PMID- 10984461 OWN - NLM STAT- MEDLINE DA - 20001016 DCOM- 20001016 LR - 20041117 PUBM- Print IS - 1067-5027 VI - 7 IP - 5 DP - 2000 Sep-Oct TI - Opportunities at the intersection of bioinformatics and health informatics: a case study. PG - 431-8 AB - This paper provides a "viewpoint discussion" based on a presentation made to the 2000 Symposium of the American College of Medical Informatics. It discusses potential opportunities for researchers in health informatics to become involved in the rapidly growing field of bioinformatics, using the activities of the Yale Center for Medical Informatics as a case study. One set of opportunities occurs where bioinformatics research itself intersects with the clinical world. Examples include the correlations between individual genetic variation with clinical risk factors, disease presentation, and differential response to treatment; and the implications of including genetic test results in the patient record, which raises clinical decision support issues as well as legal and ethical issues. A second set of opportunities occurs where bioinformatics research can benefit from the technologic expertise and approaches that informaticians have used extensively in the clinical arena. Examples include database organization and knowledge representation, data mining, and modeling and simulation. Microarray technology is discussed as a specific potential area for collaboration. Related questions concern how best to establish collaborations with bioscientists so that the interests and needs of both sets of researchers can be met in a synergistic fashion, and the most appropriate home for bioinformatics in an academic medical center. AD - Yale University, New Haven, Connecticut, USA. FAU - Miller, P L AU - Miller PL LA - eng GR - G08-LM-05583/LM/NLM PT - Journal Article PL - UNITED STATES TA - J Am Med Inform Assoc JID - 9430800 SB - IM CIN - J Am Med Inform Assoc. 2000 Sep-Oct;7(5):512-6. PMID: 10984470 MH - *Computational Biology/organization & administration MH - Cooperative Behavior MH - Decision Support Systems, Clinical MH - Gene Expression MH - Genome MH - *Medical Informatics/organization & administration MH - Neurosciences MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2000/09/14 11:00 MHDA- 2000/10/21 11:01 PST - ppublish SO - J Am Med Inform Assoc 2000 Sep-Oct;7(5):431-8. PMID- 10984462 OWN - NLM STAT- MEDLINE DA - 20001016 DCOM- 20001016 LR - 20041117 PUBM- Print IS - 1067-5027 VI - 7 IP - 5 DP - 2000 Sep-Oct TI - The interactions between clinical informatics and bioinformatics: a case study. PG - 439-43 AB - For the past decade, Stanford Medical Informatics has combined clinical informatics and bioinformatics research and training in an explicit way. The interest in applying informatics techniques to both clinical problems and problems in basic science can be traced to the Dendral project in the 1960s. Having bioinformatics and clinical informatics in the same academic unit is still somewhat unusual and can lead to clashes of clinical and basic science cultures. Nevertheless, the benefits of this organization have recently become clear, as the landscape of academic medicine in the next decades has begun to emerge. The author provides examples of technology transfer between clinical informatics and bioinformatics that illustrate how they complement each other. AD - Stanford University, Stanford, California 94305-5479, USA. russ.altman@stanford.edu FAU - Altman, R B AU - Altman RB LA - eng GR - GM-61374/GM/NIGMS GR - LM-05652/LM/NLM GR - LM-06422/LM/NLM PT - Journal Article PL - UNITED STATES TA - J Am Med Inform Assoc JID - 9430800 SB - IM CIN - J Am Med Inform Assoc. 2000 Sep-Oct;7(5):512-6. PMID: 10984470 MH - Academic Medical Centers/organization & administration MH - California MH - *Computational Biology/education/methods/organization & administration MH - Cooperative Behavior MH - *Medical Informatics/organization & administration MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - *Technology Transfer EDAT- 2000/09/14 11:00 MHDA- 2000/10/21 11:01 PST - ppublish SO - J Am Med Inform Assoc 2000 Sep-Oct;7(5):439-43. PMID- 10984463 OWN - NLM STAT- MEDLINE DA - 20001016 DCOM- 20001016 LR - 20001218 PUBM- Print IS - 1067-5027 VI - 7 IP - 5 DP - 2000 Sep-Oct TI - Electronic health record meets digital library: a new environment for achieving an old goal. PG - 444-52 AB - Linking the electronic health record to the digital library is a Web-era reformulation of the long-standing informatics goal of seamless integration of automated clinical data and relevant knowledge-based information to support informed decisions. The spread of the Internet, the development of the World Wide Web, and converging format standards for electronic health data and digital publications make effective linking increasingly feasible. Some existing systems link electronic health data and knowledge-based information in limited settings or limited ways. Yet many challenging informatics research problems remain to be solved before flexible and seamless linking becomes a reality and before systems become capable of delivering the specific piece of information needed at the time and place a decision must be made. Connecting the electronic health record to the digital library also requires positive resolution of important policy issues, including health data privacy, government encouragement of high-speed communications, electronic intellectual property rights, and standards for health data and for digital libraries. Both the research problems and the policy issues should be important priorities for the field of medical informatics. AD - Library of Medicine, Bethesda, Maryland 20894, USA. blh@nlm.nih.gov FAU - Humphreys, B L AU - Humphreys BL LA - eng PT - Journal Article PL - UNITED STATES TA - J Am Med Inform Assoc JID - 9430800 SB - IM MH - *Internet/standards MH - Libraries/*organization & administration/standards MH - Medical Records Systems, Computerized/*organization & administration MH - Publishing/*organization & administration/standards MH - Systems Integration EDAT- 2000/09/14 11:00 MHDA- 2000/10/21 11:01 PST - ppublish SO - J Am Med Inform Assoc 2000 Sep-Oct;7(5):444-52. PMID- 10984464 OWN - NLM STAT- MEDLINE DA - 20001016 DCOM- 20001016 LR - 20001218 PUBM- Print IS - 1067-5027 VI - 7 IP - 5 DP - 2000 Sep-Oct TI - Improving clinical communication: a view from psychology. PG - 453-61 AB - Recent research has studied the communication behaviors of clinical hospital workers and observed a tendency for these workers to use communication behaviors that were often inefficient. Workers were observed to favor synchronous forms of communication, such as telephone calls and chance face-to-face meetings with colleagues, even when these channels were not effective. Synchronous communication also contributes to a highly interruptive working environment, increasing the potential for clinical errors to be made. This paper reviews these findings from a cognitive psychological perspective, focusing on current understandings of how human memory functions and on the potential consequences of interruptions on the ability to work effectively. It concludes by discussing possible communication technology interventions that could be introduced to improve the clinical communication environment and suggests directions for future research. AD - Hewlett-Packard Research Laboratories, Bristol, England (JSP). jsp@hplb.hpl.hp.com FAU - Parker, J AU - Parker J FAU - Coiera, E AU - Coiera E LA - eng PT - Journal Article PL - UNITED STATES TA - J Am Med Inform Assoc JID - 9430800 SB - IM MH - Attitude of Health Personnel MH - Cognitive Science MH - *Communication MH - Memory MH - Personnel, Hospital/*psychology EDAT- 2000/09/14 11:00 MHDA- 2000/10/21 11:01 PST - ppublish SO - J Am Med Inform Assoc 2000 Sep-Oct;7(5):453-61. PMID- 10984465 OWN - NLM STAT- MEDLINE DA - 20001016 DCOM- 20001016 LR - 20041117 PUBM- Print IS - 1067-5027 VI - 7 IP - 5 DP - 2000 Sep-Oct TI - Comparative evaluation of three continuous speech recognition software packages in the generation of medical reports. PG - 462-8 AB - OBJECTIVE: To compare out-of-box performance of three commercially available continuous speech recognition software packages: IBM ViaVoice 98 with General Medicine Vocabulary; Dragon Systems NaturallySpeaking Medical Suite, version 3.0; and L&H Voice Xpress for Medicine, General Medicine Edition, version 1.2. DESIGN: Twelve physicians completed minimal training with each software package and then dictated a medical progress note and discharge summary drawn from actual records. MEASUREMENTS: Errors in recognition of medical vocabulary, medical abbreviations, and general English vocabulary were compared across packages using a rigorous, standardized approach to scoring. RESULTS: The IBM software was found to have the lowest mean error rate for vocabulary recognition (7.0 to 9.1 percent) followed by the L&H software (13.4 to 15.1 percent) and then Dragon software (14.1 to 15.2 percent). The IBM software was found to perform better than both the Dragon and the L&H software in the recognition of general English vocabulary and medical abbreviations. CONCLUSION: This study is one of a few attempts at a robust evaluation of the performance of continuous speech recognition software. Results of this study suggest that with minimal training, the IBM software outperforms the other products in the domain of general medicine; however, results may vary with domain. Additional training is likely to improve the out-of-box performance of all three products. Although the IBM software was found to have the lowest overall error rate, successive generations of speech recognition software are likely to surpass the accuracy rates found in this investigation. AD - Boston Veterans Administration Medical Center, Boston, Massachusetts 02130, USA. devine.eric@boston.va.gov FAU - Devine, E G AU - Devine EG FAU - Gaehde, S A AU - Gaehde SA FAU - Curtis, A C AU - Curtis AC LA - eng PT - Journal Article PL - UNITED STATES TA - J Am Med Inform Assoc JID - 9430800 SB - IM MH - Comparative Study MH - Evaluation Studies MH - Humans MH - *Medical Records Systems, Computerized MH - Research Support, U.S. Gov't, Non-P.H.S. MH - *Software MH - *Speech EDAT- 2000/09/14 11:00 MHDA- 2000/10/21 11:01 PST - ppublish SO - J Am Med Inform Assoc 2000 Sep-Oct;7(5):462-8. PMID- 10984466 OWN - NLM STAT- MEDLINE DA - 20001016 DCOM- 20001016 LR - 20041117 PUBM- Print IS - 1067-5027 VI - 7 IP - 5 DP - 2000 Sep-Oct TI - Variables that may enhance medical students' perceived preparedness for computer-based testing. PG - 469-74 AB - OBJECTIVE: To identify variables that may enhance medical student's preparedness for computer-based administration of the United States Medical Licensing Examination (USMLE). DESIGN: A cross-sectional survey of 301 medical students who completed a self-administered questionnaire. MEASUREMENTS: The questionnaire was designed to obtain information about students' computer resources, personal experience with computers, computer expertise, opinions about computers, experience with computer-based testing, perceived preparedness for the computer-based USMLE, and demographic variables. Variables related to students' perceived preparedness for the computer-based USMLE were identified by ordinal logistic regression. RESULTS: A significant regression model yielded four significant predictors: perceived preparedness for USMLE content (P: < 0.0001), opinions about computers (P: < 0.0012), gender (P: < 0.0001), and a gender by computer-based testing experience interaction (P: < 0. 0004). Computer resources, personal experience with computers, computer expertise, age, race, and year of medical school were not significant predictors. CONCLUSION: Students' perceived preparedness for computer-based administration of high-stakes examinations may be facilitated by preparing them for examination content, by enhancing their opinions about computers, and by increasing their computer-based testing experiences. AD - East Carolina University, Greenville, North Carolina, USA. dlynch@med-scape.com FAU - Lynch, D C AU - Lynch DC FAU - Whitley, T W AU - Whitley TW FAU - Emmerling, D A AU - Emmerling DA FAU - Brinn, J E AU - Brinn JE LA - eng PT - Journal Article PL - UNITED STATES TA - J Am Med Inform Assoc JID - 9430800 SB - IM MH - Attitude to Computers MH - *Computer Literacy MH - Computer User Training MH - Continental Population Groups MH - Cross-Sectional Studies MH - Educational Measurement/*methods MH - Female MH - Humans MH - Licensure, Medical MH - Logistic Models MH - Male MH - Questionnaires MH - Sex Factors MH - *Students, Medical/psychology MH - United States EDAT- 2000/09/14 11:00 MHDA- 2000/10/21 11:01 PST - ppublish SO - J Am Med Inform Assoc 2000 Sep-Oct;7(5):469-74. PMID- 10984467 OWN - NLM STAT- MEDLINE DA - 20001016 DCOM- 20001016 LR - 20041117 PUBM- Print IS - 1067-5027 VI - 7 IP - 5 DP - 2000 Sep-Oct TI - Exploring performance issues for a clinical database organized using an entity-attribute-value representation. PG - 475-87 AB - BACKGROUND: The entity-attribute-value representation with classes and relationships (EAV/CR) provides a flexible and simple database schema to store heterogeneous biomedical data. In certain circumstances, however, the EAV/CR model is known to retrieve data less efficiently than conventionally based database schemas. OBJECTIVE: To perform a pilot study that systematically quantifies performance differences for database queries directed at real-world microbiology data modeled with EAV/CR and conventional representations, and to explore the relative merits of different EAV/CR query implementation strategies. METHODS: Clinical microbiology data obtained over a ten-year period were stored using both database models. Query execution times were compared for four clinically oriented attribute-centered and entity-centered queries operating under varying conditions of database size and system memory. The performance characteristics of three different EAV/CR query strategies were also examined. RESULTS: Performance was similar for entity-centered queries in the two database models. Performance in the EAV/CR model was approximately three to five times less efficient than its conventional counterpart for attribute-centered queries. The differences in query efficiency became slightly greater as database size increased, although they were reduced with the addition of system memory. The authors found that EAV/CR queries formulated using multiple, simple SQL statements executed in batch were more efficient than single, large SQL statements. CONCLUSION: This paper describes a pilot project to explore issues in and compare query performance for EAV/CR and conventional database representations. Although attribute-centered queries were less efficient in the EAV/CR model, these inefficiencies may be addressable, at least in part, by the use of more powerful hardware or more memory, or both. AD - Yale University, New Haven, Connecticut, USA. FAU - Chen, R S AU - Chen RS FAU - Nadkarni, P AU - Nadkarni P FAU - Marenco, L AU - Marenco L FAU - Levin, F AU - Levin F FAU - Erdos, J AU - Erdos J FAU - Miller, P L AU - Miller PL LA - eng GR - G08-LM05583/LM/NLM GR - T15-LM07056/LM/NLM GR - U01-CA78266/CA/NCI PT - Journal Article PL - UNITED STATES TA - J Am Med Inform Assoc JID - 9430800 SB - IM MH - Comparative Study MH - *Database Management Systems MH - Databases/organization & administration MH - *Databases, Factual MH - Information Storage and Retrieval/*methods MH - Microbiology MH - Pilot Projects MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2000/09/14 11:00 MHDA- 2000/10/21 11:01 PST - ppublish SO - J Am Med Inform Assoc 2000 Sep-Oct;7(5):475-87. PMID- 10984468 OWN - NLM STAT- MEDLINE DA - 20001016 DCOM- 20001016 LR - 20041117 PUBM- Print IS - 1067-5027 VI - 7 IP - 5 DP - 2000 Sep-Oct TI - GEM: a proposal for a more comprehensive guideline document model using XML. PG - 488-98 AB - OBJECTIVE: To develop a guideline document model that includes a sufficiently broad set of concepts to be useful throughout the guideline life cycle. DESIGN: Current guideline document models are limited in that they reflect the specific orientation of the stakeholder who created them; thus, developers and disseminators often provide few constructs for conceptualizing recommendations, while implementers de-emphasize concepts related to establishing guideline validity. The authors developed the Guideline Elements Model (GEM) using XML to better represent the heterogeneous knowledge contained in practice guidelines. Core constructs were derived from the Institute of Medicine's Guideline Appraisal Instrument, the National Guideline Clearinghouse, and the augmented decision table guideline representation. These were supplemented by additional concepts from a literature review. RESULTS: The GEM hierarchy includes more than 100 elements. Major concepts relate to a guideline's identity, developer, purpose, intended audience, method of development, target population, knowledge components, testing, and review plan. Knowledge components in guideline documents include recommendations (which in turn comprise conditionals and imperatives), definitions, and algorithms. CONCLUSION: GEM is more comprehensive than existing models and is expressively adequate to represent the heterogeneous information contained in guidelines. Use of XML contributes to a flexible, comprehensible, shareable, and reusable knowledge representation that is both readable by human beings and processible by computers. AD - Yale University, New Haven, Connecticut, USA. richard.shiffman@yale.edu FAU - Shiffman, R N AU - Shiffman RN FAU - Karras, B T AU - Karras BT FAU - Agrawal, A AU - Agrawal A FAU - Chen, R AU - Chen R FAU - Marenco, L AU - Marenco L FAU - Nath, S AU - Nath S LA - eng GR - 1-R29-LM-05552/LM/NLM GR - T15-LM07056/LM/NLM PT - Journal Article PL - UNITED STATES TA - J Am Med Inform Assoc JID - 9430800 SB - IM MH - Algorithms MH - *Hypermedia MH - Models, Theoretical MH - *Practice Guidelines MH - *Programming Languages MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2000/09/14 11:00 MHDA- 2000/10/21 11:01 PST - ppublish SO - J Am Med Inform Assoc 2000 Sep-Oct;7(5):488-98. PMID- 10984469 OWN - NLM STAT- MEDLINE DA - 20001016 DCOM- 20001016 LR - 20001218 PUBM- Print IS - 1067-5027 VI - 7 IP - 5 DP - 2000 Sep-Oct TI - Corpus-based statistical screening for phrase identification. PG - 499-511 AB - PURPOSE: The authors study the extraction of useful phrases from a natural language database by statistical methods. The aim is to leverage human effort by providing preprocessed phrase lists with a high percentage of useful material. METHOD: The approach is to develop six different scoring methods that are based on different aspects of phrase occurrence. The emphasis here is not on lexical information or syntactic structure but rather on the statistical properties of word pairs and triples that can be obtained from a large database. MEASUREMENTS: The Unified Medical Language System (UMLS) incorporates a large list of humanly acceptable phrases in the medical field as a part of its structure. The authors use this list of phrases as a gold standard for validating their methods. A good method is one that ranks the UMLS phrases high among all phrases studied. Measurements are 11-point average precision values and precision-recall curves based on the rankings. RESULT: The authors find of six different scoring methods that each proves effective in identifying UMLS quality phrases in a large subset of MEDLINE. These methods are applicable both to word pairs and word triples. All six methods are optimally combined to produce composite scoring methods that are more effective than any single method. The quality of the composite methods appears sufficient to support the automatic placement of hyperlinks in text at the site of highly ranked phrases. CONCLUSION: Statistical scoring methods provide a promising approach to the extraction of useful phrases from a natural language database for the purpose of indexing or providing hyperlinks in text. AD - National Library of Medicine, Bethesda, Maryland 20894, USA. wonkim@ncbi.nlm.nih.gov FAU - Kim, W AU - Kim W FAU - Wilbur, W J AU - Wilbur WJ LA - eng PT - Journal Article PL - UNITED STATES TA - J Am Med Inform Assoc JID - 9430800 SB - IM MH - *Abstracting and Indexing MH - *Hypermedia MH - Information Storage and Retrieval/*methods MH - MEDLINE MH - *Natural Language Processing MH - Statistics MH - *Unified Medical Language System MH - Vocabulary, Controlled EDAT- 2000/09/14 11:00 MHDA- 2000/10/21 11:01 PST - ppublish SO - J Am Med Inform Assoc 2000 Sep-Oct;7(5):499-511. PMID- 10984470 OWN - NLM STAT- MEDLINE DA - 20001016 DCOM- 20001016 LR - 20041117 PUBM- Print IS - 1067-5027 VI - 7 IP - 5 DP - 2000 Sep-Oct TI - Bioinformatics and clinical informatics: the imperative to collaborate. PG - 512-6 FAU - Kohane, I S AU - Kohane IS LA - eng PT - Comment PT - Editorial PL - UNITED STATES TA - J Am Med Inform Assoc JID - 9430800 SB - IM CON - J Am Med Inform Assoc. 