. A control marker of up to 2% of the parasites that were fluorescence positive was set up on the FITC-conjugated internal control histogram . This marker was used to determine the percentage of positive fluorescent parasites (PPFP) for each sample. Figures show representative histograms for the negative and positive controls, respectively. Data analysis was performed by establishing 20% PPFP as the cutoff between negative and positive results as described by Martins-Filho et al. . Thus, samples were considered positive when the PPFP was >20% and negative when the PPFP was <=20%. Positive results were additionally classified according to the method of Cordeiro et al. as low positive (20% < PPFP <= 50%) and high positive (PPFP > 50%). Statistics. | Statistical analysis was performed using one-way analysis of variance followed by Student's t test. Differences were considered significant when the P value was <0.05. FIG. 1. | FC-ALTA. FC-ALTA. (a) Dot plot analysis of a representative trypomastigote distribution (R1) based on size and granularity. Single histograms represent PPFP values for FITC-conjugated anti-human IgG internal control (b), negative control (c), and positive control (d). TABLE 1 | Patients, conventional serology, and xenodiagnosis PMID- 12204967_RESULTS TI - AB - Optimization of original method for FC-ALTA. (i) Effect of incubation temperature on PPFP values. | In order to evaluate the effect of incubation conditions on the performance of FITC-conjugated anti-human IgG antibody binding, we performed a parallel study in which a 1-h incubation at 4C was compared to a 30-min incubation at 37C. Figure shows representative histogram analyses demonstrating that the latter condition led to higher PPFP values for samples from one NT patient, whereas no change was detected for a sample from a TC patient . Using a range of samples, our data demonstrated that incubation of parasites for 30 min at 37C increases the sensitivity of FC-ALTA (P = 0.0007 for NT patients and P = 0.0001 for TNC patients) with no effect on its ability to discriminate samples with PPFP values of <=20% (P = 0.68) . Therefore, we have adjusted this condition in the original method in order to improve the sensitivity of FC-ALTA. (ii) Comparison of PPFP values between pure and GPS. | With the purpose of fitting the method for field studies, in which storage of frozen samples (FS) may not be possible, we tested the applicability of our method to glycerol-preserved samples (GPS) that can easily be maintained at room temperature. Parallel studies which used 70 pairs of FS and GPS samples were carried out, and the PPFP values for each individual sample are presented in Fig. . Data analysis was performed by grouping the samples based on the PPFP values obtained with FS as negative (PPFP <= 20%), low positive (20% < PPFP <= 50%), or high positive (PPFP > 50%) followed by comparison with the PPFP values obtained with GPS. One out of 29 samples with negative results (PPFP = 20.0%) became low positive (PPFP = 25.0%) when tested as GPS. Two out of 11 samples with low-positive results showed a distinct result when tested as GPS. One became negative (PPFP = 26.0 to 17.0%) and another one became high positive (PPFP = 50.0 to 66.0%). No changes were observed in the performance of FC-ALTA for all 40 samples that showed high-positive results. Statistical analysis did not show any differences between the mean PPFP values obtained from GPS and FS (P = 0.89 for results with PPFP <= 20%, P = 0.66 for results with 20% < PPFP <= 50%, and P = 0.68 for results with PPFP > 50%). Performance of FC-ALTA with treated and nontreated patient samples. (i) PPFP values from NT and TNC patients. | PPFP values from chagasic patients with positive serology and xenodiagnosis are presented in Fig. . Our data demonstrated that all NT patients showed positive PPFP values. Moreover, it was interesting that NT samples were always high positive (PPFP > 50%) . The TNC patients were subdivided into two groups as they received treatment during the acute or chronic phase of the disease . In the same manner, all TNC patients presented PPFP values greater than the 20% cutoff, and then they did not differ from NT patients. However, in this group we found two low-positive samples with PPFP values of >20% and <=50% (samples 38 and 76), suggesting that the therapy was able to decrease parasitemia and also lower antibody titers but not enough to make them free of infection . (ii) PPFP values from TC individuals. | PPFP results for TC individuals are presented in Fig. . The patients were subdivided into three groups as they received treatment during the acute, subacute, or chronic phase of the disease. All but one (sample 17) TC individual had PPFP values lower than 20%. With the aim of further investigating this unexpected result, after decoding the patient identification number, we retested this sample from 1997 along with two other previous samples from 1981 and 1989, taken before treatment and early after treatment, respectively. The results of this analysis confirm the high reactivity of this patient before and at different times after treatment (PPFP = 95.5, 94.3, and 95.1%, respectively). (iii) PPFP values from TUE patients. | PPFP results for TUE individuals are presented in Fig. . The data demonstrated a wide degree of reactivity, including 12 samples classified as high positive (PPFP > 50%), 9 samples classified as low positive (20% < PPFP <= 50%), and 7 samples presenting negative results (PPFP <= 20%) . These seven samples with negative PPFP results are classified as dissociated according to the Krettli and Brener criteria . As previously described , these individuals should be considered cured since they presented negative results for xenodiagnosis and PPFP. Additional analysis demonstrated that all patients with less than 10 years of follow-up after treatment (9 of 9) showed positive PPFP values and 78.0% of them (7 of 9) had high-positive results (PFPF > 50%) . Patients with more than 10 years of follow-up after treatment presented a wide range of results. The frequency of samples with high positive results dropped to 31.5% (6 of 19), and the frequency of low-positive and negative results increased to 31.5% (6 of 19) and 37.0% (7 of 19), respectively. Statistical analysis demonstrated that results for patients in the <=10 years after treatment group differed significantly from those for patients in the >10 years after treatment group (P < 0.05). To further address the differential reactivity of the TUE patient group, we subdivided the patients based on their results from CSA analysis . Then the PPFP values from patients with positive CSA and those with oscillating CSA were compared. The data demonstrated that both subgroups had negative, low-positive, and high-positive results. The percentages of samples with negative, low-positive, and high-positive PPFP values were 36.5% (4 of 11), 36.5% (4 of 11), and 27.0% (3 of 11) for patients with oscillating CSA and 18.0% (3 of 17), 29.0% (5 of 17), and 53.0% (9 of 17) for those with positive CSA, respectively. Interestingly, the frequency of high-positive results was higher within the group with positive CSA (53.0 versus 27.0%). Together, our data support the hypothesis that high-positive results with FC-ALTA are in general associated with positive CSA, regardless of the clinical group analyzed (NT, TNC, or TUE). FIG. 2. | Representative histograms showing effect of incubation temperature on PPFP values. Representative histograms showing effect of incubation temperature on PPFP values. Parasites were incubated in parallel study with a 1:256 dilution of PBS-10% FBS-diluted human sera (from NT and TC patients) followed by a 1-h incubation at 4C (a) or a 30-min incubation at 37C (b) with FITC-conjugated anti-human IgG (whole-molecule) antibody. (c) A range of samples were tested for both protocols (-4C for 60 min and 37C for 30 min [*]), including samples from the NT (n = 12), TNC (n = 9), and TC (n = 4) patient groups. Statistical analysis demonstrated significant differences between PPFP values obtained at 37C for 60 min in comparison to those obtained at 4C for 60 min for the NT and TNC patient groups but not for the TC patient group. FIG. 3. | Comparison of PPFP values between pure and GPS. Comparison of PPFP values between pure and GPS. Parallel studies with pairs of FS (*) and GPS samples were carried out, and the results were presented as PPFP values. Samples were grouped based on the PPFP values obtained with FS followed by comparison with the PPFP values obtained with GPS. No significant differences were observed between mean PPFP values obtained from FS and GPS. FIG. 4. | Analysis of PPFP values from patients with positive xenodiagnosis and positive conventional serology. Analysis of PPFP values from patients with positive xenodiagnosis and positive conventional serology. The performance of FC-ALTA with samples from the NT (a) and TNC (b) patient groups was investigated by using FS, and data analysis was performed by establishing a 20% PPFP value as the cutoff between negative and positive results, as described by Martins-Filho et al. . Positive results were additionally classified according to the method of Cordeiro et al. as low positive (20% < PPFP <= 50%) and high positive (PPFP > 50%). The TNC patients were subdivided into two groups, as they received treatment during the acute (A) or chronic (C) phase of the disease. FIG. 5. | Analysis of PPFP values from patients with negative xenodiagnosis and negative conventional serology. Analysis of PPFP values from patients with negative xenodiagnosis and negative conventional serology. The performance of FC-ALTA for samples from the TC patient group was investigated by using FS, and data analysis was performed by establishing a 20% PPFP value as the cutoff between negative and positive results, as described by Martins-Filho et al. . Positive results were additionally classified according to the method of Cordeiro et al. as low positive (20% < PPFP <= 50%) and high positive (PPFP > 50%). The TC patients were subdivided into three groups, as they received treatment during the acute (A), subacute (SA), or chronic (C) phase of the disease. FIG. 6. | Analysis of PPFP values from patients with negative xenodiagnosis but positive or oscillating conventional serology. Analysis of PPFP values from patients with negative xenodiagnosis but positive or oscillating conventional serology. The performance of FC-ALTA for samples from the TUE patient group was investigated by using FS, and data analysis was performed by establishing a 20% PPFP value as the cutoff between negative and positive results, as described by Martins-Filho et al. . Positive results were additionally classified according to the method of Cordeiro et al. as low positive (20% < PPFP <= 50%) and high positive (PPFP > 50%). The TC patients were subdivided into groups, as they received treatment during the acute (A) or chronic (C) phase of the disease (b), had positive or oscillating serology (a), and had less or more than 10 years of follow-up after treatment (c). PMID- 12204967_DISCUSSION TI - AB - Different studies performed with either human or murine models have focused on strategies to evaluate the efficacy of specific chemotherapy against Chagas' disease . Conflicting reports have arisen because of differences in the approaches applied as cure criteria . Positive parasitological tests indicate the persistence of circulating parasites and represent a definitive marker of ongoing infection and treatment failure . However, lack of sensitivity is the major feature that leads to a low-negative predictive value of these methods . On the other hand, most types of CSA tests, evaluating the presence of specific antibodies, remain positive for long periods after treatment. Therefore, despite their high sensitivity, CSA tests are not recommended as cure criteria . For this reason, there is an urgent need for the development of serological tests, which avoid the occurrence of positive serology after parasite elimination. One of the strategies is to search for new immunological techniques, which are more reliable, to be used in follow-up studies after treatment. The finding of two types of anti-T. cruzi antibodies (LA and CSA) enabled Krettli and Brener to describe a phenomenon of serology dissociation and postulate that the absence of LA should be considered a cure marker, despite residual positive CSA tests. Considering the technical laboriousness of CoML, we have used an alternative tool (FC-ALTA) in our laboratory that was shown to be comparable to CoML for monitoring the treatment effectiveness in cases of human Chagas' disease . In fact, the sensitivity of FC-ALTA was much higher than that of CoML, as shown by the optimal dilutions required, 1:256 and 1:4, respectively. Furthermore, the capacity of flow cytometry to count thousands of trypomastigotes per assay improved the data confidence. Like CoML, the original FC-ALTA method allows for distinguishing sera from NT and TNC patients from both TC and noninfected individuals. However, the original FC-ALTA let two out of nine TNC patients with positive CoML results fall below the 20% cutoff level. For this reason, in the present investigation we designed an optimization of the original method in order to improve its performance and sensitivity. Our data demonstrated that incubation with FITC-conjugated anti-human IgG antibody for 30 min at 37C led to higher PPFP values for samples from chagasic patients, with no changes for samples from cured individuals. Therefore, we have incorporated this adjustment in the original method for further studies. Another great advance achieved by this study was the perspective of fitting the method for field surveys since the PPFP values were not adversely affected when testing GPS. Parallel analysis of FS and GPS demonstrated that only 3 out of 70 samples showed discrepancies in PPFP values. From those, only two samples interchanged between negative and low-positive results within PPFP intervals of 20% +- 10%. Then we recommended that when testing GPS, the interpretation of PPFP values should take this variation into account. In the study described here, the major goal of the research was to conduct a double-blind investigation to reinforce the use of FC-ALTA for clinical survey. For this purpose, we tested coded serum samples from four different categories of chagasic patients, including TC, TNC, TUE, and NT patients. Our data confirmed, in another study population, our previous statement that patients with ongoing infection (NT and TNC), in this case identified by positive xenodiagnosis, had positive results with FC-ALTA. It was interesting that the optimization of the original method, as described above, held all TNC patient samples with PPFP values higher than 20%, improving the performance of the original technique, in which 2 out of 9 TNC patient samples fell bellow the cutoff level. In our pioneer study, we suggested that those low PPFP values should be the result of antibody shedding from the parasite membrane and that shorter incubation times at a lower temperature should resolve this question. Here, we confirm that lower incubation time but higher temperature was the optimal condition to improve the sensitivity of FA-ALTA with no effect on its ability to discriminate negative samples. Together, the data from NT and TNC patient samples reemphasizes that the persistence of circulating parasites is associated with the presence of ALTA, which can be used as a valid immunological marker for active infection. Furthermore, it should be stressed that, in general, patients with negative CSA and negative xenodiagnosis persistently had PPFP values under 20%. Unexpectedly, we found that one cured patient had PPFP values over 50% in sequential samples and repeated tests. Interestingly, we found that this patient had been treated for tuberculosis 10 years before inclusion in this study. This patient had more than 140 negative xenodiagnoses and persistent negative CSA results over the past 10 years (data not shown). Several studies have reported the possibility of cross-reactivity in the serology for Chagas' disease in patients with tuberculosis . We are now testing to see if samples from patients with tuberculosis may present cross-reactivity in FC-ALTA assay. Considering these observations, we suggest the association of FC-ALTA with other tests already indicated as cure criteria to evaluate therapeutic efficacy in Chagas' disease. Parasitological and CSA tests should be performed repeatedly before a single FC-ALTA. Individuals presenting negative results on conventional tests should be considered cured, without requirement of confirmation by FC-ALTA. However, the FC-ALTA should be indicated, to confirm cure, in cases of individuals with several negative parasitological tests and positive or oscillating CSA tests (TUE patients), since this approach could save several years of inconclusive diagnosis. In fact, we have found that seven samples within the TUE patient population had negative PPFP results and could be classified as dissociated according to the Krettli and Brener criteria . As previously described , these individuals should be considered cured since they had negative results for xenodiagnosis and PPFP. We therefore postulated that patients who have been treated and show negative parasitological tests and the absence of ALTA should be considered cured despite residual positive or oscillating CSA. Here, using a 1:256 serum dilution from a TUE patient, we have observed that a period of 10 years after treatment was required to identify negative results with FC-ALTA for patients treated during the chronic phase of the disease. Then, we hypothesized that FC-ALTA follow-up with serial dilution could identify the antibody clearance sooner by the shift of positive PPFP values to lower titers. This hypothesis is currently under investigation in our laboratory. Finally, our study optimized and validated our method, offering another tool, less laborious than CoML and parasitological tests, for investigating and monitoring results of specific chemotherapy. Furthermore, these data reinforce the relevance of studies regarding etiological treatment of chronic patients since a considerable number of successful treatments were achieved during the chronic phase of Chagas' disease. PMID- 12204965 TI - Gamma Interferon Inhibits Production of Anti-OspA Borreliacidal Antibody In Vitro AB - | The ability of a Lyme borreliosis vaccine to induce and maintain sustained levels of borreliacidal antibody is necessary for prolonged protection against infection with Borrelia burgdorferi. Vaccination against infection with B. burgdorferi could be improved by determining the mechanism(s) that influences the production of protective borreliacidal antibody. Borreliacidal antibody was inhibited in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi and cultured with macrophages and B. burgdorferi and treated with recombinant gamma interferon (rIFN-gamma). The suppression of production of outer surface protein A (OspA) borreliacidal antibody by rIFN-gamma was not affected by the time of treatment. In addition, treatment with rIFN-gamma inhibited the production of other anti-B. burgdorferi antibodies. By contrast, treatment of cultures of immune lymph node cells with anti-IFN-gamma marginally increased the production of borreliacidal antibody and enhanced the production of other antibodies directed against B. burgdorferi. These results show that IFN-gamma does not play a major role in the production of anti-OspA borreliacidal antibody. Additional studies are needed to determine which cytokine(s) will enhance production of borreliacidal antibody. PMID- 12204965_Introduction TI - AB - Public health concerns about the morbidity associated with Lyme borreliosis have prompted the development of several vaccines to prevent infection with Borrelia burgdorferi . The efficacies of the vaccines are based on the ability of a major outer surface protein (Osp) of B. burgdorferi, specifically OspA, to induce antibody that can kill the Lyme spirochete in vaccinees or ticks . In an extensive field trial involving 10,936 participants, the vaccine was 76% effective in preventing infection with B. burgdorferi after three inoculations . However, the duration of protection had not been determined . In addition, we showed previously that vaccination of humans with recombinant OspA (rOspA) induced only low levels of anti-OspA borreliacidal antibody and that the borreliacidal response waned rapidly . Only one individual had detectable anti-OspA borreliacidal antibody after 180 days. A similar anti-OspA borreliacidal antibody response was detected in hamsters vaccinated with rOspA . The poor antibody response induced by vaccination may have contributed to the withdrawal of the vaccine. The ability of rOspA or other protective immunogens to induce high and sustained levels of borreliacidal antibody is necessary to ensure prolonged protection against infection with B. burgdorferi. Recently, we developed an in vitro system to determine the effects that immunologic mediators have on the production and regulation of borreliacidal antibody. Anti-OspA borreliacidal antibody was readily produced when lymph node cells obtained from vaccinated mice were cultured with macrophages and B. burgdorferi . When OspA borreliacidal antibody-producing cells were exposed to a known B-lymphocyte-stimulating factor , interleukin 4 (IL-4), borreliacidal-antibody production was inhibited. Furthermore, treatment of the immune lymph cell cultures with anti-murine IL-4 did not alter the production of anti-OspA borreliacidal antibody. These results suggested that IL-4 plays a minor role in the production and up-regulation of borreliacidal antibody. The inability of IL-4-stimulated immune lymph node cells to increase production of borreliacidal antibody may be due to down-regulation of gamma interferon (IFN-gamma). It is known that IL-4 strongly down-regulates functions promoted by IFN-gamma , especially class switching to immunoglobulin G2a (IgG2a) by B lymphocytes . Since B. burgdorferi organisms are killed by IgG2a and complement , we sought evidence of whether IFN-gamma augments anti-OspA borreliacidal-antibody production. Such information could provide insight into the mechanism of borreliacidal-antibody production and contribute to the development of a more efficacious Lyme borreliosis vaccine. PMID- 12204965_MATERIALS AND METHODS TI - AB - Mice. | Eight- to 12-week-old inbred C3H/HeJ mice were obtained from our breeding colony located at the Wisconsin State Laboratory of Hygiene. Mice weighing 20 to 40 g were housed four per cage at an ambient temperature of 21C. Food and acidified water were provided ad libitum. Organism. | B. burgdorferi sensu stricto isolate 297 was originally isolated from human spinal fluid . Low-passage (<6) organisms were cultured once in modified Barbour-Stoenner-Kelly (BSK) medium containing screened lots of bovine serum albumin to a concentration of 5 x 107 spirochetes per ml. Five-hundred-microliter samples were then dispensed into 1.5-ml screw-cap tubes (Sarstedt, Newton, N.C.) containing 500 mul of BSK supplemented with 10% glycerol (Sigma Chemical Co., St. Louis, Mo.), sealed, and stored at -70C. When necessary, a frozen suspension of spirochetes was thawed and used to inoculate fresh BSK medium. The spirochetes were viewed by dark-field microscopy and enumerated using a Petroff-Hausser counting chamber. Preparation of vaccine. | B. burgdorferi organisms were grown in 1 liter of BSK medium for 6 days, pelleted by centrifugation (10,000 x g; 15C; 10 min), and washed three times with phosphate-buffered saline (PBS; pH 7.4). The washed pellet was resuspended in 1% formalin, incubated at 32C for 30 min with periodic mixing, washed three times by centrifugation with PBS (12,000 x g; 10C; 15 min), and resuspended in PBS. Subsequently, the formalin-inactivated spirochetes were mixed in a volume of a 1% suspension of aluminum hydroxide (alum; Reheis, Berkeley Heights, N.J.) to yield 4 x 106 spirochetes/ml. Vaccination of mice. | We showed previously that mice vaccinated with whole cells of B. burgdorferi with or without alum yielded higher levels of anti-OspA borreliacidal antibody than those vaccinated with rOspA in the presence or absence of alum. Therefore, whole cells of B. burgdorferi were used in this investigation. Whole cells are not recommended as a vaccine for human usage. The ability of whole cells to consistently induce anti-OspA borreliacidal antibody in mice permits evaluation of cytokine mechanisms responsible for control of anti-OspA borreliacidal ability. Sixty mice were mildly anesthetized with methoxyflurane contained in a mouth-and-nose cup and vaccinated subcutaneously in the inguinal region with 0.25 ml (similar106B. burgdorferi organisms) of the formalin-inactivated vaccine preparation. The suspension contained approximately 100 mug of borrelial protein. Sham-vaccinated mice were injected with BSK medium or alum alone. Recovery of macrophages. | Five to 10 mice per experimental protocol were mildly anesthetized with methoxyflurane contained in a mouth-and-nose cup and injected intraperitoneally with 2 ml of 3% 3-week-old thioglycolate in PBS. Four days after injection, the mice were euthanized by CO2 asphyxiation, and 8 ml of cold Hanks' balanced salt solution (Sigma) was injected intraperitoneally. The peritoneal cavity was massaged for similar1 min, and the exudate cells were recovered by aspiration with a syringe. The suspension of peritoneal exudate cells was centrifuged at 1,500 rpm (IEC Centra-7) for 10 min at 4C. The supernatant was decanted, and the cells were resuspended in Dulbecco's modified Eagle's medium (DMEM; Sigma) that was free of antimicrobial agents but supplemented with 10% heat-inactivated (56C; 45 min) fetal bovine serum (HyClone Laboratories, Logan, Utah), 5 x 10-5 M 2-mercaptoethanol (Sigma), and l-glutamine (2.92 mg/ml; Sigma). Aliquots of the cell suspension were then poured over polystyrene tissue culture dishes (100 by 20 mm; Corning Glass Works, Corning, N.Y.) and incubated at 37C in a humidified atmosphere of 5.0% CO2 for 4 to 6 h. After incubation, the nonadherent cells were aspirated from the tissue culture dishes. The dishes were gently rinsed twice with 8-ml portions of warm Hanks' balanced salt solution to further eliminate nonadherent cells. Five milliliters of cold, nonenzymatic cell lifter (Sigma) was then added to each tissue culture dish and incubated at 4C for 30 min. The macrophages were detached from the surfaces of the dishes by vigorously tapping and gently scraping the inside of the tissue culture dishes with a sterile rubber policeman. Suspensions of macrophages from several tissue culture dishes were aspirated, pooled, and centrifuged at 1,500 rpm for 10 min at 4C. The supernatant was decanted, and the pellet was resuspended in 1 ml of DMEM. Cell viability was determined by trypan blue exclusion. The preparations of macrophages obtained by this method were 98% free of lymphocyte contamination. Isolation of lymph node cells. | Mice were euthanized by CO2 inhalation 17 days after vaccination with formalin-inactivated B. burgdorferi. The inguinal lymph nodes were removed from vaccinated and nonvaccinated mice and placed in cold DMEM. Single-cell suspensions of lymph node cells were prepared by teasing apart the lymph nodes with forceps and gently pressing them through a sterile stainless steel 60-mesh screen into antimicrobial-free cold DMEM supplemented with 10% heat-inactivated fetal bovine serum, l-glutamine, and 2-mercaptoethanol. The lymph node cells were washed twice by centrifugation (1,500 rpm; 4C; 10 min) with DMEM. The supernatants were decanted, and the pellets were resuspended in 1 ml of cold DMEM. Cell viability was assessed by trypan blue exclusion. Production of antibody in vitro. | Sterile six-well flat-bottom tissue culture dishes (Becton Dickinson, Lincoln Park, N.J.) were inoculated with lymph node cells (5 x 106) obtained from vaccinated or nonvaccinated mice, macrophages (105), and 106 live B. burgdorferi organisms. DMEM was added to the suspensions of cells to bring the final volume to 3 ml. On days 3, 6, 9, 12, and 15 after cultivation at 37C in the presence of 5.0% CO2, 1.0-ml samples of the supernatants were removed after gentle agitation and replaced with equal volumes of warm DMEM. In some experiments, rIFN-gamma at quantities ranging from 0.1 to 10 mug or rat anti-murine IFN-gamma at quantities ranging from 25 to 75 mug (R&D Systems, Minneapolis, Minn.) was added to cultures of immune lymph node cells, macrophages, and B. burgdorferi at 10 min and 2 and 4 days of incubation. In other experiments, rat anti-murine CD119 at quantities ranging from 1 to 100 mug (PharMingen, San Diego, Calif.) was added to cultures of immune lymph node cells, macrophages, and B. burgdorferi at 10 min of incubation. In similar fashion, control cultures were treated with a rat isotype-nonspecific antibody. Supernatants were collected after centrifugation at 13,000 rpm for 8 min to remove spirochetes and other cellular debris. Supernatants were stored at -70C until they were used. Detection of borreliacidal antibody by membrane filtration. | The frozen supernatants were thawed, heat inactivated (56C, 30 min), sterilized with a 0.22-mum-pore-size filter (Acrodisk; Gelman Sciences, Ann Arbor, Mich.), and serially twofold diluted (from undiluted to 1:8,192) with fresh BSK medium. One hundred-microliter aliquots of each dilution were transferred to 1.5-ml screw-cap tubes (Sarstedt), and 100 mul of BSK containing 104B. burgdorferi organisms per ml was added along with 20 mul of sterile guinea pig complement (Sigma). The tubes were then gently shaken and incubated for 3 days at 32C. Controls included filter-sterilized supernatants obtained from suspensions of nonimmune lymph node cells with macrophages and B. burgdorferi. Other controls included supernatants from nonimmune lymph node cells, macrophages alone, and DMEM. After incubation, 100 mul of each suspension was removed and placed in individual 1.5-ml screw-cap tubes (Sarstedt). Subsequently, 100 mul of a solution of propidium iodide (1.0 mg/ml; Molecular Probes, Eugene, Oreg.) diluted 1:20 in sterile PBS was added. The suspensions were briefly mixed before being incubated at 56C for 30 min to permit intercalation of propidium iodide into the spirochetes. One hundred microliters of each sample was then filtered through 0.22-mum-pore-size Nuclepore polycarbonate membrane filters (47-mm diameter; Whatman Nuclepore, Clifton, N.J.) under negative pressure with a single-place sterility test manifold (Millipore Corporation, Bedford, Mass.) attached to a vacuum pump. The membrane filters were washed with similar8 ml of sterile double-distilled H2O (ddH2O), removed from the vacuum apparatus, allowed to dry, and placed onto glass microscope slides. Coverslips were placed on the filters before they were viewed with a Laborlux S fluorescence microscope (Leitz, Wetzlar, Germany) using an x50 oil immersion objective. The number of spirochetes on each filter was determined by viewing similar30 fields. The borreliacidal-antibody titer was defined as the reciprocal of the dilution preceding the dilution at which the number of spirochetes or clumping was equal to that of the control. Generally, individual spirochetes with a few clumps were uniformly distributed throughout the fields on filters of the control supernatants. Western immunoblotting. | B. burgdorferi 297 organisms were grown in 1 liter of BSK medium for 6 days, pelleted by centrifugation (10,000 x g; 15C; 10 min), and washed three times with PBS at pH 7.4. The washed pellet was resuspended in 1% formalin and incubated at 32C for 30 min with periodic mixing and then washed three times by centrifugation with PBS (12,000 x g; 10C; 15 min) and resuspended in PBS. The spirochetes were suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer and boiled for similar3 min. One hundred twenty micrograms of B. burgdorferi lysate was loaded onto a preparative 12% acrylamide gel, and the proteins were resolved by overnight electrophoresis at similar7 mA constant current with the buffer system of Laemmli . The proteins were transferred onto a nitrocellulose membrane for 1 h at 20 V, using a semidry blotting apparatus (Bio-Rad Laboratories, Hercules, Calif.). The nitrocellulose membrane was incubated overnight at 4C in 5% milk dissolved in Tris-buffered saline with 0.05% Tween 20 (TBS-T; pH 7.4) to block nonspecific reactivity, washed two times each with TBS-T and ddH2O, allowed to dry, and finally cut into strips. The strips were washed three times with TBS and subsequently incubated for 1 h with a 1:1,000 dilution of an alkaline phosphatase-conjugated goat anti-murine IgG (heavy and light chain specific; Kirkegaard & Perry Laboratories, Gaithersburg, Md.) in 5% milk in TBS-T. This was followed by four washes with TBS. The strips were developed by the addition of 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium substrate (Kirkegaard & Perry). The reactions were stopped after 2 min with several large volumes of chilled ddH2O. Flow cytometric analysis of immune supernatants. | Suspensions of immune lymph node cells containing macrophages and B. burgdorferi in the presence or absence of IFN-gamma, anti-IFN-gamma, or isotype-nonspecific antibody were analyzed for the numbers of cells, B lymphocytes, and T lymphocytes on day 4 of incubation by using flow cytometry. Briefly, suspensions (1 ml) of immune lymph node cells were placed in chilled centrifuge tubes, and the total number of lymphocytes was determined. The suspensions of cells were then mixed with both phycoerythrin-conjugated anti-murine CD3 (5 mul of a 1:5 dilution; PharMingen) and fluorescein isothiocyanate-conjugated anti-murine CD45R/B220 (10 mul of a 1:50 dilution; PharMingen) and incubated at 4C for 15 min. The cells were washed twice by centrifugation with PBS containing 0.1% bovine serum albumin (1,500 rpm; 4C; 10 min). The pellets of cells were resuspended in 250 mul of cold DMEM and kept in the dark at 4C until they were analyzed by flow cytometry. One hundred microliters of 50-mug/ml propidium iodide (Sigma) was then added to each tube just prior to acquisition by the flow cytometer to discriminate viable and nonviable cells. Data were acquired on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.) using CellQuest acquisition and analysis software (Becton Dickinson). Twenty thousand events were detected by forward and side angle light scatter and by propidium iodide, phycoerythrin, and fluorescein isothiocyanate fluorescence. A dot blot profile of forward angle light scatter and propidium iodide fluorescence enabled identification and gating of live lymphocyte populations. The gated events were subsequently analyzed by quadrant dot blots of phycoerythrin and fluorescein isothiocyanate fluorescence for enumeration of CD3+ and B220+ lymphocytes in a given sample. Statistical analysis. | A t test was used to determine significant differences in the titers of borreliacidal antibody and in lymphocyte counts among supernatants. In addition, titers that were determined during kinetic studies of in vitro borreliacidal antibody production were tested by analysis of variance, utilizing the Minitab statistical analysis program. The Fisher least-significant-difference test was used to examine pairs of means when a significant F ratio indicated reliable mean differences. The alpha level was set at 0.05 before the experiments were started. PMID- 12204965_RESULTS TI - AB - Modulation of in vitro production of anti-OspA borreliacidal antibody by IFN-gamma. | We showed previously that anti-OspA borreliacidal antibody is readily detected in supernatants of lymph node cells obtained from mice vaccinated with formalin-treated B. burgdorferi in adjuvant and cultured with macrophages and B. burgdorferi for several days. The data presented in Fig. confirms and extends these findings. High levels of anti-OspA borreliacidal antibody (titer, 256) were detected in supernatants on day 6 of culture of immune lymph node cells with macrophages and B. burgdorferi. The peak anti-OspA borreliacidal activity (titer, 1,024) was detected with supernatants obtained on days 9 to 15 of culture. When cultures of immune lymph node cells with macrophages and B. burgdorferi were exposed to 0.1, 1.0, or 10 mug of rIFN-gamma, the levels of anti-OspA borreliacidal antibody were significantly reduced 16-fold or more. Maximum suppression of anti-OspA borreliacidal antibody occurred in cultures of lymph node cells exposed to 0.1 mug of rIFN-gamma followed by 1.0 and 10 mug of rIFN-gamma. The borreliacidal activity was due to the production of anti-OspA antibody. Adsorption of the supernatant from immune lymph node cells with rOspA reduced the borreliacidal-antibody titer. No borreliacidal antibody was detected in supernatants obtained from nonimmune lymph node cells cultured with macrophages and B. burgdorferi. When these studies were repeated three times, similar results were obtained. In other studies, immune lymph node cells were treated with 25, 50, or 75 mug of anti-murine IFN-gamma. Anti-OspA borreliacidal-antibody production was increased two- to fourfold in supernatants obtained from immune lymph node cell cultures treated with 50 or 75 mug of anti-IFN-gamma for 9 days or more compared to the levels of anti-OspA borreliacidal antibody detected in cultures of untreated immune lymph node cells . However, treatment of immune lymph node cells with 25 mug of anti-IFN-gamma did not affect the production of borreliacidal antibody. No borreliacidal antibody was detected in supernatants obtained from nonimmune lymph node cells cultured with macrophages and B. burgdorferi. Similar results were obtained when these studies were repeated. Temporal effects of rIFN-gamma and anti-IFN-gamma on the production of borreliacidal antibody. | Immune lymph node cell cultures were treated with rIFN-gamma or anti-IFN-gamma at 10 min or 2 or 4 days after cultivation. Production of anti-OspA borreliacidal antibody was inhibited by rIFN-gamma at all treatment intervals, although less effect was detected with rIFN-gamma treatment on day 4 of cultivation . Treatment of immune lymph node cell cultures with anti-IFN-gamma slightly enhanced production of anti-OspA borreliacidal antibody at all treatment intervals . When these studies were repeated, similar results were obtained. Effects of rIFN-gamma and anti-IFN-gamma on production of anti-B. burgdorferi antibody. | Figure shows that supernatant obtained from immune lymph node cells (day 17 after vaccination) cultured with macrophages and B. burgdorferi for 9 days produced predominately anti-OspA (31-kDa) antibody. Treatment of the lymph node cell cultures with rIFN-gamma abrogated the antibody response . By contrast, treatment of immune lymph node cell cultures with anti-IFN-gamma induced production of antibodies to multiple B. burgdorferi antigens . Furthermore, supernatant from anti-IFN-gamma-treated lymph node cell cultures had an anti-OspA Western immunoblot titer of 40,960 compared to a titer of 2,560 for supernatant obtained from untreated immune lymph node cell cultures. Flow cytometric analysis of lymph node cell cultures. | Immune lymph node cells cultured with macrophages and B. burgdorferi in the presence or absence of rIFN-gamma or anti-IFN-gamma for 4 days were analyzed for viability and numbers of T and B lymphocytes by using flow cytometry. Figure shows that the number of viable cells in lymph node cell cultures treated with rIFN-gamma decreased approximately 25% from the number of viable cells detected in cultures of lymph node cells treated with an isotype-nonspecific antibody or anti-IFN-gamma . In addition, the number of B lymphocytes (B220 marker) decreased approximately 50% in lymph node cell cultures treated with rIFN-gamma compared to the control . By contrast, a 10% increase in B lymphocytes was detected in lymph node cell cultures treated with anti-IFN-gamma . Similarly, the number of T lymphocytes (CD3 marker) decreased approximately 25% in cultures of lymph node cells treated with rIFN-gamma , while a 6% decrease was detected in cultures treated with anti-IFN-gamma compared to control cultures . When these studies were repeated, similar results were obtained. Effect of rIFN-gamma on macrophages. | In other studies, the lymph node cell cultures treated with an isotype-nonspecific antibody (control), rIFN-gamma, or anti-IFN-gamma were gently washed to remove nonadherent cells and viewed by light microscopy. The macrophages cultured with rIFN-gamma were rounded and lacked pseudopodia , whereas macrophages treated with the isotype-nonspecific antibody or anti-IFN-gamma were spindle shaped with long pseudopodia . FIG. 1. | Effect of 0.1 , 1 , or 10 mug (*) of rIFN-gamma on production of borreliacidal antibody by lymph node cells obtained from 17-day-vaccinated mice cultured with macrophages and B. burgdorferi Effect of 0.1 , 1 , or 10 mug (*) of rIFN-gamma on production of borreliacidal antibody by lymph node cells obtained from 17-day-vaccinated mice cultured with macrophages and B. burgdorferi for 3, 6, 9, 12, and 15 days. Controls included lymph node cells obtained from vaccinated and nonvaccinated mice cultured with macrophages and B. burgdorferi. The error bars indicate standard deviations. FIG. 2. | Effect of 25 , 50 , or 75 mug (*) of anti-IFN-gamma on production of borreliacidal antibody by lymph node cells obtained from 17-day-vaccinated mice cultured with macrophages and B. burgdorferi Effect of 25 , 50 , or 75 mug (*) of anti-IFN-gamma on production of borreliacidal antibody by lymph node cells obtained from 17-day-vaccinated mice cultured with macrophages and B. burgdorferi for 3, 6, 9, 12, and 15 days. Controls included lymph node cells obtained from vaccinated and nonvaccinated mice cultured with macrophages and B. burgdorferi. The error bars indicate standard deviations. FIG. 3. | Borreliacidal-antibody response of lymph node cells obtained from 17-day-vaccinated mice cultured with B. burgdorferi Borreliacidal-antibody response of lymph node cells obtained from 17-day-vaccinated mice cultured with B. burgdorferi and macrophages and treated with rIFN-gamma (A) or anti-IFN-gamma (B) 10 min (large-grid bars) or 2 (small-grid bars) or 4 (cross-hatched bars) days after cultivation. Control cultures (solid bars) were treated with PBS (A) or an isotype-nonspecific antibody (B). The error bars indicate standard deviations. FIG. 4. | Western immunoblots obtained with supernatants from cultures of immune lymph node cells (day 17 after vaccination) cultured with macrophages and B. burgdorferi Western immunoblots obtained with supernatants from cultures of immune lymph node cells (day 17 after vaccination) cultured with macrophages and B. burgdorferi without treatment (A) or treated with IFN-gamma (B) or anti-IFN-gamma (C). The supernatants were diluted 1:160 (lanes I) or 1:640 (lanes II). FIG. 5. | Flow cytometric analysis of lymph node cells (day 17 after vaccination) cultured with macrophages and B. burgdorferi Flow cytometric analysis of lymph node cells (day 17 after vaccination) cultured with macrophages and B. burgdorferi in the presence of isotype-nonspecific antibody (A), rIFN-gamma (B), or anti-IFN-gamma (C) for 4 days. The cells were labeled with both phycoerythrin-conjugated anti-murine CD3 (T cells) and fluorescein isothiocyanate-conjugated anti-murine CD45R/B220 (B cells). Viability was determined by exclusion of propidium iodide. FIG. 6. | Macrophages (adherent cells) detected in cultures of lymph node cells cultured with macrophages and B. burgdorferi Macrophages (adherent cells) detected in cultures of lymph node cells cultured with macrophages and B. burgdorferi and treated with isotype-nonspecific antibody (A) or rIFN-gamma (B) for 4 days after removal of nonadherent cells. Macrophages detected in cultures of lymph node cells treated with anti-IFN-gamma were similar to those pictured in panel A. PMID- 12204965_DISCUSSION TI - AB - B. burgdorferi organisms are easily killed when they bind specific antibody that activates complement . Several outer surface proteins of B. burgdorferi, including OspA, OspB, OspC, and the 39-kDa protein (, , -, ), induce complement-dependent borreliacidal antibody. This feature makes these outer surface proteins ideal candidates for development of a vaccine to prevent infection with B. burgdorferi. Of these proteins, OspA has been used to vaccinate humans and animals . Although rOspA is immunogenic, the antibody response is dominated by production of antibody that is not protective . Most importantly, vaccinated animals challenged with B. burgdorferi during periods with concomitant high levels of nonbactericidal anti-OspA and low levels of anti-OspA borreliacidal antibodies develop arthritis . Recently, we showed that hamsters vaccinated with 30, 60, or 120 mug of rOspA with or without alum developed severe destructive arthritis when challenged with B. burgdorferi . Similarly, hamsters vaccinated with a commercially available canine rOspA vaccine developed severe destructive arthritis after challenge with the Lyme spirochete . Only the presence of high levels of borreliacidal antibody prevented infection with B. burgdorferi from inducing arthritis in vaccinated animals . Maximum protection due to borreliacidal antibody, however, is restricted to 7 to 9 weeks after vaccination. One approach to increase the duration of protection and lessen the potential for adverse effects is to determine the immune mediators responsible for the production and maintenance of borreliacidal antibody. We showed previously that IL-4 does not augment the production of anti-OspA borreliacidal antibody, even though IL-4 is known to up-regulate B-lymphocyte growth and differentiation . In fact, IL-4 inhibited the production of anti-OspA borreliacidal antibody, including IgG2a, by lymph node cells obtained from vaccinated mice . It is known that IgG2a is negatively regulated by IL-4 but up-regulated by IFN-gamma . Our data and those of others suggested that IL-4 counteracted the effects of IFN-gamma on the production of borreliacidal antibody. When lymph node cells producing borreliacidal antibody were exposed to rIFN-gamma, borreliacidal antibody production was inhibited. The time of exposure (10 min to 4 days) of immune lymph node cells to rIFN-gamma did not affect the inhibition of production of borreliacidal antibody. In addition, treatment with rIFN-gamma inhibited the production of other anti-B. burgdorferi antibodies. The suppression of borreliacidal and other anti-B. burgdorferi antibodies by rIFN-gamma was unexpected. Flow cytometric analysis of rIFN-gamma-treated and untreated immune lymph node cells showed that treatment with rIFN-gamma reduced the number of viable lymphocytes, especially B lymphocytes. In addition, macrophages cultured in the presence of exogenous rIFN-gamma were rounded and exhibited rare pseudopodia. By contrast, macrophages obtained from cultures of untreated lymph node cells producing borreliacidal antibody were spindle shaped with many pseudopodia. IFN-gamma has been shown to induce apoptosis in T and B lymphocytes. These events could have affected the activation of macrophages and their ability to process borrelial antigen. It is possible that inhibition of borreliacidal antibody was due to the use of toxic concentrations of rIFN-gamma. However, when more physiologically relevant concentrations (1.0 or 0.1 mug) of rIFN-gamma were added to immune lymph node cells, borreliacidal activity also failed to increase. In addition, immune lymph node cells were treated with anti-CD119, which blocks the binding of murine IFN-gamma to cellular receptors . No significant decrease in borreliacidal-antibody production was detected in these cultures compared to immune lymph node cells treated with an isotype-nonspecific antibody (data not shown). These results suggest that IFN-gamma is not a major force in driving the production of borreliacidal antibody. Perhaps the most compelling evidence that IFN-gamma is not responsible for production of borreliacidal antibody came from experiments utilizing neutralizing antibody to IFN-gamma. Treatment of immune lymph node cells with various concentrations of anti-IFN-gamma failed to suppress borreliacidal activity. In fact, borreliacidal antibody production was marginally enhanced, especially when treatment with anti-IFN-gamma occurred early in cultivation of the immune lymph node cells. Furthermore, treatment with anti-IFN-gamma resulted in polyclonal expansion of the anti-B. burgdorferi antibody response. In support of this idea, by using flow cytometric analysis, we also detected a significant increase in the number of B lymphocytes in cultures of immune lymph node cells treated with anti-IFN-gamma. IFN-gamma performs numerous immunologic functions, including T helper lymphocyte differentiation and stabilization , enhancement of major histocompatibility complex expression on both B lymphocytes and macrophages , antiviral effects , and amelioration of production of IgG2a . Our results, however, show that IFN-gamma suppresses antibody production, including IgG2a borreliacidal antibody . An explanation may be that IgG2a antibody expression is not completely dependent upon IFN-gamma . In addition, IFN-gamma may prevent the expression of IL-4 functions . However, we showed previously that IL-4 suppressed the production of borreliacidal antibody . This suggests that other cytokines, but not IL-4 or IFN-gamma, may be responsible for the induction, production, and maintenance of borreliacidal antibody. In support of this idea, treatment with anti-IFN-gamma augmented not only borreliacidal antibody but other antibodies directed against B. burgdorferi. Additional studies are needed to determine which cytokines are responsible for the production of borreliacidal antibody. In conclusion, we showed that IFN-gamma plays a major role in suppression of the production of borreliacidal antibody. Effective neutralization of endogenous IFN-gamma slightly augmented the production of borreliacidal antibody and expanded the anti-B. burgdorferi antibody responses. Determination of the mechanism that inhibits the production of borreliacidal antibody by rIFN-gamma may lead to the development of a safe and effective Lyme borreliosis vaccine. PMID- 12204945 TI - Active Surveillance for Scrapie by Third Eyelid Biopsy and Genetic Susceptibility Testing of Flocks of Sheep in Wyoming AB - | Control of scrapie, an ovine transmissible spongiform encephalopathy or prion disorder, has been hampered by the lack of conventional antemortem diagnostic tests. Currently, scrapie is diagnosed by postmortem examination of the brain and lymphoid tissues for PrPSc, the protein marker for this group of disorders. For live, asymptomatic sheep, diagnosis using tonsil or third-eyelid lymphoid tissue biopsy and PrPSc assay has been described. To evaluate the feasibility and efficacy of third-eyelid testing for identification of infected flocks and individual infected sheep, 690 sheep from 22 flocks were sampled by third-eyelid lymphoid tissue biopsy and immunohistochemistry. Sheep were further evaluated for relative genetic susceptibility and potential contact exposure to scrapie. Third-eyelid testing yielded suitable samples for 80% of the sheep tested, with a mean of 18.1 lymphoid follicles (germinal centers) per histologic section. Three hundred eleven of the sheep were sampled through passive surveillance programs, in which only sheep with potential contact with an infected sheep at a lambing event were tested, regardless of their scrapie susceptibility genotype. In addition, 141 genetically susceptible sheep with no record of contact with an infected animal at a lambing event were sampled through a targeted active surveillance program. Ten PrPSc-positive sheep were identified through the passive surveillance program, and an additional three PrPSc-positive sheep, including two from flocks with no history of scrapie, were identified through the active surveillance program. All PrPSc-positive sheep had the highly susceptible PrP genotype. Third-eyelid testing is a useful adjunct to flock monitoring programs, slaughter surveillance, and mandatory disease reporting in a comprehensive scrapie eradication and research program. PMID- 12204945_Introduction TI - AB - Scrapie is a fatal neurologic disease of sheep and goats, introduced into the United States in 1947 and now endemic in many states . Scrapie is a member of the heterogeneous group of prion diseases, or transmissible spongiform encephalopathies (TSE), which includes bovine spongiform encephalopathy (BSE), cervid chronic wasting disease, and transmissible mink encephalopathy. Although scrapie is not a zoonotic disease, the apparent transmission of BSE to humans in the United Kingdom has resulted in a call for eradication of all TSE in food-producing animals. The transmissible agent in scrapie remains controversial because no conventional bacterial, viral, fungal, or toxic source has been identified. The major component in infectious tissue homogenates is a relatively denaturation-resistant conformational isoform (PrPSc) of the highly conserved mammalian PrP protein (PrPC) . The prion model proposes that PrPSc and other less well characterized cofactors induce conversion of PrPC to the pathogenic isoform PrPSc through aggregation and posttranslational structural changes. Regardless of the etiology of scrapie, PrPSc is a reliable marker for infection. Scrapie transmission is thought to occur by oral exposure to the causative agent, uptake through the intestinal wall, amplification of the agent in the gut-associated lymphoid and nervous tissue , and transport to the brain through the autonomic fibers of the vagus nerve . In U.S. sheep, PrPSc detected by immunoassay or a transmissible agent detected by rodent bioassay is found in some lymphoid tissue by the age of 14 months and in the brain by 2.5 years, approximately 6 months before onset of clinical signs. Early accumulation of PrPSc in lymphoid tissue is the basis for preclinical scrapie diagnosis by immunohistochemistry (IHC) assay of postmortem tonsil and antemortem third-eyelid lymphoid tissue or tonsil biopsy samples . Disease transmission depends on both exposure to the infectious agent and susceptible genetics in the host. Sheep housed with an infected postparturient ewe (within 60 days of lambing or abortion) are considered to be at increased risk of scrapie, presumably because they are exposed to the infectious agent in placental and fetal tissues or fluids . Following contact with an infected postparturient ewe, disease occurs almost exclusively in sheep with particular polymorphisms in the host PrP gene . In the U.S. sheep population, scrapie (whether associated with clinical disease or with PrPSc accumulation in the absence of clinical signs) has been confirmed only in sheep homozygous for the PrP allele encoding glutamine at codon 171 (QQ171) , regardless of breed. In Europe and the United Kingdom, relative susceptibility of sheep with several other PrP genotypes is reported in various breeds, a finding useful in national scrapie control programs . Scrapie is typically diagnosed by IHC assay of brain tissue from sheep with clinical signs of scrapie, notably weight loss, wool loss, and ataxia. Following diagnosis of an index animal, control measures in the flock include culling or quarantine of sheep at risk due to susceptible genetics and/or possible contact with the infected postparturient ewe . However, many infected sheep die from intercurrent diseases or routine culling without diagnostic testing, and identification of infected flocks by surveillance for clinical scrapie alone may not be sufficient for disease eradication. Cooperative state-federal programs that meet or exceed the current control measures and that are designed to test or improve program procedures or facilitate research are allowed as pilot programs . The state of Wyoming has proposed a pilot program in which passive surveillance (quarantine, testing, or removal of sheep having potential contact with an infected ewe) is supplemented with active surveillance, including live-animal testing of genetically susceptible, clinically normal sheep with no reported contact with an infected sheep during a lambing event. The objective of this study was to investigate the feasibility and efficacy of including third-eyelid testing and susceptibility genetics in active and passive surveillance programs. PMID- 12204945_MATERIALS AND METHODS TI - AB - Study flocks. | Twenty-two flocks in Wyoming were included in this study. Ninety sheep from the University of Wyoming Animal Science Department were sampled by third-eyelid biopsy to estimate the efficiency of eyelid biopsy sampling (percentage of sheep with suitable biopsy samples) for sheep of various ages and breeds. The sheep were aged 14 months to 7 years and represented four breeds (Suffolk, Columbia, Hampshire, and Rambouillet) . In addition, 600 sheep from privately owned flocks in Wyoming were tested . These sheep were of Suffolk and Hampshire origin. All sheep over the age of 14 months at the time of testing were sampled in flocks 1 to 5 and 7 to 13. Only sheep over the age of 14 months and considered at risk due to potential contact with a postparturient ewe subsequently diagnosed with scrapie (see "Relative risk designation" below) were sampled in flocks 6 and 14 to 16. The group designated flock 16 represents one sheep with potential contact risk from each of five flocks and two sheep with potential contact risk from a sixth flock. Flocks were included in the study following identification of an infected animal or an animal with potential contact exposure, either born into the flock (flocks 3 to 5) or purchased (flocks 6 to 16). Flocks 1 and 2 were volunteered by the producers for educational purposes or due to industry concerns about scrapie. Restraint. | One eye on each sheep, usually the left eye, was pretreated with topical 0.5% sterile ophthalmic proparacaine HCl (Bausch and Lomb, Tampa, Fla.) several minutes before the procedure was performed. The sheep was haltered and restrained manually. All sheep were sampled on the farm of origin, and equipment for restraint of the sheep varied among the facilities. Many farms were equipped with a fitting stand, in which the head was immobilized by resting the chin on a horizontal support and securing a restraint strap behind the head. Alternative restraint devices included a tilt table in which the sheep was restrained in a squeeze chute, or a head catch in which partial restraint was afforded by a stanchion. In all cases, immobilization of the head was achieved through traction on the halter strap. Optimally, one operator restrained the sheep's body, one operator maintained control of the halter, one person retracted the third eyelid, and a fourth person collected the biopsy specimen. If only two operators were available, equipment suitable for restraint of the sheep was needed; one operator maintained control of the halter and retracted the third eyelid, and one operator collected the sample. Biopsy sample collection. | After adequate immobilization of the sheep, the third eyelid was retracted by using 1 by 2 toothed forceps (Econo Thumb Tissue forceps 21-762; Sklar Instruments, West Chester, Pa.) and treated again with proparacaine. The lymphoid tissue was visualized on the bulbar surface as a slightly raised pink tissue on either side of the midline (Fig. , lower arrow). The color, location, and size of the tissue varied slightly among individual sheep. Sheep were sampled even if there was no gross evidence of lymphoid tissue. The tissue was grasped with a second set of forceps, raised slightly from the underlying connective tissue, and excised with single-use curved Metzenbaum scissors (Econo Metzenbaum scissors 21-336; Sklar Instruments) by using a medial to lateral cut; typically, two or three sequential cuts with the scissors were required to acquire a single biopsy specimen of sufficient size containing the entire area of lymphoid tissue on one side of the midline (Fig. , upper arrow). The biopsy sample was fixed in 10% neutral buffered formalin for at least 24 h. Tissue processing. | Typically, fixed biopsy samples were observed to be folded in half with the lymphoid tissue facing outward, secured by the indentation formed by the forceps teeth. Biopsy samples were opened using iris scissors and placed flat between biopsy sponges in plastic histopathology cassettes. The lymphoid tissue was placed down in the cassettes. Cassettes with properly positioned biopsy samples were immersed in 95 to 98% formic acid for 1 h to decontaminate the tissue . Samples were rinsed in three changes of deionized water and for at least 10 min in running deionized water, then reequilibrated in 10% neutral buffered formalin. Tissues were processed conventionally and embedded in paraffin; care was taken to position the biopsy samples in the paraffin blocks so that the lymphoid tissue was facing and on the same plane as the surface of the block, to provide the largest possible section when cut. Adhesion of formic acid-decontaminated tissue to glass slides required additional attention during preparation. Three- to five-micrometer-thick sections were cut with a microtome and applied to adhesive-coated aminosilane-treated slides (Newcomer Supply, Middleton, Wis.). Residual moisture under the section was removed with absorbent laboratory tissue paper, and slides were positioned vertically to dry for at least 1 h. Slides were baked at 57C for approximately 15 h (overnight) to remove excess paraffin. Immunostaining. | Sections were rehydrated through xylene substitutes and graded alcohols, then treated with 95% formic acid for 5 min and neutralized by three changes of 0.1 M Tris-HCl, pH 7.6. Antigen retrieval was performed by incubation at 120C in a medical pressure cooker (BioCare Medical, Walnut Creek, Calif.) for 20 min in a modified citrate buffer (Target Retrieval solution, pH 6.1; Dako Corporation, Carpinteria, Calif.). IHC was performed on an automated immunostainer (NexES; Ventana Medical Systems, Tucson, Ariz.) by using the manufacturer's detection reagents (antibody diluent, AEC detection kit, hematoxylin, bluing, and mouse immunoglobulin G1 [IgG1] negative control antibody). The primary antibody was a cocktail of two monoclonal antibodies (MAbs) to separate epitopes on the PrP molecule (MAb F99/97.6.1 and MAb F89/160.1.5 ; Veterinary Medical Research Development, Inc., Pullman, Wash.) at 5 mug/ml each in antibody diluent (Ventana Medical Systems) filtered through a 0.22-mum-pore-size syringe top filter. The primary antibody was applied for the maximum time available with the automated system (32 min), followed by biotinylated goat anti-mouse IgG, streptavidin-conjugated horseradish peroxidase, and 3-amino-9-ethylcarbazole as a chromogen. The default settings for incubation time and temperature provided by the manufacturer were used for all detection steps. The slides were counterstained with hematoxylin, rinsed, and covered with coverslips by use of a compatible mounting medium (Faramount aqueous mounting medium; Dako). Lymphoid tissue (tonsil or retropharyngeal lymph node) from an infected sheep was used as a positive control. Negative controls included replacement of the primary anti-PrP antibodies with an isotype control antibody (Ventana) and assay of tissues from uninfected sheep. Lymphoid follicles appearing as discrete oval or round areas with a darker border were counted on each section. Sections were considered "PrPSc detected" if a granular red precipitate was noted in lymphoid follicles (germinal centers) , regardless of the number of follicles in the section. Sections were considered "no PrPSc detected" if no precipitate was noted in a section. A minimum of six lymphoid follicles per section were needed for this designation. Sections with no immunostaining and fewer than six lymphoid follicles were scored "insufficient lymphoid tissue for determination." All IHC assays were scored by trained observers with no knowledge of the potential contact or genetic susceptibility status of the sheep. Relative risk designation. | Sheep were scored for potential contact risk by using producer records and the definitions established in the Code of Federal Regulations 79.4 . Briefly, sheep were defined as high risk by potential contact if they were born in a flock during the same lambing season as a lambing event by a sheep subsequently diagnosed with scrapie. Lambing events include the birth of an infected sheep as well as parturition or abortion by an infected ewe in any year of her life. Sheep were considered to be at lower risk by contact if they were born on the farm at other times, in different lambing facilities on the same farm, or on farms without reported cases of scrapie. If an infected ewe had produced at least one lamb at the facility and no birth records were available for the flock, all sheep born in the flock during the years that the infected ewe lambed or aborted were designated high risk. Relative contact risk status was based on producer records, and no outside measure of the accuracy of those records is available. No producer could rule out a previously undetected scrapie case, because some of the clinical signs (particularly weight loss) occur frequently in these flocks and result in culling of the animal without diagnostic testing. Sheep were scored for relative genetic susceptibility based on the deduced amino acid sequence at residue 171. Blood was collected by jugular venipuncture into EDTA-treated vacuum tubes and shipped to a commercial laboratory for scrapie susceptibility testing (GeneCheck, Fort Collins, Colo.). DNA was extracted from the buffy coat, amplified by PCR, and analyzed by a DNA mismatch binding assay. The diploid genotype was reported as QQ, QR, or RR. However, this assay does not distinguish between sequences encoding glutamine (Q) and sequences encoding histidine (H) (both are reported as 171Q), and samples scored QQ may actually be QQ, QH, or HH. Samples scored QR may be QR or HR. Samples scored RR are unequivocally homozygous for arginine (R) at position 171. Sheep were considered to have high genetic susceptibility if their genotype was 171QQ, 171QH, or 171HH and to have lower genetic susceptibility if their genotype was 171QR, 171HR, or 171RR. Surveillance programs. | Under the current program, passive surveillance includes testing of all sheep in flocks in which an infected animal was born or produced a lamb and all sheep sold from those flocks if they were born in a year in which the infected ewe produced a lamb. The passive surveillance program therefore includes sheep with potential contact with the transmissible agent shed at parturition, even if those sheep are at low genetic risk. Active surveillance includes only genetically susceptible sheep with no reported contact risk. In some flocks, all sheep over the age of 14 months were sampled, even if they were not eligible for either program. The number of sheep sampled in each surveillance program (or sampled as whole-flock testing but enrolled in neither program) is shown in Table . FIG. 1. | Bulbar surface, nictitating membrane of a Suffolk sheep. Bulbar surface, nictitating membrane of a Suffolk sheep. The lymphoid tissue is typically visualized as a slightly raised, red to pink area (lower arrow). Complete excision of the lymphoid tissue (upper arrow) is visible approximately 2 months after biopsy. The lymphoid tissue at this site does not regenerate. FIG. 2. | IHC detection of PrPSc in the nictitating membrane lymphoid tissue. IHC detection of PrPSc in the nictitating membrane lymphoid tissue. A third-eyelid biopsy specimen from a live sheep with preclinical scrapie is shown. Lymphoid follicles (discrete round to oval accumulations of lymphocytes, macrophages, and dendritic cells with a dark border) show granular PrPSc immunoreactivity (red) when immunostained with a cocktail of MAbs F89/160.1. and F99/97.6.1. Bar, 200 mum. TABLE 1 | Suitability of eyelid biopsy samples from sheep of four breeds, aged 1 to 7 years TABLE 2 | Flocks and sheep sampled by third-eyelid biopsy and PrP genotyping PMID- 12204945_RESULTS TI - AB - Third-eyelid biopsy samples were collected from a university-owned flock to determine if particular breeds of sheep or age groups were unsuitable for this procedure. Ninety sheep of four breeds were available for sampling . Suitable samples were those with at least six lymphoid follicles per section. Nine sheep were sampled twice because the left eye did not appear to have adequate lymphoid tissue when examined grossly. Five of these nine animals had suitable biopsy specimens from both eyes. One animal, a 5-year-old Columbia, had unsuitable samples from both eyelids. Three animals had one suitable sample and one unsuitable sample; this group included a 3-year-old Hampshire, a 4-year-old Columbia, and a 2-year-old Suffolk. The overall efficiency of testing was 76% (68 of 90) when the results from either eyelid were used to score an individual sheep. There was no breed for which all samples were unsuitable. There was a trend toward a lower percentage of suitable samples from sheep older than 3 years, but a larger study population is needed before conclusions on the feasibility of testing older sheep can be drawn. A total of 600 sheep from 21 producer-owned flocks were assessed for evidence of infection with the scrapie agent by IHC of third-eyelid biopsy samples and for relative genetic susceptibility to scrapie by DNA genotyping. Sampled sheep were over the age of 14 months and were primarily of the Suffolk or Hampshire breed. Sheep were housed in flocks with no reported cases of scrapie, flocks in which an infected ewe was born or had produced at least one lamb, and flocks with at least one purchased animal with potential contact exposure in the flock of origin . The PrP genotypic ratio in this sample was 0.48:0.52 (290 sheep with the highly susceptible 171QQ, -QH, or -HH genotype and 310 sheep with the lower-susceptibility 171QR, -RH, or -RR genotype). This ratio is not significantly different from that reported for an independent sample of 1,000 U.S. Suffolk sheep in 1996 . Three hundred eleven of the 600 sheep in the study were tested under passive surveillance programs , representing sheep with potential contact risk from exposure to an infected ewe at parturition. Eighty-five of 311 had the highly susceptible genotype. One hundred forty-one of the 600 sheep were tested under the Wyoming active surveillance program and represent genetically susceptible sheep with no record of contact with an infected sheep at a lambing event, whether they resided in flocks with a purchased animal with potential contact risk at a different facility (n = 96) or in flocks with no history of scrapie contact in either native or purchased animals (n = 45). One hundred forty-eight sheep in the lower-genetic-susceptibility group and with no reported contact with an infected postparturient ewe were tested because they were housed with sheep in the active surveillance trial. Sheep were tested for PrPSc, a marker for scrapie, by third-eyelid lymphoid tissue biopsy and IHC analysis . Biopsy samples of the third eyelid from 481 (80%) of the sheep tested contained at least six lymphoid follicles and were considered suitable for diagnosis, although all biopsy samples were analyzed by IHC. The mean follicle count in suitable biopsy samples was 18.1 (standard deviation, 13.3). PrPSc was detected in 13 of the biopsy samples, collected from sheep on seven farms . All 13 sheep with PrPSc detected in lymphoid tissue had the highly susceptible PrP 171 genotype QQ, QH, or HH. Of the 13 sheep with positive eyelid biopsy samples, 4 were removed for immediate necropsy, and scrapie was confirmed by IHC of brain and/or lymphoid tissues. Nine sheep were moved to quarantine. Scrapie has been confirmed in seven of these nine sheep by postmortem analysis following development of clinical signs and in one of these nine sheep following acute loss (found dead with no clinical signs). The last of the nine sheep brought to quarantine is asymptomatic at the age of approximately 36 months. Ten of the 13 PrPSc-positive sheep were identified by the passive surveillance program, and 3 were identified by the active surveillance program. The positive samples were collected from sheep in flocks in which an infected sheep had been born (n = 7), flocks with no reported cases of scrapie (n = 2), and flocks with at least one sheep purchased from an infected flock (n = 4), although in one case, the PrPSc-positive sheep was not the purchased sheep with contact risk but an unrelated animal purchased from a different flock. The efficiency of the surveillance programs was estimated as the number of PrPSc-positive sheep per number of sheep in each program (10 of 311, or 3.2%, in the passive surveillance program and 3 of 141, or 2.1%, in the active surveillance program). TABLE 3 | Flock status at the time of testing and risk status of PrPSc-positive sheep PMID- 12204945_DISCUSSION TI - AB - Scrapie was reported as a clinical entity nearly 300 years ago and was introduced into the United States in 1947. In the United States, control programs have relied on total or partial flock depopulation following diagnosis of the disease in a sheep with clinical signs of scrapie. The insidious nature of the disease, the long preclinical incubation period, the lack of a preclinical test, loss of infected sheep from intercurrent disease before development of clinical signs, and underreporting of scrapie due to the negative economic effect of a diagnosis have contributed to the failure of these programs to control the prevalence of scrapie, which is estimated at 0.07% in the United States (based on a mailed producer survey conducted in 1996 by the National Animal Health Monitoring System of the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service). State and federal flock certification programs, in which producers of monitored flocks agree to purchase breeding stock from similarly monitored flocks, permanent identification of individual sheep and/or flock of origin, and restrictions on interstate movement of suspect and high-risk animals have been in place since 1992. The USDA Animal and Plant Health Inspection Service recently expanded the program to include identification of breeding and mature sheep and breeding goats in interstate commerce, uniform standards for state scrapie control programs, indemnity payments, slaughter surveillance, and third-eyelid testing of exposed animals and/or flocks. These programs are being implemented to eradicate scrapie from the United States. The development of the third-eyelid test for antemortem diagnosis of scrapie and the identification of PrP genotypes associated with clinical and subclinical PrPSc accumulation have provided additional means for controlling scrapie in the U.S. sheep population. The estimated specificity of the third-eyelid test approaches 100% . The estimated sensitivity of the test is 85 to 90%. The false-negative results are due to a number of factors. Some infected sheep have PrPSc detectable in the brain but not in lymphoid tissues; in other cases, PrPSc is detectable in tonsil but not in antemortem eyelid biopsy specimens, due in part to inadequate biopsy sample size. Optimal sampling conditions include a restraint device such as a tilt table, fitting stand, or head catch and adequately trained personnel for immobilization of the head, retraction of the eyelid, and biopsy sample collection. Laboratory handling of the tissue to maximize the number of follicles available for inspection in a single histologic section requires laboratory personnel trained in recognizing the lymphoid side of the tissue and embedding a flat sample in the plane of the microtome cut. In other trials, techniques such as embedding strips of lymphoid tissue at right angles to the plane of the microtome cut or embedding folded or rolled tissues typically resulted in small numbers of follicles available for inspection in each section. In the present study, sampling was performed by one of three veterinarians (J.V.D., J.R.L., and E.S.W.), and IHC of samples from producer flocks was performed by one person (A.K.A.). When sampling, tissue handling, and assay were performed under these circumstances, approximately 80% of the sheep in each flock could be assessed. In this study, the third-eyelid test identified PrPSc-positive sheep in flocks with previous scrapie exposure, although in two cases the individual sheep identified by eyelid biopsy had no reported contact with an infected ewe at parturition. The efficiency of the programs (2 to 3% in this relatively small sample) exceeds the estimated scrapie prevalence level of 0.07% nationally and illustrates the potential value of preclinical surveillance. However, there are some limitations to the use of the eyelid test in a scrapie eradication program. One hundred to 150 sheep can be sampled per day in a single location with proficient personnel using restraint equipment available on most facilities; testing small numbers of sheep at multiple locations decreases the efficiency of the testing personnel. The costs of personnel, equipment, topical anesthetics, and sample testing are borne by the state and federal governments through a joint scrapie eradication program. Although the procedure is performed under local anesthesia, third-eyelid sampling may be perceived as objectionable by some producers. The third-eyelid test is most useful in animals between the ages of 14 and 36 months, because older animals tend to have smaller areas of lymphoid tissue. Further, the estimated test sensitivity is approximately 85 to 90%. Therefore, the third-eyelid test is not suitable as a stand-alone eradication tool. The test is more appropriately considered an adjunct surveillance tool to identify infected flocks which could then be managed through an integrated program of testing, necropsy or quarantine, genetic selection, and husbandry modifications. The test is also useful for identification of naturally infected sheep for transfer to research facilities. Although active surveillance is expensive, cost-effectiveness is increased by targeting the testing program to 14- to 24-month-old sheep of heavily impacted breeds in flocks with a history of purchasing breeding ewes from multiple sources. Active surveillance is a justifiable addition to live-animal passive surveillance, in which flocks are identified only after submission of a clinical suspect. One of the sheep quarantined after selection by this program was found dead with no history of clinical signs, and another remains clinically normal at the age of 36 months. Inapparently infected sheep such as these produce lambs annually and serve as a continuing source of the transmissible agent. Slaughter surveillance and active live-animal surveillance programs such as the Wyoming pilot program will be critical for identification of infected flocks and eventual disease eradication. This study demonstrated that identification of scrapie-infected flocks by third-eyelid testing is technically feasible with proficient personnel and adequate field and laboratory resources. Third-eyelid testing and genotype analysis are useful in identifying infected flocks for regulatory intervention and infected animals suitable for research purposes. Active surveillance for scrapie is a useful component of an integrated scrapie control program based on permanent identification, flock monitoring, slaughter surveillance, and passive surveillance by diagnosis of clinical suspects with testing of their scrapie-exposed flockmates. PMID- 12204969 TI - Immunoblot Analysis of the Humoral Immune Response to Leishmania donovani Polypeptides in Cases of Human Visceral Leishmaniasis: Its Usefulness in Prognosis AB - | Sera from Indian patients with parasitologically confirmed visceral leishmaniasis were studied by immunoblot analysis in order to identify a specific pattern for Leishmania infection. A soluble extract of Leishmania donovani was used as antigen. At diagnosis the sera from patients with visceral leishmaniasis specifically recognized fractions represented by bands of 201 kDa (50% of serum samples), 193 kDa (60%), 147 kDa (50%), 120 kDa (60%), 100 kDa (50%), 80 kDa (80%), 70 kDa (70%), 65 kDa (100%), 50 kDa (50%), 36 kDa (50%), 20 kDa (70%), and 18 kDa (50%). The 65-kDa band, common to all patients infected with Leishmania parasites, was found at the time of diagnosis. However, the immunoblot pattern changed after patients were treated and cured with sodium antimony gluconate (SAG; n =10) or miltefosine (n =10), as was evident from blots of sera obtained pretreatment and at 1, 3, and 6 months posttreatment. At 6 months posttreatment, immunoblots of sera from patients on the SAG regimen showed the disappearance of all bands except the 70-kDa band. Similarly, sera from those on the miltefosine regimen showed the disappearance of all bands except the 65- and 70-kDa bands. This study shows that Western blot analysis is a sensitive test for detection of anti-Leishmania antibodies. Moreover, the persistence of reactivity with the 65- and 70-kDa bands in the sera of all groups shows its promise as a diagnostic and prognostic tool. PMID- 12204969_Introduction TI - AB - Human visceral leishmaniasis is caused by a protozoan parasite of the Leishmania donovani complex, namely, Leishmania donovani. This infection is characterized clinically by fever, hepatosplenomegaly, anemia, and weight loss. A marked hypergammaglobulinemia and absence of detectable cell-mediated immunity are the principal immunological features of the disease. Although patients generate high levels of nonspecific immunoglobulin as well as specific antileishmanial antibodies, they fail to respond to parasite antigen in the delayed-type hypersensitivity skin test . A kinesin-related protein-encoding gene has been discovered in Leishmania chagasi and contains a repetitive 117-bp sequence encoding 39 amino acid residues (K39) conserved at the C-terminal end in all of the visceral leishmaniasis-causing isolates examined so far . The recombinant product of K39 (rK39) has proved to be a very sensitive and specific antigen in an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of visceral leishmaniasis from the foci of endemicity in Brazil, China, Pakistan, and Sudan . Kumar et al. reported extremely high levels of anti-rK39 antibodies in patients with visceral leishmaniasis, suggesting the application of rK39 for a sensitive and specific means of serodiagnosis and the potential of rK39 ELISA in the monitoring of drug therapy and the detection of disease relapses. To date, several L. donovani-specific proteins, including species-specific and subspecies-specific molecules, have been identified with monoclonal antibodies or polyvalent antisera. Several of these proteins elicit a strong humoral immune response during visceral leishmaniasis, allowing the specific diagnosis of visceral leishmaniasis by a competitive ELISA or a direct dot blot assay . Immunoblots have been useful in studies of host serological responses during infection, and a few antigens with potential diagnostic value have been selected . The humoral immune response occurs during the active phase of infection, with the appearance of low titers of antibodies that disappear some months after the end of treatment representing a temporary response . This fact has made the assessment of antibody titers a useful tool for patient follow-up after therapy and for defining the period during which clinicians should observe patients in order to make a prognosis. In the present study, we have examined patients' antibody responses at the time of diagnosis and various stages posttreatment using the immunoblot technique with soluble L. donovani antigen. We had the double objective of identifying a particular band pattern present in the patients affected by visceral leishmaniasis (confirmed by the presence of parasites in a sample taken from the spleen) and describing any possible variation in this band pattern following antileishmanial therapy with sodium antimony gluconate (SAG) or miltefosine. PMID- 12204969_MATERIALS AND METHODS TI - AB - Antigen. | L. donovani (MHOM/IN/96/B.H.U.70) promastigotes were cultivated in tissue culture flasks with RPMI 1640 medium (Hi-Media, Mumbai, India) supplemented with 10% fetal calf serum (Gibco, Grand Island, N.Y.) and antibiotics (gentamicin) . Parasites were taken at the late-logarithmic phase of growth, washed five times at 4C with sterile phosphate-buffered saline (PBS), and centrifuged at 1,400 x g for 15 min. The parasite pellet was resuspended in 1 ml of PBS, and the mixture was immediately frozen at -70C. In order to make up only one batch of antigen for Western blot analysis, all parasites were kept frozen at this temperature until there were sufficient parasites from which soluble antigen could be obtained. To prepare the soluble antigen, a method described by Isaza et al. was used, with slight modifications. Briefly, the parasites were defrosted and resuspended in 2 ml of lysis buffer (20 mM Tris HCl [pH 7.4] containing 40 mM NaCl, 10 mM EDTA, 2 mM phenylmethylsufonyl fluoride [BDH, Mumbai, India], and 0.4% sodium dodecyl sulfate [BDH]) . The mixture was left on ice for 30 min, with vortex agitation every 10 min. It was then centrifuged at 6,000 x g for 20 min at 4C. The supernatant was removed and kept at -70C until use. A small sample was used for protein determination by a method modified from that of Lowry et al. . By this method the final antigen protein concentration was found to be 9.4 mg/ml. Human sera. | Serum samples were collected from patients with parasitologically confirmed visceral leishmaniasis (kala azar) (L. donovani body score in splenic aspirate of 2+ to 4+, i.e., between >1 to 10 parasites/100 field and >1 to 10 parasites/field) at the time of diagnosis. Sera were obtained by venipuncture from patients and controls registered at Kala-azar Medical Research Centre, Muzaffarpur, India, and Sir Sundar Lal Hospital, Banaras Hindu University, Varanasi, India. Blood was allowed to coagulate at room temperature and was then centrifuged at 1,400 x g for 5 min. All sera were stored at -70C until required. Western blot analysis. | SDS-polyacrylamide gel electrophoresis was done with a vertical (Bangalore Genei, Peenya Bangalore, India) gel apparatus. The antigen was boiled for 5 min in sample buffer (two times) and was immediately subjected to electrophoresis in an SDS-10% polyacrylamide gel containing 0.1% SDS as described by Laemmli . The slab gel was run with two lanes per comb: a 100-mm lane for the parasite antigen sample and a 7-mm lane for a wide-range molecular mass marker (kind gift of David Sacks, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.). Three hundred micrograms of protein was used for gel electrophoresis. The gels were run at 15 mA in the stacking gel and 30 mA in the resolving gel until the bromophenol blue dye migrated 1 cm from the bottom of the gel in Tris-glycine-SDS buffer (pH 8.3) . Immunoblotting. | Transfer of polypeptides from SDS-polyacrylamide gels onto nitrocellulose membrane (pore size, 0.45 mum; kind gift of R. T. Kenney, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Md.) was done in a transblotting chamber at 4 V/cm and 4C for overnight in 25 mM Tris-192 mM glycine-20% (vol/vol) methanol . Following the blotting, the membranes were stained with 0.5% ponceau red for 5 min and were destained with distilled water. The blotted gels were stained with Coomassie brilliant blue in order to make sure that all polypeptides had been completely transferred. Detection of antibodies from patients' sera bound to antigens was done with a 5-bromo-4-chloro-3-indolylphosphate-Nitro Blue Tetrazolium system (BCIP-NBT; Kirkegaard & Perry Laboratories, Gaithersburg, Md.). Four-millimeter-wide strips were cut from the previously blotted membrane and blocked in 5% bovine serum albumin-0.3% Tween 20 in 100 mM Tris-buffered saline (TBS; pH 7.4) for an hour at room temperature. After six washes of 15 min each in TBS-0.1% Tween 20, the strips were incubated with serum (diluted 1:100 in 1% bovine serum albumin-TBS) for 2 h at 37C with constant stirring. After incubation with the primary antibody, the strips were washed four times for 15 min each time with Tween 20-TBS. After the last wash, immune complexes were detected with a 1:500 dilution of alkaline phosphatase (Bangalore Genei) conjugated with goat anti-human immunoglobulin G (IgG), and the mixture was incubated with constant shaking for 2 h at room temperature. After incubation the membrane was washed with Tween 20-TBS three or four times and then washed with TBS only to remove the Tween 20. The membranes were developed with the BCIP-NBT system (Kirkegaard & Perry Laboratories) for 5 min. The reaction was stopped with PBS-EDTA (20 mM EDTA). Statistical analysis. | Analysis of the corresponding polypeptides in the blotted membranes was done by using an exponential regression curve with the molecular mass markers of each membrane, calculating the relative mobility (Rf) of each protein , and looking for its corresponding molecular mass. PMID- 12204969_RESULTS TI - AB - Study population. | The clinical and laboratory features of the visceral leishmaniasis patients on admission are summarized in Table . The response to SAG or miltefosine therapy was prompt, and the splenic aspirate smears of all patients showed no parasites (L. donovani bodies) at the end of treatment (the patients were parasitologically cured). These patients were subsequently monitored. Pretreatment sera. | In general, the sera from patients (n = 10) with visceral leishmaniasis specifically recognized polypeptides ranging from 18 to more than 201 kDa, with the frequently occurring bands being of 201 kDa (50% of serum samples), 193 kDa (60%), 147 kDa (50%), 120 kDa (60%), 100 kDa (50%), 80 kDa (80%), 70 kDa (70%), 65 kDa (100%), 50 kDa (50%), 36 kDa (50%), 20 kDa (70%), and 18 kDa (50%). We found one band of 65 kDa in sera from 100% of patients with active disease . Immunoblot evaluation. (i) SAG treatment group. | We studied the sera of patients (n = 10) who were being treated with SAG. We did antibody tests at regular intervals for up to 6 months posttreatment. All patients were cured after the end of treatment, as confirmed by laboratory and parasitological tests . At 1 month after treatment, the bands of 201, 193, 100, and 36 kDa disappeared. At 3 and 6 months posttreatment all bands except the 70-kDa band disappeared . (ii) Miltefosine treatment group. | We also studied patients (n = 10) who were being treated with the oral drug miltefosine. We compared pretreatment sera with sera obtained at 1, 3, and 6 months posttreatment. At 1 month posttreatment only the 20- and 80-kDa bands had disappeared. At 3 months posttreatment all bands except the bands at 36, 65, and 70 kDa had disappeared; and at 6 months posttreatment all bands except the 65- and 70-kDa bands had disappeared. The 65- and 70-kDa bands remained at all time points. Sera from all patients had antibodies to the 65- and 70-kDa bands in common at pretreatment and 1, 3, and 6 months (follow up) posttreatment . Interestingly, all patients in both the SAG and miltefosine treatment groups showed the exact same response pattern after treatment and follow-up. FIG. 1. | Immunoblotting with L. donovani Immunoblotting with L. donovani soluble antigen with the alkaline phosphatase system. VL lanes 1 to 10, sera from patients with visceral leishmaniasis at the time of diagnosis; EC lanes 1 to 3, sera from controls from an area of endemicity; and NEC lanes 1 to 3, sera from controls from an area of nonendemicity. FIG. 2. | Immunoblotting with soluble L. donovani Immunoblotting with soluble L. donovani antigen with the alkaline phosphatase system. Lane 1, serum from patients at the time of diagnosis; lanes 2, 3, and 4, patterns obtained for all patients (n = 10) with serum taken on days 30, 90, and 180, respectively, after treatment with SAG. FIG. 3. | Immunoblotting with soluble L. donovani Immunoblotting with soluble L. donovani antigen with the alkaline phosphatase system from a patient with visceral leishmaniasis. Lane 1, serum from patients at the time of diagnosis; lanes 2, 3, and 4, the patterns obtained for all patients (n = 10) with serum taken on days 30, 90, and 180, respectively, after treatment with miltefosine. TABLE 1 | Clinical and laboratory features of patients with visceral leishmaniasis PMID- 12204969_DISCUSSION TI - AB - Parasitological methods have always been considered first-choice procedures for the diagnosis and prognosis of leishmaniasis due to their 100% specificities, although their sensitivities may vary depending upon the experience of the diagnostic team. However, in some instances it is very difficult to demonstrate the presence of the parasite, such as in patients with mucocutaneous leishmaniasis and visceral leishmaniasis. In these patients, serodiagnosis becomes an important alternative for demonstrating the presence of the parasite . Specific serological techniques are based on the determination of antibodies produced against the circulating Leishmania-specific antigens. Both clinical and epidemiological data provide presumptive evidence in the diagnosis of visceral leishmaniasis. However, on the basis of these same criteria, other diseases must be considered in the differential diagnosis; among these are malaria, typhoid fever, tuberculosis, and leukemias. Definitive diagnosis is based on demonstration of the parasites, which may be found in aspirates of the spleen (98% of the aspirates are positive), (sternal) bone marrow (85% of the aspirates are positive), and liver (60% of the aspirates are positive). Splenic aspiration is the most reliable procedure, but it is a high-risk procedure. Because in many cases demonstration of parasites is difficult, serological tests have provided a useful alternative. Among the most recently described methods, the Western blot technique is highly sensitive and specific and provides broader information about the parasite's antigenic profile . In the present study, a 65-kDa antigenic component was recognized by 100% of serum specimens from patients with clinically and pasitologically confirmed visceral leishmaniasis. It was never identified in the control sera tested (100% specificity). There was little variation in the patterns of the bands recognized by each of the serum specimens . Comparing our results with those of other groups, we found differences in the band patterns recognized by sera from patients with visceral leishmaniasis and cutaneous leishmaniasis using several Leishmania isolates as antigen. The studies of Dos Santos et al. showed reactivities with the 119- and 123-kDa bands when an extract of L. infantum was used as the antigen with sera from patients with visceral leishmaniasis. In additon, Rolland-Burger et al. found that the sera of patients with visceral leishmaniasis reacted to a 119-kDa band when L. infantum was used as the antigen. If antibodies remain in the patient for years and the patient's serum has a Western blot pattern similar to the one obtained at the time of diagnosis, then serious doubts arise as to whether a patient is ever completely cured (i.e., the patient has no remaining parasites) or whether treatment has induced a chronic subpatent infection, leaving the patient susceptible to relapse later on; for example, immunosuppression can lead to the reemergence of active infection, as in the case of visceral leishmaniasis in patients with AIDS . A test as sensitive as Western blotting may be able to identify immunosuppressed patients who could be at risk of a relapse of leishmaniasis. The only difference between the pre- and posttreatment sera of the groups treated with SAG and miltefosine was the disappearance of all protein bands except those of 65 and 70 kDa, respectively. We could detect a 65-kDa polypeptide in 100% of the pretreatment sera from kala azar patients . Vinayak et al. reported on a 63- to 68-kDa complex which is an immunodominent surface molecule of L. donovani that can be used as an immunoprophylactic agent. Investigations have indicated that major surface glycoproteins with molecular masses ranging from 63 to 68 kDa (known as the glycopeptide 63 [gp63] complex) are important ligands in cell-to-cell interactions and cell infectivity . Vinayak et al. have shown that promastigotes of L. donovani use the gp66 molecule not only for identification but also for attachment to macrophages. Sera from patients with confirmed leishmaniasis have been shown to identify the gp63 complex molecule in Western immunoblots of Leishmania crude antigen . Further work is needed to define the possible value of the 65-kDa band as a prognostic indicator. We suggest that the band of 65 kDa could be useful for diagnosis of Indian visceral leishmaniasis. Furthermore, the results of our studies suggest that Western blotting could be a sensitive technique for differentiation between the acute and chronic stages of disease. The presence of bands of 201, 193, 147, 100, 80, 70, 65, 50, 36, 20, and 18 kDa (Fig. and ) and the subsequent disappearance of all bands except those of 65 and 70 kDa during the course of treatment suggest the usefulness of Western blotting in making a clear-cut prognosis. The most interesting observation was the disappearance of protein bands unique to both the SAG and the miltefosine treatment groups, as the sera of all patients in the trial groups showed the same band patterns at the respective time points. This suggests that the type of drug used for treatment probably has a bearing on the immune response. Our data also suggest that Western blotting could successfully be used when there is no splenomegaly. Western blotting could also be a prognostic tool, as the disappearance or reappearance of specific bands could predict cure or relapse. Moreover, Western blotting analysis showed a large diversity in the antibody response to visceral leishmaniasis and permitted detection of antibodies to Leishmania in serum. PMID- 12204946 TI - Augmentation of the Lipopolysaccharide-Neutralizing Activities of Human Cathelicidin CAP18/LL-37-Derived Antimicrobial Peptides by Replacement with Hydrophobic and Cationic Amino Acid Residues AB - | Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L1 to S37) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K15 to V32) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14+ murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E16 and K25 with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q22, D26, and N30 with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14+ cells and attenuating production of tumor necrosis factor alpha (TNF-alpha) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-alpha production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock. PMID- 12204946_Introduction TI - AB - Peptide antibiotics exhibit potent antimicrobial activities against both gram-positive and gram-negative bacteria, fungi, and viruses, and they form one group of effector components in the innate host defense system . The peptide-based defense in mammals against invading microbes relies on two evolutionarily distinct groups of antimicrobial peptides, defensins and cathelicidins, which have been identified in several epithelial tissues and in the granules of phagocytes (, , -, , , ). Defensins contain six conserved cysteine residues in their sequences and exhibit characteristic beta-sheet structures stabilized by three intramolecular disulfide bonds . In contrast, cathelicidins are characterized by highly conserved cathelin-like prosequences and variable carboxyl-terminal sequences that correspond to the mature antibacterial peptides . About 30 cathelicidin members from various mammalian species have been identified; however, only one cathelicidin, hCAP18 (human cationic antibacterial protein of 18 kDa), has been found in humans, and its carboxyl-terminal antibacterial peptide, called LL-37, which comprises 37 amino acid residues (L1LGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES37), has recently been identified . In addition to exhibiting potent antibacterial activities against gram-positive and gram-negative bacteria, peptides derived from LL-37 can bind to lipopolysaccharide (LPS) and neutralize its biological activities . LPS is a major constituent of the outer membranes of gram-negative bacteria and is recognized as a key molecule in the pathogenesis of endotoxin shock associated with gram-negative bacterial infections . Recently, we have shown that LL-37 exerts protective action against endotoxin shock by blocking the binding of LPS to CD14+ cells, thereby suppressing the production of cytokines by these cells . Thus, LL-37 and its related derivatives could be attractive candidates for therapeutic agents that can be used for endotoxin shock and sepsis caused by gram-negative bacterial infections . Although antibacterial peptides are diverse in their sizes, structures, and activities, they are mostly amphipathic, retaining both cationic (positively charged) and hydrophobic faces . These features facilitate interactions with negatively charged microbial surface membranes, followed by insertion into the microbial lipid membrane, which alters membrane permeability and impairs internal homeostasis . Moreover, the positively charged amphipathic structures are assumed to be important for interaction with a negatively charged amphipathic LPS . Interestingly, secondary-structure predictions indicate that antibacterial peptides of some cathelicidin members, such as hCAP18/LL-37, rabbit CAP18-derived peptide, and guinea pig CAP11 (cationic antibacterial polypeptide of 11 kDa), adopt an alpha-helical amphipathic conformation ; the helical wheel regions are clearly amphipathic and subtended by the hydrophilic (positively charged) and hydrophobic sectors . Interestingly, structure-activity relationship (SAR) studies using different kinds of natural and synthetic model peptides have revealed that the potencies and spectra of the antibacterial activities of amphipathic alpha-helical antimicrobial peptides can be influenced by interrelated structural and physicochemical parameters such as charge (cationicity), hydrophobicity, and amphipathicity . Thus, it could be anticipated that by changing these parameters one could design novel antimicrobial peptides with increased potency and directed activity which would be effective as therapeutic agents for bacterial infections and their related symptoms. FIG. 1. | Helical wheel projections for LL-37 and its 18-mer peptide derivatives. Helical wheel projections for LL-37 and its 18-mer peptide derivatives. The sequences of alpha-helical peptides LL-37, 18-mer K15-V32, 18-mer LL, and 18-mer LLKKK are presented according to the Shiffer-Edmundson wheel projection analyzed with a Genetyx-Mac computer system (Software Development, Tokyo, Japan). To increase hydrophobicity, E16 and K25 in 18-mer K15-V32 were replaced by L16 and L25 in 18-mer LL, respectively. Furthermore, to increase cationicity, Q22, D26, and N30 in 18-mer LL were replaced by K22, K26, and K30 in 18-mer LLKKK, respectively. Positively charged residues are circled, whereas negatively charged residues are boxed. Hydrophobic residues are outlined, while neutral hydrophilic residues are not. The hydrophilic and hydrophobic sectors are divided by dashed lines. During analysis of the biological activities of hCAP18/LL-37-derived antimicrobial peptides of different sizes , it was found that a short fragment peptide (18-mer; K15 to V32 [K15-V32]) of LL-37 displayed an amphipathic alpha-helical structure and possessed LPS-neutralizing activity almost equal to that of the parent peptide, LL-37; like LL-37, the 18-mer peptide inhibited the binding of LPS to CD14+ cells and suppressed LPS-induced cytokine production by these cells. Thus, the 18-mer peptide may be a good template for development of therapeutic agents that can be used for prevention of gram-negative bacterial sepsis and endotoxin shock. In this study, therefore, to develop 18-mer peptides with improved LPS-neutralizing activities, we modified the hydrophobicity and cationicity of the peptide by substitution of leucine and lysine residues, respectively, and evaluated the activities of those peptide derivatives by using the CD14+ murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. PMID- 12204946_MATERIALS AND METHODS TI - AB - Reagents. | Fluorescein isothiocyanate (FITC)-conjugated or unconjugated LPS (from Escherichia coli O111:B4), the 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate system, and d-galactosamine were purchased from Sigma (St. Louis, Mo.). In some experiments, LPS was biotinylated with biotin-LC-hydrazide, based on the manufacturer's protocol (Pierce, Rockford, Ill.). A 37-mer peptide of hCAP18 (LL-37; L1LGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES37) and its 18-mer derivatives (18-mer K15-V32, 18-mer LL, and 18-mer LLKKK) were synthesized by the solid-phase method on a peptide synthesizer (model PSSM-8; Shimadzu, Kyoto, Japan) by fluorenylmethoxycarbonyl (Fmoc) chemistry. The peptides were eluted from the resin and purified to homogeneity by reversed-phase high-performance liquid chromatography on a Cosmosil 5C18 column (Nacalai Tesque, Kyoto, Japan) by use of a 0 to 70% acetonitrile gradient in 0.1% trifluoroacetic acid. The molecular masses of the peptides synthesized were confirmed on a mass spectrometer (model TSQ 700; Thermo Quest Finnigan, Manchester, United Kingdom). The sequences of the peptides were as follows: 18-mer K15-V32, K15EFKRIVQRIKDFLRNLV32; 18-mer LL, K15LFKRIVQRILDFLRNLV32; 18-mer LLKKK, K15LFKRIVKRILKFLRKLV32 (underlining indicates amino acid substitutions introduced into the original 18-mer peptide to increase hydrophobicity or cationicity). Tissue culture supplies were obtained from Iwaki Glass (Tokyo, Japan). Antibodies. | As anti-LPS-binding protein (LBP) antibodies, mouse anti-human LBP monoclonal antibody (MAb) 6G3 (HyCult Biotechnology, Uden, The Netherlands), which can cross-react with bovine LBP, and rat anti-mouse LBP MAb clone 39 (class 2) were used. These anti-LBP MAbs can recognize both free LBP and LBP-LPS complexes, and they inhibit the transfer of LPS to CD14. As anti-CD14 antibodies, FITC-conjugated rat anti-mouse CD14 MAb rmC5-3 (BD PharMingen, San Diego, Calif.) and FITC-conjugated or unconjugated rat anti-mouse CD14 MAb 4C1 were utilized. MAb 4C1 can block the binding of LPS to CD14+ cells, whereas rmC5-3 has little effect on LPS binding . Cells. | The murine macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (Manassas, Va.) and cultured in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Sanko Junyaku, Tokyo, Japan) at 37C under 5% CO2. Confluent RAW 264.7 cells were detached by washing with 0.05% EDTA in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4 [pH 7.4]) and suspended in RPMI 1640 containing 10% FBS. Assay for binding of FITC-conjugated LPS to RAW 264.7 cells. | RAW 264.7 cells (5 x 105/ml) were incubated with FITC-conjugated LPS (100 ng/ml) in the absence or presence of 18-mer peptides or LL-37 (0.01 to 10 mug/ml) in RPMI 1640 containing 10% FBS for 15 min at 37C. After cells were washed with PBS, the binding of FITC-conjugated LPS was analyzed by flow cytometry (FACScan; Becton Dickinson, Rutherford, N.J.), and median fluorescence intensity was determined. Alternatively, RAW 264.7 cells were incubated with FITC-conjugated LPS in the presence of anti-human LBP MAb 6G3 or anti-mouse CD14 MAb 4C1 at 5 mug/ml in RPMI 1640 containing 10% FBS, and the binding of FITC-conjugated LPS was analyzed as described above. In some experiments, RAW 264.7 cells were preincubated with 18-mer peptides or LL-37 at 1 mug/ml in RPMI 1640 containing 10% FBS for 10 min at 37C. After a wash, cells were incubated with FITC-conjugated LPS (100 ng/ml) in RPMI 1640 containing 10% FBS for 15 min at 37C, and LPS binding was evaluated by flow cytometry. In separate experiments, using RPMI 1640 containing 10% mouse serum, we confirmed that FITC-conjugated LPS could bind to RAW 264.7 cells, and the binding was completely suppressed by anti-mouse LBP MAb clone 39 and anti-mouse CD14 MAb 4C1. Moreover, we observed that 18-mer peptides and LL-37 inhibited the binding of FITC-conjugated LPS to RAW 264.7 cells and LPS-induced tumor necrosis factor alpha (TNF-alpha) expression by these cells (see below) in the medium containing 10% mouse serum, as in the medium containing 10% FBS (data not shown). These findings suggest the possibility that the peptides could function in mice. Evaluation of TNF-alpha expression. | RAW 264.7 cells (106/well in a 24-well microplate) were incubated with LPS (100 ng/ml) in the absence or presence of 18-mer peptides or LL-37 at 1 mug/ml in 500 mul of RPMI 1640 containing 10% FBS for 4 h at 37C. After incubation, cells were detached by a wash with 0.05% EDTA-PBS, and expression of TNF-alpha mRNA and protein was analyzed by Northern and Western blotting, respectively . In brief, total cellular RNA (2.5 mug) was separated by electrophoresis on a 1% agarose-formaldehyde gel, and Northern blot hybridization was performed by using cDNA probes labeled with a digoxigenin-High Prime DNA labeling kit (Roche Diagnostics, Mannheim, Germany); probes used were the 0.39-kb mouse TNF-alpha cDNA (encompassing nucleotides 427 to 819) and the 2.1-kb beta-actin cDNA (pHFbetaA-1) . In Western blot analysis, cell sonicates in PBS containing 1 mM diisopropyl fluorophosphate (3 x 105 cells for TNF-alpha and 3 x 104 cells for beta-actin) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 7.5 to 20% linear gradient of polyacrylamide under reducing conditions. Resolved proteins were electrotransferred to an Immobilon-P membrane (Millipore, Bedford, Mass.), and blots were probed with a rabbit anti-mouse TNF-alpha antibody (Ab) (Genzyme Diagnostics, Cambridge, Mass.) or an anti-beta-actin MAb (Sigma). Blots were further probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (Organon Teknika, Durham, N.C.) or goat anti-mouse IgG/IgM (Chemicon International, Temecula, Calif.), and proteins were finally detected with SuperSignal West Pico chemiluminescent substrate (Pierce). In some experiments, RAW 264.7 cells were incubated with LPS in the medium containing 10% mouse serum, and the effects of 18-mer peptides and LL-37 on LPS-induced TNF-alpha expression were investigated as described above. Measurement of LPS-binding activities of 18-mer peptides. | Ninety-six-well microtiter plates (Immulon 2H; Dynex Technologies, Ashford, United Kingdom) were coated with LPS (100 ng/well) as described previously . Excess binding sites were blocked with 100 mul of PBS containing 1% bovine serum albumin (BSA)/well, and 18-mer peptides and LL-37 (0.02 to 0.5 mug/well) were incubated in the plates for 1 h at 37C in 50 mul of RPMI 1640 without phenol red (Life Technologies, Grand Island, N.Y). Alternatively, 18-mer peptides and LL-37 (0.1 mug/well) were incubated in the presence of LPS (0.05 to 2.5 mug/well) in 50 mul of RPMI 1640. After a wash, an affinity-purified rabbit anti-CAP18 Ab (50 mul/well; 2 mug/ml in PBS containing 0.1% BSA) was added and incubated for 1 h at 37C in the plates. The Ab solution was then rinsed out, and HRP-conjugated goat anti-rabbit IgG (50 mul/well; diluted 2,000-fold in PBS containing 0.1% BSA) was incubated for 1 h at room temperature. Finally, a TMB liquid substrate (100 mul/well) was incubated for 5 to 15 min. The reaction was stopped by addition of 100 mul of 0.18 M sulfuric acid/well, and absorbances at 450 and 560 nm were quantitated in a microtiter plate reader. An anti-CAP18 serum was raised in rabbits by use of the LL-37 peptide covalently coupled to keyhole limpet hemocyanin, and the Ab was purified by affinity chromatography using LL-37 peptide-conjugated, epoxy-activated Sepharose (Amersham Pharmacia Biotech). In preliminary experiments, we confirmed that the anti-CAP18 Ab could recognize not only LL-37 but also 18-mer K15-V32, 18-mer LL, and 18-mer LLKKK (data not shown). Assay for interaction of LPS with LBP. | LPS (100 ng/well) was used to coat the 96-well-microtiter plates as described above. After blocking, RPMI 1640 containing 0.1, 1, or 10% FBS (50 mul/well) was added and incubated for 1 h at 37C. Plates were then washed, and 50 mul of anti-LBP MAb 6G3 (25 nM in PBS containing 0.1% BSA)/well was incubated in the plates for 1 h at 37C. The MAb solution was rinsed out, and 50 mul of HRP-conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark; diluted 1,000-fold in PBS containing 0.1% BSA)/well was incubated for 1 h at room temperature. Finally, binding of LBP to the immobilized LPS was detected by incubation with TMB liquid substrate (100 mul/well). Alternatively, the LPS microtiter plates were preincubated with 18-mer peptides or LL-37 (0.025 to 0.2 mug/well) in 50 mul of RPMI 1640/well for 1 h at 37C. After a wash, RPMI 1640 containing 10% FBS (50 mul/well) was added, and binding of LBP was determined as described above. Assay for binding of LBP to 18-mer peptides. | Microtiter plates were coated with 18-mer peptides or LL-37 (2.5 mug/well) by incubating 50 mul of 50-mug/ml peptides in PBS/well overnight at room temperature. After blocking, RPMI 1640 containing 10% FBS (50 mul/well) was added and incubated for 1 h at 37C. Plates were then washed, and 50 mul of anti-LBP MAb 6G3 (25 nM in PBS containing 0.1% BSA)/well was incubated in the plates for 1 h at 37C. The MAb solution was rinsed out and replaced with 50 mul of HRP-conjugated rabbit anti-mouse IgG (diluted 1,000-fold in PBS containing 0.1% BSA)/well for 1 h at room temperature. Finally, binding of LBP to the immobilized peptides was detected by TMB reaction. As a positive control, biotinylated LPS (100 ng/well) was added to plates coated with 18-mer peptides or LL-37, and the plates were incubated for 1 h at 37C in 50 mul of RPMI 1640/well. The LPS solution was then rinsed out, and 50 mul of HRP-conjugated streptavidin (Dako; diluted 5,000-fold in PBS containing 0.1% BSA)/well was incubated for 1 h at 37C. Binding of biotinylated LPS to the peptides that had been used to coat the wells was finally detected by TMB reaction. Flow cytometric assay for expression of CD14 and binding of 18-mer peptides to CD14. | To analyze the effects of 18-mer peptides and LL-37 on CD14 expression, RAW 264.7 cells (5 x 105/ml) were incubated without or with the peptides (1 mug/ml) or LPS (100 ng/ml) in RPMI 1640 containing 10% FBS for 15 min at 37C and were further incubated with FITC-conjugated rat anti-mouse CD14 MAb rmC5-3 (2.5 mug/ml), or with FITC-conjugated rabbit anti-mouse IgG (Dako) as a negative control, for 15 min at 37C. After a wash, the binding of the anti-CD14 MAb was measured by flow cytometry. Furthermore, the binding of 18-mer peptides and LL-37 to CD14 was examined by using the neutralizing anti-mouse CD14 MAb 4C1, which recognizes the murine CD14 epitope located near the LPS-binding site . RAW 264.7 cells (5 x 105/ml) were preincubated without or with the peptides (1 mug/ml), or with LPS (100 ng/ml) as a positive control, for 15 min at 37C in RPMI 1640 containing 10% FBS. Cells were then added with 50 ng of FITC-conjugated anti-CD14 MAb 4C1/ml, or with 50 ng of FITC-conjugated rabbit anti-mouse IgG/ml as a negative control, and were further incubated for 15 min at 37C. After a wash, the binding of FITC-conjugated anti-CD14 MAb 4C1 was analyzed by flow cytometry. Evaluation of effects of 18-mer peptides on the murine endotoxin shock model. | To determine the protective activities of 18-mer peptides against lethal LPS activity, we utilized d-galactosamine-sensitized mice, which are highly susceptible to LPS . d-Galactosamine (18 mg/0.3 ml of saline), LPS (200 ng/0.2 ml of saline), and 18-mer peptides or LL-37 (1 mug/0.2 ml of saline) were sequentially injected intraperitoneally (i.p.) into male C57BL/6 mice aged 10 weeks (Charles River Japan, Kanagawa, Japan), and deaths were recorded every 24 h until day 6 after injection. Furthermore, 75 min after LPS challenge, mice were sacrificed by drawing blood from the heart, and serum samples were prepared. Serum TNF-alpha levels were determined by using a commercially available mouse TNF-alpha enzyme-linked immunosorbent assay kit (Endogen, Woburn, Mass.) that can detect <9 pg of TNF-alpha/ml. In some experiments, 75 min after challenge with d-galactosamine (18 mg), FITC-conjugated LPS (200 ng), and 18-mer peptides or LL-37 (1 mug), peritoneal fluids were harvested by washing the peritoneal cavities with PBS, and peritoneal macrophages were recovered. Binding of FITC-conjugated LPS to peritoneal macrophages was analyzed by flow cytometry, and expression of TNF-alpha in peritoneal macrophages was investigated by Northern and Western blotting, as described above. The experiments were carried out in accordance with institutional guidelines, and mice received proper care and maintenance. Statistical analysis. | Data are shown as means +- standard deviations (SD). Statistical significance was determined by one-way analysis of variance with a multiple-comparison test (StatView; Abacus Concepts, Berkeley, Calif.) unless otherwise noted. Survival in mice after LPS administration was assessed by a chi2 test (StatView). A P value of <0.05 was considered to be significant. PMID- 12204946_RESULTS TI - AB - Effects of 18-mer peptides on binding of FITC-conjugated LPS to RAW 264.7 cells. | We first examined the effects of 18-mer peptides on the binding of FITC-conjugated LPS to CD14+ cells by flow cytometry using the murine macrophage cell line RAW 264.7. When FITC-conjugated LPS was incubated with RAW 264.7 cells in the presence of serum, it bound to the cells . However, when FITC-conjugated LPS was incubated with RAW 264.7 cells in the presence of a neutralizing anti-LBP MAb or anti-CD14 MAb, LPS binding was completely inhibited to the background level (the level without FITC-conjugated LPS), indicating that LPS binds to the cellular CD14 molecule via the action of LBP in serum. Interestingly, 18-mer peptides as well as LL-37 (1 mug/ml each) markedly suppressed the binding of FITC-conjugated LPS to RAW 264.7 cells . Their effects were dose dependent , and 18-mer LLKKK (50% inhibitory concentration [IC50], 0.09 mug/ml, or 0.043 muM) was the most potent of the peptides tested at inhibiting the binding of FITC-conjugated LPS to RAW 264.7 cells (for 18-mer LL, the IC50 was 0.23 mug/ml, or 0.102 muM; for 18-mer K15-V32, the IC50 was 0.31 mug/ml, or 0.136 muM; for LL-37, the IC50 was 0.31 mug/ml, or 0.070 muM). Furthermore, the effects of 18-mer peptides on TNF-alpha expression were examined by Northern and Western blot analyses . The 18-mer peptides and LL-37 (1 mug/ml) apparently suppressed LPS-induced TNF-alpha expression by RAW 264.7 cells at both the mRNA and protein levels, and the effect of 18-mer LLKKK was the most prominent. Eighteen-mer LLKKK also exerted the most suppressive action on LPS-induced TNF-alpha expression by RAW 264.7 cells at 0.1 mug/ml among the peptide derivatives investigated (data not shown). LPS-binding activities of 18-mer peptides and their effects on the binding of LPS to LBP. | In order to clarify the mechanisms by which 18-mer peptides inhibit the binding of LPS to CD14+ cells, we investigated the LPS-binding activities of these peptides by using LPS-coated microtiter plates. The 18-mer peptides and LL-37 bound to the LPS-coated plates in a dose-dependent fashion , and the binding was dose-dependently inhibited by addition of LPS to the plates . Notably, the binding of 18-mer LLKKK was more potently inhibited by LPS addition (IC50, 0.08 mug of LPS/well) than that of the other peptides; IC50s of LPS, in micrograms per well, were 0.16 for18-mer LL, 0.25 for 18-mer K15-V32, and 0.34 for LL-37. Using the LPS-coated plates, we next examined the effects of 18-mer peptides on the interaction between LPS and LBP, which catalyzes the transfer of LPS to CD14 . When LPS-coated plates were incubated with FBS, the LBP in the serum bound to the LPS-coated plates proportionately to the concentrations of serum used . When the LPS-coated plates were pretreated with the peptide derivatives, binding of LBP to the LPS-coated plates was inhibited in a dose-dependent manner by addition of 18-mer peptides or LL-37 to the plates . Importantly, 18-mer LLKKK (IC50, 0.018 mug/well, or 7.9 pmol/well) was the most potent of the 18-mer peptides at inhibiting LPS-LBP binding; other IC50s were 0.024 mug/well, or 10.9 pmol/well, for 18-mer LL; 0.035 mug/well, or 15.2 pmol/well, for 18-mer K15-V32; and 0.025 mug/well, or 5.4 pmol/well, for LL-37. Furthermore, we evaluated the interaction of LBP with the peptides by using 18-mer peptide- or LL-37-coated microtiter plates. Apparently, substantial amounts of biotinylated LPS, a positive control, bound to the 18-mer peptide- or LL-37-coated plates. In contrast, LBP hardly bound to the plates (data not shown). The observations described above indicate that the 18-mer K15-V32, 18-mer LL, and 18-mer LLKKK peptides have potential to bind to LPS, but not LBP, thereby inhibiting the interaction of LPS with LBP. Moreover, 18-mer LLKKK appears to be the most potent at binding to LPS and inhibiting LPS-LBP interaction. Effects of 18-mer peptides on CD14 expression and binding of 18-mer peptides to CD14+ cells. | It is possible that antimicrobial peptides may alter CD14 expression, thereby affecting the binding of LPS to CD14+ cells. To check this, we investigated the expression of CD14 after treatment of RAW 264.7 cells with 18-mer peptides by flow cytometry using FITC-conjugated anti-mouse CD14 MAb rmC5-3. Neither 18-mer peptides LL-37 (1 mug/ml each), nor LPS (100 ng/ml) altered CD14 expression on RAW 264.7 cells . Likewise, it is conceivable that antimicrobial peptides bind to the CD14 molecule on the cells and influence the binding of LPS to CD14+ cells without affecting CD14 expression. To confirm this, RAW 264.7 cells were preincubated with 18-mer peptides, and after a wash, the binding of FITC-conjugated LPS was analyzed by flow cytometry. Interestingly, preincubation with 18-mer LLKKK or 18-mer LL (1 mug/ml) inhibited the binding of FITC-conjugated LPS to RAW 264.7 cells by 33.9% +- 0.1% or 22.5% +- 2.6%, respectively (n = 3; P < 0.001 for comparison with binding of FITC-conjugated LPS in the absence of peptides), while preincubation with 18-mer K15-V32 or LL-37 inhibited LPS binding by only 5 to 7% . To further clarify the binding of peptides to the CD14 molecule, we utilized anti-CD14 MAb 4C1, which can recognize the murine CD14 epitope and inhibit the binding of LPS to CD14+ cells . As expected, when RAW 264.7 cells were preincubated with LPS (100 ng/ml), binding of anti-CD14 MAb 4C1 (50 ng/ml) to the cells was inhibited by 40.2% +- 11.9% (n = 3) , suggesting that the epitope, which can be recognized by MAb 4C1, is located on murine CD14 next to the LPS-binding site. Importantly, preincubation with 18-mer LLKKK or 18-mer LL (1 mug/ml each) inhibited the binding of FITC-conjugated anti-CD14 MAb 4C1 to RAW 264.7 cells by 33.5% +- 5.2% or 26.2% +- 9.8%, respectively (n = 3 or n = 5; P < 0.05 for comparison with the binding of FITC-conjugated anti-CD14 MAb 4C1 in the absence of peptides), whereas preincubation with 18-mer K15-V32 or LL-37 inhibited the binding by only 10 to 14% . These observations likely suggest that among 18-mer peptides, 18-mer LLKKK has the most potent activity to bind to CD14 near the LPS-binding site, thereby influencing the binding of LPS to CD14. Effects of 18-mer peptides on the murine endotoxin shock model. | Using d-galactosamine-sensitized mice, we assessed the effects of 18-mer peptides on lethal LPS activity in vivo . d-Galactosamine administration sensitized mice to the lethal effect of LPS, and 94% of the mice died within 24 h after i.p. injection of 200 ng of LPS . Notably, administration of 18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37 (1 mug/mouse) increased the survival rate to 33, 50, 67, or 47%, respectively, all significantly higher than that of the group with d-galactosamine and LPS administration alone (P < 0.05); 18-mer LLKKK administration was the most protective. In agreement with this, administration of an 18-mer peptide or LL-37 markedly lowered the LPS-induced increase in serum TNF-alpha levels (P < 0.001) , and 18-mer LLKKK had the greatest effect. In addition, the effects of 18-mer peptides on the binding of FITC-conjugated LPS to peritoneal macrophages (CD14+ cells) were analyzed by flow cytometry. Administration of the peptides, especially 18-mer LLKKK, markedly suppressed the binding of FITC-conjugated LPS to peritoneal macrophages (P < 0.001) . Concurrently, TNF-alpha expression in peritoneal macrophages was investigated. Northern and Western blot analyses indicated that administration of the peptides, particularly 18-mer LLKKK, strongly suppressed LPS-induced TNF-alpha mRNA and protein expression by peritoneal macrophages (data not shown), just as was observed in LPS-stimulated RAW 264.7 cells . Together, these observations likely suggest that among 18-mer peptides, 18-mer LLKKK exerts the most protective action against murine endotoxin shock by blocking the binding of LPS to CD14+ cells, thereby suppressing the production of cytokines (such as TNF-alpha) by these cells, possibly via its potent binding to LPS and CD14. FIG. 2. | Effects of 18-mer peptides on binding of FITC-conjugated LPS to RAW 264. Effects of 18-mer peptides on binding of FITC-conjugated LPS to RAW 264.7 cells. (A) RAW 264.7 cells (5 x 105/ml) were incubated with 100 ng of FITC-conjugated LPS/ml in the absence or presence of 5 mug of anti-LBP MAb 6G3 or anti-CD14 MAb 4C1/ml in RPMI 1640 containing 10% FBS for 15 min at 37C. (B) Alternatively, RAW 264.7 cells were incubated with FITC-conjugated LPS in the presence of 1 mug of peptide (18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37)/ml in RPMI 1640 containing 10% FBS. After a wash, the binding of FITC-LPS was analyzed by flow cytometry. Background (BG) was assessed by using RAW 264.7 cells incubated without FITC-LPS. Data are from one of five separate experiments. FIG. 3. | Dose-dependent inhibition of the binding of FITC-conjugated LPS to RAW 264. Dose-dependent inhibition of the binding of FITC-conjugated LPS to RAW 264.7 cells by 18-mer peptides. RAW 264.7 cells (5 x 105/ml) were incubated with 100 ng of FITC-conjugated LPS/ml in the absence or presence of 0.01 to 10 mug of 18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37/ml in RPMI 1640 containing 10% FBS for 15 min at 37C. After a wash, the binding of FITC-LPS was analyzed by flow cytometry, and median fluorescence intensity was determined. Binding of LPS was expressed as a percentage of that obtained by using RAW 264.7 cells incubated with FITC-conjugated LPS alone. Data are means +- SD from three to five separate experiments. Values for 18-mer K15-V32 are compared with those for 18-mer LL, 18-mer LLKKK, or LL-37. *, P < 0.05; **, P < 0.01; ***, P < 0.001. FIG. 4. | Effects of 18-mer peptides on LPS-induced TNF-alpha expression by RAW 264. Effects of 18-mer peptides on LPS-induced TNF-alpha expression by RAW 264.7 cells. RAW 264.7 cells (106/well in a 24-well microplate) were incubated without (Control) or with 100 ng of LPS/ml in the absence (LPS) or presence of 1 mug of peptide (18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37)/ml in 500 mul of RPMI 1640 containing 10% FBS for 4 h at 37C. After incubation, cells were recovered, and expression of TNF-alpha mRNA and protein was analyzed by Northern (A) and Western (B) blotting, respectively. As a control, expression of beta-actin mRNA and protein was also analyzed. Data are from one of three separate experiments. FIG. 5. | Evaluation of LPS-binding activities of 18-mer peptides. Evaluation of LPS-binding activities of 18-mer peptides. (A) LPS-binding activities of the peptides were investigated by incubating 0.02 to 0.5 mug of 18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37 in LPS-coated 96-well microtiter plates (100 ng of LPS/well) for 1 h at 37C in 50 mul of RPMI 1640. Bound peptides were detected by TMB reaction by using a rabbit anti-CAP18 Ab and HRP-conjugated goat anti-rabbit IgG. (B) The peptide (0.1 mug of either 18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37) was incubated in LPS-coated 96-well microtiter plates for 1 h at 37C in the absence or presence of added LPS (0.05 to 2.5 mug/well) in 50 mul of RPMI 1640, and bound peptides were detected as described above. Binding of peptides to LPS-coated plates was expressed as a percentage of that observed when 0.5 mug (for panel A) or 0.1 mug (for panel B) of each peptide was incubated in the absence of added LPS. Data are means +- SD from three to five separate experiments. Values for 18-mer K15-V32 are compared with those for 18-mer LL, 18-mer LLKKK, or LL-37. *, P < 0.05; ***, P < 0.001. FIG. 6. | Effects of 18-mer peptides on the interaction of LPS with LBP. Effects of 18-mer peptides on the interaction of LPS with LBP. (A) LPS-LBP binding was examined by incubating 50 mul of RPMI 1640 containing 0.1, 1, or 10% FBS in LPS-coated 96-well microtiter plates (100 ng of LPS/well) for 1 h at 37C. After incubation, bound LBP was detected by TMB reaction using anti-LBP MAb 6G3 and HRP-conjugated rabbit anti-mouse IgG. (B) LPS-coated microtiter plates were preincubated with either 18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37 at 0.025 to 0.2 mug/well in 50 mul of RPMI 1640 for 1 h at 37C. Thereafter, LPS-LBP binding was determined by incubating 50 mul of RPMI 1640 containing 10% FBS in the microtiter plates as described above. LPS-LBP binding was expressed as a percentage of that obtained by incubation with RPMI 1640 containing 10% FBS in the absence of added peptide. Data are means +- SD from three to eight separate experiments. Values for 18-mer K15-V32 are compared with those for 18-mer LL, 18-mer LLKKK, or LL-37. *, P < 0.05; **, P < 0.01; ***, P < 0.001. FIG. 7. | Effects of 18-mer peptides on expression of CD14 by RAW 264. Effects of 18-mer peptides on expression of CD14 by RAW 264.7 cells. RAW 264.7 cells (5 x 105/ml) were incubated without or with 100 ng of LPS/ml (A) or 1 mug of peptide (18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37)/ml (B) in RPMI 1640 containing 10% FBS for 15 min at 37C, after which cells were further incubated with 2.5 mug of FITC-conjugated rat anti-mouse CD14 MAb rmC5-3/ml for 15 min at 37C. After a wash, the binding of FITC-anti-CD14 MAb rmC5-3 was measured by flow cytometry. Background (BG) was assessed by using RAW 264.7 cells incubated with FITC-conjugated rabbit anti-mouse IgG (a negative control for nonspecific binding). Data are from one of three separate experiments. FIG. 8. | Evaluation of the binding of 18-mer peptides to CD14+ cells. Evaluation of the binding of 18-mer peptides to CD14+ cells. (A) RAW 264.7 cells (5 x 105/ml) were preincubated with 1 mug of peptide (18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37)/ml in RPMI 1640 containing 10% FBS for 10 min at 37C. After a wash, cells were incubated with 100 ng of FITC-conjugated LPS/ml in RPMI 1640 containing 10% FBS for 15 min at 37C, and the binding of FITC-conjugated LPS was analyzed by flow cytometry. Background (BG) was assessed by using RAW 264.7 cells incubated without FITC-conjugated LPS. (B and C) RAW 264.7 cells (5 x 105/ml) were preincubated without or with 100 ng of LPS/ml or 1 mug of peptide (18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37)/ml for 15 min at 37C in RPMI 1640 containing 10% FBS. Cells were further incubated with 50 ng of FITC-conjugated neutralizing anti-CD14 MAb 4C1/ml for 15 min at 37C, and after a wash, the binding of FITC-conjugated anti-CD14 MAb 4C1 was analyzed by flow cytometry. Background (BG) was assessed by using RAW 264.7 cells incubated with FITC-conjugated rabbit anti-mouse IgG (a negative control for nonspecific binding). Data are from one of three to six separate experiments. FIG. 9. | Protective effects of 18-mer peptides on survival and serum TNF-alpha levels in LPS-challenged mice. Protective effects of 18-mer peptides on survival and serum TNF-alpha levels in LPS-challenged mice. (A) Mice were i.p. injected with d-galactosamine alone at 18 mg/mouse (Control) or with LPS at 200 ng/mouse with or without 1 mug of peptide (18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37)/mouse. After injection, deaths were recorded every 24 h until day 6 (16 mice in each group). (B) After LPS challenge (75 min), serum samples were prepared, and serum TNF-alpha levels were determined by using a commercially available mouse TNF-alpha enzyme-linked immunosorbent assay kit. Data are means +- SD for 16 mice in each group. Values without and with peptide administration are compared, as are values with and without LPS administration. *, P < 0.05; **, P < 0.01; ***, P < 0.001. FIG. 10. | Effects of 18-mer peptide administration on the binding of FITC-conjugated LPS to mouse peritoneal macrophages. Effects of 18-mer peptide administration on the binding of FITC-conjugated LPS to mouse peritoneal macrophages. Mice were i.p. injected with d-galactosamine alone at 18 mg/mouse (Control) or with FITC-conjugated LPS at 200 ng/mouse with or without 1 mug of peptide (18-mer K15-V32, 18-mer LL, 18-mer LLKKK, or LL-37)/mouse. After LPS challenge (75 min), peritoneal macrophages were recovered, and binding of FITC-conjugated LPS to the cells was analyzed by flow cytometry. (A) Data are from 1 of 16 mice in each group. (B) Data are means +- SD for 16 mice in each group. Values without and with peptide administration are compared, as are values with and without LPS administration. ***, P < 0.001. PMID- 12204946_DISCUSSION TI - AB - For prevention of bacterial infections and their related symptoms (e.g., gram-negative bacterial septic shock), much attention has been focused on the low-molecular-weight cationic antimicrobial peptides that possess both antibacterial and LPS-neutralizing activities . Previously, it was demonstrated that in addition to exhibiting potent antibacterial activity against gram-positive and gram-negative bacteria , hCAP18/LL-37 could bind to the lipid-A moiety of LPS and inhibit the interaction of LPS with LBP, which transports LPS to CD14+ cells, thereby suppressing the binding of LPS to CD14+ cells and possibly attenuating Toll-like receptor-mediated CD14+ cell activation . In this study, we have revealed that the LPS-neutralizing activity of the amphipathic hCAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK, with increased hydrophobicity and cationicity, is the most potent of the peptide derivatives. Defensins and cathelicidins are the two major families of mammalian antimicrobial peptides, which contribute to the innate host defense against invading microorganisms by virtue of their broad spectra and potent antimicrobial activities (, , -, , ). Defensins are characterized by the presence of six invariant cysteine residues in their sequences and exhibit beta-sheet structure . In contrast, antimicrobial peptides of cathelicidins display remarkable structural variety; some are alpha-helical (e.g., hCAP18/LL-37, rabbit CAP18-derived peptide, and guinea pig CAP11), and others are proline and arginine rich, showing a polyproline type structure (e.g., porcine PR-39 and bactenecins), whereas porcine protegrins exhibit beta-sheet structure . Notably, alpha-helical cathelicidin peptides such as hCAP18/LL-37- and rabbit CAP18-derived peptides and guinea pig CAP11 have been shown to exert antimicrobial activities even under physiological conditions in which salt (150 mM NaCl) and serum are present, where the antimicrobial activities of defensins are substantially diminished . In addition, among cathelicidin peptides, only CAP18-derived peptides (human and rabbit) and CAP11 have been revealed to possess LPS-binding and -neutralizing activities . In contrast, defensins exhibit little, if any, LPS-binding or -neutralizing activity . Thus, CAP18- and CAP11-derived peptides could have therapeutic potential for bacterial infections and gram-negative bacterial endotoxin shock in vivo. In this study, to develop cathelicidin-derived antimicrobial peptides with improved LPS-neutralizing activities, we utilized the 18-mer peptide (K15-V32) of human cathelicidin hCAP18/LL-37 as a template and evaluated the activities of its peptide derivatives. By replacement of E16 and K25 with two L residues, the hydrophobicity of the peptide was increased and the hydrophobic sector in the helix was extended (Fig. and ; compare 18-mer K15-V32 with 18-mer LL). Concomitantly, the pI was increased from 11.50 to 12.22. Furthermore, by replacement of Q22, D26, and N30 with three K residues, the hydrophilicity of the peptide was enhanced and the positively charged hydrophilic sector in the helix was expanded (18-mer LL versus 18-mer LLKKK), accompanied by a further increase in pI from 12.22 to 12.82. Among 18-mer peptides, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting LPS-LBP interaction, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14+ cells and attenuating the production of TNF-alpha, an important inflammatory mediator for the development of endotoxin shock , by these cells. Furthermore, 18-mer LLKKK most effectively protected mice from lethal endotoxin shock. Thus, the enhanced hydrophobicity and cationicity (positive charge) of 18-mer LLKKK are likely important for the expression of its augmented activity in binding with and neutralizing a negatively charged amphipathic LPS. Furthermore, these features may facilitate the interaction of the peptide with the CD14 molecule. Taken together, these observations likely indicate that the alpha-helical amphipathic antimicrobial peptides with increased hydrophobicity and cationicity, such as 18-mer LLKKK, can interact more potently with negatively charged amphipathic LPS than the parent 18-mer peptide, thereby exerting augmented LPS-neutralizing activities. In agreement with this, SAR studies have indicated that the antibacterial activities of alpha-helical antimicrobial peptides could be modulated by interrelated structural and physicochemical parameters (such as charge, hydrophobicity, and amphipathicity) and that the maximum antibacterial potency of alpha-helical antimicrobial peptides could be obtained when a high charge (cationicity) and amphipathicity were achieved . Thus, our present findings suggest that the results of SAR studies on the antibacterial activities of alpha-helical antimicrobial peptides could be applied to the estimation of the LPS-neutralizing activities of these peptides and that novel antimicrobial peptides with enhanced LPS-neutralizing activity and therapeutic potential may be evolved by changing those parameters. Cationic antimicrobial peptides target cell surface anionic lipids such as phosphatidyl glycerol and cardiolipin that are abundant in microorganisms; the action is not receptor based but involves a less specific interaction with microbial membrane components . In contrast, the mammalian cell membrane is mainly composed of electrically neutral phospholipids such as phosphatidylcholine and sphingomyelin, for which the affinity of the antimicrobial peptides is generally low . The simple electrostatic interaction between cationic antimicrobial peptides and microbial membrane lipids provides selective toxicity (bacteria versus mammalian cells) as well as a broad spectrum of antimicrobial activities. Moreover, development of microbial resistance is assumed to be low, because the target molecules (anionic lipids) are important components conserved among microorganisms, and the molecular recognition between cationic peptides and target molecules is rather lenient . In addition, the peptides are small and relatively easy to synthesize. From these points of view, cationic antimicrobial peptides could be promising candidates for new antibiotics with therapeutic value. In this study, we have revealed that by modifying the hydrophobicity and cationicity of the amphipathic alpha-helical antimicrobial peptide (hCAP18/LL-37-derived 18-mer peptide), the in vitro and in vivo biological activities of the peptide could be enhanced. Among the peptide derivatives, 18-mer LLKKK obviously possessed more potent LPS-neutralizing activities than the parent peptide and appeared to have therapeutic potential for gram-negative bacterial endotoxin shock. However, it should be noted that as cationic antimicrobial peptides act principally via electrostatic attraction with, and hydrophobic partitioning into, the membrane targets, they could also bind to various host components such as plasma lipoproteins and anionic constituents of host cell membranes, leading to potentially harmful side effects on the host . In this context, it has been demonstrated that high concentrations of cationic antimicrobial peptides are occasionally toxic to host cells and that cytotoxicity is correlated with the extent of the hydrophobic regions in the peptides . Actually, we observed in the present study that 18-mer LL and 18-mer LLKKK with increased hydrophobicity were toxic to mammalian cells such as peritoneal macrophages and RAW 264.7 cells at >10 mug/ml; however, they exerted strong anti-LPS activities without exhibiting any cytotoxic effect on these cells at <10 mug/ml (data not shown). Thus, cationic antimicrobial peptides should be cautiously administered in vivo, considering their toxic effects on host cells. Although many problems still need to be solved , antimicrobial peptides could become one of the new classes of antibiotics that can be used for treatment of bacterial infections and their related symptoms in the future. FIG. 11. | Hydrophilicity/hydrophobicity plots of LL-37 and its 18-mer peptide derivatives. Hydrophilicity/hydrophobicity plots of LL-37 and its 18-mer peptide derivatives. Hydropathy indices (+, hydrophilicity; -, hydrophobicity) of LL-37 and its derivatives (18-mer K15 to V32, 18-mer LL, and 18-mer LLKKK) were calculated by the algorithm of Hopp and Woods by use of a Genetyx-Mac computer system, and the pIs of the peptides were also determined by the same system. Horizontal axes display the amino acid position number. PMID- 12204942 TI - Biological and Clinical Significance of Lipids as Modulators of Immune System Functions AB - PMID- 12204942_ TI - AB - It is widely recognized that malnutrition is the commonest cause of immunodeficiency worldwide . In fact, a recent investigation has determined that food intake increases the levels of gamma interferon (IFN-gamma) but not interleukin-4 (IL-4) production, whereas starvation enhances the IL-4 response but not IFN-gamma levels of T lymphocytes . Therefore, the interactions between certain nutrients and immunity exert a crucial role that should be analyzed from a biological and a clinical point of view. In recent years, a large number of studies have been conducted to investigate the relevance of certain fatty acids in the alteration of immune system functions in both humans and animals. At the beginning, the epidemiological findings contributed to demonstrating that certain fatty acids supplied in their diets (particularly long-chain n-3 polyunsaturated fatty acids contained in fish oil) affect the immune responses of Greenland Eskimos, as indicated by the low prevalence of inflammatory disorders in this population . Subsequently, on the basis of these epidemiological studies, experimental investigations determined the main processes by which several polyunsaturated or monounsaturated fatty acids are capable of altering the immune system, as well as the mechanisms of action involved in the regulation of immune functions. At least four modes of action have been proposed to explain the potential action of fatty acids on the modulation of immune system in both animals and humans. Accordingly, immune system modulation by dietary lipids may be attributed to changes in the composition of membrane phospholipids, lipid peroxidation, alteration of gene expression, or eicosanoid production. In addition, recent in vitro and ex vivo studies have demonstrated the involvement of several fatty acids (such as eicosapentaenoic acid [EPA], docosahexaenoic acid [DHA], arachidonic acid [AA], or palmitic acid [PA]) in the induction or inhibition of programmed cell death or apoptosis . The participation of different fatty acids in apoptosis opens new approaches for the study of the properties that different fatty acids exert as modulators of immune system functions. In this review, we summarize the effects of dietary lipids on the immune functions and the mechanisms responsible for these alterations as well as the crucial aspects that relate fatty acids to the triggering of apoptotic mechanisms. Similarly, we analyze the clinical consequences derived from dietary lipid administration, the effects of these substances on tumor promotion, and finally, the adverse effects that several dietary lipids may exhibit as a direct consequence of an immunosuppressive process . PMID- 12204942_TYPES OF FATTY ACIDS THAT ALTER IMMUNE SYSTEM FUNCTIONS TI - AB - Overall, the integrated action of several polyunsaturated or monounsaturated fatty acids on the immune system includes the alteration of different immune functions in both humans and animals. Therefore, lymphocyte proliferation , production of cytokines , modification of cell surface molecules , changes in phagocytic activity , and alteration of natural killer (NK) cell activity are susceptible to be modulated by dietary lipid administration or by culture of cells in the presence of several free fatty acids. However, it is important to note that not all of the fatty acids have the same immunomodulatory properties. Thus, long-chain n-3 polyunsaturated fatty acids, such as EPA (20:5n-3) or DHA (22:6n-3) (which belong to n-3 fatty acid series derived from fish oil) are particularly known to exert a profound influence on the immune system, reducing many of these functions previously described for both humans and animals . Other fatty acids belonging to n-6 fatty acid series such as AA (20:4n-6) have also been reported to affect the immune system; although AA has not been shown to produce an immunosuppression as relevant as long-chain n-3 fatty acids, it is also considered an immunosuppressive agent. Nevertheless, both n-6 polyunsaturated fatty acids and saturated fatty acids have been associated to a chemopromotive role in cancer, probably due in part to a suppression of apoptosis . However, oleic acid (a monounsaturated fatty acid [18:1n-9] that is the main component of olive oil belonging to n-9 fatty acid series) is also capable of acting as a modulator of immune functions . Accordingly, olive oil (a traditional and essential component of the Mediterranean diet) also plays an important role in the modulation of immune functions, and therefore it has been efficiently applied in the alleviation of the symptomatology caused by rheumatoid arthritis . PMID- 12204942_BIOLOGICAL CONSEQUENCES ON IMMUNE FUNCTIONS ATTRIBUTED TO FATTY ACID ADMINISTRATION TI - AB - Differential properties of fatty acids and their role in the modulation of immune system functions. | The changes attributed to fatty acid administration include the reduction of lymphocyte proliferation, which is modified by polyunsaturated fatty acids (n-3 or n-6) or monounsaturated fatty acids (n-9). Studies carried out in both humans and animals have revealed that the administration of high levels of dietary n-3 polyunsaturated fatty acids or the inclusion in parenteral regimens of lipid emulsions rich in n-3 or n-9 fatty acids are related to the reduction of lymphocyte proliferation during the supplementation . Thus, concanavalin-A or lipopolysaccharide-stimulated lymphocytes have reduced the cellular proliferation in assays carried out in vitro or ex vivo in the presence of free fatty acids or in cell cultures from both animals and humans fed dietary lipids, respectively . Cytokine production is reduced by the action of n-3 polyunsaturated fatty acids or n-9 monounsaturated fatty acids , cytokine receptor expression is also affected , natural killer (NK) activity is significantly suppressed , phagocytic activity of macrophages is modified , and the antigen-presenting function of human monocytes is inhibited . Based on these experimental observations, we can affirm that n-3 polyunsaturated fatty acids rather than n-6 polyunsaturated or n-9 monounsaturated fatty acids are directly associated to the alteration of immune and inflammatory response. This modulation depends on different factors, such as the nature of fatty acids added to diets, the concentration of fatty acids, the duration of supplementation with dietary lipids, or differences among animal species fed dietary lipids . Mechanisms of action. | As mentioned previously, for several years numerous studies have attempted to elucidate the mechanisms by which some dietary lipids produce a potential effect on immune system functions . Several hypotheses have been suggested as possible mechanisms. Experimental investigations have confirmed that several fatty acids exert changes in the phospholipids of plasma membrane which affect the membrane fluidity ; they also alter eicosanoid production , produce lipid peroxides , or regulate the transcription factors . Overall, long-chain n-3 polyunsaturated fatty acids, such as EPA, are incorporated into cell membranes, replacing AA (the most important of the eicosanoid precursors). This process reduces the production of a biological mediator as prostaglandin E2 responsible for inhibiting IL-1 and tumor necrosis factor (TNF) production . Similarly, EPA inhibits gene transcription because it reduces the translocation of NF-kappaB . In addition to these important considerations, a crucial role of lymphocyte subsets has been suggested to explain the effects of different dietary polyunsaturated fatty acids on the immune system, because they alter the number of lymphocyte subsets as well as the proliferation of these cells . Thus, antigen presentation and the proportion of T-cell subsets are modified after dietary lipid manipulation. In fact, recent studies have determined that polyunsaturated fatty acids inhibit the surface expression of major histocompatibility complex (MHC) class II molecules, as well as some adhesion molecules on human monocytes . Similarly, consumption of a monounsaturated fatty acid-rich diet by humans is also involved in the reduction of adhesion molecules from peripheral blood mononuclear cells . Overall, these actions may explain the different expression of CD4 or CD8 on the T-cell surfaces of peripheral blood mononuclear cells of mice that were fed diets supplemented with DHA . The biological consequences of these changes are still unclear. It is probable that the alteration experimented by the lymphocyte population enhances the host susceptibility against opportunistic infections. In other words, the modulation of lymphocyte subsets may lead in part to a modification of cytokine production, NK cell activity, antibody production, etc. Accordingly, Th1-type cytokines such as IL-2 are more susceptible to polyunsaturated fatty acid effects than are Th2-type cytokines, such as IL-4, which indicates that these fatty acids are capable of altering the balance of Th1- and Th2-type cytokines in mice . As a direct consequence of a reduction of IL-2 production, Th1 cell activation is suppressed, whereas an increase of Th2 cell activation due to an enhancement of IL-4 production suppresses Th1 cell proliferation in animals fed a diet containing fish oil . Therefore, the anti-inflammatory effects derived from long-chain n-3 polyunsaturated fatty acid administration may be the result of these two interconnected mechanisms. Hence, it is obvious that these alterations may affect significantly the interaction between host natural resistance and infectious agents due to the crucial importance of Th1-mediated response in the infectious processes promoted by viruses or bacteria . Although results from epidemiological, experimental, or clinical studies have demonstrated an important association between n-3 polyunsaturated fatty acids and immunosuppressive properties in both humans and animals, their effects are complex and still not clearly known. In fact, some discrepant results have suggested that n-3 polyunsaturated fatty acids have stimulatory effects on TNF and IL-1 production . Evidence for the involvement of fatty acids in apoptosis modulation. | Programmed cell death or apoptosis has been defined as an essential mechanism responsible for the regulation of homeostasis, tissue development, or immune functions. Thus, damaged, aberrant, or unnecessary cells must be eliminated to ensure the correct development of multicellular organisms. Irrespective of its crucial role in normal cell control, apoptosis regulates pathological processes including human clinical disorders such as cancer, autoimmune diseases, viral or bacterial infections, and neurodegenerative disorders. Several biological factors (many viruses, bacteria, or parasites), chemical factors (glucocorticoids, ceramides, etc.), and physical factors (irradiation) are responsible for apoptosis induction . Moreover, different studies with both animals and humans, involving in vitro assays or administration of dietary lipids, have recently described an important role of several fatty acids in the induction or inhibition of apoptosis. As a result, different mechanisms of action have been proposed in order to explain the action of fatty acids on apoptosis modulation. Thus, polyunsaturated fatty acids, such as EPA and DHA, saturated fatty acids, such as PA, or fats such as fish oil administered in the diets have been defined as substances capable of inducing cell death via a mitochondrial process or by downregulation of Bcl-2 . Different mechanisms have been proposed in order to explain the induction of apoptosis by certain fatty acids. Thus, PA (a saturated fatty acid) added to cellular cultures in in vitro assays induces apoptosis via a direct effect on mitochondria, because it causes a dissipation of the mitochondrial transmembrane potential (DeltaPsim), an event that precedes nuclear apoptosis . Another possible partial explanation for the modulation of apoptosis by long-chain n-3 polyunsaturated fatty acids is that it is caused by direct action of these substances on the cells and by activation of the caspase cascade through cytochrome c release coupled with a modulation of mitochondrial membrane depolarization . Recent studies have also determined the crucial importance of dietary fatty acids in the reduction of Bcl-2 expression as well as an increase of Fas ligand (Fas-L) expression. Therefore, when the concentration of polyunsaturated fatty acids augments, Bcl-2 expression is reduced and cell death occurs . In other words, long-chain fatty acids contained in fish oil trigger apoptosis by suppressing Bcl-2 expression and increasing Fas-L expression. On the other hand, it has been recently determined that Ras membrane localization (a protein that plays a critical role in the cell growth and apoptosis) is reduced after administration of fish oil, which indicates that dietary fish oil may play a protective role against colon cancer development . Finally, it is important to note that lipid peroxidation is of crucial importance in apoptosis induction. This argument suggests that the effects of polyunsaturated fatty acids can be directly associated to specific alterations on the expression of Bcl-2, Fas, and Ras . PMID- 12204942_CLINICAL CONSEQUENCES OF FATTY ACID ADMINISTRATION TI - AB - Early studies revealed the importance of n-3 polyunsaturated fatty acids as possible therapeutic substances capable of reducing the incidence of inflammatory disorders in humans . Autoimmune diseases such as rheumatoid arthritis, psoriasis, or systemic lupus erythematosus are characterized by the production of an inflammatory response due to a marked increase of proinflammatory cytokines. In recent years, numerous epidemiological studies have described widely the reduction in the incidence of rheumatoid arthritis in populations who consume fish oil or olive oil in their diets, which are related to the prevention of inflammatory disorder incidence, although these events occur after a prolonged ingestion of these fats (, -, , ). Nevertheless, in spite of the importance of these substances as biological mediators in the inflammation and their application in the resolution of inflammatory diseases, in the present review we will summarize other important features of fatty acids from a clinical point of view, focusing on the potential effect of fatty acids in cancer incidence and the involvement of these substances in the alteration of host natural resistance against infectious agents. Crucial role of fatty acids in the incidence of cancer. | Many lines of evidence have indicated the essential role of fish oil in the incidence of cancer . Numerous reports have suggested that high intakes of long-chain n-3 fatty acids or n-9 fatty acids (the most important fatty acid contained in olive oil) may reduce the risk of breast cancer in both humans and animals. Thus, n-3 polyunsaturated fatty acids may inhibit colon cancer in rats as well as reduce the risk of colorectal cancer development . In contrast, n-6 fatty acids or saturated fatty acids may be involved in the increase of mammary or colon tumorigenesis by altering membrane phospholipid turnover. Accordingly, AA is released from plasma membrane that produces an alteration in the synthesis of prostaglandins via cyclooxygenase enzyme . Epidemiological studies have determined the protective role of n-3 polyunsaturated fatty acids against colon cancer in populations of Alaskan and Greenland Eskimos . Subsequently, experimental studies demonstrated that a fish oil diet appears to exert a protective effect against experimentally induced colon cancer . Similarly, a recent study has determined that olive oil also exerts a protective role against the development of colorectal cancer . Although the mechanisms that contribute to this protective effect have not been elucidated, a recent investigation has reported that fish oil diets are able to increase apoptosis and cell differentiation in induced rat colon tumorigenesis . Based on current knowledge, an interesting study has recently determined that the Western diet (which consists predominantly of a mixture of saturated, monounsaturated, and polyunsaturated fats) produces dysplastic lesions in the colon, which is indicative of a tumorigenic process. As a result, the administration of a diet containing mixed lipids and high levels of saturated fatty acids (similar to the lipids contained in the Western diet) induces colon carcinogenesis in mice by an unknown mechanism . Nevertheless, the most interesting results from this investigation reveal that an alteration of cyclooxygenase activity as well as apoptosis suppression appears to play a crucial role in the colon tumorigenesis induced by this diet in animal models . In addition, our experimental observations have indicated that the survival of mice fed a diet containing fish oil and transplanted with a T-lymphoma cell line is reduced in comparison to that of mice fed a diet containing low fat. We speculate that this event may be attributed to the impairment in the function of T cells or NK cells due to polyunsaturated fatty acid administration, which reduces the cytotoxic activity of NK cells . Recently, an interesting investigation has shown that n-3 fatty acids alter cell transformation in a mouse epidermal cell line because DHA or EPA inhibited transcription activator protein AP-1 whereas AA did not produce any effect on AP-1 activity. Therefore, this finding confirms previous reports of the chemoprotective role of diets containing n-3 fatty acids . In contrast, the most significant relationship between fatty acids and reduction of cancer incidence is found in the Mediterranean diet, which could decrease the development of cancer by up to 10 to 15% in the populations of highly developed Western countries . In spite of the wide information currently available, the potential role of dietary lipids in cancer etiology remains controversial. In fact, although most of the studies have shown that diets containing n-3 polyunsaturated fatty acids might be beneficial during inflammatory conditions, their effects are not elucidated because they promote an impairment of the host defense to infection. Therefore, future studies are needed in order to confirm or refute the different hypotheses and arguments suggested hitherto. Relevance of dietary lipids in the reduction of host natural resistance and susceptibility to infection. | As mentioned above, nutrient intake is considered a critical determinant of immunocompetence. Many investigations have reported the modulatory functions exerted by fatty acids and the clinical benefits of dietary lipid supplementation with fish oil or olive oil in both humans and animals. As a consequence, this dietary supplementation has been applied in the treatment of patients suffering from inflammatory diseases, because unsaturated fatty acids (mainly n-3 or n-9 fatty acids) reduce the levels of many biological mediators associated to the promotion of the inflammatory events that participate in an inappropriate immune response. However, the reported reduction of immune response caused by the administration in the diet of some fatty acids may exert a detrimental effect, and therefore, they may impair the cellular immunity to pathogenic agents . For obvious reasons, the altered resistance to bacterial infection has been analyzed in animal models in which the administration of diets containing fish oil reduces the clearance of bacteria from liver or spleen and significantly reduces the survival during the course of Listeria monocytogenes infection. As a result, the elimination of pathogenic agents (bacteria, viruses, or parasites) is more difficult. Different reports have described the clinical consequences of dietary supplementation rich in n-3 fatty acids that suppress the immune system functions. Thus, a significant reduction of survival percentage after feeding experimental mice with a diet containing fish oil has been determined whereas an increase of survival percentage was observed after challenge with L. monocytogenes in animals fed diets containing either lard oil, hydrogenated coconut oil, or palm oil . These mice were inoculated with a lethal dose of a virulent L. monocytogenes strain, a facultative intracellularly growing bacterium that has been used as a model of infectious and pathogenic processes. After the administration of this diet different results were observed. Bacterial clearance from liver or spleen was increased in these animals , bactericidal activity of peritoneal cells was significantly reduced , and cytotoxic effects due to bacterial infection were increased , whereas the susceptibility of cells to adhesion or invasion by L. monocytogenes infection was substantially modified . These observations indicate the loss of capacity of the immune system from animals fed a diet containing fish oil to destroy and eliminate the infectious agents . A recent investigation has contributed to explaining in part the reasons for which n-3 polyunsaturated fatty acids reduce host defense against L. monocytogenes: consumption of EPA or DHA (both contained in fish oil) impairs the production of IL-12 and IFN-gamma, cytokines that play an essential role in the innate and adaptive responses of host immune system . Hence, the reduction of IL-12 levels may explain the impaired bacterial clearance and the reduction of survival to L. monocytogenes infection . Another possible explanation for the reduction of host resistance is based on the inhibition of the expression of MHC class II (called Ia in mice) that is reduced in mice fed a fish oil diet and infected with L. monocytogenes . Similarly, it is important to note that these effects may be associated with the reported increase of reactive oxygen species, whose numbers were significantly increased in mice after the animals were experimentally infected with this bacterium and fed a diet containing hydrogenated coconut oil . In fact, the role of cellular oxidative processes requires considerable attention because they play an important role in the elimination of infectious agents. However, not all polyunsaturated fatty acids are capable of exerting the same effects, since many studies are not in accordance with the previously described arguments. Thus, a recent investigation has determined that conjugated linoleic acid does not alter the resistance of mice after L. monocytogenes infection . Similarly, a previous study indicated the irrelevant effect of dietary lipid manipulation on the survival of mice in two models of peritonitis, one with Pseudomonas aeruginosa and the other with Salmonella enterica serovar Typhimurium . Irrespective of these findings, other experimental studies have demonstrated the adverse effects of dietary lipids containing n-3 or n-6 polyunsaturated fatty acids in the impairment of immune functions in animals experimentally infected with different pathogenic agents such as S. enterica serovar Typhimurium , P. aeruginosa , Staphylocococcus aureus , or Mycobacterium tuberculosis . By contrast, it has been reported that the administration of diets containing fish oil does not reduce the survival percentage after experimental infection with Klebsiella pneumoniae . In fact, dietary fish oil supplementation increased resistance to infection due to an enhancement of IL-1 and TNF production by peritoneal cells in mice . It is probable that the discrepancy in these findings is due to different factors that include the duration of the supplementation period, the concentration of fatty acids used, or the cell population affected by fatty acid administration. Finally, experimental observations with viruses have also demonstrated that influenza virus infection in animals fed fish oil diet delays the clearance due to an impairment of primary virus-specific T-cell cytotoxicity but has no effect on NK cytotoxicity . PMID- 12204942_CONCLUDING REMARKS TI - AB - Nutritional status is generally recognized as an essential factor involved in the modulation of immune response, which may be determinant in the development of the clinical effects derived from a malnutrition process . Thus, in recent years the effects that different dietary lipids exert upon immune functions have received considerable attention, because different functions of immune system are altered after dietary lipid administration. Hence, lymphocyte proliferation, cytokine production, phagocytic activity, adhesion molecule expression, and NK cell activity are susceptible to modification by the action of certain lipids in both animals and humans. Different mechanisms of action have been proposed to be involved in these processes, such as lipid peroxidation, changes in the plasma membrane, eicosanoid production, or alteration of gene expression. Both epidemiological and experimental studies have applied the beneficial properties associated to a modulation of the immune functions. Thus, different fats such as fish oil or olive oil have been applied in the amelioration of symptoms related to rheumatoid arthritis and other inflammatory disorders due to their anti-inflammatory properties. Similarly, they have also been applied as biological mediators capable of exerting a protective effect against colon or breast cancer, whereas other fatty acids such as n-6 polyunsaturated fatty acids exert a chemopromotive role in tumorigenic processes. In addition, these effects may be related to the ability of several fatty acids to modulate apoptosis by different pathways. Nevertheless, the immunosuppressant role associated with dietary polyunsaturated fatty acids containing fish oil may produce adverse effects because they reduce the host's natural resistance against pathogenic agents. Therefore, in the light of current experimental observations, the benefits of administering these dietary lipids should be weighed before they are applied as immunosuppression factors in order to prevent detrimental or adverse effects caused by an excessive suppression of immune functions. PMID- 12204942_ TI - FIG. 1. AB - Schematic representation of potential role of dietary lipids and biological and clinical consequences of the administration of several dietary lipids. Schematic representation of potential role of dietary lipids and biological and clinical consequences of the administration of several dietary lipids. PUFAs, polyunsaturated fatty acids; MUFAs, monounsaturated fatty acids; SFAs, saturated fatty acids. PMID- 12204975 TI - Centrifugation of Human Lung Epithelial Carcinoma A549 Cells Up-Regulates Interleukin-1beta Gene Expression AB - PMID- 12204975_ TI - AB - Cytokines are important mediators for host defense and inflammation to bacterial infections. Chlamydia pneumoniae, an obligate intracellular bacterium that causes acute respiratory infections in human hosts, has been shown to induce cytokine expression in various human and murine model systems . In most in vitro studies of C. pneumoniae, a centrifugation step is employed, and it has been demonstrated that the addition of this centrifugation step can increase the formation of inclusion bodies . However, the stress of centrifugation may also affect some cellular activities, such as proliferation and gene expression; certain cytokines, such as interleukin-1beta (IL-1beta), can also be induced under stress conditions . Therefore, the effects of centrifugation of cells should be considered when performing in vitro studies of C. pneumoniae, particularly studies using lung epithelial cells, which serve as a major target for C. pneumoniae infection. We intended to determine if centrifugation stress can affect the expression of proinflammatory cytokine IL-1beta in a human lung epithelial carcinoma A549 cell line. Centrifugation of A549 cells for 1 h at 2,000 rpm (similar760 x g) at room temperature did not affect cell proliferation or cell viability. Furthermore, C. pneumoniae infection with or without centrifugation (direct inoculation) also did not alter cell proliferation or viability. The effect of centrifugation stress on IL-1beta expression was then examined by reverse transcription-PCR (RT-PCR). The primers used were 5'-AAA CAG ATG AAG TGC TCC TTC CAG G-3' and 5'-TGG AGA ACA CCA CTT GTT GCT CCA-3' . beta-Actin was used as an amplification control, detected by primer sequences 5'-CGG GAC CTG ACT GAC TAC-3' and 5'-GAA GGA AGG CTG GAA GAG-3' . As shown in Fig. , the stress of centrifugation clearly increased the expression of the IL-1beta gene. This increase in expression of IL-1beta was present even at 24 h after centrifugation. FIG. 1. | Centrifugation stress up-regulates IL-1beta gene expression. Centrifugation stress up-regulates IL-1beta gene expression. A549 cells were centrifuged at 427 x g, 760 x g, or 1,187 x g for 1 h at room temperature. Four hours later, total RNA was extracted, and cellular levels of IL-1beta were measured by RT-PCR. beta-Actin was used as internal control (Con). (A) Electrophoretogram of RT-PCR. The RT-PCR products were run on agarose gels, stained with ethidium bromide, and visualized using UV. (B) Densitometric quantification of the electrophoretogram in panel A after normalization to beta-actin. The inclusion bodies of C. pneumoniae in both centrifuged and uncentrifuged A549 cells were also counted, and the inclusion body formation units (IFU) were calculated as (3.58 +- 0.61) x 108 and (1.03 +- 0.49) x 108 IFU/ml, respectively. There was a significant difference between the two values as determined by the Student t test (P < 0.01), suggesting that centrifugation does increase the formation of inclusion bodies. However, even without the centrifugation, C. pneumoniae could still efficiently infect A549 cells. Although centrifugation has been shown to improve inclusion body formation, it is always better to eliminate as many confounding factors as possible that may interfere with the host-pathogen interactions. In this case, we have shown that the stress of centrifugation alone can up-regulate the expression of the IL-1beta gene without affecting cell proliferation and viability. Thus, the IL-1beta induction data from some earlier studies where centrifugation was used to infect cells may be compromised. Therefore, reevaluation of earlier data to identify the true effects of C. pneumoniae infection on cytokine expression may be warranted. Although we did not test the effects of centrifugation on cytokines other than IL-1beta, they may also be affected by centrifugation. Cellular responses to stress are very complex, and different cell types have differing responses to the same stressor. Thus, it is noted that the results we observed using A549 cells may or may not be applicable to other cell types. Proper controls should always be run to clarify the role that the stress of centrifugation plays in cellular cytokine gene expression. PMID- 12204947 TI - Impaired Macrophage Phagocytosis of Apoptotic Neutrophils in Patients with Human Immunodeficiency Virus Type 1 Infection AB - | Dysfunction of neutrophils (polymorphonuclear leukocytes [PMNL]) and macrophagic cells occurs as a consequence of human immunodeficiency virus type 1 (HIV-1) infection. Macrophages contribute to the resolution of early inflammation ingesting PMNL apoptotic bodies. This study investigated macrophage ability to phagocytose PMNL apoptotic bodies in patients with HIV-1 infection in comparison with uninfected individuals and the effect of HIV Nef protein on apoptotic body phagocytosis to determine if phagocytic activity is impaired by HIV infection. Monocytes/macrophages were isolated from 10 HIV-1-infected patients and from five healthy volunteers, whereas PMNL were isolated from healthy volunteers. Macrophage phagocytosis of apoptotic PMNL was determined by staining of apoptotic bodies with fluorescein-conjugated concanavalin A or with fluorescein-labeled phalloidin. Our data show significant impairment of PMNL apoptotic body macrophage phagocytosis in subjects with HIV-1 infection presenting a concentration of CD4+ T lymphocytes of >200/mm3 and in particular in those with <200 CD4+ T lymphocyte cells/mm3. In addition, HIV-1 recombinant Nef protein is able to decrease phagocytosis of apoptotic PMNL from normal human macrophages in a dose-dependent manner. The results of our study suggest that impaired macrophage phagocytosis of PMNL apoptotic bodies may contribute to the persistence of the inflammatory state in HIV-infected subjects, especially during opportunistic infections that are often favored by defective phagocytic activity. PMID- 12204947_Introduction TI - AB - Neutrophil (polymorphonuclear leukocytes [PMNL]) function, including chemotaxis, phagocytosis, oxidative burst capacity, and bacterial killing, is impaired in the course of human immunodeficiency virus type 1 (HIV-1) infection, particularly in the later stages of the disease, and this abnormal function may predispose to some secondary bacterial infections and/or to opportunistic infections . PMNL have the shortest half-life of all circulating leukocytes and are programmed to die within 1 day. These aging leukocytes spontaneously undergo apoptosis and are recognized and phagocytosed by macrophages . Pitrak et al. have demonstrated that the rate of PMNL apoptosis is accelerated in AIDS patients, and this defect is intrinsic and not an effect of endogenous serum factors. Moreover, it has been proposed that the ingestion of apoptotic PMNL triggers production of anti-inflammatory mediators from macrophages , whereas persistent PMNL-rich inflammatory infiltrates have been associated with unresolved inflammatory reactions, including adult respiratory distress syndrome and rheumatoid arthritis . Thus, the removal of apoptotic cells appears to be critical in the resolution of inflammation. We have previously demonstrated that macrophages from HIV-positive subjects have a reduced ability to phagocytose Candida albicans cells, and there is a significant decrease in oxidative processes for the intracellular killing. These phenomena seem to be induced, at least in part, by HIV Nef protein . Since the effects of macrophage phagocytosis of apoptotic PMNL have not been completely investigated, especially in HIV-positive subjects, the purpose of this study was to evaluate phagocytosis of PMNL apoptotic bodies performed by macrophagic cells obtained from HIV-1-positive subjects and in parallel by the macrophages obtained from healthy individuals. Furthermore, we studied the effect of Nef protein on PMNL apoptotic body macrophagic phagocytosis, since this viral protein is able to depress both specific and nonspecific immune responses in HIV-infected patients, particularly microbial phagocytosis . PMID- 12204947_MATERIALS AND METHODS TI - AB - Subjects. | Ten HIV-1-infected subjects (mean age, 35.3 +- 5.8 years) were enrolled and five healthy volunteers (mean age, 37.1 +- 4.4 years), without HIV-1 risk factors, served as controls. Five of the HIV-1-infected subjects presented more than 200 CD4+ T lymphocytes/mm3 (mean = 517 +- 225), had a CD4/CD8 T-cell ratio of 0.5 +- 0.1, and had mean HIV-1 RNA levels in plasma of 32,628 +- 42,188 copies/ml. Five patients had less than 200 CD4+ T lymphocytes/mm3 (mean = 101 +- 70), a ratio of 0.2 +- 0.2, and mean HIV-1 RNA levels of 375,000 +- 246,815/ml. All these patients had a moderate anemic state and were generally studied before receiving antiretroviral therapy. In fact, a significant part of our study population included individuals who had ignored their seropositive condition for a long time and in consequence came to medical evaluation late. Monocyte and PMNL isolation and apoptotic body preparation. | Monocytes obtained from peripheral blood of healthy subjects and HIV-1-infected patients were selected as adhering cells after separation of peripheral blood mononuclear cells with a Ficoll Paque gradient (Pharmacia, Uppsala, Sweden). After repeated washes, the adherent cells were harvested by trypsinization and resuspended at a concentration of 106/ml in RPMI-1640 medium (Gibco, Paisley, Scotland), supplemented with 10% fetal calf serum (Celbio, Milan, Italy). Then, macrophage colony-stimulating factor (10 ng/ml; Genzyme, Milan, Italy) was added to the medium in order to differentiate monocytic cells into activated macrophages, and the cultures were maintained at 37C with 5% CO2. Cellular cultures were enriched for macrophages by adherence and repeated trypsinization and, to obtain a pure population, by phenotypic evaluation (more than 98% of cells). After 7 days of incubation in flasks (25 cm2; Nunc Kamstrup, Baltimore, Md.), the cells were harvested by trypsin treatment and transferred, at a final concentration of 5 x 105/ml, to four-well chamber slides (Lab-Tek; Nunc). Cultures of PMNL were obtained from peripheral blood of healthy subjects by separation with a Polymorphoprep gradient (Pharmacia). PMNL cells spontaneously go to apoptosis after 24 h of culture, and this method was adopted to induce apoptosis. In order to separate the apoptotic bodies of PMNL, after centrifugation at 600 rpm for 10 min (to remove nonapoptotic cells), the supernatants were centrifuged at 3,000 rpm, and the pellets containing PMNL apoptotic bodies were resuspended at the indicated concentrations. Fluorescent microscopy for phagocytosis detection. | Two staining methods were used to detect apoptotic bodies of PMNL in macrophagic cells. In the first method, 5 x 106 apoptotic bodies/ml were incubated for 20 min at room temperature with fluorescein-conjugated concanavalin A (ConA; Calbiochem Corp., La Jolla, Calif.) at a final concentration of 150 mug/ml. Then the apoptotic bodies were washed twice by centrifugation for 10 min at 3,000 rpm and resuspended in RPMI-1640 with 10% fetal calf serum at a concentration of 5 x 106/ml to obtain a ratio of 5:1 per macrophagic cell (this preparation was diluted 1:2 in the wells containing macrophagic cells). This method allows uniform staining of the apoptotic body surface, making the apoptotic bodies easily detectable in macrophagic cells without having any significant influence on phagocytosing activity. In the second method, fluorescein-labeled phalloidin (Sigma-Aldrich Chemicals, Milan, Italy) was used at a final concentration of 5 mug/ml. This product is able to stain the interior of the apoptotic bodies, binding firmly to the microtubular structures. This staining method was used like the first one. In a subsequent set of experiments, macrophage phagocytosis of PMNL apoptotic bodies was also investigated in the presence of various concentrations of recombinant Nef protein (American Biotechnologies, Inc., Cambridge, Mass.), using macrophagic cells obtained from four healthy individuals and performing the experiments in triplicate. The challenge with stained apoptotic bodies, using both the first and second methods, was made at a ratio of 5:1. After 2 h of incubation in the presence of apoptotic bodies, the supernatants were removed, and slides were detached from their supports and washed twice with phosphate-buffered saline (PBS). Then the slides were fixed with 10% ethyl alcohol solution, mounted in phosphate-buffered glycerol (30% PBS and 70% glycerol, vol/vol), and examined under a fluorescence microscope at x400 by four blinded microscopists. At least five microscopic fields were observed for each sample. Macrophage oxidative phenomenon evaluation. | We indirectly studied the oxidative phenomena of macrophages in the absence or presence of Nef protein, by evaluating the antioxidant power of the supernatants, as previously described . For this purpose we used the antioxidant power (PAO) kit furnished by Med. Dia S.r.l. (San Germano, Vercelli, Italy). The test is based on the detection of Cu+ ions produced by the reduction of a known amount of Cu2+; this reduction is induced by the antioxidant factors present in the culture, the activity of which decreases in an inversely proportional manner to the activity of macrophage oxidative phenomena. The concentration of Cu2+ ion was detected through the formation of complexes consisting of Cu+ and the chelating agent betacuproine disulfonate (2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonate), and it was measured spectrophotometrically at 490 nm (Metertech spectrophotometer; Medical System, Genoa, Italy). The data are expressed as microequivalents per liter of reducing equivalents +- standard deviation, corresponding to PAO units. Statistical analysis. | The results were expressed as mean and standard deviation. The differences among the experimental and control groups were statistically evaluated using Student's t test. Statistical significance was defined as P <= 0.05. Moreover, a linear correlation test was employed for the correlation study reported in the results. PMID- 12204947_RESULTS TI - AB - In the first set of experiments, we studied the phagocytosis of PMNL apoptotic bodies stained with fluoresceinated ConA from macrophages of patients with HIV-1 infection and from those of uninfected healthy individuals. A significant decrease in the percentage of phagocytosing cells was observed in HIV-1-positive subjects presenting a CD4+ T-cell concentration of >200 cells/mm3 (24.2% +- 12.3%; P = 0.002) and in those with <200 CD4+ T cells/mm3 (23.7% +- 6.9%; P < 0.001), compared to healthy control subjects (50.1% +- 3.7%) . We point out that by analyzing all the HIV-positive subjects, the mean number of phagocytosing cells was 23.9% +- 10% in 52% of the controls (P < 0.001). Since fluoresceinated ConA only stains the surface of apoptotic bodies, we performed further experiments with fluoresceinated phalloidin, which is endowed with a specific ability to bind to microtubular structures. We also observed a reduction of about 50% in phagocytosing cells in HIV-positive subjects compared to the controls (unreported data). As reported in Table , a significant decrease in the number of PMNL apoptotic bodies phagocytosed per macrophagic cell was observed in HIV-1 subjects with >200 CD4 T cells/mm3 (0.6 +- 0.4; P = 0.001) and in those with <200 CD4 T cells/mm3 (0.3 +- 0.1; P < 0.001) compared to the controls (2.4 +- 0.7). Evaluating all the HIV-positive subjects, the mean number of apoptotic bodies phagocytosed per cell was 0.45 +- 0.3 (P < 0.001). As can be seen in Table , the reduction of the mean number of PMNL apoptotic bodies phagocytosed per cell in HIV-positive subjects compared to the controls is fivefold, and the percent reduction in phagocytosing cells in infected patients is twofold. This observation suggests a decreased ability of single phagocytosing cells to swallow apoptotic bodies. In a further set of experiments, we studied the phagocytic activity of PMNL apoptotic bodies in normal human macrophages preincubated with recombinant Nef protein. The results obtained demonstrate that this protein is able to inhibit the phagocytosis of PMNL apoptotic bodies. In particular, preincubation with Nef (2 h before the challenge with apoptotic bodies) inhibited macrophage phagocytosis of PMNL apoptotic bodies by about 50% (P = 0.01) at the concentration of 1 mug/ml and 65% at the concentration of 2 mug/ml (P < 0.001). When the preincubation time was prolonged to overnight, inhibition of macrophage phagocytosis was 80% at a Nef protein concentration of 2 mug/ml. Table reports the mean number of PMNL apoptotic bodies stained with fluoresceinated phalloidin phagocytosed per cell in the absence or presence of Nef protein. As shown, the reduction was about 34%, 90%, and 92% with Nef at 0.5, 1, and 2 mug/ml, respectively. Consequently, in these experiments the reductions induced by Nef were also more evident when evaluating the number of PMNL apoptotic bodies phagocytosed per cell than the percentage of phagocytosing cells. Moreover, we observed a negative and significant correlation between the Nef protein concentration and the number of PMNL apoptotic bodies phagocytosed per cell (r = -0.65; P = 0.0221). The mean number of apoptotic bodies phagocytosed related to 106 macrophages was 1.18 x 106 in the absence of HIV infection (23% of the total) and 48,000 with a viral load in plasma of 8 x 105 HIV-1 RNA copies/ml (about 1%). A negative correlation was found between the HIV RNA load and the number of apoptotic bodies phagocytosed (r = -0.623; P < 0.05). Finally, we evaluated the antioxidant power (PAO) of the macrophage supernatants in the absence or presence of various concentrations of Nef, and we found a significant increase in PAO that meant a reduction of oxidative phenomena in the presence of the viral protein compared to the control values, especially at a concentration of 2 mug/ml (PAO units = 164.1 +- 8.0 with Nef at 2 mug/ml versus 105.6 +- 12.9 without Nef; P < 0.001). TABLE 1 | Percentage of macrophages phagocytosing PMNL apoptotic bodies stained with fluoresceinated ConA: ex vivo test of macrophages from uninfected and HIV-1-infected subjects TABLE 2 | Number of PMNL apoptotic bodies phagocytosed per macrophage TABLE 3 | Effect of Nef viral protein on phagocytosis of PMNL apoptotic bodies PMID- 12204947_DISCUSSION TI - AB - The results of this study show that phagocytosis of PMNL apoptotic bodies by macrophages of HIV-1-infected patients is impaired and that Nef, a regulatory viral protein of HIV, is able to decrease phagocytosis of PMNL apoptotic bodies by human normal macrophages. In addition, macrophagic oxidative phenomena are depressed by Nef protein. This is relevant because these effects are involved in intracellular killing processes and consequently in the destruction of phagocytosed particles . The decrease in oxidative processes and the inhibition of macrophagic function seem to occur in parallel. Aging PMNL spontaneously undergo apoptosis and are recognized and phagocytosed by monocytes and macrophages . PMNL function is impaired in all stages of HIV-1 infection and especially in the terminal stage of the disease . Pitrak et al. have shown that abnormalities of PMNL function observed in HIV-positive subjects might partly depend on the accelerated apoptosis induced by HIV infection. In fact, during all stages of HIV-1 infection, there is an increased number of apoptotic PMNL which are unable to function as host defenders . The removal of apoptotic cells appears to be central to the resolution of inflammation. In fact, the clearance of apoptotic PMNL not only prevents the release of toxic and immunogenic intracellular contents, but also stimulates the macrophages to produce inflammatory mediators, including transforming growth factor beta1, prostaglandin E2, and platelet-activating factor, and inhibits the production of tumor necrosis factor, interleukin-1beta, and interleukin-8 . Dysregulation of PMNL function, along with that of monocytic macrophagic cells, in HIV-1-infected patients is reflected in the increased incidence of some microbial infections among these patients . During several microbial infections, PMNL migrate and accumulate at the inflammatory sites, followed by removal of inflammatory cells. This occurs mainly by apoptosis and by phagocytosis of apoptotic bodies. In parallel, some antimicrobial pathogens can be phagocytosed and killed by macrophages. These phenomena appears to be critical to the resolution of inflammation and infection. However, the decreased phagocytosis of apoptotic PMNL by macrophages in HIV-1-infected patients and the accelerated apoptosis of PMNL lead to accumulation of apoptotic inflammatory PMNL and a decrease in their clearance. The persistence of apoptotic PMNL and their apoptotic bodies at the inflammatory site may maintain the inflammatory state through persistent stimulation of proinflammatory cytokines . This can explain some of the pathological conditions in the gastrointestinal tract or bronchoalveolar tract that have been reported . PMID- 12204948 TI - Evaluation of an In-House-Developed Radioassay Kit for Antibody Detection in Cases of Pulmonary Tuberculosis and Tuberculous Meningitis AB - | A radioassay for the detection of antitubercular antibody has been developed. The technique involves the addition of 125I-labeled Mycobacterium tuberculosis antigen as a tracer, diluted clinical sample (serum or cerebrospinal fluid [CSF]), and heat-inactivated Staphylococcus aureus to capture the antibody, incubation for 4 h, and quantitation of the amount of antibody present in the sample. A total of 330 serum samples from patients with pulmonary tuberculosis and 138 control serum samples from individuals who were vaccinated with M. bovis BCG and from patients with pulmonary disorders of nontubercular origin were analyzed. Also, 26 CSF samples from patients with tuberculous meningitis and 24 CSF samples as controls from patients with central nervous system disorders of nontuberculous origin were analyzed. Sensitivities of 80 and 73% were observed for patients with pulmonary tuberculosis and tuberculous meningitis, respectively, and specificities of 90 and 88% were seen for the two groups of patients, respectively. The sensitivity was lower, however, for human immunodeficiency virus-infected patients coinfected with M. tuberculosis. The control population could be differentiated from the patient population. This assay is rapid and user friendly and, with its good sensitivity and specificity, should benefit the population by providing diagnoses early in the course of disease and, hence, permit the early administration of appropriate chemotherapy. PMID- 12204948_Introduction TI - AB - Tuberculosis, caused by Mycobacterium tuberculosis, represents a major health problem worldwide. It is estimated that almost one-third of the entire world's population is exposed to this disease . In 1993, the World Health Organization declared tuberculosis a global emergency and estimated that by the start of the new millennium there would be more than 1 billion newly infected individuals and more than 17 million deaths due to this disease if control programs were not implemented properly. These control programs are dependent on good tests for early diagnosis, constant monitoring of disease status, and response to and completion of treatment. The diagnosis of tuberculosis has never been simple, especially in children and in patients with extrapulmonary tuberculosis, from whom clinical specimens for microscopy and culture are not easily obtained. Even in patients with pulmonary tuberculosis, diagnosis by microscopy is rather insensitive and culture is time-consuming. Since the mid-1970s radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) techniques have been developed for the detection of M. tuberculosis antigen and anti-M. tuberculosis antibody from clinical specimens, and these assays have various degrees of sensitivity and specificity (, , , 7, , , -, , -, , ). Almost all the assays developed at the Laboratory Nuclear Medicine Section of the Bhabha Atomic Research Centre require the detection of both antigen and antibody from clinical samples (, , -, -). With the incidence of tuberculosis being on the rise and with the added risk of coinfection with human immunodeficiency virus (HIV), the need at this time is for a test with good sensitivity and specificity for rapid and early diagnosis. With this as the aim, we have modified the antibody test and describe here the evaluation of a single test kit for the diagnosis of tuberculosis. PMID- 12204948_MATERIALS AND METHODS TI - AB - Antigens. | M. tuberculosis H37Rv was grown in Youman's synthetic medium for 4 to 6 weeks. Cultures were heat inactivated by autoclaving at 120C for 20 min, followed by sonication for extraction of antigen from the cells. The suspension was ultracentrifuged at 105,000 x g for 90 min, and the supernatant was used as the source of antigen. This sonicate antigen was iodinated with 125I by the iodogen method . The iodinated antigen was purified from free iodide by chromatography on a Sepharose 6B column. The immunoreactive peak was used as the radioactive antigen or tracer. Antibody. | Rabbit anti-M. bovis BCG antibody (M/S Dakopatts, Copenhagen, Denmark) was commercially obtained and was used for the preparation of quality control (QC) samples. Solid phase. | Staphylococcus aureus (strain Cowan I) was used as the solid phase. S. aureus has protein A on its surface and can bind to immunoglobulins (immunoglobulin G [IgG]) efficiently through the Fc region. The S. aureus cultures were grown in nutrient medium for 3 to 4 days. After the cells were harvested, they were heat inactivated at 80C for 40 min. A 10% suspension was made and was used as the solid phase. Assay procedure. | The antibody assay developed is a single-step assay. A total of 0.1 ml of clinical sample (a serum sample diluted 1:25 or a neat cerebrospinal fluid [CSF] sample) whose antibody levels are to be determined was incubated with 0.1 ml of an S. aureus suspension diluted 1:3 and 0.3 ml of 125I-labeled M. tuberculosis antigen. Assay control samples and QC samples were also treated in the same way as the patient samples. The reaction mixture was incubated for 4 h at room temperature with constant shaking. Subsequently, 2 ml of 0.02 M barbitone buffer (pH 7.6) was added, and the entire mixture was centrifuged at 2,000 x g for 40 min. The supernatant was aspirated, the pellet was counted in a gamma counter, and the antibody concentration in the samples was determined by calculating the ratio as described in the Results. Determination of assay cutoff and assay control samples. | In order to determine a cutoff limit for negativity above which the clinical samples could be classified as positive, a standard curve was constructed by using anti-BCG IgG as the standard, with concentrations ranging from 5 to 200 mug/dl, as shown in Fig. . The assay was performed as described above with 77 control serum samples. These samples were taken from healthy individuals and patients with pulmonary infections of nontubercular nature. Antibody levels were quantitated by referring to the standard curve and are expressed as micrograms per deciliter. The values obtained were calculated for the upper limit of negativity as the mean + 3 standard deviations (SDs), which was found to be 25 mug of IgG per dl, as shown in Fig. . With this as the basis, two assay control samples were included in the kit and for calculation purposes. Control C1, which represented nonspecific binding, was the assay buffer with no antibody, and control C2 represented a concentration of 25 mug of anti-BCG IgG per dl in the same assay buffer. Stability of the kit. | The stability of the kit was determined for a period of 7 weeks both at room temperature (RT) and at 4C. In this case, the kits were kept at RT and at 4C and were evaluated at 0, 1, 2, 3, 4, and 7 weeks. QC samples Q1 and Q2 were also included to determine the reproducibility of the assay. Sample selection. (i) Pulmonary tuberculosis. | Three hundred and thirty blood samples were collected from patients attending a tuberculosis clinic. The patients had pulmonary tuberculosis at various stages of the disease and were at various stages of treatment. For all patients, the diagnosis of tuberculosis was made on the basis of the following criteria, determined by taking a detailed history: (i) presenting clinical signs and symptoms such as cough and fever; (ii) BCG vaccination status, past history of tuberculosis, or presence of a close contact with tuberculosis; (iii) routine laboratory tests which included assay of sputum for acid-fast bacilli (AFB), culture of sputum, skin test, and chest X ray; and (iv) response to antituberculosis treatment, as against specific treatment for chronic obstructive pulmonary disease (COPD) and asthma. On the basis of the criteria described above, the patients were diagnosed as having tuberculosis and classified into the groups described below. Group I comprised 86 patients who were positive by both smear for AFB and culture. Of these, 20 patients had fresh cases of tuberculosis, 14 had been treated for tuberculosis with antituberculosis drugs for less than 6 months, 11 had been treated for 6 months to 1 year, and 33 had been treated for more than 1 year; data on the treatment given were not available for 8 patients. Group II comprised 126 patients who were negative by smear for AFB but positive by culture. In this group, 23 patients had fresh cases of tuberculosis, 15 had been treated for less than 6 months, 16 had been treated for 6 months to 1 year, and 57 had been treated for more than a year; data on the type of treatment given were not available for 15 patients. Group III comprised 118 patients who were negative by both smear for AFB and culture. However, 99 of these patients had previously received a diagnosis of tuberculosis on the basis of a positive smear for AFB and culture and were receiving antituberculosis treatment. Nine patients had been treated with antituberculosis drugs for less than 6 months, 14 had been treated for 6 months to 1 year, and 45 had been treated for more than 1 year; data on the type of treatment given were not available for 31 patients. Only 19 patients in this group had fresh cases of tuberculosis, and diagnosis was made on the basis of a combination of tests and criteria such as X ray, skin test, and presence of a contact. Further analysis indicated that 33 of the 330 tuberculosis patients were HIV positive. Of these, 7 patients were in group I, 16 were in group II, and 10 were in group III. The control group comprised 138 individuals between the ages of 21 and 50 years who had no prior history of tuberculosis but who had all been vaccinated with BCG. Thirty-one of these were healthy volunteers, 75 had bronchitis leading to COPD, and 28 were asthma patients. Diagnosis of COPD and asthma was done on the basis of the criteria given earlier, such as X ray, sputum cultures, clinical presentation, and response to antibacterial treatment. COPD patients were treated with aminophylline and theophylline, and asthma patients were treated with bronchodilators. (ii) Extrapulmonary tuberculosis. | Fifty CSF samples were collected from individuals with signs and symptoms of meningitis and other neurological disorders. Of these, 26 were diagnosed as having tuberculous meningitis on the basis of the results of clinical and biochemical investigations. Twenty-four of the patients had central nervous system (CNS) disorders of nontuberculous origin. FIG. 1. | (a) Standard curve for the antibody assay; (b) determination of the cutoff value. (a) Standard curve for the antibody assay; (b) determination of the cutoff value. PMID- 12204948_RESULTS TI - AB - Calculation. | The amount of antibody in the clinical samples was determined by using the two assay controls (C1 and C2) included in the kit. Control C1 represented nonspecific binding with no antibody, and control C2 represented an anti-BCG IgG concentration of 25 mug/dl. The ratio X (as a percentage) was calculated by using the counts in C1, C2, and the unknown, as follows: [(counts in unknown - counts in control C1)/(counts in control C2 - counts in control C1)] x 100. Samples that gave values of more than 100 were considered positive. Stability of the kit. | In order to ascertain the stability of the kit, the reproducibilities of values for the QC samples in the kit were determined. The expected value of the ratio for QC Q1 was 200, and the mean observed values over a period of 7 weeks were 198 and 205 at RT and 4C, respectively. The SD and percent coefficient of variation (CV) were 22.8 and 11.5%, respectively, at RT and 18.7 and 9.1%, respectively, at 4C. The expected value of the ratio for the second QC, Q2, was 400, with mean values of 344 and 355 at RT and 4C, respectively. The SD and CV were 50.8 and 14.7%, respectively, at RT and 41.9 and 11.8%, respectively, at 4C. These CVs were within the statistically acceptable range for interassay variability. These results are represented graphically in Fig. Pulmonary tuberculosis. | Table presents the antibody responses in patients with pulmonary tuberculosis with respect to the period of treatment given, and Table presents the antibody responses in patients with HIV and M. tuberculosis coinfection. It was observed that 14 of the 138 control samples had antibody levels above the ratio cutoff value of 100, contributing to a rate of nonspecific detection of 10% and a specificity of 90%. Group I comprised 86 patients who were positive by both smear and culture and had 69 patients who had high antibody ratios, giving the test a sensitivity of 80.2% and a positive predictive value of 83.1%. Of the 7 HIV-positive patients in this group, 4 patients had high titers of antitubercular antibody. Group II had 126 patients who were negative by smear but positive by culture. Of these, 92 were positive for antibody, giving the test a sensitivity of 73% and a positive predictive value of 86.8%. Six of the 16 patients in this group were positive for both HIV and M. tuberculosis and had high titers of antibodies against M. tuberculosis. Group III comprised 118 patients who were negative by both smear and culture and had 71 patients with high antibody titers, giving the test a sensitivity of 60.2% and positive predictive value of 83.5%. Three of the 10 HIV-positive patients in this group also had high antibody levels. The detailed analysis of each subgroup is presented in Tables and 2. Figure provides a graphical representation of the response ratios for the different groups of patients with pulmonary tuberculosis. Compared to the antitubercular antibody levels in the control group, the patient groups had high levels of antitubercular antibody. The patient population was further classified as those who had received antituberculous treatment at the time of sample collection and those who had not received any treatment at the time of sample collection (fresh cases). The test showed a sensitivity of 62.9% and a positive predictive value of 73.6% for the patients with fresh cases. For the treated patients, the sensitivity was 72% and the positive predictive value was 93.2%, as depicted in Table . Extrapulmonary tuberculosis. | Table represents the percent positivity of antibody detection in patients with tuberculous meningitis. Of the 26 patients with clinically proven tuberculous meningitis, 19 had high antibody levels by this test, giving a sensitivity of 73%. A false-positivity rate of 12% was observed for the control group, giving a specificity of 88% and a positive predictive value of 91%. Figure is a graphical representation of the antibody response ratios for patients with CNS disorders of tuberculous and nontuberculous origin. FIG. 2. | Stability of the kit at RT (a) and at 4C (b). Stability of the kit at RT (a) and at 4C (b). *, C1; , C2; , QC1; , QC2. FIG. 3. | Antibody levels in patients with pulmonary tuberculosis. Antibody levels in patients with pulmonary tuberculosis. , group I; , group II; , group III; *, group IV (controls). FIG. 4. | Antibody levels in patients with tuberculous meningitis. Antibody levels in patients with tuberculous meningitis. , patients with tuberculous meningitis; , controls (patients with CNS disorders of a nontubercular nature). TABLE 1 | Antibody response in pulmonary tuberculosis TABLE 2 | Antibody responses in patients with HIV and M. tuberculosis coinfection TABLE 3 | Predictive values for treated and nontreated patients TABLE 4 | Antibody response in patients with tuberculous meningitis PMID- 12204948_DISCUSSION TI - AB - The need for a serological test for the diagnosis of tuberculosis is emphasized on the grounds of potential benefits for rapid and differential diagnosis, particularly in culture- and/or smear-negative patients. During bacterial diseases like tuberculosis, the immune response leads to a rise in the titers of antibodies against different antigenic determinants of the organisms, and hence, the specific antibodies present may range from those that are species specific to those that share certain common antigenic determinants with the causative organisms. The antibody response in tuberculosis has been studied for a long time (, , , , 13, , -, , -, , ). Although one of the major drawbacks, especially in serological tests, is the persistence of antibodies even after effective treatment, measurement of the antibody response to 38-kDa antigen has been found to be useful in assessing the effectiveness of treatment in patients with tuberculous meningitis . A radioimmunoassay for the detection of M. tuberculosis antigen and anti-M. tuberculosis antibodies developed at the Laboratory Nuclear Medicine Section of the Bhabha Atomic Research Centre has been tested with samples from patients with various clinical manifestations of tuberculosis (, , , , -). It was demonstrated that detection of antigen and antibody gave a good sensitivity and specificity for diagnosis. It was also shown that antibody detection alone could be useful for the diagnosis of tuberculous meningitis. Antigen and antibody detection in patients with pulmonary tuberculosis was done from immune complexes, not serum. In this particular study, antibody levels in serum samples from patients with pulmonary tuberculosis were measured. The aim of the present work was to make the test user friendly and evaluate detection of antibody from serum rather than immune complexes in the case of pulmonary tuberculosis. The modified antibody assay described here is simple and rapid and involves the addition and incubation of all reagents together for 4 h at room temperature with a final centrifugation step. This not only reduces the pipetting errors and the number of centrifugation steps required but also reduces the incubation time, facilitating early diagnosis and hence the early institution of treatment. The most significant aspect of this test was that it was useful for the diagnosis of tuberculosis in those patients for whom the rapid test for AFB was negative, which included patients from group II and group III. Although a false-positivity rate of 10% was observed for the control population, this was acceptable, as India is an area with a high prevalence of tuberculosis and the entire population is vaccinated with BCG. Early diagnosis and early treatment of tuberculosis in patients coinfected with HIV are mandatory, as delayed therapy results in increased rates of mortality . In the present study, in the case of patients coinfected with M. tuberculosis and HIV, antibodies against M. tuberculosis persisted even after antituberculosis treatment. However, the percent positivity of antibody detection for the HIV-infected tuberculous patient population is lower than that for the entire tuberculous patient population (Table and Table ). These findings were similar to those presented in other reports, in which the sensitivity dropped from 62% for non-HIV-infected tuberculous patients to 28% for HIV-infected tuberculous patients . It has been reported that no antibody is detected by an immunochromatographic test (ICT) for tuberculosis in any of the serum samples of HIV-infected patients coinfected with M. tuberculosis or mycobacteria other than M. tuberculosis . Evaluation of the Mycodot immunodiagnostic test for the diagnosis of tuberculosis, which detects antibodies to lipoarabinomannan antigen, showed that it has very good specificity. The sensitivity of detection for HIV-negative tuberculous patients was 56% and much lower, as low as 25%, for HIV-positive patients . In general, serological assays have high negative values for HIV-positive individuals, low sensitivities, and low negative predictive values, which markedly decreases their utility in populations in which HIV infection is prevalent . Diagnosis of tuberculous meningitis has always been a challenge, especially in children. Data from a previous study in which the 38-kDa antigen in the ELISA system was used for the detection of antibodies in CSF samples showed sensitivities of 60% for patients proven to have tuberculosis postmortem, 80% for patients with culture-proven tuberculous meningitis, and 62.5% for patients with clinically proven tuberculous meningitis . The sensitivity in that study was superior to those reported elsewhere . Use of the 19-kDa antigen for the detection of antibodies in CSF of patients with tuberculous meningitis could clearly differentiate the patient group from the control group in studies with a European population, but no such differentiation was observed in the study with an Indian population . The modified antibody assay mentioned here has a fairly good sensitivity of 73% for patients with tuberculous meningitis and a specificity of 88%, and above all, it could differentiate between the patient group and control group in the Indian population. In Western countries, the prevalence of tuberculosis is low and a clear distinction between patient and control populations is possible. An ELISA with the 38-kDa antigen specific for the M. tuberculosis complex showed a sensitivity of 83% and a specificity of 99% . In a study with a Chinese population, however, the sensitivity of detection of IgG antibodies to the 38-kDa antigen was 64% and the specificity was 81% . Similarly, an ELISA performed with the Ag5 antigen to study the antibody response in patients with pulmonary tuberculosis showed a sensitivity of 84% and a specificity of 96% for populations in low-prevalence areas, while the sensitivity was 94% and the specificity was 80% for populations in high-prevalence areas . The same ELISA for the detection of antibodies used elsewhere showed sensitivities that varied from 49 to 89% and specificities that ranged from 88 to 98% . The A60 antigen, which is a thermostable component of the purified protein derivative, has been used in the serodiagnosis of tuberculosis. In patients with smear-negative but culture-positive pulmonary tuberculosis, measurement of both IgM and IgG by ELISA revealed a sensitivity of 68% and a specificity of 100% . In the pediatric group, at a chosen specificity of 98%, anti-A60 IgG antibodies were observed in 68% of children with clinically active tuberculosis . In the Indian population, an ELISA with the A60 antigen for the detection of IgM antibodies in patients with pulmonary tuberculosis had a sensitivity of 77.5% and a specificity of 87.5% , while quantification of antibodies in patients with neurotuberculosis showed a sensitivity of 63% and a specificity of 100% . When titers of antibodies to the 19-kDa antigen in the Indian population were measured by ELISA, the test was found to have a poor sensitivity (8%), but the same test used for a population in the United Kingdom was found to have a sensitivity of 58% and a specificity of 97% . An ELISA for the detection of antilipoarabinomannan antibodies in sera from patients with pulmonary, miliary, and pleural tuberculosis and pleural lymphadenitis and in CSF from patients with tuberculous meningitis had an overall sensitivity of 61 to 72% and a specificity of 72% . Among the HIV-negative patients with active tuberculosis, the sensitivity of antilipoarabinomannan antibody detection ranged from 21.5 to 89%, but the sensitivity of detection was generally poor, about 7 to 40%, for HIV-positive patients . In developing countries like India, where the prevalence of tuberculosis is high and where individuals are by and large vaccinated with BCG or exposed to environmental mycobacteria, antibodies are present even in the healthy control population, so that the cutoff for any diagnostic test is high. The antibody test with partially purified antigen developed in the study described here shows a sensitivity of 80% and a specificity of 90% for patients with pulmonary tuberculosis. These results are quite acceptable and compare well with those of tests from Western countries. Previous reports have indicated that ELISA kits for the detection of antibody for the diagnosis of tuberculosis have poor sensitivities and specificities . Clinical evaluation of the Andelisa IgG kit, which uses the A60 antigen derived from M. bovis BCG, gave a sensitivity of 48.3% and a specificity of 92%, while the Kreatech IgA kit, which uses the KP90 antigen derived from M. tuberculosis, showed a sensitivity of 49.7% and a specificity of 84% . Simultaneous evaluation of seven serological tests, i.e., two ICTs and five ELISAs, with the same clinical samples also showed poor to modest sensitivities that ranged from 16 to 57%. The specificities of these tests ranged from 80 to 97% with sera from the Mantoux test controls and 62 to 100% with sera from the anonymous controls . These results show lower sensitivities and specificities, indicating considerable overlap in antibody levels in the healthy control group and the patient group and indicating that none of the tests has utility for the diagnosis of tuberculosis. Evaluation of the validity of the ICT kit in a study with a Korean population showed a sensitivity of 73% for patients with fresh cases of tuberculosis and 87% for patients with reactivated tuberculosis, while the specificity was 88% for the healthy group and 94% for the nontuberculous patients and hospital workers . A similar evaluation of the ICT kit in Madagascar showed sensitivities of 68.2% for patients with pulmonary tuberculosis and 65.2% for patients with extrapulmonary tuberculosis and a specificity of 83.3% . However, evaluation of the ICT kit with the Indian population showed an overall sensitivity of 20% for patients in the first month of the disease (fresh cases) and a very low sensitivity of 4%. The test was also positive for 30% of cases of disease caused by mycobacteria other than M. tuberculosis, indicating cross-reactivity. The overall specificity of the test was 89% . The ELISA kit for the detection of IgG antibodies against tuberculous glycolipid antigen containing cord factor (TBGL Ag) had a sensitivity of 87% and a specificity of 100%. The other findings included in the report were an increase in the antibody titers after the start of antituberculosis chemotherapy and the persistence of antibodies in the circulation even after the completion of treatment . The ICT kit developed for detection of antibodies against the 38-kDa antigen of M. tuberculosis had an overall sensitivity of 70 to 92% and an overall specificity of 92 to 93% for patients with pulmonary tuberculosis. For patients with extrapulmonary tuberculosis, the overall sensitivity was 76% and the overall specificity was 92% . Results from a study conducted in the Europe demonstrated a low sensitivity of 50% but a high specificity of 100% . This low sensitivity will result in many false-negative results, whereby the negative predictive value will be unacceptably low even in countries with a high prevalence of tuberculosis . A specificity of 100% is not possible in developing countries, particularly in tropical areas where people are permanently in contact with various pathogens and develop cross-reacting antibodies, resulting in poor test specificities. Hence, every new serodiagnostic test should be validated with controls from countries where the test would be applied . In this context a cutoff of 100 was determined on the basis of the values obtained for the control population, which included contacts and patients with nontubercular disease. It was observed, however, that if the cutoff were raised to 125, the specificity would go up to 97% and the sensitivities would be 69.7, 66.6, and 51.6%, for groups I to III, respectively, as shown in Table , with a positive predictive value of 94%. Thus, it is possible to alter the cutoff and obtain appropriate sensitivity, specificity, and predictive values according to the prevalence of the disease in different areas. The antibody test kit described here involves very few steps in terms of the addition of reagents and a shorter incubation time, which make it user friendly. Above all it has a good sensitivity and a good specificity. This kit should benefit the population by providing diagnoses early in the course of disease and, hence, permitting the early administration of appropriate chemotherapy. PMID- 12204974 TI - Diagnostic Techniques To Detect Cryptic Leishmaniasis in Dogs AB - | This study of several techniques for detecting cryptic leishmaniasis in dogs from areas in Spain where Leishmania infantum is highly endemic concludes that immunological techniques (enzyme-linked immunosorbent assay, immunofluorescence antibody test, Western blotting, delayed-type hypersensitivity reaction, and in vitro lymphocyte proliferation assay) do not clearly differentiate between noninfected and infected asymptomatic dogs and that culture and PCR are more reliable diagnostic tools. PMID- 12204974_Introduction TI - AB - Seroepidemiological studies of canine leishmaniasis have revealed a large number of asymptomatic seropositive animals . Moreover, in areas where leishmaniasis is highly endemic, a high proportion of apparently healthy animals show low levels of anti-Leishmania antibodies. Serological follow-up of these animals has revealed that some of them are in the prepatent infection period, which will lead to increased antibody titers in subsequent blood extractions. Others have regressive forms of the disease, and their antibody levels will decrease in the following months or years; still others maintain low levels of antibodies without developing the disease for many years . However, the total number of infected animals is unknown. The detection of the extent of the infection, particularly among asymptomatic dogs, is of great importance for the control of leishmaniasis. Most epidemiological and control studies of canine leishmaniasis are performed by serological methods. Although such methods are traditionally considered to be more sensitive than parasitological techniques for the diagnosis of the disease, they underestimate the prevalence and incidence of the infection relative to those estimated by culture and PCR . Indeed, experimentally infected dogs that develop the disease have an anti-Leishmania humoral immunoresponse while those that remain asymptomatic present a cellular response . The application of highly sensitive techniques, such as PCR and Western blotting (WB) , as well as the optimization of culture , have improved the rate of detection of leishmaniasis. Here, we compare the results obtained by various diagnostic methods (direct examination, culture, PCR, enzyme-linked immunosorbent assay [ELISA], WB, delayed-type hypersensitivity reaction [DTH], and a lymphocyte proliferation assay [LPA]) for dogs from two areas in Spain where canine leishmaniasis is endemic, and we consider the results in the context of the clinical status of the animals. The study was carried out with 72 animals, 38 of which were from the Priorat, an area in northeast Spain where leishmaniasis is highly endemic. The remaining 34 dogs were from an animal protection society in Madrid, Spain. Animals were selected for the study after extensive serological screening for anti-Leishmania-specific antibodies, and subjects were sometimes chosen for screening because of the presence of sick animals in the kennel. In other cases, we picked asymptomatic animals with specific antibodies or under other irregular circumstances. Thus, the dog samples were biased and cannot be considered representative of the whole dog population in the areas studied. Clinical symptoms and lesions consistent with canine leishmaniasis (skin abnormalities, onychogryposis, weight loss, epistaxis, apathy, ocular and other lesions, lymph node and spleen enlargement, etc.) were recorded for all dogs. Blood was collected by cephalic or jugular venipuncture for complete blood count and biochemical analysis (serum proteins, the renal markers urea and creatinine, and the hepatic markers aspartate aminotransferase and alanine aminotransferase), for detection of anti-Leishmania antibodies, and for PCR and LPA. Popliteal lymph node aspirates were obtained for direct examination, culture, and PCR. Needle aspiration skin microbiopsy was also performed to obtain samples for PCR. Anti-Leishmania antibodies were quantitatively detected by an immunofluorescence antibody test (IFAT) and an ELISA for immunoglobulin G2 (ELISA-IgG2) for dogs from Madrid and by ELISAs for protein A (ELISA-protein A) and for IgG2 for dogs from the Priorat. The IFAT was performed by standard methods using a fluorescent conjugated anti-dog IgG (heavy plus light chains; Jackson ImmunoResearch, West Grove, Pa.) at a 1:50 dilution. ELISAs were performed as described elsewhere . Horseradish peroxidase-conjugated protein A (dilution, 1:30,000; Sigma, St. Louis, Mo.) and anti-dog IgG2 (dilutions, 1:2,000 to 1:5,000; Bethyl Laboratories, Montgomery, Tex.) were used as second antibodies. The reaction results were quantified in units relative to those of a positive serum sample that was used as a calibrator and arbitrarily set at 100 U. WB analyses of sera from the Priorat area were performed at the Faculty of Pharmacy, Barcelona, Spain, as described elsewhere . Those sera that developed any of the polypeptide fractions of 14 or 16 kDa, previously reported as 12 and 14 kDa , were considered positive. WB analyses of sera from the Madrid area were performed at the Faculty of Veterinary Science, Madrid, as described previously . The sera that revealed the polypeptide fractions of 30, 42, 50, and 57 kDa were considered positive, as reported previously . The correspondence between positive results detected by each method has been established (S. Mendez, M. J. Aisa, F. J. Fernandez-Perez, L. Iniesta, M. Portus, J. M. Alunda, and M. T. Gomez-Munoz, submitted for publication). DNA for PCR analysis was extracted with a PCR template preparation kit (Boehringer Mannheim, Mannheim, Germany). Leishmania-specific oligonucleotide primers A2 (5'-GGGAGAAGCTCTATTGTG-3') and B1 (5'-ACACTCAGGTCTGTAAAC-3') were used to amplify a 650-bp fragment in the internal transcribed spacer region of genomic DNA . The DTH was performed by inoculation into the skin of the groin of 0.1 ml of Leishmania infantum antigen (3 x 108 promastigotes/ml), which was kindly supplied by the Instituto de Salud Carlos III, Majadahonda, Spain. Reactions were read after 48 h and measured by the ballpoint pen method. The LPA was performed with peripheral blood mononuclear cells as described elsewhere . Crude results are shown in Table . Each laboratory (Madrid and Barcelona) applied its current serological tests in addition to the ELISA-IgG2, which was performed in both laboratories with the same conjugates, the same quantification system, and the same calibrator serum. The IgG2 results correlated highly with the ELISA-protein A results (Spearman's rank correlation coefficient, 0.950; P = 0.000) and the IFAT results (Spearman's rank correlation coefficient, 0.627; P = 0.000). Therefore, to simplify this discussion, only results from the ELISA-IgG2 were included in the statistical analysis and comparison of techniques. In contrast, no correlation was observed between the techniques for detecting the cellular immunoresponse, DTH, and LPA (Spearman's rank correlation coefficient, 0.052; P = 0.75), and so the results obtained in each determination were analyzed independently. Attempts to link DTH and LPA findings for use in detecting human leishmaniasis have led to contradictory results. DTH and LPA results correlated in L. major-infected children of Jericho and Tunisia . Nevertheless, the lymphoproliferative response to the L. major antigen by peripheral blood mononuclear cells from Sudanese individuals with a positive leishmania skin test and no history of cutaneous leishmaniasis was similar to the response observed in Danish people with no reported exposure to Leishmania parasites . TABLE 1 | Clinical and laboratory findings and results of diagnostic methods for visceral leishmaniasis in dogs The dogs that were positive by direct examination, culture, PCR, or at least two immunological methods were considered probably infected. Among them, those with external signs of leishmanial disease were considered to be symptomatic and those without external signs of disease, despite the detection of biochemical and hematological (analytical) disorders, were considered to be asymptomatic or to have cryptic infections. The specific IgG2 levels