2000 Sep-Oct;7(5):431-8. PMID: 10984461 CON - J Am Med Inform Assoc. 2000 Sep-Oct;7(5):439-43. PMID: 10984462 MH - *Computational Biology/organization & administration MH - Confidentiality MH - Cooperative Behavior MH - Costs and Cost Analysis MH - False Positive Reactions MH - Genome, Human MH - Humans MH - *Medical Informatics/organization & administration MH - Vocabulary, Controlled EDAT- 2000/09/14 11:00 MHDA- 2000/10/21 11:01 PST - ppublish SO - J Am Med Inform Assoc 2000 Sep-Oct;7(5):512-6. PMID- 11818032 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021125 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 TI - Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome. PG - 2 AB - BACKGROUND: In humans, there are four alkaline phosphatases, and each form exhibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnant with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. RESULTS: To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity. CONCLUSION: Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome. AD - Laboratoire de Synthese et Physicochimie de Molecules d'Interet Biologique, Universite Paul Sabatier (UMR 5068), 118 route de Narbonne, 31 062, Toulouse, France. denier@cict.fr FAU - Denier, Colette C AU - Denier CC FAU - Brisson-Lougarre, Andree A AU - Brisson-Lougarre AA FAU - Biasini, Ghislaine G AU - Biasini GG FAU - Grozdea, Jean J AU - Grozdea JJ FAU - Fournier, Didier D AU - Fournier DD LA - eng PT - Journal Article DEP - 20020115 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Enzyme Inhibitors) RN - 0 (Isoenzymes) RN - 0 (Nitrophenols) RN - 0 (Organophosphorus Compounds) RN - 0 (Phosphates) RN - 0 (carcinoplacental isoenzymes) RN - 13598-51-1 (thiophosphoric acid) RN - 330-13-2 (nitrophenylphosphate) RN - 5036-02-2 (Tetramisole) RN - 6646-46-4 (4-bromotetramisole) RN - EC 3.1.3.1 (Alkaline Phosphatase) SB - IM MH - Alkaline Phosphatase/*analysis/genetics/metabolism MH - Baculoviridae/genetics MH - Comparative Study MH - Down Syndrome/diagnosis MH - Enzyme Inhibitors/chemistry/pharmacology MH - Enzyme Stability MH - Enzyme Tests MH - Female MH - Humans MH - Isoenzymes/*analysis/genetics/metabolism MH - Kinetics MH - Neutrophils/enzymology MH - Nitrophenols/chemistry/metabolism MH - Organophosphorus Compounds/chemistry/metabolism MH - Phosphates/chemistry/pharmacology MH - Placenta/*enzymology MH - Pregnancy MH - Protein Denaturation MH - Research Support, Non-U.S. Gov't MH - Tetramisole/*analogs & derivatives/chemistry/pharmacology EDAT- 2002/01/31 10:00 MHDA- 2002/11/26 04:00 PHST- 2001/08/23 [received] PHST- 2002/01/15 [accepted] PHST- 2002/01/15 [aheadofprint] PST - ppublish SO - BMC Biochem 2002;3(1):2. Epub 2002 Jan 15. PMID- 11825341 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021125 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 TI - Decreased insulin binding to mononuclear leucocytes and erythrocytes from dogs after S-nitroso-N-acetypenicillamine administration. PG - 1 AB - BACKGROUND: Nitric oxide (NO) and oxygen free-radicals play an important part in the destruction of beta-cells in auto- immune diabetes although the precise mechanism of interaction is still not known. This study was designed to examine any possible diabetogenic effect of NO by investigating any differences in cellular binding of insulin to its receptor on the cell membranes of erythrocytes and mononuclear leucocytes of dogs treated with the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) and controls treated with captopril. RESULTS: The result obtained showed decreased binding of insulin to its receptor on the cell membranes of erythrocytes and mononuclear leucocytes. Mononuclear leucocytes from SNAP-treated dogs had decreased ability to bind insulin (16.30 +/- 1.24 %) when compared to mononuclear leucocytes from captopril-treated controls (20.30 +/- 1.93 %). Similar results were obtained for erythrocytes from dogs treated with SNAP (27.20 +/- 1.33 %) compared with dogs treated with captopril (34.70 +/- 3.58 %). Scatchard analysis demonstrated that this decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with mononuclear leucocytes of SNAP-treated dogs having 55 % less insulin receptor sites per cell compared with those of captopril-treated controls (P < 0.05). Average affinity and kinetic analysis revealed a 35 % decrease in the average receptor affinity, with mononuclear leucocytes from captopril-treated controls having an empty site affinity of 12.36 +/- 1.12 x 10(-8) M(-1) compared with 9.64 +/- 0.11 x 10(-8) M(-1) in SNAP-treated dogs (P < 0.05). CONCLUSION: These results suggest that acute alteration of the insulin receptor on the membranes of mononuclear leucocytes and erythrocytes occurred in dogs treated with S-nitroso-N-acetylpenicillamine. These findings suggest the first evidence of the novel role of NO as a modulator of insulin binding and the involvement of NO in the aetiology of diabetes mellitus. AD - Department of Basic Medical Sciences, University of the West Indies, Kingston, Jamaica. dmcgrowd@yahoo.com FAU - McGrowder, Donovan AU - McGrowder D FAU - Ragoobirsingh, Dalip AU - Ragoobirsingh D FAU - Dasgupta, Tara AU - Dasgupta T LA - eng PT - Journal Article DEP - 20020102 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Nitric Oxide Donors) RN - 11061-68-0 (Insulin) RN - 62571-86-2 (Captopril) RN - 79032-48-7 (S-Nitroso-N-Acetylpenicillamine) RN - EC 2.7.1.112 (Receptor, Insulin) SB - IM MH - Animals MH - Binding, Competitive MH - Captopril/pharmacology MH - Cell Membrane/metabolism MH - Dogs MH - Erythrocytes/drug effects/metabolism MH - Female MH - Insulin/*metabolism MH - Kinetics MH - Leukocytes, Mononuclear/drug effects/metabolism MH - Male MH - Nitric Oxide Donors/*pharmacology MH - Receptor, Insulin/*metabolism MH - Research Support, Non-U.S. Gov't MH - S-Nitroso-N-Acetylpenicillamine/*pharmacology EDAT- 2002/02/05 10:00 MHDA- 2002/11/26 04:00 PHST- 2001/08/06 [received] PHST- 2002/01/02 [accepted] PHST- 2002/01/02 [aheadofprint] PST - ppublish SO - BMC Biochem 2002;3(1):1. Epub 2002 Jan 2. PMID- 11825346 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030424 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 TI - Quality and methods of developing practice guidelines. PG - 1 AB - BACKGROUND: It is not known whether there are differences in the quality and recommendations between evidence-based (EB) and consensus-based (CB) guidelines. We used breast cancer guidelines as a case study to assess for these differences. METHODS: Five different instruments to evaluate the quality of guidelines were identified by a literature search. We also searched MEDLINE and the Internet to locate 8 breast cancer guidelines. These guidelines were classified in three categories: evidence based, consensus based and consensus based with no explicit consideration of evidence (CB-EB). Each guideline was evaluated by three of the authors using each of the instruments. For each guideline we assessed the agreement among 14 decision points which were selected from the NCCN (National Cancer Comprehensive Network) guidelines algorithm. For each decision point we recorded the level of the quality of the information used to support it. A regression analysis was performed to assess if the percentage of high quality evidence used in the guidelines development was related to the overall quality of the guidelines. RESULTS: Three guidelines were classified as EB, three as CB-EB and two as CB. The EB guidelines scored better than CB, with the CB-EB scoring in the middle among all instruments for guidelines quality assessment. No major disagreement in recommendations was detected among the guidelines regardless of the method used for development, but the EB guidelines had a better agreement with the benchmark guideline for any decision point. When the source of evidence used to support decision were of high quality, we found a higher level of full agreement among the guidelines' recommendations. Up to 94% of variation in the quality score among guidelines could be explained by the quality of evidence used for guidelines development. CONCLUSION: EB guidelines have a better quality than CB guidelines and CB-EB guidelines. Explicit use of high quality evidence can lead to a better agreement among recommendations. However, no major disagreement among guidelines was noted regardless of the method for their development. AD - H Lee Moffitt Cancer Center and Research institute, the University of South Florida, Tampa, FL, USA. crusehh@moffitt.usf.edu FAU - Cruse, Hugh AU - Cruse H FAU - Winiarek, Magdalena AU - Winiarek M FAU - Marshburn, Jan AU - Marshburn J FAU - Clark, Otavio AU - Clark O FAU - Djulbegovic, Benjamin AU - Djulbegovic B LA - eng PT - Evaluation Studies PT - Journal Article DEP - 20020111 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Algorithms MH - Benchmarking MH - Breast Neoplasms/*therapy MH - Comparative Study MH - *Consensus MH - *Evidence-Based Medicine MH - Female MH - Humans MH - Observer Variation MH - Peer Review, Research MH - Practice Guidelines/*standards MH - Quality Control MH - Regional Medical Programs/standards MH - Reproducibility of Results MH - United States EDAT- 2002/02/05 10:00 MHDA- 2003/04/25 05:00 PHST- 2001/10/20 [received] PHST- 2002/01/11 [accepted] PHST- 2002/01/11 [aheadofprint] PST - ppublish SO - BMC Health Serv Res 2002;2(1):1. Epub 2002 Jan 11. PMID- 11825347 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030424 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 TI - Resource use data by patient report or hospital records: do they agree? PG - 2 AB - BACKGROUND: Economic evaluations alongside clinical trials are becoming increasingly common. Cost data are often collected through the use of postal questionnaires; however, the accuracy of this method is uncertain. We compared postal questionnaires with hospital records for collecting data on physiotherapy service use. METHODS: As part of a randomised trial of orthopaedic medicine compared with orthopaedic surgery we collected physiotherapy use data on a group of patients from retrospective postal questionnaires and from hospital records. RESULTS: 315 patients were referred for physiotherapy. Hospital data on attendances was available for 30% (n = 96), compared with 48% (n = 150) of patients completing questionnaire data (95% Cl for difference = 10% to 24%); 19% (n = 59) had data available from both sources. The two methods produced an intraclass correlation coefficient of 0.54 (95% Cl 0.31 to 0.70). However, the two methods produced significantly different estimates of resource use with patient self report recalling a mean of 1.3 extra visits (95% Cl 0.4 to 2.2) compared with hospital records. CONCLUSIONS: Using questionnaires in this study produced data on a greater number of patients compared with examination of hospital records. However, the two data sources did differ in the quantity of physiotherapy used and this should be taken into account in any analysis. AD - Health Economics Research Group, Brunel University, Uxbridge, Middlesex, UK. hesradk@brunel.ac.uk FAU - Kennedy, Andrew D M AU - Kennedy AD FAU - Leigh-Brown, Anne P AU - Leigh-Brown AP FAU - Torgerson, David J AU - Torgerson DJ FAU - Campbell, James AU - Campbell J FAU - Grant, Adrian AU - Grant A LA - eng PT - Clinical Trial PT - Journal Article PT - Randomized Controlled Trial DEP - 20020117 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Adult MH - Comparative Study MH - Female MH - Great Britain MH - Health Resources/*utilization MH - *Hospital Records MH - Humans MH - Male MH - Middle Aged MH - Musculoskeletal Diseases/*therapy MH - Orthopedics/statistics & numerical data MH - Patient Compliance/*statistics & numerical data MH - Physical Therapy Department, Hospital/*utilization MH - Questionnaires MH - Referral and Consultation MH - Reproducibility of Results MH - *Self Disclosure EDAT- 2002/02/05 10:00 MHDA- 2003/04/25 05:00 PHST- 2001/11/26 [received] PHST- 2002/01/17 [accepted] PHST- 2002/01/17 [aheadofprint] PST - ppublish SO - BMC Health Serv Res 2002;2(1):2. Epub 2002 Jan 17. PMID- 11835696 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021125 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 TI - Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches' broom disease. PG - 3 AB - BACKGROUND: Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes has been considered as the main mechanism involved in the antagonistic process. However, although Trichoderma strains were found to impair development of Crinipellis perniciosa, the causal agent of cocoa plant witches' broom disease, no fungal strain is available for effective control of this disease. We have then undertaken a program of construction of hydrolytic enzyme-overproducing Trichoderma strains aiming improvement of the fungal antagonistic capacity. The protease of an indian Trichoderma isolate showing antagonistic activity against C. perniciosa was purified to homogeneity and characterized for its kinetic properties and action on the phytopathogen cell wall. RESULTS: A protease produced by the Trichoderma harzianum isolate 1051 was purified to homogeneity by precipitation with ammonium sulfate followed by hydrophobic chromatography. The molecular mass of this protease as determined by SDS-polyacrylamide gel electrophoresis was about 18.8 kDa. Its N-terminal amino acid sequence shares no homology with any other protease. The purified enzyme substantially affected the cell wall of the phytopathogen C. perniciosa. Western-blotting analysis showed that the enzyme was present in the culture supernatant 24 h after the Trichoderma started to grow in casein-containing liquid medium. CONCLUSIONS: The capacity of the Trichoderma harzianum protease to hydrolyze the cell wall of C. perniciosa indicates that this enzyme may be actually involved in the antagonistic process between the two fungi. This fact strongly suggest that hydrolytic enzyme over-producing transgenic fungi may show superior biocontrol capacity. AD - Departamento de Biologia Celular, Universidade de Brasilia, 70910-900, Brasilia, Brazil. jdemarco@uol.com.br FAU - De Marco, Janice L AU - De Marco JL FAU - Felix, Carlos Roberto AU - Felix CR LA - eng PT - Journal Article DEP - 20020122 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Antifungal Agents) RN - EC 3.4.- (Endopeptidases) SB - IM MH - Agaricales/*drug effects/ultrastructure MH - Antifungal Agents/*analysis/isolation & purification/pharmacology MH - Cacao/microbiology MH - Cell Wall/drug effects MH - Endopeptidases/*analysis/isolation & purification/pharmacology MH - Hydrogen-Ion Concentration MH - Microscopy, Electron, Scanning MH - Pest Control, Biological MH - Plant Diseases/microbiology MH - Research Support, Non-U.S. Gov't MH - Temperature MH - Trichoderma/*enzymology/isolation & purification EDAT- 2002/02/12 10:00 MHDA- 2002/11/26 04:00 PHST- 2001/09/17 [received] PHST- 2002/01/22 [accepted] PHST- 2002/01/22 [aheadofprint] PST - ppublish SO - BMC Biochem 2002;3(1):3. Epub 2002 Jan 22. PMID- 11876827 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021125 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 TI - Probing substrate binding to metallo-beta-lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis. PG - 4 AB - BACKGROUND: The metallo-beta-lactamases are Zn(II)-containing enzymes that hydrolyze the beta-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-beta-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-beta-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics. RESULTS: Site-directed mutations were generated of amino acids previously predicted to be important in substrate binding. Steady-state kinetic studies using the mutant enzymes and 9 different substrates demonstrated varying Km and kcat values for the different enzymes and substrates and that no direct correlation between Km and the effect of the mutation on substrate binding could be drawn. Stopped-flow fluorescence studies using nitrocefin as the substrate showed that only the S224D and Y228A mutants exhibited weaker nitrocefin binding. CONCLUSIONS: The data presented herein indicate that Ser224, Ile164, Phe158, Tyr228, and Asn233 are not essential for tight binding of substrate to metallo-beta-lactamase L1. The results in this work also show that Km values are not reliable for showing substrate binding, and there is no correlation between substrate binding and the amount of reaction intermediate formed during the reaction. This work represents the first experimental testing of one of the computational models of the metallo-beta-lactamases. AD - Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA. sutsong@yahoo.com FAU - Carenbauer, Anne L AU - Carenbauer AL FAU - Garrity, James D AU - Garrity JD FAU - Periyannan, Gopal AU - Periyannan G FAU - Yates, Robert B AU - Yates RB FAU - Crowder, Michael W AU - Crowder MW LA - eng GR - R29 AI40052/AI/NIAID PT - Journal Article DEP - 20020213 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Carbapenems) RN - 0 (Cephalosporins) RN - 0 (Metals) RN - 0 (Penicillins) RN - 41906-86-9 (nitrocefin) RN - 55520-40-6 (Tyrosine) RN - 56-45-1 (Serine) RN - 63-91-2 (Phenylalanine) RN - 7006-34-0 (Asparagine) RN - 73-32-5 (Isoleucine) RN - EC 3.5.2.