detected by ELISA were directly related to the pathophysiological disorders detected during the physical and analytical examinations . All symptomatic dogs (10 of 10) had high antibody titers as determined by the ELISA-IgG2, while only 35 of 51 animals with cryptic infections were positive by this technique. FIG. 1. | IgG2 expression in L. infantum IgG2 expression in L. infantum-infected dogs is related to the pathophysiology. Columns indicate mean values, and error bars indicate standard deviations. Dogs are divided into cohorts as follows: (column 1) symptomatic dogs, i.e., infected animals with external signs of leishmanial disease; (column 2) infected dogs, i.e., animals found positive by direct examination, culture, PCR, or at least two immunological techniques; (column 3) infected asymptomatic dogs with biochemical alterations; (column 4) infected asymptomatic dogs without biochemical alterations; (column 5) noninfected dogs. n, number of dogs in each cohort. The dotted line indicates the cutoff separating positive and negative results. The positive and negative results obtained with the techniques were compared two by two by the McNemar and Pearson tests in the whole dog sample studied, in animals considered to be infected (symptomatic and asymptomatic animals) and in those found to have cryptic infections . Culture and PCR provided very similar results; they gave the same proportion of positives (McNemar test, P = 0.629) and a high degree of association (Pearson test, P < 0.001). The culture and PCR results differed for 17 of the 72 dogs studied (10 animals positive by PCR and negative by culture and 7 animals positive by culture and negative by PCR). In 11 of these animals, immunological techniques confirmed the presence of parasites. In three cases PCR was the only positive test, and in two cases only the culture was positive, as confirmed by new samples and repeated analyses. TABLE 2 | Statistical analysis of results (positive/negative) obtained by various diagnostic techniques in three dog cohorts: (i) the whole dog population studied, (ii) dogs considered to be infected with L. infantum, and (iii) asymptomatic dogs with a cryptic Leishmania infection The humoral immunoresponse, detected as L. infantum-specific IgG2 expression, was also associated with PCR and culture results when the whole dog population was considered (Pearson test, P <= 0.001) but was independent of those results when only the animals with cryptic infections were considered. The results obtained by immunological techniques were independent when compared two by two, even when these techniques were considered to have the same target as those of antibody detection (ELISA-IgG2 and WB) or cellular immunoresponse (DTH and LPA) techniques. The lack of a "gold standard" for diagnosis of asymptomatic infections caused by L. infantum is a drawback to epidemiological studies of the disease. Parasitological techniques like direct microscopic examination and culture offer the only reliable evidence of the presence of parasites in a sample. However, direct examination lacks sensitivity when the parasite number is small, and the growth capacity in vitro varies from one Leishmania strain to another. Other analytical techniques, such as PCR and immunological methods, lack some specificity and sensitivity. There is evidence of Leishmania persistence inside the host after recovery from the disease , and a positive response to Leishmania antigen detected by DTH and LPA has been associated with asymptomatic infection . However, cellular immunoresponse or low antibody levels may be due to immunological memory rather than to the presence of the parasite in the host, especially in areas of endemicity. Moreover, positive lymphoproliferative responses to Leishmania antigen have been observed in nonexposed humans . Whether a dog can be considered infected or uninfected is the key determinant of parameters such as sensitivity, specificity, and predictive values for diagnostic techniques. If we consider that only those animals that are positive by direct examination or culture are parasitized, the sensitivities and specificities of other methods such as PCR and immunological techniques are affected. Therefore, these parameters were not determined in the present study. The arbitrary definition of a cutoff in the ELISA and the lack of a clear separation between positive and negative results entail a lack of specificity when this cutoff is established to improve the sensitivity of the technique. The low humoral immunoresponse characteristic of asymptomatic animals frequently places their antibody levels at the borderline of detectability, and positive results may be caused by artifacts and cross-reactions. This may account for the association between the PCR and ELISA-IgG2 results for the total dog sample (Pearson test, P = 0.001) and for their independence for the sample comprising only animals with cryptic infections. WB analysis is regarded as a highly specific technique, when the specific bands are considered, and is very useful with samples with low antibody concentrations . However, bands are difficult to identify, since several antigen fractions have similar molecular weights and subjectivity in reading the results cannot be avoided when very weak bands are detected. This may explain the independent results (Pearson test, P > 0.05) provided by the ELISA-IgG2 and WB. Moreover, since the antigen treatments in the ELISA and WB differed strongly, each technique identified a different set of epitopes, and this explains the lack of association in animal cohorts with a predominance of individuals with low humoral immunoresponses. In conclusion, visceral leishmaniasis in sick dogs from areas of endemicity can be easily diagnosed. It is characterized by a high humoral immunoresponse that is measurable by conventional serological techniques (e.g., IFAT and ELISA) and a high parasite burden that is detectable by parasitological and PCR methods. However, when we deal with asymptomatic animals, particularly in epidemiological studies, immunological techniques do not discriminate between infected and noninfected animals. Antibody detection alone and DTH or LPA, which are used to estimate the infected dog population , are not suitable tools,and parasitological methods and PCR offer more accurate results. PMID- 12204949 TI - Functional and Phenotypic Changes in Circulating Lymphocytes from Hospitalized Zambian Children with Measles AB - | Measles is associated with immunosuppression and increased susceptibility to secondary infections and is a particular problem in developing countries. Lymphocyte changes accompanying immune activation and regulation of the immune response may contribute to immunosuppression. To evaluate lymphocyte changes during measles, children (n = 274) hospitalized with measles in Lusaka, Zambia, were evaluated at entry, discharge, and 1-month follow-up and compared to healthy Zambian children (n = 98). Lymphopenia was present on hospital admission and reflected decreased CD4 and CD8 T cells but resolved quickly. Lymphopenia was most marked in girls, in those with temperatures of >38.5C, and in malnourished children. CD4/CD8 ratios were decreased at all time points and were lower in boys than in girls at discharge and follow-up. Spontaneous death occurred in cultured lymphocytes, and the proportions of freshly isolated cells undergoing apoptosis, based on annexin V and propidium iodide staining, were increased. Surface Fas was increased on both CD4 and CD8 T cells compared to controls, and expression was greater on CD4 T cells and was inversely correlated with lymphocyte viability in culture at study entry. Mitogen stimulation of lymphocytes improved viability, but inhibitors of Fas, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, and TNF did not. Plasma levels of beta2 microglobulin and soluble Fas, Fas ligand, CD8, CD4, and TNF receptor were increased, and soluble CD8 was higher in boys than in girls. The multiple effects of measles on lymphocytes from Zambian children include decreased numbers in circulation, increased activation, and increased susceptibility to cell death, with substantive differences in the magnitude of these changes between boys and girls. PMID- 12204949_Introduction TI - AB - Measles causes nearly 1 million deaths per year worldwide, with the heaviest burden occurring in sub-Saharan Africa . The high morbidity and mortality associated with measles virus (MV) infection is due primarily to secondary infections, particularly of the respiratory and gastrointestinal tracts . Increased susceptibility to other infectious diseases has been linked to the immune suppression associated with this infection. Cell-mediated immunity, manifested by decreased delayed-type hypersensitivity responses to skin test antigens and decreased proliferation of lymphocytes after stimulation in vitro, is suppressed, and this suppression may persist for several months after recovery . The mechanism of MV-induced immune suppression is incompletely understood, and few studies of children in developing countries, where mortality rates are highest, have been done. Lymphocyte activation is a consistent feature of acute measles in North and South American children, and we have proposed that the observed cytokine "shift" from type 1 cytokines early during infection to type 2 cytokines during and after recovery inhibits development of effective type 1 cytokine-mediated cell-mediated immunity responses upon subsequent exposure to new pathogens . In addition, lymphocyte apoptosis, often associated with immune activation, is a feature of acute infections with other immunosuppressive murine and human viruses , and it has been hypothesized that increased apoptosis of lymphocytes in individuals with acute measles may contribute to immune suppression . To better understand these processes in African children, we have evaluated the functional and phenotypic changes that occur during acute MV infection and recovery from measles in children hospitalized with measles in Lusaka, Zambia. PMID- 12204949_MATERIALS AND METHODS TI - AB - Study population. | The children studied (n = 274; mean age, 3.05 +- 0.21 years; median age, 1.4 years; range, 2 months to 14.3 years) were a subgroup of children enrolled in a study of the clinical manifestations and immune responses of human immunodeficiency virus (HIV)-infected and non-HIV-infected Zambian children hospitalized with measles between January 1998 and August 2000 . Children admitted to the infectious-disease isolation ward at the University Teaching Hospital (UTH) in Lusaka, Zambia, with the clinical diagnosis of measles were prospectively enrolled. Measles was confirmed by the presence of immunoglobulin M (IgM) antibody against MV (Wampole, Cranbury, N.J.), and children who were HIV infected, as determined by the presence of antibody to HIV (Organon Technika, Boxtel, The Netherlands) and reverse transcription-PCR detection of HIV type 1 RNA (Amplicor version 1.5; Roche Pharmaceuticals Inc., Branchburg, N.J.) were excluded. Children who were moribund at the time of hospitalization were not enrolled in the study, although many of those enrolled had severe or complicated infections and there were four deaths among the study children. Samples from all children were not included in all assays or at all time points due to the limited amounts of blood available, difficulty with blood drawing, or parental withdrawal from the study. Not all children returned for the 1-month follow-up visit after discharge, and active tracing was not performed. Healthy control children (n = 98; mean age, 3.47 +- 0.31 years; median age, 1.75 years; range, 6 months to 9.4 years; P = 0.03) were enrolled from two sources located within 2.5 miles of the UTH: the clinic for well-child care at Chilenge Health Center (<5 year olds) and the Regiment Primary School (6 to 7 year olds). Control children were documented to be negative for MV IgM and for HIV antibody and RNA. The study populations were equally matched for sex, with 49% boys in the measles group and 50% boys in the control group. Written informed consent was obtained from the parents or guardians of the children studied. The study was reviewed and approved by the Committee on Human Research of the Johns Hopkins Bloomberg School of Public Health and the Ethics Committee of the UTH. Nutritional status. | Weight-for-height and height-for-age z scores were determined with Epi-Info software (Centers for Disease Control and Prevention). Wasting was defined as a weight-for-height >2 standard deviations below the norm, and stunting was defined as a height-for-age >2 standard deviations below the norm (defined by National Center for Health Statistics reference values). Nine percent of the children with measles and 2.2% of the control children were wasted (P < 0.05), and 48.6% of the children with measles and 33.7% of the control children were stunted (P < 0.02; chi2 test) Blood collection and processing. | Peripheral blood, collected in a sterile tube containing EDTA, was obtained at study entry (mean, 3.6 +- 0.1 days after onset of rash; range, 1 to 10 days after onset of rash; n = 252), at hospital discharge (mean, 6.5 +- 0.18 days after onset of rash; range, 2 to 17 days after onset of rash; n = 185), and at a 1-month follow-up visit (mean, 37.6 +- 0.5 days after onset of rash; range, 25 to 61 days after onset of rash; n = 78). White blood cell counts were determined manually. Differential counts were performed on Wright-Giemsa-stained smears by the hematology laboratory at the UTH. Mononuclear cells were separated by Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) density gradient centrifugation. Plasma was frozen in aliquots at -80C. Lymphocyte viability studies. | Washed fresh peripheral blood mononuclear cells (PBMCs) were suspended at a concentration of 106/ml in RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 mM HEPES, and 10 mug of gentamicin/ml. The cells were cultured, with or without stimulation with phytohemagglutinin (PHA; 5 mug/ml; Sigma Chemical Co., St. Louis, Mo.), in 96-well round-bottom microtiter plates at a volume of 200 mul/well. For some studies, cells were cultured with the following inhibitors of apoptosis: tumor necrosis factor (TNF)-related apoptosis-inducing ligand-Fc IgG chimera (20 ng/ml), Fas-Fc IgG chimera (10 ng/ml), TNF receptor II (TNFRII)-Fc IgG chimera (20 ng/ml) (R&D Systems, Minneapolis, Minn.), and mouse anti-human Fas ligand (FasL) monoclonal antibody (1 mug/ml; PharMingen, San Diego, Calif.). Cell viability was determined by trypan blue exclusion. All studies were performed on fresh samples at the Virology Laboratory in Lusaka. Flow cytometry. | All analyses were performed with fresh samples at the Virology Laboratory in Lusaka on a FACScan flow cytometer using Cell Quest software (Becton Dickinson, San Jose, Calif.). Directly conjugated mouse monoclonal antibodies to the following human antigens were used: CD3-fluorescein isothiocyanate (FITC), CD4-phycoerythrin (PE), CD8-PE (Becton Dickinson), and Fas-FITC (Antigenix America, Huntington Station, N.Y.). In each case, staining was compared with that of the appropriately labeled isotype control antibody. The percentages of CD4 and CD8 T cells were determined after staining whole blood. CD3+/CD4+ and CD3+/CD8+ cells were enumerated with lymphocyte gates set on the basis of forward and side scatter information adjusted to exclude monocytes based on percent CD3-/CD4+ cells. Fas expression was measured on CD4+ and CD8+ subsets using the same gates. The percentage of freshly isolated mononuclear cells staining with annexin V only was determined after simultaneous staining with annexin V-FITC and propidium iodide (PI) (R&D Systems) using a wide gate based on forward and side scatter information which included dead and dying cells. The percentage of annexin V-positive cells at entry among subsets was assessed after 18 h of culture by dual staining for annexin V and CD4 or CD8. Soluble factors in plasma. | The levels of gamma interferon (IFN-gamma; BD PharMingen), soluble CD8 (sCD8), sCD4 (Endogen, Woburn, Mass.), sTNFRII (R&D Systems), sFas, and sFasL (Medical Biological Laboratories, Nagoya, Japan) in plasma were measured by enzyme immunoassay according to the manufacturer's instructions. beta2 microglobulin was measured by radioimmunoassay (Pharmacia & Upjohn, Uppsala, Sweden). Values below the limit of detection were assigned values midway between zero and the lower limit of detection for the purposes of analysis. Statistical analysis. | Data are presented as means +- standard errors of the mean unless otherwise indicated. All analyses were performed using STATVIEW or STATA software (SAS, Cary, N.C.). Mann-Whitney U, Wilcoxon signed-rank, and Spearman rank correlation coefficient tests were used as appropriate, with the level of significance set at a P value of 0.05. No corrections were made for multiple comparisons. PMID- 12204949_RESULTS TI - AB - Lymphopenia. | The numbers and types of leukocytes in the peripheral blood were measured at study entry (usually the day after admission to the hospital), at discharge from the hospital, and approximately 1 month after discharge, and the values were compared to those of healthy children . On entry, total leukocyte counts were not different between measles patients (8,400 +- 287 cells/mul) and controls (8,300 +- 345 cells/mul), but they were increased in measles patients by the time of discharge (10,579 +- 425 cells/mul; P < 0.0001) and at follow-up (10,610 +- 498 cells/mul; P = 0.0005) . Monocyte counts were increased at all times in measles patients compared to controls . Lymphocyte counts were decreased for measles patients at study entry (3,681 +- 164 cells/mul) compared to controls (5,650 +- 240 cells/mul; P < 0.0001) and were lower in girls than in boys . The lymphocyte counts rose to 5,835 +- 298 cells/mul at the time of discharge and to 6,362 +- 359 cells/mul (P = 0.27) at follow-up . The lymphocyte counts in the four children who died (girls aged 6, 8, and 34 months; boy aged 8 months) were 1,200, 2,196, 2,280, and 2,500 cells/mul, all in the lowest quartile at study entry (306 to 2,873 cells/mul) for children with measles. However, the lymphocyte count on entry did not predict the disease severity as measured by days of hospitalization (rs = 0.013; P = 0.85; Spearman rank correlation test). As in control children (rs = -0.549; P < 0.0001), younger children with measles tended to have higher lymphocyte counts than older children at study entry (rs = -0.456; P < 0.0001) and higher numbers of CD4 T cells (rs = -0.418; P < 0.0001; Spearman rank correlation test for all comparisons). Lymphopenia was not prolonged in children <1 year of age (4,652 +- 316 cells/mul on entry [n = 85; P < 0.0001], 7,118 +- 489 cells/mul on discharge [n = 50], and 7,417 +- 580 cells/mul on follow-up [n = 40]) compared to age-matched controls at 7,323 +- 530 cells/mul [n = 24]; Mann-Whitney U test). Lymphocyte counts on entry were lower in children with evidence of malnutrition (wasted, 2,821 +- 317 cells/mul [n = 20]; not wasted, 3,869 +- 191 cells/mul [n = 181; P = 0.0668]) and were higher in children with stunted growth (stunted, 3,911 +- 233 cells/mul [n = 124]; not stunted, 3,184 +- 225 cells/mul [n = 99; P = 0.0209]; Mann-Whitney U test). Fever and the release of proinflammatory cytokines induced by infection can lead to rapid depletion of lymphocytes from the blood by increasing the rate of lymphocyte entry into secondary lymphoid tissue and reducing the rate of return to the circulation . To determine if fever played a role in lymphopenia during measles, patients from this and the larger cohort were stratified according to temperature at the time of study entry . The lymphocyte count was significantly lower in children with fever of >38.5C (2,782 +- 261 cells/mul) than in children with temperatures of <38.5C (3,766 +- 131 cells/mul; P = 0.007; Student's t test). The lymphocyte count did not correlate with plasma IFN-gamma levels on entry (rs = -0.16; P = 0.549; n = 15; Spearman rank correlation test). The absolute numbers of CD4 and CD8 T lymphocytes were determined by flow cytometry . The numbers of both CD4 T cells (1,013 +- 67 cells/mul) and CD8 T cells (782 +- 56 cells/mul) were depressed at study entry compared to controls (CD4, 2,007 +- 93 cells/mul [P < 0.0001]; CD8, 1,347 +- 73 cells/mul [P < 0.0001]). CD4 T-cell counts remained depressed at discharge (1,813 +- 155 cells/mul [P = 0.022]) but had returned to normal (2,117 +- 157 cells/mul) at the 1-month follow-up visit. These changes in CD4 and CD8 T-cell numbers led to lower CD4/CD8 ratios at study entry (1.492 +- 0.1; P = 0.0024), hospital discharge (1.243 +- 0.07; P < 0.0001), and follow-up (1.527 +- 0.11; P = 0.041) compared to controls (1.679 +- 0.072). Because CD8 T cells tended to increase more rapidly in boys than in girls, the CD4/CD8 ratios were significantly different by sex at discharge and at follow-up . Lymphocyte viability. | To determine whether lymphocytes from measles patients were more likely to undergo spontaneous cell death in culture, the viability of fresh PBMCs cultured without stimulation for 3 days was assessed . Fewer cells from measles patients at study entry than from control children were viable when assessed 1, 2, and 3 days after being placed in culture (P < 0.0001 for all comparisons). On day 3, viability was 74.7% +- 1.3% in cultures of cells from measles patients compared to 85.2% +- 0.9% in cultures of cells from control children. The tendency to undergo spontaneous cell death improved with time after acute measles, but differences from controls persisted at hospital discharge (day 3 viability, 78.7% +- 1.5%; P = 0.0007) and at the follow-up evaluation 1 month later (day 3 viability, 81.5% +- 1%; P = 0.0122). To determine whether stimulation of lymphocytes in vitro would alter this pattern of cell death, the viabilities of cells cultured in the presence of PHA were assessed . Cells from children with measles had lower day 3 viability (79.1% +- 1.2%) at study entry than cells obtained from control children (85% +- 0.7%; P = 0.0001). However, at later times during recovery there were no differences in the viabilities of PHA-stimulated cells obtained from children with measles and those obtained from control children. Surface binding of annexin V. | To determine whether lymphocyte death was apoptotic and whether cells were primed for apoptosis in vivo, PBMCs from patients with measles were stained simultaneously with annexin V and PI . Annexin V binds phosphatidyl serine residues exposed on the surfaces of apoptotic and necrotic cells, while PI enters only necrotic cells. The percentage of annexin V+/PI- cells was increased in cells isolated from measles patients at the time of study entry (28.7% +- 2.2%) and at hospital discharge (30.8% +- 3.1%) compared to cells obtained from control children (22.2% +- 2.2%; P = 0.0289 and P = 0.0134, respectively). Both CD4 and CD8 T cells from measles patients at the time of study entry bound annexin V , with a larger proportion of annexin V-positive CD8 T cells (17.7% +- 1.6%) than CD4 T cells (8.7% +- 1.4%; P = 0.0007) after overnight culture. Surface expression of Fas. | Surface expression of Fas (CD95), a member of the TNFR family, is often upregulated during T-cell activation and can render lymphocytes susceptible to apoptosis upon interaction with cells expressing FasL . There was a marked increase in the percentage of CD4 T cells expressing Fas at the time of study entry (25.9% +- 3%) in measles patients compared to control children (1.1% +- 0.16%; P < 0.0001), and this remained elevated at discharge (22.5% +- 3.4%; P < 0.0001) and 1 month later (8.2% +- 2.3%; P = 0.0012) . The percentage of CD4 T cells expressing Fas at study entry was inversely correlated with the viabilities of cultured PBMCs on days 1 (rs = -0.752; P = 0.0067) and (rs = -0.598; P = 0.0384; Spearman rank correlation test). There was no significant correlation of viability with Fas expression on CD4 T cells at discharge or follow-up (data not shown). A smaller percentage of CD8 T cells expressed Fas . This percentage was elevated in measles patients at the time of study entry (11.8% +- 1.9%) compared to control children (0.8% +- 0.3%; P < 0.0001) and at the time of discharge (11.5% +- 4.4%; P = 0.0004), but it had returned nearly to normal (1.95% +- 0.6%; P = 0.0358) by the 1-month follow-up visit. There was no correlation of lymphocyte viability in culture with Fas expression on CD8 T cells (data not shown). Levels of soluble cell surface proteins in plasma. | Soluble forms of Fas are produced by activated PBMCs and inhibit Fas-induced cell death . Plasma sFas levels were elevated (1,910 +- 132 ng/ml) in measles patients at the time of hospital discharge compared to control levels (1,342 +- 481; P = 0.0006) but did not differ significantly from that of controls at entry (1,463 +- 124) or at 1-month follow-up (1,491 +- 115) . sFasL is produced by proteolytic cleavage of FasL from the surfaces of activated FasL-expressing T cells . sFasL is postulated to suppress Fas-mediated death signals by competitive binding to Fas without inducing apoptosis . Children with measles had normal levels of sFasL in their plasma at study entry (379 +- 36 ng/ml). These levels rose by hospital discharge (432 +- 42; P = 0.0776) and were significantly increased at the 1-month follow-up visit (518 +- 0.047 ng/ml; P = 0.0048) compared to the levels in the plasma of control children (345 +- 36) . Soluble forms of CD4, CD8, and TNFRII are shed from the surfaces of activated lymphocytes, and levels of beta2 microglobulin are increased as a consequence of immune activation . Plasma levels of sCD8, sTNFRII, and beta2 microglobulin were elevated at entry and discharge compared to those in controls , and levels of sTNFRII and beta2 microglobulin remained elevated at follow-up. At entry, sCD8 levels were higher in boys (1,056 +- 599 U/ml; n = 45) than in girls (761 +- 468; n = 31; P = 0.0169; Mann-Whitney U test), while none of the other molecules measured had levels that were significantly different in boys and girls (data not shown). Effects of inhibitors of receptor-induced apoptosis on viability of cultured cells. | Several cell surface receptors in addition to Fas can induce cell death after ligand binding. To determine if blocking any of these receptor-ligand interactions would protect cultured lymphocytes from measles patients from spontaneous cell death, Fc IgG chimeras of TNF-related apoptosis-inducing ligand, Fas, and TNFRII and a FasL-blocking antibody were added to cultured PBMCs. Viability was determined after 3 days in culture. None of these inhibitors prevented the spontaneous death of PBMCs obtained from children with measles (data not shown). FIG. 1. | Effect of measles on numbers of leukocytes in the peripheral blood. Effect of measles on numbers of leukocytes in the peripheral blood. The mean absolute numbers of leukocytes (A), monocytes (B), lymphocytes (C), CD4 T cells (D), and CD8 T cells (E) and CD4/CD8 T-cell ratios (F) in children with measles at study entry, hospital discharge, and 1-month follow-up compared to controls are shown. The numbers of CD4 and CD8 T cells were calculated using the absolute lymphocyte count and percentages of CD3+ T cells coexpressing CD4 or CD8, as measured by flow cytometry. The number of children in each group is indicated at the base of each bar. The error bars indicate standard errors of the mean. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05 (Mann-Whitney U test). FIG. 2. | Association of fever with numbers of lymphocytes in circulation; mean lymphocyte counts on entry for children with measles stratified by presence or absence of fever of >38. Association of fever with numbers of lymphocytes in circulation; mean lymphocyte counts on entry for children with measles stratified by presence or absence of fever of >38.5C. The number of children in each group is indicated at the base of each bar. The error bars indicate standard errors of the mean. **, P = 0.007 (Student's t test). FIG. 3. | Viability of mononuclear cells in culture; mean percent viability of mononuclear cells isolated from patients with measles compared to controls after culture without stimulation (A) and with PHA stimulation (B). Viability of mononuclear cells in culture; mean percent viability of mononuclear cells isolated from patients with measles compared to controls after culture without stimulation (A) and with PHA stimulation (B). Mononuclear cells were cultured with or without 10 mug of PHA/ml. Viability was determined by trypan blue exclusion on days 1, 2, and 3 after initiation of cultures. The numbers of unstimulated samples assessed were 68 to 73 at entry, 35 to 47 at discharge, 42 to 46 at follow-up, and 54 to 71 for controls. The numbers of PHA-stimulated samples assessed were 53 to 57 at entry, 20 to 29 at discharge, 32 to 36 at follow-up, and 54 to 70 for controls. The error bars indicate the standard error of the mean. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05 (Mann-Whitney U test). FIG. 4. | Annexin V binding to lymphocytes. Annexin V binding to lymphocytes. Annexin V binding to the cell surfaces of PI-negative PBMCs (A) and of CD4-PE- or CD8-PE-stained T lymphocytes obtained at entry and cultured overnight (B) was measured by flow cytometry using a wide forward and side scatter gate. The number of samples in each group is indicated at the base of each bar. The error bars indicate the standard error of the mean. *, P < 0.05 (Mann-Whitney U test); ***, P < 0.001 (Wilcoxon rank test). FIG. 5. | Fas (CD95) expression on the surfaces of lymphocytes during measles. Fas (CD95) expression on the surfaces of lymphocytes during measles. (A and B) Mean percentages of CD4 (A) and CD8 (B) T cells from measles patients and control children with surface expression of Fas. Fas expression was determined by flow cytometry of freshly isolated CD4+ and CD8+ lymphocytes gated by forward and side scatter. The number of samples in each group is indicated at the base of each bar. The error bars indicate the standard error of the mean. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05 (Mann-Whitney U test). (C) Correlation of percent Fas-positive CD4 T cells at entry with lymphocyte death in culture. P = 0.0067 (Spearman rank correlation). FIG. 6. | Levels of soluble forms of cell surface proteins, sFas(A), sFasL (B), sTNFRII (C), sCD4 (D), and sCD8 (E), and beta2 microglobulin (F) in plasma of measles patients and controls as determined by enzyme or radio immunoassay. Levels of soluble forms of cell surface proteins, sFas(A), sFasL (B), sTNFRII (C), sCD4 (D), and sCD8 (E), and beta2 microglobulin (F) in plasma of measles patients and controls as determined by enzyme or radio immunoassay. The number of samples in each group is indicated at the base of each bar. The error bars indicate the standard error of the mean. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 (Mann-Whitney U test). TABLE 1 | Changes in numbers of lymphocytes, CD4 T cells, and CD8 T cells and in CD4/CD8 ratios in boys and girls with measles and controls PMID- 12204949_DISCUSSION TI - AB - During measles, the rash marks the onset of the virus-specific immune response, the initiation of virus clearance, and the appearance of a variety of immunologic abnormalities that often persist for weeks to months after recovery . In this study, we have evaluated the functional and phenotypic changes in lymphocytes from Zambian children with measles during acute infection and after recovery from infection. Lymphopenia occurred early and affected both CD4 and CD8 T cells. Cells in circulation were activated and susceptible to apoptosis as indicated by increased expression of Fas, binding of annexin-V, and spontaneous death in culture. During the recovery phase, lymphocyte counts normalized rapidly, expression of Fas decreased, sFas and sFasL levels in plasma became elevated, and mitogen stimulation protected cells from death in culture. Lymphocyte responses differed between boys and girls. In boys, lymphopenia was less marked, CD8 T cells in circulation increased more rapidly, and the sCD8 level was higher than in girls. Therefore, in children with measles, T lymphocytes undergo profound changes associated with altered peripheral circulation, activation, apoptosis, and susceptibility to death in culture. The differences in responses between boys and girls may be linked to sex-based differences in measles mortality and complications associated with measles immunization. Lymphopenia is a characteristic of many acute viral infections, including measles . Profound lymphopenia has previously been associated with severe measles and high mortality in African children , and the four children in our study who died during hospitalization had lymphocyte counts in the lowest quartile at study entry. More profound decreases in T-cell counts during acute measles have also been associated with malnutrition , and the acutely malnourished (wasted) children enrolled in our study had lymphocyte counts lower than those of children who were not wasted. Interestingly, stunted children had higher lymphocyte counts, but the reason for and importance of this observation are unclear. In our study, profound lymphopenia was also associated with female sex. Lymphocyte counts did not predict the severity of disease as measured by days of hospitalization; however, this parameter does not accurately measure the disease severity of those who died during hospitalization. Furthermore, some of the most severely ill children were not enrolled in the study, limiting our ability to detect these relationships. Lymphopenia was transient in this study, and counts normalized quickly as the fever and rash resolved during hospitalization. Although persistent lymphopenia has been associated with ages of <1 year in Japanese children , this was not seen in Zambian infants. Genetic factors may contribute to these differences. In addition, environmental factors, such as exposure to other infections, nutritional status, and prior immunization, may also play as yet undefined roles. The mechanism by which the numbers of lymphocytes in circulation are decreased during the acute phases of viral diseases is not clear. Although lymphopenia has been postulated to be due to MV-induced death of T cells , MV replicates preferentially in macrophages , and circulating lymphocytes represent only a small proportion of the total lymphocyte population. Therefore, alterations in lymphocyte trafficking are more likely to be responsible for profound, transient changes in the numbers of circulating lymphocytes. Increased exit of naive T cells from the circulation into secondary lymphoid tissue will improve the chances that lymphocytes capable of recognizing viral antigens will contact appropriate antigen-presenting cells . In macaques with measles, lymphocyte counts decrease during viremia and increase after the onset of the rash . One potential common mechanism for induction of altered trafficking is the effect of increased temperature. Fever can be associated with induction of inflammatory cytokines, increased adhesion of lymphocytes to lymph node vessels, and depletion of lymphocytes from the circulation . Fever is a universal feature of measles, typically beginning 2 to 3 days before the onset of the rash as a part of the prodrome , a time when lymphocyte counts are decreased . In our study, fever at the time of study entry was specifically associated with low lymphocyte counts. Lymphopenia during acute simian immunodeficiency virus infection of macaques has been attributed to alterations in lymphocyte trafficking associated with production of IFN-gamma . Although we did not find a correlation between plasma IFN-gamma and lymphocyte counts in measles, IFN-gamma levels are highest early during the rash and could contribute to altered trafficking and a reduction in the numbers of circulating lymphocytes. Thus, lymphopenia during measles is likely to involve increased trafficking of peripheral blood lymphocytes into secondary lymphoid tissue during the acute phase of the immune response to infection. The increasing numbers of lymphocytes in circulation during recovery probably correspond with the release of activated T cells from secondary lymphoid tissue for dissemination to sites of virus infection Our previous studies have shown that activated, proliferating CD4 and CD8 T cells are found in the circulation of measles patients after the onset of the rash , and the present studies show that Fas, another marker of lymphocyte activation , is also highest during the rash. Decreases in the numbers of both CD4 and CD8 T cells contributed to the lymphopenia, but CD4 T cells were more depressed for a longer time than CD8 T cells, resulting in decreased CD4/CD8 T-cell ratios at all time points. Late in infection, boys were the major contributors to the overall CD4/CD8 T-cell ratio suppression. A previous study of measles patients in Senegal also noted the tendency for girls to have higher percentages of CD4 T cells and higher CD4/CD8 T-cell ratios during convalescence . In our study, boys also had higher levels of sCD8 in plasma than girls, further suggesting greater stimulation and proliferation of CD8 T cells in response to infection. There is intense interest in understanding how girls and boys differ in their responses to measles and measles immunization. Studies from Guinea-Bissau and South Africa have observed increased mortality due to measles in girls , and three of the four deaths in our study were those of girls, reflecting the higher mortality in girls in the larger study . Studies of a high-titer measles vaccine showed long-term increased mortality in girls , and girls are more likely to develop fever and rash after routine measles-mumps-rubella immunization . Our data showing more profound lymphopenia and less evidence of CD8 T-cell activation in girls add to the accumulating evidence for an important effect of sex on responses to MV and suggest that the CD8 T-cell response may be an important aspect of the differences. The vigor and timeliness of the CD8 T-cell response is likely to be important for recovery from measles. Other studies have shown that circulating CD8 T cells have cytotoxic activity against MV-infected cells , and CD8 T cells are present at sites of MV replication and are presumed to play an important role in the clearance of MV from infected tissues . Lymphocytes were susceptible to cell death when placed in culture long after lymphocyte numbers in the blood had returned to normal, and in other studies this abnormality was present for up to 6 months after recovery . The propensity for lymphocytes to die in culture is assumed to reflect the in vivo process whereby virus-specific cells are eliminated after virus clearance . The death of activated T cells can be induced by at least two general pathways: an active antigen-dependent pathway that is mediated through death receptors, such as Fas and TNFR, and a passive, antigen-independent lymphokine withdrawal pathway regulated by members of the Bcl-2 family . During acute measles in our study, large percentages of lymphocytes were annexin V positive and PI negative, indicating apoptotic cell death. The process of deletion of virus-specific CD8 T cells is the best studied, involves multiple cell death-inducing mechanisms, and may be preceded by functional inactivation . Over 17% of CD8 T cells bound annexin, suggesting that these cells were apoptotic. Expansion of CD4 T cells is generally slower and downregulation is a much more prolonged process than for CD8 T cells. Therefore, during convalescence, virus-specific CD4 T cells may be more numerous than CD8 T cells. This pattern is consistent with our observations that indicators of CD8 T-cell activation, such as levels of sCD8 and IFN-gamma in plasma, are increased during the first 7 to 10 days after the onset of rash and then decrease, while evidence of CD4 T-cell activation persists (, , -, ). During measles, CD4 T cells were more likely to be Fas-positive at all time points than CD8 T cells, and 8% of circulating CD4 T cells were still expressing Fas a month after discharge. The activation-induced death of CD4 T cells is often mediated by FasL , but expression of Fas does not necessarily correlate with susceptibility to FasL-induced death. Lymphocyte death in culture was associated with Fas expression on CD4 T cells at study entry, suggesting that Fas may be involved in CD4 T-cell death early in infection. Although FasL antagonists did not block death, it is possible that apoptosis initiated in vivo is not influenced by attempts at blocking death stimuli in vitro. Increased circulating levels of sFas during hospitalization and of sFasL at follow-up suggest that this pathway is active in vivo. However, the mechanism for death in culture during recovery from measles may be different, since there was no association with Fas expression at later times. In previous studies of PBMCs from measles patients, stimulation with anti-CD3 enhanced or had no effect on death in culture, while stimulation with phorbol ester and ionomycin reduced lymphocyte death . In the present study, PHA stimulation partially protected cells from measles patients against death in culture. Lymphocyte viability was improved at the earliest time points and was completely restored at later times, again suggesting that more than one mechanism is involved in spontaneous death and that the mechanisms may change during the acute and recovery phases of measles. As in other viral infections (, , -, , , , , ), lymphokine deprivation may play a role in spontaneous death, since we have shown that lymphocytes from measles patients have increased expression of CD25, the alpha chain of the interleukin 2 (IL-2) receptor , and levels of IL-2 are increased in plasma for the first 2 weeks after the onset of the rash and then rapidly decline . IL-2 is particularly important for maintaining CD4 T cells and partially reverses the in vitro defect in mitogen-induced proliferation seen in measles . PHA-induced protection may occur by inducing production of growth factors or transcription factors that promote viability without inducing proliferation . In summary, we have shown that MV infection of hospitalized African children causes transient lymphopenia that involves CD4 and CD8 T cells and is associated with fever, malnutrition, and the female sex. During hospitalization and at the time of discharge, subpopulations of both CD4 and CD8 T cells are apoptotic, but other types of cell death, as well as alterations in lymphocyte trafficking, may contribute to lymphopenia. The magnitude of these changes differs between boys and girls, providing new insights into potential mechanisms of sex-based differences in the outcome of MV infection and immunization. PMID- 12204958 TI - Spontaneous Cytokine Production and Its Effect on Induced Production AB - | Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) by peripheral-blood B lymphocytes, T cells, CD8- T cells, CD8+ T cells, CD3- CD16/56+ lymphocytes (natural killer [NK] cells), CD3+ CD16/56+ lymphocytes (natural T [NT] cells), and/or monocytes of 316 acutely ill hospitalized persons and 62 healthy adults in Malawi, Africa. We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-alpha production occurred most frequently, followed in descending order by IFN-gamma, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-alpha; for 12 of these patients, TNF-alpha was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-alpha and IFN-gamma, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states. PMID- 12204958_Introduction TI - AB - Cytokines regulate cellular immune interactions and are produced by lymphocytes, monocytes, macrophages, and, for some cytokines, also fibroblasts, neutrophils, endothelial cells, or mast cells (for a review, see reference ). Cytokines function on a microenvironmental level, but human cytokines are most commonly assessed at the macro level by measuring their levels in serum or plasma or in the supernatant of in vitro-stimulated blood cells. In recent years, improved reagents have permitted flow cytometric cell-specific cytokine assessments in which peripheral blood mononuclear cells (PBMCs) are stimulated ex vivo in the presence of an agent to inhibit protein secretion. This stimulation is followed by cell permeabilization, fixation, fluorescent staining, and cytokine detection . With multiparameter flow cytometry, specific cell populations can be identified by surface antigen staining and examined for cytokine production without cell separation or cloning. Unstimulated cells are usually run in parallel, as a negative control and to assist in setting quadrants for assessing positivity, much as is done with an isotype control. Spontaneous ex vivo cytokine production by PBMCs has been reported not to occur in healthy individuals or in persons with various disorders, including allergic asthma , active and inactive systemic lupus erythematosus , and sarcoidosis , and those recovering from vascular surgery . We ourselves have rarely, if ever, noted spontaneous cytokine production in PBMCs of healthy U.S. blood donors (reference and unpublished data). However, in a multicenter, multiphase study of the immune correlates of bloodstream infections in developing countries , we noticed individuals whose PBMCs produced cytokines without stimulation. Other researchers, measuring either protein or mRNA, have detected spontaneous cytokine production by bronchoalveolar lavage fluid cells of patients with asthma , interacting umbilical vein endothelial cells-monocyte cells , human milk mononuclear cells , peripheral blood lymphocytes of persons with atopic dermatitis , peripheral blood monocytes of patients with multiple sclerosis , or CD4+ T cells of persons with active Mycobacterium tuberculosis pulmonary infection . The presence of spontaneous cytokine production ex vivo implies prior in vivo stimulation. Since the process of cell stimulation often leads to programmed cell death, in vivo stimulation, preceding in vitro stimulation, is not only of potential physiologic importance but may also be of practical importance in terms of interpreting the results of in vitro stimulation of cytokine production. At our study site in Malawi, Africa, we acquired a sufficient number of participants to examine spontaneous cytokine production in some depth. For this study, we defined spontaneous cytokine production in a simple, quantitative manner and then assessed the cell types spontaneously producing cytokines, the cytokines and cytokine arrays being produced, the relationships between spontaneous production and subsequent induced cytokine production, and the relationship between spontaneous production and serum cytokine levels. PMID- 12204958_MATERIALS AND METHODS TI - AB - Participants. | During three periods in 1997 and 1998, we enrolled all 561 febrile (oral temperature, >=38C) adults (>=13 years old) and all 244 acutely ill children (<13 years old) admitted to Lilongwe Central Hospital, Malawi, Africa, into a study of bloodstream infections . All hospitalized children were included because infected children often do not present with fever. A random subset including 166 adult and 150 pediatric patients had immune studies done at admission; this subset was comparable to the general study population and was the study group analyzed here. Demographic, epidemiological, and clinical data will be presented elsewhere. We also enrolled 62 asymptomatic, healthy, employed adults; children were not included in this group for ethical reasons. Epidemiological data were not obtained for these participants. The study protocol was approved by the institutional review boards of the Centers for Disease Control and Prevention (CDC) and the Malawian Health Sciences Research Committee; informed consent was obtained from all participants and/or their guardians. As in most developing nations, human immunodeficiency virus (HIV)-infected persons in this study were neither receiving antiviral therapies nor being monitored for changes in CD4+-cell counts or HIV RNA levels in plasma. Laboratory methods. (i) Microbiological. | HIV antibody testing was done at study enrollment, using enzyme-linked immunosorbent assay kits (Murex Diagnostics Inc., Norcross, Ga.). HIV type 2 has not been reported in Malawi. Whole-blood samples were cultured as described previously . (ii) Cytokine stimulation. | Heparinized blood was either stimulated for 5 h at 37C with phorbol 12-myristate 13-acetate (200 ng/ml) (Sigma Chemical Co., St. Louis, Mo.) and ionomycin (4 mug/ml) (Sigma) in the presence of brefeldin A (40 mug/ml) (Sigma) and RPMI 1640 with l-glutamine (induced, or stimulated, cytokine expression) or retained in identical medium without phorbol 12-myristate 13-acetate and ionomycin but with brefeldin A (spontaneous, or unstimulated, cytokine expression) . No serum was added to the cultures. After being washed, the red blood cells were lysed with ammonium chloride solution, and the lymphocytes were permeabilized and fixed using Permeafix (Ortho Diagnostic Systems, Inc., Raritan, N.J.). Cell counts before and after stimulation could not be done, and markers of apoptosis were not assessed; therefore, the issue of programmed cell death could not be addressed in our analyses. After being processed, samples were shipped at 4 to 8C to CDC for further analysis. (iii) Flow cytometric reagents. | The surface antigens assessed in this study were ones previously shown in our laboratory to be stable with this permeabilization-fixation protocol, i.e., using these techniques, we had comparable results for the surface-related antigens when staining was done either pre- or postpermeabilization. Fluorescein isothiocyanate (FITC)-conjugated, phycoerythrin (PE)-conjugated, peridinin chlorophyll protein-conjugated, or allophycocyanin (APC)-conjugated murine monoclonal antibodies were obtained from the following sources: (i) Becton Dickinson Immunocytometry Systems/PharMingen (BD/PMG; San Jose, Calif.) (CD8-FITC and -PE [clone SK1], CD3-peridinin chlorophyll protein and -APC [clone SK7], CD4-APC [clone SK3], CD45-FITC [clone 2D1], CD19-APC [clone SJ25C1], CD14-PE [clone MoP9], CD16-PE [clone B73.1], CD56 [clone MY31], interleukin 4 [IL-4]-PE [clone 8D4-8], IL-8-PE [clone G265-8], and IL-10-PE [clone JES3-9D7]), (ii) Research and Diagnostics (Minneapolis, Minn.) (IL-6-PE [clone 1927.311]), and (iii) Immune Source (Reno, Nev.) (CD8-APC [clone KL.12], IL-2-APC [clone R-56.2], tumor necrosis factor alpha [TNF-alpha]-FITC [clone DTX.34], and gamma interferon [IFN-gamma]-APC [clone 13.TR]). Isotype controls were obtained from BD/PMG. Stimulation was confirmed using anti-CD69-FITC [clone L78] (BD), and permeabilization was confirmed using anti-microtubulin [clone DM1A] (Sigma) custom conjugated to FITC by CalTag, South San Francisco, Calif. (iv) Flow cytometry. | All staining was done at room temperature for 30 min in the dark after permeabilization, fixation, and shipment to CDC. Staining was followed by a buffered saline wash. Four-color cytofluorometry was done using a single FACSort (1997 and 1998) or FACSCalibur (1998) cytometer and CellQuest software (BD/PMG). Between 50,000 and 80,000 ungated events were collected from each tube in the panel. Instrument settings were standardized each day using CaliBRITE Beads (BD) and chicken red blood cells (BioSure Controls; Riese Enterprises, Inc., Grass Valley, Calif.); these settings varied little within each phase of the study. (v) Serum cytokines. | Serum samples were analyzed for IL-2, IL-4, IL-6, IL-8, and IL-10 (all provided by BD/PMG) and for IFN-gamma and TNF-alpha (both provided by Genzyme Diagnostics, Cambridge, Mass.) by enzyme-linked immunosorbent assays using pairs of cytokine-specific monoclonal antibodies according to the manufacturers' instructions. Details are provided elsewhere . No healthy participants and few children had serum cytokines assessed (see Results). Analytic techniques. (i) Definition. | For the sake of simplicity and consistency, we defined spontaneous cytokine production as occurring if the percentage of a particular cell type producing a certain cytokine was above the 95th percentile for the entire pediatric or adult patient group . For example, 7 of 158 (<5%) of the adult patients had >1.7% of unstimulated CD3+ lymphocytes read as being positive for intracellular IL-2, and 6 of the 150 (<5%) pediatric patients had >2.2% of unstimulated CD3+ lymphocytes read as being positive for intracellular IL-2. These 13 individuals were classified as having CD3+ lymphocytes spontaneously making IL-2; the remaining patients were classified as having CD3+ lymphocytes that were not spontaneously making IL-2. The number of healthy participants producing cytokines spontaneously was fairly small; the results for the healthy participants will be provided separately from those of patients. (ii) Cells assessed. | For each participant, analyses were done for all lymphocytes and various combinations of CD3+ lymphocytes (T cells), CD3+ CD8+ lymphocytes, CD3+ CD8- lymphocytes, CD3+ CD16/56+ lymphocytes (natural T [NT] cells), CD3- CD16/56+ lymphocytes (natural killer [NK] cells), CD19+ (B) lymphocytes, and monocytes, depending upon the tube configuration . The tube configurations varied slightly among the three phases of the study . (iii) Flow analyses. | For consistency, all data in this study concerning spontaneous cytokine production were based on analyses of flow data by a single individual. Quadrants were based on both isotype controls and parallel unstimulated samples; these quadrants did not tend to vary between patients. An example of an instance of spontaneous cytokine production is provided in Fig. . Lymphocytes were defined on the basis of forward and side scatter; monocytes were defined on the basis of a wide gate based on forward and side scatter of stimulated and unstimulated CD14+ cells. (iv) Statistical techniques. | Comparisons of continuous variables between those with and without spontaneous cytokine production were made using Wilcoxon rank sum tests. Correlations were assessed using Pearson and Spearman correlation coefficients (rp and rs, respectively). The significance level was set at 0.05. FIG. 1. | Dot plot of unstimulated CD3+ lymphocytes of an individual spontaneously producing IL-6. Dot plot of unstimulated CD3+ lymphocytes of an individual spontaneously producing IL-6. FL1, anti-CD8-FITC; FL2, anti-IL-6-PE. TABLE 1 | Percentages of cells staining with monoclonal antibodies to various cytokines by cell and cytokine types and spontaneous cytokine production category PMID- 12204958_RESULTS TI - AB - Individual cytokines and cell types. | Spontaneous cytokine production was defined for seven cytokines and assessed in up to seven cell types . The numbers of patients with cell-specific spontaneous cytokine production according to our definition are provided in Table , as are the median and range of the percentage of each cell type producing each cytokine. The overlap in the ranges between those defined as being with and without spontaneous production is due to differences in the ranges for adult and pediatric patients and the differences in the age distributions of those with and without spontaneous cytokine production. Except in the case of IL-6 (see below), these age-associated differences tended to be slight and were not in any consistent direction (data not shown). Our definition of spontaneous cytokine production was chosen to be quite conservative, in that the lower limit for spontaneous production was in all cases well over 1% . The highest percentages of any cells spontaneously producing a cytokine were for IL-6; the majority of these instances were produced by the PBMCs of children, and the percentages themselves were higher in children (e.g., for CD3+ lymphocytes, the figures were as follows: median, 69%; range, 68 to 71%; n = 6 [children] versus 21 and 61%, n = 2 [adults]). The percentages of cells producing IL-6 with or without stimulation were strongly associated with serum transferrin receptor levels, an indicator of chronic iron deficiency . Using the definition of spontaneous cytokine production established for this study, a slightly higher proportion of B lymphocytes than T (CD3+) lymphocytes spontaneously produced IL-4. The percentages of CD8- and CD8+ T cells producing IL-6 were comparable to one another and higher than the percentages of monocytes producing IL-6 spontaneously. Immunoregulatory NT cells were proportionately highest in producing IFN-gamma and TNF-alpha spontaneously, followed distantly by NK cells and CD8+ T cells. Using the definition provided above, 33 of the 62 healthy persons had peripheral blood cells that spontaneously produced one (n = 27 persons), two (n = 5 persons), or three (n = 1 person) cytokines. The T cells of three healthy participants spontaneously produced IL-2, and those of three spontaneously produced IL-10. The B cells of one healthy person made IL-4. The CD8- T cells of two healthy persons made IL-8, and those of nine made IL-6. The CD8+ T cells of three healthy persons made IL-8, those of three made IFN-gamma, and those of nine made IL-6. The NK cells of three healthy persons made TNF-alpha, as did the NT cells of two healthy persons. Monocytes spontaneously produced IL-6 (one healthy person), IL-8 (six persons), IL-10 (two persons), or TNF-alpha (two persons). Thus, for these healthy persons, few made more than one cytokine spontaneously and IL-6, not the proinflammatory cytokine TNF-alpha, was the cytokine most commonly made. There was no predominant cell type making cytokines spontaneously, but immunoregulatory cells predominated in spontaneous TNF-alpha production. Cytokine and cell type constellations. | Unlike the healthy participants, most patients producing cytokines spontaneously produced more than one cytokine type . For both those spontaneously producing only one and those spontaneously producing more than one cytokine, the cytokine most frequently made was the proinflammatory cytokine TNF-alpha, followed by IFN-gamma. Production of a given cytokine by multiple cell types was frequent but did not occur in the majority of cases. For example, for IL-4, 4 patients had both T and B cells spontaneously producing the cytokine while a total of 18 patients had T cells and 17 had B cells spontaneously producing it. Similarly, three patients had both T cells and monocytes producing IL-6 and five had both CD8- and CD8+ T cells making IL-6. For IL-8, these values were four and six patients, respectively. Four patients had both CD8- and CD8+ T cells spontaneously producing IFN-gamma. The overlap was greatest between NT and NK cells, with fully half (nine patients) of those producing IFN-gamma spontaneously in NT or NK cells producing it in both these immunoregulatory cell populations. This was not the case for TNF-alpha, of which only two patients had spontaneous production in both NT and NK cells. Thirteen adult patients and nine children had two to eight combinations of cells spontaneously producing type 1 cytokines without any cell type spontaneously producing any type 2 cytokine. Conversely, five adult patients and seven children had two to five combinations of cells spontaneously producing type 2 cytokines without any cell type spontaneously producing any type 1 cytokine. Nineteen adult patients and 17 children had both one to six combinations of cells spontaneously producing type 1 cytokines and one to five combinations of cells spontaneously producing type 2 cytokines. Four adult patients and six children had two to six cell types spontaneously producing TNF-alpha and IL-8; two adult patients and four children had two or three cell types spontaneously producing IL-4 and/or IL-10. For healthy participants, the mix of cell types spontaneously producing a given cytokine was similar to that of the patients. For six persons, both the CD8+ and CD8- T cells spontaneously produced IL-6; for three persons each, only CD8- or CD8+ T cells spontaneously produced IL-6; and for one person, only monocytes spontaneously produced IL-6. For two persons, both CD8+ and CD8- T cells spontaneously produced IL-8; for one person, only CD8+ T cells spontaneously produced IL-8; and for six persons, only monocytes spontaneously produced IL-8. For three healthy persons, only T cells spontaneously made IL-10, and for two persons, only monocytes spontaneously made IL-10. For three healthy persons, only NK cells spontaneously made TNF-alpha; for one person, only NT cells spontaneously made TNF-alpha; for one person, only monocytes spontaneously made TNF-alpha; and for one person, monocytes and NT cells spontaneously made TNF-alpha. Of the six healthy persons producing more than one cytokine spontaneously, two made both IL-6 and TNF-alpha, one made IL-6 and IL-10, one made IL-8 and IL-10, one made IL-8 and TNF-alpha, and one made IL-4, IL-8, and IFN-gamma. Effects of spontaneous cytokine production on induced (stimulated) production by the same cell type. | For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production by a given cell type tended to be or was significantly associated with a higher percentage of the same cell type producing the same cytokine with stimulation, with the single exception of children's T-cell production of IL-4, for which there was a nonsignificant trend in the opposite direction (data not shown). Findings for IFN-gamma varied by cell type, with NK and NT responding in a pattern similar to those of other cytokines, CD3+ CD8- cells without an obvious effect, and the percentage of CD3+ CD8+ cells producing IFN-gamma with stimulation being lower in those for whom this cell type was spontaneously producing the cytokine . For TNF-alpha, the only significant effect was found in NK cells, again in a positive direction . These trends were present in both adult and pediatric patients (data not shown). The numbers of healthy participants making cytokines spontaneously were too small for statistical analyses, except in the cases of IL-6 production by T cells and IL-8 production by monocytes (see below). The trends were in the same direction found with the patients, except that spontaneous production of IL-10 by T cells did not affect induced production (data not shown). In healthy persons, as in the patients, the percentage of each cell type producing IL-6 with induction was significantly higher in those with spontaneous IL-6 production than in those without spontaneous production (medians for CD8- T cells, 9.3 versus 0.3% [P < 0.001], and for CD8+ T cells, 7.6 versus 1.2% [P < 0.001]). As with patients, the spontaneous production of IL-8 by monocytes did not have a significant effect on the percentages of monocytes producing IL-8 with induction (74.2 versus 58.3%; P = 0.398). Effects of spontaneous cytokine production on induced (stimulated) production by other cell types. | We next sought evidence of intercellular regulation of cytokine production by examining the relationships between spontaneous production of a cytokine by one cell type and the induced production of that same cytokine by another cell type. Few relationships were found; those for IL-4, IL-6, and IFN-gamma were in a positive direction . For two proinflammatory cytokines, IL-8 and TNF-alpha, in adults but not in children, the spontaneous production of a cytokine was associated with a lower percentage of another cell type producing that cytokine with stimulation. This was especially the case for TNF-alpha . Findings for adult and pediatric patients were similar to one another in regard to the other cytokines (data not shown). For healthy participants, the effects of spontaneous production of IL-6 on induced production by other cell types were similar to those for the patients. For example, for healthy persons, the median percentages of CD8- T cells and of monocytes making IL-6 with induction were higher for those whose CD8+ T cells made IL-6 spontaneously than for those whose CD8+ T cells did not make IL-6 spontaneously (7.4 versus 0.3% [P < 0.001] and 12.2 versus 1.0% [P < 0.001], respectively). Unlike in the patients, in these healthy persons, spontaneous production of IL-8 by monocytes did not affect the percentage of CD8+ T cells making induced IL-8 (medians, 1.7 versus 2.8%; P = 0.365). Relationships between the percentages of cells spontaneously producing cytokines and serum cytokine levels. | Only one patient (each) with spontaneous cell-specific IL-2 or IL-6 production had the matching serum cytokines assessed; each had detectable levels of the matching serum cytokine: IL-2, 17 pg/ml, and IL-6, 445 pg/ml. For the other cytokines, the numbers of patients with spontaneous cell-specific production and serum cytokine levels assessed were 9 for IL-4, 18 for IL-8, 11 for IL-10, 32 for IFN-gamma, and 34 for TNF-alpha. Of these, the numbers with detectable serum cytokines were 0 for IL-4, 13 for IL-8, 8 for IL-10, 19 for IFN-gamma, and 6 for TNF-alpha. No healthy participants and few children had serum cytokines assessed. No child with spontaneous cellular production had detectable levels of IL-4, IL-10, or TNF-alpha. All three children with spontaneous cellular IL-8 production also had detectable serum IL-8, and both children with spontaneous cellular IFN-gamma production had detectable serum IFN-gamma. Similarly, of 5 adult patients with spontaneous cellular IL-4 production, none had detectable serum IL-4; of 15 adult patients with cellular IL-8, 10 had detectable serum IL-8; of 30 adult patients with cellular IFN-gamma, 17 had detectable serum IFN-gamma; and of 29 adult patients with cellular TNF-alpha, only 6 had detectable serum TNF-alpha. We did correlation analyses between the percentages of various cell types spontaneously producing a given cytokine and the levels of that cytokine in the serum, with the caveat that the numbers (see above) were quite small. In analyses where undetectable serum cytokine levels were treated as being half the detection limit, correlations were significant between the percentage of monocytes spontaneously producing IL-8 and serum IL-8 levels (rs = +0.55; P = 0.018; n = 18), between the percentages of lymphocytes and of T cells spontaneously producing IFN-gamma and serum IFN-gamma levels (rs = +0.44, P = 0.014, n = 31 and rp = +0.36, P = 0.043, n = 32, respectively), and between the percentages of lymphocytes and of NT cells spontaneously producing TNF-alpha and serum TNF-alpha levels (rs = +0.37, P = 0.030, n = 34 and rs = +0.48, P = 0.004, n = 34, respectively). When those with undetectable levels were excluded from the analyses, correlations were significant between the percentage of lymphocytes spontaneously producing IFN-gamma and serum IFN-gamma levels (rs = +0.57; P = 0.014; n = 18) and between the percentages of lymphocytes spontaneously producing TNF-alpha and serum TNF-alpha levels (rs = +0.90; P = 0.015; n = 6). TABLE 2 | Numbers of patients spontaneously making >=1 cytokine TABLE 3 | Median percentages of cells expressing cytokine with stimulation, by presence or absence of spontaneous production of that cytokine TABLE 4 | Median percentages of cells producing cytokine with stimulation, by presence or absence of spontaneous production of that cytokine by a different cell type PMID- 12204958_DISCUSSION TI - AB - We report here that the peripheral blood cells of some hospitalized and some apparently healthy persons in Malawi produced cytokines ex vivo without additional stimulation . Spontaneous ex vivo cytokine production by peripheral blood cells rarely occurs in healthy U.S. or European individuals or even in those with a wide variety of chronic and/or systemic diseases, including allergic asthma, collagen vascular diseases, and sarcoidosis . The clinical and demographic characteristic of the Malawi patients spontaneously producing cytokines will be discussed elsewhere, but suffice it to say, these participants were not analogous to healthy U.S. blood donors. HIV, malaria, numerous intestinal parasites, and mycobacterial infections are all endemic in Malawi. In addition, by the enrollment criteria, the patients in this study were acutely ill. Since there is great interest in the roles of cytokines in acute and chronic infections, spontaneous cytokine production by the peripheral blood cells of persons within our study population warranted further investigation. We therefore examined data from these Malawi patients and the ostensibly healthy individuals to characterize this previously unstudied phenomenon and to assess its relationship to induced cell-specific cytokine production. By the criteria used, spontaneous cytokine production was not rare in this study group and often involved NK and NT cells, two immunoregulatory cell populations important in mycobacterial and HIV infections but only infrequently examined in clinical studies. TNF-alpha, a proinflammatory cytokine, was the cytokine most often produced spontaneously by patients, consistent with these patients having acute inflammation. The type 1 cytokine IFN-gamma was next in frequency, often being made by NK and NT cells, followed by the chemokine IL-8, which activates neutrophils and neutrophil adhesion to vascular endothelium. This order appears reasonable given the participants' acutely ill state and the frequency of HIV and mycobacterial infections in this population . Spontaneous production of type 2 cytokines was less frequent than that of the proinflammatory and type 1 cytokines, and a higher proportion of patients spontaneously produced the type 2 cytokine IL-4 than the type 2 cytokine IL-10. Production of a given cytokine by one cell type was not necessarily associated with production by other cell types; however, most patients who produced cytokines spontaneously produced more than one type of cytokine. For the healthy individuals, IL-6 was the cytokine most frequently produced spontaneously, and spontaneous production of a single cytokine was the rule, not the exception. Spontaneous cytokine production has both physiologic and practical implications. Cytokines affect infectious-disease morbidity and mortality and the interaction between various opportunistic infections and HIV. Presumably, spontaneous ex vivo cytokine production reflects in vivo stimulation and thus represents cell-specific activity in the context of a current infection(s) and/or physical condition and in the actual natural milieu of that cell. Consistent with this assumption, in previous assessments of this Malawian study group we found spontaneous IFN-gamma production by NT cells to be associated with mortality and the spontaneous production of a number of other cytokines by various cell types to be associated with malaria parasitemia , iron deficiency , or vitamin A deficiency . In nonhospitalized HIV-infected persons in the United States, we found significant declines in spontaneous TNF-alpha and IL-4 production by T cells to be associated with the acute response to highly active antiretroviral therapy . These previously published data support the potential physiological importance of measurable spontaneous cytokine production. However, we and many others have also reported significant relationships between induced cytokine production and a wide variety of illnesses and physiologic states. Thus, we were concerned about whether spontaneous cytokine production might obscure or alter the more commonly measured induced cytokine production. Ex vivo induction theoretically expands a stimulated population to measurable levels; however, human data to support the idea that this expanded population is representative of in vivo cell-specific cytokine profiles are sparse at best. As with measurements of serum cytokine levels, the underlying assumption is that peripheral-blood findings are of importance in systemic, or even localized, diseases. This is somewhat of an assumption in regard to both measures. We have previously shown that, in general, serum and cell-specific cytokines are at best only weakly associated with one another . Correlations between cell and serum cytokine parameters were not infrequently even negative, consistent with the hypothesis that serum and cell-specific cytokines measure different things . Interestingly, for the patients with spontaneous cytokine production discussed here, the correlations between serum and peripheral-blood cell-specific cytokines were all in a positive direction but, as with the more general population, generally with only weak correlation coefficients (this study). Theoretically, if cells have been stimulated by in vivo antigen exposure, secondary ex vivo induction with phorbol esters could lead to in vitro apoptosis and obscure the role of that cell-specific cytokine in the physiologic process. This would be analogous to measuring expression of variable beta-chain families of the T-cell receptor too long after superantigen stimulation . In this Malawi study, we found that in many instances, spontaneous cytokine production was not associated with a significant difference in induced production. When a relationship was found, it was usually in a positive direction, not a negative one, as would have been expected with apoptosis. IL-6 findings were especially interesting in this regard. They strongly followed the described pattern; however, for this cytokine, the cell percentages were actually higher for unstimulated than for induced cells (e.g., for patients' CD3+ CD8+ cells, the medians were 85.9 versus 53.7%), suggesting that some degree of activation-associated cell death might have been taking place but that cell division and cytokine induction were generally keeping in balance with the negative process. Further, this IL-6 finding strongly suggests that the cytokine was far more strongly induced by events occurring in vivo, e.g., iron deficiency , than by the stimulation provided in vitro. Finally, evidence of down modulation of induced cytokine production by one cell type in the presence of spontaneous production by a different cell type was found only for TNF-alpha (among T, NT, and NK cells) for adult patients . In conclusion, spontaneous cytokine production, albeit unusual in industrialized societies, can occur in some individuals and may not be that unusual in persons in developing countries. These findings strongly suggest that in certain study populations ---e.g., those living in developing countries, with acute illness, and/or with HIV infection ---spontaneous cell-specific cytokine measurements should be recorded in addition to the measurement of induced cell-specific cytokine production. One parameter does not negate the potential importance of the other; both may provide useful information concerning the roles of cytokines in various disease states. In light of the variable effects of spontaneous cytokine production on induced production, it may also be useful to calculate and assess the subtractive or proportionate difference between the percentage of cells producing a given cytokine without stimulation and the percentage producing that cytokine with stimulation. PMID- 12204968 TI - Modulation of Human Immunodeficiency Virus (HIV)-Specific Immune Response by Using Efavirenz, Nelfinavir, and Stavudine in a Rescue Therapy Regimen for HIV-Infected, Drug-Experienced Patients AB - | Analysis of the virologic and immunomodulatory effects of an association of efavirenz (EFV), nelfinavir (NFV), and stavudine (d4T) was performed in 18 human immunodeficiency virus (HIV)-infected and highly active antiretroviral therapy (HAART)-experienced patients who failed multiple therapeutic protocols. Patients (<500 CD4+ cells/mul; >10,000 HIV copies/ml) were nonnucleoside reverse transcriptase inhibitor (NNRTI)-naive and were treated for 10 months with EFV (600 mg/day) in association with NFV (750 mg three times daily) and d4T (30 or 40 mg twice daily). Measurement of HIV peptide- and mitogen-stimulated production of interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-4, and IL-10 as well as quantitation of mRNA for the same cytokines in unstimulated peripheral blood mononuclear cells were performed at baseline and 2 weeks (t1), 2 months (t2), and 10 months (t3) into therapy. The results showed that HIV-specific (but not mitogen-stimulated) IL-2 and IFN-gamma production was augmented and IL-10 production was reduced in patients who received EFV, NFV, and d4T. Therapy was also associated with a reduction in HIV RNA in plasma and an increase in CD4+ cell count. These changes occurred in the first year of therapy (t2 and t3) and were confirmed by quantitation of cytokine-specific mRNA. Therapy with EFV, NFV, and d4T increases HIV-specific type 1 cytokine production as well as CD4 counts and reduces plasma viremia. This therapeutic regimen may be considered for use in cases of advanced HIV infection. PMID- 12204968_Introduction TI - AB - The progression of human immunodeficiency virus (HIV) infection to AIDS is associated with increasing HIV viral load and qualitative and quantitative defects affecting CD4 T lymphocytes and cell-mediated immunity . Drugs designed as therapeutic tools against HIV infection should thus ideally be capable of stimulating cell-mediated immunity and reducing HIV viremia. Nevertheless, whereas highly active antiretroviral therapy (HAART) can achieve suppression of HIV viremia in HIV-infected individuals, the efficacy of these antiretroviral therapies in reconstituting immune function has been less dramatic . Nonnucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI), and protease inhibitors (PI) are currently used in the treatment of HIV-1 infection (reviewed in references , , , and ). Efavirenz (EFV) is an NNRTI which in combination with other antiretroviral drugs induces viral suppression as well as increases in CD4 counts. Treatment with EFV may be provided on a once-a-day, no-food-interaction schedule that makes its compliance higher than that of other drugs. Furthermore, although cross-resistance within the NNRTI class is extensive and the presence of point mutations (K103N, Y181C) may exclude the efficacy of the entire class, there is some evidence that EFV may retain full activity against more than 25% of Y181C-mutated viruses , even though an NNRTI sequential treatment is still not recommended by international guidelines . Because of these interesting pharmacokinetic properties we decided to evaluate the potential usefulness of this compound in advanced HIV infection. Therefore, we investigated immune and virologic parameters in patients undergoing therapy with an EFV-containing regimen. In particular, because progression to AIDS and loss of CD4+ T cells is associated with the impairment of type 1 cytokine production, we verified that EFV-containing antiviral therapies stimulated cell-mediated immunity and a type 1 cytokine profile. PMID- 12204968_MATERIALS AND METHODS TI - AB - Patients. | Eighteen HIV-infected patients were randomly selected among those enrolled in a study to evaluate the efficacy of an EFV-, nelfinavir (NFV)-, and stavudine (d4T)-based rescue therapy . The only selection bias was the patient's willingness to be included in the study. These patients had failed previous combination therapies (including multiple nucleoside analogues and at least one PI) and they (i) were failing the current PI-containing therapy, (ii) were naive for NNRTIs, (iii) had never received NFV, (iv) had no evidence of active HIV-related infections or neoplasms; and (v) had never been treated with immunomodulants (e.g., interleukin-2 [IL-2]). EFV was administered at a dose of 600 mg daily at bedtime, NFV was given at 750 mg every 8 h, and d4T was given at 30 or 40 mg, depending on body weight, twice daily. Blood sample collection. | Whole blood was collected by venipuncture immediately before treatment (t0) and 2 weeks (t1) as well as 2 months (t2) and 10 months (t3) after the start of therapy in EDTA Vacutainer tubes (Becton Dickinson & Co., Rutherford, N.J.). Peripheral blood mononuclear cells (PBMC) were separated on lymphocyte separation medium (Organon Teknika Corp., Durham, N.C.) and washed twice in phosphate-buffered saline, and the number of viable leukocytes was determined by trypan blue exclusion. PBMC were resuspended in RPMI 1640 (Sigma, St. Louis, Mo.) plus 2% AB serum (Sigma). All experiments were performed using cryopreserved PBMC. Antigen- and mitogen-stimulated cytokine production. | Gamma interferon (IFN-gamma), IL-2, IL-4, and IL-10 production by PBMC was determined by culturing 3 x 106 PBMC per well in 24-well LINBRO plates (Flow Laboratories, Inc.) (37C; 7% CO2). PBMC were unstimulated or stimulated with phytohemagglutinin (PHA) (M form; Sigma, St. Louis, Mo.) (final dilution, 1:100) for 48 h or a pool of five synthetic peptides from the gp160 envelope (Env) of HIV type 1 (HIV-1) (2.5 mM final concentration) for 5 days. The following peptides were utilized: T1 (KQIINMWQEVGKAMYA), HIV-1/IIIb Env amino acid residues 428 to 443; T2 (HEDIISLWDQSLK), HIV-1/IIIb Env amino acid residues 112 to 124; TH4.1 (DRVIEVVQGAYRAIR), HIV-1/IIIb Env amino acid residues 834 to 848; p18-IIIb (RIQRGPGRAFVTIGK), HIV-1/III IIIB Env amino acid residues 315 to 329; and p18-MN (RIHIGPGRAFYTTKN), HIV-1 (MN) Env region homologous to p18-IIIB. Cytokine production was evaluated in the supernatants with commercially available enzyme-linked immunosorbent assays (Endogen, Woburn, Mass.). RNA extraction. | RNA was extracted from unstimulated lymphocytes with the acid guanidium thiocyanate-phenol-chloroform method and dissolved in RNase-free water; purity was determined by spectrophotometry. Genomic DNA contamination was removed by RNase-free DNase (RQ1 DNase; Promega, Madison, Wis.). Reverse transcription (RT). | One microgram of RNA was reverse transcribed into first-strand cDNA in a 20-mul final volume containing 1 mM random hexanucleotide primers, 1 mM oligo(dT), and 200 U of Moloney murine leukemia virus reverse transcriptase (Clontech, Palo Alto, Calif.); cytokine mRNA expression was normalized for beta-actin cDNA content by semiquantitative PCR (Clontech). Quantification of cytokine cDNA by PCR. | PCR was performed in 50 mul of reaction mixture containing 10 mul of RT reaction mixture, 1x PCR buffer (20 mM Tris-HCl, 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5% Tween 20, 0.5% Nonidet P-40, 50% glycerol), 200 mM dNTPs, 1.25 U of Taq polymerase (Takara, Otsu, Japan), 0.4 mM beta-actin primers, and 0.4 mM (each) cytokines. The following primers were used: IL-4 5' primer, 5'CGGCAATTTGACCACGGACACCCGTGCATA3'; IL-4 3' primer, 5'ACGTACTCTGGTTGGCTTCCTTCACAGGACAG3'; IL-10 5' primer, 5'AAGCTGAGAACCAAGACCCAGACATCAAGGCG3'; IL-10 3' primer, 5'AGCTAT CCCAGAGCCCCAGATCCGATTTTGG3'; IFN-gamma 5' primer, 5'GCATCGTTTTGGGTTCTCTTGGCTGTTACTGC3'; IFN-gamma 3' primer, 5'CTCCTTTTTCGCTTCCCTGTTTTAGCTGCTGG3'; tumor necrosis factor alpha (TNF-alpha) 5' primer, 5'GAGTGACAAGCCTGTAGCCCATGTTGTAGCA3'; TNF-alpha 3' primer, 5'GCAATGATCCCAAAGTAGACCTGCCCAGACT3'; TNF-beta 5' primer, 5'ATGACACCACCTGAACGTCTCTTC3'; TNF-beta 3' primer, 5'GAAGGCTCCAAAGAAGACAGTACT3'. Thermal cycling was performed in a Touchdown Hybaid apparatus (Celbio, Milan, Italy) as follows: initial denaturation at 95C for 10 min; 35 cycles of denaturation at 95C for 30 s, annealing at 60C for 30 s, and extension at 72C for 30 s; and a final amplification step at 72C for 10 min. PCR products were electrophoresed in a 10% acrylamide gel and stained with 0.5 mg of ethidium bromide per ml; the size of cDNA products was determined by comparison to a DNA size marker, pbR322 (Sigma). The bands on the gels were scanned by transmission densitometry to quantify relative levels of gene expression, and the areas of the peaks were calculated in arbitrary units. To evaluate the relative levels of expression of the target genes in RT-PCR, the value of the internal standard (beta-actin) was used as the baseline gene expression of that sample, and the relative value was calculated for the target genes amplified in that reaction. Statistical analysis. | Statistical analysis was based on a nonparametric Jonckheere-Terpstra test for trends. Data were also analyzed by a nonparametric Kruskal-Wallis test; P values were two sided. PMID- 12204968_RESULTS TI - AB - Demographic profile of the study cohort. | The patients enrolled in the protocol (7 females and 11 males) had a mean age of 33.8 years. Four had a diagnosis of AIDS in their past medical history and eight had a CD4+ cell count below 200 cells/mul at the baseline. None of the patients had been immunized with candidate HIV vaccines; patients had previously received a mean of 3.6 NRTI and 2 PI. All of the patients were tested at the time points shown in the text. CD4+ cell counts progressively increased in all individuals after the initiation of therapy. Thus, mean (range) CD4 cell counts were as follows: t0, 212 (115-328) CD4 cells/mul; t1, 227 (131- 350) CD4 cells/mul; t2, 291 (174-444) CD4 cells/mul; t3, 399 (312-497) CD4 cells/mul. The change was significant (P = 0.011) when the baseline value was compared with the value observed after 10 months of therapy (t3). HIV genomic RNA was detectable in all patients at the baseline (mean, 109,217; range, 62,488-198,714 copies/mul) and decreased during therapy in all cases (t1 mean, 104,100; range, 58,227-196,515 copies/mul; t2 mean, 39,744; range, 21,057-74,480 copies/mul; t3 mean, 11,518; range, 499 to 15,511 copies/mul). HIV RNA at t3 was undetectable for 12 of 18 subjects. A trend toward a greater increase in CD4 counts was seen in the 12 patients who achieved suppression of plasma viremia below the limit of detection. Modulation of immune functions. | Env-stimulated production of IL-2 and IFN-gamma was augmented whereas IL-10 production was reduced in the patients. These results (means +- standard errors) are shown in Fig. . Thus, Env-stimulated IL-2 and IFN-gamma production was significantly increased 2 months after initiation of therapy (t2); the increase was still observed at t3 (P < 0.01 in all cases). IL-10 production was significantly diminished at the last time point (t3) (P < 0.01) . Mitogen-stimulated IL-2 production was marginally improved at t3; the production of both IFN-gamma and IL-10 upon mitogen stimulation was augmented, albeit not significantly, at t1 and t2 and returned to baseline by the end of the study period . Modulation of cytokine mRNA. | Cytokine-specific mRNA was quantified in resting PBMC of all patients at the beginning and after 10 months of therapy (cytokine mRNA could not be examined at t1 and t2 because of limitations in the number of available PBMC). Because TNF-alpha exerts a direct effect on viral replication and TNF-beta is suggested to be a mediator of apoptotic cell death in HIV infection, the expression of these two cytokines was measured as well. Results showed that after 10 months of therapy, (i) the expression of IFN-gamma was higher and (ii) the expression of IL-10, IL-4, TNF-alpha, and TNF-beta was lower. IL-2 was not detectable in resting PBMC (Fig. and Table ). FIG. 1. | (A) Env-stimulated cytokine production (IL-2, IFN-gamma, IL-4, and IL-10) by PBMC of HIV-infected individuals at different time points (before treatment [t0] and 2 weeks [t1], 2 months [t2], and 10 months [t3] into therapy). (A) Env-stimulated cytokine production (IL-2, IFN-gamma, IL-4, and IL-10) by PBMC of HIV-infected individuals at different time points (before treatment [t0] and 2 weeks [t1], 2 months [t2], and 10 months [t3] into therapy). The results are shown as means +- standard errors. (B) PHA-stimulated production of the same cytokines. *, values are significantly different from those at t0. FIG. 2. | Quantification of mRNA for IL-4, IL-10, IFN-gamma, TNF-alpha, and TNF-beta in resting PBMC of one representative HIV-infected individual before (t0) and after 10 months of (t3) therapy. Quantification of mRNA for IL-4, IL-10, IFN-gamma, TNF-alpha, and TNF-beta in resting PBMC of one representative HIV-infected individual before (t0) and after 10 months of (t3) therapy. The upper bands show cDNA retrotranscribed from cytokine mRNA extracted from patient PBMC; the lower bands show cDNA retrotranscribed from beta-actin mRNA extracted from patient PBMC and used to normalize the PCRs. TABLE 1 | Cytokine-specific mRNA in HIV-infected patients undergoing a rescue therapy PMID- 12204968_DISCUSSION TI - AB - The activities of an EFV-, NFV-, and d4T-based therapy on immunologic and virologic parameters were evaluated by analyzing the effects of this association in 18 randomly selected, HIV-infected, heavily pretreated individuals who failed previous therapeutic regimens . The combination was well tolerated, resulted in increased CD4 cell counts and reduced HIV plasma viremia, and at the same time induced a positive trend in HIV-specific type 1 cytokine production as well as a significant reduction in the generation of IL-10. To verify if the variations in cytokine production were secondary to modifications in their patterns of expression, the amount of cytokine-specific mRNA was analyzed in resting PBMC. The results showed that IFN-gamma mRNA was more abundant while IL-10 and IL-4 mRNAs were expressed less after 10 months of therapy. Because the generation of type 1 and type 2 cytokines is cross-regulatory, it is not surprising that during follow-up the decreased expression and production of IL-10 coincided with an augmented generation of IL-2 and IFN-gamma. Env-stimulated IL-2 and IFN-gamma production increased before down modulation of IL-10 production could be observed. A speculative hypothesis could be that the drug regimen studied here preferentially stimulates type 1 cytokine production and that the increase of these cytokines results in the subsequent cross-regulation of IL-10 production. TNF-alpha expression was reduced in this study. Because TNF-alpha directly stimulates viral replication , the modulation of this cytokine could contribute to the reduction of HIV viral load observed in our patients. CD4 cell counts progressively improved concomitantly with a reduction of TNF-beta expression in the study presented here. Because TNF-beta is known to be a mediator of apoptotic cell death in HIV infection it is tempting to speculate that increases in CD4 counts are at least partially secondary to a reduced susceptibility of CD4 cells to apoptosis. The analysis of the efficacy data relative to the entire cohort of patients showed that plasma viral load was reduced by 1.7 log at 12 months with a sustained increase in CD4+-T-cell count . HIV plasma viremia was undetectable in 12 of the 18 patients after 1 year of therapy. In light of recent data suggesting that HIV-specific T-cell function would not be recovered in patients with fully suppressed viral replication (reviewed in reference ), we compared Env-specific cytokine production in patients in whom viremia was or was not still detectable after therapy. We did not observe any correlation between residual viremia and improvement in immune function. This result can probably be explained considering the small number of individuals studied and the fact that our patients had advanced illness and had failed previous combination therapies. It is interesting to observe that modulation of HIV-specific immune responses was detected in our patients within a few months. These results contrast with recent data showing that improvement in HIV-specific immunity may not be seen in the first year of antiviral therapy . The discrepancy could be secondary to the fact that, whereas we measured Env-specific responses, Angel et al. and Blankson et al. stimulated PBMC with Gag or Pol. Numerous studies showed that HAART positively influences quantitative and qualitative immune parameters in both acute and chronic HIV infection, but the immune reconstitution that ensues nevertheless is never complete . Thus, triple therapy is associated with improvement in CD4 counts and functions that are more easily seen in early- than in late-stage patients (, , , , , , , , , , , ; M. Albrecht, D. Katzenstein, R. Bosch, and S. Liou, Abstr. 7th Conf. Retrovir. Opportunistic Infect., abstr. 531, 2000). Our data show that partial modulation of immune functions in chronic HIV infection can be seen even in patients who had previously failed multiple regimens. NNRTIs, in addition to NRTIs and PIs, have gained a definite place in the treatment of HIV-1 infection (reviewed in references , , , and ). EFV is an NNRTI which is used in combination with other antiviral compounds in the treatment of HIV infection and is shown to knock out virus replication and to delay virus resistance from arising (; Albrecht et al., 7th Conf. Retrovir. Opportunistic Infect.). Treatment guidelines were recently modified to include NNRTI as an acceptable substitute for a PI in primary therapy . Results stemming from a number of protocols in which this therapeutic approach was used in HIV-infected individuals confirmed that NNRTI-including regimens are capable of achieving a very consistent suppression of HIV plasma viremia. For the 006 study participants were randomized to receive EFV in association either with nucleoside analogues or with PIs. The results showed that suppression of plasma HIV-1 RNA to undetectable levels was achieved in a higher percentage of patients in the NNRTI plus nucleoside analogues group than in the PI plus nucleoside analogues group . In the ACTG 302 study NNRTI- and PI-naive patients who had previously failed an NRTI therapy were randomized to receive EFV, NFV, or both drugs in combination to 1 or 2 NRTIs. By week 40 the percentage of individuals with a viral load below the detection limit was significantly higher in the EFV- than in the NFV-based arms (Albrecht et al., 7th Conf. Retrovir. Opportunistic Infect.). Finally, recent data comparing the virologic effect of NNRTI- and PI-based antiretroviral combinations suggest that the use of NNRTI is associated with a faster and more frequent initial virologic response . In conclusion, our results suggest that NNRTI-employing antiviral therapies could be useful as a rescue regimen in advanced cases of HIV infection. Because these data are based on a small group of patients and because the design of this preliminary study did not involve a control arm, further studies will be necessary to clarify the best way to utilize this strategy in advanced HIV disease. PMID- 12204971 TI - Helicobacter pylori-Specific Immune Responses of Children: Implications for Future Vaccination Strategy AB - | We analyzed the specific anti-Helicobacter pylori immunoglobulin G (IgG) antibody profile for a sample of 824 asymptomatic schoolchildren in southern Germany (mean age, 10.7 +- 0.65 years) with an H. pylori-specific IgG enzyme-linked immunosorbent assay and Western blot analysis. The prevalence of infection was 19.8% (95% confidence interval, 17.1 to 22.7%). The immunoresponses were characterized predominantly by antibodies against low-molecular-mass antigens of 14 and 29 kDa, with a significant difference between children of German and Turkish nationalities (P = 0.0012 and P < 0.0001, respectively). PMID- 12204971_Introduction TI - AB - Helicobacter pylori plays an etiologic role in gastritis, peptic ulcer disease, gastric cancer, and mucosa-associated lymphoid tissue lymphoma , and acquisition of the infection seems to occur mainly in the first years of life . Genetic typing has shown that H. pylori is extremely diverse as a species and possesses a number of antigens that induce a specific immune response in infected individuals . The specific immune response is part of the natural history of the infection in children, which remains poorly characterized, and may mirror the specific interaction of the host with the bacterium (, , , 21). This immune response may be of special importance with regard to vaccination, as it may determine the antigens considered for a vaccine. However, the few existing studies of the infection in children are based mainly on symptomatic and small clinical samples . The aim of this study was to analyze the specific anti-H. pylori immunoglobulin G (IgG) antibody profile in a sample of asymptomatic schoolchildren. This study is part of a population-based investigation performed by the Baden-Wurttemberg State Health Office between October 1999 and April 2000 with a sample of schoolchildren (fourth graders; mean age +- standard deviation, 10.7 +- 0.65 years; range, 9 to 13 years; median, 11 years) living in southern Germany in four different areas of Baden-Wurttemberg (Stuttgart, Mannheim, Kehl, and Ravensburg). Participation in the study was voluntary. The study was approved by the Ethics Board of the University of Ulm. The parents of 1,118 children filled out a questionnaire which requested information regarding sociodemographic factors, family characteristics, and medical history. Serum samples from 824 (73.7%) of those 1,118 children were available. H. pylori IgG antibodies were determined by enzyme-linked immunosorbent assay (GAP; Bio-Rad Laboratories Diagnostics Group, Munich, Germany). According to the manufacturer's instructions, levels of >20 U/ml were considered positive. This test has previously been evaluated with 310 children (compared with the histology, culture, and urease test, the sensitivity of our test was 94.9% and the specificity was 92.4%) . The serum samples of the IgG-positive children were further analyzed for the presence of antibodies against H. pylori-specific antigens with a commercial Western blot method (Helicobacter pylori Western blot; AID, Strasberg, Germany) as described previously . Of the 824 children included in the study, 53.3% were boys. About two-thirds, or 63.5%, of the schoolchildren were of German nationality, 12.5% were Turkish, and 12.7% were of other nationalities (for 89 of the children (10.8%), there were no data available concerning nationality). Information gathered on family characteristics showed that 10.2% of the children had no siblings, 43.3% had one, and 38.1% had two siblings; 45.5% of the fathers and 37.5% of the mothers had 9 or fewer years of school education. One hundred sixty-three children (19.8% of the total number, 95% confidence interval [CI], 17.1 to 22.7%) had positive serology results for H. pylori . The seroprevalence in children of German nationality was 13.9% (95% CI, 11.0 to 17.1%) and varied from 12.4% (95% CI, 9.6 to 15.6%) for the German children born in Germany to 42.3% (95% CI, 23.4 to 63.1%) for the German children born elsewhere (mostly children of families who had lived in countries of Eastern Europe for generations and who had returned to Germany quite recently). The prevalence of H. pylori in children of Turkish nationality was 37.9% (95% CI, 28.5 to 48.0%) (in Turkish children, place of birth was not associated with seroprevalence), and for children of other nationalities, it was 30.5% (95% CI, 21.9 to 40.2%). These strong differences among ethnic groups were independent of socioeconomic factors, such as family size and school education of parents, and persisted after these factors had been controlled for. On the other hand, duration of school education of parents and number of siblings were not factors associated with seroprevalence if ethnic origin was considered in the analysis (data not shown). TABLE 1 | Prevalence of H. pylori infection in children according to nationality Western blots were used to detect and analyze eight different H. pylori-specific immunoreactive bands . TABLE 2 | Prevalence of specific H. pylori IgG antibodies in H. pylori-infected children (n = 163) Table shows that antibodies to the 25-kDa antigen and the 14-kDa antigen were detected much more frequently in infected children of Turkish or other nationality than in infected children of German nationality (P values of 0.0012 and <0.0001, respectively). TABLE 3 | Prevalence of H. pylori-specific antibodies identified by immunoblotting among infected children according to nationality of the children (n = 144) This study, conducted with a population-based sample of asymptomatic schoolchildren in Germany, demonstrated that the specific systemic immune response in children to H. pylori infection is particularly pronounced against low-molecular-weight proteins of H. pylori. Furthermore, there was a large variation in both the prevalence of infection and the specific antibody patterns with respect to the nationalities of the children, although the children were living in the same geographic region. As shown in a previous study, differences between ethnic groups are not explained by differences in socioeconomic factors and most likely mirror strain differences among these groups. A recent French study of 80 pediatric patients found the highest prevalence (88.7%) for antibodies to a 26-kDa antigen . Little is known about the function of the 26-kDa antigen, which may be identical to the 25-kDa antigen in our study. However, recently it has been shown that a 25-kDa outer membrane protein acts in a lectin-like manner with lipopolysaccharide to mediate attachment of H. pylori to laminin and may therefore be important for the colonization process in the early phase of the infection . Much interest exists in the seroprevalence in children of CagA antibodies, which, according to a study by Mitchell et al. , correlated with age and increased up to the age of 15 years. There are few data available for children, and the results of recent studies have been inconsistent concerning the prevalence and association with disease of CagA-positive strains in both adults and children (, 6, , , , , ). This may be due partly to the considerable variation in the frequencies of vacAsl subtypes and iceA alleles in children originating from different geographic regions . As for adults, a high prevalence of CagA-positive H. pylori strains was found in Japanese children; however, there was no association of this prevalence with nodular gastritis or peptic ulcer disease in Japanese children (mean age, 12 +- 3 years). This result challenges the role of CagA as a marker for more serious gastroduodenal disease in children as well as in adults in Japan . The highest prevalence of CagA antibodies in Europe (80%), has been found in a study of infected children in Poland ; the prevalence in infected schoolchildren in Estonia (9 to 15 years old) was 46% . Raymond et al. described the prevalence of CagA antibodies in children in France who had abdominal pain to be 43% . In conclusion, the differences in the antibody patterns between children of various ethnic groups or found in our study may indicate strain differences and should be considered if antigens are chosen as targets for a vaccination approach. PMID- 12204963 TI - Cytokine Gene Expression by Peripheral Blood Leukocytes in Horses Experimentally Infected with Anaplasma phagocytophila AB - | Human granulocytic ehrlichiosis (HGE), a tick-borne zoonosis, is caused by an obligatory intragranulocytic bacterium, the HGE agent, a strain of Anaplasma phagocytophila. The equine model of HGE is considered valuable in understanding pathogenic and immune mechanisms of HGE. In the present study, cytokine mRNA expression by peripheral blood leukocytes (PBLs) in horses was examined during the course of infection by intravenous inoculation of A. phagocytophila or by allowing feeding by infected ticks. The p44 genes encoding the major outer membrane protein P44s of A. phagocytophila were detected by PCR in PBLs of all four horses from 4 to 20 days postexposure. During the 20-day infection period, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) mRNA expression was upregulated in PBLs of all four horses, and IL-8 mRNA expression was upregulated in three horses. Gamma interferon, IL-10, and IL-12 p35 mRNAs were weakly expressed in only one horse each. IL-2, IL-4, IL-6, and IL-12 p40 mRNA expression , however, could not be detected in the PBLs of any of the four horses. These results suggest that IL-1beta, TNF-alpha, and IL-8 generation during A. phagocytophila infection has a primary role in HGE pathogenesis and immunomodulation. PMID- 12204963_Introduction TI - AB - Human granulocytic ehrlichiosis (HGE) is characterized by fever, chills, headache, myalgia, and laboratory findings including leukopenia, anemia, and thrombocytopenia, as well as elevated liver enzyme activities . Delayed treatment, misdiagnosis, and/or the presence of immunosuppression may lead to a severe or fatal outcome . HGE has been increasingly recognized in the United States and various parts of Europe . HGE is caused by a strain of Anaplasma phagocytophila (the HGE agent), a gram-negative obligatory intragranulocytic bacterium. The white-footed mouse (Peromyscus leucopus) is considered to be a major wild-animal reservoir of A. phagocytophila in the northeastern United States, and Ixodes spp. ticks, the vector of Borrelia burgdorferi, the agent of Lyme disease, are considered to be the vector . Strains of A. phagocytophila have also been known to cause tick-borne fever in ruminants in Europe and to cause equine ehrlichiosis in the United States (it has hence formerly been called Ehrlichia phagocytophila and Ehrlichia equi). Pathogenesis and cellular immune responses of HGE are not well defined. The presence of low levels of A. phagocytophila in the blood of patients in the acute stage and autopsied patients' tissues and the nature of clinical signs and laboratory findings suggest the involvement of proinflammatory cytokines and chemokines in the pathogenesis. In vitro, we found that A. phagocytophila strain HZ, isolated from an acute-stage HGE patient, induced rapid and strong proinflammatory cytokine (interleukin-1beta [IL-1beta], tumor necrosis factor alpha [TNF-alpha], and IL-6) mRNA expression by human peripheral blood leukocytes (PBLs) and monocytes within 2 h and protein secretion within 24 h . However, only IL-1beta is upregulated in neutrophils, and the mRNA of these three cytokines is not upregulated in HL-60 cells. Within 2 h of incubation with A. phagocytophila, IL-8, IL-10, gamma interferon (IFN-gamma), IL-2, and transforming growth factor beta mRNAs are not consistently upregulated in human PBLs, suggesting that for these cytokines, gene expression either is not induced or is induced at later time points in vitro. Akkoyunlu et al. reported IL-8 production by retinoic acid-treated HL-60 cells (human promyelocytic leukemia cell line) after 24 h (negative at 12 h for both mRNA and protein) and by human neutrophils 7 h postincubation with A. phagocytophila in vitro. That report also indicated that no IL-1alpha, IL-1beta, or TNF-alpha is detected in the culture supernatant of retinoic acid-treated and nontreated HL-60 cells at 6 days after the addition of A. phagocytophila. Klein et al. reported that in infected dimethyl sulfoxide-treated HL-60 cells or human bone marrow cells, IL-8 and other chemokine levels, but not that of IL-1, IL-6, or TNF-alpha in the culture medium, were significantly elevated at 48 h postinfection . IL-8 levels are significantly increased in the blood of HGE patients . Dumler et al. reported that IFN-gamma and IL-10 levels are elevated in acute-phase (median, 4 days after onset) sera compared with convalescent (median, 31 days after onset) sera from HGE patients or normal controls whereas serum IL-1beta, TNF-alpha, and IL-4 levels are not elevated . Using C3H mice, which do not show clinical signs and spontaneously clear the infection by 15 days after intraperitoneal inoculation of A. phagocytophila, splenic IL-12 and IFN-gamma mRNA and serum IFN-gamma levels are significantly elevated starting at 2 days postinfection, although the clearance of A. phagocytophila is not delayed in IFN-gamma-/- mice . Considering apparently conflicting data in vitro using cell lines, bone marrow cells, or human PBLs which were assayed at varied time points of incubation or in vivo using a transient mouse infection model or patients' sera, an equine model of HGE may be useful for clarifying the importance of these cytokines in HGE. Several reports have suggested that the horse serves as a more useful infection and disease model of HGE than the mouse, since A. phagocytophila isolated from patients induces clinical signs in horses similar to those of HGE and equine ehrlichiosis . However, cytokine responses in horses infected with A. phagocytophila have not been described so far. In the present study, we investigated cytokine mRNA expression by PBLs in horses following transmission of A. phagocytophila by intravenous inoculation or tick bite. PMID- 12204963_MATERIALS AND METHODS TI - AB - A. phagocytophila. | HZ strain A. phagocytophila, isolated from an HGE patient , was propagated in HL-60 cells as previously described . The percentage of infected cells was determined by Diff-Quik (Baxter Scientific Products, Obetz, Ohio) staining as previously described . Horse infection. | Four horses purchased from Ohio horse farms were kept in vector-proof stalls in the Equine Isolation Unit (College of Veterinary Medicine, The Ohio State University). All horses were mixed breed. Horse 1 was a 2-year-old male (380-kg body weight [BW]), horse 2 was a 5-year-old male (200-kg BW), horse 3 was a 5-year-old male (180-kg BW), and horse 4 was a 3-year-old female (170-kg BW). Prior to A. phagocytophila infection, these animals were confirmed as seronegative and PCR negative for A. phagocytophila. Horses 1 and 2 were intravenously (i.v.) inoculated with A. phagocytophila of high passage (HP; i.e., more than 50 passages in HL-60 cells) and low passage (LP; i.e., fewer than 10 passages), respectively, at 107 infected HL-60 cells (approximately 80% infected cells) in 5 ml of RPMI 1640 medium. A total of 100 and a total of 47 infected Ixodes scapularis adults were allowed to feed on horses 3 and 4, respectively. As nymphs, these ticks were acquisition fed on experimentally infected DBA/2 mice . Uninfected nymphs were kindly provided by S. R. Telford (Harvard School of Public Health, Boston, Mass.). All horses were euthanized at 24 days postinoculation (PI) or postattachment (PA). The use of horses for this study has been approved by the Ohio State University Institutional Animal Care and Use Committee. Clinical evaluation, hematology, PBL preparation, morula examination, and indirect fluorescent antibody (IFA) testing. | Fever, depression, anorexia, diarrhea, leukopenia, laminitis, and other clinical signs were monitored daily for 20 days. For complete blood cell counts, blood samples were taken from the jugular veins of horses prior to injection or tick attachment (day 0) and every 4 days until 20 days PI or PA. To prepare the PBL fraction, the blood samples, which were collected in acid citrate dextrose anticoagulant tubes, were centrifuged at 500 x g for 5 min. Erythrocytes in the pellet were lysed in sterile 0.83% NH4Cl solution for 3 min at room temperature. Cells were washed twice by centrifugation (500 x g for 5 min) in phosphate-buffered saline (137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4 [pH 7.2]). Viabilities of PBLs were >98%, as assessed by the trypan blue dye exclusion test. Intracytoplasmic microcolonies (morulae) of A. phagocytophila were examined in PBLs by using Diff-Quik staining and IFA using monoclonal antibody (MAb) 5C11 . The IFA test was performed as previously described . The serum antibody titer was expressed as the reciprocal of the highest dilution of serum that showed a positive reaction. Reverse transcription (RT)-PCR for cytokine genes. | Total RNA was extracted from equine PBLs (107) by TRIzol reagent (GIBCO-BRL) and resuspended in 90 mul of diethyl pyrocarbonate-treated sterile water. As a positive control, PBLs (107) of uninfected healthy horses were stimulated with either concanavalin A (ConA; Sigma, St. Louis, Mo.) (10 mug/ml) or Escherichia coli lipopolysaccharide (LPS) (10 mug/ml) in RPMI 1640 medium at 37C for 4 h. Total RNA (2 mug) was reverse transcribed in a 30-mul reaction mixture containing 1x reaction buffer (50 mM Tris-HCl [pH 8.3], 75 mM KCl, 3 mM MgCl2), 0.5 mM each deoxynucleoside triphosphate (dNTP), 1 U of RNase inhibitor (GIBCO-BRL), 1.5 muM oligo(dT) primer, and 10 U of Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL) at 42C for 1 h. The reaction was terminated by heating at 94C for 5 min, and the cDNA was used in the PCR. The cDNA (2 mul) was amplified in a 50-mul reaction mixture containing 1x PCR buffer (10 mM Tris-HCl [pH 8.3] and 50 mM KCl), 1.5 mM MgCl2, 0.2 mM each dNTP, and 0.4 muM (each) 3' and 5' primers (0.5 muM each primer for equine IL-8 mRNA). The primers used for equine IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12 p35/40, TNF-alpha, IFN-gamma, and beta-actin mRNA detection were as previously described . Primer sequences for equine IL-8 mRNA expression, designed based on the equine cDNA sequence (GenBank accession number ), were kindly provided by S. Giguere (Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida). The sequences were as follows: sense, 5'-GACTTCCAAGCTGGCTGTTGC-3', and antisense, 5'-GTCCTCTTTAGAAACGCCTGC-3'. To reduce nonspecific priming, all PCRs were performed by the hot-start method: Taq DNA polymerase (2 U/reaction; GIBCO-BRL) was added after incubation of the mixture at 94C for 5 min. PCR was 25 cycles (27 cycles for equine IL-8), consisting of denaturation at 94C for 45 s, annealing at 60C (62C for IL-8) for 45 s, and extension at 72C for 2 min. The final extension was for 7 min. These conditions were within the linear range for PCR, as determined previously . PCR products (10 mul each) were electrophoresed in 1.5% agarose gel containing ethidium bromide (final concentration, 0.5 mug/ml) at 95 V for 1 h and photographed under UV illumination with a gel video system (Gel Print 2000i; Biophotonics Corporation, Ann Arbor, Mich.). DNA size markers (HaeIII fragments of phiX174 replicative-form [RF] DNA; GIBCO-BRL) providing bands from 1,353 to 72 bp were run in parallel. PCR of p44 genes of A. phagocytophila. | To detect A. phagocytophila DNA in equine PBLs, nested PCR, based on the multigene family p44 genes encoding immunodominant outer membrane protein P44s , was performed. The two primer pairs p3726 (5'-GCTAAGGAATTAGCTTATGA-3')-p4257 (5'-AGAAGATCATAACAAGCATTG-3') and p3761 (5'-CTGCTCKGCCAARACCTC-3')-p4183 (5'-CAATAGTYTTAGCTAGTAACC-3') (K = T+G; R = A+G; Y = C+T [mixed bases]) that are located in conserved regions of p44 genes (Q. Lin, N. Zhi, H.-Y. Kim, H. Horowitz, G. Wormser, and Y. Rikihisa, 101st Gen. Meet. Am. Soc. Microbiol., abstr. D-204, p. 319, 2001) were used. The first and second PCR product sizes were 531 and 422 bp, respectively. The first PCR was performed with 3 min of denaturation at 94C followed by 27 cycles consisting of 1 min each of denaturation at 94C, annealing at 55C, and extension at 72C. In the first cycle, each 50-mul PCR mixture contained 2 mug of RNA, 5 mul of 10x reaction buffer, 0.2 mM each dNTP, 1.5 mM MgCl2, 1.25 U of Taq polymerase, and 25 pmol of each primer of the primer pair p3726-p4257. The second PCR was performed the same as the first PCR, except that the annealing temperature was 52C, 1 mul of the first PCR product was used as template, and 10 pmol of each primer of the primer pair p3761-p4183 was used. PMID- 12204963_RESULTS TI - AB - Clinical signs and hematology. | Horse 1, which had been inoculated i.v. with HP A. phagocytophila, developed fever (body temperature > 38.9C; normal range, 37.5 to 38.6C ) during days 5 to 8 and 15 to 18 PI and thrombocytopenia (<1.1 x 1011 platelets/liter; normal value for total equine platelets, [2.2 +- 0.93] x 1011/liter ) during days 4 to 8 PI. Horse 2, inoculated i.v. with LP A. phagocytophila, developed fever only at day 8 and thrombocytopenia (1.1 x 1011 platelets/liter) and slight neutropenia (1.5 x 109 leukocytes/liter; normal value, [4.4 +- 2.0] x 109/liter ) on day 16. Horses 3 and 4 did not show any significant clinical signs. Total red blood cell counts (5.9 x 1012 to 13.9 x 1012 cells/liter; normal value, [7.5 +- 2.5] x 1012/liter ) of all four horses were within the normal range during the 20 days PI. However, at 8 days PI, variable sizes (anisocytosis) and abnormal shapes (poikilocytosis) were detected in less than 1% of the red blood cells in all 4 horses. Leukocyte levels (5.1 x 109 to 16.7 x 109/liter; normal value, [7.6 +- 3.0] x 109/liter ) and differential cell count levels (1.0 x 109 to 7.9 x 109 lymphocytes/liter [normal value, (3.0 +- 1.9) x 109/liter] and 0.1 x 109 to 0.8 x 109 monocytes/liter [normal value, (0.25 +- 0.25) x 109/liter] ) were within normal ranges in all four horses. The reactive lymphocytes (less than 1%) were observed for horse 1 (days 8, 12, 16, and 20), for horse 2 (day 16), for horse 3 (days 16 and 20), and for horse 4 (every day examined). Slight eosinophilia was observed during days 12 to 16 in horse 3 (0.4 x 109 to 0.5 x 109 eosinophils/liter; normal value, [0.15 +- 0.15] x 109/liter ). In horses 1 and 2, which were inoculated i.v., examination of PBLs by using Diff-Quik staining or IFA staining with MAb 5C11 revealed the presence of A. phagocytophila in neutrophils during days 4 to 12 PI (horse 1, 1.0% of neutrophils infected on day 4, 4.9% infected on day 8, and 2.2% infected on day 12; horse 2, 1% of neutrophils infected on day 4 and 3% infected during days 8 to 16) . Morulae were not detected in the granulocytes of horses 3 and 4 throughout 20 days. Detection of p44 genes of A. phagocytophila. | The presence of immunodominant major outer membrane protein P44s has been demonstrated in all strains of A. phagocytophila so far examined . Since P44 proteins are encoded by a multigene family, we have developed a highly sensitive p44-based nested-PCR method for HGE diagnosis (Lin et al., abstr. 101st Gen. Meet. Am. Soc. Microbiol., 2001). beta-Actin DNA served as a control for the amount of horse PBL DNA across the samples. In all four horses, p44 genes were detected during days 4 to 20 PI , indicating that ehrlichemia was established in all four horses. Although the nested PCR used was not quantitative, lack of progressive increase in band intensities following the day of infection in all four horses suggests an immune mechanism to contain the infection. IFA titers. | In the two horses inoculated i.v. with HP and LP A. phagocytophila, respectively, similar IFA immunoglobulin G (IgG) titers were observed . In horses 1 and 2, seroconversions (>1:20) were detected from day 8 and day 4 PI, respectively. The maximum IgG titers of the 2 horses were 1:320 on days 12 and 16 PI, respectively. The IgG titers in horse 3, which had been infected by attaching infected ticks, were less than 1:20 throughout 20 days, and horse 4 showed a peak IgG titer of 1:160 on day 12 PA . Equine cytokine mRNA expression. | Expression of several equine mRNAs (IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p35/40, TNF-alpha, IFN-gamma, and beta-actin) by PBLs in the four horses infected with A. phagocytophila by i.v. inoculation or by attaching infected ticks was measured following the time course of infection by RT-PCR at a linear range of 25 or 27 PCR cycles. Contamination of RNA preparations with the genomic DNA was negligible, because PCR products from "minus-reverse transcriptase" negative controls were not detected in all specimens. Constitutively expressed beta-actin mRNA served as a control for the amount of input RNA across the samples. As positive controls, ConA- and/or E. coli LPS-induced IL-1beta, TNF-alpha, IL-6, IFN-gamma, IL-8, IL-2, IL-4, IL-12 p35, IL-12 p40, and IL-10 mRNAs were clearly detectable in uninfected equine PBLs, using the primers and our RT-PCR conditions . For horse 1 only, the results for all 10 cytokine and beta-actin mRNAs examined are shown, and for the remaining horses, the results for only the cytokines whose mRNAs were expressed and IL-8 and beta-actin mRNAs are shown . Regardless of method of inoculation (i.e., intravenous inoculation or tick transmission), expression of IL-1beta and TNF-alpha mRNAs was induced in all four horses by A. phagocytophila infection . IL-8 mRNA expression was detected in horses 1, 2, and 4 but not in horse 3. IFN-gamma mRNA expression was weakly induced only in horse 2. A weak IL-10 mRNA expression level was also detected in horse 1. In horse 4, IL-12 p35 mRNA was weakly detected . Expression of IL-2, IL-4, IL-6, and IL-12 p40 mRNAs was not detectable in the PBLs of any infected horses. FIG. 1. | Microcolonies (morulae) of A. phagocytophila Microcolonies (morulae) of A. phagocytophila (black arrowheads) found in peripheral blood neutrophils from HP A. phagocytophila-infected horse 1 by using Diff-Quik staining and IFA with MAb 5C11 (insert, white arrowhead). Magnification, x625. FIG. 2. | p44 gene detection of A. phagocytophila p44 gene detection of A. phagocytophila in the PBLs. The levels of A. phagocytophila in PBLs in horses infected by i.v. inoculation and/or by attachment of infected ticks were examined by PCR using p44 gene-specific primers. Input DNA was normalized by beta-actin PCR. The PCR products were resolved on agarose gels containing ethidium bromide. M, DNA size markers (HaeIII fragments of phiX174 RF DNA); N, no template; P, positive-control DNA from cell-cultured A. phagocytophila HZ. FIG. 3. | IFA titers, showing development of IgG antibodies against A. phagocytophila IFA titers, showing development of IgG antibodies against A. phagocytophila in the four horses infected by intravenous inoculation of HP or LP A. phagocytophila or by attachment of HP A. phagocytophila-infected ticks. FIG. 4. | Induction of cytokine mRNAs in PBLs from four horses infected with A. phagocytophila Induction of cytokine mRNAs in PBLs from four horses infected with A. phagocytophila. Total RNA was extracted from equine PBLs and subjected to RT-PCR. The amount of cDNAs was normalized against beta-actin mRNA in corresponding samples. M, DNA size markers (HaeIII fragments of phiX174 RF DNA); N, no template. Uninfected horse PBLs were stimulated with ConA (C) or E. coli LPS (L) for 4 h in vitro. The results shown are representative of more than three assays. PMID- 12204963_DISCUSSION TI - AB - In our equine model of HGE, only IL-1beta and TNF-alpha mRNAs were consistently upregulated in all four horses, regardless of A. phagocytophila cell culture passage number or route of infection. This suggests that A. phagocytophila-induced mild fever, neutropenia, and thrombocytopenia are mediated in horses through the induction of these two major proinflammatory cytokines, which are known to cause these changes . In our previous study, A. phagocytophila or rP44 induced IL-1beta, TNF-alpha, and IL-6 mRNAs within 2 h PI and induced protein within 24 h PI in human PBLs in vitro in a dose-dependent manner . However, IL-6 mRNA expression was not detectable in our equine model. The lack of IL-6 mRNA induction by PBLs in infected horses may be due to low numbers of A. phagocytophila in the blood of horses, since IL-6 mRNA induction in vitro requires 10-fold higher levels of A. phagocytophila than TNF-alpha or IL-1beta mRNA induction does . The lack of IL-6 mRNA induction may be also related to the relatively mild nature of clinical signs of our horses. Coinfection of C3H mice with B. burgdorferi and A. phagocytophila was reported to increase the levels of A. phagocytophila and elevate IL-6 levels in the sera at 2 weeks postinfection beyond those of infection with B. burgdorferi alone . One report observed that in sera of acute-phase patients, the levels of IL-1beta, TNF-alpha, and IL-6 are not elevated . Since, unlike those of experimentally infected animals, it is difficult to preserve these rapid-turnover cytokines in the specimens of human patients , it is premature to dismiss the role these cytokines play in HGE pathogenesis. Acute-phase HGE patient sera contain significantly higher levels of IFN-gamma than convalescent-phase sera . A. phagocytophila can transiently establish infection in C3H mice without any clinical signs, and these mice develop significant IFN-gamma levels in serum during days 2 to 8 postintraperitoneal inoculation with A. phagocytophila-containing blood from infected SCID mice . Although in IFN-gamma-/- mice, initial infection levels with A. phagocytophila are greater than in control mice, the speed of clearance of A. phagocytophila does not differ . In our study, IFN-gamma mRNA expression was seen only in one horse. This finding may be related to the lack of spontaneous clearance of A. phagocytophila from the blood of all horses at even 20 days PI or PA. The role of IFN-gamma in clearance of A. phagocytophila remains to be studied. In the present study, IL-8 mRNA expression was upregulated in 3 horses; however, horse 3 had the weakest cytokine response overall and did not show IL-8 mRNA expression. Recently, Akkoyunlu et al. proposed that IL-8 generated by neutrophils in response to A. phagocytophila facilitates the infection. Klein et al. reported that in dimethyl sulfoxide-treated infected HL-60 cells or human bone marrow cells, IL-8 and other chemokine levels, but not that of IL-1, IL-6, or TNF-alpha in the culture medium, were significantly elevated at 48 h postinfection . Since findings, including those of our present horse study, indicate that IL-8 mRNA is not upregulated at early stages of infection in vitro and in vivo, IL-8 does not seem to be critical for the initial establishment of infection. Overall clinical signs for our horses, which were inoculated with a strain from an HGE patient in the state of New York, were milder than those reported with studies using the BDS or Webster strain from Wisconsin but rather similar to those using inoculation with a New York strain as reported by Chang et al. . In the study of Pusterla et al. , at day 16 after i.v. inoculation and at day 18 after inoculation by tick attachment, A. phagocytophila Webster and BDS strains became undetectable by real-time quantitative PCR assays, based on the presence of the 16S rRNA gene in the blood of horses. However, A. phagocytophila HZ strain p44 genes were detected at day 20 of infection in four horses in the present study, suggesting that A. phagocytophila establishes subclinical persistent infection in horses, as suggested by Chang et al. . A pioneering phylogenic study, comparing different A. phagocytophila strains from patients and animals in the United States and Europe by using the ank gene as a marker, showed that Wisconsin strains are closer to Ehrlichia equi (A. phagocytophila, horse isolate, 99.18 to 99.24% amino acid identity) than New York strains (97.90 to 97.92% amino acid identity) . Thus, it is possible that Wisconsin strains are more pathogenic to horses but cleared more rapidly than New York strains. Disease severity in horses inoculated i.v. with A. phagocytophila strain Webster was reported to be dependent on A. phagocytophila passage numbers in culture . This finding appears to be among those of studies that are not evident unless larger number of animals are tested, since we did not observe clear differences in clinical manifestations between horses infected with HP and LP A. phagocytophila HZ strains. Using a real-time quantitative PCR assay, Pusterla et al. reported 55-fold-lower initial ehrlichial load in PBLs from tick-infected horses than in those from horses inoculated i.v. . In the same study, however, higher ehrlichial levels in the blood of horses infected by tick attachment were observed at 7 days PI. According to the findings based on the use of PCR with p44 genes, our study appears to support their observation with respect to the lower rate of increase of ehrlichial growth with tick transmission than with intravenous inoculation. Pusterla et al. did not see significant difference in antibody titer at day 30 PI between intravenous and tick transmission. In the present study, antibody responses were lower by tick attachment than by intravenous inoculation. More studies are needed to learn whether tick inoculation modulates host immune response to facilitate the infection. PMID- 12186657 TI - A ~35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP AB - Abstract | Background | The S. cerevisiae origin recognition complex binds to the ARS consensus sequence in an ATP dependent fashion. Recently, the yeast Cdc6 has been reported to have DNA binding activity. Conservation of replication proteins among different species strongly supports their functional similarity. Here we report the results of an investigation into the DNA binding activity of human Cdc6 protein. Cdc6 was expressed and purified from baculovirus infected Sf9 (Spodoptera frugiperda) insect cells as GST fusion protein (GST-Cdc6) and its DNA binding activity was tested. Results | Partially purified fractions containing GSTCdc6 or GST showed an ACS binding activity in an ATP dependent manner. However, further purification revealed the presence of a putative 35 kDa insect cell protein (p35) which was found responsible for the DNA binding activity. A close match to the 9/11 bases of the ARS consensus sequence was sufficient for p35 binding activity. A DNA fragment from the human c-myc origin region containing yeast ACS like elements also showed p35 binding activity. Conclusions | We have identified a Spodoptera frugiperda protein with ATP dependent DNA binding activity to ACS like elements. ACS like elements have been reported to be essential for ORC binding and replication initiation in yeast but their role in higher eukaryotes still remains elusive. Like the ARS consensus sequence elements of yeast, ACS like elements found in c-myc and lamin beta 2 origin regions may play similar roles in replication and indicate a conserved role for this DNA motif among eukaryotes. PMID- 12186657_Background TI - AB - Proteins recognizing specific DNA sequences play an important role in the regulation of gene expression and in DNA replication. Nearly all eukaryotic genes transcribed by RNA Polymerase II for instance, contain the conserved TATA box which is present upstream of the transcription start site. The TATA-box binding protein, a ~30 kDa component of the TFIID complex binds specifically to the heptanucleotide A and T residues and forms the core of the transcription initiation complex. Additionally, many specific transcription factors bind to the upstream promoter in a sequence specific manner and regulate gene expression. For example in Drosophila, a heat-shock transcription factor (HSTF) can bind to consensus heat-shock response elements and regulates expression of heat/stress inducible genes. Promoter activity in later branching eukaryotes is greatly modulated by enhancer and repressor sequences which have no activity of their own but are the targets of DNA biding proteins or protein complexes which can remodel the promoter chromatin to make it more or less accessible to RNA polymerase. Biochemical assays have shown that the action of ATP-dependent chromatin remodelling activities increase the accessibility of DNA within chromatin templates. S. cerevisiae SWI/SNF , Ino80 complex , Drosophila NURF are examples of some high molecular weight chromatin remodelling factors which can facilitate transcription by binding to chromatinized DNA templates. However, none of the above chromatin remodelling factors binds to specific DNA sequences. Unlike transcription, the role of sequence specific DNA binding proteins in eukaryotic DNA replication is not well characterized. In higher eukaryotes finding of specific DNA sequences essential for DNA replication has been elusive so far. In yeast Saccharomyces cerevisiae, a six-protein origin recognition complex binds to ARS consensus sequence (ACS) in a sequence specific manner . Individual ORC subunits have not been demonstrated to show DNA binding activity in vitro. Recently, in an in vitro study ScCdc6 has been shown to bind double stranded DNA . The minimal requirement for the binding of Cdc6 to DNA has been mapped within its N-terminal 47-amino acid sequence. Saccharomyces pombe ORC4 subunit has been reported to contain DNA binding activity by using its N-terminal AT hook region . Neither ScCdc6 nor SpORC4 showed any sequence specific DNA binding activity. Recombinant six protein Drosophila ORC (DmORC) binds to ACE region of the Drosophila chorion gene . In vivo, DmORC co-localized with the amplified chorion gene locus. In Xenopus, biochemical analysis of replication and cell cycle events using egg extracts has helped to understand the mechanism of eukaryotic DNA replication . However, two dimensional gel electrophoresis analysis of the rDNA locus showed that replication initiated at all sites tested . All six human homologs of yeast and Drosophila ORC subunits have been cloned and characterized . Other replication proteins like Cdc6, Cdt1, MCMs, Cdc45 that are essential for initiation of DNA replication have also been reported . Conservation of replication factors among higher eukaryotes suggests that functionally they may play similar roles. In an attempt to identify DNA binding activity of human Cdc6, it was expressed and purified as a GST-Cdc6 fusion protein from baculovirus infected Sf9 insect cells. Partially purified fractions (reduced glutathione eluate) containing GSTCdc6 or GST showed an ACS binding activity in an ATP dependent manner. The GSTCdc6 protein fraction contained both the GSTCdc6 and a 35 KDa S. frugiperda protein. The DNA binding activity was confined to a 35 kDa polypeptide. It was latter found that the p35 has an intrinsic affinity to GST. This polypeptide bound to yeast ACS like elements in the presence of ATP. 9/11 matches to ARS consensus sequence were found to be essential for this DNA binding activity both by gel shift assay as well as by in vitro foot printing assay. A DNA fragment containing 9/11 matches from human c-myc replication origin region also showed p35 binding activity suggesting that this polypeptide has intrinsic DNA binding activity. The implications of this DNA binding activity are discussed here. PMID- 12186657_Results TI - AB - Partially purified protein fractions containing GSTCdc6 or GST contain an ACS binding activity | We infected Sf9 insect cells with the baculovirus expressing GSTCdc6. Cells were harvested 48 hours post infection and the proteins were extracted according to the procedures described in materials and methods. The GSTCdc6 protein was partially purified by pull down on glutathione beads . The partially purified protein was used in DNA binding assays with a 240 bp DNA fragment containing all three conserved boxes (A, B1 and B2) of the ARS consensus sequences (Fig. &). As a control, we used GST alone, which was purified using the same strategy used for GSTCdc6 purification. A DNA protein complex was formed in both the cases as evidenced by the retarded mobility of the free 32P phosphate labelled probe . The specificity of the DNA binding was examined in a competition reaction by increasing the amount of unlabeled DNA fragment containing ACS like elements. It was determined that the DNA-Protein complex could be competed efficiently by increasing amount of unlabelled ACS like DNA (20x, 50x, 100x and 200x respectively) . A ~350 bp DNA fragment from pBlueScript KS+ (HinfI digested and subsequently gel purified) of similar base composition did not compete with the complex formation in the Electrophoretic Mobility Shift Assay when added at a similar concentration indicating a degree of specificity in the DNA-Protein complex formation . Figure 1 | Purification of GST-Cdc6 and its DNA binding activity. Purification of GST-Cdc6 and its DNA binding activity. (A) SDS-PAGE of GST-Cdc6 purified by glutathione agarose beads purification technique from Sf9 insect cells infected with baculovirus expressing GST-Cdc6. The polypeptide was visualised by commassie stain. Molecular mass markers are indicated on the right. (B) Gel retardation assays were performed in the presence of reduced glutathione eluted GST or GST-Cdc6 fusion protein. S. cerevisiae ARS1 fragment (240 bp) was end labelled using gamma-32P ATP and incubated with GST or GST-Cdc6 as described in the materials and methods. Both GST and GST-Cdc6 showed strong band shifts. Figure 2 | Specificity of the ARS1 binding Specificity of the ARS1 binding. Gel retardation assays were performed in the absence (-) or presence (+) of GST-Cdc6 using radiolabeled ARS1 probe as described in Fig. . The binding reaction was competed either with cold ARS1 fragment (lanes 3 --6) or a 350 bp fragment from the plasmid BlueScript KS(+) digested with HinfI followed by gel purification (lanes 7 --10). Free probe and the band shifts are indicated by arrow. An unidentified ~35 kDa protein from baculovirus infected insect cells is responsible for DNA binding activity | Partially purified fractions containing GSTCdc6 or GST showed DNA binding activity. To further fractionate the proteins present in the partially purified GSTCdc6 fraction the glutathione column was washed with 200 mM and 300 mM sodium chloride prior to GSTCdc6 elution with reduced glutathione. Western blot analysis using anti GST antibodies revealed that neither 200 mM nor 300 mM fractions contained GSTCdc6 . GSTCdc6 protein was present only in the proteins eluted by reduced glutathione. Proteins released by different salt fractionation were separated on an SDS-PAGE and visualized by silver stain . A prominent band of molecular mass ~35 kDa was visualised both in the 200 mM and 300 mM salt eluate. Figure 3 | Identification of p35 Identification of p35. (A) Western blot analysis of salt eluate and reduced GSH eluate using anti GST antibody. GST-Cdc6 beads were washed using wash buffer containing NaCl (200 mM and 300 mM respectively). Beads following salt wash were treated with reduced glutathione and released proteins were run in SDS-PAGE along with salt eluate followed by western blot analysis. GST-Cdc6 was found only in GSH eluate. (B) Silver staining of salt eluate: Salt eluates were run in SDS-PAGE and proteins were visualised by silver stain. A distinct band of p35 was found both in 200 mM and 300 mM salt eluate. Molecular mass markers are shown on the right. (C) DNA binding activity of p35. Gel shift assays were performed using salt wash (300 mM NaCl eluate) and GSH eluate following 300 mM salt wash of GST-Cdc6 beads. ARS 240 bp DNA fragment was used as probe. 300 mM salt eluate gave a strong band shift whereas GSH eluate failed to do so though it contained GST-Cdc6 as shown in figure . To check whether the DNA binding activity was due to the presence of ~35 kDa band or GSTCdc6 itself, gel shift assays were performed either using 50 ng ~35 kDa protein obtained from the salt wash or GSTCdc6 by itself . Salt eluate gave a strong band shift which was identical with the band shift found with GSTCdc6 found in Fig. . GSTCdc6 eluted from the beads following high salt wash failed to give any band shift suggesting that the ~35 kDa polypeptide was responsible for the DNA binding activity. To further test whether the presence of p35 is absolutely required for DNA binding activity, the 300 mM salt eluate was dialysed against low salt buffer H/0.15 and then passed through Superose 12 gel filtration column. Each fraction was checked for DNA binding activity by gel retardation assay using a 32P labelled DNA fragment containing ACS elements. DNA binding activity was found only in high molecular weight fraction (~670 kDa) . Proteins present in the gel filtration fractions in the high molecular weight range (fractions 13 --17) were separated by SDS-PAGE followed by silver stain. Surprisingly, p35 was found to be present only in the fraction 15 which contains the DNA binding activity . The presence of p35 in the same fraction containing the DNA binding activity strongly suggests that p35 is responsible for the binding activity. Figure 4 | Purification of p35 by gel filtration chromatography Purification of p35 by gel filtration chromatography. (A) Gel retardation assay. 300 mM salt eluate (as shown in fig. ) was dialysed against Buffer H/0.15 and passed through Superose-12 Gel filtration column. Fractions 13 --25 were tested for ARS 240bp DNA binding activity. Only fraction 15 showed a band shift compared to the free probe. Molecular mass markers are shown on the top. (B) Silver stain of gel filtration fractions. 0.25 ml of each gel filtration fractions (fraction 13 --17) were precipitated using trichloro acetic acid (TCA). The samples were run in SDS-PAGE and proteins were visualised by silver stain. p35 was found only in the fraction 15. DNA binding activity is ATP dependent | One of the hallmarks of yeast ORC binding to yeast ARS consensus sequences is its ATP dependence . We were interested to see whether the DNA binding activity of the ~35 kDa protein is ATP dependent or not. Gel shift assays were performed either in the absence or in the presence of increasing amount of ATP in the reaction mixture . In the absence of ATP, a very weak binding was observed whereas with increasing amount of ATP strong binding was detected. There is a threshold of ATP concentration (6 mM and onwards) which stimulated the binding remarkably. A nonhydrolysable ATP analog, ATPgammaS was used in the binding reaction to see whether ATP hydrolysis is required for this binding. With increasing amount of ATPgammaS, the band shift was completely inhibited suggesting that ATP hydrolysis is required for this DNA binding activity. This was confirmed by adding ATP back in the reaction mixture when ATPgammaS was already present in the reaction. Under these reaction conditions, increasing amount of ATP again stimulated the DNA binding activity even in the presence of ATPgammaS suggesting that ATPgammaS can be competed with ATP and it is the ATP hydrolysis which is essential for this DNA binding activity (Fig. , lanes 13 and 14). Figure 5 | ATP hydrolysis is required for the DNA binding activity of p35 ATP hydrolysis is required for the DNA binding activity of p35. Gel shift assays were performed using 32P labelled ARS fragment either in the presence of ATP (lanes 3 --7) or ATPgammaS (lanes 8 --12) or both (lanes 13 --14). A strong band shift was observed in the presence of ATP whereas ATPgammaS completely inhibited the binding. The inhibition can be reversed by adding back ATP in the reaction mixture. p35 binds to A, B1 and B2 boxes of ARS consensus sequences as revealed by footprinting assay | After establishing the fact that a protein of approximately 35 kDa binds to DNA fragment containing ARS consensus sequences, the exact site of binding of the protein on the DNA was mapped by copper-phenanthroline footprinting assay. Unlike DNaseI, 1,10-phenanthroline-cuprous complex is a small chemical probe which can demark the boundaries of the protected region clearly. The protein was bound to a 5' 32P labelled 240 bp DNA fragment containing yeast ARS consensus sequence and separated from the free DNA by gel shift assay. The gel was then treated with copper-phenanthroline reagent as described in materials and methods. The bound and unbound DNA was purified and allowed to run in a sequencing gel . On the T-rich strand (bottom strand) two protected regions were observed. The first region entirely covered the A box of the ARS consensus sequence and the second region covered the overlapping regions of B1 and B2. Therefore the 35 kDa protein has a strong affinity to bind to A, B1 and B2 boxes of the ARS consensus sequences. The long stretch of protection could be due to multimeric form of p35 or could be due to the formation of a higher order nucleoprotein complex. Figure 6 | Copper-Phenanthroline footprint analysis of ARS DNA fragment bound to p35 Copper-Phenanthroline footprint analysis of ARS DNA fragment bound to p35. ARS DNA fragment was 5' end labelled at the T rich strand and incubated with p35 (300 mM salt eluate) and finally subjected to gel retardation assay. The gel was then treated with copper-phenanthroline mixture and DNA from bound (B) and unbound (U) fractions were extracted from gel, denatured and loaded in a sequencing gel. A+G ladder was also loaded to figure out the position of footprint. The relative positions of A, B1 and B2 boxes were indicated. The protected regions are shown by solid lines and asteric (*). The hypersensitive site is denoted by arrowhead. Figure 7 | At least one ACS like element is required for p35 binding At least one ACS like element is required for p35 binding. (A) ARS consensus sequence. (B) 240 bp yeast S. cerevisiae ARS1 fragment containing several functional elements required for ARS activity (Boxes A, B1, B2 and B3 respectively). Several oligos spanning the entire ARS1 as indicated in the figure were designed and subsequently cloned into p21N fragment. Double stranded oligo 'a' (ARSa) does not contain any ARS like element. Oligo 'b' (ARSb) contains one 11/11 match and one 9/11 match to ACS element. Oligo 'c' (ARSc) and oligo 'd' (ARSd) contain one 9/11 match to ACS element each. In the fragment dmut, 'A's and 'T's within the core ARS were mutated to 'G's and 'C's. (C) Nucleotide sequences of ARSd and ARSdmut. (D) Gel shift assay was performed either using ARSwt (lanes 1,8), or p21N (lanes 2,3 and 9) or the constructs derived from p21N by cloning fragments a-d and dmut (lanes 4 --7, 10,11) as described in (B). p35 does not bind to p21N or p21N + ARSa or p21N + ARSdmut. All the other DNA fragments showed DNA binding activity. p35 binds to A, B1 and B2 boxes and mutation in these boxes abolish DNA binding activity | Previous studies of the structure of ARS1 in both plasmid and chromosome contexts have shown that it contains one essential DNA element, A, that includes a perfect (11/11) match to the ARS consensus sequence (ACS, Fig. ), and three additional elements, B1, B2, and B3, with 9 out of 11 bases match to the ACS that are also important for ARS function . We were interested to see whether A, B1 and B2 boxes were sufficient to allow p35 binding activity. We used the p21N protein (N terminal 150 bases of coding region of p21) as a control. This DNA when incubated with ~35 kDa protein does not form a DNA protein complex . The DNA fragment was divided into four subfragments (a, b, c and d; Fig. ) and subcloned in the middle of p21N fragment. Fragment 'a' does not contain any ACS sequence whereas fragments b, c and d contain at least one ACS like element. All the ACS containing sub fragments (b, c, d) showed a mobility shift , which suggests that at least one ACS like element (either 11/11 match or 9/11 match to ARS consensus sequence) is essential and sufficient for p35 binding activity. This was further confirmed by using a subfragment d mutated at the ACS motif . Subfragment d as shown if Fig lane 10 can bind strongly to the 35 kDa protein, however mutation altering the A and Ts of the core ACS sequence to G and Cs to generate the dmut oligonucleotide results in the abolition of its ability to bind p35. Therefore the ACS sequence is essential for the p35-DNA protein complex formation. 35 kDa polypeptide binds to ARS consensus sequence found in c-myc origin of replication | In S. cerevisiae, ARS elements have been implicated to be important both for ORC binding and origin function . We looked for the availability of such sequences in known human origins of replication like c-myc, lamin beta 2 and Dnmt1. An origin of replication was mapped previously by nascent strand abundance analysis within 2.0 kb zone immediately upstream of c-myc gene . Detailed analysis of the 2.0 kb upstream sequences revealed the presence of two ACS like elements separated by 148 nucleotides (AAAAGATAAAG and AAAAGAAAAAA). A 300 bp DNA fragment containing both the ACS elements was amplified by polymerase chain reaction and the product was used subsequently for p35 binding studies. A strong band shift was observed which could be competed out using a 50 bp long double stranded oligo containing two ACS like elements (oligo 'b', Fig. ). Increasing amount of ~65 bp long unrelated (non specific) double stranded oligo did not have any effect on this binding activity suggesting that binding of p35 to c-myc origin region is specific. Interestingly, analysis of Lamin beta 2 origin of replication region also revealed the presence of two ACS (9/11 match) like elements . Further studies are required to find out whether p35 also binds to lamin beta 2 origin region. Figure 8 | p35 binds to DNA fragment containing ACS like elements from c-myc origin region p35 binds to DNA fragment containing ACS like elements from c-myc origin region. A 300 bp DNA fragment containing two ACS like elements (10/11 matches) was PCR amplified using primers specific for the zone of replication from c-myc locus. PCR product was end labelled using gamma-32P ATP and used for gel shift assays. Competition reactions were performed by using different quantities (50x, 100x and 200x) of 50 bp long double stranded oligos (oligo 'b', Fig. ) in the reaction mixtures corresponding to lanes 3 --5. Lanes 6 --8 contained 65 bp double stranded DNA fragment (plasmid BlueScript KS(+) digested with HinfI followed by gel purification). p35-ACS interaction is sensitive to high salt, temperature and EDTA | The stability of p35-ACS DNA complex was further tested either by changing NaCl concentration in the reaction mixture, or by shifting reaction temperature or by adding EDTA. The DNA binding activity was found to be sensitive to NaCl concentration . Strong band shift was obtained up to 0.2 M NaCl. NaCl concentration at 0.5 M and above completely inhibited the binding activity. Higher temperature also showed a drastic effect on the binding activity. Normal DNA binding activity was observed up to 42C. Temperature higher than 42C completely abolished the binding activity suggesting that the off rate of p35 from DNA is much faster at higher temperature. Finally inclusion of EDTA in the reaction mixture inhibited the binding reaction suggesting that the divalent cations are essential for this binding activity. Figure 9 | Effect of salt concentration, temperature and EDTA on p35 gel shift assay Effect of salt concentration, temperature and EDTA on p35 gel shift assay. Gel retardation assay was performed either using no extra salt or different concentration (lanes 1 --5) of NaCl. Band shift was completely inhibited at 0.5 M NaCl concentration and above. Effect of temperature on binding reaction was tested by incubating the reaction mixture (lanes 6 --9) at different temperature. Binding reaction was completely inhibited at 55C and above. All the other reactions were carried out at room temperature. Effect of bivalent cations were tested by incubating the reaction mixture in the presence of 50 mM and 100 mM EDTA (lanes 10 --11). Presence of EDTA completely inhibited the binding reaction. PMID- 12186657_Discussion TI - AB - Few proteins have been reported in the literature, which are capable of binding to DNA in a sequence specific ATP dependent manner. Although transcription factors bind to specific DNA sequences, the binding activity is not dependent on ATP hydrolysis. In contrast, chromatin remodelling factors like SWI/SNF, ISW1, BRG1 facilitate transcription from chromatinised templates in the presence of ATP . However, these factors do not bind to specific DNA sequences. In eukaryotic DNA replication, sequence specific ATP dependent DNA binding activity has been demonstrated in yeast S. cerevisiae where ORC, a six polypeptide complex binds to yeast ARS consensus sequence in an ATP dependent manner . The binding sites for other ORCs are not very clear at present. DmORC binds the critical elements of well-characterized, chromosome III amplification domain (ACE3 and ori-beta, though the precise sequence recognized by DmORC within ACE3 and ori-beta have not been identified . Studies of both ScORC and DmORC indicate the ATP binding by Orc1p is required for DNA binding. However ATP hydrolysis is not required for DNA binding for both the cases suggesting that ATP hydrolysis may be required for further downstream processes. Chromatin immunoprecipitation (ChIP) studies have demonstrated the association of SpORC with S. pombe origins and human ORC with the EBV OriP . Whether ORC binds to these sequences directly or indirectly with the help of other proteins are subject to in vitro DNA binding assays using purified ORC proteins. In this study, we report a ~35 kDa protein from the baculovirus infected Sf9 insect cells that binds to yeast ACS sequences in an ATP dependent fashion. p35 was purified as high salt (300 mM NaCl) eluate from the GST-Cdc6 beads. GST-Cdc6 eluted from the beads following high salt wash failed to show any DNA binding activity whereas high salt eluate containing only p35 showed strong DNA binding activity suggesting that p35 not Cdc6 is responsible for the binding activity. This experiment was repeated several times and always the protein preparations containing p35 showed DNA binding activity. p35 has an intrinsic affinity to GST moieties. Sf9 insect cells were infected with baculovirus expressing GST alone. The cell lysate was allowed to bind to GST beads. High salt eluate (300 mM NaCl) from GST beads was tested for DNA binding activity. Surprisingly, we observed a very similar band shift as obtained previously using high salt eluate from GSTCdc6 (data not shown). Further, high salt eluate from GSTORC2 and GSTORC4 (GST fusion protein containing human origin recognition complex subunit 1 and 2 respectively) also showed DNA binding activity (data not shown). Therefore, it can be concluded that p35 interacts with GST and high salt concentration is required to disrupt this interaction. The nature and the specificity of the interaction between GST and p35 are not clear at this moment. It is important to note that majority of the p35 bound to GST or GST fusion proteins are released mostly at high salt concentration (300 mM) allowing us to get rid of most of the impurities by stringent washing of the GST beads with buffer containing 250 mM NaCl. A weak DNA binding activity was found till 4 mM ATP concentration . A strong stimulation was obtained at 6 mM ATP concentration. It is possible that p35 is purified as ATP bound form but an associated weak ATPase activity does not allow it to give a strong binding activity. It is suggested that 6 mM ATP concentration may be sufficient to overcome this inhibitory effect. ATP hydrolysis is essential for DNA binding activity of p35 since ATPgamma S, a nonhydrolysable analog of ATP completely inhibited the binding activity . It is possible that ATP is required for strand opening which needs to be further explored. An insect cell factor, polyhedrin promoter binding protein has been reported previously, capable of binding to AT rich DNA sequence . However, the reported DNA binding activity was unusual in a sense that the activity was heat and salt concentration resistant. 100 mM EDTA concentration did not affect the DNA binding activity. The DNA binding activity reported in this study was found to be temperature, EDTA and salt concentration sensitive suggesting that this polypeptide is completely different from the polyhedrin promoter binding protein. p35 showed a strong DNA binding affinity towards ACS like elements. This was confirmed first by using unlabeled specific competitor DNA which completely abolished the binding of 32P labelled probe. Competition using unrelated DNA did not affect the DNA binding activity. We took p21N, which normally does not bind to p35, to further test the binding specificity. Introduction of a single ACS like element (9/11 match) in p21N (ARSc and ARSd) showed a strong band shift suggesting that only one ACS like element is sufficient for p35 binding. This was further confirmed by making mutations in the ARSd fragment. The resulting ARSdmut did not show any p35 binding activity suggesting that the ACS like element itself but not the adjacent sequences are essential for the p35 binding activity. However, a systematic mutational analysis of ACS like elements will be required to explore the exact binding specificity of p35 towards ACS like elements. ACS elements are normally AT rich. However, p35 did not bind to random AT rich sequences. p35 did not bind to p21N + ARSa which contains ARSa oligo (78% AT rich) with no ACS like element. Surprisingly, p35 showed strong binding activity in the presence of the oligo ARSb (64% AT rich), ARSc (66% AT rich) and ARSd (74% AT rich) respectively . ARSb, c and d contain at least one ACS like element . Finally the copper phenanthroline footprint analysis confirmed that p35 binds to A and B1-B2 boxes of the ARS1 DNA fragment. At this moment, the function of p35 is not very clear. It may play major role(s) in the transcription of certain insect cell genes. It may as well be responsible for DNA replication. The fact that it binds to yeast ACS and to a DNA fragment from c-myc origin of replication region containing yeast ACS like elements in an ATP dependent manner further strengthen the hypothesis. S. cerevisiae origin recognition complex (ORC) binds to ARS consensus sequences in an ATP dependent fashion and this binding is essential for both origin function and activity. It is interesting to note that a huge six protein origin recognition complex binds to yeast ARS1 whereas p35, a small protein is showing same kind of protection as evidenced by foot print analysis . We believe that p35 forms an oligomeric structure or it maintains a multimeric form which may explain the wide footprint over the ARS1 fragment. The presence of p35 in high molecular weight fraction (~670 kDa) following superose 12 gel filtration chromatography strengthen this hypothesis and clearly suggests that p35 forms an oligomeric structure. ARS consensus sequence has been found near the vicinity of c-myc, lamin beta 2 and Dnmt1 replication origin . Therefore, Identification and characterization of this protein from insect cells and finding its human counterpart will greatly help in elucidating its possible function in DNA replication. PMID- 12186657_Conclusions TI - AB - The data presented here leads to the identification and characterization of a polypeptide from insect cells with ATP dependent DNA binding activity. This is an important and unique observation. In S. cerevisiae, ACS elements have been reported to be essential for ORC binding and replication initiation. Yeast ACS like elements found in c-myc and lamin beta 2 origin region may play similar roles in replication initiation. However, it is also possible that p35 is a transcription factor which may facilitate transcription of some insect cell genes. Further characterization of p35 from insect cells and finding its human homolog will be very helpful to dissect its functional role in replication and/or transcription. PMID- 12186657_Materials and Methods TI - AB - Plasmid construction | Cloning of human Cdc6 cDNA is described elsewhere. Coding sequence of human Cdc6 was cloned in pFastBac-GST vector (Life Technologies, Inc.) to express GST fusion protein. A 240 bp DNA fragment from S. cerevisiae ARS1 chromosomal DNA replication origin containing all the key elements including boxes A, B1, B2 and B3 was subcloned in pBlueScript KS(+) between EcoRI and HindIII. Subsequently EcoRI-HindIII fragment was end labelled using gamma32P ATP and used either for gel shift assay or copper-phenanthroline foot print assay. p21N, (N terminal ~150 bp of p21) was previously cloned in pBlueScript KS(+) between EcoRI and HindIII sites. Complementary oligos corresponding to ARSa, b, c, d and dmut were synthesized (~50 bases in length) and subsequently annealed to get double stranded oligos. p21N/KS(+) construct contains only one StuI site which is present within the p21N insert. All the annealed double stranded oligos were cloned into the StuI site using blunt end ligation. The sequences of ARSa, b, c and d are followed: ARSa: ttagtttttcggtttactaaatcgtaatagaaatgtagaacaataaaatgt ARSb: tctaaaatacaaatctagaaaatacgaacgaaaagttttccggacgtccgt ARSc: cggacgtccgttcacgtgtttgttatgaatttatttatgatgagtcattat ARSd: tgagtcattattggataaagaatcgtaaaaactgctttaaacgataaaa Plasmid containing 2.5 kb DNA fragment from c-myc origin region was a kind gift from Michael Leffak, Wright State University Ohio. Forward and reverse PCR primers (5'-gaagaaaaactctcttttc-3' and 5'-atttgctgggttgaaaaatg-3' respectively) were used to amplify 300 bp region containing two ACS like elements. Expression of GSTCdc6 and GST in insect cells and purification | Baculoviruses were produced from the recombinant pFB-GST plasmid using Bac-to-Bac expression system (Life Technologies Inc.). Sf9 cells (Invitrogen) were infected with the pFB-GSTCdc6 or pFBGSTbaculovirus according to the manufacturers' recommendations. Cells were harvested 48 hours post-infection. The cell pellet was washed once in cold phosphate-buffered saline and subsequently resuspended in hypotonic lysis buffer (10 mM Tris.Cl, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 2 mug/ml pepstatin, 2 mug/ml leupeptin, 5 mug/ml aprotinin, 1 mM dithiothreitol). The cell suspension was homogenized in a Dounce homogenizer using a B-type pestle followed by centrifugation at 3000 rpm for 7 min. The pellet containing the nuclei was lysed in buffer H/0.15 (50 mM HEPES/KOH, pH 7.5, 150 mM KCl, 0.02% Nonidet P-40, 5 mM magnesium acetate, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 mug/ml pepstatin, 2 mug/ml leupeptin, 5 mug/ml aprotinin, 1 mM dithiothreitol). The resulting suspension was subjected to ammonium sulphate precipitation (starting with 10% followed by 30% and finally 50%). The pellet after the 50% ammonium sulphate cut was resuspended in buffer H/0.0 (no salt) and then dialyzed overnight against buffer H/0.15. The dialyzed sample was then bound to GST beads (Sigma) and washed three times with buffer H*/0.15 (containing 150 mM NaCl instead of 150 mM KCl). Proteins were eluted using reduced glutathione elution buffer (50 mM Tris.Cl, pH 8.0, 20 mM reduced glutathione, 0.01% Nonidet P-40, 100 mM NaCl). Immunoblotting and Silver stain | Anti-GST polyclonal antibodies were purchased from Santa Cruz Biotechnologies. Western blotting technique was carried out using standard protocol. The silver stain protocol is described elsewhere . Gel retardation assay | Gel retardation assay was performed with slight modification of the protocol used by Mukhopadhyay et al . The DNA fragments to be used for gel retardation assay were endlabeled with gamma32P ATP. The binding reactions were performed in 20 mul of T buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 50 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 30 mug/ml BSA) supplemented with 5 mM ATP and 6% (v/v) glycerol for most of the reactions. The mixture was incubated at 37C for 10 min and loaded directly on a 5% polyacrylamide gel in TBE buffer (89 mM Tris base, 89 mM boric acid, 2.5 mM EDTA, pH 8.3). The gel was run at 150 V for 2 hours, dried and autoradiographed. Copper-phenanthroline footprint assay | The Copper-phenanthroline footprint assay was performed essentially by using the protocol described by Kuwabara et al . The DNA-protein gel is run in the absence of free radical scavengers as described in gel retardation assay. The gel is placed in 200 ml of 50 mM Tris-HCl, pH 8.0. The gel is further incubated for 10 minutes in a solution containing equal volume of solution A (40 mM 1,10 Phenanthroline monohydrate in 100% EtOH and 9 mM Cupric sulphate mixed with equal volume followed by 1:10 dilution with water) and solution B (0.5% 3-Merceptopropionic acid in water). Finally the gel is soaked in solution C (28 mM 2,9 Dimethyl-1,10 Phenanthroline in 100% EtOH) for 2 min. The gel is washed twice in deionised water. After the pre-treatment of the gel, it is autoradiographed and the retarded band is cut from the gel and placed in an eppendorf tube. The DNA is eluted from the gel slice, denatured and loaded in a sequencing gel. The sequencing gel is fixed, dried and the bands were visualised by autoradiography. PMID- 12186657_Authors' contributions TI - AB - SKD designed, performed and co-ordinated the whole study. NM participated in insect cell culture and helped in making and amplification of baculovirus expressing GST-Cdc6 and GST. RKS helped in making the figures and drafting the manuscript. GM helped in analysing the data and critically reviewed the manuscript. All authors read and approved the final manuscript. PMID- 12221110 TI - Role of Fission Yeast Tup1-like Repressors and Prr1 Transcription Factor in Response to Salt Stress AB - | In Schizosaccharomyces pombe, the Sty1 mitogen-activated protein kinase and the Atf1 transcription factor control transcriptional induction in response to elevated salt concentrations. Herein, we demonstrate that two repressors, Tup11 and Tup12, and the Prr1 transcription factor also function in the response to salt shock. We find that deletion of both tup genes together results in hypersensitivity to elevated cation concentrations (K+ and Ca2+) and we identify cta3+, which encodes an intracellular cation transporter, as a novel stress gene whose expression is positively controlled by the Sty1 pathway and negatively regulated by Tup repressors. The expression of cta3+ is maintained at low levels by the Tup repressors, and relief from repression requires the Sty1, Atf1, and Prr1. Prr1 is also required for KCl-mediated induction of several other Sty1-dependent genes such as gpx1+ and ctt1+. Surprisingly, the KCl-mediated induction of cta3+ expression occurs independently of Sty1 in a tup11Delta tup12Delta mutant and so the Tup repressors link induction to the Sty1 pathway. We also report that in contrast to a number of other Sty1- and Atf1-dependent genes, the expression of cta3+ is induced only by high salt concentrations. However, in the absence of the Tup repressors this specificity is lost and a range of stresses induces cta3+ expression. PMID- 12221110_INTRODUCTION TI - AB - Exposure of cells to environmental stress triggers a rapid increase in the transcription of genes whose products have protective functions . Key to this response are stress-activated protein kinase (SAPK) pathways that transmit the signal from stress sensors to the transcription factors that regulate gene expression. These pathways are evolutionarily conserved, and homologs of the mammalian SAP kinases, p38/RK/CSBP , are present in both Saccharomyces cerevisiae (Hog1) and Schizosaccharomyces pombe (Sty1/Spc1) . The Hog1 pathway in S. cerevisiae is activated essentially by hyperosmolarity , whereas the S. pombe Sty1 pathway, like mammalian p38, is activated by a range of adverse conditions . Models of SAPK-dependent regulation of transcription have been almost exclusively based upon the positive control of activators. However, recent analysis of S. cerevisiae has demonstrated that the Sko1 repressor regulates the expression of Hog1-dependent osmostress genes, such as ENA1 and GRE2, via recruitment of the Ssn6(Cyc8)-Tup1 global corepressor complex . Ssn6-Tup1 mediates its function via the organization of repressive chromatin structures and by inhibition of the basal transcription machinery . This global repressor controls the expression of numerous genes through interaction with a variety of site-specific DNA binding proteins . Relief from this repression is achieved by control of the proteins that serve to tether the complex to DNA; for example, Sko1 is phosphorylated by Hog1 at three sites in its N-terminal region, disrupting the interaction with Ssn6-Tup1 . Therefore, a component of the osmotic induction of some genes occurs via derepression rather than by activation. In fission yeast, Sty1 operates via the transcriptional activators Atf1/Gad7 and Pap1 . Atf1 is phosphorylated in a Sty1-dependent manner and loss of Atf1 results in hypersensitivity to osmotic stress, high levels of calcium, and an inability to respond to deteriorating nutritional conditions . In addition, Atf1 forms a heterodimeric complex with Pcr1, a related ATF/CREB factor, which is also required for transcriptional induction of some stress genes . Pap1 activates transcription in response to oxidative stress, and its subcellular localization is regulated in a Sty1-dependent manner . Recently, Prr1, a homolog of Skn7 in S. cerevisiae , has also been implicated in the transcriptional response to oxidative stress . Skn7 and Prr1 have heat-shock factor-like DNA binding domains and also share homology with bacterial "two-component" response regulators that are controlled by histidine-to-aspartate phosphorelay systems . Herein, we have addressed the roles of the Tup-like repressors Tup11 and Tup12 in the response to stress in S. pombe. We find that deletion of both tup genes in combination results in hypersensitivity to KCl and CaCl2, and we also identify cta3+ as a novel stress gene that is negatively regulated by Tup11-Tup12. The expression of cta3+ is rapidly and specifically induced in response to salt shock in a Sty1- and Atf1-dependent manner, but the dependence on the Sty1 pathway for induction is lost in a tup11Delta tup12Delta mutant. Furthermore, Tup11 and Tup12 proteins function as specificity factors by preventing induction of cta3+ in response to inappropriate stresses such as heat and oxidative stress. We also reveal a new role for the "response regulator" protein Prr1 and demonstrate that it is required for proper KCl-mediated transcriptional induction of Sty1-dependent genes such as cta3+, ctt1+, and gpx1+. PMID- 12221110_MATERIALS AND METHODS TI - AB - Strains | Routine culture of S. pombe and general genetic methods were performed as described in . The strains used in this study are described in Table . The cta3+ gene was disrupted using a polymerase chain reaction (PCR)-based approach as described by . Oligonucleotides 5' KO (5'-TTTGATTTTACTTATATTTCTCCCCTTCTACTCATCCCGATATATTCTTACTTCCTTGATTCAATCTCAAATATTGTTCAGCTTAGCTACAAATCCCACT-3') and KO 3' (5'-ATAAATCCTTTACGATTTGTCGGTTCTGTGAAAACGATACACTCACGCATATTCATATACATATTCATGGCAAGAAAACATCTGACATAAAACGCCTAGG-3') were used to amplify a 1.6-kb ura4+-containing fragment from pRep42. The amplified fragment was used to transform strain NT5 strain to Ura+, creating strain SW95. Integration at the correct locus was confirmed by PCR analysis. Table 1 | Strains used in this study Plasmids | The tup11+ coding sequence was amplified by PCR from a cDNA library by using the following primers: 5'-GCACGGATCCCATGGCGTCAGTGGAGGATGC-3' and 5'-CTAGGGATCCAATTCAA-GGAGATGCAGGGTC-3'. The tup12+ coding sequence was amplified using primers 5'-GCACGGATCCCATGATTACTGTCCGCCAATC-3' and 5'-CTGCTAGGCATATGGCGCTCATGAAACAAACCG-3'. Fragments were cleaved with BamHI and cloned into the BamHI site of derivatives of pRep41 and pRep42 vectors that allow the expression of proteins as N-terminal HA or 6HisMyc fusions . RNA Analysis | RNA samples were prepared from 0.25 to 0.5 x 109 cells. Pellets were washed in H2O and resuspended in 200 mul of RNA buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 50 mM EDTA pH 8.0, and 0.25% SDS) with 200 mul of phenol/chloroform. Cells were disrupted with 0.75 ml of glass beads (0.5 mm; Biospec Products, Bartlesville, OK) in a Ribolyser (Hybaid, Middlesex, United Kingdom). A further 0.75 ml of RNA buffer was added followed by spinning in a microfuge for 10 min. The aqueous layer was subjected to two further phenol/chloroform extractions before the RNA was precipitated with 0.1 volume of sodium acetate, pH 5.2, and 0.6 volume of isopropanol. RNA pellets were washed in 70% ethanol and resuspended in H2O. RNA analysis was as described by . Briefly, a 10 --15-mug sample of total RNA was denatured with glyoxal, separated on a 1.2% agarose gel prepared in 15 mM sodium phosphate, pH 6.5, and transferred to a GeneScreen hybridization membrane (PerkinElmer Life Sciences, Boston, MA). The his3+ probe has been described previously . Other gene-specific probes were produced by PCR amplification from genomic DNA by using the appropriate primers. All probes were labeled with [alpha-32P]dCTP by using a Prime-a-Gene labeling kit (Promega, Madison, WI). Transcript levels were quantified relative to the loading control using a PhosphorImager BAS-1500 (Fuji Photo Film, Tokyo, Japan). beta-Galactosidase Assays | Assays were performed as described previously . Coprecipitations | Whole cell extracts were prepared as described by with some modification. Cultures were grown to mid-log phase (OD595 = 0.25 --0.5) in EMM medium. Cells were harvested washed once and snap frozen. Pellets were washed in 1 ml of lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 10 mM imidazole, 2 mug/ml pepstatin, 2 mug/ml leupeptin, 2 mug/ml aprotinin, and 100 mug/ml phenylmethylsulfonyl fluoride). Cells were disrupted with 2 ml of glass beads by vortexing twice for 45 s with 1-min incubation on ice in between. Protein extracts were recovered and centrifuged at 13,000 rpm for 10 min at 4C. Protein precipitations were performed by adding 25 mul of nickel-agarose (50% slurry in lysis buffer) to 1 mg of whole protein extract and incubating at 4C for 1 h with gentle agitation. Precipitates were recovered by centrifugation and washed four times with lysis buffer containing 200 mM NaCl and 20 mM imidazole. Samples were analyzed by SDS-PAGE and proteins were transferred to nitrocellulose membrane and subjected to Western blotting by using monoclonal hemagglutinin (HA) (12CA5) antibody (Babco, Berkeley, CA). Electrophoretic Mobility Shift Assays (EMSAs) | Whole cell extracts were prepared as described above except that cells were grown in YE5S medium and extracts were prepared in buffer containing 25 mM HEPES pH 7.6, 0.1 mM EDTA, 150 mM KCl, 0.1% Triton X-100, 25% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride 2 mug/ml pepstatin, 2 mug/ml leupeptin, and 2 mug/ml aprotinin. Radiolabeled DNA fragments were prepared using PCR amplification as described in . The oligonucleotides used for amplification of probe 1 were 5'-TAAAACACCGACATGTAGCC-3' and 5'-TTGAGAGAAACTAACCAAGG-3'. The oligonucleotides for probe 2 were 5'-CTCTGTCATGGAAATCCACAC-3' and 5'-ATAAGCAGCAAAGCTTGCCTG-3'. Binding reactions were performed by adding 15 mug of whole cell extract to 20-mul reactions containing 25 mM HEPES pH 7.6, 34 mM KCl, 5 mM MgCl2, and 2 mug of poly[d(I-C)]. Reactions were incubated for 10 min at room temperature before the addition of similar0.5 ng of radiolabeled probe DNA followed by a further 20-min incubation. Samples were analyzed by electrophoresis through 4% polyacrylamide gels run in 0.5x Tris-borate-EDTA buffer. Antibody supershift was performed by adding 0.2 mug of monoclonal HA antibody (12CA5) (Babco) 10 min after the addition of probe DNA. PMID- 12221110_RESULTS TI - AB - To address the role of repressors in the transcriptional response to stress in fission yeast we examined whether cells lacking the tup genes tup11+ and tup12+ exhibited any stress-related phenotypes. We found that single and double tup mutant strains exhibited an increased tolerance to cadmium but that the tup11Delta tup12Delta mutant strain had decreased tolerance to elevated levels of Ca2+ and K+ ions. The degree of sensitivity to these salt stresses was similar to that associated with loss of either the Atf1 transcription factor or the Sty1 MAP kinase that are known to control the induction of genes in response to elevated cation concentrations . The tup11Delta tup12Delta double mutant strain was only slightly less sensitive to KCl than the sty1-1 and atf1Delta strains and the tup11Delta tup12Delta strain was actually more sensitive to CaCl2 than strain lacking Sty1 (Figure A). In contrast, the double tup mutant strain had wild-type levels of tolerance to high sorbitol concentrations, indicating that although they are K+- and Ca2+-intolerant they are not osmosensitive (our unpublished data). Figure 1 | Stress-related phenotypes of cells lacking Tup repressors. Stress-related phenotypes of cells lacking Tup repressors. (A) Exponentially growing cultures (similar0.4 x 107 cells/ml) were diluted serially, spotted onto YE5S agar, and incubated for 2 --3 d at 30C or spotted onto YE5S agar supplemented with CaCl2, KCl, or CdSO4 at the indicated concentration and incubated for 3 --4 d at 30C. (B) Exponentially growing cultures of wild type (w.t.) (NT4), atf1Delta (NT147), and tup11Delta tup12Delta (SW76) strains were treated with H2O2 (to a final concentration of 50 mM), and viable cell numbers were determined by plating onto YE5S agar. The similarity in the sensitivities of tup11Delta tup12Delta and atf1Delta strains to elevated K+/Ca2+ ions was unexpected because Tup11 and Tup12 have been previously demonstrated to be repressors , whereas Atf1 is primarily a transcriptional activator. We therefore investigated whether tup11Delta tup12Delta cells shared any other phenotypes with atf1Delta cells. It has recently been demonstrated that atf1Delta cells are sensitive to an acute oxidative stress . When challenged with a high dose of H2O2 (50 mM) atf1Delta cells rapidly lose viability (Figure B). In contrast, tup11Delta tup12Delta cells were only slightly more sensitive than wild-type cells in this assay. Furthermore, although atf1Delta cells conjugate poorly , a tup11Delta tup12Delta strain conjugates in nutrient-rich media . Hence, tup11Delta tup12Delta cells and atf1Delta cells share only a subset of phenotypes. Taken together, these findings are consistent with Tup11 and Tup12 having overlapping functions and indicate that Tup11 and Tup12 play roles in the cellular response to stress. Tup11 and Tup12 Negatively Regulate Expression of Salt-Stress gene cta3+ | Cells lacking atf1+ and both tup genes have similar sensitivities to salt stress and so we examined whether the expression of genes known to be induced by exposure to salt stress, via Atf1, were also regulated by the Tup repressors (Figure , A and B). We found that expression of cta3+, which encodes a cation-transporting P-type ATPase , was markedly influenced by loss of the Tup proteins; deletion of both tup genes together resulted in a large increase in the basal level of expression (Figure , A and B). Loss of both Tup proteins also resulted in a large increase in expression after exposure to CaCl2 (Figure , A and B) and KCl (Figures , , and ). Thus, Tup11 and Tup12 function in a partially redundant manner to repress cta3+ expression and limit the level of induction. As previously observed , the induction of cta3+ expression is completely dependent upon both the Sty1 MAP kinase and the Atf1 transcription factor, and thus cta3+ displays a novel pattern of stress regulation that is positively controlled by Sty1 and Atf1 but negatively regulated by the Tup repressors. Indeed, deletion of the tup genes either singly or in combination had only minor effects on the expression levels of other Sty1-dependent genes such as gpd1+, ctt1+, and gpx1+ in unstressed cells and in cells subjected to a CaCl2 shock (Figure , A and B). Figure 2 | Tup11 and Tup12 repress the transcription of the salt stress gene cta3+. (A) Strains used are indicated above the lane and were wild type (w.t.) (NT4), tup12Delta (BSP03), tup11Delta (SW53), tup11Delta tup12Delta (SW76), atf1Delta (78 Tau147), and sty1-1 (JM1144). Tup11 and Tup12 repress the transcription of the salt stress gene cta3+. (A) Strains used are indicated above the lane and were wild type (w.t.) (NT4), tup12Delta (BSP03), tup11Delta (SW53), tup11Delta tup12Delta (SW76), atf1Delta (78 Tau147), and sty1-1 (JM1144). Log phase cultures growing at 30C in YE5S (lanes 1, 4, 7, 10, 13, and 16) were incubated with CaCl2 (to final concentration of 0.3 M) for 15 min (lanes 2, 5, 8, 11, 14, and 17) and 30 min (lanes 3, 6, 9, 12, 15, and 18). Total RNA was extracted, separated by electrophoresis, and Northern blots were analyzed with the indicated probes. The level of his3+ mRNA was used as a loading control. (B) Quantification of cta3+ mRNA levels in A. (C) Influence of Tup proteins on the activity of a cta3-lacZ reporter. beta-Galactosidase assays were performed on extracts derived from exponentially growing cells (open bars) and cells treated with KCl to 0.6 M for 60 min (black bars). The strains used were wild type (w.t.) (HAI003) and tup11Delta tup12Delta (SW107). Data is the mean of three independent cultures. (D) Deletion of cta3+ does not rescue the salt sensitivity of tup11Delta tup12Delta cells. Exponentially growing cultures (similar0.4 x 107 cells/ml) were diluted serially, spotted onto YE5S agar and incubated for 2 --3 d at 30C or spotted onto YE5S agar supplemented with CaCl2 or KCl (at the indicated concentration) and incubated for 3 --4 d at 30C. Strains used were wild type (w.t.) (NT4), tup11Delta tup12Delta (SW76), cta3Delta (SW95), and cta3Delta tup11Delta tup12Delta (SW93). To confirm that increased level of cta3+ transcripts associated with deletion of tup genes was due to an effect on transcription and not mRNA stability, we measured the expression of an integrated cta3+ promoter ---lacZ reporter . It is highly unlikely that Tup repressors would specifically influence the stability of lacZ transcripts. Consistent with the Northern analysis, deletion of both the tup genes resulted in 14-fold increase in expression of the lacZ reporter relative to the wild-type control (Figure C). Furthermore, exposure of cells to high KCl concentrations (0.6 M for 1 h) increased the level of expression sevenfold in wild-type cells and threefold in the cells lacking the Tup repressors (Figure C). These results suggest that S. pombe Tup proteins exert their effects at the level of transcription. To determine whether the high level of cta3+ expression observed in the tup11Delta tup12Delta double mutant confers the increased sensitivity of this strain to elevated K+ and Ca2+ concentrations we examined the effect of deleting the cta3+ gene in the presence or absence of the tup genes. Loss of Cta3 function has previously been reported to result in increased sensitivity to elevated Ca2+ concentrations . In contrast, found that cta3 null cells did not exhibit any detectable change in resistance to K+, Ca2+, or Na+ ions. In agreement with the latter study our cta3Delta mutant exhibited wild-type levels of resistance to both Ca2+ and K+ (Figure D). Moreover, deletion of the cta3+ gene in a tup11Delta tup12Delta strain did not rescue the salt-sensitive phenotype associated with the loss of the tup genes (Figure D), and plasmid-mediated overexpression of cta3+ in wild-type cells did not result in any increased sensitivity to KCl or CaCl2 (our unpublished data). These results indicate that the salt sensitivity of tup- cells is not simply due to the elevated expression of the cta3+ gene. To date, the fbp1+ gene encoding fructose 1,6-bisphosphatase is the only gene that has been identified as a target gene for Tup11-Tup12 --mediated repression . The expression of fbp1+ is also positively regulated by the Sty1 pathway , but its expression is induced by carbon limitation and not by other acute stresses that activate Sty1, such as heat shock, oxidative stress, and osmotic shock. Furthermore, the cAMP pathway negatively regulates the expression of fbp1+, and mutations that disrupt this pathway result in increased expression under repressing (glucose-rich) conditions . In contrast, growing cells under carbon-limiting conditions did not induce the expression of cta3+ nor were mRNA levels influenced by a deletion of git2+ that encodes adenylate cyclase (our unpublished data). Formation of Protein Complexes on the cta3+ Promoter | Studies have indicated that Atf1 binds constitutively to a CRE binding site in the gpd1+ promoter . In contrast, EMSAs of the fbp1+ promoter have demonstrated that Atf1 associates with a CRE-like element in UAS1 only under activating (glucose-limiting) conditions . We therefore examined the ability of Aft1 to bind to the cta3+ promoter. Inspection of the DNA sequence revealed the presence of a number of potential CRE-like Atf1 binding sites located between -1111 and -1401 relative to the initiation codon (Figure A). We performed EMSAs by using whole cell extracts and a DNA fragment corresponding to the -1477 to -1297 region of the promoter. This region includes a near consensus CRE element and two CRE-like sequences containing the highly conserved ACGT core sequence. A major slow-migrating complex was formed on this probe (Figure B). This binding activity was not changed by subjecting cells to stress (KCl 0.6 M for 15 min) before extract preparation. The complex was also present in extracts derived from atf1Delta cells, indicating that it does not require Atf1. Furthermore, the mobility of the complex was unchanged when HA antibody was included in reactions containing HA epitope-tagged Atf1 (our unpublished data). Next, we examined the ability of complexes to form on a probe corresponding to the -1249 and -1058 region of the promoter that contains a single CRE element. In this case, we also observed a binding activity that was Atf1-independent (Figure C). However, we also detected a slow-migrating complex that was absent in reactions lacking Atf1. Also, the mobility of this complex was reduced by the addition of the HA antibody to reactions containing HA epitope-tagged Atf1. This Atf1-dependent complex was present in reactions using extracts derived from unstressed and stressed cells, indicating that at least under these experimental conditions Atf1 binds constitutively to this region of the cta3+ promoter. We were unable to properly assess the role of Tup proteins on DNA binding activity; when tup- extracts were used a marked reduction in the level of complex formation on both probes was observed. However, this seemed to be due to difficulties in preparing extracts from these cells rather than a specific effect because we found that extracts lacking Tup proteins also showed a reduced ability to form complexes on a DNA probe unrelated to the cta3+ promoter (our unpublished data). Figure 3 | Complexes formed on the cta3+ promoter. Complexes formed on the cta3+ promoter. (A) Schematic of the CRE-like elements in the cta3+ promoter and the probes used for EMSAs. Consensus or near consensus CRE elements are shaded black, and the CRE-like sequences containing an ACGT core are shaded gray. Their locations are given relative to the initiation codon. (B) EMSAs were performed with probe 1 by using whole cell extracts derived from exponentially growing atf1HA (KS14709), w.t. (NT4), or atf1Delta (NT147) cells as indicated above the lanes. The extract used in lane 3 was derived from atf1HA6H cells treated with KCl (0.6 M for 15 min) immediately before extract preparation. Lane 1 is a probe alone control. (C) EMSAs were performed with probe 2 by using extracts derived from exponentially growing cells (lanes 2, 4, and 5) or cells treated with KCl (0.6 M for 15 min) immediately before extract preparation (lane 3). The identity of the extract is indicated above the lane. The reaction in lane 4 contained 0.2 mug of HA antibody. Lane 1 is a probe alone control. Arrows mark the positions of the Atf1-dependent shifted (S) and supershifted (SS) complexes. Tup11 and Tup12 Interact | Our data indicate that Tup11 is capable of repressing cta3+ expression in the absence of Tup12 and vice versa. It is also known that S. cerevisiae Tup1 tetramerizes through its N-terminal domain and based on homology it is very likely that the S. pombe Tup proteins form homotetramers. However, it is possible that in addition to functioning in homomeric complexes Tup11 and Tup12 may also function in a heteromeric complex. Therefore, we investigated the ability of Tup11 and Tup12 to interact using a coprecipitation assay. Whole cell extracts were prepared from wild-type cells that expressed 6His-tagged Tup11 (or Tup12) and coexpressed HA-tagged Tup11 (or Tup12). Ni2+-agarose was then used to precipitate His-tagged Tup proteins, and the presence of HA-tagged Tup proteins was examined by Western blotting . In these experiments, Tup11 copurified with Tup12 and vice versa, indicating that Tup11 and Tup12 physically interact. The specificity of this interaction was demonstrated by the absence of HA-tagged Tup proteins in control precipitates derived from cells extracts expressing the empty 6His vector (Figure , lanes 7 and 8). Thus, Tup11 and Tup12 have the potential to regulate gene expression in the same protein complex. Figure 4 | Tup11 and Tup12 coprecipitate. Tup11 and Tup12 coprecipitate. Whole cell extracts were prepared from wild-type cells containing plasmids expressing epitope-tagged Tup proteins: pRep42-HisMycTup12 (lanes 1 --3), pRep42-HisMycTup11 (lanes 4 --6), pRep42-HisMyc empty vector (lanes 7 and 8), pRep41-HATup12 (lanes 1, 4, and 7), and pRep41-HATup11 (lanes 2, 5, and 8). Extracts were precipitated with Ni2+-agarose analyzed on 8% SDS polyacrylamide gels and subjected to Western blotting by using HA monoclonal antibody. Tup Repressors Link Transcriptional Induction to the Sty1 Pathway | To test whether the high level of cta3+ expression observed in the absence of the Tup repressors was dependent upon the Sty1 MAP kinase cta3+ mRNA levels were examined in a strain that lacks both Sty1 and Tup function (sty1-1 tup11Delta tup12Delta). In this mutant the level of cta3+ transcripts was similar to that observed in tup11Delta tup12Delta cells, indicating that Sty1 is not required for basal levels of expression (Figure , A and B). Surprisingly, exposure of this strain to a KCl-mediated shock resulted in induction of cta3+ expression, indicating that in the absence of the Tup proteins the Sty1 MAP kinase is not required for the stress-mediated induction of cta3+. Consistent with these observations the expression of cta3+ was also induced by salt shock (0.6 M KCl) in a sty1Delta tup11Delta tup12Delta strain (our unpublished data). The expression of other genes such as pyp2+ and gpd1+ was not induced in the sty1-1 tup11Delta tup12Delta triple mutant, although deletion of the tup genes did restore the basal level of expression in sty1- cells (Figure A). The kinetics of KCl-mediated induction of cta3+ were similar in wild-type and tup11Delta tup12Delta cells, with mRNA levels peaking at 20 min but elevated mRNA levels persisted for a greater length of time in cells lacking the Tup proteins (Figure , C and D). In the sty1-1 tup11Delta tup12Delta triple mutant strain induction was delayed and peak mRNA levels were not observed until 30 min after the addition of KCl. Figure 5 | Deletion of tup11+ and tup12+ allows Sty1-independent transcriptional induction of cta3+. (A) Strains used are indicated above the lanes and were wild type (w.t.) (NT4), sty1-1 (JM1144), sty1-1 tup12Delta (SW88), sty1-1 tup11Delta (SW89), tup11Delta tup12Delta (SW76), and sty1-1 tup11Delta tup12Delta (SW90). Deletion of tup11+ and tup12+ allows Sty1-independent transcriptional induction of cta3+. (A) Strains used are indicated above the lanes and were wild type (w.t.) (NT4), sty1-1 (JM1144), sty1-1 tup12Delta (SW88), sty1-1 tup11Delta (SW89), tup11Delta tup12Delta (SW76), and sty1-1 tup11Delta tup12Delta (SW90). Log phase cultures growing at 30C in YE5S (lanes 1, 3, 5, 7, 9, and 11) were incubated with KCl (to a final concentration of 0.6 M) for 15 min (lanes 2, 4, 6, 8, 10, and 12). Total RNA was extracted, separated by electrophoresis, and analyzed by Northern blotting with the indicated probes. The level of his3+ mRNA was used as a loading control. (B) Quantification of the cta3+ mRNA levels in A. (C) Kinetics of the induction of cta3+ mRNA in response to KCl shock. Log phase cultures growing at 30C in YE5S (lanes 1, 7, and 13) were incubated with KCl (to a final concentration of 0.6 M) for 10 min (lanes 2, 8, and 14), 20 min (lanes 3, 9, and 15), 30 min (lanes 4, 10, and 16), 40 min (lanes 5, 11, and 17), or 50 min (lanes 6, 12 and 18). The strains used were wild type (w.t.) (NT4), tup11Delta tup12Delta (SW76), and sty1-1 tup11Delta tup12Delta (SW90). Total RNA was extracted, separated by electrophoresis, and analyzed by Northern blotting with the indicated probes. (D) Quantification of the cta3+ mRNA levels in C. We next determined whether removal of Tup11 and Tup12 rescued any of the other phenotypes associated with loss of Sty1. We examined the ability of cells to grow on medium supplemented with cadmium. Deletion of the tup genes in a sty1+ background increases resistance to cadmium (Figure A) but unexpectedly deletion of tup11+ and tup12+ in a sty1-1 background reduced cadmium tolerance (Figure A). Thus, the resistance of tup- cells to cadmium depends on Sty1 function and in its absence they become hypersensitive. The elongated cell morphology of sty1-1 cells that is indicative of a G2 cell cycle delay was slightly exacerbated by deletion of the tup genes (Figure B). Furthermore, deletion of the tup genes in an aft1Delta or a sty1-1 background resulted in a small increase in sensitivity to KCl and the tup11Delta tup12Delta atf1Delta triple mutant strain was slightly less tolerant to CaCl2 than the parental strains (Figure C). Figure 6 | Genetic interactions. Genetic interactions. (A) Deletion of the tup genes does not rescue phenotypes associated with the sty1-1 mutation. The indicated strains were subcultured onto YE5S agar (control) or subcultured onto YE5S agar supplemented with CdSO4 (to the indicated concentration) and incubated at 30C for 3 --4 d. (B) Comparison of the morphology of wild type (w.t.) (NT4), sty1-1 (JM1144), tup11Delta tup12Delta (SW76), and sty1-1 tup11Delta tup12Delta (SW90) cells. (C) Exponentially growing cultures were diluted serially, spotted onto YE5S agar or YE5S agar supplemented with CaCl2 or KCl (at the indicated concentration), and incubated for 2 d at 30C. The strains used were wild type (w.t.) (NT4), tup11Delta tup12Delta (SW76), sty1-1 (JM1144), sty1-1 tup11Delta tup12Delta (SW90) atf1Delta (NT147), and atf1Delta tup11Delta tup12Delta (SW92). We next addressed whether removal of Tup11-Tup12 repression rendered the induction of cta3+ independent of Atf1. Deletion of atf1+ in a tup11Delta tup12Delta mutant strain resulted in a further increase in cta3+ transcript levels in unstressed cells, suggesting that nonactivated Atf1 may have a repressive effect on transcription that is independent of Tup11 and Tup12 (Figure , A and B). A similar effect has been observed previously; the decrease in the basal level of ctt1+ mRNA associated with loss of Sty1 function is suppressed by deletion of atf1+ . In the atf1Delta tup11Delta tup12Delta background, cta3+ mRNA levels did not increase after exposure to KCl (0.6 M), indicating that Atf1 is absolutely required for induction of cta3+ in response to a salt shock. Figure 7 | Transcription induction of cta3+ in cells lacking Tup11 and Tup12 is Atf1 dependent. Transcription induction of cta3+ in cells lacking Tup11 and Tup12 is Atf1 dependent. (A) Strains used are indicated above the lanes and were wild type (w.t.) (NT4), atf1Delta (NT147), tup11Delta tup12Delta (SW76), and atf1Delta tup11Delta tup12Delta (SW92). Log phase cultures growing at 30C in YE5S (lanes 1, 3, 5, and 7) were incubated with KCl (to a final concentration of 0.6 M) for 15 min (lanes 2, 4, 6, and 8). Total RNA was extracted, separated by electrophoresis and subjected to Northern analysis with the indicated probes. The level of cdc2+ mRNA was used as a loading control. (B) Quantification of cta3+ mRNA levels in A. (C) As for A, except strains used were wild type (w.t.) (NT4), pcr1Delta (JX25), tup11Delta tup12Delta (SW76), and pcr1Delta tup11Delta tup12Delta (RJP59). (D) Quantification of cta3+ mRNA levels in C. The bZIP transcription factor Pcr1 that can heterodimerize with Atf1 is also required for stress-mediated induction of cta3+ expression (Figure , C and D). Examination of cta3+ mRNA levels in a pcr1Delta tup11Delta tup12Delta strain revealed that Pcr1 is not required for the high level of basal expression, and furthermore in this strain expression of cta3+ was partially induced in response to a salt shock. Thus, the Tup repressors ensure that induction of cta3+ remains dependent upon the Sty1 MAP kinase and to a lesser extent, the activator Pcr1. Prr1 Is Involved in Regulation of Gene Expression in Response to Elevated K+ Ions | Our analysis suggests that another factor may regulate transcription of cta3+ independently of Sty1. The Prr1 transcription factor is known to regulate oxidative stress responsive genes , but there is no evidence that Sty1 regulates its activity directly. Therefore, we analyzed mRNA levels in a prr1Delta strain and found that the level of cta3+ transcripts after exposure to KCl was significantly reduced in comparison with the wild-type strain (Figure , A and B). Furthermore, the influence of Prr1 was not confined to the cta3+ gene because KCl-mediated induction of both ctt1+ and gpx1+ expression was also significantly reduced in the prr1Delta mutant strain. This was surprising because Prr1 has previously been reported not to be involved in the transcriptional response to high salt and indeed KCl-mediated induction of some genes such as gpd1+ occurs independently of Prr1 (; Figure , A and B). To determine the role that Prr1 plays in control of cta3+ expression, we measured cta3+ mRNA levels in a tup11Delta tup12Delta prr1Delta triple mutant strain. In this background the expression of cta3+ was induced by exposure to high concentrations of KCl (0.6 M) (Figure , C and D). However, the deletion of prr1+ in a tup11Delta tup12Delta background resulted in a decrease in the basal level of cta3+ mRNA (Figure , C and D). Thus, Prr1 activity contributes to the high basal level of expression that is associated with loss of the Tup repressors. Figure 8 | Prr1 is involved in the regulation of gene expression in response to high salt. Prr1 is involved in the regulation of gene expression in response to high salt. (A) Strains used are indicated above the lanes and were wild type (w.t.) (NT4) and prr1Delta (SW97). Log phase cultures growing at 30C in YE5S (lanes 1 and 3) were incubated with KCl (to a final concentration of 0.6 M) for 15 min (lanes 2 and 4). Total RNA was prepared and subjected to Northern analysis with the indicated probes. The level of his3+ mRNA was used as a loading control. (B) Quantification of the mRNA levels in A. (C) Strains used are indicated above the lanes and were tup11Delta tup12Delta (SW76) and prr1Delta tup11Delta tup12Delta (SW96), and the treatment was as described in A. (D) Quantification of the mRNA levels in C. In vitro experiments have demonstrated that recombinant Prr1 binds to a heat shock-like element in the ste11+ promoter . Analysis of the cta3+ promoter revealed the presence of such an element (GGAAAATTC) located at -2068 relative to the initiation codon. However, in assays using this region of the promoter and whole cell extracts we were unable to detect a Prr1-dependent binding activity (our unpublished data). Therefore, we cannot exclude the possibility that the role of Prr1 in regulation of cta3+ expression is indirect. Tup11 and Tup12 Prevent Induction in Response to Inappropriate Stresses | Sty1, and thus in turn Atf1-Pcr1, is activated in response to a number of environmental insults. Accordingly, the expression of Atf1- and Pcr1-dependent genes such as ctt1+, pyp2+, and gpx1+ are induced in response to a variety of stresses such as UV irradiation, heat shock, and hyperosmolarity and an oxidative stress elicited by exposure to high concentrations of H2O2 . However, some Atf1-Pcr1 target genes are induced only by a subset of these stresses. For example, we found that cta3+ expression was induced specifically in response to salt shock but not by oxidative stress (6 mM H2O2) or by heat shock (15 min at 42C) (Figure , A and D). In contrast, ctt1+ and gpx1+ mRNA levels were both induced by these treatments, indicating that Atf1 (and Pcr1) were active under these conditions. This indicates that activation of the Sty1 pathway per se is not sufficient to induce the expression of cta3+ and mechanisms must exist to prevent induction of gene expression in response to such "inappropriate stresses." We wanted to examine the possibility that Tup11 and Tup12 play a role in this process. Therefore, we measured the levels of cta3+ mRNA after exposing a tup11Delta tup12Delta strain to an oxidative stress (6 mM H2O2) and a heat shock (15 min at 42C). In contrast to the wild-type strain, the expression of cta3+ was significantly induced by heat stress and by exposure to high levels of H2O2. In addition, the level of cta3+ transcripts was induced by hypotonic conditions in a tup11Delta tup12Delta strain but not in a wild-type strain (our unpublished data). We also examined cta3+ transcript levels in a sty1Delta tup11Delta tup12Delta triple mutant strain (Figure , B and D). This revealed that the induction in expression in response to heat shock was partly independent of the MAP kinase. In contrast, the induction of cta3+ expression in response to oxidative stress mediated by H2O2 was completely dependent upon Sty1, suggesting a difference in the mechanism of induction. Further analysis indicated that the response to heat shock was independent of Prr1 (Figure , C and D) but dependent upon Atf1 (our unpublished data). Taken together, these findings indicate that the Tup repressors function as part of the mechanism that ensures the specificity of stress-mediated transcriptional induction at the cta3+ promoter. Figure 9 | Tup11 and Tup12 prevent induction in response to inappropriate stresses. Tup11 and Tup12 prevent induction in response to inappropriate stresses. (A) Strains used were wild type (w.t.) (NT4) and tup11Delta tup12Delta (SW76). Mid log cultures growing at 30C in YE5S (lanes 1 and 4) were incubated with H2O2 (final concentration 6 mM) for 15 min (lanes 2 and 5) or shifted to 42C for 15 min (lanes 3 and 6). Total RNA was prepared and subjected to Northern analysis with the indicated probes. The level of cdc2+ mRNA was used as a loading control. (B) Strain used was sty1Delta tup11Delta tup12Delta (SW91) and the treatments were as described in A. (C) Strain used was prr1Delta tup11Delta tup12Delta (SW91) and the treatments were as described in A. (D) Quantification of the cta3+ mRNA levels in A, B, and C. The strains are indicated above the graphs and the treatments are indicated below. PMID- 12221110_DISCUSSION TI - AB - In this study, we reveal roles for S. pombe Tup11 and Tup12 in the cellular response to elevated K+ and Ca2+ levels. We identify cta3+ as a novel stress-induced gene whose transcription is coregulated by the Sty1 MAP kinase pathway and the Tup repressors. Our results indicate that the Tup repressors fulfill a number of functions in the control of cta3+ expression. First, they maintain low levels of basal expression and limit the level of induction. Second, they ensure that induction of expression is linked to the Sty1 pathway. And third, they maintain the specificity of induction. We also reveal a new role for the response regulator Prr1 and demonstrate that it functions to regulate gene expression in response to elevated salt concentrations. Prr1 is known to contribute to the regulation of several genes whose expression is induced by oxidative stress via the Pap1 transcription factor , and so Prr1 regulates gene expression in response to a number of stresses. Tup11 --Tup12 Interaction | Our data and that of others suggest that Tup11 and Tup12 can function in homomeric complexes. In addition, we demonstrate that Tup11 and Tup12 have the potential to form a heteromeric complex. This is significant because full function requires both repressors; some derepression of a fbp1::lacZ reporter is observed upon deletion of a single tup gene . Furthermore, single tup mutants have demonstrable phenotypes such as increased resistance to cadmium. Thus, regulation of some genes may depend upon both repressors and the formation of heteromeric Tup complexes. Relief of Tup11-Tup12 --mediated Repression | The Hog1 MAP kinase in S. cerevisiae plays a direct role in relieving Ssn6-Tup1 --mediated repression at osmostress genes; Hog1 phosphorylates Sko1, reducing its affinity for the corepressor complex . It is possible that the Sty1 MAP kinase may similarly antagonize the action of Tup11-Tup12; however, our results demonstrate that the Atf1, Pcr1, and Prr1 transcription factors are required for relief from Tup-mediated repression at the cta3+ promoter. It is probable that the S. pombe Tup proteins function, at least in part, through the organization of repressive chromatin structures , and therefore it is possible that Atf1-Pcr1 and Prr1 overcome this repression by recruiting positive-acting chromatin remodeling complexes such as Swi-Snf or histone acetylase complexes (HATs). In support of this, DNA binding by the Atf1-Pcr1 heterodimer is known to alter local nucleosome positioning at the ade6-M26 hotspot and thereby promote meiotic recombination . Moreover, genes such as SUC2 in S. cerevisiae are regulated by the interplay between Ssn6-Tup1 repression and Swi-Snf --mediated activation . In S. cerevisiae, Hog1-dependent transcriptional induction of HAL1 requires the Gcn4 activator that relieves Tup1-Ssn6 --mediated repression by competing with Sko1 for the occupancy of a single CRE binding site . This CRE element functions as a dual control element and integrates both positive and negative regulatory signals. Furthermore, analysis of the S. pombe fbp1+ promoter, which is regulated by both Atf1-Pcr1 and Tup11-Tup12, has demonstrated the presence of a control element (called UAS2) that contains a CRE-like sequence and is bound by multiple activators and repressors . Interestingly, the Atf1-Pcr1 transcription factor does not bind to UAS2 directly, but it does influence the protein complexes that assemble on it . The cta3+ promoter contains a number of CRE-like elements at least one of which mediates Atf1 binding (Figure C). It will be interesting to determine the contributions of these elements to activation and repression of cta3+ transcription. Tup Repressors Maintain Specificity of Induction | The advent of stressful conditions results in the rapid and Sty1-dependent phosphorylation of Atf1 . Although the precise role of Atf1 phosphorylation remains obscure, it is evident that transcriptional activation by Atf1 is dependent upon Sty1. However, deletion of the tup genes allows transcriptional induction of cta3+ to occur in sty1- cells. Furthermore, in a tup11Delta tup12Delta mutant induction of cta3+ expression does not require Pcr1 or Prr1. Thus, the Tup repressors function to "wire" induction to the Sty1 pathway, insulating it from interfering signals. These results also suggest that Atf1 activity can be "uncoupled" from Sty1 in this specific case and that an additional mechanism for activating transcription that requires Atf1 exists. The finding that Prr1 also controls expression of cta3+ suggests that it may function as part of this mechanism. The Sty1 pathway in S. pombe is fundamentally different to the Hog1 pathway in S. cerevisiae because it is triggered by exposure to a wide range of adverse environmental conditions. As a consequence, a large number of Sty1 target genes are up-regulated by multiple stresses. The products of such genes may comprise a set of "general stress response proteins" that are necessary because a single environmental insult may result in multiple classes of intracellular stress . Nonetheless, discrete stimuli also produce distinct transcriptional outputs, because there are subsets of Sty1-dependent genes, such as cta3+, that are induced only by specific stresses. A major question to be addressed is the mechanism by which Sty1 signaling is integrated into the regulation of such genes. Expression of cta3+ is not induced by oxidative stress, heat shock, carbon limitation, or sexual differentiation (Figure ; our unpublished data), and furthermore cta3+ is only poorly induced by an osmotic shock mediated by high sorbitol concentrations . Thus, the transcriptional response is triggered essentially by elevated intracellular cation concentrations rather than by an osmotic effect (i.e., decrease in turgor pressure across the plasma membrane). The cta3+ gene encodes a putative intracellular P-type ATPase transporter that is involved in cation extrusion or sequestration into intracellular compartments. Loss of function leads to an accumulation of cytoplasmic Ca2+ levels , although recent evidence suggests that Cta3 is primarily a K+ ion pump . It is thus consistent that it is salt stress that specifically that triggers its transcriptional induction. However, removal of the constraints imposed by Tup repressors allows cta3+ to be induced in response to other stresses such as elevated temperature and oxidative stress. Thus, the Tup repressors function as a part of a mechanism that adds specificity to Sty1-dependent transcriptional induction. Our results also indicate that activation of the Sty1 pathway alone is insufficient to induce cta3+ expression and implies that an elevated cation concentration triggers an additional pathway that is required to circumvent repression . In this respect it may be significant that Prr1 is involved in the regulation of cta3+ expression because its structure suggests that it may be one part the target of a histidine-aspartate phosphorelay pathway. Recent work has identified several of these pathways in fission yeast , and current experiments are addressing its contribution to the regulation of Prr1 in the response to stress. Figure 10 | Model for the regulation of cta3+ expression. Model for the regulation of cta3+ expression. Under nonstress conditions, cta3+ expression is repressed by Tup11 and/or Tup12 that are tethered to the promoter through interaction with a site-specific DNA binding protein "X." Activation of the Sty1 pathway alone is insufficient to induce expression, and the Tup repressors prevent activation by Atf1-Pcr1 and Prr1. Elevated Ca2+ or K+ concentrations trigger the activity of other pathways (indicated by dashed lines) that interfere with Tup repression and/or facilitate activation via the response regulator Prr1 and Atf1-Pcr1. In cells lacking the Tup repressors, specificity is lost and expression is induced in response to a range of stresses. PMID- 12221111 TI - The Roles of Bud-Site-Selection Proteins during Haploid Invasive Growth in Yeast AB - | In haploid strains of Saccharomyces cerevisiae, glucose depletion causes invasive growth, a foraging response that requires a change in budding pattern from axial to unipolar-distal. To begin to address how glucose influences budding pattern in the haploid cell, we examined the roles of bud-site-selection proteins in invasive growth. We found that proteins required for bipolar budding in diploid cells were required for haploid invasive growth. In particular, the Bud8p protein, which marks and directs bud emergence to the distal pole of diploid cells, was localized to the distal pole of haploid cells. In response to glucose limitation, Bud8p was required for the localization of the incipient bud site marker Bud2p to the distal pole. Three of the four known proteins required for axial budding, Bud3p, Bud4p, and Axl2p, were expressed and localized appropriately in glucose-limiting conditions. However, a fourth axial budding determinant, Axl1p, was absent in filamentous cells, and its abundance was controlled by glucose availability and the protein kinase Snf1p. In the bud8 mutant in glucose-limiting conditions, apical growth and bud site selection were uncoupled processes. Finally, we report that diploid cells starved for glucose also initiate the filamentous growth response. PMID- 12221111_ TI - Glossary AB - Abbreviations used: : DIC = differential interference contrast FITC = fluorescein GFP = green fluorescent protein HA = hemagglutinin Rh = rhodamine SC = synthetic complete URA = uracil YPD = yeast peptone dextrose wt = wild-type PMID- 12221111_INTRODUCTION TI - AB - Cells of the yeast S. cerevisiae can undergo a developmental switch from a yeast form of growth to a filamentous form of growth (; for reviews, see ; ). In haploid cells, one of the triggers for the switch to the filamentous form is glucose starvation . The developmental switch to filamentation has at least three components. First, cells change their budding pattern. For example, haploid cells switch from an axial budding pattern, in which new buds emerge at sites adjacent to the birth scar or the site of the preceding bud, to a unipolar-distal budding pattern, in which new buds emerge at the pole distal to the birth scar . Second, the cells become elongated. Third, the cell surface changes, enabling the cells to adhere to each other and to invade the agar substratum. In this article, we investigate the requirements for the change in budding pattern associated with filamentation in haploid cells. Are proteins required for bud site selection in yeast-form cells necessary for the budding pattern observed during filamentation? If so, which ones, and how are their activities modulated to affect the unipolar pattern during filamentation? In vegetative cells, the budding pattern is controlled by cell type (; ; ; for reviews, see ; ; ; ; ; ; ,). As described above, haploid cells bud in an axial pattern. Diploid cells, on the other hand, bud in a bipolar pattern. A new bud can emerge from either the birth scar pole or the distal pole, although there is a bias for distal pole budding in the first bud formed . A GTPase module is required for cells to display either of these budding patterns; in its absence, cells bud in a random pattern . The module is composed of a RAS-like GTPase, Rsr1p/Bud1p; its GTPase-activating protein, Bud2p; and its guanine nucleotide exchange factor, Bud5p . Bud2p and Bud5p are localized to axial positions in haploid cells and bipolar positions in diploid cells , where they direct bud emergence, in part through interaction with polarity establishment proteins . The recruitment of the GTPase module to the appropriate site is controlled by other bud-site-selection proteins. In haploid cells, axial budding requires Bud3p, Bud4p, and Axl2p/Bud10p/Sro4p . These proteins are localized to the mother-bud neck and together recruit Bud5p to the axial position . In addition, Axl1p is a haploid-specific protein required for axial budding . Loss of Bud3p, Bud4p, Axl2p, or Axl1p causes bipolar budding in haploid cells, but does not affect budding pattern in diploid cells. A different set of factors is required to orchestrate bipolar, rather than axial, budding in diploid cells. Genetic studies suggest that Bud8p and Bud9p mark the poles distal and proximal to the birth scar, respectively . For example, mutants deleted for BUD8 bud exclusively from the proximal pole. Moreover, green fluorescent protein (GFP)-tagging and immunofluorescence studies reveal that Bud8p is located at the distal pole and Bud9p at the proximal pole, implying that they may comprise part of the marks that identify these poles to the GTPase module . In addition, Bud6p and Bni1p, which form a protein complex , are also required for bipolar budding . Loss of any of these four proteins disrupts bipolar budding in diploid cells, but does not affect axial budding in haploid cells. Pea2p and Spa2p are also components of the Bud6p/Bni1p protein complex and are important determinants of bipolar budding and of polarized growth . The switch in budding pattern during filamentous growth is particularly striking in the case of the axial-to-unipolar transition of haploid cells deprived of glucose . We show herein that Bud8p is localized to the distal tip of haploid cells, and under glucose-limiting conditions it directs bud emergence to the distal pole. Glucose depletion results in the Snf1p-dependent disappearance of Axl1p, providing one mechanism by which glucose modulates budding pattern in haploid cells. PMID- 12221111_MATERIALS AND METHODS TI - AB - Strains, Plasmids, and Microbiological Techniques | The yeast strains used in this study are listed in Table . All of the strains were derived from HYL333 and HYL334 of the filamentous Sigma1978b background (provided by G. Fink, Whitehead Institute for Biomedical Research, Cambridge, MA); these strains exhibit particularly robust filamentous growth compared with other strains from the Sigma1978b background (H. Madhani, UCSF, San Francisco, CA; personal communication). To construct SY3687 and SY3688, HYL334 was made his3::URA3 or leu2::URA3, by using a polymerase chain reaction (PCR)-based method (, and references therein) and plasmid pRS306 as a template . The resulting strains were then made Ura3- by selection on 5-fluoroorotic acid (Biovectra, Oxford, CT). Disruption of BNI1 was performed using plasmid p321, provided by C. Boone . Disruptions of PEA2 and SPA2 were performed using plasmids pNV44 and p210, provided by I. Herskowitz . The plasmid used to disrupt GRR1, pBM2101, was provided by M. Johnston . Other gene disruptions were performed by PCR-based methods (, and references therein) to remove the entire open reading frame with plasmids described by , or other plasmids containing auxotrophic markers from Candida glabrata (for LEU2 and HIS3) and Kluyveromyces lactis (for URA3) and that were provided by I. Herskowitz. Integrated GFP fusions and GAL1 promoter fusions were made by PCR-based methods with plasmids provided by J. Pringle . Gene disruptions and integrated promoter and protein fusions were confirmed by PCR analysis and by phenotype. All of the GFP- and hemagglutinin (HA)-tagged fusion proteins used in this study were functional with respect to bud site selection and invasive growth phenotypes. Table 1 | Yeast strains Yeast and bacterial strains were propagated using standard methods . Yeast peptone dextrose (YPD) and synthetic complete dextrose (SCD) media have been described previously . Yeast transformations were performed as described previously . Bacterial transformations, bacterial DNA preparations, and plasmid constructions were performed by standard methods . Genes controlled by a galactose-inducible promoter were induced in SC or YP medium containing 2% galactose (Gal) as indicated. Geneticin (Biovectra) selection was performed as described previously . Protein Localization | The localization of Bud8p was determined using plasmid YEpGFP*-BUD8 (provided by J. Pringle; ), in which the GFP-Bud8p fusion was expressed from its own promoter. Wild-type cells containing YEpGFP*-BUD8 were grown in synthetic medium lacking leucine (SCD-LEU) to stationary phase and spread onto SCD-LEU or SC-LEU medium for 16 h at 25C. A coverslip was placed directly onto the plates, and GFP-Bud8p was visualized by fluorescence microscopy with a fluorescein isothiocyanate (FITC) filter at 100x. The localization of GFP-Bud2p was determined using plasmid pHP726 (provided by H.-O. Park; ) carried in bud2 and bud2 bud8 strains. The localizations of Bud3p, Bud4p, and Axl2p were determined using C-terminal GFP fusions that were integrated into the genome. Actin staining was performed as described previously . Cells were incubated in SCD or SC medium and fixed in 3.7% formaldehyde for 1 h. Fixed cells were incubated with rhodamine (Rh)-phalloidin (Molecular Probes, Eugene, OR). Cells were washed twice and visualized by florescence microscopy at 100x by using an Rh filter. Invasive Growth Assays | The single cell invasive growth assay was performed as described previously . For some experiments, cells were scraped from plates by using 4 ml of distilled water, concentrated by centrifugation, resuspended in 20 mul of water, and visualized by microscopy. For other experiments, a coverslip was placed directly on the agar medium and cells were visualized directly by microscopy. The plate-washing assay was performed essentially as described previously . Equal concentrations of cells were spotted onto YPD or YPGal medium as specified, invasion was allowed to proceed for 2 d at 30C, and then plates were washed vigorously with water and rubbed with a wet finger to remove cells that did not invade the agar. In some cases, invasion was allowed to proceed for 5 d, during which time the cells became more than twice as elongated as observed in the single cell assay. Cell-cell adhesion was assessed by a standard flocculation assay, as described previously . Microscopy | Differential interference contrast (DIC) and fluorescence microscopy with Rh and FITC filter sets were performed using an Axioplan 2 microscope (Zeiss, Jena, Germany), a black-and-white Orca II digital camera (Hamamatsu, San Jose, CA), and the Openlab software program (Improvision, Coventry, UK). Only brightness and contrast digital adjustments were performed on photographs. Budding Pattern Analysis | Budding pattern determination was performed as described previously , with the following modifications. Equal concentrations of cells were spotted onto YPD medium and incubated for 2 d at 30C. Plates were washed, and invaded cells were excised from the agar by using a toothpick. Cells were resuspended in water containing 1 mug/ml calcofluor (Sigma-Aldrich, St. Louis, MO), and after a 10-min incubation, bud scars were visualized directly by fluorescence microscopy. The enhanced cohesion of cells in the filamentous background facilitated the distinction between proximal and distal bud scars by their position relative to the cell-cell orientation. A bud scar was scored as distal if it was at the pole opposite to the birth scar, or if it was present at the distal pole of a cell that comprised a filament whose growth direction was obvious. A bud scar was scored as proximal if it was at the same pole as the birth scar or at the same pole as the attached parent cell. Bud scars in the middle third of the cell were scored as equatorial. At least 200 bud scars were scored for each experiment. Previously, wild-type cells in glucose-limiting conditions were shown to bud at the distal pole for 95% of all first buds . Subsequent buds were more frequently observed at the proximal pole. In the bud scar counts in the present work, all budding events were considered, resulting in the lower percentage of bud scars observed at the distal pole (similar70%). Budding patterns were corroborated by using the single cell invasive growth assay . Equal concentrations of cells were spread onto SCD or SC medium, and budding pattern was assessed directly by microscopic examination. Microcolonies at the 10-cell stage or less were chosen for analysis. For some experiments cells were placed onto SCD or SC medium by micromanipulation and allowed to grow to the 10-cell stage, which showed the exact lineage of cells within the microcolony. The precise position of bud placement was determined for a subset of experiments by photographing cells and aligning the photographs to an arbitrary model cell. Western Blot Analysis and Determination of Axl1p Abundance | Western blots were performed as described previously . Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose, and visualized by probing with antibodies specific to GFP (Roche Applied Science, Indianapolis, IN), HA, or Dpm1p (provided by Tom Stevens, Institute of Molecular Biology, University of Oregon, Eugene, OR), which served as a loading control. Band intensity was determined using ImageQuant software (Amersham, Piscataway, NJ), and, where indicated, the values reported were normalized to Dpm1p levels. The abundance of Axl1p was measured using plasmid p151 (provided by C. Boone; ), which expresses a functional Axl1p-HA fusion protein expressed from the AXL1 promoter. In some experiments, Axl1p-HA abundance in yeast-form and filamentous cells was determined by incubating wild-type cells containing p151 (SY3718) on SCD-URA or SC-URA solid agar medium. Cells were harvested from plates, resuspended in water, and adjusted to equal density by measuring optical density. Proteins were then extracted and subjected to Western analysis. In other experiments, Axl1p-HA abundance was measured through the course of a growth cycle in cells incubated in SD-URA liquid medium for various times. In addition, the glucose-limited disappearance of Axl1p-HA was measured in p151-containing wild-type (SY3718) and snf1 mutant (SY3720) cells. Cells were grown to early log phase in liquid SCD-URA medium at 30C, and each culture was split, washed twice with water, and incubated in liquid SC-URA or SCD-URA medium prewarmed to 30C for various times. PMID- 12221111_RESULTS TI - AB - Bud8p Is Required for Haploid Invasive Growth, whereas Bud9p Impedes Invasion | Disruption of BUD8 in a haploid strain of the filamentous background caused an invasive growth defect in the plate-washing assay (Figure A). In addition, both the single cell invasive growth assay and bud scar staining demonstrated that the bud8 mutant was defective in budding at the distal pole (Figure B; Table ). The mutant cells budded at the distal pole at a frequency of only 11%, in contrast to 70% for wild-type cells. The bud8 mutant cells were as elongated as wild-type cells (Figure C), suggesting that in the bud8 mutant, apical growth and the selection of budding sites were independent. The morphology of the bud8 mutant microcolony was a rosette, a morphology that contrasted strikingly with the linear form of wild-type filamentous cells (Figure C). Figure 1 | Bud8p is required for agar invasion and distal pole budding in haploid cells; Bud9p impedes invasion. Bud8p is required for agar invasion and distal pole budding in haploid cells; Bud9p impedes invasion. (A) Plate washing assay. Equal concentrations of wild-type (SY3687), bud8 (SY3689), and bud9 (SY3692) cells were spotted onto YPD medium and incubated for 2 d at 30C. The plate was photographed (left), washed, and photographed again (right). (B) Single cell invasive growth assay. Equal concentrations of wild-type (top) or bud8 mutant (bottom) cells were spread onto SC medium, incubated for 16 h at 25C, scraped from the plates, and photographed. Arrows indicate the first bud produced by the first daughter cell. (C) Prolonged incubation illustrates the difference between wild-type (left) and the bud8 mutant (right). Equal concentrations of cells were spotted onto YPD medium and grown for 5 d at 30C. The lower right panel shows bud scar (calcofluor) staining for the bud8 mutant . Bars, 5 mum. Table 2 | Budding patterns of mutants known to have defects in bipolar bud site selection during haploid invasive growth Disruption of BUD7 caused an invasive growth defect similar to that of the bud8 mutant in the plate-washing assay, although the bud7 mutant was slightly more invasive (our unpublished data). The bud7 mutant also exhibited a distal pole bud site selection defect . Disruption of bud7 and bud8 together had an invasive growth defect equivalent to either single mutant, and the double mutant had a similar (although slightly more severe) budding-pattern defect than either single mutant (Table ; our unpublished data). Taken together, these data suggest that Bud7p and Bud8p may be components of the same genetic pathway. In contrast to the noninvasive phenotype of bud7 and bud8 mutants, the bud9 mutant exhibited hyperinvasive growth (Figure A). The bud9 mutant also had a higher percentage of distal pole buds compared with wild-type cells , which may account for its hyperinvasive growth phenotype. Disruption of BUD8 in the bud9 mutant caused invasive-growth and distal-pole budding defects equivalent to those of the bud8 single mutant , consistent with the budding pattern observed in bud8 bud9 homozygous diploid cells during vegetative growth . Disruption of BUD7 also suppressed the hyperinvasive growth of the bud9 mutant but to a lesser extent than did disruption of BUD8, consistent with the phenotypes of the bud7 and bud8 single mutants . In glucose-rich conditions, haploid bud7, bud8, and bud9 mutants did not show a budding pattern defect (; ; our unpublished data). In summary, genes identified by virtue of their role in diploid cell budding pattern determination also have a role in haploid cells, specifically during invasive growth that occurs under glucose limitation. Bud8p Is Localized to Distal Pole in Haploid Cells | Bud8p is localized to the distal pole of diploid cells . We examined the localization of Bud8p in haploid cells by using a plasmid containing a functional GFP-Bud8p fusion under the control of the BUD8 promoter . GFP-Bud8p was observed at the distal pole of haploid cells grown in glucose-limiting conditions . GFP-Bud8p was also observed at the distal pole in glucose-rich conditions , a situation in which distal pole budding does not occur. Western blot analysis confirmed that the level of Bud8p was equivalent in glucose-rich and limiting conditions (our unpublished data). Thus, the Bud8p at the distal pole of haploid cells is apparently recognized only under glucose-limiting conditions. Figure 2 | Bud8p is localized to the distal tip of haploid cells, and its localization is dependent upon Bud6p and Bni1p. Bud8p is localized to the distal tip of haploid cells, and its localization is dependent upon Bud6p and Bni1p. Strain backgrounds (wild-type, SY3695; pea2, SY3698; bud6, SY3696; and bni1, SY3697) are as shown. Growth on SCD (+Glu) or SC medium (-Glu) is as indicated. Left, DIC. Right, FITC filter. All pictures were taken at the same scale; bar, 5 mum. In diploid cells, Bud8p's localization to the distal pole is dependent upon Bni1p . We examined the localization of Bud8p in bni1 and related mutants in haploid cells. Distal-pole localization of GFP-Bud8p was not observed in the bni1 mutant (<0.2% of cells had GFP-Bud8p at the distal pole compared with >50% for wild-type cells), but GFP-Bud8p was observed throughout the cell periphery . In the bud6 mutant, distal-pole localization of GFP-Bud8p was observed in a lower percentage of cells than wild type (5% of cells had GFP-Bud8p at the distal pole), and in bud6 cells in which GFP-Bud8p was at the distal pole, the fluorescence intensity was reduced . The abundance of GFP-Bud8p was equivalent in bud6, bni1, and wild-type cells (by Western blot). Consistent with the peripheral localization pattern of Bud8p, bud scar staining of invaded cells showed that bud6 and bni1 mutants had random budding patterns . No budding pattern defects were detected in the mutants in glucose-rich conditions, under which Bud8p was also mislocalized; the cells showed normal axial budding. The Pea2p and Spa2p proteins were not required for distal-pole localization of Bud8p (Figure ; our unpublished data), and pea2 and spa2 mutants maintained the unipolar budding pattern . However, the pea2, spa2, bud6, and bni1 mutants were all defective in the extended apical growth that results in elongated cells during haploid invasion . Consequently, the four mutants all exhibited an invasive growth defect. Bud8p Is Required for Distal Pole Localization of Bud2p and Actin in Glucose-limiting Conditions | Bud2p, the GTPase-activating protein for Rsr1p, has been shown to localize to the incipient bud site, where it presumably recruits Rsr1p to the bud site . In diploid cells, this localization is dependent upon Bud8p . We examined the localization of a functional GFP-Bud2p fusion (provided by Hay-Oak Park, Ohio State University, Columbus, OH) expressed from a high-copy plasmid in bud2 and bud2 bud8 strains. In glucose-limiting conditions, Bud2p was observed at the distal pole of the cell, directly underneath the emerging bud, and at the mother-bud neck of small distal buds . In contrast, GFP-Bud2p localization in a bud2 bud8 mutant was mostly at the proximal pole , a result consistent with the proximal budding pattern observed in the bud8 mutant. In glucose-rich conditions, Bud2p was observed adjacent to the previous bud site (our unpublished observations; ). Figure 3 | Bud8p is required for localization of Bud2p and actin to the distal pole in glucose-limiting conditions. Bud8p is required for localization of Bud2p and actin to the distal pole in glucose-limiting conditions. Left four panels, GFP-Bud2p localization. bud2 (top, SY3707) and bud2 bud8 (bottom, SY3708) strains containing a GFP-BUD2 fusion plasmid were grown in glucose-limiting conditions. Cells were photographed using DIC (left) or FITC filters (right). Right four panels, actin localization. Wild-type (SY3687) and bud8 mutant (SY3689) cells grown in glucose-limiting conditions were stained with rhodamine-phalloidin and photographed using DIC (left) or Rh filters (right). Arrows indicate regions of highly localized actin patches. All pictures were taken at the same scale; bar, 5 mum. Bud site selection components (e.g., Bud8p and Bud2p) are known to recruit actin to the incipient bud site, an event required for bud emergence . We examined actin localization in filamentous cells and found that in glucose-limiting conditions, actin localized to the distal tip of daughter cells . In contrast, actin accumulation was observed at the proximal pole in the bud8 mutant and in wild-type cells grown in glucose-rich conditions (our unpublished data). Thus, glucose limitation caused the localization of Bud2p and bud emergence machinery (e.g., actin) to the distal pole of the cell, an event that was dependent upon Bud8p. Proteins Required for Axial Budding Are Localized Appropriately in Filamentous Cells | We hypothesized that the disappearance of an axial cue in glucose-limiting conditions might result in Bud8p-dependent distal pole budding. In particular, the axial cues Bud3p and Bud4p are transient and are reported to disappear in nutrient-limiting conditions, because they are cell cycle regulated and absent in the Go phase of the cell cycle . The localization of the known axial cues (Bud3p, Bud4p, and Axl2p) was examined in cells grown in both glucose-rich and glucose-limiting conditions. Functional Bud3p-GFP, Bud4p-GFP, and Axl2p-GFP fusions were expressed from chromosomal loci and were found to be localized to the mother-bud neck in cells grown under both conditions (Figure A). Thus, an explanation other than axial cue disappearance must be invoked to explain the change in budding pattern to Bud8p-dependent bud site selection. Figure 4 | Localization and role of axial cues in haploid filamentous cells. Localization and role of axial cues in haploid filamentous cells. (A) Localization of axial cues in cells grown in glucose-rich and glucose-limiting conditions. Equal concentrations of cells (Bud3p-GFP, SY3709; Bud4p-GFP, SY3710; and Axl2p-GFP, SY3711) were spread onto either SCD (+Glu) or SC (-Glu) medium, incubated for 16 h at 25C, scraped from plates, and photographed using DIC (left) or FITC filters (right). Bar (all panels), 5 mum. (B) Loss of axial cues causes hyperinvasive growth. Strains were spotted onto YPD medium and incubated for 2 d at 30C. The plate was photographed (left), washed, and photographed again (right). Strains: wt (SY3687), bud3 (SY3712), bud4 (SY3713), and axl2 (SY3714). (C) Suppression of the bud8 invasive growth defect by disruption of axial cues. Strains were spotted onto YPD medium and incubated for 2 d at 30C. The plate was photographed (left), washed, and photographed again (right). Strains: wt (SY3687), bud8 (SY3689), bud8 bud3 (SY3715), bud8 bud4 (SY3716), and bud8 axl2 (SY3717). Although Bud8p is the primary bud site cue in haploid cells undergoing filamentous growth, genetic evidence suggests that axial cues are used to some degree. First, disruption of BUD3, BUD4, or AXL2 caused hyperinvasive growth (Figure B), due to a significant decrease in bud site selection at the proximal pole . Second, disruption of axial cues in the bud8 mutant ablated the proximal budding , correlating with partial suppression of the invasive growth defect (Figure C). The increased percentage of distal pole budding in the axl2 bud8 mutant, compared with the bud3 bud8 and bud4 bud8 mutants, may be due to the enhanced apical growth that was observed in axl2 mutant cells (our unpublished data). Table 3 | Budding patterns of mutants lacking axial-specific cues or both axial cues and Bud8p Control of Axl1p Abundance by Glucose and Snf1p | Because AXL1 is transcriptionally repressed in diploid cells, an event sufficient to prevent axial budding in wild-type cells , we considered the possibility that Axl1p protein abundance is regulated by glucose in haploid cells. Axl1p protein levels were measured in cells expressing an Axl1p-HA fusion from the AXL1 promoter (provided by C. Boone). We initially observed that Axl1p-HA was not present in filamentous cells (Figure A). Axl1p-HA abundance was examined in cells grown throughout a culture growth cycle. In reference to a control protein, the amount of Axl1p-HA increased steadily during early log phase and was highest in mid-log phase (Figure B). Axl1p-HA abundance declined as growth rate slowed and was significantly reduced in stationary phase (Figure B). To confirm that glucose influenced Axl1p abundance, cells containing the Axl1p-HA fusion were grown to early log phase and shifted to medium lacking glucose. As expected, the level of Axl1p-HA declined markedly upon the shift to glucose-limited medium (Figure C). Figure 5 | Axl1p abundance is controlled by glucose and Snf1p, and Axl1p is an inhibitor of invasive growth. Axl1p abundance is controlled by glucose and Snf1p, and Axl1p is an inhibitor of invasive growth. (A) Axl1p is absent in filamentous cells. Western blot of protein extracts from wild-type cells containing p151 (SY3718) incubated on SCD-URA (+Glu) or SC-URA (-Glu) solid agar medium for 16 h at 25C and probed with antibodies against HA (to detect Axl1p-HA) or Dpm1p (see MATERIALS AND METHODS). (B) Abundance of Axl1p correlates with the culture growth cycle. Wild-type cells containing p151 (SY3718) were grown through a growth cycle in SCD-URA liquid medium at 30C, and protein extracts were harvested from cells at the times indicated. Quantitation of Western blots of Axl1p levels (adjusted to Dpm1p levels) is shown (filled circles). Optical density at 600 nm is also shown (open squares). (C) Decrease in Axl1p abundance upon a shift to glucose-limited medium is dependent upon Snf1p. Wild-type cells containing p151 (SY3718) were grown to early log phase and shifted to SCD-URA (open squares, +Glu) or SC-URA (open triangles, -Glu) medium. A snf1 mutant containing p151 (SY3720) was grown in the same way (closed circles, -Glu). Quantitation of Western blots of Axl1p levels (adjusted to Dpm1p levels) is shown. (D) Hyperinvasive growth in axl1 mutants. Equal concentrations of wild-type (SY3687), axl1 (SY3721), axl1 bud8 (SY3722), and bud8 (SY3689) cells were spotted onto YPD medium and incubated for 2 d at 30C. The plate was photographed (left), washed, and photographed again (right). The colonies shown are all from the same plate. (E) Overproduction of Axl1p prevents invasive growth. Wild-type (SY3687) and GAL1-AXL1 ( AXL1, SY3723) cells were grown to saturation in YPGal medium, and equal concentrations of cells were spotted onto YPGal medium for 2 d at 30C. The plates were photographed (left), washed, and photographed again (right). The Snf1p protein kinase is a global regulator of glucose response , and we showed previously that Snf1p is required for unipolar budding during haploid invasive growth . We investigated the role of Snf1p in the glucose-dependent regulation of Axl1p. In contrast to the observations reported above for wild-type cells, Axl1p-HA protein abundance remained high in a snf1 mutant after a shift to glucose-limited medium (Figure C). Thus, Snf1p is required for the disappearance of Axl1p in glucose-limiting conditions. Genetic analysis also supports a role for Axl1p in the transition to filamentous growth. Disruption of AXL1 caused hyperinvasive growth (Figure D) due to distal pole budding in both glucose-rich and glucose-limiting conditions . These phenotypes were largely suppressed by disruption of BUD8 (Figure D and Table ). The axl1 bud8 double mutant invades better than the bud8 single mutant because it has constitutive nonaxial budding, whereas the bud8 single mutant buds almost exclusively from the proximal pole under both glucose-rich and glucose-limiting conditions. In contrast, overexpression of AXL1 suppressed agar invasion by wild-type cells (Figure E) due to an increase in proximal budding . Thus, the disappearance of the Axl1p protein in glucose-limiting conditions is sufficient to explain the Bud8p-dependent budding during haploid invasive growth. Table 4 | Budding patterns of strains deleted for AXL1 and/or BUD8 or overexpressing AXL1 Unipolar Budding in rsr1 Mutant Due to Increased Apical Growth during Haploid Invasion | It has been reported that ablation of the general bud-site-selection machinery does not disrupt the ability of haploid cells to undergo agar invasion . This result is seemingly at odds with our finding that mutants that are defective for distal-pole budding cannot invade agar. Therefore, we characterized the rsr1 mutant, which is lacking the core bud-site-selection GTPase, in detail. In the single cell invasive growth assay, the rsr1 mutant formed filaments composed of elongated cells emanating away from the mother cell, suggesting a unipolar-distal pattern (Figure A); however, buds emerging from the equatorial regions of cells were also observed (Figure A, black arrows). To determine more precisely the budding pattern of first and second buds for the rsr1 mutant, cells were placed onto glucose-limited medium by micromanipulation and budding pattern was assessed by microscopic examination. Strikingly, the first bud produced was at the distal pole >90% of the time . The second buds emerged uniformly around the entire surface of the cell . In glucose-rich conditions, a less dramatic distal-pole bias was observed in the rsr1 mutant , consistent with previous reports . Figure 6 | Distal pole budding in the rsr1 mutant during haploid invasive growth. Distal pole budding in the rsr1 mutant during haploid invasive growth. (A) Wild-type (SY3687) and rsr1 mutant (SY3724) cells were spread onto SC medium, incubated for 16 h at 25C, and photographed. Bar, 5 mum. (B) Diagram of precise position of buds as determined by microscopic examination. Microcolonies at the 10-cell stage (or less) were visualized by light microscopy and photographed. Photographs were analyzed for bud position; only buds whose precise locations were unambiguous were chosen for analysis. The position of 50 buds is shown for each depiction. (C) Bud8p is not required for agar-invasion in the rsr1 mutant. Wild-type (SY3687), bud8 (SY3689), rsr1 (SY3724), and rsr1 bud8 mutant (SY3725) cells were spotted onto YPD medium and grown for 2 d at 30C. Plates were photographed (top), washed, and photographed again (bottom). Table 5 | Bud position of the rsr1 mutant grown on glucose-rich or glucose-limited medium We hypothesized that the distal-pole budding observed in the rsr1 mutant was due to the lengthened period of apical growth that leads to cell elongation during haploid invasion . Precise mapping of bud placement showed that in the rsr1 mutant, buds emerged within an arc that included the distal pole, whereas in wild-type cells, bud emergence was confined to the extreme tip of the cell (Figure B), suggesting that the mechanism of distal budding in the rsr1 mutant was different than in wild-type cells. Indeed, disruption of BUD8 did not affect distal-pole budding or agar invasion of the rsr1 mutant (Figure C), demonstrating that the distal-pole budding in the rsr1 mutant was not due to Bud8p-dependent bud site selection. To gain further support for the idea that enhanced apical growth can influence budding pattern in the rsr1 mutant, we used the grr1 mutation , which causes hyperpolarized growth . grr1 mutant cells were elongated in glucose-rich conditions, but their budding pattern was axial . The rsr1 grr1 double mutant, however, had a clear distal pole bias compared with the rsr1 single mutant in glucose-rich medium , supporting the idea that in the rsr1 mutant, hyperpolarized growth leads to distal-pole budding. Contribution of Different Aspects of Filamentous Growth to Agar Invasion | Filamentous growth is characterized by several physiological events: a change in budding pattern, an increase in cell length, and enhanced cell-cell adhesion. We found that disruption of FLO11, which is required for cell-cell adhesion and haploid invasive growth , did not affect cell elongation or unipolar-distal budding (Figure A), implicating cell-cell adhesion as the primary defect in the flo11 mutant. We directly compared mutants defective primarily in a single aspect of filamentous growth to a ste20 mutant, which is defective in all three aspects. In particular, we compared a bud8 mutant (defective for distal-pole budding but not elongation or adhesion), a pea2 mutant (defective for elongation but not distal-pole budding or adhesion), and a flo11 mutant (defective for adhesion but not distal-pole budding or elongation). A defect in any single aspect of filamentous growth caused a partial invasive growth defect, although the flo11 mutant was less invasive than the pea2 and bud8 mutants (Figure B). These results were extended by examining the phenotypes of double and triple mutants. The bud8 pea2, bud8 flo11, and pea2 flo11 double mutants were less invasive than any single mutant, and in the pea2 bud8 flo11 triple mutant, no agar invasion was observed (Figure B), implying that the three physiological events affected by these mutations are the major contributors to agar invasion. Figure 7 | Genes involved primarily in a single aspect of filamentous growth contribute independently to agar invasion. Genes involved primarily in a single aspect of filamentous growth contribute independently to agar invasion. (A) Flo11p is not required for unipolar-distal budding or cell elongation. Wild-type (SY3687) and flo11 (SY3729) cells were spread onto SC medium, incubated for 16 h at 25C, and photographed. Bar, 20 mum. (B) Contribution of individual filamentation functions to agar-invasion. Equal concentrations of cells were spotted onto YPD medium and grown for 2 d or 4 d at 30C, as indicated. Plates were photographed, washed, and photographed again. Strains are as indicated: wild-type (SY3687), bud8 (SY3689), pea2 (SY3698), flo11 (SY3729), ste20 (SY3728), and triple, the pea2 bud8 flo11 triple mutant (SY3733). Diploid Cells Exhibit Filamentous Growth in Response to Glucose Depletion | Filamentous growth has been thought to be induced by different cues in haploid cells (glucose limitation; ) and diploid cells (nitrogen limitation; ). We investigated the effect of glucose depletion on diploid cells and found that on glucose-limited medium, diploid cells were more elongated than on glucose-rich medium (Figure A). The elongated phenotype was apparent within the first cell division and was observed for >80% of cells. In addition to the increase in cell length, diploid cells budded in a unipolar-distal pattern upon glucose limitation. Bud scar staining of invaded diploid cells showed unipolar-distal bud scars 85% of the time (15% had scars at both poles); in contrast, diploid cells in glucose-rich medium had unipolar-distal bud scars 51% of the time (45% had scars at both poles and 4% had at least one scar in an equatorial site). Wild-type diploid cells also exhibited robust agar invasion by the plate-washing assay (Figure B). Thus, diploid cells can undergo filamentous growth in response to limiting glucose. Figure 8 | Diploid cells exhibit filamentous growth in response to limiting glucose. Diploid cells exhibit filamentous growth in response to limiting glucose. (A) Single cell assay. Diploid cells (SY3734) were spread onto SC (-Glu) and SCD (+Glu) medium, incubated for 16 h at 25C, and photographed. Bars, 10 mum. (B) Equal concentrations of diploid cells (SY3734) and haploid cells (SY3687) were spotted onto YPD medium and grown for 2 d at 30C. Plates were photographed (left), washed, and photographed again (right). PMID- 12221111_DISCUSSION TI - AB - Bipolar Bud-Site-Selection Components Are Required for Haploid Invasive Growth | We investigated the role of bud-site-selection components in haploid invasive growth and found that proteins required for bipolar budding in diploid cells were required in haploid cells for distal pole budding during invasive growth. In particular, our data indicate that Bud8p marks the distal pole of haploid cells and is recognized upon glucose depletion to direct budding to the distal pole. Bud8p had not previously been implicated in haploid invasion , perhaps because bud8 mutants exhibit only a partial invasive growth defect. We have shown, however, that Bud8p has a profound role in bud site selection under conditions of glucose limitation, even though it has no known role when glucose is abundant. This contrasts with Bud8p's role in diploid cells, where it controls budding pattern during both yeast form and filamentous growth . Thus, an additional level of regulation is required in haploid cells to prevent Bud8p from directing budding pattern in glucose-rich conditions (see below). Our results also indicate that Bud7p, Bud9p, Bni1p, and Bud6p are required in glucose-limiting conditions for normal haploid invasive growth. As in diploid cells, Bni1p is required in haploid cells to localize Bud8p to the distal pole. We have also found that Bud6p is important, although not essential, for Bud8p localization in haploid cells, an apparent difference from previously reported results with diploid cells ; this may either represent a genuine difference between haploid and diploid cells or just be a function of different strain backgrounds. (Note that previous studies have also shown less precise use of the distal pole in bud6 mutant diploids; ; ; ). Two pieces of evidence in our study support the conclusion that the bipolar budding machinery is in place, albeit dormant, in haploid cells. First, Bud8p is present and localized appropriately in haploid cells in glucose-rich conditions. Second, Bni1p is required for localization of Bud8p to the distal pole in glucose-rich conditions. Because these proteins are not required for bud site selection during yeast-form growth in haploid cells, we suggest that the bipolar budding machinery constitutes a default program in haploid cells that becomes active in glucose-limiting conditions. Indirect evidence from a number of studies has indicated that haploid cells have properly located proximal and distal cues , and we have provided direct evidence for this conclusion. In glucose-rich environments, the axial Bud3p/Bud4p/Axl2p cues are chosen, whereas glucose limitation causes the distal cue Bud8p to be chosen in preference to Bud3p/Bud4p/Axl2p. The presence of both sets of cues enables an individual cell to reorient bud growth rapidly in response to changing nutrient availability. Glucose Controls Bud Site Selection by the Snf1p-dependent Regulation of Axl1p | How is unipolar-distal budding prevented in haploid cells growing in glucose-rich conditions? We show here that the abundance of the axial-promoting factor Axl1p is controlled by glucose. Axl1p levels decline sharply upon glucose limitation , an event that is concomitant with the appearance of unipolar buds (our unpublished data). Thus, it is reasonable to speculate that the disappearance of Axl1p is a trigger for distal-pole budding. We found that the Snf1p protein kinase, which plays a role in the derepression of glucose-repressed genes , is required for the disappearance of Axl1p in glucose-limiting conditions. Snf1p may exert its effect on Axl1p indirectly, by allowing derepression of a gene whose product regulates Axl1p. Alternatively, Snf1p may directly phosphorylate Axl1p and target it for degradation. Irrespective of the mechanism by which Snf1p regulates Axl1p abundance, the genetic evidence that we have presented identifies Axl1p as an important regulator of haploid invasive growth. Loss of Axl1p permits Bud8p-dependent unipolar-distal budding, whereas overexpression of Axl1p suppresses unipolar-distal budding and agar invasion in glucose-limiting conditions. How Axl1p functions to direct budding to sites marked by Bud3p/Bud4p/Axl2p is not known but is a question that is crucial to the ultimate understanding of bud site determination in yeast. Coordination of Unipolar Budding and Polarized Growth | A lengthened period of polarized, apical growth promotes bipolar budding in diploid cells and also promotes unipolar-distal budding in haploid cells during invasive growth . In fact, in this latter case, we showed that an extended period of apical growth could confer distal-pole budding to a mutant lacking a functional bud-site-selection system (rsr1 mutant). In wild-type cells, polarized growth and localization of the bud site machinery are coordinated. For example, Bni1p, which is involved in polarized growth, is also required for localization of Bud8p to the distal tip of the daughter cell in both haploids and diploids . These two processes are not inextricably linked, however. Pea2p (and Spa2p) were shown to be required for cell elongation during invasive growth but not for Bud8p localization or distal-pole budding. Conversely, loss of Bud8p disrupted the budding pattern but did not affect cell elongation. Thus, two distinct cues mark the distal pole of daughter cells: a bud site cue (Bud8p) to direct bud site selection and a second cue to direct polarized growth. The notion that the components of invasive growth could be genetically isolated was extended to cell-cell adhesion. Disruption of FLO11 prevented adhesion but had no effect on cell elongation or the budding pattern. Thus, haploid invasive growth can be divided into three separate processes: cell elongation, unipolar-distal budding, and cell adhesion. Loss of any one of these processes partially compromises invasive growth; loss of all three prevents it entirely. Diploid Cells Starved for Glucose Initiate the Filamentous Growth Response | Filamentous growth was first characterized in diploid cells and was described as a response to nitrogen limitation . Mutations in nitrogen-sensing and nitrogen utilization pathways confirmed that pseudohyphal growth was a response to low levels of environmental fixed nitrogen . Subsequently, haploid cells were also shown to undergo a filamentation-like process in which they invaded the agar substratum . Diploid pseudohyphal growth and haploid invasive growth were presumed to be similar processes because they required some of the same signal transduction pathways, but differences are apparent, in particular in the degree of agar invasiveness. Recently, we showed that glucose depletion was a trigger for haploid invasive growth . Hence, it was proposed that diploid cells initiate filamentous growth in response to limiting nitrogen, whereas glucose depletion triggers haploid invasive growth . However, we have shown here that diploid cells manifest all of the characteristics of filamentous growth in response to glucose limitation. Perhaps variation within the Sigma1278b background has caused confusion as to the cues that underlie filamentous growth. For example, strains of yeast capable of starch degradation have been reported to initiate filamentous growth upon either carbon or nitrogen source depletion . Diploid cells sporulate upon limitation of both carbon and nitrogen cues ; depletion of either single cue, however, triggers filamentous growth. PMID- 12221112 TI - Pkh1 and Pkh2 Differentially Phosphorylate and Activate Ypk1 and Ykr2 and Define Protein Kinase Modules Required for Maintenance of Cell Wall Integrity AB - | Saccharomyces cerevisiae Pkh1 and Pkh2 are functionally redundant homologs of mammalian protein kinase, phosphoinositide-dependent protein kinase-1. They activate two closely related, functionally redundant enzymes, Ypk1 and Ykr2 (homologs of mammalian protein kinase, serum- and glucocorticoid-inducible protein kinase). We found that Ypk1 has a more prominent role than Ykr2 in mediating their shared essential function. Considerable evidence demonstrated that Pkh1 preferentially activates Ypk1, whereas Pkh2 preferentially activates Ykr2. Loss of Pkh1 (but not Pkh2) reduced Ypk1 activity; conversely, Pkh1 overexpression increased Ypk1 activity more than Pkh2 overexpression. Loss of Pkh2 reduced Ykr2 activity; correspondingly, Pkh2 overexpression increased Ykr2 activity more than Pkh1 overexpression. When overexpressed, a catalytically active C-terminal fragment (kinase domain) of Ypk1 was growth inhibitory; loss of Pkh1 (but not Pkh2) alleviated toxicity. Loss of Pkh2 (but not Pkh1) exacerbated the slow growth phenotype of a ypk1Delta strain. This Pkh1-Ypk1 and Pkh2-Ykr2 dichotomy is not absolute because all double mutants (pkh1Delta ypk1Delta, pkh2Delta ypk1Delta, pkh1Delta ykr2Delta, and pkh2Delta ykr2Delta) were viable. Compartmentation contributes to selectivity because Pkh1 and Ypk1 were located exclusively in the cytosol, whereas Pkh2 and Ykr2 entered the nucleus. At restrictive temperature, ypk1-1tsykr2Delta cells lysed rapidly, but not in medium containing osmotic support. Dosage and extragenic suppressors were selected. Overexpression of Exg1 (major exoglucanase), or loss of Kex2 (endoprotease involved in Exg1 processing), rescued growth at high temperature. Viability was also maintained by PKC1 overexpression or an activated allele of the downstream protein kinase (BCK1-20). Conversely, absence of Mpk1 (distal mitogen-activated protein kinase of the PKC1 pathway) was lethal in ypk1-1tsykr2Delta cells. Thus, Pkh1-Ypk1 and Pkh2-Ykr2 function in a novel pathway for cell wall integrity that acts in parallel with the Pkc1-dependent pathway. PMID- 12221112_INTRODUCTION TI - AB - A cascade of protein kinases is a commonly used mechanism for amplifying and disseminating signals that control metabolism, growth, survival, and differentiation in eukaryotic cells. In animal cells, recruitment of phosphatidylinositol 3-kinase by growth factor receptors generates 3-phosphoinositides, which stimulate 3-phosphoinositide-dependent protein kinase-1 (PDK1) (for review, see ; ). Activated PDK1 phosphorylates and activates multiple downstream targets, including protein kinase B/c-Akt , p70 S6 kinase , protein kinase C (PKC) isoforms , and serum- and glucocorticoid-inducible protein kinase (SGK) isoforms , thereby eliciting physiological responses. We have demonstrated previously that, in budding yeast (Saccharomyces cerevisiae), Pkh1 and Pkh2 are the homologs and functional equivalents of mammalian PDK1. Pkh1 and Pkh2 share an essential function because pkh1Delta and pkh2Delta single mutants are viable, whereas a pkh1Delta pkh2Delta double mutant is inviable. Expression of human PDK1 rescues the lethality of a pkh1Delta pkh2Delta strain . The PDK1 enzymes from Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens all possess a C-terminal pleckstrin homology (PH) domain that binds phosphatidylinositol (PtdIns)(3,4,5)P3 and PtdIns(3,4)P2 . However, S. cerevisiae does not produce PtdIns(3,4,5)P3 or PtdIns(3,4)P2 . Moreover, Pkh1 and Pkh2 lack discernible PH domains, and PDK1 lacking its PH domain was sufficient to rescue the growth of pkh1Delta pkh2Delta cells , suggesting that the activity of Pkh1 and Pkh2 in yeast does not depend on phosphoinositides. It was shown subsequently that sphingosine (4-dehydro-sphinganine) can also stimulate mammalian PDK1 autophosphorylation and increase its ability to phosphorylate in vitro known PDK1 substrates, such as c-Akt and PKCbeta . Correspondingly, it has been reported recently that Pkh1 and Pkh2 can be activated in vitro by nanomolar concentrations of the major sphingoid base in yeast, phytosphingosine (4-hydroxy-sphinganine) . Moreover, endocytosis in yeast seems to require sphingoid base synthesis and overexpression of Pkh1 or Pkh2 can suppress this requirement , suggesting that sphingoid bases activate a signaling pathway involving Pkh1 and Pkh2. Mammalian PDK1 activates its downstream targets by phosphorylating a Thr residue (starred) in a sequence motif, Thr*-Phe-Cys-Gly-Thr-X-Glu-Tyr (where X represents any amino acid), that lies within the "activation loop" of their catalytic domains and is unique to and conserved in all known PDK1 substrates. Full activation of c-Akt/PKB and other PDK1 targets also seems to require phosphorylation at a second site (starred) situated in a hydrophobic motif, Phe-X-X-Ar-Ser*/Thr*-Ar (where Ar represents an aromatic residue), that is located near the C terminus of each of these enzymes . In S. cerevisiae, four previously characterized protein kinases possess both of these motifs, suggesting that they are physiological substrates of Pkh1 and/or Pkh2. These four protein kinases are the products of the following genes: YPK1 , YKR2/YPK2 , PKC1 , and SCH9 . Studies from this laboratory have demonstrated that Ypk1 is a direct substrate of Pkh1 and that Ykr2 is phosphorylated by Pkh2 . Similarly, it has been shown that Pkc1 can also be phosphorylated by Pkh1 and Pkh2 . Reduced Pkc1 activity was observed in a pkh1-1ts pkh2Delta strain, and the temperature sensitivity of this strain was partially suppressed by a dominant PKC1(R398P) allele, suggesting that Pkh1 and Pkh2 are required for Pkc1 function in vivo . The catalytic domains of Ypk1 and Ykr2 are 88% identical and these proteins also share extensive homology across their N- and C-terminal extensions. Moreover, the catalytic domains of Ypk1 and Ykr2 closely resemble (55% identity) that of mammalian SGK. Indeed, cells lacking Ypk1 or Ykr2 are viable, whereas cells lacking both Ypk1 and Ykr2 are inviable , and expression of mammalian SGK rescues this inviability . Furthermore, both purified PDK1 and purified Pkh1 phosphorylate the same residue (Thr504) in the consensus motif in purified Ypk1, and Ypk1 phosphorylation is significantly diminished in vivo in cells lacking Pkh1 . Thus, just as SGK is a downstream target of PDK1 in animal cells, Ypk1 and Ykr2 seem to act downstream of Pkh1 and Pkh2 in yeast. Moreover, lipid-derived signals are required as upstream activators in both pathways, 3-phosphoinositides and sphingosine in the case of PDK1 and closely related sphingoid bases in the case of Pkh1 and Pkh2. Consistent with this view, overexpression of Ypk1 confers resistance to myriocin (ISP-1), an antibiotic that specifically inhibits serine C-palmitoyltransferase (product of the LCB1 gene), which is the enzyme responsible for sphinganine biosynthesis . Herein, we describe experiments that address the genetic and biochemical interrelationships between Pkh1 and Pkh2 and Ypk1 and Ykr2, which we undertook to try to understand the reason for the redundancies within these protein kinase cascades. To provide further insight, we also investigated the subcellular localization of all four proteins. Finally, as two independent approaches for discerning the physiological function of the Ypk1 and Ykr2 enzymes, we selected for dosage suppressors and also for chromosomal mutations that suppress the lysis phenotype of ypk1-1ts ykr2Delta cells. PMID- 12221112_MATERIALS AND METHODS TI - AB - Strains and Growth Conditions | Yeast strains used in this study are listed in Table . Standard rich (YP) and defined minimal (SC) media , containing either 2% glucose (Glc), 2% raffinose (Raf), or 2% galactose (Gal) as the carbon source and supplemented with appropriate nutrients to maintain selection for plasmids, were used for yeast cultivation. For gene expression from the galactose-inducible GAL1 promoter in liquid media, cells were pregrown to mid-exponential phase in SC containing 2% raffinose-0.2% sucrose (Raf/Suc) and then Gal was added to a final concentration of 2% and incubation continued for 2 h. In experiments involving growth on solid medium containing 5-fluoroorotic acid, 5-fluoroorotic acid was used at a concentration of 0.5 mg/ml . Cells were grown routinely at 30C, except for strains carrying temperature-sensitive mutations, which were propagated at their permissive temperature (26C). Table 1 | S. cerevisiae strains used in this study Recombinant DNA Methods | Escherichia coli strain DH5alpha was used for the construction and propagation of plasmids. Conventional recombinant DNA methods were used for the construction of plasmids . The sequences of constructs that contained DNA fragments amplified by polymerase chain reaction (PCR) were verified by the dideoxy chain termination-sequencing method . Native and Turbo Pfu polymerases (Stratagene, La Jolla, CA) were used for PCR, unless noted otherwise. Plasmids | Plasmids pYPK1, pYKR2, pGAL-YPK1, pGAL-YKR2 (pAM1), pGAL-Ypk1-Myc (pAM54), pADH-YPK1, pRS316-YKR2 (pAM12), pGAL-PKH1 (pAM73), and pGAL-PKH2 (pAM79) have been described previously . To create plasmid pADH-YKR2 (pAM4), which constitutively overexpresses YKR2 from the ADH1 promoter, a 2.4-kb XhoI (blunt)-SalI fragment containing the entire YKR2 gene was excised from pYKR2, gel purified, and inserted into vector pAD4 M that had been linearized with SmaI/SalI. To generate a version of Ykr2 tagged at its C-terminal end with the c-Myc epitope , a PCR-based method for precise gene fusion was performed using the YKR2 sequence cloned in pUC18 as one template (pYKR2), and as the other template, pOGFP (E. Swartzman, this laboratory), which contains a sequence encoding the 16-residue version of the c-Myc epitope followed by a (His)6 tag cloned in pBluescript (Stratagene); with three appropriate synthetic oligonucleotide primers: T3 (Stratagene); 5'-GGA CAT ATT GCA CTG TGT G-3' (RMN5), corresponding to sequences in YKR2 overlapping a DraIII site near the C terminus; and a "joiner" primer, 5'-TTC AGA AAT CAA CTT TTG TTC ACT AAT GCT TCT CCC CTG-3' (RMC), corresponding to the 3' end of the YKR2 coding sequence and the first several residues of the c-Myc epitope. An similar1.6-kb DraIII/KpnI fragment of the resulting PCR product was used to replace the corresponding segment in pYKR2, yielding pYkr2-Myc (pAM24). An similar3-kb NcoI/HindIII fragment from pYkr2-Myc was gel purified and used to replace the corresponding similar2.2-kb NcoI/HindIII segment in pGAL-YKR2 to create a 2-mum DNA-containing, LEU2-marked plasmid, pGAL-Ykr2-Myc (pAM59), that overexpresses Ykr2-Myc upon galactose induction. To generate a catalytically inactive ("kinase-dead") version of Ypk1, a PCR-based method for site-directed mutagenesis was performed using pYPK1 as the template and three appropriate synthetic oligonucleotide primers: 5'-CTT GAA CAC AGT AAG TAA CGG-3' (PKC2), corresponding to the flanking genomic sequence commencing 68-base pairs downstream of the stop codon; 5'-CAC AAA AAG TAT ACG CCT TGG CGG CAA TCA G-3' (PKD), where the underlined nucleotide is a silent mutation to introduce a BglI site, and the bold nucleotides correspond to an introduced alanine codon (GCG) in place of the native lysine codon (AAG); and 5'-GTC CAT CGA TGA TTT CGA TC-3' (Pseq2), corresponding to the coding strand of YPK1 starting at nucleotide position 1024. The resulting similar1.1-kb PCR product was digested with ClaI and NcoI, and the resulting similar850-base pair fragment was used to replace the corresponding segment in pYPK1, yielding pYPK1(K376A-KD) (pAM46). Conversion of the Lys residue at the equivalent position in all other protein kinases examined to date eliminates their catalytic activity . To generate a catalytically inactive (kinase-dead) version of Ykr2, a similar PCR-based approach for site-directed mutagenesis was performed using pYKR2 as the template, and three appropriate synthetic oligonucleotide primers: 5'-AGT ATA GCC CTG CCC CAA C-3' (Rseq2), corresponding to the noncoding strand of YKR2 commencing at nucleotide position 1544; 5'-CCC AAA AGA TTT ACG CCT TGG CGG CTC TGA G-3' (RKD), where the underlined nucleotide is a silent mutation to introduce a BglI site, and the bold nucleotides correspond to an introduced alanine codon (GCG) in place of the native lysine codon (AAG); and 5'-CGT GGG GTA ATG GCC TG-3' (Rseq3), corresponding to the coding strand of YKR2 starting at nucleotide position 66. The resulting similar1.4-kb PCR product was digested with NcoI and DraIII and used to replace the corresponding segment in pYKR2, yielding pYKR2(K373A-KD) (pAM47). Plasmid pYPK1(K376A-KD) was digested with AlwnI, converted to flush ends by treatment with T4 polymerase (NEB) and all four dNTPs then digested with SalI. The resulting 3.3-kb YPK1(K376A-KD) --containing fragment was gel purified and ligated into YEp351GAL that had been linearized by digestion with XbaI, converted to flush ends by incubation with T4 polymerase and all four dNTPs, and then digested with SalI. The resulting plasmid, pGAL-YPK1(K376A-KD) (pAM48), expresses a catalytically inactive allele [Ypk1-(K376A-KD)] from a 2-mum DNA-containing, LEU2-marked plasmid under control of the GAL1 promoter. An similar1.2-kb NcoI/SalI fragment from pYpk1-Myc was gel purified and used to replace the corresponding NcoI/SalI segment in pGAL-YPK1(K376A-KD) to create pGAL-YPK1(K376A-KD)-Myc (pAM49). An similar2.4-kb XhoI-HindIII fragment containing the entire YKR2(K373A-KD) allele was excised from pYKR2(K373A-KD), gel purified, and inserted into YEp351GAL that had been linearized with SalI and HindIII, to create a 2-mum DNA-containing, LEU2-marked plasmid, pGAL-YKR2(K373A-KD) (pAM50), that overexpresses catalytically inactive Ykr2 upon galactose induction. Galactose-inducible expression vectors that are URA3 based were constructed as follows. An similar3.3-kb BamHI/HindIII fragment carrying YPK1 was excised from pGAL-YPK1, gel purified, and ligated into YEp352GAL , which had been linearized with BamHI/HindIII, yielding YEp352GAL-YPK1 (pAM75). An similar3.8-kb BamHI/HindIII fragment from pGAL-Ypk1-Myc was gel purified and ligated into YEp352GAL that had been linearized with BamHI/HindIII, yielding YEp352GAL-Ypk1-Myc (pAM76). An similar2.2-kb BamHI/HindIII fragment from p2GAL-YKR2 was gel purified and ligated into YEp352GAL, which had been linearized with BamHI/HindIII, yielding YEp352GAL-YKR2 (pAM77). An similar3.0-kb BamHI/HindIII fragment from p2GAL-Ykr2-Myc was gel purified and ligated into YEp352GAL, which had been linearized with BamHI/HindIII, yielding YEp352GAL-Ykr2-Myc (pAM78). To generate an amino-terminal truncation of Ypk1, the following two-step approach was taken. First, an similar1.1-kb fragment corresponding to the last 344 amino acids of Ypk1 was amplified by PCR from pYPK1 with the following oligonucleotides: 5'-GGC GGA TCCATG TCC AGA AAT AAA CCT TTG TCC-3' (PCT), corresponding to sequences in the middle of the YPK1 coding sequence, just upstream of the beginning of the catalytic domain, where the underlined nucleotides correspond to an introduced BamHI restriction site, and the bold nucleotides represent an introduced start codon (ATG); and 5'-CTT GAA CAC AGT AAG TAA CGG-3' (PKC2), corresponding to the flanking genomic sequence commencing 68-base pairs downstream of the stop codon. The resulting PCR product was digested with BamHI and NcoI, gel purified, and used to replace an similar2.6-kb BamHI/NcoI fragment in pRS315-YPK1(B/H). The resulting CEN-containing, LEU2-marked plasmid encodes an amino-terminal truncation of Ypk1, which contains only the catalytic domain, but essentially no promoter sequence, and is called pRS315-YPK1-DeltaN (pAM55). An similar1.2-kb NcoI/SalI fragment from pRS315-Ypk1-myc was gel purified and used to replace the corresponding NcoI/SalI segment in pRS315-YPK1-DeltaN to create a CEN-containing, LEU2-marked plasmid, pRS315-Ypk1-DeltaN-myc (pAM56), that encodes a myc-tagged version of the Ypk1 catalytic domain. To insert a promoter, an similar2.3-kb BamHI/SalI fragment from pRS315-YPK1-DeltaN was gel purified and inserted into YEp351GAL that had been linearized with BamHI/SalI to create a 2-mum DeltaNA-containing, LEU2-marked plasmid, pGAL-YPK1-DeltaN (pAM99), that overexpresses the amino-terminal truncation of Ypk1 upon galactose induction. Likewise, an similar2.0-kb BamHI/SalI fragment from pRS315-Ypk1-DeltaN-Myc was gel purified and inserted into YEp351GAL that had been linearized with BamHI/SalI to create a 2-mum DeltaNA-containing, LEU2-marked plasmid, pGAL-Ypk1-DeltaN-Myc (pAM100), that overexpresses a myc-tagged version of the amino-terminal truncation of Ypk1 upon galactose induction. To move these truncated Ypk1 derivatives into URA3-marked plasmids, an similar2.3-kb BamHI/SalI fragment from pGAL-YPK1-DeltaN was gel purified and inserted into YEp352GAL that had been linearized with BamHI/SalI to create a 2-mum DNA-containing, URA3-marked plasmid, YEp352GAL-YPK1-DeltaN (pAM101), that overexpresses the amino-terminal truncation of Ypk1 upon galactose induction. Similarly, an similar2.0-kb BamHI/SalI fragment from pGAL-Ypk1-DeltaN-Myc was gel purified and inserted into YEp352GAL that had been linearized with BamHI/SalI to create a 2-mum DNA-containing, URA3-marked plasmid, YEp352GAL-Ypk1-DeltaN-myc (pAM102), that overexpresses a myc-tagged version of the amino-terminal truncation of Ypk1 upon galactose induction. To generate a catalytically inactive derivative of the Ypk1-DeltaN allele, a 2.3-kb ClaI/HindIII fragment from pGAL-YPK1-KD, encoding the carboxy terminus (containing the K376A kinase-dead mutation of Ypk1) was gel purified and used to replace the corresponding segment in YEp352GAL-Ypk1-DeltaN-Myc. The resulting plasmid, YEp352GAL-Ypk1-DeltaN-KD (pFR30), overexpresses a catalytically inactive derivative of the amino-terminal truncation of Ypk1 upon galactose induction. Protein Localization by Using Chimeras Containing Green Fluorescent Protein (GFP) | To create vectors for galactose-inducible expression of YPK1, YKR2, and PKH2, each fused to the carboxy terminus of a protein comprising three tandem repeats of an enhanced (S65T V163A) mutant of GFP, the following approach was taken. Two primers, 5'-GCG AGC GGG ATC CAT G, the first 18 bases of the gene-3' (primer A), where underlined bases correspond to an introduced BamHI site and start codon in bold; and 5'-GGC ACG CGT CGA CTT A, the last 18 bases of the gene-3' (primer B), where underlined bases correspond to an introduced SalI site and stop codon in bold, were used to amplify the entire open reading frames of the corresponding genes from genomic DNA. The PCR products were digested with BamHI and SalI and ligated into vector pGS836 (YCpGAL-3GFP) that had been digested with BamHI and SalI, yielding plasmids pGAL-3GFP-YPK1 (pFR33), pGAL-3GFP-YKR2 (pER2), and pGAL-3GFP-PKH2 (pER3). To create pGAL-3GFP-PKH1 (pFR37), the same approach was used, but due to the presence of BamHI and SalI restriction sites in the gene, two PCR products were made, one with primer A and a primer corresponding to the sequence 3' to the ClaI site present in PKH1, and the other with a primer 3' of the ClaI site and primer B. A three-way ligation was then used to ligate the two PCR products digested with BamHI and ClaI, or ClaI and SalI, into pGS836 that had been digested with BamHI and SalI. Cells expressing the GFP constructs were grown to mid-exponential phase at 30C in SC-Leu containing Raf/Suc and then induced with 2% galactose for 3 h. Nuclear DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) by adding the dye directly in the medium at a concentration of 1 mug/ml for the last hour of growth. Rhodamine-labeled phalloidin was purchased from Molecular Probes (Eugene, OR). Samples of each culture were viewed directly with a TE300 fluorescence microscope (Nikon, Melville, NY) equipped with a 100x/1.4 Plan-Apo objective and a 1.4 numerical aperture condenser. Digital images were acquired with a bottom-ported Orca 100 charge-coupled device camera (Hamamatsu, Bridgewater, NJ) and Phase 3 Imaging Systems software (Northern Exposure, Inc., Glen Mills, PA). The fraction of lysed cells in cultures was assessed by direct counting of at least 200 cells after staining with a vital dye (LIVE/DEAD Yeast Viability kit; catalog no. 7009; Molecular Probes). Antibody Production | To express a glutathione S-transferase (GST)-Ypk1 fusion protein in E. coli, an similar370-base pair fragment corresponding to the first 114 amino acids of Ypk1 was amplified by PCR from pYPK1 by using the following oligonucleotides: 5'-GGG GGG GGA TCCATG TAT TCT TGG AAG TCA AAG TTT-3', where the underlined nucleotides correspond to an introduced BamHI site, and the bold nucleotides correspond to the start codon; and 5'-GGG GGG AAT TCT CAG GTG GCA TCA TTG GGT GTC CC-3', where the underlined nucleotides correspond to an introduced EcoRI site, and the bold nucleotides correspond to the reverse complement of an introduced stop codon. This PCR fragment was then digested with BamHI and EcoRI, gel purified, and ligated into the pGEX2T vector (Pharmacia, Peapack, NJ), which had been linearized with BamHI and EcoRI to create plasmid pGEX-Ypk1 (pAM5). To express a GST-Ykr2 fusion protein in E. coli, an similar360-base pair fragment corresponding to the first 111 amino acids of Ykr2 was amplified by PCR from pYKR2 by using the following olignucleotides: 5'-GGG GGG GGA TCCATG CAT TCC TGG CGA ATA TCC AAG-3', where the underlined nucleotides correspond to an introduced BamHI site, and the bold nucleotides correspond to the start codon; and 5'-GGG GGG AAT TCT CAA CTC GGT CCC TGC GTC TCA GT-3', where the underlined nucleotides correspond to an introduced EcoRI site, and the bold nucleotides correspond to the reverse complement of an introduced stop codon. This PCR fragment was then digested with BamHI and EcoRI, gel purified, and ligated into the pGEX2T vector, which had been linearized with BamHI and EcoRI to create plasmid pGEX-Ykr2 (pAM6). To prepare antigen, expression of GST-Ypk1(1-114) and GST-Ykr2(1-111) fusions from plasmids, pGEX-Ypk1 and pGEX-Ykr2, respectively, were induced in a protease-deficient E. coli strain BL21 (DE3)[pLys] by addition of isopropyl-beta-d-thiogalacto-pyranoside to a final concentration of 0.2 mM followed by incubation with aeration for 2 h at 30C. Cells were harvested, washed once with ice-cold wash buffer (50 mM Tris-HCl pH 8, 0.5 mM dithiothreitol [DTT], 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 0.05% NP-40, 1 mM EGTA, and 1 mM EDTA), and resuspended in 1/20 volume of wash buffer containing 1 M NaCl. Cells were disrupted by digestion with lysozyme (final concentration, 2 mg/ml) followed by sonication. Insoluble material was removed by centrifugation at 12,000 x g, and the soluble GST-Ypk1(1-114) or GST-Ykr2(1-111) proteins were purified by adsorption to, and elution from, glutathione-agarose beads (Pharmacia), essentially as directed by the manufacturer, except that elution was performed in the presence of 1 M NaCl and 20 mM glutathione. The purified proteins were used as immunogens to raise polyclonal antisera in adult female New Zealand White rabbits following standard immunization protocols . The resulting anti-Ypk1 antibodies (serum #1446) and anti-Ykr2 antibodies (serum #1732) are specific to Ypk1 and Ykr2 and do not display any cross-reaction against the incorrect antigen. Anti-GFP antibodies were the generous gift of Roger Tsien and Charles Zuker (Department of Cellular and Molecular Medicine, University of California, San Diego, CA). Preparation of Cell Extracts and Immunoblot Analysis | Yeast cells were grown at 30C to mid-exponential phase (A600 nm = 0.5 --1), either in SC medium supplemented in a manner appropriate for maintenance of plasmids or in rich medium (YPGlc). If cells required galactose induction for expression from the GAL1 promoter, galactose was added to a final concentration of 2% and the cultures were incubated at 30C for an additional 2 h. Cells were harvested by brief centrifugation, washed twice by resuspension and resedimentation in ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 3 mM DTT and 1 mM phenylmethylsulfonyl fluoride), and resuspended in 200 mul of the same buffer. Prechilled glass beads (0.45 --0.6 mm in diameter) were added to the meniscus of the cell suspension, and lysis was achieved by vigorous vortex mixing for six 1-min intervals, with intermittent cooling on ice. To remove the glass beads, the bottom of the Eppendorf tube was punctured with a syringe needle (<0.5 mm in diameter) and inserted into another tube; the lysate was collected into the fresh tube by brief centrifugation in a clinical centrifuge. The crude extract was subjected to centrifugation at 30,000 x g for 15 min to remove unbroken cells and large debris. The protein concentration of the crude extract was measured using a dye-binding method with a protein assay kit as instructed by the manufacturer (, Hercules, CA), by using bovine serum albumin (, Beverly, MA) as the standard. For immunoblot analysis, samples (50 mug of total protein) were diluted into SDS-PAGE sample buffer , subjected to electrophoresis in an 8 --12% gel, and then transferred to nitrocellulose . To detect Ypk1, rabbit polyclonal anti-Ypk1 antiserum #1446 was used at a dilution of 1:3000. To detect Ykr2, rabbit polyclonal anti-Ykr2 antiserum #1732 was used at a dilution of 1:3000. To detect proteins using the anti-c-Myc monoclonal antibody (mAb) 9E10, ascites fluid containing this mAb was used at a dilution of 1:10,000 . Immobilized immune complexes were detected using a commercial chemiluminescence detection system (Renaissance; PerkinElmer Life Sciences, Boston, MA) and x-ray film (Biomax MR; Eastman Kodak, Rochester, NY). Immunoprecipitations | Yeast cultures to be used for immunoprecipitation analysis were grown as described above, and then rinsed in ice-cold IP buffer (20 mM Tris-HCl pH 7.5, 125 mM potassium acetate, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 1 mug/ml leupeptin, 1 mug/ml pepstatin A, 0.1% Triton X-100, and 12.5% glycerol). Glass beads were added to the meniscus of the cell suspension, and lysis was achieved by vigorous vortex mixing for eight 30-s intervals with intermittent cooling on ice. The lysate was clarified by centrifugation at 14,000 x g at 4C for 30 min. The clarified extract was assayed for protein concentration, and a sample (1 mg of total protein) was diluted to a final volume of 200 mul in IP buffer. An aliquot (20 mul) of protein G/protein A-agarose beads (30% slurry) (Oncogene Science, Cambridge, MA) and a sample of an appropriate control antibody, either 2 mul of preimmune rabbit serum or 1 mug of purified mouse anti-T-cell receptor antibody (gift of James Allison, Department of Molecular and Cell Biology, University of California, Berkeley, CA), were added. The samples were then incubated on a roller drum for 1 h at 4C to adsorb proteins that bound nonspecifically to the solid support and to rabbit or mouse IgG (preclearing). The beads were removed by centrifugation for 10 min in a microfuge, and the supernatant fraction was transferred to a fresh tube containing another aliquot (15 mul) of protein G/protein A-agarose beads and either 2 mul of anti-Ypk1 (or anti-Ykr2) polyclonal antiserum or 1 mul of anti-c-Myc (mAb 9E10) ascites, and incubated on a roller drum for 1 to 3 h at 4C. The beads were sedimented by brief centrifugation in a microfuge and washed three times (1 ml each) with ice-cold IP buffer and collected by centrifugation for 1 min in a microfuge on maximum speed. Bead-bound immune complexes were solubilized in SDS-PAGE sample buffer and immediately boiled for 5 min in a water bath and then clarified by brief centrifugation in a Microfuge before resolution by SDS-PAGE. The proteins of interest were visualized as described above. Immune-Complex Protein Kinase Assays | Cells expressing either wild-type or kinase-dead Ypk1-myc (or Ykr2-myc) under control of the GAL1 promoter were grown in SC containing Raf/Suc to an A600 nm = 0.6, induced by addition of galactose (2% final concentration), incubated with shaking at 30C for 2 h, collected by centrifugation, washed with ice-cold 1x phosphate-buffered saline, resuspended in 0.2 ml of ice-cold IP buffer, and lysed as described above. The resulting lysates were clarified by centrifugation at 4C for 30 min at 30,000 x g. Protein concentration in the resulting crude extracts was determined by the method. A volume of extract containing 1 mg of total protein was immunoprecipitated with mAb 9E10 as described above. The immunoprecipitates were washed once with ice-cold IP buffer, once with ice-cold IP buffer containing 0.5 M NaCl, and twice with ice-cold buffer A (50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, and 0.1% [by vol] 2-mercaptoethanol). As part of the final wash, the slurry of beads was split into two equal portions. For immunoblot analysis, SDS-PAGE sample buffer (similar15 mul) was added directly to one sample of each bead suspension. For protein kinase assays, the activity of the Ypk1-myc or Ykr2-myc immune complex was assayed by adding 30 mul of a mixture containing 1 muM microcystin-LR, 10 mM Mg-acetate, 100 muM [gamma-32P]ATP (200 --400 cpm/pmol), and 100 muM Cross-tide (GRPRTSSFAEG) , which we have documented previously is an excellent peptide phospho-acceptor substrate for Ypk1 and Ykr2 . After incubation for 15 min at 30C, each reaction was terminated by spotting a portion (45 mul) of the reaction mixture onto small squares of phosphocellulose paper (P81; Whatman, Maidstone, United Kingdom), which were washed and analyzed as described in detail previously . In some experiments, samples of the immunoprecipitates were resuspended in an appropriate buffer (20 mM Tris-HCl pH 8.8 and 10 mM MgCl2) and treated with shrimp alkaline phosphatase (0.25 U; US Biochemical, Cleveland, OH) in either the absence or presence of a mixture of inhibitors of this phosphatase (25 muM Na-orthovanadate and 100 muM beta-glycerol-phosphate, adjusted to pH 8). Bioassays for Drug Sensitivity | An agar diffusion (halo) assay was performed to test the relative sensitivity of various strains to rapamycin, valinomycin, hygromycin B, cycloheximide, and polyoxin D. Nascent lawns of the strains to be tested were prepared by mixing similar2 x 106 cells from a saturated culture with 2 ml of molten (55C) 1% agar. The cell-containing agar was rapidly mixed and immediately poured evenly onto plates containing an appropriate medium. Various concentrations of rapamycin (50 and 500 ng/mul), hygromycin B (5 and 50 mug/mul), or the other drugs indicated, were spotted in the same volume (10 mul) onto sterile cellulose filter discs (0.6 cm), which were placed on the nascent lawn. The plates were incubated at 30C, and photographed after 2 d. Selection and Analysis of Dosage Suppressors | A library of restriction fragments of yeast genomic DNA cloned into a URA3-marked, 2-mum DNA-based vector, YEp352 , was introduced into strain YPT40 (ypk1-1ts ykr2Delta) by selecting transformants on SCGlc-Ura medium at 26C. Temperature-resistant clones were then selected by their ability to grow at 35C. One suppressor plasmid obtained carried the EXG1 locus as the sole open reading frame. To create a plasmid that expressed EXG1 from a high-level constitutive promoter, two primers, 5'-GCG TCT CGA GAT GCT TTC GCT TAA AA-3', where the underlined bases correspond to an introduced XhoI site, and the start codon is in bold; and 5'-CGC CGG AGC TC T TAG TTA GAA ATT GTG CC-3', where the underlined bases correspond to an introduced SacI site, and the stop codon is in bold, were used to amplify the entire open reading frame of EXG1 from the library plasmid that was originally isolated as a dosage suppressor of the ypk1-1ts ykr2Delta strain (see RESULTS). This 1.4-kb PCR product was digested with XhoI and SacI and ligated into vector pAD4 M that had been digested with SalI and SacI, yielding pADH-EXG1 (pAM88). To express PKC1 from its own promoter on a high copy number (2-mum DNA) plasmid, a 4.2-kb NsiI fragment containing the entire open reading frame of PKC1 as well as 560 base pairs upstream of the ATG and 175 base pairs downstream of the stop codon, was cut out of a genomic clone obtained by F. Owen Fields (this laboratory; ; ) and ligated into the URA3-based plasmid YEp352 that had been linearized with PstI, yielding plasmid pPKC-PN, or into the LEU2-based plasmid YEp351 that had been linearized with PstI, yielding plasmid pFR32. To express BCK1-20 in a LEU2-based plasmid, the PvuI-PvuI fragment of plasmid pRS314-BCK1 --20 was replaced by the equivalent LEU2-containing fragment of plasmid pRS315, yielding plasmid pRS315-BCK1-20. Selection and Analysis of Chromosomal Suppressor Mutations | A genomic DNA library containing Tn3::LacZ::LEU2 insertions (generous gift of Michael Snyder, Department of Biology, Yale University) was introduced into strain YPT40 (ypk1-1ts ykr2Delta) by selecting transformants on SCGlc-Leu medium at 26C. Temperature-resistant clones were then selected by their ability to grow at 35C. Plasmids carrying genomic DNA corresponding to the sites of insertion were recovered as described in detail previously and characterized by direct nucleotide sequence analysis. PMID- 12221112_RESULTS TI - AB - Ypk1 Has a More Prominent Role in Maintaining Viability Than Ykr2 | The two protein kinases encoded by the YPK1 and YKR2/YPK2 genes are very similar to each other and functionally redundant at the genetic level . Yeast cells missing either Ypk1 or Ykr2 are viable, but cells lacking both proteins are inviable, indicating that these enzymes share an essential function. However, several observations suggest that Ypk1 plays the predominant role in executing this essential function. First, ypk1Delta mutants are slow growing at 30C , whereas ykr2Delta cells do not display any obvious growth phenotype, compared with otherwise isogenic YPK1+ YKR2+ control cells . Moreover, we found that the slow-growth phenotype of ypk1Delta cells is strongly exacerbated at lower temperatures, even at 26C . In essence, ypk1Delta mutants are cold sensitive and ykr2Delta mutants are not. Figure 1 | Phenotypes of ypk1Delta mutants. Phenotypes of ypk1Delta mutants. ypk1Delta cells (but not ykr2Delta cells) grow slowly at 30C and are cold sensitive. Serial dilutions of exponentially growing wild-type (WT; YPH499) and derived ypk1Delta (YES3) and ykr2Delta (YES1) mutants were spotted on YPGlc plates and grown for 3 d at the indicated temperatures (26, 30, and 35C). Among the close mammalian relatives of Ypk1 and Ykr2 is p70 S6 kinase (50% identity in the catalytic domain). Activation of mammalian p70 S6 kinase by mitogens is blocked by rapamycin, an immunosuppressive drug . Therefore, we tested whether loss of either Ypk1 or Ykr2 conferred on yeast cells elevated sensitivity to this agent. Compared with wild-type cells, we found that ypk1Delta cells, but not ykr2Delta cells, were hypersensitive (similar10-fold) to the growth inhibitory effect of rapamycin , providing a second distinction between ypk1Delta and ykr2Delta mutants. We found that ypk1Delta cells (but not ykr2Delta cells) were also hypersensitive to hygromycin B, valinomycin, polyoxin D, cycloheximide , and caffeine (Bezman, unpublished observations). These results suggested that ypk1Delta cells are generally more permeable to drugs and provided indirect evidence for a defect or perturbation in the cell envelope in ypk1Delta cells (see below). When overexpressed, either YPK1 or YKR2 was able to restore the normal level of drug sensitivity (Roelants, unpublished observations). Given the fact that Pkh1 and Pkh2 are upstream activators of Ypk1 and Ykr2 , it was of interest to test whether loss of either Pkh1 or Pkh2 might also confer a drug-sensitive phenotype. However, neither a pkh1Delta mutant nor a pkh2Delta mutant showed any degree of hypersensitivity to the compounds mentioned above, compared with the parental strain . Catalytically inactive (kinase-dead) alleles of protein kinases frequently act as dominant-negatives . Hence, we tested whether overexpression of catalytically inactive versions of Ypk1 and Ykr2 would be toxic to cells. We constructed two such derivatives altered in the invariant Lys found in conserved kinase motif II . We showed that Ypk1(K376A) and Ykr2(K373A) are indeed catalytically nonfunctional in vitro and unable to complement the lethality of ypk1Delta ykr2Delta cells in vivo . To test whether these alleles behave in a dominant-negative manner when overexpressed from an inducible promoter, plasmids pGAL-YPK1-(K376A-KD) and pGAL-YKR2-(K373A-KD) were introduced into wild-type cells (YPH499) and into ypk1Delta (YES3) and ykr2Delta (YES1) mutants, selecting for transformants on SC-Leu medium containing Glc as the carbon source. The resulting transformants then were streaked onto SC-Leu medium containing Gal/Suc to select for maintenance of the plasmid and to induce expression of either kinase-dead Ypk1 or kinase-dead Ykr2. High-level expression of catalytically inactive Ypk1 was growth inhibitory to all three cell types; however, the strongest growth inhibition was observed in ypk1Delta cells and the mildest was observed in wild-type cells (Figure A). In contrast, high-level expression of catalytically inactive Ykr2 had no detectably detrimental effect on growth in any of the strains (Figure A). To rule out the possibility that this differential effect was due to a difference in the level of expression of these proteins, Ypk1, Ypk1(K376A), Ykr2, and Ykr2(K373A) were tagged at their C termini with a c-Myc epitope and introduced into ypk1Delta or ykr2Delta strains. Identical amounts of protein from extracts of the resulting transformants were immunoprecipitated with anti-Myc mAb 9E10 antibodies, resolved by SDS-PAGE, and visualized by immunoblotting with polyclonal anti-Ypk1 or anti-Ykr2 antibodies. This analysis verified that the proteins were expressed at equivalent levels (Figure B). The fact that kinase-dead Ypk1 was able to effectively impede its own function and that of Ypk2, and the fact the converse was not true, provided a third independent indication that Ypk1 plays the more predominant role. Figure 2 | Overexpression of catalytically inactive Ypk1 inhibits growth. Overexpression of catalytically inactive Ypk1 inhibits growth. (A) Dominant-negative effect of inactive Ypk1. Wild-type cells (WT; YPH499), and ypk1Delta (YES3) and ykr2Delta (YES1) mutants were transformed, as indicated, with either an empty vector (YEp352GAL) or the same vector expressing from the GAL1 promoter either full-length Ypk1 (pGAL-YPK1), a catalytically inactive (kinase-dead) ypk1 allele (pGAL-YPK1-KD; pAM48), full-length Ykr2 (pGAL-YKR2; pAM59), or a catalytically inactive (kinase-dead) ykr2 allele (pGAL-YKR2-KD; pAM61). Transformants were selected on glucose-containing medium and then representative isolates were streaked to single colonies on selective medium containing either Glc (left) or Gal (right) as the carbon source and grown for 3 d at 30C. (B) Equivalent overexpression confirmed by immunoblotting. Cultures of the resulting transformants were grown at 30C in SCGal/Suc-Leu medium, induced by addition of galactose (2% final concentration) for 3 h. After harvesting the cells by centrifugation, extracts were prepared, and an identical amount of total protein (1 mg) from each extract was subjected to immunoprecipitation with anti-Myc mAb 9E10, as described in MATERIALS AND METHODS. After washing, the immunoprecipitates were solubilized and resolved by electrophoresis on an SDS-slab gel. The species corresponding to Ypk1 and Ypk2 were visualized by immunoblotting with rabbit polyclonal anti-Ypk1 (left) or anti-Ykr2 (right) antibodies, respectively. Pkh1 Preferentially Activates Ypk1 and Pkh2 Preferentially Activates Ykr2 | Purified Pkh1 phosphorylates and activates purified Ypk1 in vitro . To examine the state of activation of Ypk1 and Ykr2 in cell extracts and its dependence on the function of Pkh1 and Pkh2, we developed an immune-complex kinase assay. Cell extracts were prepared from strains expressing c-Myc epitope-tagged derivatives of Ypk1 or Ykr2, immunoprecipitated with anti-Myc mAb 9E10, and samples of the resulting immunoprecipitates were examined for protein content by SDS-PAGE and for catalytic activity using a specific peptide substrate (Cross-tide) and [gamma-32P]ATP in a filter binding assay . As independent negative controls to assess the nonspecific background, extracts were prepared from cells expressing untagged Ypk1 and Ykr2 and from cells expressing kinase-dead derivatives of Ypk1 and Ykr2. Immune complexes from wild-type cells expressing wild-type Ypk1-myc showed similar10-fold increase in phosphotransferase activity compared with both negative controls: immune complexes from wild-type cells expressing untagged Ypk1 (Figure A, top) and immune complexes from wild-type cells expressing catalytically inactive Ypk1-myc . When Ypk1-myc was isolated from pkh1Delta cells, however, the increase in activity was reproducibly 40 --60% lower than that observed in wild-type cells, whereas within experimental error, the recovery of Ypk1-myc activity was unaffected in pkh2Delta cells (Figure A, top). Most tellingly, activity was greatly increased (>=4-fold) when Ypk1-myc was isolated from cells cooverexpressing Pkh1 but only modestly elevated (similar1.5-fold) when Pkh2 was cooverexpressed (Figure A, top). Because Ypk1-myc and Pkh1 (or Pkh2) were both expressed from multicopy plasmids carrying the GAL1 promoter, which compete for a limiting pool of the Gal4 transactivator, the total amount of Ypk1 produced was reduced (Figure A, bottom); thus, the increase in specific activity when Ypk1-myc and Pkh1 were co-overexpressed is even more dramatic than the observed increase in total activity. Moreover, immunoprecipitated Ypk1-myc ran as a set of multiple bands that were collapsed into a single band of faster mobility upon treatment with a phosphatase (Figure A, bottom). Phosphatase-treated samples were no longer catalytically active (Torrance, unpublished observations). These findings suggest that activation is due to phosphorylation and that activation of Ypk1 is more dependent on Pkh1 than on Pkh2. Figure 3 | Preferential activation of Ypk1 and Ykr2 by Pkh1 and Pkh2. Preferential activation of Ypk1 and Ykr2 by Pkh1 and Pkh2. (A) Pkh1 differentially activates Ypk1. Top, wild-type (WT; W303-1B), pkh1Delta (AC301), and pkh2Delta (YPT67) cells overexpressing from the GAL1 promoter on plasmids either Ypk1 (pAM75) or Ypk1-myc (pAM76), in the absence or presence of co-overexpression from the GAL1 promoter on plasmids of either Pkh1 (pAM73) or Pkh2 (pAM79), as indicated, were grown to mid-exponential phase and induced with galactose for 3 h. Extracts were prepared and identical amounts of total protein (1 mg) were immunoprecipitated with anti-Myc mAb 9E10. The immune complexes were washed extensively with protein kinase assay buffer and then incubated with a specific peptide substrate (Cross-tide) and [gamma-32P]ATP, and the resulting product measured, as described in MATERIALS AND METHODS. Bottom, samples of each immunoprecipitate were treated with phosphatase in the absence and presence of phosphatase inhibitors, and examined by SDS-PAGE, as described in MATERIALS AND METHODS. (B) Pkh2 differentially activates Ykr2. Wild-type (WT; W303-1B), pkh1Delta (AC301), and pkh2Delta (YPT67) cells overexpressing from the GAL1 promoter on plasmids either Ykr2 (pAM1) or Ykr2-Myc (pAM78), in the absence or presence of co-overexpression from the GAL1 promoter on plasmids of either Pkh1 (pAM73) or Pkh2 (pAM79), as indicated, were grown to mid-exponential phase and induced with galactose for 3 h. Extracts were prepared and activity was measured, as in A. Values shown in A and B represent the average of three independent experiments, each performed in duplicate, and the error bars represent the range of values observed. Immune complexes from wild-type cells expressing wild-type Ykr2-myc showed approximately a 10-fold increase in phosphotransferase activity compared with both negative controls: immune complexes from wild-type cells expressing untagged Ykr2 (Figure B) and immune complexes from wild-type cells expressing catalytically inactive Ykr2-myc . When Ykr2-myc was isolated from either pkh1Delta or pkh2Delta cells, however, the increase in activity was reproducibly lower than that observed in wild-type cells, suggesting that both Pkh1 and Pkh2 contribute to phosphorylation and activation of Ykr2. Total activity was stimulated approximately threefold when Ykr2-myc was recovered from cells co-overexpressing Pkh2, but only similar1.5-fold when Pkh1 was co-overexpressed (Figure B), indicating that phosphorylation and activation of Ykr2 are more responsive to Pkh2 than to Pkh1. Loss of PKH2 (but Not PKH1) Exacerbates Slow Growth of ypk1Delta Cells | The biochemical assays discussed above indicated that Pkh1 preferentially activates Ypk1 and Pkh2 preferentially activates Ykr2. Given that both phk1Delta pkh2Delta and ypk1Delta ykr2Delta double mutants are inviable , if the discrimination observed in vitro with overexpressed proteins is even more stringent in vivo when these enzymes are expressed at their normal levels then it might be expected that certain mutant combinations might display genetic interaction. To construct all possible double mutant combinations between either ypk1Delta or ykr2Delta and either pkh1Delta or pkh2Delta, a ypk1Delta haploid was crossed to a pkh1Delta strain and to a pkh2Delta strain to create two diploid strains: PKH1/pkh1Delta::TRP1 YPK1/ypk1Delta::HIS3 and PKH2/pkh2Delta::HIS3 YPK1/ypk1Delta::TRP1. Likewise, a ykr2Delta haploid was crossed to a pkh1Delta strain and to a pkh2Delta strain to create two additional diploid strains: PKH1/pkh1Delta::TRP1 YKR2/ykr2Delta::HIS3 and PKH2/pkh2Delta::HIS3 YKR2/ykr2Delta::TRP1. The four doubly heterozygous diploid strains were sporulated. After tetrad dissection, viable Trp+ His+ haploid spores were readily recovered from all four diploids, indicating that all four double mutants (pkh1Delta ypk1Delta, pkh2Delta ypk1Delta, pkh1Delta ykr2Delta, and pkh2Delta ykr2Delta) are viable (Figure A). However, all of the pkh2Delta ypk1Delta spore clones grew significantly more slowly than any of the ypk1Delta spore clones or any of the pkh2Delta spore clones, when either streaked to single colonies on plates (Figure A) or examined by more definitive spot tests (Figure B). This finding provides genetic evidence in support of the biochemical results that the primary and physiologically relevant upstream activator of Ykr2 in vivo is Pkh2 (and not Pkh1). Nonetheless, a yeast cell can survive with either member of these two tiers of protein kinases. Hence, Pkh1 and Pkh2 must each be able to phosphorylate and activate either Ypk1 or Ykr2 to at least some significant degree. Figure 4 | Slow growth of ypk1Delta cells is exacerbated by absence of Pkh2 (but not Pkh1). Slow growth of ypk1Delta cells is exacerbated by absence of Pkh2 (but not Pkh1). (A) Four spores of tetratype asci produced by doubly heterozyous diploids derived from the following four crosses were streaked to single colonies and tested for growth on YPGlc plates at 30C: top left, a ypk1Delta strain (YFR107) against a pkh1Delta strain (YFR105); top right, a ypk1Delta strain (YFR107) against a pkh2Delta strain (YFR106); bottom left, a ykr2Delta strain (YFR119) against a pkh1Delta (YFR105) strain; and bottom right, a ykr2Delta strain (YFR64) against a pkh2Delta (YFR106) strain. (B) Serial dilutions of exponentially growing cultures of the indicated genotypes derived, respectively, from the crosses ypk1Delta (YFR107) against pkh1Delta (YFR105), left side, and ypk1Delta (YFR107) against pkh2Delta (YFR106), right side, were spotted on YPGlc plates and grown for 2 d at 30C. Loss of PKH1 (but Not PKH2) Alleviates Toxicity of Hyperactive Ypk1 | Ypk1 and Ykr2 share 88% identity within their 252-residue kinase domains and 75% identity within their downstream 75-residue C-terminal extensions. Ypk1 and Ykr2 also share considerable similarity within their 350 --353-residue amino-terminal extensions: 22% identity within the first similar100 residues and strikingly, 65% identity within the next 250 residues. To investigate what the large amino-terminal domain might contribute to the function of Ypk1, a truncation allele of Ypk1 was constructed in which the entire amino terminus (residues 2 --336) was deleted. The YPK1(Delta2--336) allele, encoding Ypk1-DeltaN, commences with Ser337; in Ypk1, the first Gly of the GxGxxG motif conserved in all protein kinases lies at residue 354. To permit its conditional expression, YPK1(Delta2-336) was inserted in a multicopy vector under control of the GAL1 promoter. Induction of the resulting plasmid, pGAL-YPK1-DeltaN (pAM101), in a wild-type strain (W303-1B) on galactose-containing medium was toxic as judged by the exceedingly slow growth of single colonies . Despite its toxicity, overexpression of Ypk1-DeltaN was capable of restoring growth (albeit very slowly) to an otherwise inviable ypk1Delta ykr2Delta double mutant . The observed toxicity required the catalytic activity of Ypk1-DeltaN because a kinase-dead derivative, Ypk1(K376A)-DeltaN, was not detectably growth inhibitory , even though immunoblotting indicated that, after induction, Ypk1-DeltaN and Ypk1(K376A)-DeltaN were expressed at equivalently high levels (Torrance, unpublished observations). Revealingly, the toxicity of Ypk1-DeltaN was also alleviated in cells lacking Pkh1, but not in cells lacking Pkh2 . The fact that Pkh1 was required for the dominant toxicity of Ypk1-DeltaN provides genetic evidence in support of the biochemical results that the primary and physiologically relevant upstream activator of Ypk1 in vivo is Pkh1 (and not Pkh2). Figure 5 | Absence of Pkh1 (but not Pkh2) alleviates toxicity of constitutively active Ypk1. Absence of Pkh1 (but not Pkh2) alleviates toxicity of constitutively active Ypk1. Wild-type (WT; W303-1B), pkh1Delta (AC301), or pkh2Delta (YPT67) strains were transformed with either an empty vector (YEp352GAL) or the same vector expressing from the GAL1 promoter either normal Ypk1 (pAM75), the C-terminal catalytic domain of Ypk1 (Ypk1-DeltaN; pAM101), or a catalytically inactive derivative of the C-terminal catalytic domain of Ypk1 (Ypk1-KD-DeltaN; pFR30). The resulting transformants were selected on glucose-containing medium and then representative isolates were streaked onto selective medium containing either Glc (left) or Gal (right) as the carbon source. Growth was assessed after 3 d at 30C. Differential Subcellular Localization of Pkh1, Pkh2, Ypk1, and Ykr2 | One explanation for the observed preferential phosphorylation of Ypk1 by Pkh1, and of Ykr2 by Pkh2, is that the activating enzyme and its downstream target are confined to the same subcellular compartment. As an initial approach to examine localization, each of these four proteins was tagged at its N terminus with three tandem in-frame repeats of GFP and expressed from the GAL1 promoter in a multicopy plasmid. Both 3GFP-Pkh1 and 3GFP-Pkh2 were able to complement the temperature sensitivity of a pkh1ts pkh2Delta strain at restrictive temperature (37C) on galactose-containing medium and even on glucose-containing medium (Roelants, unpublished observations), indicating that each construct was functional. Similarly, 3GFP-Ypk1 and 3GFP-Ykr2 retained their biological function (and transcriptional control was tighter) because each construct was able to complement the temperature sensitivity of ypk1-1ts ykr2Delta cells at the nonpermissive temperature (37C) on galactose-containing medium (but not on glucose-containing medium) (Roelants, unpublished observations). In addition, as judged by immunoblotting with anti-GFP antibodies, each of the four tagged proteins was expressed intact and had the molecular weight expected for the full-length chimeric protein (Roelants, unpublished observations). Live wild-type cells expressing each of the four fusions to 3GFP were examined under the fluorescence microscope . The 3GFP-Ypk1 chimera was found exclusively in the cytosol and was excluded from both the vacuole (whose position was observed by phase contrast microscopy of the same field) and the nucleus (whose position was revealed by growing the cells in the DNA-specific dye DAPI). In contrast, the 3GFP-Ykr2 chimera accumulated in the nucleus, congruent with the DAPI-stained DNA, although it was also readily detectable in the cytoplasm. Interestingly, when fused to 3GFP, the catalytic domain of Ypk1 (Ypk1DeltaN), which by itself is toxic when overexpressed (see above), was located predominantly in the nucleus, unlike full-length 3GFP-Ypk1, suggesting that its toxicity may arise largely from its mislocalization. The same patterns of distribution for Ypk1 and Ykr2 were also observed if the cells were fixed, permeabilized, and stained, respectively, with polyclonal anti-Ypk1 and anti-Ykr2 antibodies (Torrance, unpublished observations). Likewise, identical patterns of distribution were observed when cells expressing Ypk1-myc or Ykr2-myc were examined by indirect immunofluorescence using anti-c-Myc mAb 9E10 (Roelants, unpublished observations). Figure 6 | Subcellular localization of GFP-tagged Ypk1, Ykr2, Pkh1, and Pkh2. Subcellular localization of GFP-tagged Ypk1, Ykr2, Pkh1, and Pkh2. Wild-type (YPH499) cells were transformed with low copy number (CEN DNA-based) plasmids expressing from the GAL1 promoter either 3GFP-Ypk1 (pFR33), 3GFP-Ypk1-DeltaN (pFR34), 3GFP-Ykr2 (pER2), 3GFP-Pkh1 (pFR37), or 3GFP-Pkh2 (pER3), as indicated. The transformants were grown to mid-exponential phase at 30C in SCRaf/Suc-Leu, induced with galactose (2%) for 3 h, and samples of each culture were viewed directly under a fluorescence microscope. To permit visualization of the position of the nucleus, DAPI was added to the medium (1 mug/ml final concentration) during the last hour of induction. As observed for 3GFP-Ypk1, 3GFP-Pkh1 was localized exclusively to the cytosol, and clearly excluded from both the vacuole and the nucleus . The most prominent feature of the 3GFP-Pkh1 staining was, however, bright puncta or larger patches situated at the cell cortex. These dots are not congruent with actin patches, as was revealed by costaining with rhodamine-labeled phalloidin (Roelants, unpublished observations). The same distribution pattern was observed if cells expressing Pkh1-(HA)3 were fixed, permeabilized, and examined by indirect immunofluorescence by using an anti-HA mAb and an appropriate fluorescently tagged secondary antibody (Roelants, unpublished observations). Unlike 3GFP-Pkh1, 3GFP-Pkh2 was not excluded from the nucleus, but like 3GFP-Pkh1, the most prominent feature of the staining was a large number of punctate bodies immediately subtending the plasma membrane , which were also distinct from actin patches (Roelants, unpublished observations). Thus, taken together, these observations indicate that Pkh2 and Ykr2 are able to enter a compartment (the nucleus) from which Pkh1 and Ypk1 are normally excluded. Thus, these findings help to explain, at least in part, the greater dependence of Ypk1 activation on Pkh1 and the greater dependence of Ykr2 activation on Pkh2. Genetic Analysis of Ypk1 and Ykr2 Function by Selection of Dosage Suppressors | To identify gene products that may be involved in processes both upstream and downstream of Ypk1 and Ykr2, we selected, first, for genes that when overexpressed from a URA3-marked multicopy vector, were able to restore growth to ypk1-1ts ykr2Delta cells at an otherwise nonpermissive temperature (35C), as described in MATERIALS AND METHODS. From 20,000 Ura+ transformants, we recovered 18 plasmids that were able to support growth reproducibly at the restrictive temperature . As expected, seven independent isolates of YKR2 and one isolate of YPK1 were obtained. Two of the other suppressor genes obtained, one encoding a putative chaperone (HLJ1) and the other encoding a component of RNA polymerase II holoenzyme (SRB4), may rescue because they stabilize or elevate expression of the temperature-sensitive Ypk1-1 enzyme, although this hypothesis was not tested directly. Another suppressor plasmid carried the YPC1 gene, which encodes an enzyme that can generate phytosphingosine from the corresponding phytoceramide and hence presumably rescues by hyperstimulating Pkh1 and Pkh2. Indeed, we have shown previously that elevated Pkh1 can restore growth to ypk1-1ts ykr2Delta cells at otherwise restrictive temperature . Indeed, when excised from the original isolate and overexpressed from a completely different vector, YPC1 rescues the temperature sensitivity of ypk1-1ts ykr2Delta cells (Roelants, unpublished observations). In contrast, at least one other of the genes from the same insert, RPS6B, was not a suppressor on its own (Roelants, unpublished observations). Table 2 | Dosage suppressors of the temperature-sensitive lethality of ypk1-1ts ykr2Deltacells Revealingly, among the seven remaining dosage suppressors, two plasmids carried a single intact open reading frame corresponding to the EXG1 gene, which encodes the major exo-beta(1,3)-glucanase involved in cell wall remodeling . Indeed, when excised from the original isolate and expressed from a constitutive promoter (ADH1) in a completely different multicopy vector (see MATERIALS AND METHODS), elevated expression of EXG1 reproducibly suppressed, albeit weakly, the temperature-sensitive growth defect of ypk1-1ts ykr2Delta cells, even at 37C (Figure A). Also obtained were two isolates of a locus (YBL104c) of unknown function, but which seems from the phenotype of a null allele to also have effects on cell wall structure . Another suppressor plasmid isolated carries multiple open reading frames, one of which is a candidate chitinase , which may also influence cell wall structure. At least one of the other genes carried on this same plasmid, FRQ1 , is not responsible for the suppression and does not contribute to the suppression (Roelants, unpublished observations). The final two dosage suppressors encoded proteins that might act by enhancing the efficiency with which enzymes involved in cell wall biosynthesis or remodeling are delivered to their final destination and/or are activated there. One plasmid carried GOT1, which specifies a membrane protein thought to enhance the function of a t-SNARE heavy chain, Sed5, involved in vesicle-mediated protein transport from the endoplasmic reticulum (ER) to the Golgi . The other plasmid carried only PLB1, which specifies the phospholipase B that is primarily responsible for the conversion of phosphatidylcholine and phosphatidylethanolamine in the exocellular leaflet of the plasma membrane to lysophosphatidylcholine (and glycerophosphocholine) and lyso-phosphatidylethanolamine (and glycerophosphoethanolamine), respectively . It is well documented that the activity of many classes of membrane-associated enzymes can be influenced dramatically (either stimulated or inhibited), depending on the nature of the phospholipids (or their derivatives) with which those enzymes associate . Figure 7 | Relationship between dosage suppressor (EXG1) and extragenic suppressor (kex2Delta). Relationship between dosage suppressor (EXG1) and extragenic suppressor (kex2Delta). (A) Overexpression of EXG1 suppresses the temperature sensitivity of ypk1-1ts ykr2Delta cells. A temperature-sensitive ypk1-1tsykr2Delta strain (YPT40) was transformed with either an empty vector (pAD4M) or the same vector expressing from the ADH1 promoter either YPK1 (pADH-YPK1) or EXG1 (pAM88). Growth of the resulting transformants was assessed after 2 d at 30 and 37C, as indicated. (B) Deletion of KEX2 suppresses the temperature sensitivity of ypk1-1tsykr2Delta cells. A ypk1-1tsykr2Delta strain (YAN2) was crossed to a kex2Delta strain (KRY24), and the resulting diploid cells (YFR66) were sporulated and dissected. The four spores of a tetratype ascus derived from this diploid were recovered and tested for growth on YPGlc at 30 and 37C, as indicated. (C) Suppression by kex2Delta requires EXG1. A MATaypk1-1tsykr2Delta kex2Delta strain, derived as described in B, was crossed to an exg1Delta strain (YFR84) and the resulting diploid cells were sporulated and dissected. Serial dilutions of cultures of the indicated genotypes were spotted on YPGlc plates and growth was assessed after 3 d at 30 and 37C, respectively. (D) Either kex2Delta cells or rot2Delta cells lacking Mpk1 are inviable. A kex2Delta strain (KRY24) was crossed to a mpk1Delta strain (YFR128), and the resulting diploid cells were sporulated and dissected on plates containing 1.2 M sorbitol. The four spores of a tetratype ascus derived from this diploid were recovered and tested for growth on YPGlc and YPGlc containing 1 M sorbitol at 30C. The same procedure was applied to diploid cells resulting from crossing a rot2Delta strain (YFR129) and an mpk1Delta strain (YFR127). Taken together, the nature of the dosage suppressors obtained, along with our observation that ypk1Delta cells showed a general increase in permeability to inhibitory drugs of several different chemical classes (see above), strongly suggested that the primary defect in Ypk1- and Ykr2-deficient cells involved some aspect of cell wall biosynthesis and/or structure. Genetic Analysis of Ypk1 and Ykr2 Function by Selection of Suppressor Mutations | To gain further insight, and to corroborate the conclusion that absence of Ypk1 and Ykr2 compromises some aspect of cell wall structure, we also performed a selection for genes that, when interrupted by insertion of a transposon (Tn3::LacZ::LEU2), were able to restore the ability of ypk1-1ts ykr2Delta cells (strain YPT40) to grow at high temperature (35C), as described in MATERIALS AND METHODS. From 80,000 Leu+ transformants, 16 haploid isolates were obtained that contained a transposon insertion and for which Leu+ segregated with the ability to grow at high temperature when the isolate was backcrossed to a ypk1-1ts ykr2Delta cell of opposite mating type (strain YAN2) . One extragenic suppressor obtained inactivated a gene (SRN2) that interacts genetically with the machinery involved in nucleocytoplasmic transport. This mutation may simply enhance export of YPK1 mRNA and thus expression of the temperature-sensitive Ypk1-1 enzyme, although this hypothesis was not tested directly. Two other insertions disrupted uncharacterized loci of unknown function. Reassuringly, however, the 13 remaining extragenic suppressors fell in genes of known function, which were all involved in processes required for biosynthesis of normal cell wall glycoproteins. Table 3 | Transposon insertions that suppress the temperature-sensitive lethality of ypk1-1ts ykr2 cells Four independent isolates represented transposon insertions in the ALG5 gene. Alg5 is an integral membrane enzyme that transfers glucose from UDP-Glc to the dolichol carrier that is used to attach the Glc residues to the immature Asn-linked (GlcNAc)2(Man)9(Glc)3 core oligosaccharide, which is added en bloc to cell wall mannoproteins and other secreted glycoproteins in the lumen of the ER . Three independent suppressors were insertions in a gene (ROT2) encoding the integral membrane enzyme that trims two alpha(1,3)-linked glucose residues from the (GlcNAc)2(Man)9(Glc)3 core during subsequent maturation of secreted glycoproteins . Two additional extragenic suppressors inactivated two other genes, ALG3/RHK1 and ALG9 , that encode mannosyltransferases involved in adding the sixth and seventh mannose residues, respectively, to the (GlcNAc)2(Man)9(Glc)3 core during its biosynthesis. Two other suppressor mutations fell in the gene (DNF3), which encodes an apparent transport ATPase for aminophospholipids (phosphatidylethanolamine and phosphatidylserine) , which may affect the composition of the ER membrane and thereby influence the activity of one or more of the enzymes involved in Asn-linked oligosaccharide biosynthesis mentioned above. Strikingly, two more independent isolates corresponded to transposon insertions in the KEX2 gene, which encodes a Golgi-localized endoprotease that participates in maturation of secreted precursor glycoproteins by cleaving on the C-terminal side of pairs of basic residues . To verify that suppression was due to Kex2 loss of function, we crossed a ypk1-1ts ykr2Delta strain against a kex2-Delta2::LEU2 strain, in which the entire KEX2 open reading frame was deleted and replaced by the LEU2 gene, and examined the phenotype of the spores derived from resulting tetratype asci. Just like the original transposon insertions, a standard kex2 null allele also rescued the growth of ypk1-1ts ykr2Delta cells at the restrictive temperature (Figure B). One possible explanation for this suppression is that, normally, Ypk1 and/or Ykr2 are negative regulators of Kex2 synthesis, function, or intracellular trafficking. In this regard, it was noteworthy that Kex2 is reportedly involved in processing of the precursor of the Exg1 exoglucanase . Thus, the fact that overexpression of Exg1 also rescued the temperature sensitivity of a ypk1-1ts ykr2Delta strain (Figure A) suggested that, perhaps, it is the unprocessed form of Exg1 that is responsible for the suppression, because this precursor form presumably accumulates when Kex2 is absent (due to mutation) or if Kex2 is limiting (when Exg1 is overproduced). To determine whether amelioration of the temperature sensitivity of ypk1-1ts ykr2Delta cells by loss of Kex2 involved Exg1, we deleted EXG1 in the ypk1-1ts ykr2Delta kex2Delta strain. Indeed, the absence of Exg1 greatly reduced the ability of the ypk1-1ts ykr2Delta kex2Delta cells to grow at nonpermissive temperature (Figure C), suggesting that an intact EXG1 gene is required for mediating, at least in part, the suppressive effect of loss of Kex2. The residual growth observed could be explained by the fact that the S. cerevisiae genome encodes 12 other demonstrated and presumptive glucanases whose precursors may also require Kex2-mediated processing. However, there was another equally plausible explanation for the ability of kex2 mutations to suppress the temperature sensitivity of ypk1-1ts ykr2Delta cells that was consistent with all of the above-mentioned observations. Specifically, absence of Kex2 prevents processing of certain secreted cell wall mannoproteins, causing defects in the cell wall . Moreover, various defects in the cell wall trigger activation of the Pkc1-dependent mitogen-activated protein (MAP) kinase Mpk1/Slt2 and induction of genes under its control , including EXG1 . Indeed, in agreement with the hypothesis that the Pkc1-Mpk1 pathway is induced when cell wall structure is perturbed by a kex2Delta mutation and by at least one other of the extragenic suppressors (rot2Delta) we isolated, we found that these mutants are inviable when Mpk1 is absent but rescued on medium containing an osmotic support (Figure D). In fact, this synthetic lethality suggests that the only reason that kex2Delta and rot2Delta mutants are able to survive is that they induce the Pkc1 --Mpk1 pathway, which up-regulates glucan synthases and many other enzymes necessary to repair, modify, and maintain the otherwise abnormal cell wall . Thus, collectively, the above-mentioned findings suggested that Ypk1 and Ypk2 participate in a signaling pathway required for optimal cell wall integrity and that all of the dosage suppressors and extragenic suppressors rescue the lethality of ypk1-1ts ykr2Delta cells because they cause additional cell wall perturbations that induce the alternative Pkc1 --Mpk1 cell wall integrity signaling pathway, and therefore bypass the need for efficient Ypk1- and Ykr2-dependent signaling. Ypk1 and Ykr2 Are Involved in a Novel Cell Wall Integrity Signaling Pathway | One diagnostic property of many of the conditional mutations whose primary defect is perturbation of cell wall structure is that the cells lose viability rapidly at the restrictive temperature because they undergo lysis (for review, see ). Indeed, we found that >50% of the population of ypk1-1ts ykr2Delta cells underwent lysis by 2 h after shift to nonpermissive temperature (37C), and >90% of the cells were lysed by 4 h after the shift, as judged by staining with a commercial vital dye and by plating the cells for viable titer, whereas <2% of control cells were lysed under the same conditions . A second hallmark of mutations that lead directly or indirectly to defects in cell wall structure is that inviability can be rescued in medium containing an osmotic support . Again, consistent with a primary defect in cell wall integrity, the lysis phenotype of ypk1-1ts ykr2Delta cell could be completely prevented by the presence of an osmotic support (1.2 M sorbitol) in the growth medium (Figure A). Third, there is coupling between normal cell wall assembly and proper organization of the actin cytoskeleton . In normal cells, actin patches are confined to the bud and bundles of actin cables are found only in the mother cell . We found, first, that compared with isogenic wild-type cells, ypk1Delta cells showed a marked depolarization of the actin cytoskeleton even at 30C, with pronounced actin patches in the mother cell and no detectable bundles of actin cables (Figure B). Likewise, at 30C, ypk1-1ts ykr2Delta cells displayed normal actin polarization, whereas after shift to restrictive temperature (37C) and before lysis, ypk1-1ts ykr2Delta displayed a pronounced defect in actin polarization, with numerous actin patches present in the mother cell at all stages of the cell cycle and no detectable bundles of actin cables (Figure B). Figure 8 | Ypk1 and Ykr2 are required for maintenance of cell wall integrity. Ypk1 and Ykr2 are required for maintenance of cell wall integrity. (A) Osmotic support permits growth of ypk1-1tsykr2Delta cells at restrictive temperature. Either ypk1-1tsykr2Delta cells (YPT40) or wild-type cells (WT; YPH499) were streaked onto YPGlc plates or YPGlc plates containing 1.2 M sorbitol and incubated for 2 d at 30 and 37C, respectively. (B) Loss of Ypk1 causes defects in actin organization. Wild-type cells (WT; YPH499), top line, and ypk1Delta cells (YES3), second line, were grown to mid exponential phase at 30C. Also, ypk1-1tsykr2Delta cells (YPT40) were grown in YPGlc at 26C for 18 h to an A600 nm = 0.5, third line, and then a portion of the same culture was shifted to 37C for 2 h, bottom line. Samples of all four cultures were then fixed, stained with a reagent specific for visualizing F-actin (rhodamine-labeled phalloidin), and examined under the fluorescence microscope. (C) Hyperactivation of the Pkc1-dependent Mpk1 MAP kinase pathway rescues the temperature sensitivity of a ypk1-1tsykr2Delta cells. Strain YPT40 (ypk1-1ts ykr2Delta) was transformed with an empty 2-mum DNA-based vector (YEp351) or with the same vector expressing from their native promoters either YPK1 (pAM21), PKC1 (pFR32), or BCK1-20 (pRS315-BCK1-20), or a different 2-mum DNA-based vector expressing EXG1 from the ADH1 promoter (pAM88). The resulting transformants were streaked onto SCGlc-Leu and growth was assessed after 3 d at the indicated temperatures (26, 35, and 36C). (D) ypk1-1tsykr2Delta cells lacking Mpk1 are inviable. A heterozygous ypk1-1ts/YPK1 ykr2Delta/YKR2 kex2Delta/KEX2 diploid strain (YFR66) was deleted for MPK1, and the resulting diploid strain was sporulated and dissected on YPGlc containing 1.2 M sorbitol. The indicated strains were streaked onto YPGlc plates and growth was assessed after 3 d at 26 and 30C. All of the above-mentioned phenotypes are also displayed by mutations that compromise the Pkc1-dependent cell wall integrity signaling pathway (for review, see ). In this pathway, Pkc1 (a protein kinase C-like enzyme) activates a MAP kinase cascade composed of a mitogen-activated protein kinase kinase kinase (Bck1), two redundant mitogen-activated protein kinase kinases (Mkk1 and Mkk2), and the Mpk1/Slt2 MAP kinase. Therefore, we tested directly the hypothesis that induction of the Pkc1-Mpk1 signaling pathway can bypass the defect in cells deficient in Ypk1- and Ykr2-mediated signaling. Consistent with this idea, we found that overexpression of PKC1 from its own promoter on a multicopy (2 mum DNA) plasmid was sufficient to rescue the temperature sensitivity of ypk1-1ts ykr2Delta cells (strain YPT40) and did so more efficiently than any of the dosage suppressors we selected directly, including EXG1 (Figure C). Similarly, PKC1 overexpression, like expression of YKR2 (as a control), was able to rescue the actin polarization defects of ypk1-1ts ykr2Delta cells at nonpermissive temperature (Roelants, unpublished observations). Likewise, expression of a constitutively active allele of BCK1 (BCK1-20) was also able to restore growth at high temperature, although somewhat less efficaciously than PKC1 (Figure C). Thus, in some senses, Pkc1 and the Mpk1 MAP kinase pathway act downstream of Ypk1 and Ykr2. On the other hand, several observations indicated that Ypk1 and Ykr2 do not act upstream of Pkc1, but rather in a parallel pathway. First, none of the dosage suppressors, none of the extragenic suppressors, and neither osmotic support nor overexpression of PKC1 or BCK1 --20 was able to rescue the inviability of ypk1Delta ykr2Delta cells (Roelants and Torrance, unpublished observations). Thus, it seems that ypk1-1ts ykr2Delta cells retain some low level of throughput at the restrictive temperature and that elevation of Pkc1-induced processes can act in conjunction with that small signal but cannot substitute completely for it. Second, just as overexpression of PKC1 was unable to rescue ypk1Delta ykr2Delta cells, overexpression of YPK1 or YKR2 could not suppress the temperature sensitivity of the pkc1-2ts mutation (Roelants, unpublished observations). Third, and most revealingly, we found that a ypk1-1ts ykr2Delta mpk1Delta triple mutant was inviable on YPGlc medium at 30C, conditions under which otherwise congenic ypk1-1ts ykr2Delta mutants, ykr2Delta mutants, mpk1Delta mutants, and ykr2Delta mpk1Delta mutants all grow well (Figure D). This synthetic lethality indicates that efficient Ypk1- and Ykr2-dependent signaling and signaling via the MAP kinase pathway downstream of Pkc1 are both required for viability. Hence, Ypk1 and Ykr2 most likely function in a pathway that acts in parallel to the Pkc1 pathway (or perhaps through Pkc1 effectors distinct from Mpk1). PMID- 12221112_DISCUSSION TI - AB - Relative Roles of Ypk1 and Ykr2 Protein Kinases | Although either Ypk1 or Ykr2 is able to perform their shared, essential function at normal growth temperature (30C), several lines of evidence support the conclusion that Ypk1 is the primary enzyme. First, although both ypk1Delta and ykr2Delta single mutants are alive , we found that ypk1Delta cells grow slowly at 30C, are cold sensitive, and are hypersensitive to antibiotics and caffeine, whereas ykr2Delta cells display none of these phenotypes. The dramatic cold sensitivity of ypk1Delta cells is probably explained by the fact that recent analysis of global transcription patterns by using DNA microarrays has clearly shown that YKR2 (but not YPK1) is a gene strongly induced by heat stress . Inspection of the 5'-flanking region of the YKR2 locus yields three matches to the consensus nucleotide sequence TTC(N)2 --3GAA for the binding of the heat shock transcription factor (Hsf1) at -497, -404, and -104 from the ATG initiator codon. Thus, at lower temperatures, ypk1Delta cells presumably express YKR2 poorly and behave, therefore, like cells deficient in both enzymes, which are inviable. Other data also support the conclusion that, under normal growth conditions, Ypk1 is the enzyme most important for carrying out the function(s) essential for cell viability. Overexpression of catalytically inactive Ypk1 (Ypk1-KD) had detrimental effects on cell growth, whereas an equivalent level of overexpression of the analogous kinase-dead allele of Ykr2 did not. These detrimental effects were significantly exacerbated when there was no wild-type Ypk1 present to compete with the mutant protein. In other words, the presence of wild-type Ykr2 (in a ypk1Delta cell) was insufficient to overcome the toxicity imposed by Ypk1-KD, whereas the presence of wild-type Ypk1 (in a ykr2Delta cell) was able to ameliorate this toxicity to some extent. Furthermore, at steady state, Ypk1 was located exclusively in the cytosol, whereas Ykr2 was largely sequestered in the nucleus, suggesting that cytosolic targets of these enzymes are more important for normal growth than nuclear targets. Indeed, overexpression of the catalytic domain of Ypk1 was toxic, and this toxicity may be due to mislocalization because, when tagged with 3GFP, this constitutively active fragment accumulated in the nucleus. Nonetheless, overexpression of Ykr2 can overcome all of the phenotypes of ypk1Delta cells, providing evidence that both enzymes are able to perform the same essential function(s). Thus, the phenotypic differences between ypk1Delta and ykr2Delta mutants presumably arise primarily from differential expression of the corresponding genes, as discussed above, and from the differential subcellular localization of these proteins, rather than from differences in the intrinsic specificity of these enzymes for their substrates. Division of Labor between Upstream Protein Kinases Pkh1 and Pkh2 | Pkh1 and Pkh2 are responsible for the activation of several downstream protein kinases, and we have shown that either enzyme can fulfill this function . This role is sufficient to explain why Pkh1 and Pkh2 are essential gene products because the targets of Pkh1 and Pkh2 include protein kinases that are themselves known to be required for cell viability, such as Pkc1 and, together, Ypk1 and Ykr2 . Our findings suggest, however, that Pkh1 and Pkh2 have differential roles with regard to phosphorylation and activation of Ypk1 and Ykr2. First, as measured by immune-complex kinase assays, absence of Pkh1 substantially reduced Ypk1 activity in cell extracts, whereas absence of Pkh2 did not; in contrast, absence of Pkh2 did reduce Ykr2 activity. Conversely, overproduction of Pkh1 increased Ypk1 activity in cell extracts much more than overproduction of Pkh2, whereas overproduction of Pkh2 increased Ykr2 activity more than overproduction of Pkh1. Second, as expected if Pkh1 is the primary activator of Ypk1, absence of Pkh1 (but not Pkh2) suppressed the toxicity resulting from overexpression of a constitutively active, carboxy-terminal fragment containing the catalytic domain of Ypk1. This result is in accord with our previous observations that overexpression of Pkh1 (but not Pkh2) was able to suppress the temperature sensitivity of ypk1-1ts ykr2Delta cells and that absence of Pkh1 (but not Pkh2) resulted in a detectable decrease in incorporation of [32PO43-] into Ypk1 that was immunoprecipitated from extracts of metabolically labeled cells . Third, as expected if Pkh2 is the primary activator of Ykr2, the slow growth and drug sensitivity of ypk1Delta cells were exacerbated by absence of Pkh2 (but not Pkh1). Fourth, at steady state, Pkh1 and Ypk1 are confined to the cytosol and excluded from the nucleus, whereas Pkh2 and Ykr2 are not excluded from the nucleus. Taken together, these findings support the conclusion that, in the cell, Pkh1 preferentially activates Ypk1 and Pkh2 preferentially activates Ykr2. However, several other findings indicate that this separation is by no means complete. First, when overexpressed, either Pkh1 or Pkh2 was able to stimulate either Ypk1 or Ykr2 activity over that observed in the controls. Second, in the case of Ykr2, absence of either Pkh1 or Pkh2 caused a similar reduction in Ykr2 activity. Finally, and most convincingly, all four double mutant combinations (pkh1Delta ypk1Delta, pkh1Delta ykr2Delta, pkh2Delta ypk1Delta, and pkh2Delta ykr2Delta) were recovered as viable haploids at the expected frequency after tetrad dissection of the appropriate doubly heterozygous diploid strains. Therefore, despite the apparent Pkh1-Ypk1 and Pkh2-Ykr2 dichotomy, the capacity exists for significant cross talk between these enzyme-substrate pairs. Physiological Function of Pkh1-Ypk1 and Pkh2-Ykr2 Cascades | There is compelling evidence in both yeast and mammalian cells that membrane microdomains enriched in sphingolipids and sterols, referred to as rafts, are involved in the biosynthetic delivery of certain proteins to the cell surface. Likewise, there is substantial evidence both in yeast and in animal cells that sphingolipid rafts also play a role in a clathrin-independent route of endocytosis. It has been reported recently that the sphingoid base requirement for the internalization step of endocytosis may be to activate Pkh1 and Pkh2, and that Pkc1, a known Pkh1 and Pkh2 target , acts as a downstream effector in this signaling pathway. In this regard, it is noteworthy that we found GFP-tagged Pkh1 and Pkh2 both localized primarily to prominent cortical puncta, distinct from actin patches, that seem to be membrane-associated, as judged by three-dimensional reconstruction of images taken using deconvolution fluorescence microscopy (Roelants, unpublished observations). We are currently exploring whether these structures represent microdomains enriched in sphingolipids, although there are, to our knowledge, no reliable cytological markers for such structures currently available. While our studies were in progress, it was also reported that the growth inhibitory effect of an antibiotic myriocyn (also known as ISP-1) that causes sphingolipid depletion can be overcome by overexpression of Ypk1 , suggesting that Ypk1 may also be a downstream target in response to phytosphingosine-dependent activation of Pkh1 and Pkh2, in agreement with our previous findings demonstrating that Pkh1 and Pkh2 act upstream of phosphorylate and activate Ypk1 and Ykr2 . Even more recently, it has been claimed that Ypk1 (but not Ykr2) is involved directly in endocytosis . However, proper actin assembly is critical for endocytosis in yeast , and we found that loss of Ypk1 alone had profound effects on actin organization. Thus, the apparent role of Ypk1 in endocytosis may have been inferred from a rather indirect effect that is secondary to its primary function. Several properties of ypk1-1ts ykr2Delta cells, including rapid lysis at restrictive temperature, rescue of the lysis phenotype by osmotic support, altered actin organization, and increased sensitivity to many different toxic agents, were all consistent with a cell wall defect. To gain further insight about the physiological role of Ypk1 and Ykr2, we selected both dosage suppressors and transposon insertions that restored viability to ypk1-1ts ykr2Delta cells at an otherwise nonpermissive temperature. The majority of both classes of suppressors provided additional evidence for a direct connection between Ypk1 and Ykr2 and cell wall biosynthesis. In most cases, suppression could be attributed to imposition of further cell wall damage sufficient to trigger the Pkc1-mediated activation of the Mpk1 MAP kinase-dependent cell wall integrity pathway, suggesting that Ypk1 and Ykr2 themselves are components of a novel pathway also responsible for activation of the transcription of genes involved in cell wall maintenance and remodeling. Indeed, while our article was in preparation, a study was published that also linked Ypk1 and Ykr2 to cell integrity signaling on the basis of a completely independent approach . Because the rate of phytosphingosine generation will depend on the phytoceramide concentration in the plasma membrane, the Pkh1-Ypk1 and Pkh2-Ykr2 cascades could represent a feedback control mechanism whereby membrane growth via insertion of sphingolipid-enriched vesicles is monitored and coordinately coupled to appropriate expansion of the cell wall . This signaling route seems to be distinct from, but work in parallel with, the Pkc1 pathway for cell wall maintenance, in which plasma membrane proteins (such as Wsc1/Slg1 and Mid2) serve as sensors of cell turgor pressure and function by stimulating guanine nucleotide exchange factors for the small GTPase Rho1, a known activator of Pkc1 (for review, see ). Figure 9 | Pkh1-Ypk1 and Pkh2-Ykr2 cascades and cell wall integrity signaling. Pkh1-Ypk1 and Pkh2-Ykr2 cascades and cell wall integrity signaling. For proper cell enlargement, plasma membrane growth needs to be accommodated by expansion of the cell wall. This coordinated coupling is achieved through at least two pathways. As shown previously, one route for monitoring plasma membrane growth is via the insertion of transmembrane proteins that affect the state of activation of the small GTPase, Rho1. Rho1, in turn, stimulates the protein kinase, Pkc1, and the downstream MAP kinase kinase cascade (Bck1 -> Mkk1 and Mkk2 -> Mpk1). The Mpk1 MAP kinase kinase activates transcription factors, including Rlm1, that regulate