- (beta-lactamase L1) RN - EC 3.5.2.6 (beta-Lactamases) SB - IM MH - Asparagine/genetics MH - Binding Sites MH - Carbapenems/metabolism MH - Cephalosporins/metabolism MH - Computational Biology MH - Isoleucine/genetics MH - Kinetics MH - Metals/analysis MH - Models, Molecular MH - Mutagenesis, Site-Directed MH - Penicillins/metabolism MH - Phenylalanine/genetics MH - Protein Binding MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Serine/genetics MH - Stenotrophomonas maltophilia/*enzymology MH - Tyrosine/genetics MH - beta-Lactamases/chemistry/*genetics/*metabolism EDAT- 2002/03/06 10:00 MHDA- 2002/11/26 04:00 PHST- 2001/11/06 [received] PHST- 2002/02/13 [accepted] PHST- 2002/02/13 [aheadofprint] PST - ppublish SO - BMC Biochem 2002;3(1):4. Epub 2002 Feb 13. PMID- 11882257 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030424 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 TI - What kind of evidence is it that Evidence-Based Medicine advocates want health care providers and consumers to pay attention to? PG - 3 AB - BACKGROUND: In 1992, Evidence-Based Medicine advocates proclaimed a "new paradigm", in which evidence from health care research is the best basis for decisions for individual patients and health systems. Hailed in New York Times Magazine in 2001 as one of the most influential ideas of the year, this approach was initially and provocatively pitted against the traditional teaching of medicine, in which the key elements of knowing for clinical purposes are understanding of basic pathophysiologic mechanisms of disease coupled with clinical experience. This paper reviews the origins, aspirations, philosophical limitations, and practical challenges of evidence-based medicine. DISCUSSION: EBM has long since evolved beyond its initial (mis)conception, that EBM might replace traditional medicine. EBM is now attempting to augment rather than replace individual clinical experience and understanding of basic disease mechanisms. EBM must continue to evolve, however, to address a number of issues including scientific underpinnings, moral stance and consequences, and practical matters of dissemination and application. For example, accelerating the transfer of research findings into clinical practice is often based on incomplete evidence from selected groups of people, who experience a marginal benefit from an expensive technology, raising issues of the generalizability of the findings, and increasing problems with how many and who can afford the new innovations in care. SUMMARY: Advocates of evidence-based medicine want clinicians and consumers to pay attention to the best findings from health care research that are both valid and ready for clinical application. Much remains to be done to reach this goal. AD - Department of Clinical Epidemiology and Biostatistics Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada. bhaynes@mcmaster.ca FAU - Haynes, R Brian AU - Haynes RB LA - eng PT - Journal Article DEP - 20020306 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - *Decision Making MH - Diffusion of Innovation MH - *Evidence-Based Medicine/ethics/trends MH - *Health Services Research MH - Humans MH - Knowledge MH - Patient Participation MH - Physician's Practice Patterns MH - Practice Guidelines MH - Probability MH - Randomized Controlled Trials MH - Resource Allocation/ethics MH - Uncertainty EDAT- 2002/03/08 10:00 MHDA- 2003/04/25 05:00 PHST- 2001/12/24 [received] PHST- 2002/03/06 [accepted] PHST- 2002/03/06 [aheadofprint] PST - ppublish SO - BMC Health Serv Res 2002;2(1):3. Epub 2002 Mar 6. PMID- 11884248 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030424 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 TI - Reporting of measures of accuracy in systematic reviews of diagnostic literature. PG - 4 AB - 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. AD - Academic Dept, of Obstetrics & Gynaecology, Birmingham Women's Hospital, Birmingham B15 2TG, United Kingdom. h.honest@bham.ac.uk FAU - Honest, Honest AU - Honest H FAU - Khan, Khalid S AU - Khan KS LA - eng PT - Journal Article DEP - 20020307 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Bias (Epidemiology) MH - *Data Interpretation, Statistical MH - Databases, Bibliographic MH - Humans MH - Laboratory Techniques and Procedures/*standards MH - *Meta-Analysis MH - Pathology, Clinical/*standards MH - Quality Control MH - Reproducibility of Results MH - Research Design/*standards MH - Research Support, Non-U.S. Gov't MH - *Review Literature MH - Sensitivity and Specificity EDAT- 2002/03/09 10:00 MHDA- 2003/04/25 05:00 PHST- 2001/09/05 [received] PHST- 2002/03/07 [accepted] PHST- 2002/03/07 [aheadofprint] PST - ppublish SO - BMC Health Serv Res 2002;2(1):4. Epub 2002 Mar 7. PMID- 11914161 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030422 LR - 20041117 PUBM- Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Mar 21 TI - A randomised controlled trial of a patient based Diabetes Recall and Management System: the DREAM trial: a study protocol [ISRCTN32042030]. PG - 5 AB - 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. AD - Centre for Health Services Research, University of Newcastle, Newcastle upon Tyne, UK. martin.eccles@ncl.ac.uk FAU - Eccles, Martin AU - Eccles M FAU - Hawthorne, Gillian AU - Hawthorne G FAU - Whitty, Paula AU - Whitty P FAU - Steen, Nick AU - Steen N FAU - Vanoli, Alessandra AU - Vanoli A FAU - Grimshaw, Jeremy AU - Grimshaw J FAU - Wood, Linda AU - Wood L LA - eng PT - Clinical Trial PT - Journal Article PT - Randomized Controlled Trial DEP - 20020321 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - *Database Management Systems MH - Diabetes Mellitus/diagnosis/*prevention & control MH - Efficiency, Organizational MH - England MH - *Evidence-Based Medicine MH - Family Practice/organization & administration/*standards MH - Guideline Adherence MH - Humans MH - Medical Audit MH - Patient Compliance MH - Preventive Health Services/standards/utilization MH - Primary Health Care/organization & administration/standards MH - Quality Assurance, Health Care/*organization & administration MH - Registries MH - *Reminder Systems EDAT- 2002/03/27 10:00 MHDA- 2003/04/23 05:00 PHST- 2001/12/14 [received] PHST- 2002/03/21 [accepted] PHST- 2002/03/21 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Mar 21;2(1):5. PMID- 11914162 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030423 LR - 20041117 PUBM- Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Mar 25 TI - Organization specific predictors of job satisfaction: findings from a Canadian multi-site quality of work life cross-sectional survey. PG - 6 AB - 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. AD - St, Joseph's Health System Research Network, Father Sean O'Sullivan Research Centre, Hamilton, Ontario. kruegerp@mcmaster.ca FAU - Krueger, Paul AU - Krueger P FAU - Brazil, Kevin AU - Brazil K FAU - Lohfeld, Lynne AU - Lohfeld L FAU - Edward, H Gayle AU - Edward HG FAU - Lewis, David AU - Lewis D FAU - Tjam, Erin AU - Tjam E LA - eng PT - Journal Article DEP - 20020325 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Adult MH - *Attitude of Health Personnel MH - Communication MH - Community Health Centers/organization & administration MH - Community Health Nursing/organization & administration MH - Decision Making, Organizational MH - Delivery of Health Care, Integrated/*organization & administration MH - Female MH - Forecasting MH - Hospitals, Community/organization & administration MH - Humans MH - *Job Satisfaction MH - Logistic Models MH - Male MH - Middle Aged MH - Ontario MH - Patient Care Team MH - Personnel Management/*methods MH - Questionnaires MH - Research Support, Non-U.S. Gov't MH - Residential Facilities/organization & administration EDAT- 2002/03/27 10:00 MHDA- 2003/04/24 05:00 PHST- 2001/10/01 [received] PHST- 2002/03/25 [accepted] PHST- 2002/03/25 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Mar 25;2(1):6. PMID- 11914163 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030423 LR - 20041117 PUBM- Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Mar 25 TI - Outcomes research in the development and evaluation of practice guidelines. PG - 7 AB - 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. AD - Division of Clinical Epidemiology, Research Institute of the McGill University Health Center, Montreal, Canada. louise.pilote@mcgill.ca FAU - Pilote, Louise AU - Pilote L FAU - Tager, Ira B AU - Tager IB LA - eng PT - Journal Article DEP - 20020325 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Biomedical Research MH - Chronic Disease/*epidemiology/therapy MH - Confounding Factors (Epidemiology) MH - Data Collection MH - Databases MH - Epidemiologic Methods MH - Evaluation Studies MH - Humans MH - Outcome Assessment (Health Care)/*methods MH - Physician's Practice Patterns/*statistics & numerical data MH - Practice Guidelines/*standards MH - Selection Bias MH - Treatment Outcome MH - Uncertainty EDAT- 2002/03/27 10:00 MHDA- 2003/04/24 05:00 PHST- 2001/10/01 [received] PHST- 2002/03/25 [accepted] PHST- 2002/03/25 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Mar 25;2(1):7. PMID- 11914164 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030423 LR - 20041117 PUBM- Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Mar 26 TI - Inter-rater agreement in the scoring of abstracts submitted to a primary care research conference. PG - 8 AB - 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. AD - Division of Primary Health Care, University of Bristol, Cotham House, Cotham Hill, Bristol BS8 2PR, UK. alan.a.montgomery@bristol.ac.uk FAU - Montgomery, Alan A AU - Montgomery AA FAU - Graham, Anna AU - Graham A FAU - Evans, Philip H AU - Evans PH FAU - Fahey, Tom AU - Fahey T LA - eng PT - Journal Article DEP - 20020326 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Abstracting and Indexing/classification/*standards MH - Congresses MH - *Consensus MH - Data Interpretation, Statistical MH - Great Britain MH - Humans MH - Judgment MH - Manuscripts, Medical MH - Observer Variation MH - Peer Review, Research/methods/*standards MH - Primary Health Care MH - Research Support, Non-U.S. Gov't EDAT- 2002/03/27 10:00 MHDA- 2003/04/24 05:00 PHST- 2001/12/03 [received] PHST- 2002/03/26 [accepted] PHST- 2002/03/26 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Mar 26;2(1):8. PMID- 11943069 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021122 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Mar 27 TI - The Caenorhabditis elegans Y87G2A.14 Nudix hydrolase is a peroxisomal coenzyme A diphosphatase. PG - 5 AB - 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 microM 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 microM. 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. AD - School of Biological Sciences, Life Sciences Building, University of Liverpool, P,O, Box 147, Liverpool L69 7ZB, UK. salamara@liv.ac.uk FAU - AbdelRaheim, Salama R AU - AbdelRaheim SR FAU - McLennan, Alexander G AU - McLennan AG LA - eng PT - Journal Article DEP - 20020327 PL - England TA - BMC Biochem JID - 101084098 RN - 85-61-0 (Coenzyme A) RN - EC 3.6.1.- (Pyrophosphatases) RN - EC 3.6.1.- (nudix hydrolase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Caenorhabditis elegans/*enzymology MH - Cloning, Molecular MH - Coenzyme A/*metabolism MH - Kinetics MH - Molecular Sequence Data MH - Peroxisomes/*metabolism MH - Pyrophosphatases/genetics/isolation & purification/*metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Alignment MH - Substrate Specificity EDAT- 2002/04/12 10:00 MHDA- 2002/11/26 04:00 PHST- 2002/02/12 [received] PHST- 2002/03/27 [accepted] PHST- 2002/03/27 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Mar 27;3(1):5. PMID- 11972899 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021122 LR - 20041215 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Apr 24 TI - Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis. PG - 7 AB - 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. AD - Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117997 Moscow, Russia. biomasha@mail.ru FAU - Bulina, Maria E AU - Bulina ME FAU - Chudakov, Dmitry M AU - Chudakov DM FAU - Mudrik, Nikolay N AU - Mudrik NN FAU - Lukyanov, Konstantin A AU - Lukyanov KA LA - eng PT - Journal Article DEP - 20020424 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (FP595 protein, Anemonia sulcata) RN - 0 (Luminescent Proteins) RN - 0 (fluorescent protein 583) RN - 147336-22-9 (Green Fluorescent Proteins) SB - IM MH - Amino Acid Sequence MH - *Anthozoa MH - Fluorescence MH - Green Fluorescent Proteins MH - Luminescent Proteins/*chemistry/*genetics MH - Models, Molecular MH - Molecular Sequence Data MH - Mutagenesis MH - Mutagenesis, Site-Directed MH - Research Support, Non-U.S. Gov't MH - Sequence Alignment MH - Spectrometry, Fluorescence EDAT- 2002/04/26 10:00 MHDA- 2002/11/26 04:00 PHST- 2001/12/14 [received] PHST- 2002/04/24 [accepted] PHST- 2002/04/24 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Apr 24;3(1):7. PMID- 11996675 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021218 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 May 7 TI - Hisactophilin is involved in osmoprotection in Dictyostelium. PG - 10 AB - 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. AD - Max-Planck-Institute for Biochemistry, 82152 Martinsried, Germany. pintsch@switch-biotech.com FAU - Pintsch, Tanja AU - Pintsch T FAU - Zischka, Hans AU - Zischka H FAU - Schuster, Stephan C AU - Schuster SC LA - eng PT - Journal Article DEP - 20020507 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Carrier Proteins) RN - 0 (Hypertonic Solutions) RN - 0 (Microfilament Proteins) RN - 0 (Protozoan Proteins) RN - 122962-20-3 (hisactophilin protein, Protozoan) RN - 50-70-4 (Sorbitol) RN - 56-45-1 (Serine) SB - IM MH - Animals MH - Carrier Proteins/drug effects/genetics/*metabolism MH - Cell Division/drug effects/genetics MH - Cytoskeleton/drug effects/metabolism MH - Dictyostelium/cytology/*drug effects/metabolism MH - Dose-Response Relationship, Drug MH - Electrophoresis, Gel, Two-Dimensional MH - Hypertonic Solutions/pharmacology MH - *Microfilament Proteins MH - Mutation MH - Osmotic Pressure MH - Phosphorylation/drug effects MH - Protozoan Proteins/drug effects/genetics/*metabolism MH - Research Support, Non-U.S. Gov't MH - Serine/metabolism MH - Sorbitol/*pharmacology EDAT- 2002/05/09 10:00 MHDA- 2002/12/19 04:00 PHST- 2001/12/19 [received] PHST- 2002/05/07 [accepted] PHST- 2002/05/07 [aheadofprint] PST - epublish SO - BMC Biochem 2002 May 7;3(1):10. PMID- 11997336 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - A complete sequence of the T. tengcongensis genome. PG - 689-700 AB - Thermoanaerobacter tengcongensis is a rod-shaped, gram-negative, anaerobic eubacterium that was isolated from a freshwater hot spring in Tengchong, China. Using a whole-genome-shotgun method, we sequenced its 2,689,445-bp genome from an isolate, MB4(T) (Genbank accession no. AE008691). The genome encodes 2588 predicted coding sequences (CDS). Among them, 1764 (68.2%) are classified according to homology to other documented proteins, and the rest, 824 CDS (31.8%), are functionally unknown. One of the interesting features of the T. tengcongensis genome is that 86.7% of its genes are encoded on the leading strand of DNA replication. Based on protein sequence similarity, the T. tengcongensis genome is most similar to that of Bacillus halodurans, a mesophilic eubacterium, among all fully sequenced prokaryotic genomes up to date. Computational analysis on genes involved in basic metabolic pathways supports the experimental discovery that T. tengcongensis metabolizes sugars as principal energy and carbon source and utilizes thiosulfate and element sulfur, but not sulfate, as electron acceptors. T. tengcongensis, as a gram-negative rod by empirical definitions (such as staining), shares many genes that are characteristics of gram-positive bacteria whereas it is missing molecular components unique to gram-negative bacteria. A strong correlation between the G + C content of tDNA and rDNA genes and the optimal growth temperature is found among the sequenced thermophiles. It is concluded that thermophiles are a biologically and phylogenetically divergent group of prokaryotes that have converged to sustain extreme environmental conditions over evolutionary timescale. AD - Beijing Genomics Institute/Genomics and Bioinformatics Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (CAS), Beijing 100101, China. FAU - Bao, Qiyu AU - Bao Q FAU - Tian, Yuqing AU - Tian Y FAU - Li, Wei AU - Li W FAU - Xu, Zuyuan AU - Xu Z FAU - Xuan, Zhenyu AU - Xuan Z FAU - Hu, Songnian AU - Hu S FAU - Dong, Wei AU - Dong W FAU - Yang, Jian AU - Yang J FAU - Chen, Yanjiong AU - Chen Y FAU - Xue, Yanfen AU - Xue Y FAU - Xu, Yi AU - Xu Y FAU - Lai, Xiaoqin AU - Lai X FAU - Huang, Li AU - Huang L FAU - Dong, Xiuzhu AU - Dong X FAU - Ma, Yanhe AU - Ma Y FAU - Ling, Lunjiang AU - Ling L FAU - Tan, Huarong AU - Tan H FAU - Chen, Runsheng AU - Chen R FAU - Wang, Jian AU - Wang J FAU - Yu, Jun AU - Yu J FAU - Yang, Huanming AU - Yang H LA - eng SI - GENBANK/AE008691 PT - Journal Article PL - United States TA - Genome Res JID - 9518021 RN - 0 (Codon) RN - 7704-34-9 (Sulfur) SB - IM MH - Bacillaceae/cytology/*genetics/metabolism/physiology MH - Base Composition/genetics MH - Codon/genetics MH - DNA Repair/genetics MH - DNA Replication/genetics MH - GC Rich Sequence/genetics MH - Genes, Structural, Bacterial/genetics MH - *Genome, Bacterial MH - Genomics/methods MH - Heat MH - Ion Transport/genetics MH - Molecular Sequence Data MH - Oxygen Consumption/genetics MH - Protein Biosynthesis/genetics MH - Recombination, Genetic/genetics MH - Repetitive Sequences, Nucleic Acid/genetics MH - Replication Origin/genetics MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA/methods MH - Sulfur/metabolism MH - Transcription, Genetic EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.219302 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):689-700. PMID- 11997337 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041215 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods. PG - 701-12 AB - We report here the identification of a previously unknown transcription regulatory element for heat shock (HS) genes in Caenorhabditis elegans. We monitored the expression pattern of 11,917 genes from C. elegans to determine the genes that were up-regulated on HS. Twenty eight genes were observed to be consistently up-regulated in several different repetitions of the experiments. We analyzed the upstream regions of these genes using computational DNA pattern recognition methods. Two potential cis-regulatory motifs were identified in this way. One of these motifs (TTCTAGAA) was the DNA binding motif for the heat shock factor (HSF), whereas the other (GGGTGTC) was previously unreported in the