genes for cell wall enzymes. As shown in this study, a two-tiered cascade of functionally redundant protein kinases (Pkh1-Ypk1 and Pkh2-Ypk2) appears to monitor growth of the plasma membrane via a distinct mechanism. In this novel pathway, a product (phytosphingosine) derived from the vesicle-mediated insertion of sphinolipids into the plasma membrane stimulates Pkh1 and Pkh2. These enzymes, in turn, phosphorylate and activate Ypk1 and Ykr2, primarily in the cytosol and the nucleus, respectively. Genetic evidence indicates that Ypk1 and Ykr2 may act through the transcription factor, Smp1, which shares DNA-binding specificity with Rlm1. Nuclear entry is only one potential level at which the activity of these transcription factors might be regulated by these signaling pathways. Cross talk between these pathways probably occurs at two levels: full activation of Pkc1 requires phosphorylation by Pkh1 and/or Pkh2 and Ypk1 (and perhaps Ykr2) may contribute (directly or indirectly) to Mpk1 activation . See DISCUSSION for additional details. One possibility is that Ypk1 and Ykr2 feed into the known cell integrity signaling pathway by providing a Pkc1-independent route to activate Mpk1 itself. Consistent with this idea is our observation that ypk1-1ts ykr2Delta cells are inviable when Mpk1 is absent and with the fact that Mpk1 phosphorylation induced by heat stress is reduced in a ypk1Delta mutant . Moreover, the strongest suppressors we obtained were loss-of-function mutations in the Kex2-processing enzyme, which is known to cause abnormalities in the cell wall , and such abnormalities evoke Pkc1- and Mpk1-dependent signaling . Not all Pkc1-dependent responses are achieved via the Mpk1 MAP kinase pathway . However, we found that kex2Delta mutants are inviable when Mpk1 is absent, suggesting that Kex2-deficient cells can only survive because genes under Mpk1 control are activated. Likewise, it has been observed by others that kex2Delta mutants seem to have cell wall defects (Fuller, personal communication) and are inviable when they are defective in components of a signaling pathway that monitors extracellular Ca2+, including calmodulin (Cmd1) and calmodulin-activated phosphoprotein phosphatase 2B/calcineurin (Cna1, Cna2, and Cnb1) (Davis, personal communication), or the calcineurin-activated C2H2-type zinc finger transcription factor (Crz1/Tcn1) (Cunningham, personal communication), or are exposed to known calcineurin inhibitors, cyclosporin A, or FK506 (Fuller, personal communication). Strikingly, it has been shown previously that Crz1 provides an independent means to regulate many of the same genes that are under the control of the Pkc1- and Mpk1-dependent pathway . Additional evidence that absence of Kex2 leads to up-regulation of the transcriptional initiation of genes essential for viability (including, presumably, cell wall synthesis) is provided by two previously obscure findings. First, it was observed that kex2Delta mutations bypass temperature-sensitive mutations in the largest subunit of RNA polymerase II (Rpo21) and also temperature-sensitive mutations in other RNA polymerase II subunits . Second, and conversely, kex2Delta mutations are synthetically lethal when combined with otherwise viable null mutations (ppr2Delta) in the gene encoding a factor (TFIIS) needed for efficient transcriptional elongation by RNA polymerase II . However, for several reasons, we currently favor the idea that Ypk1 and Ykr2 activate genes for cell wall remodeling independently of the Pkc1-activated Mpk1 MAP kinase kinase pathway. One of the transcription factors under Mpk1 control is Rlm1 (676 residues), a member of the Mcm1, Agamous, Deficiens, Serum Response Factor-box family of transactivators . In this regard, it seems more than a coincidence that one of the dosage suppressors of the temperature-sensitive lethality of ypk1-1ts ykr2Delta cells that we isolated carries the SMP1 gene, which encodes another Mcm1, Agamous, Deficiens, Serum Response Factor-box transcription factor related to Rlm1. Smp1 (452 residues) shares near identity to Rlm1 in its similar60 residue, N-terminal, DNA-binding domain (but bears little similarity to Rlm1 beyond that), recognizes the same (but a somewhat more extended) sequence motif as Rlm1, and is even able to form heterodimers with Rlm1 . Indeed, when excised from the original isolate and expressed from a completely different multi-copy vector, elevated SMP1 expression reproducibly suppressed the temperature-sensitive growth defect of ypk1-1ts ykr2Delta cells and did so better than YPC1 and as well as EXG1 (Roelants, unpublished observations). Moreover, Smp1 contains three consensus Ypk1 phosphorylation sites (-R-x-R-x-x-S/T-Hyd-, where Hyd indicates any bulky hydrophobic residue; ), and in preliminary experiments, Smp1 can be phosphorylated by Ypk1 in vitro (Roelants, unpublished observations). Hence, currently we favor the idea that Ypk1 and Ykr2 do not act by leading to Mpk1 activation per se, but rather by providing an independent input through Smp1 that acts in parallel to or in conjunction with Rlm1 to activate genes involved in cell wall metabolism, including EXG1 . Experiments to test the above-mentioned ideas, including analysis of global transcription profiles with DNA microarrays, are underway. PMID- 12221113 TI - Distinct Chromosome Segregation Roles for Spindle Checkpoint Proteins AB - | The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage. PMID- 12221113_INTRODUCTION TI - AB - Protein components of the spindle checkpoint were first defined genetically through studies in the budding yeast Saccharomyces cerevisiae by analysis of mutants that lack the ability to arrest in the presence of spindle damage introduced by antimicrotubule drug exposure or by manipulation of temperature conditional spindle proteins . The spindle checkpoint thus defined has been shown to control at least two functionally distinct steps within mitosis. First, at metaphase, the checkpoint acts to detect a lack of bipolar attachment or tension for any sister chromatid pair. This condition delays anaphase in the presence of even a single unattached kinetochore or a lack of tension on a single chromatid pair . Second, entry into G1 (mitotic exit) is prevented in cells that have suffered spindle damage sufficient to preclude the delivery of a daughter nucleus into the bud (for review, see ; ). Metaphase arrest due to activation of the spindle checkpoint depends upon a well-conserved pathway that regulates the degradation of the anaphase inhibitor protein Securin (budding yeast Pds1p; for review, see ; ). Anaphase is normally initiated as Separin (Esp1p) is liberated from its binding partner Securin (Pds1p) after Securin is targeted for degradation by the Cdc20-associated form of the anaphase promoting complex. Cdc20p is a target of the metaphase checkpoint arrest pathway and physically interacts with other checkpoint proteins during metaphase arrest (for review, see ; ; ). Maintenance of the arrest induced by kinetochore damage also requires arrest of the mitotic exit pathway , indicating a functional connection between the distinct control pathways that operate at anaphase initiation and mitotic exit. In budding yeast, both of these steps are inhibited by the presence of Pds1 protein , and thus anaphase and exit control may be related to one another by a key role played by Pds1p or Esp1p at both cell cycle positions . Initiation of metaphase arrest in response to a lack of bipolar attachment requires at least six proteins in S. cerevisiae: Mad1p, Mad2p, Mad3p, Bub1p, Bub3p, and Mps1p . These proteins function together in kinetochore surveillance, activating a checkpoint-governed arrest in response to microtubule defects, kinetochore protein defects, or centromere DNA mutations . The proteins involved in kinetochore surveillance are remarkably conserved in eukaryotes, and homologs have been found in fission yeast, flies, maize, frog, mouse, and human (for review, see ). In systems with robust cytology, homologus of Mad1p, Mad2p, Bub1p, Bub3p, and Mps1p have been observed to concentrate at unattached kinetochores in prometaphase. Thus, these proteins behave as expected components of a molecular structure that broadcasts an inhibitory signal that will be extinguished upon achievement of bipolar attachment and/or associated tension from spindle forces exerted in opposite directions (for review, see ; ; ). Physical association studies have shown that the metaphase arrest proteins reside in several complexes that contain overlapping components, and that these complexes exhibit alterations in a cell cycle-regulated manner. In budding yeast, Mad1p/Mad2p, Bub1p/Bub3p, and Mad3p/Bub3p complexes are detected in interphase, whereas cells in damage-induced metaphase arrest contain a Bub1p/Bub3p/Mad1p complex, as well as a Cdc20p/Mad2p/Mad3p/Bub3p complex . Moreover, at metaphase arrest, both Bub1p and Mad1p exhibit shifts in gel migration consistent with hyperphosphorylated states . Movement of constituents among protein complexes may represent the spatial communication from an activated (unattached) kinetochore to site(s) where the Cdc20-associated form of the anaphase promoting complex is poised to initiate the degradation of Pds1p. Although biochemical characterization of protein complexes has provided insight into features of checkpoint activation, the nature of the spatial regulation imposed at metaphase by the presence of unattached kinetochores has not been precisely elucidated. Indeed, it is possible that different kinetochore states, such as kinetochore-microtubule attachment or the presence of tension, may be handled at metaphase by either overlapping or distinct signaling pathways . Chromosome missegregation associated with loss of kinetochore surveillance by the spindle checkpoint has been observed. In budding yeast, observed an increase in chromosome missegregation in mad1, mad2, and mad3 mutants upon recovery from aberrant mitoses induced by exposure to the antimicrotubule drug nocodazole. In the absence of intentional spindle damage, chromosome missegregation has been detected in budding yeast bub1 and mad2 mutants as well as in Drosophila melanogaster bub1 , Schizosaccharomyces pombe Deltabub1 , and Caenorhabditis elegans mdf-1 and mdf-2 mutants . The chromosome missegregation phenotypes observed suggest that the spindle checkpoint plays a role in many cell cycles (even in the absence of induced damage), or that Bub1 and Mad2 checkpoint proteins have additional roles in kinetochore function. In this report, we present a quantitative survey of the segregation roles of five nonessential metaphase checkpoint proteins that govern kinetochore surveillance (Mad1, Mad2, Mad3, Bub1, and Bub3) in cells without additional spindle damage. We find that these spindle checkpoint proteins differ in their contributions, and that the absence of Bub1p or Bub3p has the greatest impact on segregation. Further analysis of the role of Bub1p leads to a model in which Bub1 protein provides chromosome stability through two separate mechanisms. PMID- 12221113_MATERIALS AND METHODS TI - AB - Yeast Media | All yeast media are as described in . Yeast Strains | Strains used in this study are listed in Table . Except where noted, experiments were conducted in an S288c laboratory background and are related by DNA-mediated transformation or isogenic mating and sporulation. Figure B, C, and E show data for strains that are derivatives of W303-1a. The one-hybrid assay was carried out in YJL128 and transformants derived from it. Table 1 | Yeast strains used in this study Figure 1 | Spindle checkpoint mutants exhibit different rates of chromosome loss. Spindle checkpoint mutants exhibit different rates of chromosome loss. (A) Null mutant sectoring phenotypes. The strains shown are wild type (YPH278), bub1Delta (YFP2), bub3Delta (YFS1100), mad1Delta (YFS1120), mad2Delta (YCD173), and mad3Delta (YFS1205). (B) Chromosome loss rates in null mutants determined by half-sector analysis. Wild type: 49 half-sectored colonies/61,276 total colonies (YPH278); 8/9,305 (YKH231). bub1Delta: 192/4,784 (YFP2); 89/2,121 (YRJ112). bub2Delta: 17/22,101 (YCD165); 3/3,440 (YRJ113). bub3Delta: 137/3,362 (YFS1100); 63/2,237 (YRJ114). mad1Delta: 139/12,394 (YFS1120); 32/8,552 (YMB111). mad2Delta: 87/10,153 (YCD173); 9/3,106 (YMB113). mad3Delta: 57/29,364 (YFS1205); 29/13,186 (YRJ111). (C) Immunoblots showing overexpression from a MET25 promoter. Extracts were taken after 2 h of inductionin media lacking methionine and were analyzed by Western blot using antibody specific for each protein. The left lane (vector, p423MET) shows the endogenous Bub1p expression level where detected; the right lane ( --MET) shows protein expressed from the MET25 promoter. All strains were generated from YKH231 by introduction of p423MET-derived plasmids containing full-length open reading frames cloned adjacent to the MET25 promoter. (D) Chromosome loss associated with overexpression of checkpoint genes. Half-sector analysis was performed after plating the strains in C on plates lacking methionine. Vector: 14/19,030. METpBUB1: 195/17,640. METpBUB3: 17/17,875. METpMAD1: 28/11,920. METpMAD2: 92/17,065. METpMAD3: 137/12,115. Two or more additional independent transformants tested for each construct showed the same chromosome instability phenotype by colony sectoring assay. (E) Benomyl sensitivity of checkpoint null mutants. Log phase cultures were spotted in a 10-fold dilution series on rich medium (YPD) or rich medium plus Benomyl. Strains were mad1Delta (YMB111), mad2Delta (YMB113), mad3Delta (YRJ111), bub1Delta (YRJ112), and bub3Delta (YRJ114). Chromosome Loss Rate | This assay was performed as previously described . Strains containing a nonessential SUP11-marked test chromosome and plasmids were grown in selective media and were plated at a density of similar200 colonies per plate on minimal (SD) medium, including 20 mug/ml uracil, 40 mug/ml l-lysine, 6 mug/ml adenine sulfate, 20 mug/ml l-histidine, 30 mug/ml l-tryptophan, and 220 mug/ml l-leucine when required to cover auxotrophies. The limiting adenine supplementation was used to facilitate red pigment development in ade2-101 cells. Chromosome loss events during the first cell division were visualized as colonies that were at least one-half red. The loss rate for the SUP11-marked chromosome is expressed as loss per chromosome per cell division, and is calculated by dividing the number of half-sectored colonies by the total number of colonies scored. Determination of the bub1-1 Mutation | The bub1-1 allele was captured on a yeast-bacterial shuttle vector by gap repair from MAY1726 , and the entire open reading frame was sequenced from two independent transformants. The sole change observed was G to A at position 997, which substitutes a conserved glutamic acid with lysine in the putative Bub3p binding region. Therefore, bub1-1 is referred to as bub1-E333K. Introduction of bub1 Mutations at the Genomic Locus | Plasmid-borne mutant alleles were created adjacent to a HIS3 marker, amplified in a single DNA fragment with the selectable marker by high-fidelity polymerase chain reaction (PCR), and integrated by homologous recombination into the native BUB1 locus. The resulting genomic structure contained the native BUB1 promoter, a mutant bub1 allele, an HIS3 downstream marker gene, and finally, natural BUB1 3'-flanking sequence. Details of the constructions are available upon request. One-Hybrid Assay | The one-hybrid assay was performed essentially as described . YJL128 was transformed with activation domain fusion constructs, and multiple independent transformants were plated on SD-LEU supplemented with 5 mM 3-amino-triazole (3-AT). Plates were incubated at 30C for up to 2 wk. MPS1 Overexpression | A GAL-MPS1 allele was created by integration of pAFS120 at the MPS1 locus of YFS589 yielding yeast strain YML101. BUB1 overexpression plasmids were introduced into YML101, and two independent transformants were picked. Cultures were grown overnight in selective media lacking histidine and uracil supplemented with 2% raffinose, diluted into selective media lacking histidine, uracil, and methionine (to derepress the MET25 promoter) supplemented with 2% raffinose and were grown to early log phase. To induce MPS1 overexpression, galactose was added to a final concentration of 3%. Samples taken at t = 0 and t = 4 h were fixed in1 M sorbitol, 50 mM KPO4, pH 7.5, and 3.7% formaldehyde, 4,6-diamidino-2-phenylindole (DAPI) stained, and scored for bud and nuclear morphology. A minimum of 200 cells was scored for each sample. Plasmids | All overexpression constructs were made in either p423MET (2mu HIS3) or p415MET (CEN/ARS/LEU2) vectors containing the methionine-repressible MET25 promoter and the CYC1 terminator sequence flanking the multiple cloning site . For one-hybrid analysis, GAL4-AD fusions were constructed by cloning each PCR-generated open reading frame into pGADT7 (, Palo Alto, CA). All plasmids generated by PCR were verified by sequence analysis. Details are available upon request. Immunoblotting and Coimmunoprecipitation | Immunoblotting and coimmunoprecipitation were carried out as described previously . The lysis buffer for coimmunoprecipitation was 50 mM HEPES, pH 7.6, 75 mM KCl, 50 mM NaF, 1 mM Na vanadate, 1 mM MgCl2, 1 mM EGTA, 0.1% Na Deoxycholate, 1 mM phenyl methyl sulfoxide, "complete EDTA-free protease inhibitor cocktail" (Roche, Indianapolis, IN), and 1 mM dithiothreitol. Rabbit alpha-Mad1, Mad2, Mad3, Bub1, and Bub3 antibodies have been previously described . PMID- 12221113_RESULTS TI - AB - Spindle Checkpoint Mutants Exhibit Different Rates of Chromosome Loss | In previous work, it was apparent that chromosome loss of bub1 and mad2 mutants differed from one another . To determine the requirement for spindle checkpoint proteins in accurate chromosome segregation during normal unperturbed mitosis, null mutants of six checkpoint genes (BUB1, BUB2, BUB3, MAD1, MAD2, and MAD3) were generated in an otherwise isogenic background. To follow chromosome segregation fidelity, the loss of a nonessential test chromosome was ascertained by a colony color assay . In this assay, haploid colonies containing the test chromosome bearing a SUP11 (ochre-suppressing tRNA) gene are white, whereas cells that have lost the test chromosome accumulate a red pigment due to the host ade2-101 (ochre) mutation. Thus, loss events give rise to red sectors during colony growth. The checkpoint mutant strains were plated on color indicator plates and chromosome loss rates were evaluated visually by colony sectoring morphology and by half-sector analysis . In half-sector analysis, the rate of first division missegregation events is directly measured by observing the number of colonies that are at least one-half red, and dividing by the total number of colonies that were established by cells with a test chromosome. In an S288c background, bub1Delta and bub3Delta cells exhibited the highest rates of chromosome loss, 50-fold higher than the wild-type rate of 0.8 loss events per 1000 divisions (Figure , A and B). mad1Delta and mad2Delta strains also showed an increased chromosome loss rate, but at a level two- to threefold lower than bub1Delta and bub3Delta. mad3Delta exhibited a slight increase above wild type, whereas bub2Delta was indistinguishable from control. To test the generality of this result, the null mutants were characterized in a different laboratory strain background, W303-1a. The strong phenotypes for bub1Delta and bub3Delta were again observed, but the smaller differences among the mad null mutants were less apparent in W303-1a strains. At a minimum, the chromosome loss phenotypes indicate that Bub1 and Bub3 proteins have an additional role that is important to chromosome segregation during culture in the absence of intentional spindle damage. Kinetochore surveillance checkpoint proteins perform their functions in the context of multiprotein complexes. To test whether cells are sensitive to protein dosage, each full-length open reading frame was placed under the control of the MET25 promoter (MET25p), whose transcriptional strength is controlled by altering the environmental methionine concentration . MET25p-controlled expression of the five checkpoint proteins led to steady-state protein levels in excess of wild type (Figure C). The MET25p-controlled alleles were introduced into a wild-type yeast strain containing the test chromosome for monitoring chromosome segregation. Cultures grown in the presence of methionine were diluted in water and plated at similar200 cells/plate on media lacking methionine. Half-sector analysis indicated that overexpression of Bub1p and Mad3p led to a 15-fold increase in test chromosome missegregation over the wild-type rate (0.7 loss events per 1000 divisions; Figure D). High-level expression of Mad1p and Mad2p caused a smaller increase in chromosome missegregation (three- and sevenfold), whereas high level expression of Bub3p had no effect. Commonly used assays for the presence of checkpoint deficiency measure cell survival in the presence of antimicrotubule drugs such as Benomyl. The Benomyl sensitivity elicited by the absence of each kinetochore surveillance checkpoint protein was determined using the panel of null alleles. Strain viability was tested in the presence of a concentration of drug that delays but does not arrest wild-type cell growth. Cells containing bub1 and bub3 mutations were more Benomyl sensitive than mad1, mad2, or mad3 mutants by an order of magnitude (Figure E). Thus, checkpoint proteins differ in their contribution to the maintenance of cell viability in response to mild spindle damage. Note that the order of Benomyl sensitivity correlates with the relative intrinsic chromosome loss rates observed (Figure A). In principle, Benomyl sensitivity of mutants in this assay may reflect a sum of defective mechanisms contributing to cell death, including drug-induced hindrance of microtubule dynamics, null mutant kinetochore structural defects, and inappropriate cell cycle progression. BUB1 and BUB3 Cooperate in a Chromosome Segregation Role | To determine whether the overexpression phenotype of Bub1 was due to discrete domain(s), a series of BUB1 truncation alleles was constructed capable of expressing the N-terminal 210, 367, or 608 amino acids as well as amino acid segments 211-1021 and 211 --367 (Figure A). Western analysis using a Bub1p-specific antibody raised to the N-terminal 216 amino acids indicated that MET25-promoted expression led to significant protein accumulation in cells grown in the absence of methionine (Figure B). A serial dilution analysis indicated that the full-length overexpression product reached similar50-fold that of wild type. Figure 2 | The N terminus of Bub1p contributes to the overexpression phenotype and is counterbalanced by additional BUB3. The N terminus of Bub1p contributes to the overexpression phenotype and is counterbalanced by additional BUB3. (A) Diagram of BUB1 protein and protein fragments. The boxes indicate positions of conserved regions of BUB1p. Black: Mad3 like. White: Bub3 binding. Hatched: kinase domain. Star: E333K mutation. (B) Western blot detection of BUB1 overexpression alleles. Left: Wild-type cells (YPH278) containing MET25-promoted Bub1 alleles in p423MET were grown in the absence of methionine. Western blot analysis using an antibody raised to the N-terminal 216 amino acids of Bub1p detects protein bands with migrations consistent with eachconstruct, in addition to faster migrating degradation products. Endogenous Bub1p is not detected at this exposure (vector lane). Right: A dilution series Western blot indicates that the overexpression level for full-length BUB1 is similar50-fold. (C) Chromosome missegregation induced by BUB1 overexpression alleles. Left: Full-length and partial BUB1 alleles expressed from the MET25 promoter of p423MET were introduced into a wild-type strain (YPH278). Chromosome loss was determined by half-sector analysis after plating to methionine-free medium. Vector (no insert) and pBUB1 data are from D. p[1-210]: 25/14,087. p[1-367]: 127/5,741. p[1-608]: 96/6,592. p[211-1021]: 157/10,697. p[211-367]: 247/13,781. p[211-367*]: 9/5,117. Right: Chromosome loss was determined in YPH278 containing plasmid pairs as shown. p423MET + p415MET: 1/2,616. p423MET + pBUB3: 7/4,998. p[211-367] + p415MET: 37/3,015. p[211-367] + pBUB3: 6/4,510. At least two additional independent transformants of each construct were tested by visual sectoring assay and showed the same chromosome instability phenotype. (D) Chromosome loss in bub1 and bub3 null mutants. Left: Half sector analysis was used to compare chromosome loss rates of bub1Delta, bub3Delta, and bub1Delta bub3Delta mutant strains derived from sporulation of a wild-type diploid (YCD251) into which heterozygous bub1Delta::natMX and bub3Delta::kanMX alleles were introduced by transformation. bub1Delta: 186/5019 (one spore); bub3Delta: 262/5406 (one spore); bub1Delta bub3Delta: 601/12566 (four spores). Right: Chromosome missegration in a bub3Delta strain (YFS1100) containing the vector p423MET (284/5,675) or overexpression plasmid p[211-367] (289/6,069). Each of the truncation constructs was introduced into wild-type cells on high-methionine medium, where expression is suppressed. Chromosome loss was quantitated for several independent transformants by half-sector analysis after plating on low-methionine medium (Figure C, left). Under these conditions, the full-length construct exhibits a 15-fold increase in loss (from Figure D). Overexpression of the N-terminal 367 or 608 amino acids of Bub1p from plasmids (p[1 --367] and p[1 --608], respectively) caused a 30- and 20-fold increase in chromosome loss. Overexpression of the N-terminal 210 amino acids had little effect (2.4-fold, Figure C). The segment common to p[1 --367] and p[1 --608], but absent from p[1 --210], contains a well-conserved homology box predicted to mediate association between Bub1p and Bub3p . This result suggested that overexpression of a Bub3-binding region of Bub1p might cause the chromosome loss. To test this hypothesis, a construct expressing only amino acids 211 --367 under the control of the MET25 promoter was created. It too was found to induce chromosome loss at high expression levels (26-fold greater than the vector control). Several additional lines of in vivo evidence now strongly support the interpretation that the overexpression phenotype is mediated through disruption of a Bub1p/Bub3p interaction. First, the bub1-1 point mutation , which is suppressed by a low-level increase in BUB3 gene dosage, was cloned and identified as E333K (see "Materials and Methods"). This mutation is located within the 211 --367 segment. Second, when the E333K mutation was introduced into the p[211 --367] plasmid, this allele failed to induce chromosome instability (Figure C, left panel). Third, expression of additional BUB3 from a MET25-controlled allele (from plasmid pBUB3) on a centromere vector (p415MET) reversed the chromosome instability phenotype of p[211 --367] (Figure C, right). Fourth, a BUB1/BUB3 cooperative role in chromosome segregation implied by this interpretation was tested by analyzing the chromosome loss rate of a bub1Delta bub3Delta mutant (Figure D, left). The rate observed in the double mutant (48 events in 1000 divisions) is consistent with a shared role for Bub1p and Bub3p. Finally, if the presence of excessive bub1[211 --367]p interferes with a Bub1p-Bub3p association, then this protein fragment should not elicit additional missegregation in the absence of the complex. Indeed, its overexpression does not augment chromosome loss in a bub3Delta null mutant (Figure D, right). We conclude that an interaction between Bub1 and Bub3 proteins is likely to be mediated by amino acids 211 --367 of Bub1p in vivo, and disruption of this interaction contributes significantly to the overexpression phenotype associated with excessive Bub1p. Yeast Bub1p Can Associate with Kinetochores in a One-Hybrid Assay | Previous experiments have demonstrated kinetochore localization of Bub1 protein in experimental systems where these structures are cytologically visible . To date, localization of checkpoint proteins to budding yeast kinetochore structures has not been achieved. In a one-hybrid assay , kinetochore protein components can activate a HIS3 reporter gene located immediately adjacent to the centromere of chromosome III. This reporter system depends on the presence of an active centromere, reveals association of known kinetochore components, and has been successfully used to identify new kinetochore proteins . We used this assay to ask whether kinetochore association of full-length Bub1p or truncation alleles can be detected in budding yeast. Independent transformants containing Gal4-activation domain fusions of Bub1 and Bub1 fragments were spotted onto minimal medium lacking leucine and histidine supplemented with 5 mM 3-AT. Figure shows that fusions of BUB1[1 --367], BUB1[1 --608], and BUB1[211 --367] can activate transcription of the centromere reporter, indicating that these proteins can localize to kinetochores. Reporter activation with the full-length BUB1 fusion protein is not observed, likely due to a lower level of fusion protein accumulation or the presence of a nonfunctional conformation. Figure 3 | Localization of activation domain fusions to kinetochores in a one-hybrid assay. Localization of activation domain fusions to kinetochores in a one-hybrid assay. In the one-hybrid assay, fusion of the GAL4-activation domain to a kinetochore-binding protein induces transcription of a centromere-adjacent HIS3 reporter allele . GAL4AD fusion constructs were introduced into strain YJL128. The fusion moiety is indicated to the right; GAL4AD-CTF13 (top) served as a positive control. Four independent transformants were grown to saturation in SD-LEU, diluted to 1.5 x 107 cells/ml, and spotted (3 mul) on SD-HIS, LEU + 5 mM 3-AT. The spots shown were incubated at 30C for 14 d. The large papillae that appear occasionally in the GAL4AD-BUB1 transformants (observed in similar25% of transformants) may reflect the occurrence of truncating mutations. All constructs were similarly tested in YJL148, a strain containing a mutant centromere sequence adjacent to the HIS3 reporter . No growth above vector background was observed in these controls. All fusion constructs shown were functional in a two-hybrid assay. Activation domain fusions of other kinetochore surveillance proteins were also tested. MAD1 and MAD3 fusions activated the reporter , whereas BUB3 and MAD2 fusions did not. CTF13-AD is shown as a control: it activates the HIS3 reporter and supports growth on 3-AT within 3 --4 d at 30C, whereas the checkpoint fusion proteins tested require up to 14 d to show evidence of activation over vector background. This weak signal is consistent with a transient association, as would be expected for proteins that associate with a subset of kinetochores for a subset of the cell cycle. An unequal transcriptional activation efficiency for different fusion proteins may also be a contributing factor. We conclude from these experiments that the budding yeast kinetochore surveillance proteins Bub1, Mad1, and Mad3 can associate with yeast kinetochores, as is predicted from localizations of their studied orthologs. The BUB1 Overexpression Phenotype Includes Disruption of Both Checkpoint and Segregation Functions | High-level expression of Bub1p (and fragments of this protein) may disrupt kinetochore checkpoint signaling, a segregation function, or both. To address whether checkpoint signaling was disrupted, strains overexpressing full-length Bub1p or protein fragments were tested for checkpoint competence in two different assays. The first took advantage of the spindle checkpoint-dependent delay exhibited by ctf18Delta cells, which is associated with a partial defect in sister chromatid cohesion . This delay is detected as an accumulation of G2/M phase cells during early log phase using flow cytometry (Figure A, left column). MET25-controlled alleles were introduced into ctf18Delta cells and transformants were selected on high-methionine medium. Four independent transformants were then grown in medium without methionine for 18 --24 h (O.D. similar0.4) and were analyzed for DNA content using flow cytometry (Figure A). Diminution of the G2/M phase peak indicated that the delay can be disrupted by overexpression of full-length Bub1p, Bub1[1 --367]p, Bub1[1 --608]p, and Bub1p[211 --367]p. The G2/M reduction is consistent with an observed decrease in the proportion of budded cells (Figure A), as well as a reduction in viability determined by growth on solid medium with or without methionine (Figure B). The degree of delay diminution and reduced viability correlates with the amount of chromosome loss induced by the overexpression alleles (see Figure B). We observed similar loss of delay and viability in cells lacking CTF19 (C.D. Warren, unpublished data), a gene that encodes a nonessential kinetochore protein . We conclude from these experiments that overexpression of Bub1p, and Bub1p fragments that cause chromosome loss, does have the capacity to disrupt a spindle checkpoint-dependent delay. Figure 4 | Overexpression of Bub1p or Bub1p fragments both disrupts the spindle checkpoint and causes damage. Overexpression of Bub1p or Bub1p fragments both disrupts the spindle checkpoint and causes damage. (A) Disruption of a ctf18Delta-induced checkpoint delay. ctf18Delta strains containing p423MET plasmids expressing no (vector), full-length (BUB1), or partial (1 --210, 1 --367, 211 --367, 1 --608) alleles of BUB1 were created by transformation of YFS377. Cells were grown to early log phase in the absence of methionine for 18 --24 h, and were prepared for flow cytometry (as in ). A representative histogram is given for each genotype; four independent transformants were analyzed. The fractions of budded and unbudded cells were determined and are shown as mean +- SD (3 d.f.). (B) Disruption of the ctf18Delta delay results in decreased viability. Two independent isolates from each of the ctf18Delta strains described in A were grown to early log phase in media containing methionine. Cells were spotted onto solid medium without methionine in a 10-fold serial dilution series. Overexpression of bub1-[211-367]p in wild-type or bub3Delta cells did not result in a significant reduction in mortality (bottom). (C) Competence to arrest in response to MPS1 overexpression. Log phase cells grown in raffinose media lacking methionine (to induce expression of the BUB1 alleles) were treated with 3% galactose to induce MPS1 expression. Samples were taken at t = 0 and t = 4 h, formaldehyde fixed, DAPI stained, and scored for bud and nuclear morphology. The graph shows the percentage arrested (large-budded uninucleate) cells at each time point. Two independent transformants were analyzed (average +- range indicated). All strains were derived from YML101 (GAL-MPS1). All plasmids overexpressing BUB1 alleles in this strain were derived from p423MET (vector). YCD362 (bub1Delta) was included as a control for the assay. (D) Chromosome missegregation in a mad3Delta host. Chromosome loss rates were determined by half-sector analysis on plates lacking methionine. Vector: 5/3,327. pBUB1: 18/2,295. p[1 --210]: 4/2,408. p[1 --367]: 51/2,500. p[1 --608]: 42/2,766. p[211 --367]: 37/2,349. Data for a representative transformant are shown; at least two independent transformants were analyzed for each construct. The strains were YFS1205 derivatives created by introduction of p423MET (vector) and related BUB1 allele overexpression plasmids. In a second system, we obtained evidence that the checkpoint arrest pathway is not completely dysfunctional. In this experiment, checkpoint activation by overexpression of the MPS1 protein kinase was used to cause cell cycle arrest . MPS1-induced arrest is dependent on each of the known BUB and MAD checkpoint genes . If overexpression of Bub1p or Bub1p fragments completely disrupts the checkpoint pathway, similar to a null mutant, then MPS1 overexpression will not cause cell cycle arrest. Wild-type strains containing integrated GAL1-MPS1 as well as MET-controlled BUB1 overexpression plasmids were grown in medium lacking methionine to induce high-level expression of the BUB1 truncation alleles. Samples were taken before and 4 h after addition of galactose (for overexpression of MPS1). Formaldehyde-fixed cells were stained with DAPI and were scored for morphological evidence of metaphase arrest. Strains overexpressing the full-length Bub1p, Bub1[1 --367]p, or Bub1[1 --608]p arrested in response to MPS1 overexpression, whereas control bub1Delta cells did not (Figure C). The presence of an MPS1-induced arrest indicates that the overexpression of Bub1 full-length protein or the truncation alleles does not fully abrogate spindle checkpoint function. We speculate that even a single remaining active kinetochore may be sufficient to arrest cells in response to MPS1 overexpression. Both of the experiments above address the checkpoint competence of strains containing extra Bub1p or Bub1p fragments. Neither addresses whether disruption of checkpoint control is responsible for the chromosome loss introduced by overexpression of Bub1p, or whether a separate mechanism causes missegregation (e.g., competition for a kinetochore structural component). Note that mad3Delta null cells have a quite modest chromosome instability phenotype (Figure , A and B), although they are markedly defective in preanaphase arrest (; ; our unpublished data). To explore the cause of chromosome loss, overexpression interference of BUB1 alleles was tested in a mad3Delta yeast host (Figure D). The chromosome loss rates observed closely parallel those induced by the Bub1 overexpression alleles in wild-type cells. This result indicates that the mechanism responsible for chromosome loss incorporates a defect distinct from loss of a functional checkpoint pathway. Two Domains of Bub1p Play Distinct Roles in Chromosome Segregation | Because the molecular defects engendered by overexpression may be complex, genomic loss-of-function alleles have also been characterized. Chromosome loss rates of two bub1 missense mutants were measured by half-sector analysis (Figure , A and B). The bub1-1 allele (i.e., bub1-E333K) has apparent partial function because its checkpoint defect is suppressible by additional copies of the BUB3 gene on a centromere plasmid, whereas that of a bub1 null mutation is not . However, the chromosome missegregation rate measured for bub1-1 (Figure , A and B) is similar to that of bub1Delta (Figure , A and B). Like the bub1-1 checkpoint defect, the bub1-1 chromosome missegregation phenotype is suppressible by extra copies of the BUB3 gene introduced on a centromere plasmid (Figure B). Figure 5 | Chromosome missegregation associated with genomic alleles of BUB1. Chromosome missegregation associated with genomic alleles of BUB1. (A) Colony sectoring phenotypes of bub1-E333K (bub1-1) and bub1-K733R. Strains were YCD280 and YCD281. (B) Chromosome loss caused by genomic alleles. Left: Half sector analysis was used to analyze the mutants as shown. WT: 8/8,503. bub1-E333K: 248/5,964. bub1-K733R: 83/6,576. bub1[1-210]: 354/6,769. bub1[1-367]: 477/9,019. bub1[1-608]: 222/14,105. bub1[211-1021]: 355/14,837. Strains were YCD279, YCD280, YCD281, YCD371, YCD358, and YCD407. Right: A centromere plasmid containing a MET25-inducible BUB3 gene (pBUB3) or vector alone (p415MET) was introduced into wild-type (WT, YPH278), bub1-E333K (YCD280), or bub1-K733R (YCD281) strains. Half sector analysis was performed after plating on methionine-free media. WT + p415MET: 1/3,728. WT + pBUB3: 3/4,925. bub1-E333K + p415MET: 104/1,567. bub1-E333K + pBUB3: 48/3,941. bub1-K733R + p415MET: 53/3,654. bub1-K733R + pBUB3: 36/4,102. The bub1-K733R allele, which alters a conserved lysine residue in the Bub1 protein kinase domain, has been previously characterized as deficient in checkpoint competence and protein kinase activity . The chromosome stability defect of bub1-K733R is less severe than that of bub1-E333K (Figure , A and B). The addition of extra BUB3 gene copies to bub1-K733R does not markedly alter its chromosome segregation phenotype. BUB1 truncation alleles were tested at single copy in the native genomic locus under the control of the BUB1 promoter in haploid cells. Although the genomic bub1[1-367] allele exhibits a phenotype similar to the null mutant, the bub1[1-608] allele supports a chromosome loss rate that is intermediate (16 events per 1000 divisions, Figure B). This rate is similar to that observed for the kinase region missense mutant bub1-K733R (13 events per 1000). Thus, an intermediate level of chromosome stability is observed for two alleles of BUB1, with defective or absent protein kinase activity, indicating a role for the N-terminal portion of Bub1p in segregation. Note that the chromosome segregation competence conferred by the N-terminus of Bub1p does not account for the very high fidelity of segregation in wild-type cells. To test if chromosome stability can be provided by a Bub1p C-terminal protein fragment, a deletion allele expressing amino acids 211-1021 was constructed at the genomic locus. This mutant has a chromosome stability phenotype that is between wild-type and bub1Delta, at 24 events per 1000 (Figure B). We conclude that the C-terminal portion of Bub1p also contributes to chromosome stability. In summary, the bub1-E333K allele appears to be virtually null for both checkpoint and chromosome segregation activities, although it encodes a protein whose functions are rescued by additional expression of its binding partner Bub3p. This argues that an association with Bub3p is required for both checkpoint and segregation activities. Separate N-terminal (bub1[1-608]) and C-terminal (bub1[211-1021]) protein fragments contribute to chromosome stability, each providing an intermediate level of segregation fidelity. We note that the loss rates of these two partial protein alleles sum to a value that is the same as the loss rate observed in the null mutant (40 per 1000, Figure B). An N-Terminal Segment of Bub1p Is Necessary and Sufficient for Its Checkpoint Function | Genomic loss-of-function alleles were tested for checkpoint competence by evaluating arrest after spindle damage. An arresting concentration of the antimicrotubule drug nocodazole (15 mug/ml) was added to asynchronous cultures grown in rich medium at t = 0. Samples at 4, 6, and 8 h were fixed in formaldehyde, DAPI stained, and scored for the frequency of uninuclear large-budded (arrested) or multibudded (inappropriately progressing) cellular phenotypes (Figure , A and B). The bub1[1-367] and bub1-E333K mutants behaved like bub1Delta, consistent with their null chromosome instability phenotypes. However, bub1[1-608] gave results similar to wild type for both arrest and inappropriate progression tests. This indicates that the kinase domain can be deleted without loss of the checkpoint arrest function of Bub1p. Figure 6 | Checkpoint competence of BUB1 genomic alleles. Checkpoint competence of BUB1 genomic alleles. (A) Cell cycle arrest. Logarithmically growing cultures of strains containing integrated alleles were transferred to YPD + 15 mug/ml nocodazole. At t = 0, 4, 6, and 8 h after shift into nocodazole, aliquots were formaldehyde fixed and stained with DAPI. Two hundred cells from each were scored for the arrested fraction (large-budded uninucleate cells), and the mean +- standard deviation for three independent integrants is shown. The strains were YPH278, YFP2, YCD371, YCD358, YCD281, and YCD280. (B) Failure of cell cycle arrest. The same samples were scored for the fraction of cells that exhibited multibudded uninucleate cells, an indication mitotic exit in the absence of nuclear division. (C) Anti-Bub1p immunoprecipitates from the genotypes indicated were analyzed by Western blot for the presence and abundance of Bub1 protein (as described in ). Immunoprecipitation product from the wild-type cells was loaded in a dilution series (1, 0.5, and 0.25) for comparison with lanes containing immunoprecipitations from mutant extracts. The strains were YFP2, YPH278, YCD280, and YCD281. The previous report that bub1-K733R is checkpoint deficient led to the hypothesis that the kinase-encoding portion of BUB1 is the checkpoint-functional moiety of the protein. We observe that although bub1-K733R cells are indeed checkpoint deficient, the timing of arrest failure indicates the presence of partial function (Figure A). Moreover, the bub1-[211-1021] allele failed to exhibit a checkpoint arrest in nocodazole. These results, taken together with the arrest competence of bub1-[1-608], indicate that the protein kinase activity of Bub1p is not responsible for nocodazole-induced arrest. The proteins encoded by bub1-E333K and bub1-K733R alleles were further investigated. Western blot analysis of anti-Bub1p immunoprecipitates reveals the presence of a stable protein pool in bub1-E333K mutant cells (Figure C). The bub1-E333K protein is significantly underphosphorylated, strongly suggesting that function of the wild-type Bub1 protein depends upon its phosphorylation. In contrast, bub1-K733Rp appears to be less abundant, and modified forms are readily detected (Figure C). The low steady-state abundance of bub1-K733Rp may reflect a high protein turnover rate. This prediction suggests an hypothesis in which the inability of bub1-K733Rp to maintain a checkpoint arrest is in part due to a gradual loss of the mutant protein in arrested cells. Formation of a Mad1p-Bub1p-Bub3p complex is crucial for spindle checkpoint function . Therefore, we tested whether the Bub1 protein fragment alleles could form such a complex by assaying for coimmunoprecipitation with Mad1p or myc-tagged Bub3p . First, immunoprecipitates prepared with an alpha-Bub1p antibody were characterized for the presence of Bub1p and Mad1p (Figure A). Full-length Bub1p and bub1[1-608]p expressed from the genomic locus were found to coprecipitate Mad1p in nocodazole-arrested cells, whereas bub1[1-367]p did not. Second, immunoprecipitation was carried out to test for association between a genomic myc-tagged BUB3 allele and Bub1 truncation proteins expressed from the MET25 promoter on a 2-mum plasmid (Figure B). Anti-myc precipitates containing equivalent amounts of Bub3-myc protein (Figure B, bottom) also contained appreciable amounts of full-length Bub1p, bub1[1-367]p, and bub1[1-608]p, but not bub1[1-210]p. Figure 7 | Bub1-[1-608]p associates with Mad1p and Bub3p. Bub1-[1-608]p associates with Mad1p and Bub3p. (A) Coimmunoprecipitation of full-length Bub1p and bub1-[1-608]p with Mad1p. The strains shown, containing integrated Bub1 alleles expressed from the wild-type BUB1 promoter, were grown to log phase and were incubated with +- 15 mug/ml nocodazole for 2 h at 24C. Immunoprecipitates were prepared using an alpha-Bub1p antibody, separated by SDS-PAGE, and transferred to nitrocellulose. The immunoblots were then probed with alpha-Bub1p and alpha-Mad1p rabbit antibodies as indicated. The strong band labeled (*) in the Bub1 blot is IgG heavy chain from the immunoprecipitation. Strains shown are YPH278, YFP2, YCD358, and YCD371. (B) Coimmunoprecipitation of full-length Bub1p, bub1-[1-367]p, and bub1-[1-608]p with Bub3p. All strains contained a BUB3-myc allele in the genome. The experimental strains contained a wild-type BUB1 gene in addition to episomal MET25-promoted alleles as indicated. A bub1Delta strain served as control. Left: Bub1p Western blot using a rabbit alpha-Bub1p antibody. Right: Immunoprecipitation with an alpha-myc antibody recovered an equivalent amount of myc-tagged Bub3 protein (bottom). The immunoprecipitates were probed with rabbit alpha-Bub1p antibody (top). The strains were YKH300 (bub1Delta) or YKH238 with pBUB1, p[1-210], p[1-367], or p[1-608]. In summary, bub1[1-608]p exhibits biochemical characteristics of a functional Bub1 protein capable of coprecipitation with both Mad1p and Bub3p. Moreover, bub1[1-608]p is heavily phosphorylated in all of our Western blots (Figures B and ; confirmed by lambda protein phosphatase treatment; K.G. Hardwick, unpublished data), whereas bub1[1-367]p and bub1[1-210]p are not. In a functional assay, a genomic allele of bub1[1-608] supports a robust checkpoint arrest in the presence of nocodazole. Thus, we conclude that the bub1[1-608] protein is sufficient for BUB1 checkpoint arrest function, and exhibits biochemical properties expected for this activity. PMID- 12221113_DISCUSSION TI - AB - Nonessential spindle checkpoint proteins from budding yeast differ in their importance to chromosome stability in cells where spindle assembly dynamics are not challenged by intentional introduction of damage. The disparity in chromosome loss rates observed among the checkpoint null mutants indicates the presence of functional differentiation. bub2Delta cells exhibit a wild-type chromosome loss rate, in agreement with BUB2's primary role in mitotic exit rather than in kinetochore surveillance at metaphase. Each of the other mutants conferred a chromosome loss rate higher than wild type, indicating one or more roles important for high-fidelity chromosome transmission. BUB1 and BUB3 genes in particular appear to influence chromosome segregation more strongly than MAD1, MAD2, and MAD3 genes. We speculate that differential roles among these genes may include distinct kinetochore structural contributions that influence segregation, detection of different types of kinetochore status in the context of checkpoint signaling (e.g., tension vs. attachment), or communication of checkpoint signaling to diverse target molecules that mediate different aspects of checkpoint delay or recovery. It was recently argued that although mammalian Mad2p responds to the lack of microtubule attachment, the Bub proteins respond to both microtubule attachment and a lack of tension . However, evidence from budding yeast suggests that the spindle checkpoint in this organism responds to the lack of tension in a mitotic spindle, and that this checkpoint-associated delay is Mad2 dependent . Further work is needed to clarify roles of the checkpoint proteins. In this work, we have endeavored to explain the relatively high rate of loss exhibited by bub1Delta cells and to find evidence for the presence of two distinct contributions to chromosome segregation. One is encoded within the first 608 amino acids in a protein segment that is both necessary and sufficient for a nocodazole-induced checkpoint arrest. The other is encoded in the kinase domain, which is not required for checkpoint arrest and whose function is unknown. Previous work in budding yeast has indicated that a missense allele predicted to disrupt kinase activity (bub1-K733R) was also defective in checkpoint arrest . In apparent contradiction, an in vitro experiment using a Xenopus extract system has provided evidence that a kinase-defective missense allele can support an active checkpoint . Here, we find that the genomic bub1-[1-608] allele, entirely lacking the conserved kinase domain, exhibits checkpoint competence after spindle disruption, whereas bub1-K733R exhibits a transient arrest that decays rapidly. Examination of the steady-state abundance of bub1-K733R encoded protein indicates a decreased accumulation. Taken together, these studies indicate that the checkpoint defect associated with bub1-K733R is more likely due to insufficient gene product than to a dysfunctional kinase activity. The protein encoded by bub1-[1-608] exhibits several interesting properties relevant to its checkpoint function. The immunoprecipitation experiments reveal association of this truncation product with both Bub3p and Mad1p. The BUB1 partial protein allele series indicates the involvement of specific amino acid segments of Bub1p in complex formation. The segment from amino acid 211 to 367 is required for complex formation with Bub3p. Similarly, the segment from 367 to 608 is required for Mad1p association. In our analysis of the partial protein alleles, the presence of both Bub1p and Mad1p binding correlates with the accumulation of phosphorylated forms of Bub1p, as well as the presence of checkpoint arrest competence. These observations strongly support the current model that a Bub1-Bub3-Mad1 protein complex is required for checkpoint arrest, and they suggest that the phosphorylation in the N-terminal one-half of Bub1p may also be a requirement. The 211 --367-amino acid segment can localize a GAL4 transcriptional activation domain to the yeast kinetochore in the one-hybrid assay. This activity, as well as Bub3p binding, is consistent with previous work on murine Bub1p, which defined a conserved homology with similar functions in an overexpression assay. In general, our overexpression results in budding yeast parallel studies in mammalian cells where overexpression of Bub1p mutant alleles from an ectopic promoter leads to disruption of checkpoint function . However, the mammalian studies have been controversial (see ) due to differing outcomes from similar experiments. In budding yeast, under partial induction of the checkpoint (e.g., in ctf18Delta or ctf19Delta cycling populations), overexpression of Bub1p or fragments was sufficient to "silence" checkpoint signaling. We assume that in ctf18Delta or ctf19Delta mutants, many cells experience a delay due to the failure of one kinetochore (or a few) to achieve stable bipolar attachment with normal timing. In contrast, under the same Bub1-overexpression conditions, checkpoint activation by extra Mps1p was sufficient for cell cycle arrest. We speculate that because the checkpoint is strongly induced with overexpression of Mps1p, even a single remaining active checkpoint-signaling complex may cause cell cycle arrest. Comparison of the results from these two tests for checkpoint function in yeast highlights a cautionary note where partial induction or disruption of checkpoint activity is involved. For example, in vertebrate cell culture systems, seemingly subtle variation (e.g., in genotype or culture conditions) may contribute to quantitative aspects of checkpoint competence and may affect the outcome. In the overexpression survey of checkpoint proteins, extra Mad3p caused a chromosome loss rate similar to that conferred by extra Bub1p. Although Mad3p exhibits similarity in protein alignment to the Bub1p N-terminal segment, each gene is independently required for checkpoint activity and, therefore, they are not functionally equivalent. The mad3 null chromosome loss rate is notably subtle in comparison with the MAD3 overexpression phenotype, indicating that compromise of Mad3p's overexpression binding partners is more important to segregation than Mad3 protein itself. Because the interaction between Bub1p and Bub3p contributes to the BUB1 overexpression phenotype, and because Mad3p associates with Bub3p , it is likely that interference with Bub3p function is causal for the chromosome missegregation induced by Mad3p overexpression. Interestingly, the amounts of Bub3p at a single human kinetochore have been estimated to be around 1000 copies , an abundance that is suggestive of its having a structural role as well as a signaling one. In conclusion, a quantitative study of the roles played by spindle checkpoint genes in chromosome segregation indicates the presence of functional differentiation beyond their essential contributions to the spindle checkpoint. Further studies of loss-of-function alleles that define distinct functional contributions, and overexpression alleles that disrupt in vivo relationships, hold promise for elucidating the in vivo importance of biochemical properties of checkpoint components. PMID- 12221114 TI - Activation of Mitogen-activated Protein Kinase (Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase) Cascade by Aldosterone AB - | Aldosterone in some tissues increases expression of the mRNA encoding the small monomeric G protein Ki-RasA. Renal A6 epithelial cells were used to determine whether induction of Ki-ras leads to concomitant increases in the total as well as active levels of Ki-RasA and whether this then leads to subsequent activation of its effector mitogen-activated protein kinase (MAPK/extracellular signal-regulated kinase) cascade. The molecular basis and cellular consequences of this action were specifically investigated. We identified the intron 1-exon 1 region (rasI/E1) of the mouse Ki-ras gene as sufficient to reconstitute aldosterone responsiveness to a heterologous promotor. Aldosterone increased reporter gene activity containing rasI/E1 threefold. Aldosterone increased the absolute and GTP-bound levels of Ki-RasA by a similar extent, suggesting that activation resulted from mass action and not effects on GTP binding/hydrolysis rates. Aldosterone significantly increased Ki-RasA and MAPK activity as early as 15 min with activation peaking by 2 h and waning after 4 h. Inhibitors of transcription, translation, and a glucocorticoid receptor antagonist attenuated MAPK signaling. Similarly, rasI/E1-driven luciferase expression was sensitive to glucocorticoid receptor blockade. Overexpression of dominant-negative RasN17, addition of antisense Ki-rasA and inhibition of mitogen-activated protein kinase kinase also attenuated steroid-dependent increases in MAPK signaling. Thus, activation of MAPK by aldosterone is dependent, in part, on a genomic mechanism involving induction of Ki-ras transcription and subsequent activation of its downstream effectors. This genomic mechanism has a distinct time course from activation by traditional mitogens, such as serum, which affect the GTP-binding state and not absolute levels of Ras. The result of such a genomic mechanism is that peak activation of the MAPK cascade by adrenal corticosteroids is delayed but prolonged. PMID- 12221114_INTRODUCTION TI - AB - Small, monomeric Ras GTP-binding proteins initiate pleiotropic signaling cascades to affect many aspects of cellular physiology. Ras signaling through the extracellular signal-regulated kinase (ERK) cascade mediated by mitogen-activated protein kinases (MAPKs) 1/2, for instance, is well documented to play a pivotal role in cellular growth and differentiation. Protein hormones, which target GTP exchange factors and GTPase-activating proteins via plasma membrane receptors, activate the MAPK cascade by increasing the GTP-bound state of Ras proteins and not the absolute levels of these proteins. Thus, the "classic" paradigm of Ras -> MAPK signaling involves posttranslational control of Ras. Emerging evidence suggests that numerous steroids, including aldosterone, also affect Ras signaling . The molecular basis and end effect of this steroid action remain, for the most part, not well described. Because steroids control cell activity through receptors that function as trans-acting factors to modulate gene expression, it is possible that steroids act on the Ras signaling cascade via a "genomic" mechanism that is dependent on transcription and subsequent translation to increase Ras protein levels and thus, distinct from the classic mechanism. The current study, which investigated this possibility, identifies a novel paradigm by which corticosteroid activate the Ras -> MAPK signaling cascade. The adrenal cortical steroid hormone aldosterone is the major endocrine factor regulating Na+ and K+ homeostasis. Aldosterone, consequently, plays a central role in maintaining electrolyte and water balance . Aldosterone also plays a direct role in pathological remodeling of the heart, possibly by promoting fibrosis and cellular proliferation both of which are generally known to be impacted by Ras signaling via ERK cascades . Although the systemic effects and target tissues of aldosterone are well known, little is actually known about its cellular mechanisms of action. Many integral membrane proteins involved in epithelial cell transport, such as the epithelial Na+ channel (ENaC), apical membrane potassium channel, Na+/Cl- cotransporter, H+/K+-ATPase, and Na+/K+-ATPase are end effectors of aldosterone signaling . Although aldosterone affects cell activity by modulating gene expression, the expression levels of these proteins involved in transport, however, are not themselves initially controlled by the steroid. This has led to the proposal that aldosterone must control expression of factors that initiate or impinge upon signal transduction. Adrenal corticosteroids, including aldosterone, increase the levels of the small, monomeric GTP-binding protein Kirsten Ras (Ki-Ras; ; ; ; ). Aldosterone preferentially increases expression in epithelia of the A splice variant of Ki-Ras via control of transcription with induction of Ki-ras mRNA being a primary response to steroid that is independent of de novo protein synthesis and begins within 30 min after steroid addition. Induction of Ki-RasA is necessary and sufficient for aldosterone action, in part, on Na+ transport . In addition, Ki-RasA activates ENaC when both proteins are overexpressed in a heterologous system and increases the open probability of this channel in native epithelia . Consequently, Ki-RasA is a likely candidate in some instances to transduce information form the nucleus to final effectors in response to aldosterone. Induction of Ras expression by steroids may impact more than just ENaC and epithelial transport for glucocorticoids and estrogen increase Ras expression in mammary epithelia with enhanced expression possibly being associated with tumor formation and metastasis . Indeed, it has long been recognized that in cells that lack mutant Ras, elevated levels of normal Ras can lead to cell growth and/or transformation, presumably through inappropriate stimulation of Ras effector cascades . This suggests that through mass action, induction of Ras leads to activation of this protein and subsequent signaling. The general consequences and in particular those associated with cell signaling of steroid-dependent induction of Ki-ras are not well understood. It also is not clear whether increases in Ki-RasA levels in response to aldosterone result from actions mediated by nuclear receptors and whether steroid-sensitive increases in Ki-RasA result in concomitant increases in functional GTP-bound Ki-RasA. Similar to the other Ras proteins (Ha-Ras, N-Ras, and Ki-RasB), active Ki-RasA initiates many different intracellular signaling cascades, including the MAPK cascade. This cascade is known to affect several aldosterone-target proteins, such as ENaC, Na+/K+-ATPase, Na+/H+ exchanger, Na+/Cl-, and Na+/bicarbonate cotransport; and Na+/Ca2+ exchange proteins . Thus, the MAPK cascade may play a pivotal role in signaling aldosterone action secondarily to stimulation of Ki-RasA or may ultimately be involved in a negative feedback pathway initiated by this steroid. The current work tested the hypothesis that aldosterone-stimulated Ki-RasA activates the MAPK cascade in renal epithelia. In addition, we asked whether Ki-RasA and the MAPK cascade are activated in response to aldosterone via nuclear steroid receptors, and whether increases in Ki-RasA expression in response to steroid result in increases in functional Ki-RasA:GTP levels. Through the course of this work, we also investigated possible molecular mechanisms by which aldosterone induces Ki-Ras expression and compared aldosterone effects on Ki-Ras -> MAPK signaling with that of a traditional mitogen, such as serum. PMID- 12221114_EXPERIMENTAL PROCEDURES TI - AB - Cell Culture | All experiments were performed with renal A6 epithelial cells (passages 75 --81; American Type Culture Collection, Manassas, VA). Cells were cultured on polycarbonate supports (Transwell-Clear Inserts, pore size 0.4 muM, growth area 4.7 cm2; , Cambridge, MA) and allowed to form polar monolayers by using standard methods described previously (,, , ). In brief, cells were maintained at 26C in 4% CO2 with complete amphibian medium (3/10 Coon's F-12, 7/10 Leibovitz's L-15) supplemented with fetal bovine serum (10%). Basic medium was devoid of serum and aldosterone. High-resistance (>2 KOmega), polarized A6 cell monolayers were used for all experiments. To observe the full action of aldosterone, confluent cells were treated with basic media for 48 to 72 h before experimentation. Molecular Biology | Plasmid Preparation and Isolation of Ki-ras Intron 1-Exon 1. | The pMMrasDN plasmid was a kind gift form Dr. G. Firestone (University of California at Berkeley, Berkeley, CA). In brief, this construct allows glucocorticoid-inducible expression of dominant-negative Ha-RasN17. Similar to that described previously by the Firestone laboratory for Con8 rat mammary epithelial cells , this construct in conjunction with G418 selection was used to create clonal A6 cell lines stably expressing inducible dominant-negative RasN17. The firefly luciferase reporter plasmid pGL2-TK was generated by subcloning the minimal herpes simplex virus thymidine kinase promotor from pRL-TK (Promega, Madison, WI) into pGL2 Basic Vector (Promega) with HindIII and BglII. (pGL2-TK was a kind gift from Dr. A. Firulli, University of Texas, San Antonio, TX.) The control pRL-CMV plasmid contains the cytomegalovirus promotor upstream of Renilla luciferase (Promega). Mouse c-Ki-ras2 exon 1 plus its 5'-flanking region (intron 1-exon 1 region; nucleic acids -165 --153 as labeled from the adenosine of the translation start codon ATG in exon 1; see GenBank accession numbers , 52798, , and ; ; ) were amplified with a standard polymerase chain reaction by using mouse whole tail genomic DNA and the 5'-ATGCGGTACCGACTTACAGGTTACTC (incorporating KpnI site, underlined) and 5'-GCATCTCGAGCTGCCGTCCTTTACAAGCG (incorporating XhoI site, underlined) upstream and downstream primers, respectively. The 321-base pair product from this polymerase chain reaction was subcloned into pGL2-TK with KpnI and XhoI to produce pGL2-TK-rasI/E1. Luciferase Reporter Gene Assay. | A quantitative assay with a Renilla luciferase internal control was used to measure the firefly luminescent signal in A6 cells overexpressing reporter genes. In brief, A6 cells plated at 80% confluence on 100- x 20-mm2 culture dishes were transfected with 100 ng of pRL-CMV in addition to 3 mug of the firefly luciferase reporter plasmid (either pGL2-TK or pGL2-TK-rasI/E1) by using the LipofectAMINE Plus (Invitrogen, Carlsbad, CA) system per the manufacturer's instructions with the exception that cells were exposed to transfection reagents for similar8 h. Twenty-four hours after transfection and 24 h before performing assay, cells were replated in a 96-well culture plate. Luciferase activity then was measured with the Dual-Luciferase Reporter assay system (Promega) per the manufacturer's instructions directly following the experimental treatment period (i.e., exposure to 1.5 muM aldosterone for 4 h.) and 1-h extract preparation period required with passive lysis buffer (see Dual-Luciferase Reporter instructions). An MLX microtiter plate luminometer (Dynatech Labs, Chantilly, VA) was used to record luminescent signal. For these experiments, all firefly luciferase activity data are normalized to the internal Renilla luciferase control. Biochemistry | Western Blot Analysis. | Whole A6 cell lysate was extracted after three washes with Tris-buffered saline by using standard procedures . Cells were scraped and then maintained for 1 --2 h at 4C in gentle lysis buffer (GLB) (76 mM NaCl, 50 mM HCl-Tris, 2 mM EGTA plus 1% Nonidet P-40, and 10% glycerol, pH 7.4) and protease inhibitors (phenylmethylsulfonyl fluoride, leupeptin, tosylphenylalanyl chloromethyl ketone, and 1-chloro-3-tosylamido-7-amino-2-heptanone). For Western blot analysis of phosphorylated proteins, GLB was supplemented with 0.1 mM NaPPi, 0.5 mM NaF, 0.1 mM Na2MoO4, 0.1 mM ZnCl2, and 0.04 mM Na3VO4 prepared fresh from 1000x stocks. After clearing cellular debris, standardizing total protein concentration, and addition of Laemmli sample buffer (0.005% bromphenol blue, 10% glycerol, 3% SDS, 1 mM EDTA, 77 mM HCl-Tris, and 20 mM dithiothreitol), lysates were heated to 85C for 10 min. Proteins were then separated by standard SDS-PAGE and subsequently electrophoretically transferred to nitrocellulose (0.2 muM). Western blot analysis was performed using standard techniques and appropriate antibodies (,,, ; see below; primary and secondary antibodies were used at 1/1000 and 1/20000, respectively). Tween 20 (0.1%) and 5% dried milk (Carnation) were used as blocking reagents. Band intensity was quantified with densitometric scanning using Sigmagel (Jandel Scientific, Costa Madre, CA). When possible, the flood configuration with the highest practical threshold was used to measure band density. Western blots were often stripped of primary and secondary antibody to subsequently reprobe with a control antibody. All Western blots were stripped in 100 mM 2-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.7, and 2% SDS for 30 min at 55C with constant agitation. After removal of antibodies, nonspecific interactions were reblocked by incubating in TBS-Tween, 5% milk for 2 h before reprobing with primary antibody. Ras:GTP Assay. | Raf-1 RBD agarose was from Upstate Biotechnology (Lake placid, NY). This immobilized fusion protein corresponds to the human Raf-1 Ras binding domain (RBD) (residues 1 --149). Raf-1 RBD binds Ras complexed with GTP. Pull-down experiments were performed in 400 mul (0.4 mg of total protein) of whole A6 cell lysate isolated with GLB. Lysates were incubated with 30 mul of Raf-1 RBD agarose overnight (at 4C and with constant agitation); pellets were washed five times with 2 volumes of fresh GLB each time for a total wash time of 2 h; and after resuspending in sample buffer and heating, Raf-1 RBD agarose precipitated proteins were separated by SDS-PAGE and Ki-RasA:GTP identified by immunoblotting. MAPK Assay. | MAPK activity in lysates prepared from cells treated with and without aldosterone was assayed by quantifying phosphorylation of exogenous myelin basic protein (MBP). MAPK activity was measured in whole A6 cell lysate (2 mg/ml) extracted in the presence of phosphatase inhibitors as described above. MAPK activity was measured for 30 min at 30C in the following assay dilution buffer (ADB; Upstate Biotechnology): 20 mM MOPS pH 7.2, 25 mM beta-glycerophosphate, 5 mM EGTA, 0.4 mM MnCl2, 0.4 mM CaCl2, 1 mM sodium orthovanadate, and 1 mM dithiothreitol. The final reaction contained 10 mul of substrate cocktail (from stock of 2 mg/ml dephosphorylated MBP in ADB), 10 mul of inhibitor cocktail (from stock of 20 muM PKC inhibitor peptide, 2 muM PKA inhibitor peptide, and 20 muM compound R24571 in ABD), 10 mul of A6 cell extract, and 10 mul of Mg2+/ATP cocktail (from a stock of 75 mM MgCl2, 500 muM ATP in ADB). Reactions were initiated with Mg2+/ATP and terminated with Laemmli sample buffer (described above). Phosphorylation of MBP (in 10 mul of final reaction) was assessed after SDS-PAGE by immunoblot analysis with a specific anti-phospho-MBP antibody. Electrophysiology | Transepithelial Na+ current was calculated as described previously (,,, ), from Ohm's law as the ratio of transepithelial voltage to transepithelial resistance under open circuit conditions by using a Millicel Electrical Resistance System with dual Ag/AgCl pellet electrodes (Millipore, Bedford, MA) to measure voltage and resistance. Materials | All reagents unless indicated otherwise were from either BIOMOL Research Laboratories (Plymouth Meeting, PA), Calbiochem (San Diego, CA), Invitrogen, or Sigma-Aldrich (St. Louis, MO). Phosphorothiate oligonucleotides were synthesized by the Emory University Microchemical Facility and stored frozen as 10 mM (in water) stocks. Aldosterone, dexamethasone, and mifepristone (RU486) were stored frozen as 1.5, 0.1, and 1.0 mM (in dimethyl sulfoxide [DMSO]) stocks. Cycloheximide (in MeOH) and emetine (in H2O) were stored frozen as 1.0-mg/ml stocks. Actinomycin D was stored at 4C as a 1.0-ng/ml (in MeOH) stock. PD-98059 and U-0126 were prepared fresh (in DMSO) before each experiment at stock concentrations of 10 and 5 mM, respectively. All reagents used for Western blot analysis unless noted otherwise were from (Hercules, CA) and (Rockford, IL). For each lysate, protein concentration was determined with the bicinchoninic acid protein assay. Kodak BioMax Light-1 film and Chemiluminescence Reagents Plus (PerkinElmer Life Sciences, Boston, MA) were used to develop Western blots. Antibodies | The rabbit polyclonal anti-MAPK 1/2 (Erk 1/2-CT) antibody was from Upstate Biotechnology. The mouse monoclonal anti-c-Raf-1 antibody was from Transduction Laboratories (Lexington, KY). The rabbit polyclonal anti-MKP-1 (V-15; MAPK phosphatase), anti-Fra-2 (L-15), and anti-K-Ras2A antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). This latter antibody recognizes only the Ki-RasA isoform of Ras proteins. The mouse monoclonal anti-v-Ha-Ras antibody was from Oncogene Science (Cambridge, MA). This antibody recognizes all isoforms of Ras protein, including Ha-Ras, Ki-RasA, Ki-RasB, and N-Ras. All phospho-specific antibodies were from Cell Signaling Technologies (Beverly, MA). All secondary horseradish peroxidase-conjugated antibodies were from Kirkegaard and Perry Laboratories (Gaithersburg, MD). Statistics | All values reported as mean +- SEM. Statistical significance (p <= 0.05) was determined using the t test for differences in mean values, and a one-way analysis of variance in conjunction with the Student-Newman-Keuls test for multiple comparisons. PMID- 12221114_RESULTS TI - AB - Aldosterone Increases Absolute and Active GTP-bound Levels of Ki-RasA | Aldosterone via transcriptional control increases Ki-rasA mRNA and Ki-RasA protein levels in renal A6 epithelial cells. Experiments in Figure tested the hypothesis that aldosterone in these cells also increases the amount of active Ki-RasA. Activated Ras bound by GTP interacts with the RBD of Raf . The representative Western blots of Figure A show that the addition of aldosterone for 3 h to A6 cell monolayers markedly increased Ki-RasA (middle) and Ki-RasA:GTP levels (bottom), but had little effect on total Ras levels (top). It is known that Ki-RasA is expressed at levels much lower than other Ras isoforms . Thus, the lack of a marked change in total Ras was not unexpected. For these experiments, Ras:GTP was isolated using GST-RBD agarose from whole cell lysates from cells treated with vehicle (CON; 0.1% DMSO) and aldosterone (ALDO; 1.5 muM) for 3 h. After isolating total cellular Ras:GTP, Ki-RasA:GTP was identified with anti-K-Ras2A antibody, which reacts only with Ki-RasA . Total Ras protein was identified with the anti-v-Ha-Ras antibody, which is reactive with Ki-RasA and B, Ha-Ras, and N-Ras isoforms. The summary graph in Figure B shows the relative change in response to aldosterone for the levels of Ras (1.6 +- 0.2, n = 11), Ki-RasA (3.1 +- 0.4, n = 8), and Ki-RasA:GTP (2.8 +- 0.4, n = 8). Aldosterone, compared with vehicle, significantly increased Ki-RasA, and Ki-RasA:GTP levels (p < 0.005 for both). Although aldosterone also significantly increased total Ras levels (p = 0.03) at a similar time point, as reported previously by our laboratory , the relative increase in total Ras was significantly less than that of Ki-RasA (p = 0.002) and Ki-RasA:GTP (p = 0.009). Importantly, the relative changes in Ki-RasA vs. Ki-RasA:GTP levels were not different (p = 0.60). Figure 1 | Aldosterone increases active, GTP-bound Ki-RasA levels. Aldosterone increases active, GTP-bound Ki-RasA levels. (A) Western blot analysis of total Ras (top), Ki-RasA (middle), and Ki-RasA: GTP (bottom) in A6 cells treated without (CON) and with ALDO for 3 h. The representative blot probed for Ki-RasA: GTP (bottom) contained the precipitant from whole cell lysate by using Raf-RBD agarose, which specifically binds GTP-bound Ras. Blot probed with anti-Ki-RasA antibody to detect Ki-RasA: GTP. (B) Summary graph showing the relative changes in the levels of Ras, Ki-RasA and Ki-RasA: GTP in response to aldosterone treatment for 3 --4 h. * indicates significantly greater relative increases for Ki-RasA and Ki-RasA: GTP vs. Ras. Aldosterone Increases MAPK Activity | One potential signaling pathway activated by Ki-RasA is the MAPK cascade. Aldosterone stimulation of MAPK activity in A6 cells was measured in an in vitro reaction by following phosphorylation of exogenous MBP. The typical Western blots in Figure , A and B, showing the time course of aldosterone stimulation of MAPK activity, were probed with anti-phospho-MBP antibody. For these blots, each lane contained 3.5 mug of MBP processed for 30 min at 30C by A6 whole cell lysate (equal concentrations of total cellular protein; supplemented with MBP, Mg2+/ATP, and protein kinase C, protein kinase A, and calcium-calmodulin-dependent protein kinase II, and phosphatase inhibitors) from cells treated with aldosterone for the indicated times (in minutes for Figure A and hours for Figure B). The bar graph in Figure C summarizes such experiments. Aldosterone significantly increased MAPK activity 6.6 +- 1.2-, 10.5 +- 1.8-, 11.5 +- 2.5-, 12.0 +- 4.2-, 7.1 +- 2.8-, and 3.1 +- 0.9-fold at the 15 and 30 min, and 1-, 2-, 4-, and 6-h time points (n >= 4). At 1 and 5 min, MAPK activity was 1.4 +- 0.2- and 3.3 +- 0.8-fold higher (n = 2), respectively. MAPK activity in response to aldosterone increased steadily peaking between 0.5 and 2 h and waning after 4 h. Figure 2 | Aldosterone increases MAPK activity. Aldosterone increases MAPK activity. (A and B) MAPK activity was assessed by measuring phosphorylation of exogenous MBP. Exogenous MBP was added to equal amounts of A6 cell lysate from cells treated with aldosterone for the indicated times (in minutes, A; and hours, B). These typical Western blots were probed with anti-phospho-MBP antibody. (C) Summary graph of such experiments. *p <= 0.05 vs. time 0. Time Course of Aldosterone Activation of MAPK Is Distinct from That of Classic Mitogens | Results in Figure suggested that compared with classic mitogens, aldosterone activates MAPK signaling in a distinct manner, taking longer (up to 1 --2 h) to reach maximal activity and having a persistent signal (up to 4 h). Such a time course would be consistent with aldosterone-dependent activation of the MAPK requiring a latent period necessary for transcription and translation of Ki-RasA. Experiments in Figure were performed to determine whether the time course of aldosterone-dependent activation (phosphorylation) of MAPK in A6 cells was indeed distinct from activation by the traditional mitogen, serum. For these experiments, determining phospho-MAPK levels assessed activation of MAPK. The Western blots in Figure A were probed with anti-phospho-MAPK antibody. These blots contained equal amounts of whole cell lysate from A6 cells treated with serum (10% FBS; top) and aldosterone (1.5 muM) in the absence (middle) and presence (bottom) of the corticosteroid receptor antagonist RU486 (mifepristone; 0.1 muM) for the indicated times in minutes. Western blots in Figure A were stripped and subsequently reprobed with anti-MAPK antibody (Figure B, same order). Serum and aldosterone clearly have temporally distinct effects on activation of MAPK with the effects of serum peaking in the first 15 --30 min and waning thereafter, whereas those of aldosterone rising from 30 min onwards. The actions of aldosterone on MAPK were completely reversed by RU486, suggesting that this steroid stimulates MAPK signaling via its genomic actions. As shown in Figure C, similar results were observed when MAPK activity was assessed in an in vitro reaction by following phosphorylation of exogenous MBP. This blot was probed with anti-phospho-MBP antibody and each lane contained 3.5 mug of MBP processed by A6 whole cell lysate (equal concentrations of total cellular protein) from cells treated with serum (10% FBS) for the indicated times (in minutes). Figure 3 | Aldosterone compared with serum activates MAPK signaling in a delayed manner. Aldosterone compared with serum activates MAPK signaling in a delayed manner. (A) Western blots of A6 whole cell lysate from cells treated with serum (10% FBS, top), aldosterone (1.5 muM), and aldosterone plus RU486 (0.1 muM) for the indicated time in minutes. All blots probed with anti-phospho-MAPK antibody. (B) Blots in A stripped and reprobed with anti-MAPK antibody. (C) Western blot of MBP processed by A6 whole cell lysates from cells treated with serum (10% FBS) for the indicated times in minutes. Blot probed with anti-phospho-MBP antibody. Persistent Stimulation of MAPK by Aldosterone Is Mediated by Nuclear Steroid Receptors | Experiments in Figure were performed to further characterize the relation of aldosterone-stimulated MAPK at the 2-h time point with the genomic effects of this steroid. Both the effects of aldosterone on activation (phosphorylation) of MAPK in the presence of inhibitors of transcription and translation (Figure A), and the effects of aldosterone on MAPK activity in the presence of inhibitors of nuclear corticosteroid receptors, translation and mitogen-activated protein kinase kinase (MEK) (Figures , B and C) were determined. Figure 4 | Aldosterone stimulates MAPK signaling in A6 cells through induction of gene expression mediated by the glucocorticoid receptor. Aldosterone stimulates MAPK signaling in A6 cells through induction of gene expression mediated by the glucocorticoid receptor. (A) Western blot containing A6 whole cell lysate from cells treated with aldosterone (1.5 muM), CON, actinomycin D (1 mug/ml), cycloheximide (1 mug/ml), and aldosterone plus actinomycin D or cycloheximide for 2 h. This blot was probed with anti-phospho-MAPK antibody (top), stripped, and then reprobed with anti-MAPK antibody (bottom). (B) This typical Western blot shows phosphorylation of exogenous MBP added to A6 cell lysate from cells treated with aldosterone, and steroid plus RU486 (0.1 muM), emetine (1 mug/ml), cycloheximide (1 mug/ml), PD-98059 (10 muM), and U-0126 (0.5 muM) for 2 h. This typical blot was probed with anti-phospho-MBP antibody. (C) Below is a summary graph of five such experiments showing relative MAPK activity. The typical Western blot of Figure A was probed with anti-phospho-MAPK antibody (top), stripped, and then reprobed with anti-MAPK antibody (bottom). This blot contained equal amounts of A6 whole cell lysate from cells treated with ALDO (1.5 muM), CON (vehicle), actinomycin D (1.0 mug/ml), cycloheximide (1.0 mug/ml), and aldosterone plus actinomycin D or cycloheximide for 2 h. Although actinomycin D and cycloheximide had no overt effect on activation of MAPK when added alone, when added simultaneously with aldosterone, they abolished steroid-dependent activation of MAPK, demonstrating that transcription and translation are necessary for aldosterone to activate MAPK signaling. The typical Western blot of Figure B shows phosphorylation of exogenous MBP added to A6 cell lysate from cells treated with aldosterone for 2 h in the absence and presence of the corticosteroid receptor inhibitor RU486 (mifepristone; 0.1 muM) and inhibitors of translation (cycloheximide and emetine 1.0 mug/ml) and MEK (PD-98059 and U-0126 at 10 and 0.5 muM, respectively). For these experiments, inhibitors were added simultaneously with aldosterone. This Western blot was probed with anti-phospho-MBP antibody and contained equal amounts of exogenous MBP processed by the respective A6 cell lysate (equal concentration of total protein). As shown in the summary graph of Figure C, RU486 significantly decreased relative aldosterone-stimulated MAPK activity to 0.2 +- 0.04 (n = 5). Similarly, relative aldosterone-induced MAPK activity was decreased to 0.2 +- 0.1 (n = 5) and 0.3 +- 0.2 (n = 4) by emetine and cycloheximide, respectively. Moreover, PD-98059 and U-0126 decreased relative aldosterone-sensitive MAPK activity to 0.2 +- 0.1 (n = 5) and 0.2 +- 0.1 (n = 5), respectively. At this time point (2 h), the negative control for U-0126, U-0124, did not affect MAPK signaling and Ki-RasA levels were 53, 61, 62, 96, and 108% of (aldosterone-treated) control in the RU486, emetine, cycloheximide, PD-98059, and U-0126 groups, respectively (our unpublished data). Ki-ras Gene Contains a Functional Steroid Response Element That Confers Aldosterone Responsiveness to a Heterologous Promotor | The results described above and those reported previously by us and others suggest that aldosterone via steroid receptors directly affects Ki-ras expression and that this then impinges upon MAPK signaling. However, the molecular basis of this regulation has not been studied. Glucocorticoids and aldosterone ultimately target similar cis-acting elements through either the glucocorticoid or mineralocorticoid receptor (reviewed in ). The human and rat Ki-ras genes contain partially characterized cis-acting elements within the intron 1-exon 1 region that are trans-activated by the glucocorticoid --steroid receptor complex . Conserved elements have similarly been identified in the Ha-ras gene as responsive to glucocorticoids . Using this paradigm, we prepared a reporter plasmid containing the mouse c-Ki-ras2 intron 1-exon 1 region (-165 --153 from adenosine of the translation start codon within exon 1; pGL2-TK-rasI/E1), which contains several putative steroid response elements, and as shown in Figure , tested whether this region conferred functional aldosterone responsiveness to a heterologous promotor in A6 epithelial cells. Luciferase activity in cells transfected with pGL2-TK-rasI/E1 in the presence of vehicle and aldosterone (1.5 muM, 4 h) was 1.5 +- 0.3 and 4.6 +- 0.9 (n = 6), respectively. Thus, aldosterone significantly increased (threefold) luciferase expression driven by the pGL2-TK-rasI/E1 chimeric reporter plasmid. Simultaneous addition of RU486 (0.1 muM, n = 3) with aldosterone markedly decreased luciferase activity 50% to 2.3 +- 0.9 in cells transfected with pGL2-TK-rasI/E1. Dexamethasone (DEX) (0.1 muM, 4 h) had a similar effect as aldosterone increasing luciferase activity vs. vehicle 2.5-fold to 3.7 +- 0.9 (n = 3; p = 0.06; our unpublished data). Luciferase activity in the presence of either steroid in cells transfected with pGL2-TK-rasI/E1 was significantly greater (p > 0.05) than that in cells transfected with pGL2-TK and treated similarly (ALDO = 1.0 +- 0.2, n = 6; DEX = 0.7 +- 0.2, n = 3). In contrast, luciferase activity in the presence of vehicle was not different (p = 0.3) between pGL2-TK (0.8 +- 0.3, n = 4) and pGL2-TK-rasI/E1 --transfected cells. Reporter gene activity in cells transfected with the minimal promoter thymidine kinase luciferase plasmid (pGL2-TK) or pRL-CMV control plasmid alone was unaffected by steroid treatment (our unpublished data). Figure 5 | rasI/E1 region of c-Ki-ras2 is sufficient for steroid responsiveness. rasI/E1 region of c-Ki-ras2 is sufficient for steroid responsiveness. A6 cells were transiently transfected with pRL-CMV plus either control reporter plasmid (pGL2-TK), which contained firefly luciferase expression driven by the minimal thymidine kinase promotor, or reporter plasmid that contained the rasI/E1 region (pGL2-TK-rasI/E1). Luciferase activity in cells treated with vehicle, aldosterone, and aldosterone plus RU486 was quantified 48 h after transfection. *p < 0.05 vs. pGL2-TK treated with aldosterone and vehicle, and pGL2-TK-rasI/E1 treated with vehicle. Activation of MAPK Cascade by Aldosterone Is Dependent on Ki-RasA Expression | Although all of the above-reported results support the idea that aldosterone activates Ki-RasA via transcriptional control mediated by nuclear steroid receptors, it is unclear whether the aldosterone-sensitive MAPK signaling reported in Figures -- is in fact dependent on induction of Ki-RasA. Experiments in Figure directly test the link between aldosterone-dependent induction of Ki-RasA and steroid-dependent activation of MAPK signaling. The typical Western blots in Figure A are of lysates extracted from cells treated with aldosterone (1.5 mum; control) and aldosterone after pretreatment (24 h, 10 muM) with sense and antisense Ki-rasA oligonucleotides. Use of these oligonucleotides in A6 cells has been described previously . The top blot in Figure A was probed with anti-K-Ras2A antibody and demonstrates the efficacy of the antisense oligonucleotide to decrease Ki-RasA levels. Ki-RasA levels in the antisense group were similar40% of those in the control and sense groups (n = 6). In response to Ras signaling, Raf and MAPK become phosphorylated. The top middle blot in Figure A was probed with anti-phospho-Raf antibody and demonstrates that the Ki-ras antisense oligonucleotide attenuates aldosterone-sensitive phosphorylation of Raf. Phospho-Raf levels in the antisense group were <20% of those in the control and sense groups (n = 3). Similarly, as shown by the bottom middle blot, antisense inhibited aldosterone-dependent activation of MAPK with phospho-MAPK levels in the antisense group being <25% of those in the control and sense groups (n = 3). In contrast, as shown by the bottom blot, antisense had no effect on total MAPK levels with all three groups having similar levels of MAPK. Figure 6 | Aldosterone activates the MAPK cascade through induction of Ki-RasA. Aldosterone activates the MAPK cascade through induction of Ki-RasA. (A) These typical Western blots show the effects of aldosterone on Ki-RasA (top), phospho-Raf (top middle), active (phospho)-MAPK (bottom middle), and absolute MAPK (bottom) levels in A6 cells treated with steroid alone (vehicle) and in the presence of sense (SENSE) and antisense (ANTI) Ki-ras oligonucleotide. (B) Typical Western blot probed with anti-Ras antibody containing equal amounts of whole cell lysate extracted from cells stably expressing inducible DNRasN17 treated with vehicle (CON) and aldosterone for 2 h. (C) Typical Western blot probed with anti-phospho-MAPK (top) and then stripped and reprobed with anti-MAPK (bottom) antibodies containing equal amounts of whole cell lysate extracted from control cells and cells stably expressing inducible DNRasN17 treated with vehicle (-) and aldosterone (+) for 2 h. The representative Western blot in Figure B shows the effects of aldosterone on two distinct clonal A6 cell lines (DNRas1 and DNRas2) stably expressing corticosteroid-inducible dominant-negative RasN17. For such experiments (n = 2), confluent cells were treated with vehicle (CON) or aldosterone (ALDO) for 2 h. This blot, which contains equal amounts of total protein for each lysate, was probed with anti-Ras antibody and demonstrates that aldosterone increases total Ras levels in these clonal lines. Because aldosterone has little effect on total Ras expression in untransfected A6 cells (Figure A), these results demonstrate that these clonal lines stably express corticosteroid-inducible DNRasN17. The effects of aldosterone on activation of MAPK in these two clonal lines were determined next (Figure C). This typical Western blot (n = 2), which contained lysates with equal amounts of total protein from control cells and cells stably expressing inducible DNRasN17 treated with vehicle (-) and aldosterone (+) for 2 h, was probed with anti-phospho-MAPK antibody (top). This blot was subsequently stripped and reprobed with anti-MAPK antibody (bottom). In A6 cells stably expressing inducible DNRasN17, aldosterone had less of an effect on activation of MAPK compared with untransfected cells. Interestingly, stable cells had elevated levels of phospho-MAPK in the absence of aldosterone with steroid actually decreasing these levels. This may reflect a feedback response to chronically depressed MAPK signaling due to DNRasN17 leak. Nonetheless, these results show that for aldosterone to stimulate MAPK signaling, Raf kinase must be available to Ki-RasA, which it is not in the presence of DNRasN17. These results also are consistent with those in Figure A and together suggest that Ki-RasA transduces the aldosterone signal onto the MAPK cascade. Peak Activation of MAP Kinase Cascade by Aldosterone-stimulated Ki-RasA Is Delayed and Prolonged | The experiments in Figure temporally map the actions of aldosterone on MAPK signaling at several discrete levels within the transduction cascade. The representative Western blot of Figure A shows that whereas aldosterone increases the amount of active (phosphorylated) MAPK, it does not affect the total cellular pool of MAPK. For these blots, each lane contained 50 mug of total cellular protein, and the aldosterone (1.5 muM) treatment time is indicated in hours. For this set of experiments, cells representing time 0 were washed with vehicle 2 h before extraction. No difference was observed between time zero and wash for these and all other experiments. The top blot in this experiment was stripped after being probed with anti-phospho-MAPK (ERK 1/2) antibody and subsequently reprobed with anti-MAPK 1/2 antibody (bottom blot). Figure 7 | Aldosterone activates the MAPK cascade in a delayed but prolonged manner. Aldosterone activates the MAPK cascade in a delayed but prolonged manner. (A) Western blot analysis of aldosterone effects on MAPK and active (phosphorylated) MAPK levels in A6 cells. Top blot probed with anti-phospho-MAP kinase antibody. This blot was subsequently stripped and reprobed with anti-MAPK antibody (bottom). (B) Western blot analysis testing the effects of aldosterone on c-Raf and phosphorylated c-Raf. Top and bottom blots probed with anti-phospho-Raf and anti-Raf antibody, respectively. (C) Western blot analysis testing the effects of aldosterone on the MAPK cascade effectors, RSK (top), Fra-2 (middle), and MKP-1 (bottom). Top blot probed with anti-phoshpo-p90RSK antibody; middle blot probed with anti-Fra-2 antibody; and bottom blot probed with anti-MKP-1 antibody. Each lane for blots in A --C contained equal amounts of whole A6 cell lysate extracted from cells treated with aldosterone for the indicated time in hours. (D and E) Summary graphs showing the time course of the relative (vs. time 0) actions of aldosterone on Ki-RasA, phospho-MAPK, MAPK, phospho-Raf, Raf, MKP-1, phospho-RSK, Fra-2, and Na+ transport. The blots in Figure B show that similar to MAPK, aldosterone increases the levels of phospho-Raf compared with total cellular Raf. These blots contain lysate from cells treated with aldosterone for the indicate times (in hours). Top and bottom blots were probed with anti-phospho-(Ser259)-Raf and anti-Raf antibody, respectively. Each lane contained similar50 mug of total protein. The actions of aldosterone on downstream effectors of the MAPK cascade are shown in Figure C. These Western blots are of the same lysates (similar to that for Raf and phospho-Raf in 7B from the indicated time points after aldosterone treatment. Each lane contained 50 mug of total protein. Top, middle, and bottom blots were probed with anti-phospho-RSK-1 (p90 ribosomal S6 kinase; also referred to as MAPKAP kinase-1), Fra-2, and MKP-1 antibody, respectively. Clearly, all three of these MAPK effector proteins are either activated/phosphorylated, as for RSK-1, or induced, for Fra-2 and MKP-1, in response to aldosterone. The summary plots of Figure D show relative changes in Ki-RasA (inverted triangles), phospho-MAPK (diamonds), and MAPK (squares) levels in response to aldosterone at 0-, 0.5-, 1-, 2-, 4-, and 6-h time points. Also shown in this graph are the effects of aldosterone on Na+ transport (current, circles) across A6 cell monolayers at each time point. Figure E shows diary plots of relative changes in phospho-Raf (circles) and total Raf (gray triangles) in response to aldosterone. Also shown in this graph are the temporal actions of aldosterone on expression of the MAPK effectors Fra-2 (inverted triangles) and MKP-1 (diamonds). Included, in addition, are the effects of aldosterone on phosphorylation of the MAPK effector RSK-1 (squares). An expanded time course for aldosterone actions of affecters and effectors of MAPK is included in Table . Table 1 | Temporal effects of aldosterone on MAPK signaling and current PMID- 12221114_DISCUSSION TI - AB - The present results support a novel mechanism whereby aldosterone induces Ki-RasA expression at the level of transcription and then through stoichiometric increases in the levels of active, GTP-complexed Ki-RasA stimulates the MAPK cascade. Figure compares this genomic mechanism with the classic mechanism initiated by traditional mitogens. Traditional mitogens, such as serum, in contrast, stimulate MAPK signaling via a mechanism involving posttranslational control of active, GTP-complexed Ras levels without effects on absolute Ras levels. Compared with the classic mechanism, the distinct but possibly complimentary genomic mechanism activated by aldosterone leads to delayed but prolonged MAPK signaling. It is speculated that the delayed but prolonged MAPK signaling in response steroids will differentially affect cellular activity compared with traditional mitogens. Figure 8 | Schematic diagram showing distinct but complementary mechanisms of activation of the MAPK cascade in response to classic mitogens and corticosteroids. Schematic diagram showing distinct but complementary mechanisms of activation of the MAPK cascade in response to classic mitogens and corticosteroids. Classic mitogens using a posttranslational mechanism affect the GTP-binding state of Ras compared with steroids, which through genomic actions lead to stoichiometric increases in total and active Ki-RasA levels without changes in GTP-binding kinetics. Once stimulated by either mechanism, Ki-RasA then activates effector kinases through a common pathway with the distinction being that the genomic response compared with the posttranslational mechanism has a delayed onset and prolonged signal. Aldosterone to Ki-RasA | Aldosterone increases both Ki-RasA mRNA and protein levels in amphibian renal epithelial cells . The current results in Figure are consistent with this finding. The analogous findings in mammals, however, have been more controversial (reviewed in ; ) with corticosteroids increasing Ki-ras and Ki-RasA expression in mammalian colonic and mammary epithelial cells (; ; ; ; Fuller, personal communication) and cardiac fibroblasts , but not kidney epithelia (; Verrey, personal communication). The underlying molecular and cellular basis for these apparent discrepancies are currently unclear, but may be related to similarly undetermined mechanisms resulting in tissue-selective aldosterone induction of other proteins, such as ENaC and serum and glucocorticoid-inducible kinase (reviewed in ). Nevertheless, in A6 cells and in the heterologous Xenopus laevis oocyte expression system, Ki-RasA activates ENaC, which is one final effector of aldosterone signaling in epithelial cells . The molecular basis whereby aldosterone induces Ki-RasA has not been described. Similarly, it also is unclear whether merely increasing absolute Ki-RasA levels in response to aldosterone is sufficient to activate Ki-RasA leading to dependent stimulation of its effector MAPK cascades. Results in Figure demonstrate that aldosterone increases active, GTP-bound Ki-RasA levels proportionately with absolute Ki-RasA levels. This is consistent with a mechanism where through mass action, aldosterone-induced Ki-RasA leads to concomitant increases in the active pool of Ki-RasA. Results in Figures , , , and are consistent with this aldosterone-increased active pool of Ki-RasA then subsequently stimulating effector MAPK signaling. The results in Figure identify a putative cis-acting element/region (-165 --153 for mouse c-Ki-ras2) within the Ki-ras gene that possibly bestows aldosterone-responsiveness at the level of transcription to the Ras -> MAPK signaling cascade. Sequence analysis of this region of mouse c-Ki-ras2 reveals the presence of several potential sites responsive to corticosteroids with one hexanucleotide (TGTTCT; -50 to -45) half-site identical to those modulating corticosteroid-responsiveness in other genes . This hexanucleotide half-site is the most highly conserved portion of the palindromic GRE (5'-GGTACAnnnTGTTCT-3') and has been shown to bind GR and trans-activate in response to activated receptor . It is provocative that this half-site contained in the noncoding region is absolutely conserved in sequence identity and relative position in the mouse, rat, and human Ki-ras genes (see accession numbers , , and , respectively). Sequence data for the corresponding region in the X. laevis Ki-ras gene has not been published (see accession number ); therefore, it currently is unclear whether Ki-ras in this species also contains a similar element. The Ha-ras gene also contains conserved regions similar to those in the -165 --153 region of mouse Ki-ras. In addition, Ha-ras is induced by corticosteroids in some tissues, but pointedly not induced in renal and colonic epithelia (; ; Fuller, personal communication). It currently is unclear how common aldosterone effects on Ha-Ras expression are and what are the underlying molecular bases, if any, allowing for discretionary induction of Ki-RasA vs. Ha-Ras in response to aldosterone and other corticosteroids in a tissue- and species-specific manner. The effects of Ha-Ras on aldosterone effectors, moreover, have not been investigated. Although we observe a small but significant increase in total Ras levels in response to aldosterone (Figure ; ), it is not clear what fraction of this increase results from induction of Ki-RasA vs. Ha-Ras. However, the effects of aldosterone on MAPK signaling in A6 cells reported in the current study are abolished by inhibiting Ki-RasA expression with an antisense oligonucleotide, suggesting that this species of Ras and not Ha-Ras is the primary mediator of aldosterone actions in these cells. These findings are consistent with previous findings showing that Ki-RasA is necessary and sufficient to reconstitute, in part, aldosterone actions on ENaC . Ki-RasA to MAPK | The current study is the first to directly link the effects of aldosterone on Ki-RasA expression with activation of the MAPK cascade. Figure shows, using an in vitro assay, that aldosterone increases MAPK activity in A6 cells. Activity peaked between 1 and 2 h and began to wane by 4 h. Similar results were observed when activation of MAPK signaling was assessed using an in vivo assay of MAPK phosphorylation (Figures and ). In contrast to the time course of aldosterone effects on MAPK signaling, the classic mitogen, serum, stimulated this cascade much quicker, reaching peak activation within the first 30 min and waning thereafter . An alternative to aldosterone affecting the MAPK cascade through genomic actions dependent on induction of Ki-RasA expression is that this steroid activates MAPK signaling independent of its nuclear effects. Indeed, report that aldosterone modulates Na+/H+ exchange in Madin-Darby canine kidney cells through MAPK signaling, and that due to the rapidity of this action it is likely independent of steroid effects on gene expression. Similarly, report that aldosterone, independent of modulating gene expression, increases phospho-MAPK levels within 3 to 5 min with levels waning soon thereafter. We believe that the current results are more consistent with aldosterone activating the MAPK cascade in A6 cells via a genomic mechanism for several reasons. This is particularly true when the maximal effects of steroid are considered. First, as mentioned above, absolute and active Ki-RasA levels increased by the same amount in response to aldosterone , and aldosterone's effects on MAPK signaling at peak activation are entirely dependent on Ki-RasA expression and activation of its downstream effectors, such as Raf and MEK . Second, activation (phosphorylation) of MAPK in response to aldosterone was not apparent until 30 min after treatment . Third, at all time points assayed the stimulatory effects of aldosterone on phosphorylation of MAPK were attenuated by treatment with the nuclear corticosteroid receptor antagonist RU486 . Similarly, the maximum stimulatory effects of aldosterone (at 2 h) on phosphorylation of MAPK and MAPK activity (Figures and ) were sensitive to blockade of nuclear corticosteroid receptor, transcription, and translation. These results strongly support the contention that for aldosterone to affect MAPK signaling in A6 cells, its genomic actions are absolutely required. However, it was not our intention to investigate nongenomic regulation of MAPK signaling by aldosterone and thus, our experimental design cannot definitively exclude this possibility for the earlier time points (<30 min; see below). It is possible that in the current study there was a nongenomic response superimposed on a slower developing genomic response. Further support for a genomic mechanism driving the activation of MAPK in response to aldosterone observed in the current study is provided by comparison of the aldosterone-dependent time course of MAPK activity and phosphorylation vs. that of serum (Figures and ). Presumably, as shown previously for aldosterone , the nongenomic effects of this steroid would have a rapid time course more similar to traditional mitogens. This is not the case in the current study. We find that aldosterone activates the MAPK cascade via a similar genomic mechanism dependent on induction of Ki-RasA in rat cardiac fibroblasts . It is not clear why some cells respond to aldosterone via nongenomic activation of the MAPK cascade and others with a genomic mechanism. It is likely that these complimentary mechanisms are manifested in a cell-specific manner. Importantly, the systemic and cellular effects of aldosterone on classic target tissues, such as the distal nephron and colon, are mediated primarily through the genomic actions of this steroid (reviewed in ). Further comparison of the maximal effect of aldosterone on MAPK activity and activation of MAPK (Figures and and Table ) shows that MAPK activity and phosphorylation increase similar12 and similar2-fold, respectively. This difference may reflect signal amplification where a unit increase in MAPK phosphorylation yields a higher increase in activity. Comparison also of the effects of aldosterone on MAPK activity and phosphorylation at the 5-min time point shows that although MAPK activity already has increased by 5 min, there is no apparent increase in phosphorylation of MAPK. At first glance, the rapid effects of aldosterone on MAPK activity seem to be consistent with an early nongenomic response superimposed on a genomic response. We do not believe this to be the case, as argued above. Moreover, it is not commonly accepted that A6 cells have a nongenomic response to aldosterone. It is likely that, although performed in the presence of several different kinase inhibitors, the in vitro assay used to quantify aldosterone actions on MAPK activity was somewhat biased by the presence of uninhibited kinases or other cellular factors that either directly impinge upon phosphorylation of MBP or MAPK activity when taken out of cellular context. However, it also is possible that the assay of MAPK activity was more sensitive than that assessing activation of MAPK. The results showing that MAPK activity is increased 12-fold, whereas MAPK phosphorylation increased only 2-fold by 2 h would also be consistent with this possibility. If this is the case then nongenomic actions of aldosterone on MAPK signaling in A6 cells during the first 30 min or so cannot be wholly excluded by the current results. However, if a nongenomic response was superimposed on a slower developing genomic response we would have expected aldosterone-stimulated MAPK activity to peak at two distinct time points, which was clearly not the case. Nevertheless, the current results definitively demonstrate that peak activation of MAPK signaling at the 2-h time point in response to aldosterone is absolutely dependent on a genomic event and induction of Ki-RasA. Time Course and Effects of Aldosterone-stimulated MAPK Signaling | The current results show for the first time the temporal effects of aldosterone on the active levels of several different affecters and effectors of MAPK (Figure and Table ). Similar to MAPK, phosphorylation of Raf in response to aldosterone was delayed and prolonged. Phosphorylation of Raf was dominant between 2 and 6 h and thus Raf was phosphorylated (on Ser259) at a time point later than phosphorylation of MAPK. This finding is consistent with Raf being phosphorylated in response to aldosterone in a negative feedback manner. In fact, the Western blots in Figure actually assessed negative regulation of Raf that had previously been active. The serine/threonine kinase Akt/PKB an effector of phosphatidylinositol 3-kinase (PI3-K) is responsible for phosphorylation of active Raf at Ser259, which then leads to inactivation . PI3-K is also a first effector of Ki-Ras and activated by aldosterone . Thus, this feedback regulation of Raf may reflect parallel activation by aldosterone-induced Ki-RasA of both the MAPK and PI3-K signaling cascades. Consistent with these findings are those showing that the MEK inhibitors PD-98059 and U-0126 attenuate aldosterone-sensitive phosphorylation of MAPK but not Raf; and that the PI3-K inhibitor LY 294002 decreases aldosterone-sensitive Raf phosphorylation but not phosphorylation of MAPK (our unpublished observations). In addition to MAPK, MEK, and Raf, aldosterone increased the levels of phospho-RSK1 beginning at 30 min, reaching a peak by 4 h. RSK-1 is well known to be a target regulated at the posttranslational level in response to MAPK signaling. Interestingly, aldosterone induced expression of Fra-2 and MKP-1, which are known to be regulated at the level of transcription in response to MAPK signaling. Fra-2 is a transcription factor related to Fos. Thus, this action may enable aldosterone to secondarily impinge upon subsequent rounds of transcription. An alternative mechanism is that aldosterone directly affects transcription/translation of these proteins independent of MAPK signaling. Indeed, recent results from support such a mechanism of aldosterone action on Fra-2. To date, a direct effect of aldosterone on MKP-1 transcription/translation has not been demonstrated. Interestingly, induction of MKP-1, which is a phosphatase that dephosphorylates MAPK in a negative feedback manner to dampen MAPK signaling, in conjunction with feedback phosphorylation of Raf on Ser259 by Akt may result in the deactivation of MAPK signaling after 4 h observed in the current study. All of the current results are consistent with the idea that activation of the MAPK cascade in response to aldosterone is mediated by induction of Ki-RasA and activation of signaling constituents, such as Raf and MEK, that lie between this Small, monomeric G protein and MAPK. Consequently, after increases in total Ki-RasA and active Ki-RasA:GTP levels, aldosterone-dependent MAPK signaling follows a normal progression with the major exception being that signaling is prolonged. The explanation for prolonged MAPK signaling in response to aldosterone clearly then must come from the mechanism of initiation: transcriptional control of Ki-RasA. Such a mechanism is distinct from that used by classic mitogens, and leads to a delayed but prolonged signaling event. The delay results from the latent period required for increased transcription/translation of Ki-RasA. Prolongation results from stoichiometric increases in total and active Ki-RasA with absolute but not relative levels of Ki-RasA:GTP increasing. MAPK activity in response to aldosterone would then primarily be dependent on Ki-RasA protein turnover and feedback regulation. It is predicted that prolonged steroid-dependent MAPK signaling produces unique changes in cellular activity compared with a classic response. Although it is known that activation of Ki-RasA by aldosterone is necessary and sufficient for induced Na+ transport and ENaC activity in A6 cells and for ENaC activation when this G protein is overexpressed along with the channel in X. laevis oocytes , the actions of MAPK signaling on transport and ENaC seem to be inhibitory . Thus, it can be speculated that the prolonged activation of the MAPK cascade described in the current study is either a component of a feedback system or impacts Na+-transporting epithelia independently of directly affecting Na+ transport. The possible physiological and pathophysiological roles for genomic activation of MAPK signaling by corticosteroids at the tissue and systemic levels remain to be elucidated. PMID- 12221115 TI - Functional Heterogeneity of Bone Morphogenetic Protein Receptor-II Mutants Found in Patients with Primary Pulmonary Hypertension AB - | Germline mutations in the BMPR2 gene encoding bone morphogenetic protein (BMP) type II receptor (BMPR-II) have been reported in patients with primary pulmonary hypertension (PPH), but the contribution of various types of mutations found in PPH to the pathogenesis of clinical phenotypes has not been elucidated. To determine the biological activities of these mutants, we performed functional assays testing their abilities to transduce BMP signals. We found that the reported missense mutations within the extracellular and kinase domains of BMPR-II abrogated their signal-transducing abilities. BMPR-II proteins containing mutations at the conserved cysteine residues in the extracellular and kinase domains were detected in the cytoplasm, suggesting that the loss of signaling ability of certain BMPR-II mutants is due at least in part to their altered subcellular localization. In contrast, BMPR-II mutants with truncation of the cytoplasmic tail retained the ability to transduce BMP signals. The differences in biological activities among the BMPR-II mutants observed thus suggest that additional genetic and/or environmental factors may play critical roles in the pathogenesis of PPH. PMID- 12221115_INTRODUCTION TI - AB - Vascular development and homeostasis are regulated by a number of cytokines, including the members of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily includes various proteins with similar dimeric structures, e.g., activins, nodal, bone morphogenetic proteins (BMPs), and growth/differentiation factors . BMPs were originally identified as osteoinductive cytokines at extraskeletal sites in vivo . Subsequently, BMPs have been shown to exhibit multifunctional activities in various types of cells. They regulate cell growth, apoptosis, and differentiation, and participate in patterning and specification of various tissues and organs . BMPs transduce their signals via two types of serine/threonine kinase receptors, type I and type II receptors, both of which are required for their signal transduction . BMPs bind to three different type II receptors, i.e., activin type II receptors (ActR-IIA and ActR-IIB) and BMPR-II , and three different type I receptors, i.e., activin receptor-like kinase (ALK)-3/BMPR-IA, ALK-6/BMPR-IB, and ALK-2 (,; ; ; ; ). On binding of BMPs, type II receptors phosphorylate type I receptors, which in turn phosphorylate intracellular signal-transducing molecules Smad1, 5, and 8 . ALK-3 and ALK-6 activate these three Smads, whereas ALK-2 activates only Smad1 and Smad5 but not Smad8 . Recently, heterozygous germline mutations of the BMPR2 gene encoding BMPR-II were found in patients with primary pulmonary hypertension (PPH) , suggesting that BMPs may play important roles in homeostasis of the pulmonary vascular system. PPH is a disorder of the pulmonary arteries characterized by formation of plexiform lesions and obliteration of small pulmonary arteries . Subsequently, sporadic form of PPH was also shown to be associated with germline mutations of BMPR2 in at least 26% of cases . Although BMP signals are involved in the regulation of proliferation of human pulmonary smooth muscle cells , it has not been determined whether all cases of PPH carrying mutations within the BMPR2 gene are caused by perturbation of BMP signals. Mutations are distributed throughout the coding region of the BMPR2 gene, suggesting heterogeneity of their contribution to the pathogenesis of PPH. Furthermore, many PPH kindreds carrying mutations of the BMPR2 gene do not develop any signs or symptoms, suggesting that additional environmental and/or genetic factors may be necessary for development of symptoms . These findings raised the following questions: 1) whether the signaling components of BMP/Smad pathways are present in human pulmonary endothelial and smooth muscle cells, 2) whether BMP signals are impaired by all types of mutations found in PPH patients, and 3) how signal-transducing capabilities are disrupted in the BMPR-II mutant proteins. In this study, we used various types of BMPR-II mutants found in patients with PPH to investigate their ability to transduce BMP signals and the biochemical mechanisms by which BMPR-II mutants interfere with BMP signaling. First, we showed that human pulmonary artery endothelial cells (HPAECs) and smooth muscle cells (PASMCs) expressed BMP/TGF-beta signaling components, suggesting that these cells may potentially transduce their signals. Next, we showed that some BMPR-II mutants lost most signal-transducing abilities, such as transcriptional activity and phosphorylation of Smad proteins, whereas others retained most of them. Some of the mutants with defects in signaling activities were predominantly located in cytoplasm and may bind a cytoplasmic pool of type I receptors. Taken together, the findings of the present study suggest that perturbation of BMP signaling in the pulmonary vascular system by some types of mutations may be involved in the pathogenesis of PPH, whereas with other types of mutations signals can still be transduced, suggesting that additional factors may be required for the development of PPH. PMID- 12221115_MATERIALS AND METHODS TI - AB - Cell Culture | HPAECs and PASMCs were obtained from Clonetics (San Diego, CA) and were maintained in EGM-2 and SmGM-2 (Clonetics), respectively. COS-7 and R-mutant mink lung epithelial cells were maintained in DMEM (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 mug/ml streptomycin. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis | Total RNA was isolated from HPAECs and PASMCs with ISOGEN (NipponGene, Tokyo, Japan), and first-strand cDNA was synthesized using the Superscript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) with random hexamer primers. Expression of various signaling components was compared by semiquantitative RT-PCR analysis. A human beta-actin primer set was used to normalize the amount of total cDNA in each sample. PCR products were separated by electrophoresis in agarose gel (1%) and visualized with ethidium bromide. The primer sequences, PCR programs, and expected sizes of PCR products are available online as indicated in Table 1. As controls, RNAs from HPAECs and PASMCs were analyzed for beta-actin expression without the prior generation of cDNA, and a PCR reaction for each set of primers was run against H2O. Plasmid Construction | Plasmids of the BMPR-II, ALKs, and Smads were described previously . Various mutant forms of BMPR-II were constructed by a PCR-based approach. An EcoRI and an XhoI site were added to the N terminus and C terminus of the BMPR-II cDNA, respectively, and the resulting fragments were subcloned into pcDNA3-FLAG and pcDNA3-HA, which add a FLAG-tag and hemagglutinin (HA)-tag, respectively, C-terminally to the insert . To increase levels of expression, inserts were subcloned into another expression vector, pcDEF3 . All of the PCR products were sequenced. The sequences of the mutagenesis primers are available upon request. Transfection, Immunoprecipitation, and Immunoblotting | COS-7 cells were transiently transfected using FuGENE6 (Roche Applied Science, Mannheim, Germany). The amounts of plasmids transfected are available online in Table 2. Immunoprecipitation and immunoblotting were performed as described previously using anti-HA 12CA5 (for immunoprecipitation; Roche Applied Science), anti-HA 3F10 (for immunoblotting; Roche Applied Science), anti-FLAG M2 (Sigma-Aldrich), and anti-phosphoserine antibodies (Zymed Laboratories, South San Francisco, CA). Luciferase Assay | R-mutant mink lung epithelial cells were transiently transfected with an appropriate combination of reporter constructs, expression plasmids, and pcDNA3. Total amounts of transfected DNAs were the same in each experiment. Luciferase activities were normalized using cotransfected sea pansy luciferase activity under the control of thymidine kinase promoter. Affinity Cross-Linking and Immunoprecipitation | Iodination of BMP-6, affinity cross-linking, and subsequent immunoprecipitation were performed as described previously . Briefly, recombinant BMP-6 was iodinated using the chloramine T method, and cross-linking was performed with disuccinimidyl suberate (, Rockford, IL). Cells were lysed and subjected to immunoprecipitation with anti-FLAG antibody followed by SDS-PAGE. Cross-linked receptor complexes were visualized by using a BAS 1800 Bio-Image Analyzer (Fuji Photo Film, Tokyo, Japan). Immunofluorescence Labeling | Immunohistochemical staining of FLAG-tagged BMPR-II in transiently transfected COS-7 cells was performed using anti-FLAG M2 antibody (Sigma-Aldrich), followed by incubation with fluorescein isothiocyanate-labeled goat anti-mouse IgG as described previously . Nuclei of the cells were stained by 4,6-diamidino-2-phenylindole. Subcellular localization was determined by confocal laser scanning microscopy (, Hercules, CA). PMID- 12221115_RESULTS TI - AB - Profiles of Expression of TGF-beta Superfamily Signaling Components in Pulmonary Vascular Cells | Recently, showed that PASMCs express type I (ALK-1, 4, 5, and 6) and type II (TGF-beta type II receptor [TbetaR-II], ActR-II, and BMPR-II) receptors for the TGF-beta superfamily. To further evaluate the expression of TGF-beta superfamily signaling components in HPAECs and PASMCs, we performed RT-PCR analysis to detect mRNA transcripts for ligands (BMP-2 and TGF-beta1), type I (ALK-1, 2, 3, 4, 5, and 6), type II receptors (BMPR-II, ActR-IIA, ActR-IIB, and TbetaR-II), endoglin, betaglycan, and Smads (Smad1, 2, 3, 4, and 5) . Figure 1 | Expression of TGF-beta superfamily signaling components in HPAECs and PASMCs. Expression of TGF-beta superfamily signaling components in HPAECs and PASMCs. RNA samples from HPAECs and PASMCs were analyzed by RT-PCR for expression of TGF-beta superfamily-signaling components and the housekeeping gene beta-actin. Two alternatively spliced forms, WT and SH, of BMPR-II mRNA transcripts were detected. As controls, RNAs from HPAECs and PASMCs were analyzed for beta-actin expression without the prior generation of cDNA, and a PCR reaction for each set of primers was run against H2O. Transcripts for both BMP-2 and TGF-beta1 were present in HPAECs and PASMCs. Among BMP type I receptors, ALK-2 and ALK-6 were expressed in both types of cells, whereas ALK-3 was expressed only in PASMCs. ALK-1 is a TGF-beta type I receptor that has been reported to be predominantly expressed in endothelial cells . We detected mRNA transcripts for ALK-1 in HPAECs but only very weakly in PASMCs, whereas we detected those for ALK-5 in both types of cells. Two alternatively spliced forms of BMPR-II mRNA transcripts have been reported . To examine which forms of BMPR-II are expressed in pulmonary vascular cells, we designed PCR primers that are able to generate distinct PCR products from the two spliced variants. As shown in Figure , transcripts for both the wild-type (WT) and short (SH) form of BMPR-II were detected in both types of cells, although intensities of the bands of BMPR-II (SH) were much weaker than those of BMPR-II (WT) for both types of cells. We also detected transcripts for other type II receptors, i.e., ActR-IIA, ActR-IIB, and TbetaR-II, and endoglin and betaglycan in both types of cells. Finally, the expression of Smads was examined in HPAECs and PASMCs. We detected mRNA transcripts for receptor-regulated Smads specific for BMPs (Smads 1, 5, amd 8), and those for TGF-betas and activins (Smads 2 and 3), and common-partner Smad (Smad4), in both of the cell types. Thus, both HPAECs and PASMCs express transcripts for most components of BMP and TGF-beta --signaling pathways, suggesting that pulmonary vascular cells are capable of responding to BMPs and TGF-betas. However, responses to these ligands may differ between HPAECs and PASMCs, because of their differences in expression profiles of type I receptors ALK-1 and ALK-3. Construction of BMPR-II Mutants Found in Patients with PPH | Because it seemed that BMP signals are intact in pulmonary vascular cells, we attempted to characterize the biological activities of the mutant forms of BMPR-II found in patients with PPH . BMPR-II has a structure essentially similar to those of other type II receptors for members of the TGF-beta superfamily. However, BMPR-II (WT) has a long cytoplasmic tail, the roles of which are not well understood (Figure A). In addition, an alternatively spliced form (SH) lacking the cytoplasmic tail exhibited no functional differences from BMPR-II (WT) when assayed using Xenopus embryos . Figure 2 | Biological activities of wild-type and mutant BMPR-II. Biological activities of wild-type and mutant BMPR-II. (A) Structure and location of mutations of WT, SH, and mutant BMPR-II used in the following experiments. Numbers indicate amino acid positions. Mutations are denoted by asterisks. Missense mutations in extracellular (E1) and kinase (K1 and K2) domain mutants and substituted amino acid residues are shown. Cytoplasmic tail mutants (T1 and T2) have frameshift or nonsense mutations resulting in truncated tails. (B and C) Transcriptional activation by wild-type and mutant BMPR-II. p3TP-Lux reporter gene was cotransfected into R-mutant mink lung epithelial cells with ALK-3 and wild-type and/or mutant forms of BMPR-II as indicated, and cells were stimulated with or without BMP-2 (100 ng/ml for B and 50 ng/ml for C). Luciferase activity was normalized against cotransfected sea pansy luciferase activity. Expression of cotransfected BMPR-II mutants was confirmed by immunoblotting of cell lysates with anti-FLAG antibodies (C, right). At least four types of germline mutations of the BMPR2 gene have been reported . The first type (type X) has nonsense or frameshift mutations in the extracellular domain, which lead to premature truncation of the transcripts and absence of the production of transmembrane BMPR-II proteins. The second type (type E) has missense mutations in the extracellular domain, most of which involve highly conserved cysteine residues. The third type (type K) has either missense or frameshift mutations in the kinase domain. The fourth type (type T) has frameshift or nonsense mutations within the cytoplasmic tail, resulting in cytoplasmic truncation of the receptor protein. To investigate the biological activities of the BMPR-II mutants, we constructed one or two of each of the three types of BMPR-II mutant (E1, K1, K2, T1, and T2) reported by the International PPH Consortium (Figure A). BMPR-II Mutants Found in PPH Patients Exhibited Differences in Transcriptional Activities | We first examined the transcriptional activities mediated by wild-type or mutant forms of BMPR-II by using p3TP-Lux, a TGF-beta --responsive promoter-reporter construct, which weakly responds to BMP signals . Coexpression of a BMP type I receptor (ALK-3) and WT or SH of BMPR-II induced transcriptional activation of p3TP-Lux, which was further enhanced in the presence of BMP-2 (Figure B). None of the E1, K1, or K2 mutants induced transcriptional activation of the reporter gene. In contrast, the T1 and T2 mutants maintained the ability to induce transcription from p3TP-Lux, suggesting that truncation of the cytoplasmic tail does not efficiently disrupt the transcriptional activity of BMPR-II. Essentially similar results were obtained using 3GC2-lux , a BMP-specific promoter-reporter construct (our unpublished data), suggesting that the transcriptional activities induced by BMPR-II mutants found in patients with PPH differ between the type E and K mutants and type T mutants. Because heterozygous mutations of the BMPR2 gene were reported to cause PPH, we examined the effects of the BMPR-II mutants on the p3TP-Lux transcriptional activity induced by BMPR-II (WT) (Figure C, left). When the E1 or K1 mutants were cotransfected with BMPR-II (WT), they repressed the transcriptional activity induced by BMPR-II (WT) in a dose-dependent manner, suggesting that the E1 and K1 mutants behave as dominant negative mutants. In contrast, the T1 or T2 mutant that retained transcriptional activities exhibited less dominant negative effect than the E1 and K1 mutants. In addition, the K2 mutant also showed less dominant negative effect, suggesting the functional heterogeneity within the type K mutants. BMPR-II Mutants Differentially Induce Phosphorylation of Smad5 | BMP receptor complexes propagate signals mainly through phosphorylation of Smads 1, 5, and 8, although there is evidence for involvement of Smad-independent pathways in this propagation . To elucidate whether the differences in transcriptional activities induced by BMPR-II mutants involve the activation of Smads, we analyzed the phosphorylation of Smad5 cotransfected with wild-type or mutant forms of BMPR-II into COS-7 cells . The WT and SH forms of BMPR-II phosphorylated Smad5, whereas the E1 and K1 mutants failed to do so. Phosphorylation of Smad5 by the K2 mutant was also significantly reduced (our unpublished data). In agreement with the transcriptional activities, the T1 mutant phosphorylated Smad5, although less efficiently than BMPR-II (WT). These findings suggest that the differences in transcriptional activities mediated by BMPR-II mutants found in PPH patients are due to their abilities to activate BMP-specific Smads. Figure 3 | Phosphorylation of FLAG-tagged Smad5 mediated by HA-tagged wild-type or mutant BMPR-IIs in transfected COS-7 cells. Phosphorylation of FLAG-tagged Smad5 mediated by HA-tagged wild-type or mutant BMPR-IIs in transfected COS-7 cells. Top, cell lysates were immunoprecipitated (IP) with anti-FLAG antibody followed by immunoblotting with anti-phosphoserine (P-Ser) antibody. Expression of Smad5 (middle) and BMPR-II (bottom) was confirmed by immunoblotting of cell lysates with anti-FLAG and anti-HA antibodies, respectively. Ligand-binding Abilities of E1 and K1 Mutants Are Decreased | To investigate the biochemical mechanisms by which the E1 and K1 mutants lost signal-transducing abilities, we examined the ligand-binding abilities of the wild-type and mutant forms of BMPR-II. COS-7 cells were cotransfected with ALK-3 and wild-type or mutant forms of BMPR-II, affinity cross-linked using 125I-BMP-6, and subjected to immunoprecipitation by using anti-FLAG antibody for BMPR-II. As shown in Figure , WT, SH, K2, T1, and T2 mutant receptors bound BMP-6 efficiently in the presence of ALK-3. In contrast, the E1 mutant carrying a mutation in the extracellular ligand-binding domain did not bind BMP-6, suggesting that its loss of ligand-binding ability resulted in the loss of Smad-phosphorylating ability. Intriguingly, we also found significant reduction of the ligand-binding ability of the K1 mutant carrying a mutation in the kinase domain, which may have, at least in part, caused its loss of Smad-phosphorylating ability. Figure 4 | Ligand-binding abilities of BMPR-II mutants. Ligand-binding abilities of BMPR-II mutants. COS-7 cells were transfected with FLAG-tagged BMPR-II (BMPR-II-FLAG) and HA-tagged ALK-3 (ALK-3-HA), followed by affinity cross-linking with 125I-BMP-6, and lysates were immunoprecipitated (IP) with anti-FLAG M2 antibody. Immuno-complexes were subjected to SDS-PAGE and visualized by Fuji BAS bio-image analyzer (top). Expression of BMPR-II (middle) and ALK-3 (bottom) was confirmed by immunoblotting of cell lysates with anti-FLAG and anti-HA antibodies, respectively. E1 and K1 Mutants Exhibit Altered Subcellular Localization | To determine how the ligand-binding abilities of the E1 and K1 mutants were reduced, we examined the subcellular localization of wild-type and mutant forms of BMPR-II. COS-7 cells transfected with the wild-type or mutant forms of BMPR-II were subjected to immunofluorescence staining. WT (Figure A) and the T1 mutant (Figure E) exhibited intense staining of the plasma membrane as well as the cytoplasm. In contrast, the E1 and K1 mutants carrying missense mutations of cysteine residues within the extracellular and kinase domains, respectively, were observed mostly in the cytoplasm (Figure , B and C), suggesting that reduction of the ligand binding abilities of the E1 and K1 mutants was due to their altered subcellular localization. The K2 mutant, carrying a missense mutation of aspartic acid within the kinase domain, was mainly located on the plasma membrane, suggesting that the mechanism of its loss of signal-transducing ability may be due to perturbation of kinase activity. Figure 5 | Differential subcellular localization of wild-type and mutant BMPR-II. Differential subcellular localization of wild-type and mutant BMPR-II. Subcellular distribution of FLAG-tagged wild-type (A), E1 (B), K1 (C), K2 (D), or T1 (E) mutant BMPR-II in transfected COS-7 cells. Permeabilized cells were subjected to immunofluorescence (fluorescein isothiocyanate; green) staining and observation by confocal laser scanning microscopy after nuclear staining with 4,6-diamidino-2-phenylindole (red). E1 and K1 Mutants Are Retained in the Intracellular Compartments with Type I Receptors | Many membrane and secreted proteins are posttranslationally modified by the addition of N-linked oligosaccharides. We expected that the altered subcellular localization of E1 and K1 mutants would be confirmed by their posttranslational modification. The E1 mutant protein was observed as a fast-migrating band compared with WT (Figure , bottom), suggesting that the E1 protein is retained in the intracellular compartments as a glycoprotein containing high-mannose-type oligosaccharides. The K1 mutant was observed as two bands, i.e., a fast-migrating band similar to the E1 mutant and a slowly migrating band similar to the BMPR-II (WT) protein. This finding suggests that a considerable portion of the K1 mutant is also retained in the intracellular compartments. Figure 6 | Hetero-oligomerization of FLAG-tagged BMPR-II with HA-tagged ALK-3 in transfected COS-7 cells. Hetero-oligomerization of FLAG-tagged BMPR-II with HA-tagged ALK-3 in transfected COS-7 cells. Top, cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-HA antibody. Expression of ALK-3 (middle) and BMPR-II (bottom) was confirmed by immunoblotting of cell lysates with anti-FLAG and anti-HA antibodies, respectively. Fast- and slowly migrating bands of BMPR-II and ALK-3 are indicated by open and closed triangles, respectively. To determine how the altered subcellular localization of the BMPR-II mutants affects complex formation with type I receptors, we examined the hetero-oligomerization of BMPR-II mutants with ALK-3. COS-7 cells cotransfected with ALK-3 and wild-type or mutant forms of BMPR-II were subjected to FLAG-immunoprecipitation for BMPR-II, followed by HA-immunoblotting for ALK-3. BMPR-II (WT), BMPR-II (SH), and the T1 mutant formed complexes with slowly migrating forms of ALK-3, whereas the E1 and K1 mutants formed complexes predominantly with fast-migrating forms of ALK-3, which may contain high-mannose-type oligosaccharides (Figure , top). These results suggest that the E1 and K1 mutants are located in the intracellular compartments and that they may preferentially form complexes with the type I receptors located in the same compartments. PMID- 12221115_DISCUSSION TI - AB - Roles of BMP and TGF-beta Signaling in Maintenance of Vascular Systems | TGF-beta plays important roles during yolk sac vasculogenesis as well as late stages of angiogenesis by growth inhibition and production of extracellular matrix of endothelial cells . In endothelial cells, two types of TGF-beta type I receptors, ALK-1 and ALK-5, mediate TGF-beta signaling. ALK-5 is ubiquitously expressed in TGF-beta --responsive cells and activates Smad2 and Smad3. In contrast, ALK-1 is predominantly expressed in endothelial cells and activates BMP-specific Smad1 and Smad5. These observations suggest that balance between Smad1/5/8 and Smad2/3 pathways is important in determining vascular endothelial properties during angiogenesis . Endoglin is a dimeric glycoprotein with a short intracellular region that is structurally similar to betaglycan (also known as TGF-beta type III receptor). Endoglin binds TGF-beta as well as BMP-2 and BMP-7, suggesting that it may regulate both TGF-beta and BMP signaling pathways . Interestingly, mutations of ALK-1 and endoglin have been found in patients with hereditary hemorrhagic telangiectasia . Taken together with the findings that the BMPR2 gene is mutated in PPH patients, our findings suggest that TGF-beta/BMP signals mediated by Smad1, 5, and 8 may play important roles in maintenance of vascular homeostasis. Recently, showed that PASMCs express receptors for TGF-beta and BMPs, and that BMP suppressed the DNA synthesis and proliferation of PASMCs from patients with secondary pulmonary hypertensions, but did not suppress those from patients with PPH . The present study showed that both HPAECs and PASMCs express most of the signaling components required for TGF-beta/BMP signal transduction, including ligands, receptors, and Smads . However, response to TGF-beta and BMPs may differ between HPAECs and PASMCs. Because HPAECs express both ALK-5 and ALK-1, TGF-beta may activate Smad2/3 and Smad1/5 pathways, similar to other endothelial cells. Because PASMCs do not express ALK-1, the Smad1/5 pathways may not be activated by TGF-beta. Intriguingly, HPAECs express ALK-2 and ALK-6, but not ALK-3, suggesting that they respond to BMP-6 and BMP-7 through ALK-2 and ALK-6, but not to BMP-4, which binds to ALK-3 . In contrast, PASMCs express ALK-2, 3, and 6, suggesting that they respond to BMP-6 and -7 as well as to BMP-4. One of the features of PPH is overproliferation of endothelial cells and smooth muscle cells. Taken together with results of previous studies showing that BMPs have growth inhibitory effects on smooth muscle cells , these findings suggest that it is likely that BMP signals maintain pulmonary vascular integrity by suppressing the overproliferation of cells and that reduction of BMP signals caused by mutations of the BMPR2 gene eventually results in symptoms of PPH. How Did Type E and K Mutants Lose Their Signal-transducing Abilities? | In the present study, we generated five BMPR-II mutants, i.e., those mutated in the extracellular domain (E1), kinase domain (K1 and K2), or cytoplasmic tail (T1 and T2) (Figure A), to examine the biological activities of the BMPR-II mutants found in PPH patients. We found that the type E and K mutants lost their transcriptional activities, whereas the type T mutants maintained transcriptional activities although they were less potent than those of BMPR-II (WT). This suggests that these BMPR-II mutants have different biological activities. To date, all missense mutations within the extracellular domain of BMPR-II have been found at cysteine residues in PPH patients . Interestingly, extracellular cysteine residues have been shown to be essential for formation of proper three-dimensional structure and to be required for membrane targeting of some receptors . Consistent with this, we found that most of the E1 mutant proteins mutated at cysteine-118 were present in the cytoplasm (Figure B). These results suggest that loss of signal-transducing abilities due to missense mutations in the extracellular ligand-binding region is due not only to loss of ligand-binding ability of the extracellular domain but also to altered subcellular localization. Notably, the E1 mutant protein migrated faster than the BMPR-II (WT) protein (Figure , bottom), implying differential posttranslational modification due to abnormal subcellular localization of the E1 protein. When ALK-3 was coexpressed with the E1 mutant, only fast-migrating protein bands of ALK-3 formed complexes with the E1 mutant proteins (Figure , top). Many membrane-targeted proteins are posttranslationally modified by addition of N-linked oligosaccharides during transport through the Golgi apparatus. Treatment of ALK-3 and BMPR-II with N-glycosidase F resulted in shift of slowly migrating bands of ALK-3 and BMPR-II to fast-migrating bands (our unpublished data), suggesting that the fast-migrating proteins of the E1 mutant and ALK-3 may contain high-mannose-type oligosaccharides and that they are retained in the cytoplasm as a complex. These results raised the possibility that dominant negative effects of the E1 mutant against BMPR-II (WT) may be due to sequestration of BMP type I receptors in the intracellular compartments. On the other hand, missense mutations within the kinase region were identified at various amino acid residues, including cysteine, aspartic acid, and arginine residues . The K1 mutant, with substitution of cysteine-347 by tyrosine, exhibited a reduced ligand-binding ability than BMPR-II (WT). This can be explained by the distribution of mutant proteins partially in cytoplasm, as demonstrated by immunohistochemical analysis and by the presence of fast-migrating bands on immunoblot analysis (Figures C and , bottom). However, this distribution profile of the K1 mutant proteins cannot fully explain the loss of signal-transducing ability and gain of dominant negative activity by them, which were equivalent to those of the E1 mutant. Kinase activity was probably lost in the K1 mutant, resulting in the potent dominant negative effects of this mutant. In agreement with this, a BMPR-II kinase negative mutant exhibited a dominant negative effect against ActR-II in transcriptional activation activity . The K2 mutant, with substitution of aspartic acid-485 by glycine, exhibited normal ligand-binding ability and subcellular localization (Figure D), but lost signal-transducing ability (Figure B). Kinase activity was probably lost in the K2 mutant, which may have caused the loss of transcriptional activity; however, how the K2 mutant has less dominant negative effect remains unknown. Role of BMPR-II Mutants with Truncation of Cytoplasmic Tail in Pathogenesis of PPH | BMPR-II is structurally similar to other type II receptors of the TGF-beta superfamily, e.g., TbetaR-II, ActR-IIA, and ActR-IIB. However, BMPR-II has a long cytoplasmic tail that is not found in other type II receptors in mammals. The functions of the cytoplasmic tail of BMPR-II are not yet clear. The fact that truncation of the cytoplasmic tail of BMPR-II was found in type T mutants from patients with PPH suggests novel functions for this region. Compared with the E1 and K1 mutants, however, the T1 mutant retained most of its biological activity with the exception that it phosphorylated Smad5 less efficiently than WT or SH forms of BMPR-II. analyzed the transcriptional activities of BMPR-II mutants K2 and T1 according to our nomenclature, in NMuMG cells in which endogenous BMP signaling pathways are intact. Although they concluded that both of mutants lost their signaling capabilities, their results showed that only the K2 mutant, but not the T1 mutant, inhibited endogenous BMP signals. Thus, there may be significant differences in biological activities between the K2 and T1 mutants, consistent with our results. Recently, showed that BMPR-II mutants completely lacking the cytoplasmic tail were capable of transducing BMP-2 signals similar to BMPR-II (SH). Taken together with the present findings, these results suggest that the cytoplasmic tail of BMPR-II may not be essential for transduction of BMP signals through Smads, although it is possible that it has yet unidentified functions in BMP signaling. It will be important to determine whether other factors, such as additional genetic mutations and/or environmental factors, play important roles in the pathogenesis of PPH. PMID- 12221116 TI - Aurora B Kinase Exists in a Complex with Survivin and INCENP and Its Kinase Activity Is Stimulated by Survivin Binding and Phosphorylation AB - | Aurora B regulates chromosome segregation and cytokinesis and is the first protein to be implicated as a regulator of bipolar attachment of spindle microtubules to kinetochores. Evidence from several systems suggests that Aurora B is physically associated with inner centromere protein (INCENP) in mitosis and has genetic interactions with Survivin. It is unclear whether the Aurora B and INCENP interaction is cell cycle regulated and if Survivin physically interacts in this complex. In this study, we cloned the Xenopus Survivin gene, examined its association with Aurora B and INCENP, and determined the effect of its binding on Aurora B kinase activity. We demonstrate that in the Xenopus early embryo, all of the detectable Survivin is in a complex with both Aurora B and INCENP throughout the cell cycle. Survivin and Aurora B bind different domains on INCENP. Aurora B activity is stimulated >10-fold in mitotic extracts; this activation is phosphatase sensitive, and the binding of Survivin is required for full Aurora B activity. We also find the hydrodynamic properties of the Aurora B/Survivin/INCENP complex are cell cycle regulated. Our data indicate that Aurora B kinase activity is regulated by both Survivin binding and cell cycle-dependent phosphorylation. PMID- 12221116_ TI - Glossary AB - Abbreviations used: : EST = expressed sequence tag GST = glutathione S-transferase I = interphase INCENP = inner centromere protein IP = immunoprecipitation MBP = myelin basic protein PBS = phosphate-buffered saline PBS-T = PBS containing 0.1% Tween 20 PCR = polymerase chain reaction Pre-I = preimmune sera xAurora B and xAurB = Xenopus Aurora B xINCENP = Xenopus inner centromere protein xSurvivin = Xenopus Survivin PMID- 12221116_INTRODUCTION TI - AB - Defects in chromosome segregation can generate aneuploidy, a condition that is found in almost all human tumors and is a major cause of miscarriages and birth defects. The complex process of chromosome segregation must be highly regulated to ensure fidelity and to prevent aneuploidy. Many of the mitotic events are regulated by the kinetochore, a proteinaceous structure assembled on centromeric DNA that coordinates at least three mitotic functions (for review, see ). First, the kinetochore is the chromosomal site of microtubule attachment and movement. Second, the kinetochore is the major site of cohesion between sister chromatids. This cohesion must be maintained through metaphase and its dissolution is the critical event that triggers anaphase. Third, kinetochores that are not attached to microtubules send signals to the cell cycle machinery to prevent this dissolution of cohesion, a process referred to as the spindle assembly checkpoint. This checkpoint ensures that all chromatids are attached before the onset of anaphase. How the kinetochore coordinates these various functions is a critical unanswered question. A group of mitotic regulators that includes Aurora B kinase and the inner centromere protein (INCENP) has been given the name chromosomal passengers . The passenger proteins are defined by a dynamic pattern of localization . In G2 and prophase, the passenger proteins have a general chromosomal localization. During prometaphase and metaphase, they move from chromosomes to inner centromeres. When chromosomes move to the poles during anaphase, passenger proteins remain at the spindle midzone where they eventually become part of the midbody . The phenotypes of the chromosomal passenger proteins suggest that they are critical regulators of various steps of chromosome segregation. RNAi experiments in Caenorhabditis elegans embryos and Drosophila cell lines suggest that cells lacking Aurora or INCENP have similar mitotic defects. First, the passenger proteins are necessary for the proper segregation of DNA. During anaphase, the chromosome masses do not properly segregate, leaving a chromatin bridge between the major DNA masses . Second, cytokinetic furrows begin but fail to fully progress in cells lacking either Aurora or INCENP . This suggests a second role for Aurora and INCENP in cytokinesis. Third, studies from budding yeast suggest that Aurora is involved in bipolar attachment of microtubules to kinetochores. The Aurora homolog Ipl1 is required during chromosome segregation, as cells without Ipl1 activity have massive nondisjunction and often segregate both sisters' chromosomes to the same pole. Ipl1 is required for kinetochores to release spindle microtubules both in vitro and in vivo . Current models propose that Ipl1 ensures that each chromosome obtains bipolar attachment of microtubules by coordinating the release of kinetochores attached from a single pole, thereby allowing kinetochores to rebind microtubules until proper bipolar attachment is achieved. This model predicts that Aurora B kinase activity is inactivated by bipolar attachment, but we presently know little about how Aurora B activity is regulated. Some overlapping phenotypes are seen in embryos lacking the Survivin/Bir1 protein (Survivin). Survivin is required in both budding and fission yeast for proper chromosome segregation . RNAi experiments in C. elegans have shown that embryos lacking Survivin display abnormal chromosome condensation, disrupted mitotic spindles, and were ultimately unable to complete cytokinesis, resulting in multinucleate embryos . Survivin-null mouse embryos displayed polyploidy, abnormal mitotic spindles, and failed cytokinesis . The similarities in phenotypes suggest that Aurora, INCENP, and Survivin could function together. INCENP and Survivin have both been shown to genetically interact with Aurora B kinases. Chan and colleagues first identified mutants of the budding yeast INCENP homolog (Sli15) that are synthetically lethal with temperature-sensitive mutants of the Aurora homolog Ipl1. Moreover, they showed that sli15 cells had phenotypes identical to those of ipl1 yeast. As discussed earlier, similar phenotypes are also seen in fission yeast, C. elegans, and Drosophila cells lacking either Survivin, Aurora, or INCENP (for review, see ). Aurora B kinase is not localized to the kinetochores in fission yeast or C. elegans embryos lacking Survivin . Similarly, in C. elegans embryos and Drosophila cells, loss of INCENP by RNAi also leads to the mislocalization of Aurora B kinase . Biochemical evidence has shown that Aurora B physically interacts with INCENP. Sli15p and Ipl1p form a complex in budding yeast , and a complex containing both INCENP and Aurora B kinase has been purified from Xenopus laevis mitotic extracts . There is no conclusive biochemical evidence that Aurora kinases or INCENP are physically associated with Survivin, although a recent report has shown that human Survivin can interact with either Xenopus INCENP (xINCENP) or Aurora B kinase in both two-hybrid and in vitro pull-down assays . Therefore, some essential questions are whether Aurora, INCENP, and Survivin physically interact in vivo, whether complex formation is cell cycle regulated, and how each subunit interacts in the complex. Moreover, it is critical to identify the molecular function(s) of each protein in the complex. To understand the interrelationship of the passenger proteins and to further understand how Aurora B kinase is regulated, we have cloned the Xenopus Survivin (xSurvivin) gene. xSurvivin is shown to exist in a complex with both xINCENP and Xenopus Aurora B kinase (xAurora B) in S-phase (interphase) and mitotic Xenopus extracts. Moreover, immunodepletion of xAurora B kinase can completely remove xSurvivin and xINCENP from Xenopus extracts, suggesting that all of the xSurvivin and xINCENP is physically associated with xAurora B kinase. We show that the N terminus of xAurora B kinase interacts with the conserved C terminus of xINCENP, whereas xSurvivin interacts with the N terminus of xINCENP. Furthermore, xAurora B activity is stimulated at least 10-fold in mitotic extracts, and this stimulation is shown to be phosphatase sensitive. Adding recombinant xSurvivin protein to xAurora B immunoprecipitations (IPs) stimulates the mitotic kinase activity an additional 10-fold, suggesting that xSurvivin binding to Aurora B plays a regulatory role similar to cyclin binding of CDKs. Therefore, our data suggests that xAurora B kinase is regulated by both complex formation and phosphorylation. PMID- 12221116_MATERIALS AND METHODS TI - AB - Materials | All chemicals were purchased from Sigma (St. Louis, MO) unless stated otherwise. All DNA restriction enzymes were purchased from (Beverly, MA). Adult wild-type Xenopus laevis were purchased from Nasco (Fort Atkinson, WI). Xenopus Interphase and Mitotic Extracts | Interphase extracts were prepared as previously described . Interphase extracts were driven into mitosis by the addition of 66 nM nondegradable glutathione S-transferase (GST)-cyclin B for 20 min at room temperature. Fusion Constructs and Protein Purification | Based on the sequence of xAurora B , primers 1292098 (5'-GCGGATCCATGGAGTACAAAGAGAATCTCAACCC) and 1292100 (5'-CGGCGGCCGCTTTTGATTGGGTGGACTGGTAGAC) were used to amplify xAurora B from a Xenopus stage 11.5 --14 cDNA library. This polymerase chain reaction (PCR) fragment was subcloned into the NcoI-NotI sites of pET 28 (Novagen, Madison, WI) to create pET 28-xAurB that expresses C-terminal 6His-tagged xAurora B. xAurora B was also amplified using primers 1292098 and 1292099 (5'-GGCTCGAGAAGCTTCATTTTGATTGGGTGGACTGG). The resulting PCR fragment was subcloned into the NcoI-HindIII sites of pET 41 (Novagen) to yield pET 41-xAurB that expresses N-terminal GST-tagged xAurora B. The gene was sequenced by the University of Virginia Biomolecular Research Facility (Charlottesville, VA), and its sequence was deposited into GenBank (accession no. ). Our isolated gene has only two amino acid changes from the published gene. As both of these changes are also found on each of the GenBank deposited expressed sequence tags (ESTs), they are unlikely to be mutations caused by the PCR isolation. Primers 1292099 and 1984819 (5'-GCCCATGGAATTCCCATTGGGGAAGGGG) were used to amplify the xAurora B kinase domain. The resulting PCR fragment was subcloned into the NcoI-HindIII sites of pFastBac and was subsequently subcloned into the NcoI-HindIII sites of pET 41B to create pET 41-xAurB (99 --384) that expresses N-terminal GST-tagged xAurora B kinase domain. To clone xSurvivin, a BLAST search of the EST database was conducted to find a Xenopus EST with high homology to human and mouse Survivin. xSurvivin was then amplified from a Xenopus stage 11.5 --14 cDNA library using primers 1242204 (5'-CTGGCCGGCCCCATATGTATTCTGCCAAGAACAGG) and 1242206 (5'-CGCTCGGGTGGTCGAGATCTATGGAGCACTG). This PCR fragment was subcloned into the NdeI-XhoI sites of pET 41 (Novagen) to yield pET 41-xSurvivin that expresses C-terminal 6His-tagged xSurvivin. xSurvivin was also amplified using primers 1242204 and 1242205 (5'-CCGGCGCGCCTCAGTGGTCAAGATCTATGGAGCAA). The resulting PCR fragment was subcloned into the NdeI-Asc1 sites of pGEXcsFA (a generous gift from Ethan Lee, Harvard Medical School, Boston, MA) to yield pGEXcsFA-xSurvivin that expresses N-terminal GST-tagged xSurvivin. xSurvivin sequence was deposited into GenBank (accession no. ). The modeled crystal structure coordinates were generated by SWISS-MODEL . The figures were constructed with RasMol. A XhoI site was engineered into the N terminus of our pCS2+xINCENP clone using PCR with the oligonucleotides (5'-CCGCGCTCGAGAACGATGCAGA-GTGCCGTGCCC) and (5'CCGGCGGGGCCCTCTAGAGGATCCTCGTATTTGAGGCCATAACC). The resulting product was cloned into the XhoI-Xba sites of Super GFP-Wee1 to form a GFP-xINCENP fusion protein. A HindIII to ApaI fragment containing GFP and the N terminus of xINCENP was cloned back into pCS2+xINCENP, and the remaining PCR product was confirmed by sequence to generate pCS2+GFP-xINCENP. The XhoI to XbaI fragment was cloned into pET 28B in the XhoI and Bpu1103 sites, and finally a NheI to PstI fragment of pET 28B-xINCENP was removed to generate pET 28-xINCENP (677 --874) that expresses N-terminal 6His-tagged xINCENP (677 --874). All proteins were expressed in the Escherichia coli strain BL21 (DE3 pLysS; Novagen). 6His-tagged proteins were purified on Ni2+-NTA agarose (Qiagen, Valencia, CA) as instructed by the manufacturer. GST-tagged proteins were purified on glutathione agarose . Antibody Production, IP, and Immunoblotting | All polyclonal antibodies were made by Covance Research Products (Denver, PA). To make anti-xAurora B antibodies, rabbits 315 and 316 were immunized with purified C-terminal 6His-tagged xAurora B. To make anti-xSurvivin antibodies, rabbits 342 and 343 were immunized with purified C-terminal 6His-tagged xSurvivin. Anti-xINCENP antibodies were produced in rabbits 354 and 355 immunized with N-terminal 6His-tagged xINCENP (677 --874) encoding the C-terminal fragment of xINCENP. All antibodies were affinity purified on the corresponding immunizing protein coupled to a cyanogen bromide-activated Sepharose column (Amersham Biosciences, Piscataway, NJ) as described . After affinity purification, the antibodies were dialyzed into XB no Ca2+ (10 mM HEPES, pH 7.7, 1 mM MgCl2, 100 mM KCl, and 50 mM sucrose), aliquoted, and stored at -80C. Immunoblotting was performed on Immobilon-P (Millipore, Bedford, MA) and was detected using horseradish peroxidase-conjugated-anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) by chemiluminescence (ECL; Amersham Biosciences). To immunoprecipitate complexes from Xenopus extracts, 20 mug of preimmune sera (Pre-I) 1 mug of affinity-purified anti-xAurora B, anti-xSurvivin, or anti-xINCENP antibodies were bound to 50 mul of protein A Sepharose beads (Amersham Biosciences) in phosphate-buffered saline (PBS) for 1 h at room temperature. The beads were washed five times in XB no Ca2+, and one-half was added to 40 mul of interphase extract and the other one-half was added to 40 mul of interphase extract driven into mitosis by the addition of GST-cyclin B. Beads were incubated with extracts for 1 h at 4C and were subsequently washed five times in XB no Ca2+ containing an additional 200 mM NaCl and 0.1% Brij 35. The concentration of xAurora B, xSurvivin, and xINCENP in Xenopus extracts was determined by quantitatively comparing interphase extracts with recombinant GST-xSurvivin, GST-xAurora B, and 6His-xINCENP (677 --874) levels on immunoblots. For quantification, the amount of INCENP in Xenopus extracts was compared with titrations of 6His-xINCENP (677 --874) transferred to Immobilon by slot blots rather than by SDS-PAGE followed by transfer. To quantify immunoblots, films were scanned with densitometer hardware (Molecular Dynamics, Sunnyvale, CA) into ImageQuant 5.0. Data were then exported into Microsoft Excel and graphed for analysis. Cell Culture and Immunofluorescence | XTC cells were obtained from Dr. Douglas DeSimone (University of Virginia, Charlottesville, VA) and were grown at room temperature in 70% L-15 media supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate. XTC cells were grown on coverslips and fixed with 4% paraformaldehyde in PHEM buffer (60 mM PIPES, pH 6.9, 25 mM HEPES, 10 mM EGTA, and 4 mM MgCl2) containing 0.75% Triton-X-100. Coverslips were subsequently washed in PBS containing 0.1% Tween 20 (PBS-T). Fixed cells were blocked in 20% heat-inactivated goat serum (Invitrogen, Carlsbad, CA) and were then incubated for 1 h at room temperature with monoclonal anti-tubulin antibody at 1:500 and either affinity-purified anti-xAurora B (1 mug/ml) or anti-xINCENP (1 mug/ml) antibodies diluted in PBS-T. Coverslips were then washed three times with PBS-T and then incubated with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G at 1:1000 and Cy3-conjugated anti-rabbit immunoglobulin G (Jackson ImmunoResearch Laboratories) at 1:1000, washed three times in PBS-T, stained with 1 mug/ml Hoechst No. 33342 for 30 s, and mounted with Vectashield (Vector Laboratories, Burlingame, CA). Images were collected at x100 using an E600 Eclipse microscope (Nikon, Tokyo, Japan) equipped with a CH350 cooled charged-coupled device camera (Photometrics, Tucson, AZ). Images were acquired and analyzed using version 4.5 Meta View software. Gel Filtration Chromatography and Sucrose Gradient Sedimentation | For gel filtration analysis, 100 mul of interphase extract was diluted 1:5 in XB no Ca2+. In parallel, microcystin (5 muM) was added to 100 mul of mitotic extracts and the mitotic extract was diluted 1:5 in 80 mM beta-glycerophosphate, 20 mM EGTA, 10 mM MgCl2, and 5 mM NaF. Clarified interphase and mitotic extracts were prepared by ultracentrifugation at 40,000 rpm for 1 h at 4C in an S120-AT2 rotor (Sorvall, Kendro Laboratory Products, Newtown, CT). The extracts were then concentrated in a Microcon (Millipore) and were subsequently loaded onto a Superose 6 column (Amersham Biosciences). Thirty-two fractions of 500 mul were collected and trichloroacetic acid precipitated as described . Alternating fractions were loaded onto 8 and 15% SDS-PAGE gels and immunoblotted for xINCENP, xAurora B, and xSurvivin. Similarly clarified extracts were loaded onto 5 --30% sucrose gradients and centrifuged at 30,000 rpm for 30 h at 4C in a SW-40 rotor. Sixteen fractions were collected, trichloroacetic acid precipitated, and equal volumes of sample were separated on 8 and 15% gels for immunoblot analysis. Molecular weight markers were run in a parallel gradient. Mapping of Passenger Protein-Binding Domains | To identify binding domains among the passenger proteins, the indicated truncated or wild-type proteins were translated in vitro in the presence of [35S]methionine using the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI). These proteins were allowed to integrate into endogenous complexes by mixing 10 mul of the in vitro translation reaction with 20 mul of Xenopus extract for 30 min, and then the endogenous protein was immunoprecipitated. Labeled proteins were detected by phosphorimager analysis of dried SDS-PAGE gels. Proteins were scored as interacting if the amount of labeled protein immunoprecipitated was fivefold higher than the amount precipitated by Pre-I; typically >80% of a labeled protein was precipitated in these experiments. The deletion set of xINCENP was a generous gift of Susannah Rankin and Marc Kirschner (Harvard Medical School). Kinase Assays | For each kinase assay, xAurora B kinase was immunoprecipitated from 50 mul of Xenopus extract on similar10 mul of protein A Sepharose beads (Amersham Biosciences). The beads were washed four times in XB no Ca2+ containing an additional 300 mM NaCl and 0.1% Triton-X-100, followed by an additional wash in kinase buffer (20 mM Tris, pH 7.5, 1 mM MgCl2, 25 mM KCl, 1 mM dithiothreitol, and 40 mug/ml bovine serum albumin). Beads were aliquoted into tubes containing the indicated recombinant protein in 25 mul of kinase buffer supplemented with 100 muM gamma[32P]ATP (2 muCi; Perkin Elmer-Cetus Life Sciences, Norwalk, CT) and 1 mug of myelin basic protein (MBP; Invitrogen). Reactions were incubated in a Mixer 5432 (Eppendorf; Brinkmann Instruments, Westbury, NY) at room temperature, and after 10 min, the reactions were stopped by the addition of sample buffer. One-half of the reaction was loaded onto a gel that was Coomassie stained, dried on 3MM paper (Whatman, Clifton, NJ), and analyzed for 32PO4 incorporation with phosphorimager hardware (Molecular Dynamics) and ImageQuant 5.0 software. The other one-half of the reaction was loaded onto a second gel and the amount of xAurora kinase was quantified by immunoblot. In kinase reactions involving lambda phosphatase, beads were washed into lambda phosphatase buffer, treated at room temperature for 30 min with either 200 U of lambda phosphatase or buffer, subsequently washed four times in kinase buffer, and assayed for kinase activity. PMID- 12221116_RESULTS TI - AB - Cloning the Xenopus Survivin Gene | To biochemically dissect the regulation of passenger proteins, we used embryonic extracts of the frog Xenopus laevis. These extracts are excellent tools for studying mitotic regulators for three reasons. First, cell cycle regulators are stockpiled in Xenopus eggs and are more abundant than in somatic cells. Second, the cell cycle state of these extracts can be easily controlled and, third, the extracts can recapitulate mitotic events in vitro. We have previously cloned the xINCENP gene and demonstrated that the protein is specifically phosphorylated in mitosis . A gene encoding the xAurora B protein was recently identified , and a highly related gene was isolated from a Xenopus stage 11.5 --14 plasmid cDNA library by PCR. The xSurvivin cDNA was cloned by identifying a Xenopus EST with high homology to human and mouse Survivin. The Survivin cDNA was then isolated from a Xenopus stage 11.5 --14 cDNA library by PCR. The Xenopus Survivin cDNA encodes a protein that is 46% identical to human Survivin (Figure A). The predominant motif on the Survivin protein is an inhibitor of apoptosis domain, a motif that has been shown in a related baculoviral protein to inhibit Caspase-3 activity (for review, see ; ), although the Survivin family does not inhibit Caspase-3 . The xSurvivin protein has the recognizable motifs of Survivin including the inhibitor of apoptosis repeat and a Cdc2 kinase phosphorylation site. Two independent groups have recently published the crystal structure of human Survivin . The Xenopus sequence was modeled into these crystal structures by the program SWISS-MODEL. The program predicts that the backbone structures of the Xenopus and the human proteins are highly similar (Figure , B and C). A prominent motif that is not included on the model is a C-terminal extended alpha helix (Figure , B and C). This is not included because this region is divergent in the primary sequence (Figure A); however, a SOPM secondary structure of this region predicted a long C-terminal alpha helix. Therefore, there is structural similarity between the Survivin homologs throughout the protein. Noel and colleagues identified two potential interaction regions (an acidic patch and a basic patch) on the surface of human Survivin. To determine if these regions are conserved in xSurvivin, we compared the charged amino acid residues of the xSurvivin model (Figure D) with the placement of charged residues in the solved crystal structure of human Survivin (Figure E). The placement of surface charge on xSurvivin is predicted to be almost identical to human Survivin. Figure 1 | Structural and sequence homology of Xenopus Survivin. Structural and sequence homology of Xenopus Survivin. (A) Clustal-W alignment of the amino acids encoding sequenced vertebrate Survivins. Significant residues from the crystal structure of human Survivin are conserved and are indicated by letters or symbols above the alignment: P-cdc2 phosphorylation site; star-residues that coordinate the Zn2+ in the Bir domain. (B-E) Comparison of solved human Survivin structure and a structural prediction of Xenopus Survivin protein produced by the SWISS-MODEL server . Ribbon diagram of Xenopus Survivin (B) and human Survivin (C). Charge distribution in Xenopus (D) and human (E) space-filled Survivin models where acidic charged residues are shown in red and basic residues are shown in blue. Note that the long C-terminal alpha helix is present in the Xenopus sequence; however, the homology is too low to fit in the modeled structure. Characterization of Antibodies | After affinity purification, anti-xAurora B and anti-xINCENP antibodies are specific as they predominantly recognize a band of 41 and 130 kDa, respectively, in a Xenopus interphase extract and a Xenopus tissue culture cell line (XTC) lysate (Figure , B-C). Anti-xSurvivin antibody recognizes a predominant band at 18 kDa (Figure A). Even after affinity purification, the anti-xSurvivin antibody recognizes six additional bands; therefore, it was not used for immunofluorescence. The antibodies are highly sensitive as they all recognize <1 ng of recombinant protein (unpublished data, M. Bolton, P.T. Stukenberg). By comparing immunoblot signals of Xenopus interphase extracts with known amounts of recombinant protein, we estimate that the concentration of xAurora B protein in these extracts is 500 nM, the concentration of xSurvivin is similar250 nM, and the concentration of xINCENP is 100 nM (unpublished data, M. Bolton, P.T. Stukenberg). Figure 2 | Specificity of affinity-purified anti-xSurvivin, anti-xAurora B, and anti-xINCENP antibodies in Xenopus interphase extracts and XTC cells. Specificity of affinity-purified anti-xSurvivin, anti-xAurora B, and anti-xINCENP antibodies in Xenopus interphase extracts and XTC cells. (A) Affinity-purified anti-xSurvivin antibody recognizes a band of 18 kDa in an interphase extract. (B) Affinity-purified anti-xAurora B antibody recognizes a band of 41 kDa in an interphase extract and in XTC cells. (C) Affinity-purified anti-xINCENP antibody recognizes a band of 130 kDa in an interphase extract and in XTC cells. (D-AA) Immunofluorescence localization of xAurora B (D-O) and xINCENP (P-AA) in XTC cells. (D-I) Immunolocalization of xAurora B during interphase (D) and throughout mitosis (E-I). (P-U) Immunolocalization of xINCENP during interphase (P) and throughout mitosis (Q-U). Colocalization of either xAurora B (J-O) or xINCENP (V-AA) with DNA and microtubules. Blue, DAPI; green, microtubules; red, xAurora B in (K-M), xINCENP in (W-Y); yellow and orange, overlap of xAurora B or xINCENP with microtubules. XTC cells stained with the anti-xAurora B antibody (Figure , D-O) and anti-xINCENP antibody (Figure , P-AA) display typical passenger protein immunolocalization. The concentration of both proteins is low in interphase (Figure , D, J, P, and V), but by prophase, the antibodies recognize both chromatin and kinetochores (Figure , E, K, Q, and W). Both proteins move to kinetochores at prometaphase-metaphase (Figure , F-G, L-M, R-S, and X-Y), and finally to bundles of overlapping microtubules in anaphase (Figure , H, N, T, and Z) where they remain throughout cytokinesis until they are discarded with the midbody (Figure , I, O, U, and AA). These data suggest that the roles of passenger proteins are conserved in Xenopus and demonstrate that the anti-xAurora B and anti-xINCENP antibodies are highly specific. xAurora B, xINCENP, and xSurvivin Are Physically Associated In Vivo | To determine the native molecular weight of the passenger proteins, Xenopus interphase and mitotic extracts were size separated by both Superose 6 gel filtration chromatography and 5 --30% sucrose gradients, and the location of each of the proteins was detected by immunoblot. In interphase egg extracts, xAurora B, xINCENP, and xSurvivin comigrate in a broad peak from 900 to 600 kDa, whereas all three proteins migrate in a sharp peak at 900 kDa in mitotic extracts (Figure A). A slower migrating form of xSurvivin is often seen in SDS-PAGE gels. Detection of this form depends on phosphatase inhibitors and its appearance is not cell cycle dependent (unpublished data, M. Bolton, W. Lan, and P.T. Stuken-berg). xAurora B, xINCENP, and xSurvivin also comigrate in a sucrose gradient, sedimenting at 9.5S in interphase and at 4.5S in mitosis (Figure B). A number of control proteins, including Eg2, the Xenopus Aurora A homolog, migrated similarly in interphase and mitosis, demonstrating that the differences in migration of the passenger protein complex were caused by cell cycle changes and not due to differences in the manipulations of the sucrose gradients (Figure B). Because of the contributions of shape, neither gel filtration nor sedimentation can accurately predict native molecular weight. Gel filtration measures the Stokes radius, which is determined by the overall length of the molecule, whereas sucrose gradients measure sedimentation, which is retarded by a long thin shape. Therefore, highly elongated molecules such as myosin migrate faster than spherical proteins of similar molecular weight in gel filtration, whereas they sediment more slowly than spheres in sucrose gradients . have derived equations using the hydrodynamic characteristics of molecules to overcome the contributions of shape and more accurately measure molecular weight. Such calculations suggest that the mitotic complex is one-half the size (similar250 kDa) of the interphase complex (similar490 kDa). Moreover, in both complexes, the overall length is comparable because they migrate similarly by gel filtration. The predicted axial ratio is >20, indicating a size and shape reminiscent of fibrinogen, but not as elongated as myosin. INCENP has a long coiled-coil domain, which is a motif that often contributes to such elongated shapes in proteins. Thus, the passenger proteins complex has a highly elongated shape in both interphase and mitosis, and the mass of the complex doubles in interphase. Figure 3 | xSurvivin, xAurora B, and xINCENP cofractionate on gel filtration columns and sucrose density gradients during interphase and mitosis. xSurvivin, xAurora B, and xINCENP cofractionate on gel filtration columns and sucrose density gradients during interphase and mitosis. (A) Interphase and mitotic extracts were separated by Superose 6 gel filtration chromatography and equal volumes of alternate fractions were run on SDS-PAGE gels. The fractions were then immunoblotted for xINCENP, xAurora B, and xSurvivin to determine the molecular weight of the passenger protein complex. The molecular weights of the void (2000 kDa), thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), and equine myoglobin (17 kDa) are shown. (B) xINCENP, xAurora B, and xSurvivin cofractionate on a sucrose density gradient, but the interphase complex migrates faster than the mitotic complex. Interphase and mitotic extracts were sedimented on 5 --30% sucrose density gradients. Equal volumes of each fraction were run on SDS-PAGE gels and immunoblotted for xINCENP, xAurora B, xAurora A (Eg2), and xSurvivin. Thyroglobulin (19S), bovine gamma globulin (7S), chicken ovalbumin (3.5S), and equine myoglobin (2S) were sedimented in a parallel gradient as markers. To directly test if the passenger proteins are in the same complex in vivo, we immunoprecipitated each of the passenger proteins from Xenopus extracts and immunoblotted the precipitates to determine if other passenger proteins were bound. After IP with xAurora B antibodies, both xINCENP and xSurvivin are also precipitated (Figure A). Because equivalent amounts of these three passenger proteins are detected in both interphase and mitotic extracts, it does not appear that their association is cell cycle regulated in the early embryo. Pre-I controls do not precipitate any of the passenger proteins. Moreover, neither xINCENP nor xSurvivin is immunoprecipitated by the xAurora B antibodies if recombinant xAurora B protein is added to the extract before the IP, demonstrating that the interaction is specific to xAurora B antibodies (unpublished data, M. Bolton and P.T. Stukenberg). In xINCENP IPs, both xSurvivin and xAurora B (Figure A) are detected by immunoblot. Finally, in xSurvivin IPs, both xAurora B and xINCENP are detected (Figure A). Therefore, in Xenopus embryos during both interphase and mitosis, there is a physical interaction between xSurvivin, xINCENP, and xAurora B kinase. Figure 4 | xSurvivin, xAurora B, and xINCENP are physically associated in vivo during both interphase and mitosis. xSurvivin, xAurora B, and xINCENP are physically associated in vivo during both interphase and mitosis. (A) xAurora B (xAurB), xINCENP, and xSurvivin were immunoprecipitated from interphase and mitotic Xenopus extracts. To determine if the chromosomal passenger proteins are physically associated, the immunoprecipitated samples were loaded onto 8 and 15% gels and were immunoblotted for xINCENP, xAurora B, and xSurvivin. Note that xINCENP is phosphorylated during mitosis and therefore exhibits retarded mobility. (B) In Xenopus extracts, the majority of xINCENP, xAurora B, and xSurvivin is physically associated. Interphase and mitotic extracts were immunodepleted with anti-xAurora B antibodies. Samples of the immunodepleted extract (xAurB Depleted Extract), beads, and interphase and mitotic whole cell extracts (WCE) were run on 8 and 15% gels and were immunoblotted for xINCENP, xAurora B, and xSurvivin. xINCENP and xSurvivin were depleted below detection levels (<1 ng) and were detected on the beads. Because the three proteins comigrate on a gel filtration column and sucrose gradient , it is possible that the majority of each passenger protein exists in this complex. This hypothesis was directly tested by immunodepleting xAurora B complex from a Xenopus low-speed extracts and immunoblotting the depleted extracts for xINCENP and xSurvivin. When 95% of the xAurora B is removed from extracts, xINCENP and xSurvivin are depleted to similar levels (Figure B). This experiment demonstrates that in the Xenopus embryo, most, if not all, of xINCENP and xSurvivin is physically associated with xAurora B kinase. Mapping the Chromosomal Passenger Complex Interactions | INCENP Is a Scaffold Protein with xSurvivin Binding Its N Terminus and xAurora B Binding Its C Terminus. An assay was developed to identify the domains on xINCENP that interact with xAurora B and xSurvivin (Figure A). 35S-labeled full-length xINCENP protein was translated in vitro and incubated in a Xenopus extract. This protein incorporates into the endogenous complex because it can be immunoprecipitated with our anti-xAurora B antibody (Figure B) and anti-xSurvivin antibody (unpublished data, M. Bolton, W. Lan, and P.T. Stukenberg). These interactions are specific, as they are not detected after IP with Pre-I controls (Figure B). The interaction between xAurora B and in vitro translated xINCENP is quite robust as the xINCENP is quantitatively immunoprecipitated from the extract (unpublished data, M. Bolton and P.T. Stukenberg), and, like the endogenous complex, the interaction is detected in both interphase and mitotic extracts (Figure , B and C). We determined whether xAurora B interacted with a series of deletion and truncated xINCENP proteins using this assay (Figure C). xAurora B interacts with all of the constructs that contain the C terminus of xINCENP except for the construct xINCENP Delta(119 --242). We do not understand why the xINCENP Delta(119 --242) construct does not interact with Aurora B, but it is likely that either the C terminus is misfolded or a regulatory region is missing. The simplest interpretation of the data is that there is an xAurora B interaction domain in the C-terminal 200 amino acids of xINCENP that is both necessary and sufficient for xAurora B binding. This region contains the IN-box that was previously shown to be an xAurora B binding domain in C. elegans and mouse . More recently, it was shown that yeast Aurora (Ipl1p) binds directly to the C terminus of the INCENP homolog Sli15 . Figure 5 | xAurora B, xINCENP and xSurvivin complex interactions. xAurora B, xINCENP and xSurvivin complex interactions. (A) Schematic of an assay used to study xAurora B, xINCENP, and xSurvivin interaction. Plasmids encoding full-length xINCENP as well as xINCENP deletion constructs (pictured in C) were in vitro translated in the presence of [35S]methionine and were subsequently incubated with Xenopus interphase and mitotic extracts. Anti-xAurora B antibodies, anti-xSurvivin antibodies, and Pre-I were used to immunoprecipitate the immunogenic protein and interacting xINCENP fragments from the extract. (B) An example of the assay where full-length xINCENP was immunoprecipitated with anti-xAurora B antibodies, but not with Pre-I. (C) The xINCENP constructs pictured were tested for their ability to coimmunoprecipitate with xAurora B and xSurvivin. Precipitation greater than fivefold above the amount detected by Pre-I controls is indicated by a + whereas less than fivefold was indicated by a -. Full-length xINCENP includes the following domains (N-terminal to C-terminal): the centromere-interacting domain, the chromosome-binding domain, the coiled-coil/microtubule-binding domain, and the IN-box (see Figure ). (D) The interaction of xSurvivin with the complex is NaCl sensitive. xAurora B was immunoprecipitated from a mitotic extract and was washed in XB buffer containing the indicated amount of additional NaCl. After washing, the concentration of xAurora B and xSurvivin in the IPs was determined by quantitative immunoblot. Densitometric analysis of this experiment indicates that after washing with 300 mM NaCl, there is 10-fold more xAurora B kinase than xSurvivin. (E) The N terminus of Aurora B is required to interact with xINCENP. Full-length xAurora B (circles) and xAurora B (99 --384) (triangles) were in vitro translated in the presence of [35S]methionine and were subsequently incubated with Xenopus interphase extracts. Anti-xINCENP antibodies, anti-xSurvivin antibodies, and Pre-I were used to immunoprecipitate the resulting complex from the extract, and the IPs were washed with XB + 0.1% Triton-X 100 and the indicated extra NaCl (mM). The presence of either full-length or kinase domain xAurora was detected by SDS-PAGE and subsequent phosphorimager analysis. Using this assay, we also mapped the xSurvivin interaction domain to the N-terminal 119 amino acids of xINCENP (Figure C). The three constructs that contained the first 119 amino acids of INCENP could be immunoprecipitated by xSurvivin antibodies, whereas the two constructs that lacked this region do not. Again, we see no cell cycle differences in the xINCENP/xSurvivin interaction. The N terminus of chicken INCENP has been implicated in centromere targeting as well as chromosome and midzone binding . We are currently testing if these biochemical activities are mediated by xSurvivin. xSurvivin Binding to the Complex Is NaCl Sensitive. The salt sensitivity of the interaction between the endogenous xSurvivin and xAurora B complex was examined. xAurora B IPs were washed with buffers containing 0.1% Triton-X 100 and varying NaCl concentrations and were subjected to quantitative Western blot (Figure D). The endogenous complex of xAurora B and xINCENP is stable in high salt. However, the interaction between xAurora B and xSurvivin is salt sensitive. There is twofold more xAurora B kinase in Xenopus extracts than xSurvivin, but after washing with only 100 mM NaCl, the ratio of the two proteins is 6:1. This ratio increases to 10:1 after a 300 mM NaCl wash, and no xSurvivin is detected after washing the complex with 1 M NaCl (Figure D). These data indicate that the interaction between xINCENP and xAurora B does not require xSurvivin because xAurora B and xINCENP remain tightly associated after removal of xSurvivin. Reciprocal IPs were performed by in vitro translating xAurora B, incubating the translation mix with Xenopus interphase extracts to allow labeled xAurora B to incorporate into the endogenous complex, and immunoprecipitating with either anti-xSurvivin or anti-xINCENP antibodies. The resulting IPs were washed with buffers containing 0.1% Triton-X 100 and varying NaCl concentrations. The amount of xAurora B bound was quantified by phosphorimager and plotted (Figure E). As seen in Figure D, the xAurora-xINCENP interaction is highly stable; however, the xAurora-xSurvivin interaction is NaCl sensitive. These data confirm two conclusions made from Figure D: the Survivin interaction is salt sensitive, suggesting that it is not tightly associated with the complex; and the interaction between xINCENP and xAurora B does not require xSurvivin. The N Terminus and Kinase Domain of xAurora B Both Interact with INCENP and the N Terminus May Weakly Bind xSurvivin. The first 98 amino acids of xAurora B define an N-terminal region of unknown function and the rest of the protein is mostly kinase domain. We demonstrate that the N terminus of xAurora B is required for the highly stable interaction with xINCENP (Figure E). A construct encoding the C-terminal amino acids 99 --384 of xAurora B was radioactively translated in vitro and was mixed with interphase Xenopus extracts. Unlike full-length xAurora B, which efficiently immunoprecipitates with xINCENP, the xAurora B kinase domain construct is not quantitatively precipitated. Whereas 70% of the full-length xAurora B coimmunoprecipitates with xINCENP, only 30% of the xAurora kinase domain coimmunoprecipitates. After washes with increasing concentrations of NaCl, the amount of xAurora kinase domain bound to xINCENP falls from 30 to 5%. The weak, salt-sensitive interaction between xAurora B kinase domain and xINCENP suggests that the N terminus of xAurora B provides the majority of the binding to xINCENP, whereas the kinase domain only weakly interacts. It has been reported that xAurora B kinase interacts with both xINCENP and human Survivin in both two-hybrid and in vitro pull-down assays . Therefore, it is surprising that we have identified xINCENP constructs that robustly bind either xSurvivin or xAurora B, suggesting that xAurora and xSurvivin do not interact directly but rather bind opposite ends of xINCENP. If this were true, we would expect that anti-xSurvivin antibodies would pull down xAurora B kinase domain through its interaction with xINCENP (Figure E). However, we find that xSurvivin antibodies were not able to precipitate any xAurora B kinase domain, even though at low NaCl concentrations, there was some interaction between the kinase domain and xINCENP. The simplest interpretation of this data is that there may be a weak direct interaction between the N terminus of xAurora B and xSurvivin that stabilizes the complex. xSurvivin Protein Can Stimulate Mitotic xAurora B Kinase Activity | Because xSurvivin could be washed off xAurora B kinase IPs, the dependence of xAurora B kinase activity on complex formation was examined. xAurora B kinase was immunoprecipitated from interphase and mitotic extracts, washed with a buffer containing 300 mM NaCl so that the ratio of xAurora B to xSurvivin was 10:1, and the kinase activity was measured by the incorporation of 32PO4 from gamma[32P]ATP onto a MBP substrate (Figure A). Kinase activity is readily detected when the IP is performed with Aurora B antibodies, but not if Pre-I is used. In correlation with previous studies, we find that this activity is cell cycle regulated, as at least 10-fold greater activity is detected if the xAurora B is precipitated from mitotic extracts as compared with interphase extracts . The kinase stimulation is due to an increase in specific activity of the enzyme as similar amounts of protein are detected in the IP by Coomassie stain of the gel (Figure , A and C) or xAurora B immunoblot (Figure B). Figure 6 | The activity of xAurora B is stimulated by stoichiometric xSurvivin binding and mitotic phosphorylation. The activity of xAurora B is stimulated by stoichiometric xSurvivin binding and mitotic phosphorylation. (A) Recombinant GST-Survivin can stimulate an xAurora B IP kinase assay. xAurora B IP kinase assay from either interphase or mitotic extracts was conducted in the presence or absence of 10 ng of recombinant GST-Survivin. The top panel is an autoradiogram of [32P]O4 incorporation into a MBP substrate; the bottom panel is a Coomassie-stained gel showing similar amounts of xAurora B in each reaction. (B) Titration of the amount of xSurvivin needed to activate the xAurora B kinase immunoprecipitated from interphase and mitotic extracts. The top panel is a graph of the kinase activity of the immunoprecipitated xAurora B in the presence of the indicated amount of GST-Survivin. The bottom panel is an xAurora B immunoblot analysis demonstrating similar amounts of xAurora B kinase in the xAurora B IP kinase assay. (C) xAurora B kinase was immunopurified from either interphase or mitotic Xenopus egg extracts; Pre-I was used as a negative control. The IPs were washed extensively and either incubated in phosphatase buffer (-) or phosphatase buffer and lambda phosphatase (+), and were then washed again extensively and the kinase activity was assayed on MBP. A Coomassie-stained gel of the IP kinase assay demonstrates that there are similar amounts of xAurora B kinase in each reaction (bottom panel). (D) The phosphorylation state of xAurora B directly regulates its kinase activity. The xAurora B IPs were washed extensively, incubated in either phosphatase buffer (-) or phosphatase buffer and lambda phosphatase (+), washed again, and assayed for kinase activity on MBP substrate. Samples were also immunoblotted for xINCENP, xAurora B, and xSurvivin to determine relative amounts of the proteins. We find that adding 10 ng of recombinant GST-Survivin stimulates the mitotic kinase activity similareightfold, but has limited affect on xAurora B isolated from interphase extracts (Figure A). This stimulation of xAurora B kinase activity was titrated in Figure B and was saturated at 10 ng of recombinant GST-Survivin. As a negative control, we added 10 ng of GST-cyclin B, which did not stimulate kinase activity (unpublished data, S. Powers, P.T. Stukenberg). We estimate that there is similar20 --30 ng of xAurora B in these IPs (Figure D), suggesting that xSurvivin is stoichiometrically required for xAurora B kinase activity. xAurora B Kinase Activity Is also Regulated by Phosphorylation | The kinase activity of Aurora A is regulated by phosphorylation . To determine if Aurora B kinases are also regulated by phosphorylation, xAurora B IPs from interphase and mitotic extracts were washed with low-salt buffer, which allows most of the endogenous xSurvivin to remain bound. This reaction was split, and one-half of the reaction was treated with lambda phosphatase, subsequently washed to remove the phosphatase, and assayed for kinase activity (Figure C). The mitotically stimulated xAurora B kinase activity is sensitive to treatment with lambda phosphatase, demonstrating that cell cycle-specific phosphorylation regulates the specific activity of xAurora B (Figure C; ). The simplest interpretation of the phosphatase sensitivity of Aurora B kinase activity is that, as in many other kinases, the phosphorylation state directly regulates kinase activity. However, dephosphorylation may also disturb the xSurvivin interaction. To distinguish between these two possibilities, we phosphatase treated an IP of xAurora B and assayed for both kinase activity and for the relative amount of the three subunits by immunoblot (Figure D). Phosphatase treatment eliminated both the kinase activity and greatly increased the gel mobility of xINCENP, demonstrating that the phosphatase treatment had worked efficiently. However, the concentration of each subunit in the complex did not change. Therefore, maximum kinase activity of xAurora B requires both interaction with xSurvivin and the mitotic phosphorylation state of the complex. PMID- 12221116_DISCUSSION TI - AB - During both interphase and mitosis, xAurora B kinase is in a complex with the majority of xINCENP and xSurvivin protein in the Xenopus embryo. The complex is cell cycle regulated as the kinase is activated >10-fold in mitosis, and hydrodynamic characterization suggests that there is a dramatic change in size that corresponds to changes in the cell cycle. We have determined two independent steps for mitotic xAurora B kinase activation: stoichiometric xSurvivin binding and a phosphorylation requirement. Therefore, like the CDK1 mitotic kinase, xAurora B activation can be regulated by both its phosphorylation state and the binding of a regulatory subunit. Our demonstration that xSurvivin, xINCENP, and xAurora B kinase are in a complex can simply explain the molecular details underpinning a number of in vivo observations. First, in C. elegans double-stranded RNA-mediated interference (RNAi) of the INCENP homolog can mislocalize the Aurora B kinase and loss of either protein causes both chromosome segregation and cytokinetic defects . Second, after RNAi of the Survivin homolog in C. elegans, the Aurora B homolog is mislocalized and embryos lacking either protein have similar phenotypes . Our data suggest that these phenotypes could either be caused by mislocalization of the proteins when the complex is not properly formed or by inactive Aurora B kinase in the absence of the Survivin protein. The N terminus of human INCENP (1 --405) has dominant-negative activity and can inhibit the final stages of cytokinesis when it is overexpressed in tissue culture cells . Our mapping of xSurvivin binding to the N terminus of INCENP and xAurora B binding to the C terminus suggests that overexpression of INCENP (1 --405) would sequester Survivin away from Aurora B and inhibit its kinase activity. A Complex of Chromosomal Passenger Proteins in the Xenopus Early Embryo | An in vivo passenger protein complex of xSurvivin, xINCENP, and xAurora B kinase has been identified in Xenopus early embryos. Our mapping data are consistent with a scaffold protein role for xINCENP, which binds xSurvivin on its N terminus and xAurora B on its C terminus (Figure A). The N terminus of INCENP has been implicated in centromere targeting in metaphase and midzone localization in anaphase . Our localization of the Survivin interaction to this region suggests that these targeting events could be mediated through the Survivin protein. The C terminus of INCENP contains the IN-box, which appears to be a highly conserved Aurora B interaction motif as it also interacts with Aurora B homologs in C. elegans, mouse, and budding yeast . We have shown that both the kinase domain of xAurora B and the N terminus provide interactions with the C terminus of xINCENP. We have also shown that the N terminus of Aurora B stabilizes xSurvivin binding in high-salt conditions. The simplest interpretation of this finding is that the major interaction between xSurvivin and xAurora is mediated through xINCENP, although there is a weak direct interaction between xSurvivin and the N terminus of xAurora B (Figure B). Figure 7 | Model of xAurora B, xINCENP, and xSurvivin interactions and regulation. Model of xAurora B, xINCENP, and xSurvivin interactions and regulation. (A) Interactions within the passenger protein complex. xSurvivin binds the N terminus of xINCENP, whereas the N terminus of xAurora B binds the C terminus of xINCENP. (B) Cell cycle regulation of the passenger protein complex. The interphase complex is hypophosphorylated and its migration in a sucrose gradient is consistent with either additional subunits (X) or a dimerization of the complex. A weak interaction between xSurvivin and xAurora may also exist. In mitosis, all three proteins are both present and phosphorylated, but the complex is smaller. Both the mitotic phosphorylation of the complex and the presence of the xSurvivin subunit are required for high kinase activity (On). (C) We present a highly speculative model in which the interaction between xSurvivin and xAurora is regulated within mitosis to control kinase activity. We propose that during mitosis, xAurora B kinase activity is inactivated by disrupting the interaction of xAurora B with xSurvivin (Off), perhaps through a mechanical force. It is also possible that Survivin binding to the complex could be spatially controlled. Such a mechanism could permit spatial regulation within parts of the same mitotic cell, allowing for pools of active Aurora kinase with Survivin-bound (On) versus inactive Aurora kinase with no Survivin interaction (Off). It has been reported that Survivin and Aurora B interact directly in a two-hybrid assay and that in an in vitro pull-down assay, the complex is stable in treatments as harsh as 3 M NaCl . Perhaps high concentrations of interacting proteins in the two-hybrid and pull-down systems uncovered the weak interaction between N terminus of Aurora B and Survivin. Alternatively, the difference between the two results may be a problem with Aurora B folding in overexpression systems. We have found that soluble recombinant Aurora B can be purified from E. coli; however, protein from these preparations migrates in the void fractions of subsequent gel filtration columns (unpublished data, W. Lan, P.T. Stukenberg). We feel that it is likely that recombinant Aurora B purified from E. coli is at least partially denatured and therefore the high NaCl sensitivity in the pull-down assay may be an artifact of an in vitro experiment. Because of this problem with recombinant protein, we have been forced to design all activity and interaction experiments with endogenous xAurora B. For further biochemical examination of the passenger protein complex, it will be necessary to develop overexpression systems that can generate the complex in its native folded state. Although a physical interaction between xAurora B, xINCENP, and xSurvivin is detected during both interphase and mitosis, the hydrodynamic properties of the complex change dramatically during the cell cycle. Specifically, the peak off a gel filtration column is much broader in interphase than in mitosis, but peaks in the same fractions (migrating at 107 A). Also, our sucrose density gradient data indicate that the interphase complex sediments significantly faster than the mitotic complex (9.5S and 4.5S, respectively). The simplest model to explain such hydrodynamic characteristics is that the active complex in mitosis is a highly elongated molecule that contains at least one molecule of xINCENP, xSurvivin, and xAurora B; in interphase, two of these complexes dimerize to double the molecular weight (Figure B). It is equally possible that there are unknown subunits that are bound in interphase. There is a 2:1 M ratio of xAurora B:xSurvivin in extracts, and all of the xSurvivin protein is complexed with xAurora B. This finding was surprising because Survivin has been shown to have two functions: one as a mitotic spindle regulator and another as a chromosome passenger protein required for chromosome segregation and cytokinesis. Because Aurora B kinase has not been localized to the metaphase mitotic spindle, we expected xSurvivin to exist in at least two complexes. It is likely that the mitotic spindle requirement of Survivin is not required in the early embryo, but becomes essential during the mitosis of somatic cells. Interestingly, the metaphase spindle/centrosome localization of Survivin has been implicated to regulate apoptosis, a process that is not present in the Xenopus early embryo until after the midblastula transition (12th division). xAurora B Kinase Activity Is Regulated by xSurvivin Binding and Phosphorylation | Mitotic kinases are highly regulated to ensure that their activity is restricted to the proper time and location of the cell cycle. For example, both the activity and localization of CDK1 kinase is regulated by binding of a cyclin subunit . CDK1 kinase activity is also both positively and negatively regulated by phosphorylation . Our goal in beginning this study was to describe in molecular detail the mechanisms of Aurora B kinase activation in mitosis. To this end, we have developed a robust IP kinase assay in Xenopus extracts to measure xAurora B kinase activity and found that the kinase is stimulated >10-fold in mitosis. Using this assay, we have made two significant contributions to our understanding of xAurora B kinase regulation. First, we have demonstrated that xAurora B kinase activity is stimulated by xSurvivin binding. Second, we have confirmed that the phosphorylation state of the mitotic complex is critical for the stimulation of kinase activity (Figure B; ). Survivin binding could increase Aurora kinase activity by two mechanisms. It could either change the substrate recognition of xAurora kinase or it could increase the rate of catalysis. Our data cannot distinguish between these two models. During the preparation of this manuscript, Chan and colleagues published that the in vitro activity of the budding yeast Aurora (Ipl1p) is stimulated 10- to 20-fold by direct binding of the INCENP homolog (Sli15p). Thus, it is either possible that Aurora B activity requires binding by both INCENP and Survivin or that Sli15p has acquired Survivin's activation role. We have not detected stimulation of Aurora kinase activity by the addition of the C-terminal fragment of xINCENP to our Aurora B IPs. However, this is not surprising as xINCENP, unlike Survivin, is not washed off in the salt washes. Unfortunately, recombinant xINCENP and xAurora B from both E. coli and baculovirus appear highly misfolded after purification, making direct activation experiments in the Xenopus system impossible at this time. We have also shown that activation of the xAurora B kinase depends upon mitotic phosphorylation. This corroborates a recent study of Xenopus Aurora B kinase and is consistent with previous work that suggests that Aurora A kinase is regulated by phosphorylation . Currently, it is unknown if the activating mitotic phosphorylation is on the xAurora B kinase or an interacting protein. Recent data from our laboratory indicate that all three proteins are phosphorylated in mitosis, and we have previously demonstrated that the xINCENP protein is specifically hyperphosphorylated in mitosis . We have extended this knowledge by mapping 26 cell cycle-specific phosphorylation sites on Xenopus INCENP (F. White, D. Hunt, W. Lan, and P.T. Stukenberg, unpublished data). Survivin has been shown previously to be a substrate of CDK1 in mitosis , and if phosphatase inhibitors are used, we often see multiple bands in Survivin blots consistent with heterogeneous phosphorylation . We have mapped seven cell cycle-specific phosphorylation sites on the xAurora B protein that are currently being characterized (F. White, D. Hunt, W. Lan, and P.T. Stukenberg, unpublished data). Therefore, the phosphorylation-dependent stimulation of Aurora B activity is likely to be very complex. Why does Survivin binding regulate kinase activity? The simplest model is that the kinase is not active until the entire complex is properly assembled. The most provocative interpretation of our data is a highly speculative model that we propose in Figure C. In this model, the interaction between Survivin and Aurora is regulated within mitosis to control kinase activity. One way to regulate the interaction of Survivin with Aurora could be through a mechanical force, which could separate Aurora and Survivin in the same complex, or Survivin could simply dissociate from INCENP. Such a mechanism could permit spatial regulation within parts of the same mitotic cell, allowing for pools of active Aurora kinase with Survivin bound versus inactive Aurora kinase with no Survivin interaction. However, we have yet to find conditions where xSurvivin binding to xAurora B is used as a regulatory mechanism in the Xenopus early embryo. Similar amounts of xSurvivin are bound to xAurora B in both interphase and mitotic extracts . We have also immunoblotted Xenopus embryos traversing the early embryonic cell cycles and we do not detect a gross difference of xSurvivin levels (M. Bolton, P.T. Stukenberg, unpublished data). However, the fact that xSurvivin is stable in interphase is probably a unique feature of embryonic systems, as the protein levels peak during mitosis in somatic cells (; Figure ). PMID- 12221117 TI - ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization AB - | Golgi-localized gamma-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only similar25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs and VPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function. PMID- 12221117_ TI - Glossary AB - Abbreviations used: : ARF = ADP-ribosylation factor Gal4AD = Gal4 activation domain Gal4BD = Gal4 binding domain GAT = GGA and Tom1 GFP = green fluorescent protein Golgi-localized = GGA, Golgi-localized, gamma-ear homology domain, ARF-binding protein GST = glutathione S-transferase M6PR = mannose 6-phosphate receptor PVC = prevacuolar compartment RFP = red fluorescent protein TGN = trans-Golgi network VHS = domain present in Vps27p, HRS, and STAM proteins VPS = vacuolar protein sorting PMID- 12221117_INTRODUCTION TI - AB - The Golgi-localized, gamma-ear homology domain, ARF-binding protein (GGA) family of proteins facilitates the sorting and transport of proteins out of the trans-Golgi network (for review, see ). GGA proteins interact directly with ADP-ribosylation factor (ARF) proteins, a family of 21-kDa GTP-binding proteins that have been implicated as regulators of membrane traffic at many steps in the secretory, endocytic, and recycling pathways (,; ; ; ). GGA proteins interact with the activated, GTP-bound form of ARF, establishing them as effectors of ARF signaling . Three GGA genes have been described in mammalian cells: GGA1, GGA2, and GGA3. Alternative splicing of GGA3 results in a long form and a short form of the GGA3 protein. The yeast Saccharomyces cerevisiae has two GGA genes, GGA1 and GGA2. The expressed yeast proteins, Gga1p and Gga2p, share 50% amino acid identity. The human and yeast GGA proteins are similar25% identical. Although the sequence identity is low, the domain structure of GGA proteins is conserved between species. Four domains are apparent when the amino acid sequences of yeast and human GGA proteins are aligned (see Figure A). An N-terminal VHS domain (150 residues) resembles the VHS domain present in Vps27p, HRS, and STAM proteins . The 170-residue GAT domain is the most highly conserved (65% identity between human GGA proteins) and contains two predicted coiled-coil regions. The GAT domain was so named for its sequence homology to a protein named Tom1 (GGA and Tom1). A "hinge" region of variable length contains one or more clathrin-binding domains but no other significant homology to each other or other known proteins. A C-terminal ear domain (120 amino acids; 40% identity between human GGA proteins) is homologous to the ear domain of gamma-adaptin. Figure 1 | ARF-binding region of GGA proteins and effects of mutations. ARF-binding region of GGA proteins and effects of mutations. (A) Comparison of ARF-binding domains from yeast and human GGA proteins. GGA proteins have four domains, with the highest identity between yeast and human GGA proteins in the ARF-binding domain. Residues identical in at least three proteins are highlighted in red. Dashes indicate gaps in the alignment. The numbers indicate the first and last residues shown for each GGA protein. Asterisks indicate mutated residues. (B) Loss of ARF binding by two-hybrid analysis. Gal4AD fusions of wild-type and mutant GGA proteins were assayed for binding to Gal4BD fusions of activated ARF in the two-hybrid assay. Serial 10-fold dilutions of each strain were replica plated on SD plates lacking tryptophan and leucine (-trp-leu) and SD plates lacking histidine, tryptophan, and leucine with 25 mM 3AT. Growth on -trp-leu shows that all strains are viable. Interaction is positive when colonies grow on 3AT and turn blue in X-gal assays. Scores in the right-hand column are defined in MATERIALS AND METHODS. (C) Loss of Arf binding by using affinity chromatography. Increasing concentrations of Arf1pGDP or Arf1pGTPgammaS (as indicated) were incubated with GST fusion proteins immobilized on glutathione agarose beads: GST-Gga2p (lanes 1 --6), GST-Gga2pI207N (lanes 7 --12), and GST only (lanes 13 --14). Retained proteins were eluted and analyzed by Coomassie staining to visualize the GST fusion proteins (bottom) or by immunoblotting to detect Arf1p (top). Arf1p was retained by GST-Gga2p, but not by mutant GST-Gga2pI207N or GST only. Work in both mammalian cells and S. cerevisiae suggests that GGA proteins function in trafficking at the TGN. In yeast, deletion of either gene alone causes no (GGA1 deletion) or minor (GGA2 deletion) defects, whereas deletion of both genes disrupts distinct post-Golgi trafficking events . Several proteins that are transported between the TGN and early or late endosomes, including carboxypeptidase Y (CPY), carboxy peptidase S, Pep12p, and Kex2p, are at least partially missorted in cells lacking both GGA1 and GGA2. For example, similar40% of newly synthesized CPY is secreted in gga1gga2 cells . These data support a role for Gga proteins in a TGN-to-early endosome pathway, in a TGN-to-late endosome pathway, or both. The function of mammalian GGA proteins has been deduced based on the protein interactions of each domain. Mammalian GGA proteins interact directly with a defined subset of cargo that trafficks between the TGN and lysosomes. Sortilin, LRP3, and both the cation-independent and cation-dependent forms of mannose 6-phosphate receptor (M6PR) contain an acidic cluster-dileucine motif that interacts with the VHS domain of human GGA proteins . Many other transmembrane proteins that lack this motif do not interact with GGA proteins, suggesting that GGAs are specific for a subset of transported proteins. It is not known whether a similar motif (acidic cluster-dileucine) is recognized by yeast Gga proteins, or even whether cargo is sorted via direct interaction with Gga proteins in yeast. The hinge domain of mammalian and yeast GGA proteins interacts directly with clathrin in vitro. The ear domain strengthens this interaction, and the ear domains of certain GGA proteins also interact with clathrin on their own . These findings suggest that GGA proteins can recruit clathrin to sites of vesicle budding on the TGN. Overexpression in cultured cells of a GGA3 construct lacking the hinge and ear domains causes M6PR to accumulate in the TGN, supporting a role for GGA-dependent clathrin recruitment in the packaging of these cargo into vesicles . Thus, it is likely that both yeast and mammalian GGA proteins are monomeric clathrin adaptors that function at the TGN. The ear domain of human GGA proteins interacts with gamma-synergin , a Golgi-localized protein partner of gamma-adaptin that has an unknown function . Thus, the function of the GGA ear was accurately predicted by its homology to gamma-adaptin. However, gamma-synergin has no obvious homolog in yeast, and the ear domains of yeast and human GGA proteins are not well conserved; hence, the role of the ear domain in yeast remains unclear. In mammalian cells, endogenous GGA1, GGA2, and GGA3 localize predominantly to the trans-Golgi region . Three lines of evidence show that TGN localization of GGAs requires interaction between the GAT domain and activated ARF proteins. First, treatment with brefeldin A causes rapid translocation of GGA proteins to the cytosol in a time frame indistinguishable from that of ARF itself . Second, the interaction between the GAT domain and ARF is strong enough to direct a green fluorescent protein (GFP) fusion of GAT onto the Golgi, even in the absence of the VHS, hinge, and ear domains . Third, and most convincingly, point mutations within the ARF-binding domain that abolish ARF interaction also cause loss of Golgi localization . Together, these data suggest that GTP-bound ARF recruits mammalian GGA proteins from the cytosol onto the late Golgi membrane by interacting with the GAT domain. The function of GGA proteins in mammalian cells is also intimately tied to activated ARF. First, expression of the constitutively active ARF1Q71L in mammalian cells causes dramatic expansion of the Golgi apparatus . Overexpression of GGA1 or GGA3 in these same cells prevents Golgi expansion . This alteration of ARF1 function indicates a functional interaction between ARF1 and GGAs in vivo. Second, as described above, mutations within the GAT domain of GGA3 disrupt its Golgi localization. These same mutations prevent GGA3 overexpression from displacing adaptor complexes or fragmenting the Golgi apparatus , which usually results from GGA overexpression in mammalian cells. Although these mutations in GGA3 might disrupt other interactions, current evidence supports the model that loss of ARF interaction disrupts GGA function in mammalian cells. Together, these data suggest that GGA proteins are ARF-dependent, monomeric clathrin adaptors that facilitate the sorting of specific cargo at the TGN into vesicles destined for endosomes. Much of the data comparing yeast and mammalian GGA function assumes that the mechanisms of GGA function are well conserved between these organisms. However, the mechanism of yeast GGA function has not been analyzed. In particular, it is not known whether Gga function or localization in yeast depends on its binding to Arf (via the GAT domain) or other proteins (via the VHS and/or hinge and ear domains). In this article, we show that yeast Gga1p and Gga2p do not require Arf for their localization or function, although in vivo binding to Arf is detectable. We propose that yeast Ggas are targeted to the Golgi membrane through interactions with Golgi-localized proteins other than Arf, which then stabilize the Gga --Arf interaction. PMID- 12221117_MATERIALS AND METHODS TI - AB - Materials | Unless otherwise specified, chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Fair Lawn, NJ). Yeast and bacterial media reagents were purchased from Difco (Detroit, MI). Restriction and other enzymes were purchased from Promega (Madison, WI) or Invitrogen (Carlsbad, CA). TA cloning vector pGEMT-easy was purchased from Promega. Unique Site Elimination Mutagenesis kit was purchased from (Palo Alto, CA). Yeast Methods | Yeast were grown under standard conditions at 30C unless otherwise noted. Transformations into yeast were performed using the LiOAc method, with herring sperm DNA as a carrier . Yeast Strains | Yeast strains used in this study are listed in Table . Diploid strains YAB538 and YAB539 were generated by mating YAB531 (gga1::TRP1) and YAB532 (gga2::HIS3) . Tetrad dissections generated several gga1gga2 strains used for trafficking assays and generation of triple knockout strains (YAB538 T6a, YAB538 T2d, YAB539 T3d; ). Strain YAB650 (gga1gga2apm1) was generated by mating and tetrad dissections of YAB539 T3d and Piper1878. Strain YAB658 (gga1gga2apm3) was generated by mating and tetrad dissections of YAB538 T2d and Piper1022. Strain YAB667 (gga1gga2vps1) was generated by transforming plasmid pCKR3A, digested with SacI and XbaI, into strain YAB538 T6a and selecting for LEU auxotrophy. Strains YAB679 and YAB680 (gga1gga2vps27 in SEY6210 background) were generated by mating and tetrad dissections of ABC114 T6b and vps27 strain JUY68. Strain YAB677 (gga1gga2vps27 in S288C background) was generated by transforming plasmid pKJH2, digested with BamHI and PstI, into strain YAB538 T6a and selecting for LEU auxotrophy. Gene deletions were confirmed by Western blotting for Gga1p and Gga2p, and by phenotype for VPS27 (class E compartment), APM3 (ALP processing), and VPS1 (CPY secretion). Multiple isolates of gga1gga2apm1 were analyzed. All other strains were generated by transformation. Plasmid Construction | Plasmids used in this study are listed in Table . Oligonucleotide primers used for polymerase chain reaction (PCR) amplification are listed in Table . All constructs generated by PCR amplification were verified by automated sequencing (Advanced Genetic Analysis Center, University of Minnesota). Table 1 | Plasmids used in this study Two-Hybrid Plasmids. | Gal4 binding domain (Gal4BD) fusions of yeast and human ARFs were generated from plasmid pBG4D (a kind gift from Rob Brazas, University of California, San Francisco, CA), as described previously . Gal4 activation domain (Gal4AD) fusions of yeast and human GGA proteins were generated from plasmid pACT2 (a kind gift from Steve Elledge, Baylor University, Waco, TX), as described previously . A single point mutation within the Arf-binding domain of yeast Gga1p was fortuitously identified after PCR amplification of the GGA1 VHS/GAT domain and subcloning into the two-hybrid vector pACT2, resulting in plasmid pAB362. This single-base substitution changed leucine at position 203 to a glutamine (L203Q). Homologous mutations in yeast Gga2p (I207N), human GGA1 (L182Q), and human GGA3 (L149Q) were generated by two-step PCR mutagenesis with oligos specified in Table . The PCR products were generated with BamHI and XhoI sites at the 5' and 3' ends of the open reading frames, respectively, and subcloned into vector pACT2 to generate Gal4AD fusion proteins of mutant Gga2p (pAB462), GGA1 (pAB418), and GGA3 (pAB503). Alanine substitutions in Gga2p (PEDL->AAAL and ANKL->AAAL) were generated using Unique Site Elimination mutagenesis in plasmid pAB473 (see below). The open reading frames were then subcloned into pACT2 by using NcoI and XhoI, resulting in plasmids pAB580 and pAB581. Yeast Expression Vectors. | Plasmids for expression of Gga1p (pAB470) and Gga2p pAB441) were described in . An NcoI site was generated at the start ATG of GGA2 in pAB441 by using two-step PCR to generate plasmid pAB471. The GGA2(NcoI) gene was then subcloned into vector pRS315 by using BamHI and XhoI to generate pAB473. Unique site elimination mutagenesis of pAB473 was used to generate PEDL->AAAL (pAB493) and ANKL->AAAL (pAB509). The mutated BamHI-XhoI fragments of pAB493 and pAB509 were then subcloned into pRS316 to generate pAB529 and pAB530, respectively. The mutated NcoI-XhoI fragments of pAB493 and pAB509 were also subcloned into pACT2, as described above. For expression of Gga1pL203Q, the mutated region from pAB362 was subcloned into pAB470, resulting in pAB469. For expression of Gga2pI207N, two-step PCR mutagenesis was used to generate the I207N mutation from plasmid pAB441. The PCR product was then subcloned into pRS316, resulting in pAB456. The amino terminal truncation of Gga2p(110 --585) was generated by PCR (oligos A053 and A038) followed by subcloning the NcoI-XhoI --digested products into pAB473. Then the NotI-XhoI cassette was subcloned into pRS316 to generate pAB555. The carboxy-terminal truncation of Gga2p(1 --326) was generated by PCR (oligos A037 and A049) and subcloned with BamHI and XhoI into pRS316, resulting in pAB468. Hemagglutinin (HA)-tagged GGA Proteins. | Unique site elimination was used to generate an NheI site immediately preceding the stop codon of GGA1 and GGA2, resulting in plasmids pAB487 and pAB483. The XbaI fragment from pMR2654 (a kind gift from Mark Rose, Princeton University, Princeton, NJ) containing a triple HA repeat was inserted into the NheI-digested plasmids, resulting in plasmids pAB492 and pAB491. Orientation of the HA tag was determined by sequencing. Mutant alleles were generated by subcloning the mutated portion of the open reading frames into the HA-tagged plasmids, resulting in plasmids pAB579 (GGA1) and pAB578 (GGA2). Glutathione S-Transferase (GST) Fusion Proteins. | All constructs were subcloned from pACT2 (described above) into pGEX5X-2 (Stratagene, La Jolla, CA) by using BamHI and XhoI sites. The following constructs were used for these studies: GST-Gga1p1 --331 (pAB391), GST-Gga1p1 --331, L203Q (pAB476), GST-Gga2p1 --326 (pAB382), and GST-Gga2p1 --326, I207N (pAB472). GFP Fusion Proteins. | Plasmids for expression of GFP-Gga1p (pCS135) and GFP-Gga2p (pCS136) were obtained from Chris Stefan and Scott Emr (University of California, San Diego, CA). Both are pRS426-based plasmids with the CPY promoter driving expression of the GFP open reading frame. A multiple cloning site follows GFP. Plasmid pAB505 for expression of GFP-Gga1pL203Q was generated by subcloning the KpnI-KpnI fragment of pAB469 into pCS135. Plasmid pAB504 for expression of GFP-Gga2pI207N was generated by PCR amplification of the open reading frame from pAB456 to incorporate an SalI site at the 5' end then subcloning the SalI-KpnI fragment into pCS136. The truncated forms of GFP-Gga1p were all generated by PCR amplification from either pCS135 or pAB505 then subcloned into pCS135 by using SalI-KpnI or KpnI-KpnI sites. Mammalian Expression Vectors. | Plasmids for expression of human GGA proteins in normal rat kidney (NRK) cells were generated from pcDNA3 as described in . GGA1L182Q was generated by two-step PCR mutagenesis with mutant oligos A025 and A026 and flanking oligos 797 and A028 then subcloned into pcDNA3-HA with EcoRI and XbaI, resulting in pAB443. GGA3L149Q was generated by subcloning the mutated region of GGA3 (HindIII-XhoI) from pAB503 into vector pAB366 (pcDNA3-HA-GGA3; ), resulting in pAB506. Truncated GGA1DeltaVHS (pAB318) begins at residue 145, as described previously . GGA1DeltaVHS,L182Q (pAB582) was generated by subcloning the BamHI-XhoI fragment of pAB418 into pcDNA3. An ATG immediately following the BamHI site acts as the start ATG. Two-Hybrid Assays | Filter assays for beta-galactosidase activity were performed as described previously with 1 mg/ml X-gal as the substrate. Colony color was scored every hour for 3 h then allowed to develop overnight. Strong positives (+++) turned blue within 1 h, moderate positives (++) turned blue within 3 h, weak positives (+) turned blue overnight, and negatives (-) remained white throughout the development period. Assays for histidine auxotrophy were performed by replica plating yeast strains on SD-trp-leu plates and 3AT plates (SD plates lacking tryptophan, leucine, and histidine, and containing 25 mM 3-amino triazole) and scoring growth after 3 --4 d. GST-Affinity Chromatography | Bacterially expressed Arf1pQ71L was purified as described previously . Bacterially expressed GST fusion proteins were purified on glutathione agarose beads as described previously . The beads were washed three times with TND (25 mM Tris pH 7.5, 100 mM NaCl, and 1 mM dithiothreitol) and once with binding buffer (20 mM HEPES pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.5 mM MgCl2, 1 mM dithiothreitol, 50 mug/ml bovine serum albumin, and 1% Triton); 30 mul of bead volume was used for each binding reaction. Bacterially expressed yeast Arf1pQ71L (6 muM) was incubated for 30 min at 30C in binding buffer with 10 muM guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) or GDP. GST fusions were mixed with 100 mul of 0.6, 1.8, or 6 muM Arf1p for 30 min. The beads were then washed three times with 1 ml of binding buffer containing 10 muM nucleotide. Bound proteins were eluted from the beads with an equal volume of 2x Laemmli sample buffer and run on a 12% SDS-PAGE gel. The final wash was also analyzed to ensure complete washing. Duplicate gels were either stained with Coomassie brilliant blue or developed by immunoblotting, by using a polyclonal rabbit antiserum (R23; ) to detect yeast Arf1p. Antibodies and Immunoblotting | Immunoblotting was performed as described previously . Antibodies were used at the following dilutions, as indicated for each experiment: 12CA5 (to HA epitope; BabCo, Berkeley, CA), 1:10,000; R89675 (to Gga1p), 1:10,000; R89013 (to Gga2p), 1:10,000; R23 (to Arf1p, a gift from R. Kahn, Emory University, Atlanta, GA), 1:2000; 10A5-B5 (to yeast CPY; Molecular Probes, Eugene, OR), 1:5000; and anti-ALP (a gift from S. Nothwehr, University of Missouri, Columbia, MO), 1:5000. Bound antibodies were detected with horseradish peroxidase-linked secondary antibodies and enhanced chemiluminescence detection reagents (Amersham Biosciences, Piscataway, NJ). Films were scanned using a flatbed scanner and figures were prepared using Adobe PhotoShop 5.0 (Adobe Systems, Mountain View, CA). Affinity Purification of Antibodies to Gga1p and Gga2p. | For Gga1p, antibodies were adsorbed onto a nitrocellulose strip to which GST-Gga1p was transferred. After washing, bound antibodies were stripped with 0.2 M glycine, pH 2.8. For Gga2p, antibodies were mixed with GST-Gga2p bound to glutathione agarose, washed, and eluted with 0.2 M glycine. The efficacy of the affinity purification was analyzed by immunoblotting (our unpublished data). Yeast Fractionation | Yeast cells (0.15 g) were suspended in 100 mul of fractionation buffer (0.4 M sucrose, 25 mM KPO4 pH 7.0, and 2 mM EDTA) and 5 mul of protease inhibitor cocktail for yeast (Sigma-Aldrich). Cells were lysed by vortexing with glass beads for 2x 3 min, on ice between. Mixture was centrifuged for 5 min at 3000 x g to remove nuclei and unbroken cells; the supernatant from this step is the lysate. The lysate was centrifuged at 100,000 x g for 30 min at 4C to generate supernatant (S100) and pellet (P100) fractions. Equal fractions of lysate, S100, and P100 were loaded on 12% acrylamide gels and immunoblotted as described above. Secretion Assays | CPY Pulse-Chase Assay. | Processing and sorting of CPY was assayed as described previously by using anti-CPY antisera (provided by Tom Stevens, University of Oregon, Eugene, OR; or Elizabeth Jones, Carnegie Mellon University, Pittsburgh, PA). Films were scanned on a flatbed scanner and processed using Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA). CPY Colony Immunoblotting. | Serial dilutions of yeast cultures were replica plated on YPD plates and incubated at 30C for 12 --24 h. Nitrocellulose filters (0.45-mum BA85; Schleicher & Schuell, Keene, NH) were overlaid for an additional 12 h at 30C. Cells were thoroughly rinsed from the filters with distilled H2O. Immunoblotting with CPY monoclonal antibodies (1:1500; Molecular Probes) or Arf1p and alkaline phosphatase polyclonal antibodies (to control for cell lysis) was performed as described above. Staining with FM 4-64. | Cultures were grown in YPD to an optical density of 0.5 --0.8. FM 4-64 (10 mM stock in dimethyl sulfoxide; Molecular Probes) was added to 500 nM; cells were incubated at 30C for 30 min. Cells were washed with water, resuspended in YPD lacking FM 4-64, and incubated an additional 30 min at 30C. Cells were concentrated 10-fold by brief centrifugation, mounted with AntiFade (Molecular Probes), and observed immediately. To assay for temperature sensitivity, strain YAB679 was shifted to 37C for 2 h before labeling. Labeling with FM 4-64 and chase incubations were performed at 37C. Cells were observed and photographed on a E600 microscope (Nikon, Melville, NY) equipped with a Spot II digital camera and MetaMorph 3.6 software (Universal Imaging, Downingtown, PA). Images were processed using Adobe Photoshop 5.0. Indirect Immunofluorescence Microscopy | pcDNA3-based plasmids for expression of GGA1, GGA1L182Q, GGA3, and GGA3L149Q with an HA epitope tag at the amino terminus were transiently transfected into NRK cells by using FuGENE 6 (Roche Applied Science, Indianapolis, IN). The cells were analyzed by indirect immunofluorescence, as described previously by using antibodies to GGA1 (R79709), the HA epitope (12CA5; BabCo), or mannosidase II (53FC3; Covance Research Products, Richmond, CA). GFP Fluorescence Microscopy | For analysis of GFP-tagged proteins by fluorescence microscopy, mid-log cultures were diluted with 