literature. We determined the significance of these motifs for the HS genes using different statistical tests and parameters. Comparative sequence analysis of orthologous HS genes from C. elegans and Caenorhabditis briggsae indicated that the identified DNA regulatory motifs are conserved across related species. The role of the identified DNA sites in regulation of HS genes was tested by in vitro mutagenesis of a green fluorescent protein (GFP) reporter transgene driven by the C. elegans hsp-16-2 promoter. DNA sites corresponding to both motifs are shown to play a significant role in up-regulation of the hsp-16-2 gene on HS. This is one of the rare instances in which a novel regulatory element, identified using computational methods, is shown to be biologically active. The contributions of individual sites toward induction of transcription on HS are nonadditive, which indicates interaction and cross-talk between the sites, possibly through the transcription factors (TFs) binding to these sites. AD - Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63114, USA. FAU - GuhaThakurta, Debraj AU - GuhaThakurta D FAU - Palomar, Lisanne AU - Palomar L FAU - Stormo, Gary D AU - Stormo GD FAU - Tedesco, Pat AU - Tedesco P FAU - Johnson, Thomas E AU - Johnson TE FAU - Walker, David W AU - Walker DW FAU - Lithgow, Gordon AU - Lithgow G FAU - Kim, Stuart AU - Kim S FAU - Link, Christopher D AU - Link CD LA - eng GR - AG12423/AG/NIA GR - HG00249/HG/NHGRI GR - R25 GM62495-01/GM/NIGMS PT - Journal Article PL - United States TA - Genome Res JID - 9518021 RN - 0 (Luminescent Proteins) RN - 147336-22-9 (Green Fluorescent Proteins) RN - 9007-49-2 (DNA) SB - IM EIN - Genome Res 2002 Aug;12(8):1301 MH - Animals MH - Base Sequence MH - Binding Sites/genetics MH - Caenorhabditis elegans/*genetics MH - Comparative Study MH - Computational Biology/*methods MH - Conserved Sequence/genetics MH - DNA/metabolism MH - Gene Expression Profiling/*methods MH - Genes, Structural, Helminth/genetics MH - Green Fluorescent Proteins MH - Heat-Shock Response/*genetics MH - Luminescent Proteins/biosynthesis MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed/genetics MH - Oligonucleotide Array Sequence Analysis/*methods MH - Probability MH - Promoter Regions (Genetics)/genetics MH - Regulatory Sequences, Nucleic Acid/*physiology MH - Research Support, U.S. Gov't, P.H.S. MH - Species Specificity MH - Statistics, Nonparametric MH - Up-Regulation/genetics EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.228902 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):701-12. PMID- 11997338 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041203 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Genes in a refined Smith-Magenis syndrome critical deletion interval on chromosome 17p11.2 and the syntenic region of the mouse. PG - 713-28 AB - Smith-Magenis syndrome (SMS) is a multiple congenital anomaly/mental retardation syndrome associated with behavioral abnormalities and sleep disturbance. Most patients have the same approximately 4 Mb interstitial genomic deletion within chromosome 17p11.2. To investigate the molecular bases of the SMS phenotype, we constructed BAC/PAC contigs covering the SMS common deletion interval and its syntenic region on mouse chromosome 11. Comparative genome analysis reveals the absence of all three approximately 200-kb SMS-REP low-copy repeats in the mouse and indicates that the evolution of SMS-REPs was accompanied by transposition of adjacent genes. Physical and genetic map comparisons in humans reveal reduced recombination in both sexes. Moreover, by examining the deleted regions in SMS patients with unusual-sized deletions, we refined the minimal Smith-Magenis critical region (SMCR) to an approximately 1.1-Mb genomic interval that is syntenic to an approxiamtely 1.0-Mb region in the mouse. Genes within the SMCR and its mouse syntenic region were identified by homology searches and by gene prediction programs, and their gene structures and expression profiles were characterized. In addition to 12 genes previously mapped, we identified 8 new genes and 10 predicted genes in the SMCR. In the mouse syntenic region of the human SMCR, 16 genes and 6 predicted genes were identified. The SMCR is highly conserved between humans and mice, including 19 genes with the same gene order and orientation. Our findings will facilitate both the identification of gene(s) responsible for the SMS phenotype and the engineering of an SMS mouse model. AD - Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA. FAU - Bi, Weimin AU - Bi W FAU - Yan, Jiong AU - Yan J FAU - Stankiewicz, Pawe AU - Stankiewicz P FAU - Park, Sung-Sup AU - Park SS FAU - Walz, Katherina AU - Walz K FAU - Boerkoel, Cornelius F AU - Boerkoel CF FAU - Potocki, Lorraine AU - Potocki L FAU - Shaffer, Lisa G AU - Shaffer LG FAU - Devriendt, Koen AU - Devriendt K FAU - Nowaczyk, Magorzata J M AU - Nowaczyk MJ FAU - Inoue, Ken AU - Inoue K FAU - Lupski, James R AU - Lupski JR LA - eng GR - P01 CA75719/CA/NCI GR - P01 HD38420/HD/NICHD GR - R01 NS27042/NS/NINDS PT - Journal Article PL - United States TA - Genome Res JID - 9518021 RN - 0 (Chaperonins) RN - 0 (Chromosomes, Artificial, Bacterial) RN - 0 (Chromosomes, Artificial, P1 Bacteriophage) RN - 0 (Fungal Proteins) RN - 0 (Saccharomyces cerevisiae Proteins) RN - EC 3.6.3.14 (ATPAF2 protein, human) RN - EC 3.6.3.14 (Proton-Translocating ATPases) SB - IM MH - Abnormalities, Multiple/*genetics MH - Animals MH - *Chaperonins MH - *Chromosome Deletion MH - Chromosomes, Artificial, Bacterial/genetics MH - Chromosomes, Artificial, P1 Bacteriophage/genetics MH - Chromosomes, Human, Pair 17/*genetics MH - Comparative Study MH - Contig Mapping/methods MH - Female MH - Fungal Proteins/genetics MH - Gene Order/genetics MH - Humans MH - Male MH - Mental Retardation/*genetics MH - Mice MH - Physical Chromosome Mapping/methods MH - *Proton-Translocating ATPases MH - Pseudogenes/genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - *Saccharomyces cerevisiae Proteins MH - Syndrome MH - Synteny/*genetics EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.73702 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):713-28. PMID- 11997339 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Structure and evolution of the Smith-Magenis syndrome repeat gene clusters, SMS-REPs. PG - 729-38 AB - An approximately 4-Mb genomic segment on chromosome 17p11.2, commonly deleted in patients with the Smith-Magenis syndrome (SMS) and duplicated in patients with dup(17)(p11.2p11.2) syndrome, is flanked by large, complex low-copy repeats (LCRs), termed proximal and distal SMS-REP. A third copy, the middle SMS-REP, is located between them. SMS-REPs are believed to mediate nonallelic homologous recombination, resulting in both SMS deletions and reciprocal duplications. To delineate the genomic structure and evolutionary origin of SMS-REPs, we constructed a bacterial artificial chromosome/P1 artificial chromosome contig spanning the entire SMS region, including the SMS-REPs, determined its genomic sequence, and used fluorescence in situ hybridization to study the evolution of SMS-REP in several primate species. Our analysis shows that both the proximal SMS-REP (approximately 256 kb) and the distal copy (approximately 176 kb) are located in the same orientation and derived from a progenitor copy, whereas the middle SMS-REP (approximately 241 kb) is inverted and appears to have been derived from the proximal copy. The SMS-REP LCRs are highly homologous (>98%) and contain at least 14 genes/pseudogenes each. SMS-REPs are not present in mice and were duplicated after the divergence of New World monkeys from pre-monkeys approximately 40-65 million years ago. Our findings potentially explain why the vast majority of SMS deletions and dup(17)(p11.2p11.2) occur at proximal and distal SMS-REPs and further support previous observations that higher-order genomic architecture involving LCRs arose recently during primate speciation and may predispose the human genome to both meiotic and mitotic rearrangements. AD - Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, 77030, USA. FAU - Park, Sung-Sup AU - Park SS FAU - Stankiewicz, Pawel AU - Stankiewicz P FAU - Bi, Weimin AU - Bi W FAU - Shaw, Christine AU - Shaw C FAU - Lehoczky, Jessica AU - Lehoczky J FAU - Dewar, Ken AU - Dewar K FAU - Birren, Bruce AU - Birren B FAU - Lupski, James R AU - Lupski JR LA - eng GR - PO1 HD39420/HD/NICHD GR - R01 NS27042/NS/NINDS GR - U54 HG02045/HG/NHGRI PT - Journal Article PL - United States TA - Genome Res JID - 9518021 SB - IM MH - Abnormalities, Multiple/*genetics MH - Base Composition/genetics MH - Cell Line MH - Cell Line, Transformed MH - Chromosomes, Human, Pair 17/genetics MH - Cloning, Molecular/methods MH - Contig Mapping/methods MH - DNA Fingerprinting/methods MH - *Evolution, Molecular MH - Gene Dosage MH - Gene Duplication MH - Genome, Human MH - Humans MH - Mental Retardation/*genetics MH - Multigene Family/*genetics MH - Repetitive Sequences, Nucleic Acid/*genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Alignment/methods MH - Sequence Analysis, DNA/methods MH - Sequence Homology, Nucleic Acid MH - Syndrome EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.82802 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):729-38. PMID- 11997340 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Discovery of regulatory elements by a computational method for phylogenetic footprinting. PG - 739-48 AB - Phylogenetic footprinting is a method for the discovery of regulatory elements in a set of orthologous regulatory regions from multiple species. It does so by identifying the best conserved motifs in those orthologous regions. We describe a computer algorithm designed specifically for this purpose, making use of the phylogenetic relationships among the sequences under study to make more accurate predictions. The program is guaranteed to report all sets of motifs with the lowest parsimony scores, calculated with respect to the phylogenetic tree relating the input species. We report the results of this algorithm on several data sets of interest. A large number of known functional binding sites are identified by our method, but we also find several highly conserved motifs for which no function is yet known. AD - Department of Computer Science and Engineering, University of Washington, Seattle, Washington 98195-2350, USA. FAU - Blanchette, Mathieu AU - Blanchette M FAU - Tompa, Martin AU - Tompa M LA - eng PT - Letter PL - United States TA - Genome Res JID - 9518021 RN - 0 (Fish Proteins) RN - 0 (Interleukin-3) RN - 11061-68-0 (Insulin) RN - 122784-93-4 (growth hormone type I, Salmo salar) RN - 9002-72-6 (Growth Hormone) RN - 9038-94-2 (Metallothionein) SB - IM MH - Algorithms MH - Animals MH - Comparative Study MH - Computational Biology/*methods MH - DNA Footprinting/*methods MH - *Fish Proteins MH - Genes, fos/genetics MH - Genes, myc/genetics MH - Growth Hormone/genetics MH - Humans MH - Insulin/genetics MH - Interleukin-3/genetics MH - Introns/genetics MH - Metallothionein/genetics MH - Multigene Family/genetics MH - *Phylogeny MH - Promoter Regions (Genetics)/genetics MH - Regulatory Sequences, Nucleic Acid/*genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.6902 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):739-48. PMID- 11997341 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041203 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Transcriptional regulation of the stem cell leukemia gene (SCL)--comparative analysis of five vertebrate SCL loci. PG - 749-59 AB - The stem cell leukemia (SCL) gene encodes a bHLH transcription factor with a pivotal role in hematopoiesis and vasculogenesis and a pattern of expression that is highly conserved between mammals and zebrafish. Here we report the isolation and characterization of the zebrafish SCL locus together with the identification of three neighboring genes, IER5, MAP17, and MUPP1. This region spans 68 kb and comprises the longest zebrafish genomic sequence currently available for comparison with mammalian, chicken, and pufferfish sequences. Our data show conserved synteny between zebrafish and mammalian SCL and MAP17 loci, thus suggesting the likely genomic domain necessary for the conserved pattern of SCL expression. Long-range comparative sequence analysis/phylogenetic footprinting was used to identify noncoding conserved sequences representing candidate transcriptional regulatory elements. The SCL promoter/enhancer, exon 1, and the poly(A) region were highly conserved, but no homology to other known mouse SCL enhancers was detected in the zebrafish sequence. A combined homology/structure analysis of the poly(A) region predicted consistent structural features, suggesting a conserved functional role in mRNA regulation. Analysis of the SCL promoter/enhancer revealed five motifs, which were conserved from zebrafish to mammals, and each of which is essential for the appropriate pattern or level of SCL transcription. AD - Cambridge Institute for Medical Research, Cambridge University, Cambridge, CB2 2XY, United Kingdom. bg200@cam.ac.uk FAU - Gottgens, Berthold AU - Gottgens B FAU - Barton, Linda M AU - Barton LM FAU - Chapman, Michael A AU - Chapman MA FAU - Sinclair, Angus M AU - Sinclair AM FAU - Knudsen, Bjarne AU - Knudsen B FAU - Grafham, Darren AU - Grafham D FAU - Gilbert, James G R AU - Gilbert JG FAU - Rogers, Jane AU - Rogers J FAU - Bentley, David R AU - Bentley DR FAU - Green, Anthony R AU - Green AR LA - eng PT - Letter PL - United States TA - Genome Res JID - 9518021 RN - 0 (5' Untranslated Regions) RN - 0 (Chromosomes, Artificial, P1 Bacteriophage) RN - 0 (DNA-Binding Proteins) RN - 0 (Genetic Markers) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Tal1 protein, mouse) RN - 0 (Transcription Factors) RN - 0 (Zebrafish Proteins) RN - 0 (tal1 protein, zebrafish) RN - 135471-20-4 (TAL1 protein, human) RN - 24937-83-5 (Poly A) SB - IM MH - 5' Untranslated Regions/genetics MH - Amino Acid Sequence MH - Animals MH - Cell Line MH - Chickens MH - Chromosomes, Artificial, P1 Bacteriophage/genetics MH - Cloning, Molecular MH - Comparative Study MH - Conserved Sequence MH - DNA-Binding Proteins/biosynthesis/*genetics/metabolism MH - Exons/genetics MH - Gene Expression Regulation, Neoplastic/*genetics MH - Genetic Markers/genetics/physiology MH - Humans MH - Leukemia, T-Cell, Acute/*genetics MH - Mice MH - Mice, Transgenic MH - Molecular Sequence Data MH - Poly A/metabolism MH - Promoter Regions (Genetics)/genetics MH - *Proto-Oncogene Proteins MH - Rats MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Nucleic Acid MH - Tetraodontiformes MH - Transcription Factors/biosynthesis/chemistry/*genetics MH - Zebrafish/genetics MH - *Zebrafish Proteins EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.45502 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):749-59. PMID- 11997342 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Determination of redundancy and systems properties of the metabolic network of Helicobacter pylori using genome-scale extreme pathway analysis. PG - 760-9 AB - The capabilities of genome-scale metabolic networks can be described through the determination of a set of systemically independent and unique flux maps called extreme pathways. The first study of genome-scale extreme pathways for the simultaneous formation of all nonessential amino acids or ribonucleotides in Helicobacter pylori is presented. Three key results were obtained. First, the extreme pathways for the production of individual amino acids in H. pylori showed far fewer internal states per external state than previously found in Haemophilus influenzae, indicating a more rigid metabolic network. Second, the degree of pathway redundancy in H. pylori was essentially the same for the production of individual amino acids and linked amino acid sets, but was approximately twice that of the production of the ribonucleotides. Third, the metabolic network of H. pylori was unable to achieve extensive conversion of amino acids consumed to the set of either nonessential amino acids or ribonucleotides and thus diverted a large portion of its nitrogen to ammonia production, a potentially important result for pH regulation in its acidic habitat. Genome-scale extreme pathways elucidate emergent system-wide properties. Extreme pathway analysis is emerging as a potentially important method to analyze the link between the metabolic genotype and its phenotypes. AD - Department of Bioengineering, University of California at San Diego, La Jolla, California 92093, USA. FAU - Price, Nathan D AU - Price ND FAU - Papin, Jason A AU - Papin JA FAU - Palsson, Bernhard O AU - Palsson BO LA - eng GR - GM57089/GM/NIGMS PT - Letter PL - United States TA - Genome Res JID - 9518021 RN - 0 (Amino Acids) RN - 0 (Nucleotides) RN - 7440-44-0 (Carbon) RN - 7727-37-9 (Nitrogen) SB - IM MH - Amino Acids/biosynthesis/metabolism MH - Biomass MH - Carbon/metabolism MH - Comparative Study MH - Escherichia coli/metabolism MH - *Genome, Bacterial MH - Genotype MH - Helicobacter pylori/*genetics/*metabolism/physiology MH - Nitrogen/metabolism MH - Nucleotides/biosynthesis MH - Phenotype MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.218002. Article published online before print in April 2002 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):760-9. PMID- 11997343 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Evidence suggesting that a fifth of annotated Caenorhabditis elegans genes may be pseudogenes. PG - 770-5 AB - Only a minority of the genes, identified in the Caenorhabditis elegans genome sequence data by computer analysis, have been characterized experimentally. We attempted to determine the expression patterns for a random sample of the annotated genes using reporter gene fusions. A low success rate was obtained for evolutionarily recently duplicated genes. Analysis of the data suggests that this is not due to conditional or low-level expression. The remaining explanation is that most of the annotated genes in the recently duplicated category are pseudogenes, a proportion corresponding to 20% of all of the annotated C. elegans genes. Further support for this surprisingly high figure was sought by comparing sequences for families of recently duplicated C. elegans genes. Although only a preliminary analysis, clear evidence for a gene having been recently inactivated by genetic drift was found for many genes in the recently duplicated category. At least 4% of the annotated C. elegans genes can be recognized as pseudogenes simply from closer inspection of the sequence data. Lessons learned in identifying pseudogenes in C. elegans could be of value in the annotation of the genomes of other species where, although there may be fewer pseudogenes, they may be harder to detect. AD - School of Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom. FAU - Mounsey, Andrew AU - Mounsey A FAU - Bauer, Petra AU - Bauer P FAU - Hope, Ian A AU - Hope IA LA - eng PT - Letter PL - United States TA - Genome Res JID - 9518021 RN - 0 (Caenorhabditis elegans Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Caenorhabditis elegans/*genetics MH - Caenorhabditis elegans Proteins/genetics MH - Computational Biology/*methods MH - Gene Expression Regulation MH - Gene Fusion/methods MH - Genes, Reporter/genetics MH - Genes, Structural, Helminth/*genetics MH - Molecular Sequence Data MH - Pseudogenes/*genetics MH - Research Support, Non-U.S. Gov't EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr208802. Article published online before print in April 2002 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):770-5. PMID- 11997344 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Analyses of the extent of shared synteny and conserved gene orders between the genome of Fugu rubripes and human 20q. PG - 776-84 AB - Cosmid and BAC contig maps have been constructed across two Fugu genomic regions containing the orthologs of human genes mapping to human chromosome 20q. Contig gene contents have been assessed by sample sequencing and comparative database analyses. Contigs are centered around two Fugu topoisomerase1 (top1) genes that were initially identified by sequence similarity to human TOP1 (20q12). Two other genes (SNAI1 and KRML) mapping to human chromosome 20 are also duplicated in Fugu. The two contigs have been mapped to separate Fugu chromosomes. Our data indicate that these linkage groups result from the duplication of an ancestral chromosome segment containing at least 40 genes that now map to the long arm of human chromosome 20. Although there is considerable conservation of synteny, gene orders are not well conserved between Fugu and human, with only very short sections of two to three adjacent genes being maintained in both organisms. Comparative analyses have allowed this duplication event to be dated before the separation of Fugu and zebrafish. Our data (which are best explained by regional duplication, followed by substantial gene loss) support the hypothesis that there have been a large number of gene and regional duplications (and corresponding gene loss) in the fish lineage, possibly resulting from a single whole genome duplication event. AD - Fugu Genomics, United Kingdom Human Genome Mapping Project Resource Centre, Wellcome Genome Campus, Hinxton Hall, Hinxton, Cambridgeshire, CB10 1SB, United Kingdom. sfsmith@hgmp.mrc.ac.uk FAU - Smith, Sarah F AU - Smith SF FAU - Snell, Philip AU - Snell P FAU - Gruetzner, Frank AU - Gruetzner F FAU - Bench, Anthony J AU - Bench AJ FAU - Haaf, Thomas AU - Haaf T FAU - Metcalfe, Judith A AU - Metcalfe JA FAU - Green, Anthony R AU - Green AR FAU - Elgar, Greg AU - Elgar G LA - eng PT - Letter PL - United States TA - Genome Res JID - 9518021 RN - 0 (Cosmids) SB - IM MH - Animals MH - Cell Line MH - Chromosomes, Human, Pair 20/*genetics MH - Comparative Study MH - Conserved Sequence/*genetics MH - Contig Mapping/methods MH - Cosmids/genetics MH - Gene Duplication MH - Gene Order/*genetics MH - *Genome MH - Genome, Human MH - Humans MH - Microscopy, Fluorescence MH - Sequence Tagged Sites MH - Synteny/*genetics MH - Takifugu/*genetics MH - Zebrafish/genetics EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.221802. Article published online before print in April 2002 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):776-84. PMID- 11997345 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Large-scale protein annotation through gene ontology. PG - 785-94 AB - Recent progress in genomic sequencing, computational biology, and ontology development has presented an opportunity to investigate biological systems from a unique perspective, that is, examining genomes and transcriptomes through the multiple and hierarchical structure of Gene Ontology (GO). We report here our development of GO Engine, a computational platform for GO annotation, and analysis of the resultant GO annotations of human proteins. Protein annotation was centered on sequence homology with GO-annotated proteins and protein domain analysis. Text information analysis and a multiparameter cellular localization predictive tool were also used to increase the annotation accuracy, and to predict novel annotations. The majority of proteins corresponding to full-length mRNA in GenBank, and the majority of proteins in the NR database (nonredundant database of proteins) were annotated with one or more GO nodes in each of the three GO categories. The annotations of GenBank and SWISS-PROT proteins are available to the public at the GO Consortium web site. AD - Compugen Inc., Jamesburg, New Jersey 08831, USA. han@cgen.com FAU - Xie, Hanqing AU - Xie H FAU - Wasserman, Alon AU - Wasserman A FAU - Levine, Zurit AU - Levine Z FAU - Novik, Amit AU - Novik A FAU - Grebinskiy, Vladimir AU - Grebinskiy V FAU - Shoshan, Avi AU - Shoshan A FAU - Mintz, Liat AU - Mintz L LA - eng PT - Letter PL - United States TA - Genome Res JID - 9518021 RN - 0 (Proteins) SB - IM MH - Animals MH - Computational Biology/*methods MH - Databases, Genetic MH - Databases, Protein MH - Genome, Human MH - Humans MH - Multigene Family MH - Proteins/*classification/*genetics/physiology MH - Sequence Analysis, Protein MH - Sequence Homology, Amino Acid EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.86902 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):785-94. PMID- 11997346 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery. PG - 795-807 AB - Cot-based sequence discovery represents a powerful means by which both low-copy and repetitive sequences can be selectively and efficiently fractionated, cloned, and characterized. Based upon the results of a Cot analysis, hydroxyapatite chromatography was used to fractionate sorghum (Sorghum bicolor) genomic DNA into highly repetitive (HR), moderately repetitive (MR), and single/low-copy (SL) sequence components that were consequently cloned to produce HRCot, MRCot, and SLCot genomic libraries. Filter hybridization (blotting) and sequence analysis both show that the HRCot library is enriched in sequences traditionally found in high-copy number (e.g., retroelements, rDNA, centromeric repeats), the SLCot library is enriched in low-copy sequences (e.g., genes and "nonrepetitive ESTs"), and the MRCot library contains sequences of moderate redundancy. The Cot analysis suggests that the sorghum genome is approximately 700 Mb (in agreement with previous estimates) and that HR, MR, and SL components comprise 15%, 41%, and 24% of sorghum DNA, respectively. Unlike previously described techniques to sequence the low-copy components of genomes, sequencing of Cot components is independent of expression and methylation patterns that vary widely among DNA elements, developmental stages, and taxa. High-throughput sequencing of Cot clones may be a means of "capturing" the sequence complexity of eukaryotic genomes at unprecedented efficiency. AD - Center for Applied Genetic Technologies and Department of Crop and Soil Sciences, University of Georgia, Athens, Georgia 30602, USA. dgp@arches.uga.edu FAU - Peterson, Daniel G AU - Peterson DG FAU - Schulze, Stefan R AU - Schulze SR FAU - Sciara, Erica B AU - Sciara EB FAU - Lee, Scott A AU - Lee SA FAU - Bowers, John E AU - Bowers JE FAU - Nagel, Alexander AU - Nagel A FAU - Jiang, Ning AU - Jiang N FAU - Tibbitts, Deanne C AU - Tibbitts DC FAU - Wessler, Susan R AU - Wessler SR FAU - Paterson, Andrew H AU - Paterson AH LA - eng SI - GENBANK/AZ921847 SI - GENBANK/AZ921848 SI - GENBANK/AZ921849 SI - GENBANK/AZ921850 SI - GENBANK/AZ921851 SI - GENBANK/AZ921852 SI - GENBANK/AZ921853 SI - GENBANK/AZ921854 SI - GENBANK/AZ921855 SI - GENBANK/AZ921856 SI - GENBANK/AZ921857 SI - GENBANK/AZ921858 SI - GENBANK/AZ921859 SI - GENBANK/AZ921860 SI - GENBANK/AZ921861 SI - GENBANK/AZ921862 SI - GENBANK/AZ921863 SI - GENBANK/AZ921864 SI - GENBANK/AZ921865 SI - GENBANK/AZ921866 SI - GENBANK/AZ921867 SI - 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GENBANK/AZ922834 SI - GENBANK/AZ922835 SI - GENBANK/AZ922836 SI - GENBANK/AZ922837 SI - GENBANK/AZ922838 SI - GENBANK/AZ922839 SI - GENBANK/AZ922840 SI - GENBANK/AZ922841 SI - GENBANK/AZ922842 SI - GENBANK/AZ922843 SI - GENBANK/AZ922844 SI - GENBANK/AZ922845 SI - GENBANK/AZ922846 PT - Journal Article PL - United States TA - Genome Res JID - 9518021 RN - 0 (Chromosomes, Artificial, Bacterial) RN - 0 (Genetic Markers) RN - 0 (Plant Proteins) RN - 0 (kafirin protein, Sorghum bicolor) SB - IM MH - Base Composition/genetics MH - Blotting, Southern/methods MH - Chromosomes, Artificial, Bacterial/genetics MH - Cloning, Molecular/*methods MH - Expressed Sequence Tags MH - GC Rich Sequence/genetics MH - Gene Dosage MH - *Genes, Structural, Plant MH - Genetic Markers/genetics MH - *Genome, Plant MH - Genomic Library MH - Molecular Sequence Data MH - Nucleic Acid Denaturation MH - Nucleic Acid Hybridization MH - Plant Proteins/genetics MH - Poaceae/*genetics MH - Repetitive Sequences, Nucleic Acid/genetics MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Sequence Analysis, DNA/*methods MH - Temperature EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.226102. Article published online before print in April 2002 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):795-807. PMID- 11997347 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - Protein coding palindromes are a unique but recurrent feature in Rickettsia. PG - 808-16 AB - Rickettsia are unique in inserting in-frame a number of palindromic sequences within protein coding regions. In this study, we extensively analyzed repeated sequences in the genome of Rickettsia conorii and examined their locations in regard to coding versus noncoding regions. We identified 656 interspersed repeated sequences classified into 10 distinct families. Of the 10 families, three palindromic sequence families showed clear cases of insertions into open reading frames (ORFs). The location of those in-frame insertions appears to be always compatible with the encoded protein three-dimensional (3-D) fold and function. We provide evidence for a progressive loss of the palindromic property over time after the insertions. This comprehensive study of Rickettsia repeats confirms and extends our previous observations and further indicates a significant role of selfish DNAs in the creation and modification of proteins. AD - Information Genetique & Structurale, CNRS-AVENTIS UMR 1889, 13402 Marseille Cedex 20, France. Hiroyuki.Ogata@igs.cnrs-mrs.fr FAU - Ogata, Hiroyuki AU - Ogata H FAU - Audic, Stephane AU - Audic S FAU - Abergel, Chantal AU - Abergel C FAU - Fournier, Pierre-Edouard AU - Fournier PE FAU - Claverie, Jean-Michel AU - Claverie JM LA - eng PT - Letter PL - United States TA - Genome Res JID - 9518021 RN - 0 (Bacterial Proteins) SB - IM MH - Amino Acid Sequence/genetics MH - Bacterial Proteins/chemistry/*genetics MH - Base Composition/genetics MH - Base Sequence MH - Computational Biology/methods MH - *Genome, Bacterial MH - Molecular Sequence Data MH - Protein Structure, Quaternary/genetics MH - Rickettsia conorii/chemistry/*genetics EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.227602. Article published online before print in April 2002 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):808-16. PMID- 11997348 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - A fine physical map of the rice chromosome 4. PG - 817-23 AB - As part of an international effort to completely sequence the rice genome, we have produced a fine bacterial artificial chromosome (BAC)-based physical map of the Oryza sativa japonica Nipponbare chromosome 4 through an integration of 114 sequenced BAC clones from a taxonomically related subspecies O. sativa indica Guangluai 4 and 182 RFLP and 407 expressed sequence tag (EST) markers with the fingerprinted data of the Nipponbare genome. The map consists of 11 contigs with a total length of 34.5 Mb covering 94% of the estimated chromosome size (36.8 Mb). BAC clones corresponding to telomeres, as well as to the centromere position, were determined by BAC-pachytene chromosome fluorescence in situ hybridization (FISH). This gave rise to an estimated length ratio of 5.13 for the long arm and 2.9 for the short arm (on the basis of the physical map), which indicates that the short arm is a highly condensed one. The FISH analysis and physical mapping also showed that the short arm and the pericentromeric region of the long arm are rich in heterochromatin, which occupied 45% of the chromosome, indicating that this chromosome is likely very difficult to sequence. To our knowledge, this map provides the first example of a rapid and reliable physical mapping on the basis of the integration of the data from two taxonomically related subspecies. AD - National Center for Gene Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China. FAU - Zhao, Qiang AU - Zhao Q FAU - Zhang, Yu AU - Zhang Y FAU - Cheng, Zhukuan AU - Cheng Z FAU - Chen, Mingsheng AU - Chen M FAU - Wang, Shengyue AU - Wang S FAU - Feng, Qi AU - Feng Q FAU - Huang, Yucheng AU - Huang Y FAU - Li, Ying AU - Li Y FAU - Tang, Yesheng AU - Tang Y FAU - Zhou, Bo AU - Zhou B FAU - Chen, Zhehua AU - Chen Z FAU - Yu, Shuliang AU - Yu S FAU - Zhu, Jingjie AU - Zhu J FAU - Hu, Xin AU - Hu X FAU - Mu, Jie AU - Mu J FAU - Ying, Kai AU - Ying K FAU - Hao, Pei AU - Hao P FAU - Zhang, Lei AU - Zhang L FAU - Lu, Yiqi AU - Lu Y FAU - Zhang, Lei S AU - Zhang LS FAU - Liu, Yilei AU - Liu Y FAU - Yu, Zhen AU - Yu Z FAU - Fan, Danlin AU - Fan D FAU - Weng, Qijun AU - Weng Q FAU - Chen, Ling AU - Chen L FAU - Lu, Tingting AU - Lu T FAU - Liu, Xiaohui AU - Liu X FAU - Jia, Peixin AU - Jia P FAU - Sun, Tongguo AU - Sun T FAU - Wu, Yongrui AU - Wu Y FAU - Zhang, Yujun AU - Zhang Y FAU - Lu, Ying AU - Lu Y FAU - Li, Can AU - Li C FAU - Wang, Rong AU - Wang R FAU - Lei, Haiyan AU - Lei H FAU - Li, Tao AU - Li T FAU - Hu, Hao AU - Hu H FAU - Wu, Mei AU - Wu M FAU - Zhang, Runquan AU - Zhang R FAU - Guan, Jianping AU - Guan J FAU - Zhu, Jia AU - Zhu J FAU - Fu, Gang AU - Fu G FAU - Gu, Minghong AU - Gu M FAU - Hong, Guofan AU - Hong G FAU - Xue, Yongbiao AU - Xue Y FAU - Wing, Rod AU - Wing R FAU - Jiang, Jiming AU - Jiang J FAU - Han, Bin AU - Han B LA - eng PT - Letter PL - United States TA - Genome Res JID - 9518021 RN - 0 (Chromosomes, Artificial, Bacterial) RN - 0 (DNA, Plant) SB - IM MH - Chromosomes/chemistry/*genetics MH - Chromosomes, Artificial, Bacterial MH - Contig Mapping MH - Cytogenetic Analysis/methods MH - DNA, Plant/genetics MH - In Situ Hybridization, Fluorescence/methods MH - Oryza sativa/chemistry/*genetics MH - Physical Chromosome Mapping/*methods MH - Recombination, Genetic/genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Seeds/genetics EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.48902 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):817-23. PMID- 11997349 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - RePS: a sequence assembler that masks exact repeats identified from the shotgun data. PG - 824-31 AB - We describe a sequence assembler, RePS (repeat-masked Phrap with scaffolding), that explicitly identifies exact 20mer repeats from the shotgun data and removes them prior to the assembly. The established software is used to compute meaningful error probabilities for each base. Clone-end-pairing information is used to construct scaffolds that order and orient the contigs. We show with real data for human and rice that reasonable assemblies are possible even at coverages of only 4x to 6x, despite having up to 42.2% in exact repeats. AD - Hangzhou Genomics Institute, Institute of Bioinformatics of Zhejiang University, Key Laboratory of Bioinformatics of Zhejiang Province, Hangzhou 310007, China. wangj@genomics.org.cn FAU - Wang, Jun AU - Wang J FAU - Wong, Gane Ka-Shu AU - Wong GK FAU - Ni, Peixiang AU - Ni P FAU - Han, Yujun AU - Han Y FAU - Huang, Xiangang AU - Huang X FAU - Zhang, Jianguo AU - Zhang J FAU - Ye, Chen AU - Ye C FAU - Zhang, Yong AU - Zhang Y FAU - Hu, Jianfei AU - Hu J FAU - Zhang, Kunlin AU - Zhang K FAU - Xu, Xin AU - Xu X FAU - Cong, Lijuan AU - Cong L FAU - Lu, Hong AU - Lu H FAU - Ren, Xide AU - Ren X FAU - Ren, Xiaoyu AU - Ren X FAU - He, Jun AU - He J FAU - Tao, Lin AU - Tao L FAU - Passey, Douglas A AU - Passey DA FAU - Wang, Jian AU - Wang J FAU - Yang, Huanming AU - Yang H FAU - Yu, Jun AU - Yu J FAU - Li, Songgang AU - Li S LA - eng GR - 1 R01 ES09909/ES/NIEHS PT - Journal Article PL - United States TA - Genome Res JID - 9518021 SB - IM MH - Cloning, Molecular/methods MH - Comparative Study MH - Contig Mapping/*methods MH - Humans MH - Oryza sativa/genetics MH - Repetitive Sequences, Nucleic Acid/*genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Analysis, DNA/methods MH - *Software EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.165102 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):824-31. PMID- 11997350 OWN - NLM STAT- MEDLINE DA - 20020508 DCOM- 20020605 LR - 20041117 PUBM- Print IS - 1088-9051 VI - 12 IP - 5 DP - 2002 May TI - rVista for comparative sequence-based discovery of functional transcription factor binding sites. PG - 832-9 AB - Identifying transcriptional regulatory elements represents a significant challenge in annotating the genomes of higher vertebrates. We have developed a computational tool, rVista, for high-throughput discovery of cis-regulatory elements that combines clustering of predicted transcription factor binding sites (TFBSs) and the analysis of interspecies sequence conservation to maximize the identification of functional sites. To assess the ability of rVista to discover true positive TFBSs while minimizing the prediction of false positives, we analyzed the distribution of several TFBSs across 1 Mb of the well-annotated cytokine gene cluster (Hs5q31; Mm11). Because a large number of AP-1, NFAT, and GATA-3 sites have been experimentally identified in this interval, we focused our analysis on the distribution of all binding sites specific for these transcription factors. The exploitation of the orthologous human-mouse dataset resulted in the elimination of > 95% of the approximately 58,000 binding sites predicted on analysis of the human sequence alone, whereas it identified 88% of the experimentally verified binding sites in this region. AD - Genome Sciences Department, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. ggloots@lbl.gov FAU - Loots, Gabriela G AU - Loots GG FAU - Ovcharenko, Ivan AU - Ovcharenko I FAU - Pachter, Lior AU - Pachter L FAU - Dubchak, Inna AU - Dubchak I FAU - Rubin, Edward M AU - Rubin EM LA - eng PT - Journal Article PT - Validation Studies PL - United States TA - Genome Res JID - 9518021 RN - 0 (Cytokines) RN - 0 (Transcription Factors) SB - IM MH - Animals MH - Base Sequence/genetics MH - Binding Sites/genetics MH - Comparative Study MH - Computational Biology/methods MH - Cytokines/chemistry/genetics MH - Humans MH - Mice MH - Multigene Family/genetics MH - Promoter Regions (Genetics)/genetics MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - *Software MH - Transcription Factors/*chemistry/*genetics EDAT- 2002/05/09 10:00 MHDA- 2002/06/06 10:01 AID - 10.1101/gr.225502. Article published online before print in April 2002 [doi] PST - ppublish SO - Genome Res 2002 May;12(5):832-9. PMID- 12006105 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030423 LR - 20041117 PUBM- Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 May 13 TI - Management of obstetric anal sphincter injury: a systematic review & national practice survey. PG - 9 AB - 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 > or = 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. AD - Academic Department of Obstetrics & Gynaecology North Staffordshire Hospital Trust/Keele University Stoke on Trent, England. ruwanfernando@hotmail.com FAU - Fernando, Ruwan J AU - Fernando RJ FAU - Sultan, Abdul H AU - Sultan AH FAU - Radley, Simon AU - Radley S FAU - Jones, Peter W AU - Jones PW FAU - Johanson, Richard B AU - Johanson RB LA - eng PT - Journal Article PT - Review DEP - 20020513 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Anus/*injuries MH - Clinical Competence MH - Colorectal Surgery/methods/*standards MH - Continuity of Patient Care MH - Delivery, Obstetric/*adverse effects MH - Fecal Incontinence/etiology/surgery MH - Female MH - Great Britain/epidemiology MH - Humans MH - Labor Complications/diagnosis/epidemiology/*surgery MH - Obstetrics/education/methods/*standards MH - Patient Care Management MH - *Physician's Practice Patterns MH - Pregnancy MH - Randomized Controlled Trials MH - Research Support, Non-U.S. Gov't MH - Rupture/diagnosis/epidemiology/*surgery MH - Treatment Outcome RF - 55 EDAT- 2002/05/15 10:00 MHDA- 2003/04/24 05:00 PHST- 2001/09/27 [received] PHST- 2002/05/13 [accepted] PHST- 2002/05/13 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 May 13;2(1):9. PMID- 12014993 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021216 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Apr 25 TI - Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate. PG - 8 AB - 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 microM. 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. AD - Department of Biological Sciences, Vanderbilt University, VU Station B 351634, Nashville TN 37235-1634, USA. tony.song@vanderbilt.edu FAU - Song, Qilin AU - Song Q FAU - Singleton, Charles K AU - Singleton CK LA - eng GR - AA12014-02/AA/NIAAA PT - Journal Article DEP - 20020425 PL - England TA - BMC Biochem JID - 101084098 RN - 154-87-0 (Thiamine Pyrophosphate) RN - 59-43-8 (Thiamine) SB - IM MH - Anemia, Megaloblastic/drug therapy/*metabolism MH - Animals MH - Biological Transport MH - CHO Cells MH - Cell Line MH - Hamsters MH - Humans MH - Kinetics MH - Lymphocytes/cytology/metabolism MH - Mitochondria/*metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Thiamine/*therapeutic use MH - Thiamine Pyrophosphate/*pharmacokinetics MH - Tumor Cells, Cultured EDAT- 2002/05/17 10:00 MHDA- 2002/12/18 04:00 PHST- 2002/02/06 [received] PHST- 2002/04/25 [accepted] PHST- 2002/04/25 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Apr 25;3(1):8. PMID- 12019031 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021218 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 May 4 TI - Expression of monolysocardiolipin acyltransferase activity is regulated in concert with the level of cardiolipin and cardiolipin biosynthesis in the mammalian heart. PG - 9 AB - 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. AD - Department of Pharmacology, Centre on Aging, University of Manitoba, Winnipeg, Canada. taylorw@ms.umanitoba.ca FAU - Taylor, William A AU - Taylor WA FAU - Xu, Fred Y AU - Xu FY FAU - Ma, Brian J AU - Ma BJ FAU - Mutter, Thomas C AU - Mutter TC FAU - Dolinsky, Vernon W AU - Dolinsky VW FAU - Hatch, Grant M AU - Hatch GM LA - eng PT - Journal Article DEP - 20020504 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Cardiolipins) RN - 0 (Membrane Proteins) RN - 0 (RNA, Messenger) RN - EC 2.3. (Acyltransferases) RN - EC 2.3.1.- (monolysocardiolipin acyltransferase) RN - EC 2.7.8 (Transferases (Other Substituted Phosphate Groups)) RN - EC 2.7.8.- (cardiolipin synthetase) SB - IM MH - Acyltransferases/genetics/*metabolism MH - Animals MH - Cardiolipins/*metabolism MH - Cell Differentiation MH - Diabetes Mellitus, Experimental/enzymology/metabolism MH - Gene Expression Regulation, Enzymologic MH - Hyperinsulinism/enzymology/metabolism MH - Hypothyroidism/enzymology/metabolism MH - Male MH - *Membrane Proteins MH - Myocardium/cytology/*metabolism MH - RNA, Messenger/genetics/metabolism MH - Rats MH - Rats, Sprague-Dawley MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Transferases (Other Substituted Phosphate Groups)/metabolism MH - Tumor Cells, Cultured EDAT- 2002/05/23 10:00 MHDA- 2002/12/19 04:00 PHST- 2002/02/06 [received] PHST- 2002/05/04 [accepted] PHST- 2002/05/04 [aheadofprint] PST - epublish SO - BMC Biochem 2002 May 4;3(1):9. PMID- 12022922 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021216 LR - 20041027 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Apr 4 TI - Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos. PG - 6 AB - 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. AD - Temple University, Fels Institute for Cancer Research and Molecular Biology, Philadelphia, PA, USA. alexandropolis@yahoo.com FAU - Proikas-Cezanne, Tassula AU - Proikas-Cezanne T FAU - Stabel, Silvia AU - Stabel S FAU - Riethmacher, Dieter AU - Riethmacher D LA - eng PT - Journal Article DEP - 20020404 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Caseins) RN - 0 (Peptide Fragments) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Tubulin) RN - 0 (Vimentin) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 2.7.1.37 (Proto-Oncogene Proteins c-mos) RN - EC 3.1.3.48 (Protein-Tyrosine-Phosphatase) RN - EC 3.1.3.48 (protein tyrosine phosphatase 1B) SB - IM MH - Animals MH - Binding Sites MH - Caseins/*metabolism MH - Cell Line MH - Peptide Fragments/genetics/metabolism MH - Phosphorylation MH - Protein-Serine-Threonine Kinases/metabolism MH - Protein-Tyrosine-Phosphatase/*metabolism MH - Proto-Oncogene Proteins c-mos/chemistry/genetics/*metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Substrate Specificity MH - Tubulin/metabolism MH - Vimentin/metabolism EDAT- 2002/05/23 10:00 MHDA- 2002/12/18 04:00 PHST- 2002/02/01 [received] PHST- 2002/04/04 [accepted] PHST- 2002/04/04 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Apr 4;3(1):6. PMID- 12028592 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021122 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 May 17 TI - The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes. PG - 11 AB - 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. AD - Division of Molecular Physiology, School of Life Sciences, University of Dundee, Dundee, MSI/Wellcome Trust Biocentre, DD1 5EH United Kingdom. m.scheperkleijn@dundee.ac.uk FAU - Kleijn, Miranda AU - Kleijn M FAU - Proud, Christopher G AU - Proud CG LA - eng PT - Journal Article DEP - 20020517 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Antibodies) RN - 0 (Antigens, CD28) RN - 0 (Antigens, CD3) RN - 0 (Carrier Proteins) RN - 0 (EIF4EBP1 protein, human) RN - 0 (Eukaryotic Initiation Factor-2B) RN - 0 (Eukaryotic Initiation Factor-4E) RN - 0 (Eukaryotic Initiation Factors) RN - 0 (Phosphoproteins) RN - EC 2.7.1.37 (Ribosomal Protein S6 Kinases, 70-kDa) SB - IM MH - Antibodies/pharmacology MH - Antigens, CD28/immunology/physiology MH - Antigens, CD3/immunology/physiology MH - Carrier Proteins/metabolism MH - Cells, Cultured MH - Eukaryotic Initiation Factor-2B/metabolism MH - Eukaryotic Initiation Factor-4E/metabolism MH - Eukaryotic Initiation Factors/*metabolism MH - Humans MH - Kinetics MH - *Lymphocyte Activation MH - Phosphoproteins/metabolism MH - Phosphorylation MH - *Protein Biosynthesis MH - Research Support, Non-U.S. Gov't MH - Ribosomal Protein S6 Kinases, 70-kDa/metabolism MH - Signal Transduction MH - T-Lymphocytes/immunology/*metabolism EDAT- 2002/05/25 10:00 MHDA- 2002/11/26 04:00 PHST- 2002/03/14 [received] PHST- 2002/05/17 [accepted] PHST- 2002/05/17 [aheadofprint] PST - epublish SO - BMC Biochem 2002 May 17;3(1):11. PMID- 12052258 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030423 LR - 20041117 PUBM- Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 May 27 TI - Hospital competition, resource allocation and quality of care. PG - 10 AB - 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. AD - University of Rochester Medical Center, Rochester, New York 14642, USA. dana_mukamel@urm.rochester.edu FAU - Mukamel, Dana B AU - Mukamel DB FAU - Zwanziger, Jack AU - Zwanziger J FAU - Bamezai, Anil AU - Bamezai A LA - eng GR - HS09545/HS/AHCPR PT - Journal Article DEP - 20020527 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - California/epidemiology MH - Cost Control/methods MH - Decision Making, Organizational MH - *Economic Competition MH - Health Expenditures/*trends MH - Health Services Research MH - Hospital Charges MH - Hospital Departments/classification/*economics/*standards MH - Hospital Mortality MH - Hospital-Patient Relations MH - Hospitals/classification MH - Humans MH - Managed Care Programs/legislation & jurisprudence/organization & administration MH - Quality of Health Care/*trends MH - Research Support, U.S. Gov't, P.H.S. MH - *Resource Allocation MH - Risk Adjustment MH - Treatment Outcome EDAT- 2002/06/08 10:00 MHDA- 2003/04/24 05:00 PHST- 2001/12/21 [received] PHST- 2002/05/27 [accepted] PHST- 2002/05/27 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 May 27;2(1):10. PMID- 12052259 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021122 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 May 29 TI - The role of the Zn(II) binding domain in the mechanism of E. coli DNA topoisomerase I. PG - 13 AB - 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. AD - Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, USA. a_ahumada@msn.com FAU - Ahumada, Adriana AU - Ahumada A FAU - Tse-Dinh, Yuk-Ching AU - Tse-Dinh YC LA - eng GR - GM17315/GM/NIGMS GR - GM54226/GM/NIGMS PT - Journal Article DEP - 20020529 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (DNA, Circular) RN - 0 (DNA, Single-Stranded) RN - 0 (DNA, Superhelical) RN - 7439-95-4 (Magnesium) RN - 7440-66-6 (Zinc) RN - 7647-14-5 (Sodium Chloride) RN - 9007-49-2 (DNA) RN - EC 5.99.1.- (DNA Topoisomerases, Type I, Bacterial) SB - IM MH - Binding Sites MH - Catalytic Domain MH - DNA/chemistry/metabolism MH - DNA Topoisomerases, Type I, Bacterial/*chemistry/*metabolism MH - DNA, Circular/metabolism MH - DNA, Single-Stranded/chemistry/metabolism MH - DNA, Superhelical/metabolism MH - Escherichia coli/*enzymology MH - Magnesium/pharmacology MH - Models, Genetic MH - Protein Binding MH - Protein Structure, Tertiary MH - Research Support, U.S. Gov't, P.H.S. MH - Sodium Chloride/pharmacology MH - Substrate Specificity MH - Zinc/*metabolism EDAT- 2002/06/08 10:00 MHDA- 2002/11/26 04:00 PHST- 2002/03/11 [received] PHST- 2002/05/29 [accepted] PHST- 2002/05/29 [aheadofprint] PST - epublish SO - BMC Biochem 2002 May 29;3(1):13. PMID- 12052260 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021122 LR - 20050201 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 May 28 TI - Mutational analysis of human profilin I reveals a second PI(4,5)-P2 binding site neighbouring the poly(L-proline) binding site. PG - 12 AB - 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. AD - Department of Medical Protein Research (VIB09), Flanders Interuniversity Institute of Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium. anja.lambrechts@rug.ac.be FAU - Lambrechts, Anja AU - Lambrechts A FAU - Jonckheere, Veronique AU - Jonckheere V FAU - Dewitte, Daisy AU - Dewitte D FAU - Vandekerckhove, Joel AU - Vandekerckhove J FAU - Ampe, Christophe AU - Ampe C LA - eng PT - Journal Article DEP - 20020528 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Actins) RN - 0 (Contractile Proteins) RN - 0 (Ligands) RN - 0 (Microfilament Proteins) RN - 0 (Peptides) RN - 0 (Phosphatidylinositol 4,5-Diphosphate) RN - 0 (profilins) RN - 25191-13-3 (polyproline) RN - 73-22-3 (Tryptophan) SB - IM MH - Actins/metabolism MH - Binding Sites MH - *Contractile Proteins MH - DNA Mutational Analysis MH - Humans MH - Ligands MH - Microfilament Proteins/*chemistry/*genetics/metabolism MH - Models, Molecular MH - Mutagenesis, Site-Directed MH - Peptides/*metabolism MH - Phosphatidylinositol 4,5-Diphosphate/*metabolism MH - Protein Folding MH - Protein Structure, Secondary MH - Research Support, Non-U.S. Gov't MH - Tryptophan/physiology EDAT- 2002/06/08 10:00 MHDA- 2002/11/26 04:00 PHST- 2002/02/13 [received] PHST- 2002/05/28 [accepted] PHST- 2002/05/28 [aheadofprint] PST - epublish SO - BMC Biochem 2002 May 28;3(1):12. PMID- 12057022 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030422 LR - 20041117 PUBM- Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Jun 2 TI - Training practitioners in preparing systematic reviews: a cross-sectional survey of participants in the Australasian Cochrane Centre training program. PG - 11 AB - 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. AD - Australasian Cochrane Centre, Monash Institute of Health Services Research, Monash Medical Centre, Melbourne, Australia. janet.piehl@med.monash.edu.au FAU - Piehl, Janet H AU - Piehl JH FAU - Green, Sally AU - Green S FAU - Silagy, Chris AU - Silagy C LA - eng PT - Journal Article DEP - 20020602 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - *Attitude of Health Personnel MH - Australia MH - Cross-Sectional Studies MH - Evidence-Based Medicine MH - Humans MH - *Meta-Analysis MH - Publishing/*statistics & numerical data MH - Questionnaires MH - *Randomized Controlled Trials MH - Research Personnel/*education/statistics & numerical data MH - Time EDAT- 2002/06/12 10:00 MHDA- 2003/04/23 05:00 PHST- 2002/02/20 [received] PHST- 2002/06/02 [accepted] PHST- 2002/06/02 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Jun 2;2(1):11. PMID- 12069692 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021220 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Jun 10 TI - Gene expression profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiation signals. PG - 14 AB - 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. AD - Department of Biochemistry and Molecular Biology George Washington University School of Medicine Washington DC 20037, USA. bcmfxk@gwumc.edu FAU - de la Fuente, Cynthia AU - de la Fuente C FAU - Santiago, Francisco AU - Santiago F FAU - Deng, Longwen AU - Deng L FAU - Eadie, Carolyne AU - Eadie C FAU - Zilberman, Irene AU - Zilberman I FAU - Kehn, Kylene AU - Kehn K FAU - Maddukuri, Anil AU - Maddukuri A FAU - Baylor, Shanese AU - Baylor S FAU - Wu, Kaili AU - Wu K FAU - Lee, Chee Gun AU - Lee CG FAU - Pumfery, Anne AU - Pumfery A FAU - Kashanchi, Fatah AU - Kashanchi F LA - eng GR - 13969/PHS GR - AI43894/AI/NIAID GR - AI44357/AI/NIAID PT - Journal Article DEP - 20020610 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Chromatin) RN - 0 (Gene Products, tat) RN - 0 (Transcription Factors) RN - 61512-21-8 (Thymosin) SB - IM MH - Cell Cycle/genetics MH - Cell Differentiation/genetics MH - Cell Division/genetics MH - Chromatin/genetics MH - Gene Expression Profiling/*methods MH - Gene Expression Regulation/genetics MH - Gene Products, tat/*genetics MH - HIV-1/*genetics MH - Hela Cells/cytology/metabolism/virology MH - Humans MH - Protein Biosynthesis/genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction/*genetics MH - Thymosin/genetics MH - Transcription Factors/genetics MH - Transcription, Genetic/genetics MH - Tumor Cells, Cultured EDAT- 2002/06/19 10:00 MHDA- 2002/12/21 04:00 PHST- 2002/01/29 [received] PHST- 2002/06/10 [accepted] PHST- 2002/06/10 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Jun 10;3(1):14. PMID- 12079498 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021218 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Jun 17 TI - Low solubility of unconjugated bilirubin in dimethylsulfoxide--water systems: implications for pKa determinations. PG - 17 AB - 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. AD - School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705-2222, USA. pmukerjee@aol.com FAU - Mukerjee, Pasupati AU - Mukerjee P FAU - Ostrow, J Donald AU - Ostrow JD FAU - Tiribelli, Claudio AU - Tiribelli C LA - eng PT - Journal Article DEP - 20020617 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Serum Albumin) RN - 635-65-4 (Bilirubin) RN - 67-68-5 (Dimethyl Sulfoxide) RN - 7732-18-5 (Water) SB - IM MH - Bilirubin/*chemistry MH - Dimethyl Sulfoxide/*chemistry MH - Humans MH - Hydrogen-Ion Concentration MH - Serum Albumin/chemistry MH - Solubility MH - Thermodynamics MH - Water/*chemistry EDAT- 2002/06/25 10:00 MHDA- 2002/12/19 04:00 PHST- 2002/04/23 [received] PHST- 2002/06/17 [accepted] PHST- 2002/06/17 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Jun 17;3(1):17. PMID- 12079499 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021220 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Jun 13 TI - Functional group interactions of a 5-HT3R antagonist. PG - 16 AB - 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. AD - Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208-3520, USA. padma964@hotmail.com FAU - Venkataraman, Padmavati AU - Venkataraman P FAU - Joshi, Prasad AU - Joshi P FAU - Venkatachalan, Srinivasan P AU - Venkatachalan SP FAU - Muthalagi, Mani AU - Muthalagi M FAU - Parihar, Harish S AU - Parihar HS FAU - Kirschbaum, Karen S AU - Kirschbaum KS FAU - Schulte, Marvin K AU - Schulte MK LA - eng PT - Journal Article DEP - 20020613 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Benzimidazoles) RN - 0 (Benzyl Compounds) RN - 0 (Piperazines) RN - 0 (Piperidines) RN - 0 (Receptors, Serotonin) RN - 0 (Receptors, Serotonin, 5-HT3) RN - 0 (Serotonin Antagonists) RN - 110-85-0 (piperazine) RN - 143257-98-1 (lerisetron) RN - 55520-40-6 (Tyrosine) RN - 73-22-3 (Tryptophan) RN - 74-79-3 (Arginine) RN - 7727-37-9 (Nitrogen) SB - IM MH - Animals MH - Arginine/physiology MH - Benzimidazoles/metabolism MH - Benzyl Compounds/metabolism MH - Cell Line MH - Humans MH - Kidney/cytology/embryology MH - Mice MH - Mutagenesis, Site-Directed/genetics MH - Nitrogen/metabolism MH - Patch-Clamp Techniques MH - Piperazines/chemistry/metabolism MH - Piperidines/metabolism MH - Protein Binding/physiology MH - Protein Interaction Mapping/*methods MH - Receptors, Serotonin/genetics/*metabolism MH - Receptors, Serotonin, 5-HT3 MH - Research Support, Non-U.S. Gov't MH - Serotonin Antagonists/*metabolism MH - Structure-Activity Relationship MH - Tryptophan/physiology MH - Tyrosine/physiology EDAT- 2002/06/25 10:00 MHDA- 2002/12/21 04:00 PHST- 2002/02/05 [received] PHST- 2002/06/13 [accepted] PHST- 2002/06/13 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Jun 13;3(1):16. PMID- 12079500 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021220 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Jun 13 TI - Identification of critical residues in loop E in the 5-HT3ASR binding site. PG - 15 AB - 