0.5 volumes of GFP KILL buffer (1 M Tris pH 8.0 and 5% sodium azide; ). Samples were mixed with an equal volume of AntiFade (Molecular Probes) and observed within 1 h. Cells were photographed as described for FM 4-64 staining. PMID- 12221117_RESULTS TI - AB - Mutations in GAT Domain Eliminate Interaction with ARF | The Arf-binding domain of human and yeast GGA proteins was previously mapped by truncation analysis to a highly conserved region within the GAT domain . Several residues within this domain are conserved in all known GGA proteins (Figure A, highlighted residues). To disrupt binding to ARF without disrupting overall protein folding, selected point mutations within this region were generated and tested for binding to ARF by using the two-hybrid assay. To compare between species, homologous mutations were generated in both human and yeast GGA proteins. We changed leucine at position 203 of Gga1p to glutamine (Gga1pL203Q), isoleucine 207 of Gga2p to asparagine (Gga2pI207N), leucine 182 of GGA1 to glutamine (GGA1L182Q), and leucine 149 of GGA3 short form to glutamine (GGA3L149Q). Additional mutations in the GAT domain of Gga2p were also generated: residues 212 --214 were changed to alanines (referred to as Gga2pPEDL -> AAAL) and residues 219 and 220 were changed to alanines (referred to as Gga2pANKL -> AAAL). None of these mutations affect protein expression or stability, because similar levels of protein were detected by Western blotting in yeast strains that expressed wild-type or mutant forms of each protein as either Gal4AD fusions or untagged constructs (our unpublished data). These mutations are not predicted to disrupt the coiled-coils in the GAT domain, determined using the COILS program . Wild-type and mutant human GGA1 and GGA3 as Gal4AD fusions were tested for binding to ARF3Q71L as a C-terminal Gal4BD fusion. As described previously , wild-type GGA1 and GGA3 interact strongly with ARF3Q71L, shown herein by growth on medium lacking histidine and supplemented with 3-amino triazole (3AT; Figure B) and by blue color development in the beta-galactosidase assay (X-gal; Figure B). In contrast, GGA1L182Q and GGA3L149Q show no interaction by growth on 3AT plates or in the X-gal assay (Figure B). These results showed that our mutations abolished detectable binding of mammalian GGAs to ARF. We then tested wild-type and mutant yeast Gga1p and Gga2p as Gal4AD fusions for binding to Arf2pQ71L as a C-terminal Gal4BD fusion. Arf2pQ71L was used for these experiments because Arf1pQ71L is lethal in this construct . As expected , wild-type yeast Gga1p and Gga2p interact with Arf2p, shown herein by growth on 3AT plates and blue color development in the X-gal assay (Figure B). Our mutations effectively abolished this interaction. Gga1pL203Q and Gga2pI207N showed no detectable binding to Arf2p; strains grew much slower on 3AT plates and remained white in the X-gal assay, even after extended times. The overall folding of these mutant proteins was unperturbed, because these mutants remained active for binding to a novel Gga-binding protein (our unpublished data). To confirm that the loss of signal in our two-hybrid assays was due to loss of affinity for Arf, we analyzed the interaction between Arf1p and Gga2p or Gga2pI207N by affinity chromatography (Figure C). GST alone was used as a negative control. Bacterially expressed Arf1p was incubated with GTPgammaS or GDP then mixed with purified GST fusion proteins on glutathione agarose beads. Retained Arf1p was detected by immunoblotting (top). Equal amounts of GST-Gga2p, GST-Gga2pI207N, or GST were present in each reaction, as shown by Coomassie staining (bottom). Arf1pGTPgammaS, but not Arf1pGDP, bound to GST-Gga2p; the amount of Arf1p retained by Gga2p increased linearly with the concentration of Arf1p in the binding reaction. Identical experiments were performed with GST-Gga1p, but no binding was detected at these concentrations (our unpublished data). This suggests a lower affinity of Arf1 for Gga1p than for Gga2p. Because of the unique nucleotide-binding properties of Arf proteins , we do not know the actual concentration of GTP-bound Arf1p in these samples and we cannot determine a Kd value for the binding of Arf1p. However, we can detect as little as 3 ng of Arf1p by immunoblotting, which is 10-fold less than present in lane 4 (GST-Gga2p, 0.6 muM Arf1pGTPgammaS) and 100-fold less than lane 6 (GST-Gga2p, 6 muM Arf1pGTPgammaS) as determined by densitometry. Strikingly, we could not detect any binding of Arf1pGTPgammaS to GST-Gga2pI207N or GST alone, even with 6 muM Arf1p. This suggests that the I207N mutation reduced the binding to Arf1p by at least 99% in vitro. Based on the two-hybrid and in vitro binding experiments, we concluded that these conserved residues in the GAT domain are critical for interaction with GTP-bound ARF proteins. ARF Interaction Is Required for Localization of Mammalian GGA Proteins | We next tested the mammalian GGA mutants for localization in vivo. Plasmids encoding HA-tagged wild-type or mutant GGA1 or GGA3 were transfected into normal NRK fibroblasts and localized by indirect immunofluorescence (IIF) by using antibodies to GGA1 or the HA epitope (12CA5). The Golgi apparatus was double labeled using antibodies to mannosidase II (Figure , b, d, and f) or beta-COP (Figure h). Transfected wild-type GGA1 localized predominantly to the trans-Golgi region (Figure a), as expected. In contrast, GGA1L182Q was distributed throughout the cytosol, with no detectable Golgi staining (Figure c). The staining shown in Figure , a and c, used antibodies against GGA1 at a dilution that fails to detect endogenous protein, thus this staining detected only the transfected proteins. Similar results were seen with antibodies against the HA epitope (our unpublished data). At higher concentrations of GGA1 antibody, the endogenous GGA1 was detected at the Golgi region (our unpublished data), suggesting that the Golgi localization of endogenous GGA1 was not altered by overexpression of GGA1L182Q. Similarly, transfected wild-type GGA3 localized to the trans-Golgi region (Figure e), whereas GGA3L149Q was distributed throughout the cytosol (Figure g). Double labeling with antibodies to ARF (1D9; our unpublished data), mannosidase II (Figure d), or beta-COP (Figure h) showed that ARF localization and Golgi structure were unaffected by these mutant GGA proteins. These results suggest that GGA1L182Q and GGA3L149Q fail to interact with ARF in vivo as well as in vitro, and that interaction with ARF is required for normal GGA localization to the Golgi membrane in mammalian cells. Figure 2 | Localization of wild-type and mutant human GGA proteins in NRK fibroblasts. Localization of wild-type and mutant human GGA proteins in NRK fibroblasts. Cells were double labeled by indirect immunofluorescence to visualize transfected GGA proteins (left) and Golgi markers mannosidase II or beta-COP (right). Wild-type GGA1 and GGA3 are localized to the Golgi region, whereas mutants GGA1L182Q and GGA3L149Q localized throughout the cytosol. These data are consistent with a recent report that different mutations in the GAT domain of GGA3 disrupt ARF interaction and Golgi localization. Also consistent with previous work, we found that removal of the VHS or ear domains does not alter GGA localization in NRK cells (Figure ; and our unpublished data), suggesting an exclusive role for the GAT/Arf-binding domain in Golgi localization of mammalian GGA proteins. Our new results identify a distinct residue (L182 in GGA1, L149 in GGA3) in the GAT domain that is essential for interaction with ARF in vivo. Mutant Yeast GGA Proteins Retain Golgi Localization | Based on the above-mentioned results for mutant human GGAs and the high conservation of the ARF-binding domain between human and yeast orthologs, we expected the homologous mutations in yeast Gga1p and Gga2p to disrupt their localization and function. Surprisingly, this was not the case. To investigate whether Ggas and Arf interact in vivo, we analyzed the localization of wild-type and mutant Gga proteins by fluorescence microscopy and subcellular fractionation. Antibodies specific to each Gga protein were initially used for indirect immunofluorescence of yeast cells. Affinity-purified antibodies to Gga1p and Gga2p showed faint but distinct puncta throughout the yeast cytoplasm, consistent with Golgi staining (Figure , a and c). Yeast strains deleted of both GGA genes failed to stain with either antibody, indicating specificity (Figure , b and d). Endogenous Gga1p and Gga2p did not colocalize with an HA-tagged form of the early Golgi protein Och1p (; our unpublished data). Technical difficulties have hampered our attempts to colocalize Ggas with late Golgi markers. Because our detection of endogenous proteins was weak, we next localized GFP-tagged Gga proteins. GFP-Gga1p and GFP-Gga2p fully complement the trafficking defects of gga1gga2 cells (Stefan, personal communication; Figure ). These GFP-constructs were each expressed in wild-type strain BY4735. Western blots with antibodies to Gga1p or Gga2p indicated that the GFP-Ggas are expressed at significantly higher levels than endogenous Gga1p and Gga2p (our unpublished data). When analyzed by fluorescence microscopy, we detected a distinct punctate fluorescence pattern for GFP-Gga1p (Figure e) that was very similar to, but significantly brighter than, the endogenous Gga1p signal. GFP-Gga2p gave similar staining, plus high levels of cytosolic staining not seen with endogenous Gga2p (our unpublished data). We therefore used GFP-Gga1p for the studies described below. GFP-Gga1p did not colocalize with a red fluorescent protein (RFP)-tagged form of the early Golgi protein Gyp1p (; our unpublished data), consistent with our IIF results. In vps27Delta cells, which accumulate TGN, endosomal, and vacuolar components in the class E compartment, GFP-Gga1p but not RFP-Gyp1p localizes to this compartment (Figure , m and p). These data suggest that Gga proteins are localized to the late Golgi, consistent with their function and with a recent report using HA-tagged Gga1 in a class E mutant strain . Surprisingly, the L203Q mutation in GFP-Gga1p did not alter the staining pattern (Figure f), suggesting that the Golgi localization of Gga1p did not require Arf interaction. Identical results were observed with GFP-Gga2pI207N (our unpublished data). These data were in stark contrast to their mammalian counterparts. Figure 3 | Localization of Gga1p and Gga2p in yeast. Localization of Gga1p and Gga2p in yeast. Top and middle, indirect immunofluorescence of Gga1p or Gga2p in wild-type strain BY4704 (a and c) or gga1gga2 strain YAB538 T6a (b and d). Bottom, GFP fluorescence of GFP-Gga1p or mutant GFP-Gga1pL203Q in wild-type strain BY4735. ARF proteins (except ARF6) cycle on and off their target membranes in concert with GTP binding and hydrolysis; GTP-bound ARF is membrane associated, and GDP-bound ARF is cytosolic. However, after cell lysis and centrifugation, the majority of yeast and mammalian ARF is found in a soluble fraction. Mammalian GGA proteins behave similarly , consistent with ARF-dependent membrane association. We analyzed yeast Ggas by subcellular fractionation to determine whether yeast Gga proteins fractionate with membranes or cytosol, and to determine whether mutations in the Arf-binding domain altered the fractionation profile. Lysates from stains expressing endogenous or HA-tagged Gga1p or Gga2p were centrifuged at 100,000 x g to generate soluble (S100) and membrane (P100) fractions, which were analyzed on immunoblots with antibodies against endogenous Gga1p or Gga2p (our unpublished data) or the HA tag (Figure A), with identical results. Duplicate gels were probed with R23 antibodies against Arf1p. As expected, Arf1p was found almost exclusively in the S100 fraction (Figure A). In contrast, yeast Gga1p and Gga2p partitioned equally between the soluble and membrane fractions (Figure A, left), strongly suggesting that yeast Gga proteins are bound to membranes by interactions with proteins other than, or in addition to, Arf. In addition, we found that the expression levels of HA-Gga1p and HA-Gga2p are nearly identical. Each is expressed from its own promoter, suggesting that endogenous Gga1p and Gga2p expression levels are also similar. This differs from a previous report stating that Gga1p is expressed at significantly lower levels than Gga2p. Figure 4 | Immunoblots of yeast lysate fractions. Immunoblots of yeast lysate fractions. (A) Fractionation of wild-type and mutant Gga proteins. Total lysates (T) from strains expressing HA-tagged Gga2p, Gga1p, Gga2pI207N, or Gga1pL203Q were centrifuged at 100,000 x g to generate supernatant (S) and pellet (P) fractions. Duplicate gels were probed with antibodies to the HA tag (top) or Arf1p (bottom). Arf1p was predominantly in the soluble fraction, whereas all Gga proteins fractionated equally between the soluble and pellet fractions. (B) Fractionation of Gga2p in the absence of Arf1p. Fractions from wild-type or arf1Delta strain TT104 were prepared as in A, and probed with antibodies to Gga2p. Gga2p was predominantly in the pellet fraction in both strains. In the same experiments, untagged (our unpublished data) or HA-tagged mutants Gga1pL203Q and Gga2pI207N also partitioned equally between the soluble and membrane fractions (Figure A, right), confirming that Arf is not the primary mediator of Golgi localization for yeast Gga proteins. In some of our fractionation experiments, the wild-type and mutant Gga proteins partitioned exclusively into the membrane fraction. We cannot yet explain why this was so, except to note that the wild-type and mutant Ggas behaved identically in any given experiment. To independently test a role for Arf in localizing Ggas to membranes, Gga2p was analyzed in yeast cells devoid of Arf1p (arf1Delta strain TT104; ). Consistent with our mutant analysis, Gga2p fractionated with membranes in the absence of Arf1p (Figure B). This strain expresses low levels of Arf2p (10% that of Arf1p; ), unlikely to account for all membrane localization of Gga2p. We conclude that Arf is not required for recruiting Gga proteins to membranes in yeast. GGA Mutants Complement CPY Sorting Defects | Yeast strains deleted of GGA1 and GGA2 have been analyzed for defects in trafficking of several proteins, including CPY . To determine whether defects in binding to Arf disrupt the trafficking functions of Gga proteins, we tested whether Gga1pL203Q or Gga2pI207N could complement the CPY sorting defect of gga1gga2 strains. The wild-type and mutant alleles of GGA1 and GGA2 were subcloned into low copy vectors under control of their endogenous promoters and transformed into gga1gga2 yeast strain YAB538 T6a . All constructs were expressed at levels similar to the endogenous Gga proteins, as determined by Western blotting (our unpublished data). Table 2 | Yeast strains used in this study Table 3 | Oligonucleotide primers used in this study As expected, the gga1gga2 strain was defective in sorting of CPY, as shown by pulse-chase analysis of newly synthesized CPY (Figure A) and colony immunoblot (Figure B). Also as expected, wild-type Gga2p fully complemented the defects in both assays (Figure , A and B). Surprisingly, the mutant Gga2pI207N also fully complemented these defects, as shown by pulse-chase analysis (Figure A) and colony immunoblotting (Figure B). We concluded that either Arf interaction is not required for Gga2p function, or these mutants can still bind Arf in vivo. Expression of Gga1p complemented the CPY-processing and -sorting defects only partially (Figure , A and B). Our first indication of a functional defect in our mutant Gga proteins was seen for Gga1pL203Q, which partially complemented the CPY processing defect in the pulse-chase assays (Figure A) but failed to complement the CPY sorting defect (Figure B). There was minor variability in the extent of partial complementation by Gga1pL203Q (compare Figure , A and B), but it is always less than wild-type Gga1p. This suggests that the sorting function of Gga1p is partially disrupted by the mutation that blocks Arf binding. Figure 5 | CPY sorting is complemented by Arf-binding domain mutants in yeast. CPY sorting is complemented by Arf-binding domain mutants in yeast. (A) Pulse-chase analysis of newly synthesized CPY in wild-type, gga1gga2, and gga1gga2 harboring low copy plasmids of Ggas. Time course is indicated in minutes. Expression of Gga2p or Gga2pI207N (right) fully complemented the CPY processing defect in gga1gga2 strains. Expression of Gga1p or Gga1pL203Q (middle) partially complemented this defect. P1, unglycosylated endoplasmic reticulum form of CPY; P2, glycosylated Golgi form; M, fully processed mature (vacuolar) form. (B) Detection of secreted CPY by colony immunoblotting. Serial dilutions of yeast cultures were replica plated onto YPD plates. A signal on colony immunoblots indicates that CPY is secreted from the cell, suggesting that CPY is missorted. The CPY sorting defect of gga1gga2 strains (DeltaDelta) was complemented by wild-type Gga2p and mutant Gga2pI207N. Wild-type Gga1p partially complemented the CPY sorting defect, whereas mutant Gga1pL203Q failed to complement. Although the homologous mutations in human GGA1 and GGA3 disrupt ARF interaction in vivo, this single mutation might not fully eliminate Arf binding in the yeast Gga2p mutants. To address this possibility, we made two clusters of alanine substitution mutations in the Arf-binding domain of Gga2p. We replaced three residues of a conserved PEDL sequence (P212A, E213A, and D214A), designated Gga2pPEDL -> AAAL, or two residues in a conserved ANKL sequence (N219A and K220A), designated Gga2pANKL -> AAAL. The latter includes the asparagine residue mutated by to disrupt ARF --GGA3 interaction. Neither mutant Gga2pPEDL -> AAAL nor Gga2pANKL -> AAAL interacted with Arf2p in the two-hybrid assay (Figure B). However, both mutants still complemented the CPY defect when assayed by either pulse chase or colony immunoblot (our unpublished data), showing that they were functional in yeast. These data suggest that reduction of Arf interaction does not affect the ability of Gga2p to sort CPY in yeast. Genetic Interaction between GGAs and VPS27 | As a complementary approach to understand the function of Gga proteins in yeast, we took advantage of the nonlethal phenotype of GGA gene deletions. Specifically, we tested for synthetic interactions between GGA deletions and genes that function in distinct post-Golgi trafficking pathways: APM1, APM3, VPS1, and VPS27. APM1 encodes the medium (mu) subunit of the adaptor protein (AP)-1 clathrin adaptor complex, which is thought to function in anterograde or retrograde traffic between the TGN and early endosome . However, we note that deletion of APM1 does not eliminate AP-1 function, because a related protein, Apm2p, can substitute in the AP-1 complex . APM3 encodes the medium (mu) subunit of the AP-3 clathrin adaptor complex, which mediates a direct TGN-to-vacuole pathway . Deletion of APM3 eliminates AP-3 function. VPS1 encodes yeast dynamin, a protein involved in the scission of clathrin-coated vesicles from donor membranes . Vps1p mediates both TGN-to-endosome and TGN-to-vacuole traffic, and deletion causes missorting of vacuolar proteins via the plasma membrane . VPS27 is a class E gene that regulates exit from the prevacuolar compartment (PVC) and entry into intralumenal vesicles . Strains were constructed with triple gene deletions and analyzed for growth defects at 30 or 37C. Deletion of either apm3 or vps1 in the gga1gga2 knockout strain showed no synthetic growth defects (Figure A, strains YAB658 and YAB667). Deletion of apm1 in the gga1gga2 knockout produced a very slight growth defect at 37C (Figure A, strain YAB650). Furthermore, these three triple-knockout strains showed no synthetic defects in trafficking of alkaline phosphatase, CPY, or Vps10p (our unpublished data). Figure 6 | (A) Synthetic ts growth phenotype of gga1gga2vps27 strains. (A) Synthetic ts growth phenotype of gga1gga2vps27 strains. Strains were tested for synthetic growth defects at 30 or 37C. Strains were streaked onto YPD plates and incubated for 2 d. Deletion of the AP-1 medium subunit (apm1), the AP-3 medium subunit (apm3), or the dynamin homolog VPS1 (vps1) showed no synthetic growth phenotypes with gga1gga2. In contrast, deletion of VPS27 in two different gga1gga2 strain backgrounds was (ts for growth. Strains: wild-type (BY4704), gga1gga2 (YAB538 T6a), gga1gga2apm1 (YAB650), gga1gga2apm3 (YAB658), gga1gga2vps1 (YAB667), gga1gga2vps27 in S288C background (YAB677), gga1gga2vps27 in SEY6210 background (YAB679), and vps27 (JUY68). (B) Synthetic ts growth phenotype of gga1gga2vps28 strain. As in Figure A, strains were streaked onto YPD plates and incubated for 2 d at 30 or 37C. Strains: wild-type (SEY6210), gga1gga2 (GPY2385), vps27 (JUY68), vps28 (YCS41), gga1gga2vps27 (YAB680), and gga1gga2vps28 (YCS195). All strains grew at 30C. Deletion of GGA1, GGA2, and VPS28 is ts for growth, similar to deletion of GGA1, GGA2, and VPS27. (C) Class E compartment forms at 30 and 37C in gga1gga2vps27 strain. Cells grown at 30 or 37C were stained with FM 4-64 to visualize the vacuole and prevacuolar compartment. FM4-64 fluorescence (left) and differential interference contrast images (right) are shown. Wild-type cells show vacuolar staining with no enlarged PVC (our unpublished data). In contrast, the gga1gga2vps27 cells had enlarged PVC, indicated by bright staining adjacent to the vacuole, at both 30 and 37C. In contrast, cells deleted of GGA1, GGA2, and VPS27 were temperature sensitive (ts) for growth in two different strain backgrounds (Figure A, strains YAB677 and YAB679). The slight differences in strain background are currently unexplained. This ts growth defect was reversible after at least 3 d at the restrictive temperature, indicating growth inhibition rather than lethality (our unpublished data). This tight and reversible ts growth phenotype in the SEY6210 background was found in multiple isolates of the genotype, including strain YAB680 shown in Figure B. To determine whether the ts phenotype was related to the VHS domain of Vps27p, we tested another class E gene for genetic interaction with GGA deletion. Deletion of GGA1, GGA2, and VPS28 in the SEY6210 background was synthetically ts for growth (Figure B). This suggests that the synthetic phenotypes are due to the function of class E genes rather than the VHS domain of Vps27p. A notable defect of class E mutants (such as vps27) is the enlargement of the PVC, or the class E compartment. Using FM 4-64 as a marker for the vacuole and PVC, we tested for PVC enlargement in the triple knockout strain to determine whether GGA deletion prevented the formation of this compartment. Both vps27 (our unpublished data) and gga1gga2vps27 strains (Figure C) contained characteristic class E compartments at 30 and 37C, suggesting that the PVC can form in the absence of GGA gene function. We also noted that the vacuolar morphology was altered in these strains, with a bubble-like appearance. This is consistent with a vacuolar phenotype of gga1gga2 strains reported by others and not observed in our previous experiments with a different strain background. GGA Mutants Complement gga1gga2vps27 Phenotype | To determine whether binding to Arf was required for Gga1p or Gga2p to complement the synthetic ts phenotype of the gga1gga2vps27 strain, the wild-type and mutant alleles of GGA1 and GGA2 were transformed into yeast strain YAB680. Transformants were streaked onto YPD and incubated at 30 or 37C for 2 d. The ts phenotype was fully complemented by expression of plasmid-borne wild-type GGA2 . The three mutant alleles Gga2pI207N, Gga2pPEDL->AAAL, and Gga2pANKL->AAAL also fully complemented the growth defect at 37C when expressed in this strain . Thus, these mutants seemed to complement all Gga trafficking functions. We concluded that Gga2p function is not dependent on Arf, or that these mutants do not disrupt Gga2p-Arf binding in vivo. As in the CPY assay, expression of Gga1p partially complemented the ts growth defect . High copy expression of GFP-Gga1p fully complemented the ts growth defect , showing that the partial complementation by Gga1p could be overcome by higher expression (see DISCUSSION). In contrast, expression of mutant Gga1pL203Q failed to complement the ts growth defect; only very slow growth was restored . This suggests that Arf binding is important for Gga1p function. At high expression levels, GFP-Gga1pL203Q partially complemented the ts growth defect . This suggests that the mutation reduced, but did not eliminate, Gga1p function. Figure 7 | Synthetic ts phenotype of gga1gga2vps27 is fully complemented by mutant Gga2p. Synthetic ts phenotype of gga1gga2vps27 is fully complemented by mutant Gga2p. Plasmids encoding wild-type or mutant Gga1p or Gga2p were transformed into gga1gga2vps27 strain YAB680. GFP fusions of Gga1p are expressed at high copy, all others at low copy. Serial dilutions of wild-type, YAB680, and each transformed strain were replica plated on YPD and incubated at 30 or 37C. Complementation of the ts growth defect is indicated by restored growth at 37C. N- and C-Terminal Domains Confer Golgi Localization of Yeast GGA Proteins | Because interaction with Arf was not required for Golgi localization of Gga proteins, we made a series of truncation constructs of GFP-Gga1p to test the VHS, hinge, and ear domains for roles in Gga localization. All constructs that included the GAT domain were made as wild-type and L203Q mutants to analyze the role of Arf interaction in the truncated proteins. We used Gga1p for these experiments because the L203Q mutation reduces function of the full-length protein. All proteins were expressed at levels similar to full-length GFP-Gga1p, as determined by Western blotting (our unpublished data). Each GFP fusion protein was localized in wild-type strain BY4735 by fluorescence microscopy. Full-length wild-type GFP-Gga1p and mutant GFP-Gga1pL203Q are shown for comparison (Figure , a and b). Figure 8 | Schematic of truncation constructs of GFP-Gga1p and Gga2p. Schematic of truncation constructs of GFP-Gga1p and Gga2p. Figure 9 | Localization of truncated GFP-Gga1p. Localization of truncated GFP-Gga1p. (a --h) Strains expressing wild-type or mutant versions of the indicated constructs were analyzed by GFP fluorescence. Punctate staining, characteristic of Golgi localization, was observed in a, b, e, g, and h. Cytosolic staining was observed in c, d, and f. (i --l) Localization of wild-type and mutant human GGA1DeltaVHS. NRK cells were transiently transfected with pcDNA3-based vectors for expressing GGA1DeltaVHS (residues 145 --639, i --j) or mutant GGA1DeltaVHS, L182Q (k and l). Cells were double-labeled for IIF with antibodies to GGA1 (R79709, i and k) and mannosidase II (j and l). GGA1DeltaVHS localizes to the Golgi region, whereas GGA1DeltaVHS, L182Q is cytosolic. (m --p) Localization of yeast Gga1p constructs in a class E mutant strain. The indicated constructs were expressed in a strain deleted of VPS27. Full-length GFP-Gga1p (m) and GFP-Gga1p1 --331 (n) localize to the class E compartment. In contrast, GFP-Gga1p141 --557 (o) localizes to distinct puncta throughout the cytoplasm in addition to limited staining near the vacuole. The early Golgi protein Gyp1p, expressed as an RFP fusion, localizes exclusively to Golgi puncta (p). A construct expressing the yeast GAT domain alone (GFP-Gga1p141 --331) was localized in the cytosol (Figure c), in stark contrast to the GAT domain of mammalian GGAs. A similar construct as a Gal4AD fusion protein interacts with Arf2pQ71L in the two-hybrid assay , yet Arf interaction is not sufficient to localize the yeast GAT domain to membranes in vivo. The mutant GFP-Gga1p141 --331,L203Q also localized to the cytosol (Figure d). Similarly, the VHS domain alone was cytosolic (our unpublished data). Importantly, a construct expressing the VHS/GAT domains (GFP-Gga1p1 --331) localized to the Golgi and was indistinguishable from full-length GFP-Gga1p (Figure e). When this same construct carried the L203Q mutation, GFP-Gga1p1 --331,L203Q was found exclusively in the cytosol (Figure f). We made two conclusions from these results. First, we concluded that binding to Arf contributes to stable association of Gga1p with membranes and that the L203Q mutation indeed disrupts interaction with Arf in vivo. Second, we concluded that the VHS domain confers weak Golgi localization, which is stabilized by GAT binding to Arf. A construct expressing the GAT/hinge/ear domains (GFP-Gga1p141 --557) localized to Golgi-like puncta (Figure g). This suggests that the hinge and/or ear domains also contribute to stable association with membranes. This construct carrying the L203Q mutation (GFP-Gga1p141 --557,L203Q) also localized to Golgi-like puncta (Figure h), suggesting that the hinge and/or ear domains interact with some Golgi component with high enough affinity to drive the GFP reporter onto the Golgi, regardless of Arf interaction. Consistent with this observation, a hinge/ear construct of Gga1p also seems membrane associated (our unpublished data). In contrast, comparable constructs of human GGA1 confirm that only the GAT domain confers Golgi localization in mammalian cells. IIF of GGA1DeltaVHS in NRK cells shows Golgi localization indistinguishable from full-length GGA1, whereas GGA1DeltaVHS,L182Q is entirely cytosolic (Figure , i --l). Interestingly, the GFP-Gga1p141 --557 construct lacking the VHS domain seemed to stain more puncta than did full-length Gga1p, suggesting that the VHS domain may restrict Gga1p to distinct Golgi cisternae (likely the TGN). To test for altered localization of the truncated constructs, each was expressed in a vps27Delta strain (Figure , m --p). Full-length GFP-Gga1 and GFP-Gga1p1 --331 localized exclusively to the class E compartment, suggesting that removal of the hinge/ear domain did not alter the localization of Gga1p. In contrast, GFP-Gga1p141 --557 localized to spots throughout the cytoplasm in addition to some accumulation near the vacuole (Figure o). The class E compartment formed in these cells, as determined by FM 4-64 staining (our unpublished data). Early Golgi cisternae do not collapse into the class E compartment; RFP-Gyp1p localized to puncta in vps27Delta cells (Figure p). These data suggest that removal of the VHS domain caused mislocalization of Gga1p to other organelles such as early Golgi. We concluded that at least three domains on Gga proteins interact with Golgi-associated proteins to recruit and stabilize Gga proteins at the TGN and that Arf interaction occurs in vivo but is neither sufficient nor required for Gga localization. Both VHS and Hinge/Ear Domains Are Required for GGA Function | To determine whether the VHS and hinge/ear domains are required for Gga function, we tested truncations of Gga2p and GFP-Gga1p (diagrammed in Figure ) for complementation of the CPY sorting defect of gga1gga2 or ts growth defect of gga1gga2vps27 strains. Each construct was expressed and stable, as determined by Western blotting (our unpublished data). In contrast to the GAT domain mutants, deletion of the VHS domain or the hinge and ear domains eliminated the function of Gga2p and GFP-Gga1p. A construct expressing the VHS and GAT domains of Gga2p (Gga2p1 --326) failed to complement the gga1gga2vps27 ts growth defect or the CPY sorting defect when assayed by pulse-chase or colony immunoblot (our unpublished data). In all three assays, the strains expressing Gga2p1 --326 were indistinguishable from strains lacking Gga2p entirely. This indicates that the hinge and/or ear domains are essential for Gga2p function. Similarly, a construct expressing the GAT, hinge, and ear domains of Gga2p (Gga2p110 --585) failed to complement the gga1gga2vps27 ts growth defect or the CPY sorting defect (our unpublished data). These results indicate that the VHS domain is essential for Gga2p function. Figure 10 | VHS and ear domains are required for Gga function in yeast. VHS and ear domains are required for Gga function in yeast. Indicated constructs of Gga2p or GFP-Gga1p were transformed into gga1gga2vps27 strain YAB680. Serial dilutions were replica plated on YPD and grown at 30 or 37C for 2 d. None of the truncated proteins complemented the ts growth defect, indicating that the VHS and ear domains are required for Gga function in yeast. Identical results were obtained using truncations of GFP-Gga1p to complement the gga1gga2vps27 growth defect . Neither GFP-Gga1p1 --331 nor GFP-Gga1p141 --557 complemented the defect. Not surprisingly, a construct expressing only the GAT domain (GFP-Gga1p141 --331) also failed to complement the gga1gga2vps27 growth defect. Together, these data show that the VHS, hinge, and ear domains are important not only for localization of Ggas to the Golgi but also for the function of the Ggas at the Golgi. PMID- 12221117_DISCUSSION TI - AB - Our main focus of this work was to analyze the functional significance of Gga --Arf interaction in yeast. Because of the high homology between human and yeast GAT domains, particularly in the ARF-binding domain, we expected that the function of this domain would be conserved between human and yeast GGA proteins. Likewise, we expected that mutations within this domain would have similar effects on the localization and function of yeast and human GGA proteins. Surprisingly, our data refute this hypothesis and show strong differences between human and yeast GGA proteins. In mammalian cells, ARF interaction is necessary and sufficient for recruitment of GGAs to the Golgi. In yeast, the VHS, GAT, and hinge/ear domains all contribute to Golgi localization; when expressed individually, only the hinge/ear domain is sufficient for Golgi localization. Although we can detect interaction with Arf in vivo, this interaction is not required for localization or function of yeast Gga proteins. Mutant GGA Proteins | We are confident that the mutations we generated in the GAT domain eliminate binding to activated ARF. First, our two-hybrid assays show loss of interaction with all mutations, and affinity chromatography confirms the loss of affinity between Gga2p and Arf1p. Second, the mutations in human GGA1 and GGA3 cause a complete shift from the Golgi to the cytosol, consistent with a recent report describing a mutation in the GAT domain of GGA3 . Third, in the context of a truncated GFP-Gga1p (but not in full-length GFP-Gga1p), the L203Q mutation causes a complete shift from the Golgi to the cytosol. Our results extend those in a recent report characterizing a mutation in human GGA3 that eliminates ARF --GGA interaction. Herein, we identified at least two additional amino acids in the ARF-binding domain that are also essential for the ARF --GGA interaction. Notably, showed that mutation of the aspartate in the GGA3 PEDL sequence (D189) had no effect on ARF interaction. Because our PEDL->AAAL mutation eliminates Arf2p binding, we conclude that the proline or glutamate residues must be important for ARF binding. The growing number of residues within the GAT domain now reported as essential for interaction with ARF proteins is interesting, particularly that alteration of any one residue eliminates interaction with ARF. Perhaps the affinity of the GGA --ARF interaction is low, and elimination of any contributing element reduces the affinity below detection by two-hybrid analysis. Alternatively, the mutations may alter the structure of the GAT domain sufficiently to disrupt interaction. Structures of this domain and cocrystal structures of ARF and GGAs will surely reveal much about the interaction. Differences between Yeast and Human GGA Proteins | In mammalian cells, ARF interaction is necessary for recruitment of GGAs to the Golgi. In yeast, our mutants have minimal effects on both localization and function, suggesting that Arf interaction is not required. The two most surprising results are that Gga1pL203Q remained Golgi localized and that all Gga2p mutants are fully functional. One possible explanation for our data is that yeast Ggas interact with Arf in vivo, and the mutations described herein reduce the affinity for Arf but do not fully disrupt the interaction. We note that the ARF-GGA interaction is significantly weaker in yeast than in mammalian cells, because the GAT domain of yeast Gga1p is not targeted to the Golgi. However, the GAT domain does contribute to Golgi localization: VHS/GAT is Golgi localized, whereas VHS alone or VHS/GATL203Q is cytosolic. The strength of the interactions between Ggas and other proteins (via the VHS and hinge/ear domains) could stabilize the Gga --Arf interaction at the Golgi and allow signaling from activated Arf. We favor a model in which interaction between Gga and Arf is stabilized by other protein interactions, hence reducing the effect of the mutations in vivo. We conclude that the role of Arf --Gga interaction in yeast is not to recruit Ggas to the Golgi, but rather to alter the structure of Ggas to allow sequential interactions with clathrin or other components. Unfortunately, we cannot detect expression of mammalian GGAs in yeast. We have tried multiple promoters, fusion constructs, and truncation constructs; only the two-hybrid constructs are expressed at a detectable level. Furthermore, we cannot detect yeast Ggas in mammalian cells after either plasmid transfection or protein microinjection. Hence, we cannot test for complementation of the yeast phenotypes by wild-type or mutant human GGAs. Differences between Gga1p and Gga2p | Previous reports concluded that Gga1p and Gga2p are functionally redundant, because expression of either Gga1p or Gga2p can complement the defects of gga1gga2 strains . However, our data reveal differences between Gga1p and Gga2p. Three mutant alleles of Gga2p function fully in all trafficking-related assays, whereas the function of mutant Gga1p is reduced. Also, full complementation of gga1gga2 defects by Gga1p requires high expression levels. There are several possible explanations for these differences. First, Gga1p and Gga2p may perform distinct functions in yeast and only Gga1p function is dependent upon interaction with Arf. This scenario is unlikely, because all gga1gga2 defects described to date can be rescued by expression of either Gga1p or Gga2p. Second, the different effect of the mutations may reflect differences in affinities for ARF. We feel this is the best explanation of our data. Our two-hybrid and affinity chromatography analyses indicate that the interaction between Gga1p and Arfs is weaker than that between Gga2p and Arfs. Whether these differences in affinity are a true reflection of affinity in vivo is not known. If the mutations reduce affinity for Arf, this reduction could ablate Gga1p --Arf interaction in vivo, but not fully ablate Gga2p --Arf interaction. Perhaps we would see an effect of the Gga2p mutants if we lowered the level of Gga2p expression. Third, Gga1p may interact with different proteins than Gga2p. Future screens to identify binding partners will address this possibility. Fourth, Gga1p may interact with the same proteins as Gga2p, but with different affinities. Perhaps these interactions are stronger with Gga2p than with Gga1p, thus stabilizing the Gga2p --Arf interaction and masking any effects of the mutants. High expression of Gga1p could force interaction with these proteins, thus complementing the double deletion. Fifth, the level of expression of Gga2p may be higher than that of Gga1p, as noted previously . However, our results with HA-tagged Gga1p and Gga2p argue that the expression levels are in fact very similar. Finally, the L203Q mutation may reduce interaction with another, unknown partner of Gga proteins that is involved in Gga function. Although we cannot rule out this possibility, the current evidence strongly favors Gga --Arf interaction via the GAT domain. Roles of VHS Domain | The VHS domain is required for function; a construct lacking the VHS domain fails to rescue the ts phenotype of gga1gga2vps27. This conclusion is consistent with two recent reports showing that the VHS domain is required for CPY sorting. If the yeast and mammalian VHS domains of GGA proteins have similar functions then the VHS domain of Gga1p and Gga2p should interact with cargo that is sorted into Gga-dependent pathways. The mammalian GGA proteins interact with an acidic cluster-dileucine motif in transmembrane cargo receptors that traffic from the TGN to endosomes . The closest yeast homolog to these cargoes is the CPY receptor Vps10p. We cannot detect direct interaction between the Vps10p cytoplasmic tail and Gga proteins by two-hybrid analysis (our unpublished data). It will be important to identify the VHS binding partners in yeast and to test whether the yeast VHS domain functions to recognize and sort cargo. Because the VHS domain of Vps27p cannot substitute for the Gga VHS domain , it will also be interesting to identify essential residues within the GGA VHS domain required for function. We also show that the VHS domain contributes to the Golgi localization of Gga proteins, likely through interaction with other Golgi-associated proteins. This contribution is seen most definitively with the VHS/GAT construct of GFP-Gga1p. This construct (GFP-Gga1p1 --331) is localized to Golgi-like puncta, whereas addition of the L203Q mutation causes exclusively cytosolic staining. It is possible that the L203Q mutation alters folding of the VHS/GAT construct such that interactions with the VHS domain are disrupted. We do not favor this explanation, because the L203Q mutation has only minor effects on Gga1p function. If L203Q altered VHS folding, we would expect the total loss of function phenotype of the VHS deletion. Hence, we conclude that both the VHS domain and the GAT domain interact weakly with Golgi-localized proteins, and that together these interactions confer Golgi localization. It seems that the GFP-Gga1p construct lacking the VHS domain localizes to more puncta than does full-length GFP-Gga1p. There are two possibilities for this staining pattern. First, this construct may fragment the late Golgi. Second, the truncated Gga1p may mislocalize to other membranes such as early Golgi or endosomes. Our data using vps27Delta cells show that the class E compartment forms but the construct still localizes to distinct puncta, supporting the second possibility. These data suggest that the VHS domain confers specificity to the late Golgi, perhaps by binding to cargo proteins that are present at the TGN. Because the VHS/GAT construct localizes to Golgi but GAT alone does not, a genetic screen for mutants that disrupt Golgi localization of VHS/GAT may identify the VHS-binding partners. Role of Hinge/Ear Domains | The hinge and/or ear domains are required for function; when both domains are deleted, Gga1p and Gga2p are nonfunctional. reported that deletion of the ear domain partially reduced Gga function. Hence, our results suggest that the hinge is required for Gga function. Because clathrin interacts with the hinge domain and facilitates vesicle formation , this loss of function is likely due to loss of clathrin interaction, leading to loss of vesicle formation. However, the hinge and ear domains also confer Golgi localization to yeast Gga proteins. This contrasts with the human GGA proteins, for which the hinge/ear domain is soluble. Two models could explain this result. First, clathrin is present at the TGN membrane in yeast and helps to recruit Gga proteins. Second, a trans-Golgi component interacts with the ear domain to recruit Ggas to the TGN, which in turn recruit clathrin. Many have shown that clathrin is recruited from the cytosol to the membrane by adaptor proteins (for review, see ), supporting the second model. However, the identity of these potential ear binders is unknown, and the role of the ear domain is still unclear. Perhaps the ear domain interacts with Golgi components to increase cargo specificity, stabilize the interactions during vesicle formation, or disassemble the complex after vesicle formation. As discussed for the VHS domain, it will be very informative to identify Gga ear partners. Mammalian GGA Function | We show that ARF interaction is required for the localization of human GGA1 and GGA3 to the TGN. This is consistent with a recent report showing that a mutant GGA3 is cytosolic with dramatically reduced function. We found that high levels of expression of mutant GGA3 shift M6PR to the plasma membrane (our unpublished data). This was similar to the shift we observed with wild-type GGA3 overexpression , suggesting that mutant GGA3 in fact retains some function. There are two possible explanations for this observation. First, the mutant GGA3 may interact weakly with ARF, and high levels of expression allow a functional interaction at the TGN. However, we do not see any accumulation of mutant GGA3 at the TGN in our immunofluorescence analyses. Second, mutant GGA3 may interact with binding partners in the cytosol rather than at the TGN, sequestering these components away from their functional sites on the TGN or plasma membrane. The most likely candidate is clathrin, shown to be recruited by GGAs to the TGN . This would be interesting, because it would indicate that GGA --clathrin interaction is not dependent upon an established ARF --GGA interaction, but that GGA and clathrin are recruited together to the TGN through interaction between GGA and ARF. If this sequestering occurs, then we would expect that clathrin-dependent endocytosis would be reduced. We have preliminary data showing that transferrin receptor accumulates on the plasma membrane in cells overexpressing GGA3, supporting this possibility. Further studies must be done to show interaction of the mutant GGA3 with clathrin in cytosol. Genetic Interactions | Our characterization of the synthetic growth defect in gga1gga2vps27 and gga1gga2vps28 strains supports the hypothesis that Gga proteins are involved in delivery of select cargo proteins to the endosomes. In vps27 and vps28 strains, vacuolar function is maintained due to mixing of vacuolar components into the enlarged PVC, or class E compartment, allowing cell growth . We show that the enlarged PVC is still able to form in the gga1gga2vps27 strain, even at the restrictive temperature, indicating that not all traffic to the PVC is blocked. If some essential proteins cannot reach the PVC due to loss of Gga function then vacuolar function would be further reduced. When cells are stressed by high temperature, this reduced function may be exacerbated, leading to growth inhibition. Because both Vps27p and Gga proteins contain a VHS domain, we tested whether the synthetic interaction is related to VHS function or common to all class E VPS genes. The ts phenotype of gga1gga2vps28 strains supports the latter. In our genetic analyses, we also tested for synthetic interactions with VPS1 and with the medium subunits of adaptor complexes AP-1 and AP-3. Others have recently reported genetic interactions between GGAs and the large subunits of AP-1 . We show that deletion of the medium subunit of AP-1 does not exacerbate the gga1gga2 phenotype, except for very minor ts growth inhibition. Our result is likely due to incomplete loss of AP-1 function when the medium subunit is deleted, because Apm2p can function in AP-1 complexes . We also show that VPS1 and GGAs do not display genetic interactions. It is possible that Vps1p is involved with Ggas in vesicle formation, but deletion of GGAs does not accentuate the more severe vps1 phenotype. Because a number of lipid modifying enzymes act upstream or downstream of ARF1, it will be interesting to test for genetic interactions between GGAs and these same genes. Together, our data suggest a model in which yeast Gga proteins assemble a protein complex at the TGN that is required for proper sorting and transport of proteins to endosomes. The overall function of GGA proteins is thus conserved between yeast and humans. However, the mechanics of the interactions seem to be very different. In mammalian cells, interaction between activated ARF and GGA proteins recruits GGAs to the Golgi membrane, where interactions with cargo and clathrin can occur. In yeast, it seems that interactions between Gga proteins and cargo and/or other Golgi components drive the recruitment of Ggas to the Golgi membrane, where Arf interaction can occur. We speculate that a conformational change in Gga proteins upon Arf activation then drives the formation of a functional complex. The mutant Gga proteins with reduced Arf-binding affinity still function in yeast, because Arf and Ggas are juxtaposed at the Golgi membrane by other interactions, thus stabilizing the Arf --Gga interaction. PMID- 12221118 TI - Inversin Forms a Complex with Catenins and N-Cadherin in Polarized Epithelial Cells AB - | Nephrogenesis starts with the reciprocal induction of two embryonically distinct analages, metanephric mesenchyme and ureteric bud. This complex process requires the refined and coordinated expression of numerous developmental genes, such as inv. Mice that are homozygous for a mutation in the inv gene (inv/inv) develop renal cysts resembling autosomal-recessive polycystic kidney disease. The gene locus containing inv has been proposed to serve as a common modifier for some human and rodent polycystic kidney disease phenotypes. We generated polyclonal antibodies to inversin to study its subcellular distribution, potential binding partners, and functional aspects in cultured murine proximal tubule cells. A 125-kDa inversin protein isoform was found at cell-cell junctions. Two inversin isoforms, 140- and 90-kDa, were identified in the nuclear and perinuclear compartments. Plasma membrane allocation of inversin is dependent upon cell-cell contacts and was redistributed when cell adhesion was disrupted after incubation of the cell monolayer with low-calcium/EGTA medium. We further show that the membrane-associated 125-kDa inversin forms a complex with N-cadherin and the catenins. The 90-kDa nuclear inversin complexes with beta-catenin. These findings indicate that the inv gene product functions in several cellular compartments, including the nucleus and cell-cell adhesion sites. PMID- 12221118_INTRODUCTION TI - AB - Organogenesis of the mammalian kidney involves the coordinated regulation of gene expression that occurs during the reciprocal induction of two embryonically distinct analages, mesenchymal metanephric blastema and epithelial ureteric bud. Successful nephrogenesis and maturation of renal tubules requires a combination of growth pattern control, cell fate determination, and cell cycle control. A relatively common derangement of tubule maturation is the growth of fluid-filled epithelial cysts. Mutations in one of several genetic loci can lead to a cystic phenotype characterized by epithelial cell proliferation, reversal of cell polarity, and alterations in apoptosis, extracellular matrix, and transepithelial transport of fluids and electrolytes (for review, see ). Rodent models have proved valuable in elucidating the mechanisms underlying renal epithelial cyst formation. One such model, the inversion of embryonic turning (inv/inv) mutant mouse was created by insertional mutagenesis and exhibits an autosomal recessive form of situs inversus and renal cysts , the latter resembling autosomal recessive polycystic kidney disease (PKD). The predicted sequence of inversin, the inv gene product, has 15 Ank/Swi6 motifs arranged in a tandem array located toward the amino-terminal side of the protein . The C-terminal one-half of inversin has no similarity to other proteins . Subcellular distribution and potential binding partners of inversin have yet to be discovered. Although the function of the inv gene product is unknown, inversin's potential contribution to renal cystogenesis is supported by quantitative trait localization data. Quantitative trait localization indicates that inv is one of many genes within a locus that contains one or more modifying genes in the pcy, cpk, jck, and bpk mouse models of human PKD. (; ; ; D.D. Woo, personal communication). Modifying genes have been proposed to explain the diversity of PKD phenotypes observed in humans and animal models . The wide range of PKD phenotypes in humans and animal models, combined with the numerous gene defects underlying cyst formation, suggests there is an abundance of proteins that participate in a common pathway of cystogenesis. Inversin appears to be one of these proteins. In this paper, the subcellular distribution of inversin is characterized. In a murine S1 proximal tubule cell line, affinity-purified antibodies raised against inversin's c-terminal domain show nuclear, perinuclear, and plasma membrane labeling by confocal microscopy and the detection of 140-, 125-, and 90-kDa isoforms on immunoblots. The 140- and 90-kDa isoforms were enriched in nuclear and cytoplasmic extracts, whereas the 125-kDa isoform was most abundant in membrane fractions. Inversin antibodies immunoprecipitated N-cadherin and alpha, beta, and gamma-catenins, but not E-cadherin, beta1-integrin, or vinculin. Reverse immunoprecipitations with antibodies to N-cadherin and beta-catenin confirmed that inversin forms a stable complex with these integral membrane proteins. Like inversin, N-cadherin has been implicated in defective left-right asymmetry , and transgenic mice overexpressing beta-catenin develop cystic kidneys . Inversin's localization at the lateral membrane is dependent upon cell-cell contacts and can be disrupted by incubating the monolayer with low-calcium/EGTA medium. These findings suggest that isoforms of inversin may participate in intercellular junction biogenesis and gene transcription similar to beta-catenin's cellular function. PMID- 12221118_MATERIALS AND METHODS TI - AB - Cell Culture | An immortalized cell line derived from the early segment (S1) of the proximal tubule (S1 cells) was a kind gift from Dr. G.T. Nagami (UCLA School of Medicine, Los Angeles, CA; ). Cells were maintained in a 50:50 mixture of Hams-F12:DMEM media supplemented with 2 mM l-glutamine, 10 mM Na-HEPES, 2 mM sodium pyruvate, insulin, sodium-selenite, and sodium-bicarbonate, and 7% fetal calf serum, penicillin, and streptomycin (, 2000). Cells were grown on dishes, coverslips, and polyester membrane filters (0.4-mum pore size; Transwell, , Cambridge, MA) coated with type I rat-tail collagen (Invitrogen, Carlsbad, CA; ). Reagents and Antibodies | Cell media, fetal calf serum, and reagent grade chemicals were supplied by Sigma (St. Louis, MO). Mouse monoclonal antibody to histone H1 was obtained from StressGen Biotechnologies (Victoria, British Columbia, Canada). Mouse-monoclonal antibodies to alpha-, beta-, and gamma-catenin, rat monoclonal antibody to E-cadherin, and polyclonal rabbit antibody to beta-catenin were obtained from Zymed Laboratories (South San Francisco, CA). Pan-cadherin antibody was purchased from Sigma. Polyclonal rabbit antibody to N-cadherin was obtained from Calbiochem (La Jolla, CA). Mouse monoclonal antibodies to vinculin, beta1-integrin, and E- and N-cadherin were purchased from BD Transduction Laboratories (Franklin Lakes, NJ). All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). 4',6-Diamidino-2-phenyindole (DAPI) nucleic acid stain, rhodamine-phalloidin, and SYPRO protein gel stain were purchased from Molecular Probes (Eugene, OR). All other chemicals and reagents were supplied by Fisher Scientific (Pittsburgh, PA). Inversin Polyclonal Antibody | An expressed sequence tag (EST; NCBI accession no. ) was obtained from Incyte Genomics (St. Louis, MO; ) and was sequenced to confirm homology to the 3' end of the inv gene (Lark Technologies, Houston, TX). A 459-base pair (bp) segment of the EST clone was ligated into pRSETB (Invitrogen) and was sequenced to confirm the clone was in-frame with a polyhistidine tag. The recombinant plasmid was transfected into Escherichia coli strain BL21(DE3)pLysS (Invitrogen) to synthesize a bacterial fusion-protein containing the C-terminal 153 amino acids of the EST clone. After induction with 1 mM isopropyl-beta-d-thiogalactopyranose, the recombinant protein was expressed and isolated from bacterial lysate by Ni2+ affinity chromatography (ProBond Resin; Invitrogen). Bound protein was eluted with 500 mM NaCl and 20 mM NaPO4, pH 4.0, and was then dialyzed against 50 mM Tris-Cl, pH 8.0, 1 mM CaCl2, and 0.1% Tween-20, after which the histidine tag was cleaved by enterokinase digestion (EKMax; Invitrogen). Digest products were separated on a 15% SDS-PAGE gel, and protein was recovered by gel excision under visualization using SYPRO protein gel stain. Recombinant protein (17 kDa) was electroeluted from gel slices using an electroeluter (, Richmond, CA). Successful cleavage of the histidine tag was confirmed by immunoblotting with a mouse monoclonal antibody specific for histidine epitope tags (Anti-HisG; Invitrogen). Rabbits were immunized with the purified 17-kDa inversin miniprotein by Covance Research Products (Denver, PA). Antibodies were purified by immunoaffinity chromatography using the inversin mini protein coupled to activated beads (Affigel 10; ) according to the manufacturer's directions. Antibody was eluted in 100 mM glycine, pH 2.4, and the eluate was neutralized with Tris base. Protein Extraction | For total protein extracts, confluent S1 cells were washed with cold phosphate-buffered saline (PBS), scraped from the dish with a rubber-policeman, and pelleted by centrifugation at 850 x g for 5 min at 4C. Cell pellets were resuspended and homogenized using a ball-bearing homogenizer in extraction buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 4 mM EDTA, 1 mM phenyl methyl sulfonyl fluoride [PMSF], and protease inhibitor cocktail [Sigma] with or without Triton X-100 at vol/vol of 1.0%). Cell lysates were centrifuged at 15,000 x g for 10 min at 4C. Supernatants were mixed with Laemmli buffer (2% SDS, 100 mM Tris-Cl, pH 6.8, 25% [vol/vol] glycerol, 10 mM dithiothreitol, 0.001% [wt/vol], and bromphenol blue) and boiled for 10 min . SDS-PAGE and Immunoblot Analysis | Proteins were separated on 7.5% SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ; ). Membranes were blocked with 3% newborn calf serum (NCS) dissolved in Tris-buffered saline (TBS) containing 0.1% (vol/vol) Tween-20 and incubated for 45 min with primary antibodies diluted in 3% NCS in TBS. Membranes were washed in TBS-Tween 20, incubated with horseradish peroxidase-conjugated secondary antibodies in 3% NCS in TBS for 45 min and washed as above. Chemiluminescence was used for detection (, Rockford, IL). Mass Spectrometry Analysis of Inversin Immunoprecipitate | Total protein extracts from cultured S1 cells were immunoprecipitated with the inversin antibody in 1% (vol/vol) Triton X-100, 150 mM NaCl, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 1 mM EGTA, 0.2 mM Na3VO4, 2 mM PMSF, and protease inhibitor cocktail, 1:100. Precipitates were recovered by protein A conjugated to magnetic beads (Dynal, Lake Success, NY). Beads were collected in a magnetic particle concentrator and were washed with three buffers featuring different ionic strengths (buffer I: 150 mM NaCl, 50 mM Tris-Cl, pH 8.0, and 1% [vol/vol] Triton X-100; buffer II: 500 mM NaCl and 50 mM Tris-Cl, pH 8.0; and buffer III: 50 mM Tris-Cl, pH 8.0). Precipitates were resuspended in Laemmli buffer and boiled for 10 min to elute complexes. Proteins were separated by 7.5% SDS-PAGE and visualized with Coomassie stain (Pierce). The 140-kDa band was excised, trypsin digested, and analyzed with a Finnigan (Thermoquest) LCQ mass spectrometer (Biochemistry Biotechnology Facility, Indiana University School of Medicine, Indianapolis, IN). A free-ware program was used to compute and map the cleavage fragments obtained from a virtual trypsin digest of the inv sequence (Client Paws; ProteoMetrics, New York, NY). Immunohistochemistry and Fluorescence Microscopy | Cells grown on filters were fixed in 4% paraformaldehyde in PBS for 10 min. The fixation reaction was quenched in 100 mM NH4Cl dissolved in PBS. Samples were incubated in blocking buffer (1% bovine serum albumin and 0.1% Triton X-100 in PBS) for 10 min before labeling. Cells were incubated in primary antibodies and rhodamine-phalloidin, followed by incubation with fluorescein- or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and washing in PBS. Nuclei were labeled with DAPI diluted in blocking buffer for 10 min. Filters were placed in 2% PFA in PBS, mounted with Mowoil (Calbiochem), and examined with a LSM 510 laser scanning microscope (Carl Zeiss North America, Thornwood, NY) equipped with a UV argon laser, a visible argon laser, and two helium-neon lasers. Images were collected sequentially and processed by Adobe Photoshop 5.5 (Adobe Systems, Mountain View, CA). Iodixanol Gradient Fractionation | Iodixanol subcellular fractionation was performed as previously described . Briefly, confluent S1 cells were homogenized in isotonic sucrose buffer [0.25 M sucrose, 90 mM KOAc, 2 mM Mg(OAc)2, 20 mM HEPES-KOH, pH 8.0, 2 mM PMSF, 0.5 mM 4-(2-aminoethyl)benzensulfonyl-fluoride, and protease inhibitor cocktail 1:100] by repeated passage through a ball-bearing homogenizer. Eight volumes of postnuclear supernatant were combined with 10 volumes of a 54% iodixinol solution that was prepared from a 60% stock solution [diluted with 1/10 vol of 900 mM KOAc, 20 mM Mg(OAc)2, and 200 mM HEPES-KOH, pH 8.0]. Samples were centrifuged at 354,000 x g for 1 h at 4C in a TLN-100 rotor (Beckman Instrument, Fullerton, CA). Fractions were collected and proteins were separated by 7.5% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with antibodies specific for beta-catenin, pan-cadherin, and inversin. Extraction of Nuclear Protein Complexes | Nuclear proteins were extracted by stepwise lysis of cells to ensure minimal cross-contamination, as described earlier . Briefly, confluent S1 cells were washed with cold PBS, scraped in PBS from the dish with a rubber-policeman, and pelleted by centrifugation at 500 x g for 3 min at 4C. Using NE-PER nuclear extraction reagents (Pierce), cells were chemically lysed and nuclei were isolated after centrifugation (16,000 x g for 5 min) and resuspension per the manufacturer's instructions. Nuclear cell debris and DNA were removed by centrifugation (16,000 x g for 5 min) and nuclear proteins were mixed with Laemmli buffer, boiled for 10 min, and loaded on 7.5% SDS-PAGE gels. Extraction of Membrane Protein Complexes | Plasma membranes were purified by the technique of aqueous two-phase partition . Briefly, confluent S1 cells were washed with cold PBS, scraped from the dish with a rubber-policeman, and pelleted by centrifugation at 500 x g for 3 min at 4C. Using Mem-PER extraction reagents (Pierce), cells were chemically lysed and membrane proteins were solubilized. The mixture was incubated at 37C and centrifuged at 10,000 x g for 2 min to separate hydrophobic from hydrophilic proteins. The viscous phase containing the membrane protein fraction was collected and proteins were purified by precipitation with 10% trichloroacetic acid. Recovered proteins were mixed with Laemmli buffer, boiled for 10 min, and loaded on 7.5% SDS-PAGE gels. Ca2+ Switch | S1 cells were grown in normal-calcium (1.8 mM Ca2+) medium (NCM, see cell culture for formula). Low-calcium medium (LCM) was prepared by supplementing NCM with EGTA to a final concentration of 4 mM, as previously described . The pH was adjusted to its initial value with NaOH, and the medium was sterile filtered. S1 cells were washed with PBS, and NCM was replaced with LCM. At 45 and 90 min of incubation in LCM, cells were fixed and processed for immunofluorescence microscopy. Immunoprecipitation | Confluent S1 cells grown on collagen I-coated tissue culture dishes were washed with PBS and incubated on ice for 30 min in CSK buffer [50 mM NaCl, 300 mM sucrose, 10 mM PIPES, pH 6.8, 3 mM MgCl2, 0.5% (vol/vol) Triton X-100, 2 mM PMSF, 0.5 mM 4-(2-aminoethyl)benzensulfonylfluoride, and protease inhibitor cocktail 1:100; ]. Cells were scraped from dishes and insoluble material was removed by centrifugation at 10,000 x g for 10 min at 4C. Cell extracts were incubated with primary rabbit polyclonal antibodies for 1 h. Immune complexes were recovered by incubation with protein A-Sepharose (Amersham) for 1 h. Protein A-Sepharose beads were washed three times in extraction buffer before protein complexes were released by boiling in Laemmli buffer for 10 min. Precipitated proteins were separated by SDS-PAGE followed by immunoblotting. PMID- 12221118_RESULTS TI - AB - Characterization of the Polyclonal Inversin Antibody | A 17-kDa recombinant protein fragment was expressed as a fusion protein containing 153 amino acids from the C-terminal portion of the EST that lacks significant homology with any other protein in the available databases . Affinity-purified antibody detected three bands estimated at 140, 125, and 90 kDa on immunoblots containing proteins from confluent S1 cells extracted in 1% Triton X-100 (Figure A, left lane). Antibody recognition of both bands was competitively blocked when the affinity-purified antibody was preincubated with the immunizing inversin miniprotein (Figure A, right lane). Figure 1 | Characterization of inversin antibody. Characterization of inversin antibody. (A) Total protein extracted from confluent S1 cells (1% Triton X-100 buffer) was separated by 7.5% SDS-PAGE, transferred to membranes, and incubated with affinity-purified inversin antibody. Three bands were detected at 140, 125, and 90 kDa with anti-inversin alone, but no bands were detected when inversin antibody was preincubated with the immunizing recombinant protein. (B) Postnuclear homogenate from confluent S1 cells was immunoprecipitated with affinity-purified inversin antibody and was resolved by 7.5% SDS-PAGE. The 140-kDa band was excised, trypsin digested in situ, and analyzed by mass spectrometry. Table shows peak values measured from mass spectrum as compared with calculated fragment masses after trypsin digestion of inversin. Differences between these values ranged in the expected variability. Mass Spectrometry Confirmation of Antibody Specificity | Mass spectrometry analysis of the 140-kDa protein detected by the affinity-purified antibody confirmed that the antibody specifically bound to inversin. Proteins from S1 cell extracts were immunoprecipitated with inversion antibody and separated by SDS-PAGE. The 140-kDa band was digested with trypsin and the resultant fragments were analyzed by mass spectrometry (Figure C). Measured mass-to-charge ratios (m/z) of tryptic peptides significantly correlated with calculated fragment m/z ratios obtained from computed analysis of the inversin protein sequence. Masses of fragments were also compared with the masses of a peptide database calculated from NCBI's nr protein database . The inversin protein was identified as the top ranking of the candidate proteins based on its calculated posterior probability . The next top nine candidate proteins did not produce significant alignments to the inversin protein sequence . Similarly, the antibody to inversin precipitated two proteins of 165- and 90-kDa that matched inversin when analyzed by mass spectrometry (unpublished data; ). Subcellular Localization of Inversin: Laser Confocal Microscopy | Subconfluent (Figure , A, C, and E) and confluent (Figures , B, D, and F, and 3) S1 cells were grown on filters and stained with inversin antibody (Figures , A and B and 3B) and DAPI, a nucleic acid stain (Figures , C and D and 3A). To show the cellular architecture, the actin cytoskeleton was labeled with rhodamine phalloidin (Figures , E and F and 3C; ). In subconfluent cells, inversin was predominantly distributed in nuclei (compare Figure , A with C). Weak perinuclear and membrane staining was also observed (Figure A). No signal was detected when cells were incubated with the inversin antibody in the presence of the immunizing peptide or the secondary antibody alone (Figure A, insets). In separate experiments, perinuclear inversin staining colocalized with anti-KDEL antibody staining, suggesting that inversin is in the rough endoplasmic reticulum (unpublished data). When incubated with anti-inversin, confluent cells showed distinct labeling of cell membranes (Figures B and B) that was only weakly apparent (Figure A, arrowheads) in subconfluent cells with adjacent cell borders (Figure E). A vertical image section (X-Z axis) of confluent S1 cells shows a lateral distribution of inversin (Figure B). Similar labeling by inversin antibody was also observed in other cultured renal epithelial cell lines such as MDCK cells or cells derived from the distal convoluted tubule (unpublished data; ), indicating that various cell lines carry this protein in a membrane-associated fashion. Figure 2 | Confocal microscopy of subconfluent (A, C, and E) and confluent (B, D, and F) S1 cells fixed in paraformaldehyde and triple-labeled with inversin antibody (A and B), DAPI (C and D), and phalloidin-rhodamine (E and F). Confocal microscopy of subconfluent (A, C, and E) and confluent (B, D, and F) S1 cells fixed in paraformaldehyde and triple-labeled with inversin antibody (A and B), DAPI (C and D), and phalloidin-rhodamine (E and F). In both subconfluent (A) and confluent (B) S1 cells, the inversin antibody stained nuclei and less intensely the perinuclear compartment. Cell membranes appeared uniformly stained in confluent cells (B), but were focally stained in subconfluent cells at early cell-cell contacts (A, arrowheads). Staining seen with inversin antibody (A and B) was absent in cells stained with inversin antibody preincubated with immunizing protein, and secondary antibody alone (A, insets). Figure 3 | Vertical sections (X-Z axis) of inversin staining. Vertical sections (X-Z axis) of inversin staining. Confluent S1 cells were grown on filters, fixed in paraformaldehyde, and triple-labeled with DAPI (A), inversin antibody (B), and phalloidin-rhodamine (C). The vertical sections (X-Z axis) demonstrate distribution of inversin to membranes of cell-cell contacts, but not to apical or basal plasma membranes. (D) overlay of all three channels. Subcellular Distribution of Inversin: Iodixanol Gradient Fractionation | To identify which inversin isoforms (i.e., 140-, 125-, or 90-kDa bands) are responsible for the membrane staining patterns, S1 cell extracts were fractionated by detergent-free iodixanol density gradient centrifugation . In iodixanol gradients, low-density plasma membrane and cytoplasmic particles (<1.1 g/ml) are recovered in the top layer of the gradient (fraction 1), while cytosolic proteins exhibiting a higher density (1.26 g/ml) migrate to the bottom of the gradient during centrifugation (fraction 12). Postnuclear supernatants of confluent S1 cells were fractionated by iodixanol gradient, and fractions were subsequently analyzed by immunoblotting with antibodies to inversin, pan-cadherin, and beta-catenin. Figure A shows the immunoblot analysis of fractions collected from the iodixanol gradient. The inversin antibody detected a band at 140 kDa in all fractions, with the greatest intensity in fractions rich in cytosolic proteins. The weakest 140-kDa bands were in the fractions rich in plasma membrane particles. Additionally, in fraction 1, the inversin antibody detected a 125-kDa band that was not detected in any other fraction. The 90-kDa inversin signal was detected in all fractions with progressively increasing signal intensity from fraction 1 to fraction 12. When the immunoblots were subjected to extended exposure times, a 165-kDa band was detected in the cytoplasmic-enriched fractions 11 and 12 (unpublished data). Pan-cadherin and beta-catenin antibodies detected bands in fractions 1 and 2 containing low-density plasma membrane particles, in agreement with previous findings . Taken together, these data suggest that the 125-kDa band is only found in the fraction that is enriched for plasma membranes, whereas the 90-kDa isoform is found in all fractions. Figure 4 | Subcellular distribution of inversin. Subcellular distribution of inversin. (A) Iodixinol subcellular fractionations were collected from the lightest (top fraction 1) to the heaviest (bottom fraction 12) of the gradient. Equivalent fraction volumes were resolved by 7.5% SDS-PAGE and immunoblotted with antibodies to inversin, pan-cadherin, or beta-catenin. Bands (140- and 90-kDa) were detected with anti-inversin in all fractions, but a 125-kDa band was detected only in membrane fraction 1. The bands detected by anti-pan-cadherin and anti-beta-catenin were restricted to the lightest fractions. The accompanying graph expresses phosphorimager values for each band as a percentage of the total volume of all fractions measured for each antibody. (B) Nuclear and membrane protein extracts from confluent S1 cells were separated by 7.5% SDS-PAGE and were immunoblotted with anti-inversin. The inversin antibody detected only one band of 125 kDa in the membrane protein extract and two bands of 140- and 90- kDa in the nuclear protein extracts. (C) Total protein was extracted from confluent S1 cells with and without 1% Triton X-100 followed by inversin immunoblot analysis. Anti-inversin detected bands at 140 and 90 kDa in both extracts, but only in the presence of Triton X-100 did the inversin antibody detect a band at 125 kDa. Immunoblot Analysis | To confirm the enrichment of the 125-kDa inversin protein in cell membranes, immunoblots were performed on membrane protein complexes extracted from confluent S1 cells using aqueous two-phase partitioning. The inversin antibody detected a single band at 125 kDa (Figure B, right lane) migrating at the same molecular weight as the 125-kDa band in fraction 1 of the iodixanol gradient (Figure A). Nuclear protein complexes were isolated from confluent S1 cells, separated by SDS-PAGE. and transferred to membranes for immunoblot analysis. Successful extraction of nuclear proteins was confirmed on immunoblots by monoclonal antibody detection of histone H1, an exclusive nuclear protein (unpublished data). Inversin antibody detected bands at 90 and 140 kDa in immunoblots of nuclear extracts (Figure B, left lane), but the 125-kDa band was not detectable. To confirm that the 125-kDa inversin protein is restricted to detergent-soluble membrane fractions, protein was extracted from confluent S1 cells in a solution with or without 1.0% Triton X-100. The 140- and 90-kDa bands were detected on immunoblots containing proteins extracted without detergent (Figure C, left lane). When S1 cell proteins were extracted with 1.0% Triton X-100, bands were detected at 140, 125, and 90 kDa (Figure C, right lane). Inversin Forms a Complex with Catenins and N-Cadherin | As described above, inversin localizes to the lateral plasma membrane at regions of cell-cell contacts in confluent S1 cells. To identify proteins that potentially interact with inversin, we immunoprecipitated S1 cell homogenates with the inversin antibody and probed the precipitates with mouse monoclonal antibodies to the cell adhesion proteins alpha-, beta-, and gamma-catenins, pan-, E-, and N-cadherins, vinculin, and beta1-integrin. Bands of the expected molecular weights were detected with antibodies to alpha-, beta-, and gamma-catenins, and pan- and N-cadherin, but not with antibodies to E-cadherin, vinculin, or beta1-integrin despite extended exposure times (Figure A). Figure 5 | (A) Homogenates from confluent S1 cells were immunoprecipitated with inversin antibody and precipitates were immunoblotted with a panel of antibodies. (A) Homogenates from confluent S1 cells were immunoprecipitated with inversin antibody and precipitates were immunoblotted with a panel of antibodies. Arrowheads indicate expected molecular weights. Bands were detected for alpha-, beta-, and gamma-catenin, and pan-, and N-cadherin, but no bands were detected for E-cadherin, B1-integrin, and vinculin despite long film exposure. (B) Confluent S1 cell homogenates were immunoprecipitated with anti-beta-catenin (lane 1) and anti-N-cadherin (lanes 2 and 3), and precipitates were immunoblotted with anti-inversin or anti-beta-catenin. The inversin antibody detected 125- and 90-kDa bands in both the beta-catenin precipitate (left panel) and the N-cadherin precipitate (middle panel). The N-cadherin precipitate that was immunoblotted for beta-catenin detected a 92-kDa band (right panel). (C) Iodixanol fractions 1 and 12 were collected as for Figure A, immunoprecipitated with inversin antibody, and immunoblotted with antibodies to beta-catenin and N-cadherin. Inversin precipitates from fraction 1 contained beta-catenin and N-cadherin (lanes 1 and 2), but no bands were detected in fraction 12 (lanes 3 and 4). Protein extracts in A, B, and C were separated by 7.5% SDS-PAGE and transferred to nitrocellulose membranes. Inverse immunoprecipitations were performed on S1 cell homogenates. Complexes that were precipitated using polyclonal rabbit antibodies to beta-catenin or N-cadherin contained inversin as detected in immunoblots using the inversin antibody (Figure B). Both beta-catenin and N-cadherin precipitates contained the 125- and 90-kDa inversin isoforms. Protein complexes precipitated by anti-N-cadherin contained beta-catenin, as shown in Figure B. To confirm that protein complexes containing inversin, beta-catenin, and N-cadherin are membrane associated in S1 cells, we analyzed membrane and cytosolic fractions obtained from the iodixanol density gradient as described for Figure A. Protein complexes from fractions enriched in membranes (fraction 1) or cytosol (fraction 12) were immunoprecipitated with anti-inversin followed by SDS-PAGE and immunoblot analysis with monoclonal antibodies to beta-catenin and N-cadherin. Coprecipitation of inversin with beta-catenin and N-cadherin was found only in the membrane fraction 1, but not in the cytosolic fraction 12 (Figure C). Codistribution of Inversin and the Cadherin/Catenin Complex | Confluent S1 cells were double-labeled with antibodies to inversin and beta-catenin or inversin and N-cadherin. Both inversin and alpha-catenin were located at the lateral cell membranes and in the nucleus (Figures , A and B). Most inversin staining colocalized with beta-catenin at the plasma membrane, consistent with our findings that these proteins are part of a complex (Figure C). In the nucleus, the antibody labeling of beta-catenin and inversin showed partial colocalization in S1 cells (Figure C) and was weak to absent in MDCK cells (unpublished data). Anti-N-cadherin strongly stained lateral cell membranes (Figure E). Figure F shows that membrane-associated inversin and N-cadherin colocalized exclusively at the plasma membrane (Figure F). No staining was observed when cells were labeled with secondary antibodies alone, confirming the specificity of the primary antibodies used (insets in Figure , A, B, D, and E). Figure 6 | Colabeling of inversin with beta-catenin or N-cadherin. Colabeling of inversin with beta-catenin or N-cadherin. Confluent S1 cells were double-labeled with antibodies to inversin (A) and beta-catenin (B) or inversin (D) and N-cadherin (E) and were analyzed by confocal microscopy. Inversin colocalized with beta-catenin at cell membranes, but there was partial overlap in nuclei (C, yellow overlap). Inversin colocalized with N-cadherin only at cell membranes. Inversin Membrane Assembly Is Calcium Dependent | Inversin's membrane distribution may be dependent on cell-cell adhesions. To study whether this distribution is calcium dependent, confluent S1 cells were switched from medium containing 1.8 mM calcium to media containing 1.8 mM calcium plus EGTA. Cells fixed at various time points (0, 45, and 90 min) were labeled with anti-inversin, anti-beta-catenin, and fluorescein isothiocyanate-conjugated phalloidin (Figure , A-I) or anti-inversin, anti-N-cadherin, and fluorescein isothiocyanate-conjugated phalloidin (Figure , J-R). In 1.8 mM calcium medium (0 min), cells were confluent, and staining for F-actin was continuous at regions of cell-cell contacts (Figure , G and P). Similar to the results shown in Figure , inversin and beta-catenin localized to the cell membrane and nuclei (Figure , A, D, and J). Under these conditions, anti-N-cadherin also labeled cell membranes (Figure M). Incubating the cells in calcium-chelated media induced changes in cell shape, cell adhesion, and redistribution of analyzed proteins. After 45 min in calcium-chelated media, cell contacts were discontinuous, and phalloidin staining illustrated a retraction of the F-actin belt (Figure , H and Q). Membrane labeling of inversin, beta-catenin, and N-cadherin was dramatically weakened, whereas nuclear staining of inversin and beta-catenin appeared unchanged (Figure , B, E, K, and N). After 90 min in LCM, cells were spherical and contacts with neighboring cells were minimal or absent (Figure , I and R). Staining of inversin and beta-catenin was almost exclusively confined to the nuclear compartment (Figure , C, F, and L). Only a few membrane segments showed residual staining of beta-catenin and N-cadherin (Figure , F and O). These results demonstrate that membrane assembly of inversin is calcium dependent. Changes in membrane assembly of inversin occurred over the same time course as the changes in N-cadherin and beta-catenin membrane assembly (unpublished data). Low calcium-induced redistribution of inversin was almost complete after 45 min. Similarly, complete redistribution of N-cadherin and beta-catenin occurred at the 45-min time point. Notably, intranuclear inversin staining was unaffected by changes in extracellular calcium. Figure 7 | Calcium depletion in confluent S1 cells by confocal microscopy. Calcium depletion in confluent S1 cells by confocal microscopy. Confluent S1 cells were calcium depleted by incubating with medium containing 4 mM EGTA. At time 0, 45, and 90 min, cells were triple labeled with anti-inversin (A-C), anti-beta-catenin (D-F), and phalloidin (G-I) or anti-inversin (J-L), anti-N-cadherin (M-O), and phalloidin (P-R). At 45 and 90 min of calcium depletion, cells progressively lost cell-cell contacts, as displayed by staining of the F-actin cytoskeleton (H, I, Q, and R). Calcium depletion also led to diminished staining of inversin (B, C, K and L), beta-catenin (E and F), and N-cadherin (N and O) from cell membranes. Nuclear staining of inversin and beta-catenin remained unchanged under low calcium conditions (C, F, and L). PMID- 12221118_DISCUSSION TI - AB - The inv gene was originally identified in the OVE210 model of reversal of embryonic turning and PKD . The activity of the inv gene product, inversin, is not known, but the defects seen in the inv/inv mouse suggest that inv plays a crucial role in the establishment of the left-right axis and maturation of epithelial structures such as renal tubules. Using an inversin-specific polyclonal antibody, this study identifies at least three inversin proteins of 140-, 125-, and 90- kDa. The existence of multiple isoforms is supported by the identification of alternatively spliced inv transcripts. described alternative splicing in exon 13 of the full-length inv sequence predicting isoforms of 99, 104, and 118 kDa. Using reverse transcriptase-polymerase chain reaction, we found splice variants lacking exon 4 (accounting for 6.2 kDa) or exon 10 (4 kDa) in mouse kidney and exon 11 (7.9 kDa) in MDCK cells (canine; unpublished data). Using the NCBI database, we identified 16 ESTs with 90% homology to inv, but when completely sequenced, none of these encoded the full-length (1062 amino acids) sequence. Fifteen of the ESTs had limited homology to exon 16 (unpublished data), but one EST was a splice variant that encoded a 76-kDa in-frame inversin protein that we used to generate antigen for our inversin antibody. We used web-based software to predict posttranslational modifications of inversin and we found potential phosphorylation sites (50 serine, 11 threonine, and 6 tyrosine; ), as well as several type O-glycosylation sites (11 serine and 2 threonine; ). Some of these posttranslational modifications may account for the difference between the predicted size of the 118- and 140-kDa bands detected by the inversin antibody. Potential Cellular Function of Inversin | Of the two lower molecular-weight inversin isoforms that precipitate with beta-catenin and N-cadherin, the 125-kDa band was most enriched in the cell membrane fractions. Like the cadherin/catenin complex , membrane-associated inversin is calcium dependent. Analysis of the inversin protein sequence did not reveal any known transmembrane sequence motifs; therefore, the effect of calcium depletion on inversin redistribution may be mediated via N-cadherin or beta-catenin. These findings suggest that the 125-kDa inversin may play a role in regulating the molecular architecture of cell-cell junctions. The inversin proteins responsible for the nuclear and perinuclear immunofluorescence appear to be the 140- and 90-kDa isoforms. The import of inversin into the nucleus likely involves the recognition of a nuclear localization signal sequence (NLS; ). The full-length inversin sequence exhibits one classical type of NLS (KHRR at aa 735; ) and two bipartite NLSs (RKDAAAKKREEENKRKE at aa 589 and KRQDRAARPRGASQKRR at aa 782; ). Rheinhardt's method for cytoplasmic/nuclear discrimination also predicted a nuclear localization of inversin , providing further evidence for a nuclear function of inversin. Based on PROSITE database analysis, the inversin sequence does not exhibit sequence motifs that are involved in DNA binding . Of the two inversin proteins that were enriched in nuclear fractions, only the 90-kDa protein complexed with beta-catenin, the latter a protein involved in transcription regulation . Therefore, a nuclear inversin protein may modulate gene transcription via interaction with transcription factors such as beta-catenin. Potential Role of Inversin in Determination of the Left-Right Axis | Mice lacking a functional inv gene exhibit defects in left-right patterning , suggesting a role of inversin in the determination of the left-right axis. In the earliest stages of asymmetrical development, a nodal flow generated by motile cilia is postulated to initiate expression of genes, such as Nodal, that regulate embryonic turning . Although little is known about the function of inversin, a reduced nodal flow was observed in the inv/inv mouse and has been proposed to be associated with the reversal of embryonic turning in this model . We found inversin localization at nuclei and at basolateral membranes. When MDCK cells were costained for inversin and tubulin, we did not observe inversin staining in monocilia (unpublished data). However, inversin precipitated with proteins that are involved in axis development, i.e., N-cadherin and beta-catenin. N-cadherin (neural cadherin), a classical or type I cadherin, is expressed in a developmental manner , regulates migration of cortical and neural crest cells , and plays a role in the embryonic development of the kidney . However, N-cadherin is also involved in establishment of embryonic left-right asymmetry. found that chicken embryos treated with anti-N-cadherin exhibit a randomization of left-right asymmetry. Mechanistically, N-cadherin, which is localized to the right side of the node, may restrict activation of Nodal , which in turn controls embryonic turning . A defective inv gene product may adversely impact N-cadherin function, resulting in defects of establishing the left-right axis. Inversin also interacts with beta-catenin, a protein that plays a crucial role in the Wnt/beta-catenin signaling pathway . Nuclear beta-catenin complexes with LEF/TCF transcription factors and subsequently activates LEF/TCF target genes , including the promoter of Nodal . Hence, the defect in the left-right axis in the inv/inv mouse could also result from deregulated beta-catenin signaling. However, altered left-right asymmetry has not been described in transgenic mice expressing an activated mutant of the beta-catenin gene . Potential Role of Inversin in Kidney Development | Renal cyst formation has been proposed to result from the disruption of a multicomponent membrane-spanning polycystin complex . Several proteins have been identified that participate in this complex, including polycystin-1, polycystin-2, the catenins, and E-cadherin . Defects in these proteins and deregulation of the linked Wnt/beta-catenin signaling cascade may also play a key role in the cystogenic pathway . Recently, have shown that transgenic mice expressing a mutant form of beta-catenin develop polycystic kidneys . Similarly, overexpression of c-myc, which is a target gene of beta-catenin signaling , results in a cystic phenotype in transgenic mice . In this study, we found inversin coimmunoprecipitating with molecules that complex with polycystin. As with other junctional proteins , the 125-kDa membrane-enriched inversin may modulate beta-catenin function. The finding of inversin and beta-catenin colocalization in nuclei provides further evidence of a partnership of these two proteins in pathways that may include cystogenesis. PMID- 12221119 TI - Modulation of Cellular Cholesterol Transport and Homeostasis by Rab11 AB - | To analyze the contribution of vesicular trafficking pathways in cellular cholesterol transport we examined the effects of selected endosomal Rab proteins on cholesterol distribution by filipin staining. Transient overexpression of Rab11 resulted in prominent accumulation of free cholesterol in Rab11-positive organelles that sequestered transferrin receptors and internalized transferrin. Sphingolipids were selectively redistributed as pyrene-sphingomyelin and sulfatide cosequestered with Rab11-positive endosomes, whereas globotriaosyl ceramide and GM2 ganglioside did not. Rab11 overexpression did not perturb the transport of 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine-perchlorate --labeled low-density lipoprotein (LDL) to late endosomes or the Niemann-Pick type C1 (NPC1)-induced late endosomal cholesterol clearance in NPC patient cells. However, Rab11 overexpression inhibited cellular cholesterol esterification in an LDL-independent manner. This effect could be overcome by introducing cholesterol to the plasma membrane by using cyclodextrin as a carrier. These results suggest that in Rab11-overexpressing cells, deposition of cholesterol in recycling endosomes results in its impaired esterification, presumably due to defective recycling of cholesterol to the plasma membrane. The findings point to the importance of the recycling endosomes in regulating cholesterol and sphingolipid trafficking and cellular cholesterol homeostasis. PMID- 12221119_ TI - Glossary AB - Abbreviations used: : ACAT = acyl coenzyme A-cholesterol acyltransferase CD = cyclodextrin CTxB = cholera toxin subunit B DiI-LDL = 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine-perchlorate --labeled low-density lipoprotein LBPA = lysobisphosphatidic acid LDL = low-density lipoprotein LPDS = lipoprotein-deficient serum mbeta-CD = methyl-beta-cyclodextrin NPC = Niemann-Pick type C PI-3-P = phosphatidylinositol 3-phosphate Pyr10SM = pyrenyldecanoylsphingomyelin TfR = transferrin receptor PMID- 12221119_INTRODUCTION TI - AB - Cholesterol is an essential constituent of membranes in mammalian cells and a precursor for steroid hormone and bile acid synthesis. Cellular cholesterol levels are tightly regulated at the level of synthesis, esterification, and exchange with plasma lipoproteins . The route of low-density lipoprotein (LDL)-cholesterol uptake is hitherto the best characterized cellular cholesterol-trafficking pathway. The role of the LDL receptor in LDL internalization, the breakdown of the lipoprotein particle in acidic organelles, and the homeostatic mechanisms regulating the LDL-receptor levels have been unraveled . However, the contribution of other endocytic routes on cholesterol transport and balance and their interplay with the LDL-receptor route are so far poorly understood at the molecular level. The endocytic organelles have been mainly defined based on the flow of different cargo molecules to early, recycling, and late compartments. Internalized molecules are initially transported to early endosomes (also termed sorting endosomes) from where they can be delivered to late endosomes and lysosomes for degradation or become recycled to the plasma membrane either directly or via a recycling endosomal membrane system . Recycling endosomes are considered to be cholesterol enriched . The cholesterol content of the early or late endocytic membranes has not been determined, but late endocytic circuits are considered to be important for the regulation of the cellular free cholesterol content. This is highlighted in the late endosomal/lysosomal cholesterol storage disorder Niemann-Pick type C (NPC) disease. In this disease, cholesterol as well as other lipids and proteins accumulate in late endocytic organelles due to mutations in either of two recently cloned gene products, NPC1 or NPC2/HE1 . Consequently, cholesterol homeostatic responses in the endoplasmic reticulum fail, as manifested by defective cholesterol esterification and inappropriately high cholesterol synthesis . NPC1 is a polytopic membrane protein of late endocytic membranes, whereas NPC2 is a cholesterol-binding soluble protein that is also targeted to the late endocytic organelles. The precise functions and trafficking itineraries of both NPC1 and NPC2 remain to be elucidated. We have recently reported that the clearance of lysosomal cholesterol deposits can be inhibited by Rab-GDP dissociation inhibitor . This protein controls multiple vesicular transport pathways by sequestering GDP-bound (inactive) forms of the small GTPases of the Rab family in the cytoplasm. Rab proteins and their effectors coordinate consecutive stages of membrane transport, such as vesicle formation, movement, and tethering of vesicles to their target compartment . To gain further insight into the Rab-dependent endosomal cholesterol-trafficking mechanisms, we screened the potential contribution of selected endosomal Rab proteins 1) morphologically by using filipin staining and 2) biochemically by measuring cholesterol esterification. Based on the results obtained, our further analyses focused on the role of Rab11 in controlling endocytic cholesterol routing. We provide evidence that Rab11-dependent membrane trafficking modulates endosomal cholesterol levels independent of LDL uptake and serves as an important regulator of cellular cholesterol balance. PMID- 12221119_MATERIALS AND METHODS TI - AB - Antibodies and Reagents | Mouse monoclonal anti-transferrin receptor (TfR) and rabbit polyclonal anti-Rab11 antibodies were from Zymed Laboratories (South San Francisco, CA), mouse monoclonal anti-lysosome-associated membrane protein (lamp) 1 antibody was from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA), and mouse monoclonal anti-LDL receptor antibodies (C7) were from American Type Culture Collection (Manassas, VA). IgG antibodies against sulfatide and IgM antibodies against globotriaosyl ceramide and GM2 were generous gifts from Jan-Eric Mansson (Sahlgrenska University Hospital, Molndal, Sweden); anti-lysobisphosphatidic acid (LBPA) antibody was from Jean Gruenberg (University of Geneva, Geneva, Switzerland), and anti-HE1/NPC2 antibody was from Naomichi Okamura (University of Tsukuba, Tusbuba, Japan). Anti-NPC1 antibody has been described previously . Fluorescein isothiocyanate (FITC)- and tetramethylrhodamine B isothiocyanate-conjugated anti-IgG secondary antibodies were from Immunotech (Marseille, France), and Cy3-conjugated streptavidin and tetramethylrhodamine B isothiocyanate-conjugated secondary antibodies against mouse IgM were from Jackson Immunoresearch Laboratories (West Grove, PA). FuGENE6 transfection reagent was from Roche Applied Science (Indianapolis, IN). Filipin, FITC lentil lectin, methyl-beta-cyclodextrin (mbeta-CD), fatty-acid free bovine serum albumin (BSA), chymostatin, leupeptin, antipain, and pepstatin A, cell culture media, cholesterol, and other unlabeled lipids were from Sigma-Aldrich (St. Louis, MO). [9,10(n)-3H]Oleic acid (specific activity 7.5 Ci/mmol), cholesteryl[1-14C]oleate (specific activity 57 mCi/mmol), [4-14C]cholesterol (specific activity 55 mCi/mmol), and [3H]acetic acid (specific activity 9 Ci/mmol) were from Amersham Biosciences (Piscataway, NJ). Alexa 568-conjugated anti-IgG secondary antibodies, Texas Red transferrin, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine-perchlorate --labeled low-density lipoproteins (DiI-LDLs), and Alexa 594-conjugated cholera toxin subunit B (CTxB) were from Molecular Probes (Eugene, OR). Pyrenyldecanoylsphingomyelin (Pyr10SM) was prepared as described previously . gamma-Cyclodextrin (gamma-CD) was from Cyclodextrin Technologies Development (High Springs, FL), and 3-beta-[2-(diethylamino)ethoxy]-androst-5-en-17-one (U18666A) was from Upjohn (Puurs, Belgium). Biotin-2xFYVE was a generous gift from Harald Stenmark. Cell Culture and Transfections | COS-1 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mug/ml streptomycin. F92-99 control fibroblasts and 93.41 NPC fibroblasts were obtained and cultured as described previously . Cells were transfected using FuGENE6 according to the manufacturer's instructions and used for experiments at 40 --48 h (COS-1 cells) or 65 --72 h (human primary fibroblasts) posttransfection. DNA Constructs | pEGFP-C3 was from (Palo Alto, CA). Green fluorescent protein (GFP)-wtRab5, GFP-wtRab6, GFP-wtRab7, and GFP-wtRab11 were as described previously . GFP-Rab5Q79L, GFP-Rab11Q70L, and GFP-Rab11S25N were generous gifts from Marino Zerial (Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany). The human TfR cDNA was in pCDNA3.1. Human LDL-receptor cDNA in pCB6 and human NPC1 in pCR3.1 have been described previously . Cholesterol Esterification Assays | Cells on 12-well plates were transfected, and the following day the medium was replaced with fresh culture medium. Alternatively, to deplete cholesterol the cells were incubated with medium containing 5% lipoprotein-deficient serum (LPDS), prepared as in for 24 h before labeling. To analyze esterification in the presence of LDL, cells grown in culture medium were washed with phosphate-buffered saline (PBS) and labeled with [3H]oleic acid (5 muCi/ml) in serum-free, 2% defatted BSA medium supplemented with 50 mug/ml LDL for 4 h. After labeling, the cells were washed with ice-cold PBS on ice and scraped into PBS, harvested by centrifugation, and resuspended in 2% NaCl. Aliquots were removed for determining the protein concentration. A chromatography recovery standard was added (2.5 --5 nCi of [14C]cholesteryl oleate) and the lipids extracted with 2 ml of methanol and 1 ml of chloroform as described previously . After subsequent centrifugation, 1/10 of the supernatant was removed for liquid scintillation counting to determine the [14C]cholesteryl oleate radioactivity. The extracted lipids were separated by thin layer chromatography on silica gel plates by using hexane/diethyl ether/acetic acid (80:20:1) as the solvent. The cholesteryl ester band was determined based on the comigration of a cholesteryl ester standard, scraped, and 3H and 14C radioactivity measured by liquid scintillation counting. The results were corrected for the volume and procedural losses based on the recovery of 14C radioactivity and plotted against the total amount of protein in the sample. The protein concentration was determined according to . To analyze esterification in delipidated cells, cells grown in 5% LPDS medium for 24 h were washed with PBS and labeled with [3H]oleic acid (5 muCi/ml) in serum-free, 2% defatted BSA medium for 4 h. Lipids were analyzed as described above. To analyze esterification in cells loaded with cholesterol/mbeta-CD-complex the cells were initially delipidated as described above and labeled with [3H]oleic acid (5 muCi/ml) in serum-free, 2% defatted BSA medium for 4 h. During the labeling, cholesterol/mbeta-CD-complex prepared as described previously was added at 50 mug/ml concentration of cholesterol at staggered time points to yield the final loading times indicated. The basal rate of esterification as determined by samples labeled without cholesterol/mbeta-CD-complex was subtracted from the values at all time points. Western Blot Analysis | Cells were harvested in 1% Nonidet-P40 in PBS supplemented with protease inhibitors (chymostatin, leupeptin, antipain, and pepstatin, at 25 mug/ml each). Aliquots of the cell lysate (20 mug of protein) were separated by SDS-PAGE, and the proteins were transferred to Hybond-C Extra membrane (Amersham Biosciences). After blocking with 5% nonfat milk in Tris-buffered saline containing 0.2% Tween 20 for 1 h at 37C, the membrane was incubated overnight at 4C with rabbit polyclonal anti-GFP antibodies . The membrane was then washed and incubated with horseradish peroxidase-conjugated anti-IgG secondary antibodies (, Hercules, CA). The staining was visualized using enhanced chemiluminescence Western blotting detection reagent (Amersham Biosciences). Immunocytochemistry | The cells were fixed with 4% paraformaldehyde for 20 min and quenched with 50 mM NH4Cl for 10 min. Cells were permeabilized either with 0.1% Triton X-100 for 4 min and blocked with 10% FBS for 30 min at 37C, or alternatively the blocking solution was supplemented with 0.05% filipin to permeabilize the cells. The primary antibodies were diluted in 5% FBS and incubated for 1 h at 37C or overnight at 4C and the secondary antibodies for 30 min at 37C. For filipin staining only, the fixed and quenched cells were incubated with 0.05% filipin in PBS for 15 min and washed with PBS. The coverslips were mounted with Mowiol and the antifading reagent 1,4 diazobicyclo-(2.2.2) octane and viewed with TCS SP confocal microscope (, Deerfield, IL), Axiophot photomicroscope (Carl Zeiss, Thornwood, NY), or IX70 inverted microscope (, Tokyo, Japan) equipped with a Polychrome IV monochromator (TILL Photonics, Eugene, OR) with appropriate filters. Labeling with Texas Red Transferrin | Cells double transfected with the indicated cDNAs and TfR were starved in serum-free culture medium for 1 h at 37C. Cells were then incubated with 50 mug/ml Texas Red transferrin in Eagle's minimum essential medium supplemented with 0.2% BSA, 0.35 g/l NaHCO3, 100 U/ml penicillin, 100 mug/ml streptomycin, and 10 mM HEPES, pH 7.4, for 30 min on ice at 4C. After labeling the cells were incubated in serum-free culture medium supplemented with 0.2% BSA for 30 min at 37C, fixed, and processed for immunofluorescence microscopy as described above. Labeling with Biotin-2xFYVE | Cells fixed and quenched as described above were blocked and permeabilized with 10% FBS supplemented with 0.05% filipin for 30 min. Cells were then incubated with 50 mug/ml biotin-2xFYVE in 10% FBS for 30 min at room temperature, washed 3 x 5 min with PBS, and further incubated with 1 mug/ml Cy3-conjugated streptavidin in 10% FBS for 30 min, washed 3 x 5 min with PBS, and mounted. Labeling with Alexa 594-conjugated CTxB | Cells were incubated with 2 mug/ml Alexa 594-conjugated CTxB in Eagle's minimum essential medium supplemented with 0.01% BSA, 0.35 g/l NaHCO3, 100 U/ml penicillin, 100 mug/ml streptomycin, and 10 mM HEPES, pH 7.4, for 1 h on ice at 4C. After labeling the cells were incubated in serum-free culture medium supplemented with 0.01% BSA for 2 h at 37C and fixed. Labeling with Pyr10SM | To prepare the Pyr10SM/gamma-CD-complex the lipid was dried under argon and desiccated in the vacuum for 30 min. gamma-CD (100 mM in PBS) was added on the lipid film in a molar ratio of 1000:1, and the suspension was sonicated 3 x 2 min . Cells were labeled with Pyr10SM/gamma-CD-complex at 10 nmol/ml concentration of the lipid for 10 min at 37C and incubated in culture medium for 2 h at 37C before fixation. The fluorescence was excited at 345 nm and visualized at 480/80 nm. The degradation rate of Pyr10SM in COS-1 cells was determined by high-performance liquid chromatography using on-line fluorescence detection . Electron Microscopy | Cells were fixed with 4% paraformaldehyde in 0.25 M HEPES pH 7.4, scraped, and infiltrated in 1.75 M sucrose in 0.25 M HEPES containing 4% paraformaldehyde for 48 h at 4C. Droplets of cells in sucrose were mounted on pins and frozen in liquid nitrogen. Ultrathin cryosections were labeled with polyclonal rabbit anti-GFP antibody (a generous gift from Graham Warren (Yale University School of Medicine, New Haven, CT) and David Shima (Imperial Cancer Research Fund, London, UK), followed by protein A coupled to 10-nm gold particles. Sections were examined and photographed at 80 kV with a 1200 EX electron microscope (, Tokyo, Japan). Labeling with DiI-LDL | Cells were incubated in medium containing 5% LPDS for 24 h then labeled with 10 mug/ml DiI-LDL in serum-free medium for 15 min at 37C. After washing with PBS, the cells were either fixed or further incubated in serum-free medium for 2 h at 37C before fixation. Cholesterol Biosynthesis | Cells on six-well plates were transfected and at 6 h posttranfection the culture medium was changed to medium supplemented with 5% LPDS and [14C]cholesterol (100 nCi/ml) for 41 h. The cells were washed with PBS, pulse labeled with [3H]acetic acid (250 muCi/ml) in serum-free medium for 15 min at 37C, and chased in serum-free medium supplemented with 10 muM lovastatin and 25 mM mevalonate for 90 min at 37C. The cells were washed with ice-cold PBS on ice, scraped into PBS, harvested by centrifugation, and resuspended in 2% NaCl. Aliquots were removed for determining the protein concentration. Lipids were extracted as described above, separated by thin layer chromatography, and analyzed by high-performance liquid chromatography as described previously . Nascent cholesterol was quantified as 3H radioactivity in the cholesterol peak, corrected for the volume and procedural losses based on the recovery of 14C radioactivity, and plotted against the amount of protein in the sample. PMID- 12221119_RESULTS TI - AB - Redistribution of Free Cholesterol and Inhibition of Cholesterol Esterification upon Overexpression of Endosomal Rab Proteins | COS-1 cells were transiently transfected for 40 --48 h with GFP-fusions of Rab proteins reported to regulate early, late, or recycling endocytic transport events, represented by Rab5, 7, and 11, respectively. Rab5 promotes homotypic fusion of early endosomes . Overexpression of the late endosomal Rab7, on the other hand, has been shown to affect early-to-late endosomal transport and lysosome biogenesis , whereas Rab11 regulates the function of the recycling endosomes . Rab6 that is involved in retrograde trafficking in the Golgi , and soluble GFP were used as controls. To visualize the distribution of free cholesterol, the cells were fixed and stained with the fluorescent sterol-binding antibiotic filipin. In COS cells, the perinuclear area of the cell is strongly filipin positive. In addition, the plasma membrane and punctate peripheral structures are stained, albeit at lower intensity . The prominent perinuclear filipin staining colocalizes with a Golgi marker lentil lectin but several filipin-positive punctae also colocalize with lysosomal or early endosomal markers as visualized by antibodies against lysosomal membrane protein lamp1, or labeling with peptide 2xFYVE that binds the early endosomal phosphatidylinositol-(3)-phosphate (PI-3-P) . The perinuclear aspect of endogenous Rab11 staining also partially overlaps with that of filipin. However, the small peripheral Rab11-positive dots are not resolved by filipin staining. The filipin staining pattern characteristic of untransfected cells was also seen in cells expressing soluble GFP, Rab6, or Rab7 . In Rab5-overexpressing cells, numerous brightly filipin-positive peripheral dots were observed. These structures were also positive for Rab5, indicating that they represent early endosomes . The most pronounced redistribution of filipin staining was seen in Rab11-overexpressing cells. In these cells, intensely filipin stained and Rab11-positive tubular elements extending to the cell periphery were observed . For Rab5, the effects of the GTPase-deficient mutant (Rab5Q79L) are significantly more pronounced than that of the wild-type protein, resulting in massive enlargement of early endosomes . Considering the moderate effect of wtRab5 on filipin staining pattern we also analyzed the effect of Rab5Q79L. In cells expressing this protein, early endosomes became heavily enlarged and their membranes were intensely filipin positive . Figure 1 | Distribution of free cholesterol in COS-1 cells. Distribution of free cholesterol in COS-1 cells. Left, cells were stained with FITC-conjugated lentil lectin, anti-lamp1 antibodies, biotin-2xFYVE, and Cy3-conjugated streptavidin or anti-Rab11 antibodies. Right, filipin stainings of the respective cells. The arrowheads indicate colocalization of endosomal markers with filipin staining. Images were obtained using wide-field microscope. Bar, 8 mum. Figure 2 | Distribution of free cholesterol in COS-1 cells overexpressing soluble GFP, GFP-fused wtRab5, wtRab6, wtRab7, wtRab11, and Rab5Q79L. Distribution of free cholesterol in COS-1 cells overexpressing soluble GFP, GFP-fused wtRab5, wtRab6, wtRab7, wtRab11, and Rab5Q79L. Left, cells overexpressing GFP or GFP-fused Rab proteins. Right, filipin stainings of the respective cells. The arrowheads indicate free cholesterol redistributed in GFP-Rab --positive organelles. Images were obtained using wide-field microscope. Bar 8, mum. To test whether overexpression of the Rab proteins was accompanied by biochemical effects on cholesterol homeostasis, we analyzed cholesterol esterification by measuring the incorporation of [3H]oleic acid into cholesteryl esters at 40 --48 h posttransfection. The values obtained with Rab overexpressions were compared with those obtained with overexpressed GFP alone. Strikingly, overexpression of the Rabs with the most pronounced effects on filipin distribution, Rab5Q79L and Rab11, also caused the strongest inhibition in cholesterol esterification (similar50% inhibition with both; Figure A). Overexpression of Rab7 was slightly inhibitory (25 --30% inhibition), whereas Rab6 overexpression was without effect. The expression levels of the individual Rabs were closely similar with the 50 --70% transfection frequencies obtained, as assessed by Western blotting with anti-GFP antibodies (Figure B). The effects of the individual Rabs on cholesterol esterification could be observed already at 24 h posttransfection (our unpublished data). Furthermore, the Rab11-induced redistribution of cholesterol was morphologically apparent already at 8 h posttransfection, at a stage when the GFP-Rab11 decorated small punctate structures throughout the cytoplasm (Figure A). By 16 h of transfection, the Rab11- and filipin-positive organelles had attained a more tubular appearance and by 24 h, larger vesicular and tubular profiles containing both Rab11 and cholesterol were generated (Figure A). Figure 3 | Rate of cholesterol esterification in COS-1 cells overexpressing soluble GFP, GFP-fused Rab5Q79L, wtRab6, wtRab7, and wtRab11. Rate of cholesterol esterification in COS-1 cells overexpressing soluble GFP, GFP-fused Rab5Q79L, wtRab6, wtRab7, and wtRab11. (A) Transfected cells grown in culture medium were pulsed for 4 h with [3H]oleic acid in the presence of 50 mug/ml LDL. The rate of esterification is shown as percentage of esterification in cells transfected with GFP alone. Each bar represents three to nine samples from one to four individual experiments; the SEs are indicated. The mean rate of esterification in the GFP control sample was 34 dpm/mug protein/h. (B) Western blot analysis of GFP and different GFP-fused Rab proteins. The cells were transfected for 40 h, and the cell lysate was separated by SDS-PAGE and analyzed by immunoblotting with anti-GFP antibodies. Figure 4 | (A) Formation of GFP-Rab11 --positive organelles with increasing expression times. (A) Formation of GFP-Rab11 --positive organelles with increasing expression times. COS-1 cells overexpressing GFP-wtRab11 were fixed 8, 16, or 24 h posttransfection and stained with filipin. The areas indicated in the top panels are shown in the bottom panels. The arrowheads indicate GFP-wtRab11 -- and filipin-positive organelles. (B) Distribution of TfR and Texas Red transferrin in COS-1 cells coexpressing GFP-wtRab11 and TfR. Cells overexpressing GFP or GFP-wtRab11 (left) and TfR as visualized by anti-TfR antibody. (C) Localization of transferrin in the cells overexpressing GFP-wtRab11 and TfR (unpublished data). The cells were labeled with Texas Red transferrin for 30 min on ice and transferrin internalized for 30 min at 37C. Images in A were obtained using wide-field microscope. Images in B and C are confocal and represent a single focal plane. Bar, 8 mum. Rab11-positive Organelles Accumulate Transferrin Receptor and Internalized Transferrin | In baby hamster kidney and Chinese hamster ovary cells, Rab11 localizes with internalized transferrin in the pericentriolar recycling compartment and in HeLa cells, Rab11 overexpression leads to morphological alterations of