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. AD - Department of Neurobiology, Northwestern University, Evanston, IL 60201. USA. padma964@hotmail.com FAU - Venkataraman, Padmavati AU - Venkataraman P FAU - Venkatachalan, Srinivasan P AU - Venkatachalan SP FAU - Joshi, Prasad R AU - Joshi PR FAU - Muthalagi, Mani AU - Muthalagi M FAU - Schulte, Marvin K AU - Schulte MK LA - eng PT - Journal Article DEP - 20020613 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Amino Acids) RN - 0 (Receptors, Serotonin) RN - 0 (Receptors, Serotonin, 5-HT3) RN - 55520-40-6 (Tyrosine) RN - 56-87-1 (Lysine) SB - IM MH - Amino Acid Sequence MH - Amino Acids/*analysis/genetics MH - Animals MH - Binding Sites/genetics MH - Cell Line MH - Humans MH - Kidney/chemistry/cytology/embryology/metabolism MH - Lysine/analysis/genetics MH - Mice MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed/genetics MH - Protein Structure, Tertiary/genetics MH - Receptors, Serotonin/*chemistry/genetics/*physiology MH - Receptors, Serotonin, 5-HT3 MH - Research Support, Non-U.S. Gov't MH - Sequence Alignment/methods MH - Sequence Homology, Amino Acid MH - Transfection MH - Tyrosine/analysis/genetics EDAT- 2002/06/25 10:00 MHDA- 2002/12/21 04:00 PHST- 2002/01/30 [received] PHST- 2002/06/13 [accepted] PHST- 2002/06/13 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Jun 13;3(1):15. PMID- 12084180 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030422 LR - 20041117 PUBM- Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Jun 25 TI - A comparison of hospital readmission rates between two general physicians with different outpatient review practices. PG - 12 AB - 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. AD - Birmingham Heartlands and Solihull NHS Trust (Teaching), Birmingham, UK. Raynerh@heartsol.wmids.nhs.uk FAU - Rayner, Hugh C AU - Rayner HC FAU - Temple, R Mark AU - Temple RM FAU - Marshall, Tim AU - Marshall T FAU - Clarke, Dianne AU - Clarke D LA - eng PT - Journal Article DEP - 20020625 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Adolescent MH - Adult MH - Age Factors MH - Aged MH - Chronic Disease MH - Cohort Studies MH - Comparative Study MH - *Continuity of Patient Care MH - *Diagnosis-Related Groups MH - Episode of Care MH - Family Practice/*organization & administration/standards/statistics & numerical data MH - Great Britain/epidemiology MH - Humans MH - International Classification of Diseases MH - Length of Stay/statistics & numerical data MH - *Medical Audit MH - Middle Aged MH - Outpatient Clinics, Hospital/*utilization MH - Patient Readmission/*statistics & numerical data MH - Risk Factors MH - *Utilization Review EDAT- 2002/06/27 10:00 MHDA- 2003/04/23 05:00 PHST- 2001/10/22 [received] PHST- 2002/06/25 [accepted] PHST- 2002/06/25 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Jun 25;2(1):12. PMID- 12086585 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021218 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Jun 18 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. PG - 18 AB - 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. AD - Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA. pandeypk@umdnj.edu FAU - Pandey, Pradeep K AU - Pandey PK FAU - Kaushik, Neerja AU - Kaushik N FAU - Singh, Kamalendra AU - Singh K FAU - Sharma, Bechan AU - Sharma B FAU - Upadhyay, Alok K AU - Upadhyay AK FAU - Kumar, Suriender AU - Kumar S FAU - Harris, Dylan AU - Harris D FAU - Pandey, Virendra N AU - Pandey VN LA - eng GR - CA72821/CA/NCI PT - Journal Article DEP - 20020618 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Amino Acids) RN - 0 (DNA-Binding Proteins) RN - 0 (Protein Subunits) RN - 9007-49-2 (DNA) RN - EC 2.7.7.- (HIV-1 Reverse Transcriptase) RN - EC 2.7.7.49 (RNA-Directed DNA Polymerase) RN - EC 2.7.7.7 (DNA-Directed DNA Polymerase) RN - EC 3.1.26.4 (Ribonuclease H, Calf Thymus) SB - IM MH - Amino Acids/genetics MH - DNA/metabolism MH - DNA-Binding Proteins/chemistry/genetics/metabolism MH - DNA-Directed DNA Polymerase/*metabolism MH - Dimerization MH - HIV-1 Reverse Transcriptase/chemistry/genetics/*metabolism MH - Models, Molecular MH - Mutagenesis, Insertional MH - Mutation MH - Protein Binding MH - *Protein Conformation MH - Protein Structure, Tertiary MH - Protein Subunits MH - RNA-Directed DNA Polymerase/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Ribonuclease H, Calf Thymus/*metabolism MH - Ultracentrifugation/methods EDAT- 2002/06/28 10:00 MHDA- 2002/12/19 04:00 PHST- 2002/02/26 [received] PHST- 2002/06/18 [accepted] PHST- 2002/06/18 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Jun 18;3(1):18. PMID- 12097150 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021218 LR - 20040924 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Jun 25 TI - Molecular cloning and tissue distribution of mammalian L-threonine 3-dehydrogenases. PG - 19 AB - 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. AD - Tissue Engineering and Regenerative Medicine Centre, Division of Investigative Science, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Chelsea & Westminster Hospital, London, United Kingdom. alasdair.edgar@ic.ac.uk FAU - Edgar, Alasdair J AU - Edgar AJ LA - eng SI - GENBANK/AY095535 SI - GENBANK/AY116662 PT - Journal Article DEP - 20020625 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (DNA, Complementary) RN - 0 (RNA, Messenger) RN - 53-84-9 (NAD) RN - EC 1.1 (Alcohol Oxidoreductases) RN - EC 1.1.1.103 (L-threonine 3-dehydrogenase) SB - IM MH - Alcohol Oxidoreductases/*genetics/metabolism MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Binding Sites MH - Biological Transport MH - DNA, Complementary/chemistry/genetics MH - Gene Expression MH - Mice MH - Mitochondria/metabolism MH - Molecular Sequence Data MH - NAD/metabolism MH - RNA, Messenger/genetics/*metabolism MH - Sequence Alignment MH - Sequence Analysis, DNA MH - Sequence Homology, Amino Acid MH - Swine EDAT- 2002/07/05 10:00 MHDA- 2002/12/19 04:00 PHST- 2002/06/17 [received] PHST- 2002/06/25 [accepted] PHST- 2002/06/25 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Jun 25;3(1):19. PMID- 12110157 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030422 LR - 20041117 PUBM- Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Jul 11 TI - Application of the development stages of a cluster randomized trial to a framework for valuating complex health interventions. PG - 13 AB - 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. AD - Department of Pathology, McMaster University, Hamilton, Ontario, Canada. loebm@mcmaster.ca FAU - Loeb, Mark B AU - Loeb MB LA - eng PT - Journal Article DEP - 20020711 PL - England TA - BMC Health Serv Res JID - 101088677 RN - 0 (Anti-Bacterial Agents) SB - IM MH - Aged MH - Algorithms MH - Anti-Bacterial Agents/*therapeutic use MH - Cluster Analysis MH - Critical Pathways MH - Drug Resistance, Bacterial MH - Female MH - Health Services Research/*methods MH - Humans MH - *Intervention Studies MH - Male MH - Nursing Homes/standards/*statistics & numerical data MH - Nursing Staff/education MH - Ontario MH - Qualitative Research MH - Randomized Controlled Trials MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Urinary Tract Infections/*drug therapy/nursing/urine EDAT- 2002/07/12 10:00 MHDA- 2003/04/23 05:00 PHST- 2002/03/13 [received] PHST- 2002/07/11 [accepted] PHST- 2002/07/11 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Jul 11;2(1):13. PMID- 12121577 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021220 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Jul 16 TI - Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases. PG - 20 AB - 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 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2) and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 x 10(3) M(-1) x s(-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. AD - Division of Cell Signalling, School of Life Sciences, The University of Dundee, Dundee, DD1 5EH, UK. n.r.leslie@dundee.ac.uk FAU - Leslie, Nick R AU - Leslie NR FAU - McLennan, Alexander G AU - McLennan AG FAU - Safrany, Stephen T AU - Safrany ST LA - eng PT - Journal Article DEP - 20020716 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (RNA, Messenger) RN - EC 3.6 (Acid Anhydride Hydrolases) RN - EC 3.6.1.- (Pyrophosphatases) RN - EC 3.6.1.- (diadenosine polyphosphate hydrolase) RN - EC 3.6.1.- (nudix hydrolase) RN - EC 3.6.1.- (nudix hydrolase Aps1, human) RN - EC 3.6.1.- (nudix hydrolase Aps2, human) SB - IM MH - *Acid Anhydride Hydrolases/analysis/genetics/metabolism MH - Amino Acid Sequence MH - Animals MH - Cattle MH - Cloning, Molecular MH - Cytosol/enzymology MH - Humans MH - Mice MH - Molecular Sequence Data MH - *Pyrophosphatases/analysis/genetics/metabolism MH - RNA, Messenger/biosynthesis MH - Research Support, Non-U.S. Gov't MH - Sequence Alignment MH - Substrate Specificity MH - Tissue Distribution MH - X Chromosome EDAT- 2002/07/18 10:00 MHDA- 2002/12/21 04:00 PHST- 2002/05/17 [received] PHST- 2002/07/16 [accepted] PHST- 2002/07/16 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Jul 16;3(1):20. PMID- 12126482 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030422 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Jul 18 TI - Priority setting for new technologies in medicine: a transdisciplinary study. PG - 14 AB - 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. AD - University of Toronto Joint Centre for Bioethics, 88 College St, Toronto, Canada M5G-1L4. jegibson@chass.utoronto.ca FAU - Gibson, Jennifer L AU - Gibson JL FAU - Martin, Douglas K AU - Martin DK FAU - Singer, Peter A AU - Singer PA LA - eng PT - Journal Article DEP - 20020718 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Consensus MH - *Decision Making, Organizational MH - *Ethics, Institutional MH - Health Priorities/*ethics MH - Humans MH - Managed Care Programs/ethics MH - Models, Organizational MH - Research Support, Non-U.S. Gov't MH - Resource Allocation MH - Social Justice MH - Social Responsibility MH - Social Values MH - Technology Assessment, Biomedical/*ethics MH - United States EDAT- 2002/07/20 10:00 MHDA- 2003/04/23 05:00 PHST- 2001/12/21 [received] PHST- 2002/07/18 [accepted] PHST- 2002/07/18 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Jul 18;2(1):14. PMID- 12149129 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021122 LR - 20041117 PUBM- Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Jul 30 TI - Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine. PG - 21 AB - 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. AD - Laboratoire de Synthese et Physicochimie des Molecules d'Interet Biologique, UMR 5068, Universite Paul Sabatier, 31062, Toulouse, France. isa10yann@yahoo.fr FAU - Fremaux, Isabelle AU - Fremaux I FAU - Mazeres, Serge AU - Mazeres S FAU - Brisson-Lougarre, Andree AU - Brisson-Lougarre A FAU - Arnaud, Muriel AU - Arnaud M FAU - Ladurantie, Caroline AU - Ladurantie C FAU - Fournier, Didier AU - Fournier D LA - eng PT - Journal Article DEP - 20020730 PL - England TA - BMC Biochem JID - 101084098 RN - 52-90-4 (Cysteine) RN - 7004-03-7 (Valine) RN - EC 3.1.1.7 (Acetylcholinesterase) SB - IM MH - Acetylcholinesterase/*chemistry/genetics/*metabolism MH - Animals MH - Cysteine/*chemistry/genetics MH - Drosophila melanogaster/*enzymology MH - Enzyme Stability MH - Hydrophobicity MH - Models, Molecular MH - Mutagenesis MH - Protein Denaturation MH - Research Support, Non-U.S. Gov't MH - Valine/genetics EDAT- 2002/08/01 10:00 MHDA- 2002/11/26 04:00 PHST- 2002/05/18 [received] PHST- 2002/07/30 [accepted] PHST- 2002/07/30 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Jul 30;3(1):21. PMID- 12153701 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030422 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Aug 1 TI - Accuracy of responses from postal surveys about continuing medical education and information behavior: experiences from a survey among German diabetologists. PG - 15 AB - BACKGROUND: Postal surveys are a popular instrument for studies about continuing medical education habits. But little is known about the accuracy of responses in such surveys. The objective of this study was to quantify the magnitude of inaccurate responses in a postal survey among physicians. METHODS: A sub-analysis of a questionnaire about continuing medical education habits and information management was performed. The five variables used for the quantitative analysis are based on a question about the knowledge of a fictitious technical term and on inconsistencies in contingency tables of answers to logically connected questions. RESULTS: Response rate was 52%. Non-response bias is possible but seems not very likely since an association between demographic variables and inconsistent responses could not be found. About 10% of responses were inaccurate according to the definition. CONCLUSION: It was shown that a sub-analysis of a questionnaire makes a quantification of inaccurate responses in postal surveys possible. This sub-analysis revealed that a notable portion of responses in a postal survey about continuing medical education habits and information management was inaccurate. AD - Research Group Medicine/Research Unit Biotechnology, Society, and Environment, University of Hamburg, Germany. trelle@uni-hamburg.de FAU - Trelle, Sven AU - Trelle S LA - eng PT - Journal Article PT - Validation Studies DEP - 20020801 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Clinical Competence MH - Decision Making MH - Diabetes Mellitus/*therapy MH - Education, Medical, Continuing/*statistics & numerical data MH - Evidence-Based Medicine/*statistics & numerical data MH - Family Practice/education/standards MH - Female MH - Germany MH - Humans MH - Internal Medicine/education/standards MH - Male MH - Meta-Analysis MH - Pediatrics/education/standards MH - Physician's Practice Patterns/*statistics & numerical data MH - Postal Service MH - Primary Health Care/*standards MH - Problem Solving MH - *Questionnaires MH - Reproducibility of Results MH - Risk Reduction Behavior EDAT- 2002/08/03 10:00 MHDA- 2003/04/23 05:00 PHST- 2002/04/12 [received] PHST- 2002/08/01 [accepted] PHST- 2002/08/01 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Aug 1;2(1):15. PMID- 12154231 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041215 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - Restriction of lentivirus in monkeys. PG - 11920-5 AB - Retroviruses are able to cross species barriers and have done so many times throughout evolution. Perhaps as a consequence, dominant mechanisms have arisen to block infection by murine retroviruses in mice (restriction factor Fv1) and humans (restriction factor Ref1), as well as in other mammals. Here we describe a block to HIV and simian immunodeficiency virus in monkeys. Like previously described restrictions the block is saturable and gives rise to multiple-hit infection kinetics. Furthermore, like restriction of murine leukemia virus in humans, the block is before reverse transcription. Intriguingly, African green monkey cells are able to block both HIV and simian immunodeficiency virus, and each virus is able to saturate and abrogate the restriction of the other, suggesting that a common factor is responsible. AD - Wohl Virion Centre, Department of Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London W1T 4JF, United Kingdom. FAU - Besnier, Caroline AU - Besnier C FAU - Takeuchi, Yasuhiro AU - Takeuchi Y FAU - Towers, Greg AU - Towers G LA - eng PT - Journal Article DEP - 20020801 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (DNA Primers) RN - 0 (Luminescent Proteins) RN - 0 (Virus Inhibitors) RN - 147336-22-9 (Green Fluorescent Proteins) SB - IM CIN - Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11549-51. PMID: 12195025 MH - Animals MH - Base Sequence MH - Cell Line MH - DNA Primers MH - DNA Replication MH - Green Fluorescent Proteins MH - HIV-1/genetics/*physiology MH - Haplorhini MH - Luminescent Proteins/genetics MH - Rabbits MH - Research Support, Non-U.S. Gov't MH - SIV/genetics/*physiology MH - Transcription, Genetic MH - *Virus Inhibitors EDAT- 2002/08/03 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/01 [aheadofprint] AID - 10.1073/pnas.172384599 [doi] AID - 172384599 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11920-5. Epub 2002 Aug 1. PMID- 12167173 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021122 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Aug 7 TI - Combinatorial diversity of fission yeast SCF ubiquitin ligases by homo- and heterooligomeric assemblies of the F-box proteins Pop1p and Pop2p. PG - 22 AB - BACKGROUND: SCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins. F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways. Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p. Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes. RESULTS: We have identified Psh1p and Pip1p, the fission yeast homologues of human SKP1 and HRT1/RBX1/ROC1, and show that both associate with Pop1p, Pop2p, and Pcu1p into a ~500 kDa SCFPop1p-Pop2p complex, which supports polyubiquitylation of Rum1p. Only the F-box of Pop1p is required for SCFPop1p-Pop2p function, while Pop2p seems to be attracted into the complex through binding to Pop1p. Since all SCFPop1p-Pop2p subunits, except for Pop1p, which is exclusively nuclear, localize to both the nucleus and the cytoplasm, the F-box of Pop2p may be critical for the assembly of cytoplasmic SCFPop2p complexes. In support of this notion, we demonstrate individual SCFPop1p and SCFPop2p complexes bearing ubiquitin ligase activity. CONCLUSION: Our data suggest that distinct homo- and heterooligomeric assemblies of Pop1p and Pop2p generate combinatorial diversity of SCFPop function in fission yeast. Whereas a heterooligomeric SCFPop1p-Pop2p complex mediates polyubiquitylation of Rum1p, homooligomeric SCFPop1p and SCFPop2p complexes may target unknown nuclear and cytoplasmic substrates. AD - Department of Cancer Cell Biology, Harvard School of Public Health, Boston, MA, USA. vseibert@europroteome.com FAU - Seibert, Volker AU - Seibert V FAU - Prohl, Corinna AU - Prohl C FAU - Schoultz, Ida AU - Schoultz I FAU - Rhee, Edward AU - Rhee E FAU - Lopez, Rebecca AU - Lopez R FAU - Abderazzaq, Kareem AU - Abderazzaq K FAU - Zhou, Chunshui AU - Zhou C FAU - Wolf, Dieter A AU - Wolf DA LA - eng GR - ES-00002/ES/NIEHS GR - GM50780/GM/NIGMS PT - Journal Article DEP - 20020807 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Macromolecular Substances) RN - 0 (Protein Subunits) RN - 0 (Rum1 protein, S pombe) RN - 0 (Schizosaccharomyces pombe Proteins) RN - 0 (Ubiquitins) RN - EC 6.3.2. (Peptide Synthases) RN - EC 6.3.2.19 (SKP Cullin F-Box Protein Ligases) SB - IM MH - Binding Sites MH - Cell Compartmentation MH - Macromolecular Substances MH - Mutation MH - Peptide Synthases/chemistry/genetics/*physiology MH - Protein Structure, Tertiary MH - Protein Subunits MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - SKP Cullin F-Box Protein Ligases MH - Schizosaccharomyces/*enzymology/metabolism MH - Schizosaccharomyces pombe Proteins/chemistry/genetics/metabolism/*physiology MH - Ubiquitins/metabolism EDAT- 2002/08/09 10:00 MHDA- 2002/11/26 04:00 PHST- 2002/06/07 [received] PHST- 2002/08/07 [accepted] PHST- 2002/08/07 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Aug 7;3(1):22. PMID- 12183222 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20031114 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Resistance to macrolides and related antibiotics in Streptococcus pneumoniae. PG - 2727-34 AD - Laboratoire de Microbiologie, CHRU de Caen, Caen, France. leclercq-r@chu-caen.fr FAU - Leclercq, Roland AU - Leclercq R FAU - Courvalin, Patrice AU - Courvalin P LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (RNA, Bacterial) RN - 114-07-8 (Erythromycin) RN - EC 2.1.1. (Methyltransferases) RN - EC 2.1.1.66 (rRNA (adenosine-O-2'-)methyltransferase) SB - IM MH - Anti-Bacterial Agents/metabolism/*pharmacology MH - Base Sequence MH - Binding Sites/drug effects MH - Drug Resistance MH - Erythromycin/metabolism/pharmacology MH - Methylation MH - Methyltransferases/genetics MH - Nucleic Acid Conformation MH - Phenotype MH - RNA, Bacterial/chemistry/genetics MH - Ribosomes/drug effects/genetics/metabolism MH - Streptococcus pneumoniae/*drug effects/genetics EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2727-34. PMID- 12183223 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Importance of the fourth alpha-helix within the CAP homology domain of type II topoisomerase for DNA cleavage site recognition and quinolone action. PG - 2735-46 AB - We report that point mutations causing alteration of the fourth alpha-helix (alpha4-helix) of the CAP homology domain of eukaryotic (Saccharomyces cerevisiae) type II topoisomerases (Ser(740)Trp, Gln(743)Pro, and Thr(744)Pro) change the selection of type II topoisomerase-mediated DNA cleavage sites promoted by Ca(2+) or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, Thr(744)Ala substitution had minimal effect on Ca(2+)- and drug-stimulated DNA cleavage sites, indicating the selectivity of single amino acid substitutions within the alpha4-helix on type II topoisomerase-mediated DNA cleavage. The equivalent mutation in the gene for Escherichia coli gyrase causing Ser(83)Trp also changed the DNA cleavage pattern generated by Ca(2+) or quinolones. Finally, Thr(744)Pro substitution in the yeast type II topoisomerase rendered the enzyme sensitive to antibacterial quinolones. This study shows that the alpha4-helix within the conserved CAP homology domain of type II topoisomerases is critical for selecting the sites of DNA cleavage. It also demonstrates that selective amino acid residues in the alpha4-helix are important in determining the activity and possibly the binding of quinolones to the topoisomerase II-DNA complexes. AD - Department of Internal Medicine and Medical Oncology, West German Cancer Center, University Medical School of Essen, 45122 Essen, Germany. dirk.strumberg@uni-essen.de FAU - Strumberg, Dirk AU - Strumberg D FAU - Nitiss, John L AU - Nitiss JL FAU - Dong, Jiaowang AU - Dong J FAU - Walker, Jerrylaine AU - Walker J FAU - Nicklaus, Marc C AU - Nicklaus MC FAU - Kohn, Kurt W AU - Kohn KW FAU - Heddle, Jonathan G AU - Heddle JG FAU - Maxwell, Anthony AU - Maxwell A FAU - Seeber, Siegfried AU - Seeber S FAU - Pommier, Yves AU - Pommier Y LA - eng GR - CA21765/CA/NCI GR - CA52814/CA/NCI PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (4-Quinolones) RN - 0 (Anti-Infective Agents) RN - 0 (DNA, Bacterial) RN - 0 (Fluoroquinolones) RN - 0 (RNA Cap-Binding Proteins) RN - 136440-70-5 (CP 115953) RN - 7440-70-2 (Calcium) RN - EC 5.99.1.- (DNA Gyrase) RN - EC 5.99.1.- (DNA Topoisomerases, Type II, Bacterial) SB - IM MH - 4-Quinolones MH - Amino Acid Sequence MH - Anti-Infective Agents/*pharmacology MH - Calcium/pharmacology MH - DNA Fragmentation MH - DNA Gyrase/antagonists & inhibitors/metabolism MH - DNA Topoisomerases, Type II, Bacterial/biosynthesis/*metabolism MH - DNA, Bacterial/chemistry/*metabolism MH - Electrophoresis, Polyacrylamide Gel MH - Escherichia coli/enzymology MH - *Fluoroquinolones MH - Protein Conformation MH - RNA Cap-Binding Proteins/*metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Reverse Transcriptase Polymerase Chain Reaction MH - Saccharomyces cerevisiae/enzymology MH - Sequence Homology, Nucleic Acid EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2735-46. PMID- 12183224 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20031114 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Evaluation of antibiotic susceptibilities of three rickettsial species including Rickettsia felis by a quantitative PCR DNA assay. PG - 2747-51 AB - Rickettsiae grow only intracellularly, and the antibiotic susceptibilities of these bacteria have been assessed by either plaque, dye uptake, or immunofluorescence assays, which are time-consuming. We used a quantitative PCR (with the LightCycler instrument) to assess the levels of inhibition of Rickettisa felis, R. conorii, and R. typhi DNA synthesis in the presence of various antibiotics. We established the kinetics of rickettsial DNA during growth and showed that R. conorii grows more quickly than R. typhi in cell culture, with maximum replication occurring after 5 and 7 days, respectively. The MICs of the antibiotics tested for R. conorii and R. typhi by the quantitative PCR assay were similar to those previously obtained by plaque and dye uptake assays. We found that R. felis is susceptible to doxycycline, rifampin, thiamphenicol, and fluoroquinolones but not to gentamicin, erythromycin, amoxicillin, or trimethoprim-sulfamethoxazole. The resistance of this new species to erythromycin is consistent with its current taxonomic position within the spotted fever group. We believe that quantitative PCR could be used in the future to simplify and shorten antibiotic susceptibility assays of other rickettsiae and other strict intracellular pathogens. AD - Unite des Rickettsies CNRS UPRES-A 6020, Faculte de Medecine, Universite de la Mediterranee, 13385 Marseille Cedex 05, France. FAU - Rolain, Jean-Marc AU - Rolain JM FAU - Stuhl, Laetitia AU - Stuhl L FAU - Maurin, Max AU - Maurin M FAU - Raoult, Didier AU - Raoult D LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (DNA, Bacterial) RN - 0 (Dyes) SB - IM MH - Anti-Bacterial Agents/*pharmacology MH - Calibration MH - DNA, Bacterial/*drug effects/genetics/isolation & purification MH - Dyes MH - Kinetics MH - Microbial Sensitivity Tests/instrumentation/*methods MH - Reverse Transcriptase Polymerase Chain Reaction/instrumentation/*methods MH - Rickettsia/*drug effects MH - Rickettsia conorii/drug effects MH - Rickettsia felis/drug effects MH - Rickettsia typhi/drug effects EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2747-51. PMID- 12183225 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - N-alkyl urea hydroxamic acids as a new class of peptide deformylase inhibitors with antibacterial activity. PG - 2752-64 AB - Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P(1)' site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P(1)' site. Compounds with MICs of 200 micro M for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 A. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents. AD - Versicor, Inc., Fremont, California 94555, USA. FAU - Hackbarth, Corinne J AU - Hackbarth CJ FAU - Chen, Dawn Z AU - Chen DZ FAU - Lewis, Jason G AU - Lewis JG FAU - Clark, Kirk AU - Clark K FAU - Mangold, James B AU - Mangold JB FAU - Cramer, Jeffrey A AU - Cramer JA FAU - Margolis, Peter S AU - Margolis PS FAU - Wang, Wen AU - Wang W FAU - Koehn, Jim AU - Koehn J FAU - Wu, Charlotte AU - Wu C FAU - Lopez, S AU - Lopez S FAU - Withers, George 3rd AU - Withers G 3rd FAU - Gu, Helen AU - Gu H FAU - Dunn, Elina AU - Dunn E FAU - Kulathila, R AU - Kulathila R FAU - Pan, Shi-Hao AU - Pan SH FAU - Porter, Wilma L AU - Porter WL FAU - Jacobs, Jeff AU - Jacobs J FAU - Trias, Joaquim AU - Trias J FAU - Patel, Dinesh V AU - Patel DV FAU - Weidmann, Beat AU - Weidmann B FAU - White, Richard J AU - White RJ FAU - Yuan, Zhengyu AU - Yuan Z LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (DNA Primers) RN - 0 (Hydroxamic Acids) RN - 0 (Protease Inhibitors) RN - 57-13-6 (Urea) RN - EC 3.4.11 (Aminopeptidases) RN - EC 3.5. (Amidohydrolases) RN - EC 3.5.1.88 (peptide deformylase) SB - IM MH - *Amidohydrolases MH - Aminopeptidases/*antagonists & inhibitors MH - Animals MH - Bacteria/drug effects MH - Biotransformation MH - Crystallography, X-Ray MH - DNA Primers MH - Drug Resistance MH - Drug Screening Assays, Antitumor MH - Escherichia coli/metabolism MH - Female MH - Haemophilus influenzae/drug effects/genetics MH - Humans MH - Hydroxamic Acids/*chemical synthesis/pharmacokinetics/*pharmacology MH - In Vitro MH - Male MH - Mice MH - Microbial Sensitivity Tests MH - Microsomes, Liver/metabolism MH - Molecular Conformation MH - Protease Inhibitors/*chemical synthesis/pharmacokinetics/*pharmacology MH - Rats MH - Rats, Sprague-Dawley MH - Septicemia/drug therapy/microbiology MH - Streptococcus pneumoniae/drug effects/genetics MH - Structure-Activity Relationship MH - Tumor Cells, Cultured MH - Urea/*analogs & derivatives/chemical synthesis/pharmacokinetics/pharmacology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2752-64. PMID- 12183226 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Oxidative stress increases susceptibility of Mycobacterium tuberculosis to isoniazid. PG - 2765-71 AB - Isoniazid is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. Isoniazid is a prodrug requiring oxidative activation by the catalase-peroxidase hemoprotein, KatG. Resistance to isoniazid can be obtained by point mutations in the katG gene, with one of the most common being a threonine-for-serine substitution at position 315 (S315T). The S315T mutation is found in more than 50% of isoniazid-resistant clinical isolates and results in an approximately 200-fold increase in the MIC of isoniazid compared to that for M. tuberculosis H37Rv. In the present study we investigated the hypothesis that superoxide plays a role in KatG-mediated isoniazid activation. Plumbagin and clofazimine, compounds capable of generating superoxide anion, resulted in a lower MIC of isoniazid for M. tuberculosis H37Rv and a strain carrying the S315T mutation. These agents did not cause as great of an increase in isoniazid susceptibility in the mutant strain when the susceptibilities were assessed by using the inhibitory concentration that causes a 50% decrease in growth. These results provide evidence that superoxide can play a role in isoniazid activation. Since clofazimine alone has antitubercular activity, the observation of synergism between clofazimine and isoniazid raises the interesting possibility of using both drugs in combination to treat M. tuberculosis infections. AD - Section of Hematology Research and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA. FAU - Bulatovic, Vanja M AU - Bulatovic VM FAU - Wengenack, Nancy L AU - Wengenack NL FAU - Uhl, James R AU - Uhl JR FAU - Hall, Leslie AU - Hall L FAU - Roberts, Glenn D AU - Roberts GD FAU - Cockerill, Franklin R 3rd AU - Cockerill FR 3rd FAU - Rusnak, Frank AU - Rusnak F LA - eng GR - AI47142/AI/NIAID PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antitubercular Agents) RN - 0 (Bacterial Proteins) RN - 0 (DNA, Bacterial) RN - 0 (Naphthoquinones) RN - 0 (Oxidants) RN - 11062-77-4 (Superoxides) RN - 2030-63-9 (Clofazimine) RN - 481-42-5 (plumbagin) RN - 54-85-3 (Isoniazid) RN - EC 1.11.1. (Peroxidases) RN - EC 1.11.1.6 (catalase HPI) SB - IM MH - Antitubercular Agents/*pharmacology MH - *Bacterial Proteins MH - Base Sequence MH - Clofazimine/pharmacology MH - DNA, Bacterial/chemistry MH - Drug Resistance, Microbial MH - Isoniazid/*pharmacology MH - Microbial Sensitivity Tests MH - Molecular Sequence Data MH - Mycobacterium tuberculosis/*drug effects/genetics MH - Naphthoquinones/pharmacology MH - Oxidants/metabolism MH - Oxidative Stress/*physiology MH - Peroxidases/genetics MH - Research Support, U.S. Gov't, P.H.S. MH - Superoxides/metabolism EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2765-71. PMID- 12183227 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Heterologous expression of epothilone biosynthetic genes in Myxococcus xanthus. PG - 2772-8 AB - Epothilones are potential anticancer drugs that stabilize microtubules in a manner similar to paclitaxel (Taxol). Epothilones are produced from the myxobacterium Sorangium cellulosum, which has a 16-h doubling time and produces only milligram-per-liter amounts of epothilone A and epothilone B. Furthermore, genetic manipulation of S. cellulosum is difficult. To produce epothilones in a more genetically amenable and rapidly growing host, we chose the closely related and best-characterized myxobacteria Myxococcus xanthus. We inserted 65.4 kb of S. cellulosum DNA that encompassed the entire epothilone gene cluster into the chromosome of M. xanthus by a series of homologous recombination events. The resulting strain produced epothilones A and B. Construction of a strain that contained a mutation in epoK, the P450 epoxidase, resulted in production of epothilones C and D. AD - Kosan Biosciences, Inc., Hayward, California 94545, USA. julien@kosan.com FAU - Julien, Bryan AU - Julien B FAU - Shah, Sanjay AU - Shah S LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Bacterial Proteins) RN - 0 (Culture Media) RN - 0 (DNA, Bacterial) RN - 0 (Epothilones) RN - 0 (Plasmids) RN - 0 (epothilone C) RN - 0 (epothilone D) RN - 9035-51-2 (Cytochrome P-450 Enzyme System) RN - EC 1. (Oxidoreductases) RN - EC 1.- (EpoK protein, Polyangium cellulosum) SB - IM MH - Bacterial Proteins/genetics MH - Chromosomes, Bacterial/genetics MH - Culture Media MH - Cytochrome P-450 Enzyme System/genetics MH - DNA, Bacterial/genetics MH - Epothilones/*biosynthesis/*genetics MH - Escherichia coli/genetics MH - Gene Expression Regulation, Bacterial/*genetics MH - Multigene Family/genetics MH - Mutation/genetics MH - Myxococcus xanthus/*genetics/*metabolism MH - Oxidoreductases/genetics MH - Plasmids/genetics MH - Protein Engineering EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2772-8. PMID- 12183228 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Different levels of genetic homogeneity in vancomycin-resistant and -susceptible Enterococcus faecium isolates from different human and animal sources analyzed by amplified-fragment length polymorphism. PG - 2779-83 AB - The genetic relationship among fecal vancomycin-resistant Enterococcus faecium (VREF) and vancomycin-susceptible E. faecium (VSEF) isolates (n = 178) from the same populations of pigs, human healthy volunteers, and hospitalized patients (from The Netherlands) and chickens (from The Netherlands and Greece) was studied by amplified-fragment length polymorphism (AFLP). The majority of VREF isolates from pigs, healthy volunteers, and hospitalized patients grouped together (genetic similarity, >or=65%). In a previous AFLP study by our group the VREF isolates from hospitalized patients grouped separately, most likely because these were clinical and not fecal isolates as in the present study. Furthermore, VSEF isolates from humans and pigs were found much more genetically diverse than VREF isolates, whereas VREF and VSEF isolates from chickens clustered together in a separate genogroup (genetic similarity, >or=65%), a pattern clearly distinct from the patterns for human and pig isolates. The present study suggests that pigs are a more important source of VREF for humans than chickens and that human- and pig-derived VSEF isolates seem much more heterogeneous than VREF isolates. AD - Department of Medical Microbiology, University Hospital Maastricht, The Netherlands. FAU - Bruinsma, Nienke AU - Bruinsma N FAU - Willems, Rob J L AU - Willems RJ FAU - van den Bogaard, Anthony E AU - van den Bogaard AE FAU - van Santen-Verheuvel, Marga AU - van Santen-Verheuvel M FAU - London, Nancy AU - London N FAU - Driessen, Christel AU - Driessen C FAU - Stobberingh, Ellen E AU - Stobberingh EE LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antibiotics, Glycopeptide) RN - 1404-90-6 (Vancomycin) SB - IM MH - Algorithms MH - Animals MH - Antibiotics, Glycopeptide/pharmacology MH - Enterococcus faecium/*drug effects/*genetics MH - Feces/microbiology MH - Genotype MH - Gram-Positive Bacterial Infections/microbiology/transmission MH - Humans MH - Phenotype MH - Polymorphism, Restriction Fragment Length MH - Poultry MH - Research Support, Non-U.S. Gov't MH - Swine MH - Vancomycin/pharmacology MH - Vancomycin Resistance/*genetics EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2779-83. PMID- 12183229 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - The LisRK signal transduction system determines the sensitivity of Listeria monocytogenes to nisin and cephalosporins. PG - 2784-90 AB - The Listeria monocytogenes two-component signal transduction system, LisRK, initially identified in strain LO28, plays a significant role in the virulence potential of this important food-borne pathogen. Here, it is shown that, in addition to its major contribution in responding to ethanol, pH, and hydrogen peroxide stresses, LisRK is involved in the ability of the cell to tolerate important antimicrobials used in food and in medicine, e.g., the lantibiotic nisin and the cephalosporin family of antibiotics. A (Delta)lisK mutant (lacking the LisK histidine kinase sensor component) displays significantly enhanced resistance to the lantibiotic nisin, a greatly enhanced sensitivity to the cephalosporins, and a large reduction in the expression of three genes thought to encode a penicillin-binding protein, another histidine kinase (other than LisK), and a protein of unknown function. Confirmation of the role of LisRK was obtained when the response regulator, LisR, was overexpressed using both constitutive and inducible (nisin-controlled expression) systems. Under these conditions we observed a reversion of the (Delta)lisK mutant to wild-type growth kinetics in the presence of nisin. It was also found that overexpression of LisR complemented the reduced expression of two of the aforementioned genes. These results demonstrate the important role of LisRK in the response of L. monocytogenes to a number of antimicrobial agents. AD - Department of Microbiology and National Food Biotechnology Centre, University College Cork, Cork, Ireland. FAU - Cotter, Paul D AU - Cotter PD FAU - Guinane, Caitriona M AU - Guinane CM FAU - Hill, Colin AU - Hill C LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antibiotics, Peptide) RN - 0 (Cephalosporins) RN - 0 (Culture Media) RN - 0 (Plasmids) RN - 0 (Transcription Factors) RN - 1414-45-5 (Nisin) RN - EC 2.7.1.37 (Protein Kinases) RN - EC 2.7.3.- (protein-histidine kinase) SB - IM MH - Antibiotics, Peptide/*pharmacology MH - Cephalosporins/*pharmacology MH - Culture Media MH - Gene Expression Regulation, Bacterial/drug effects/genetics MH - Listeria monocytogenes/*drug effects/genetics MH - Microbial Sensitivity Tests MH - Nisin/*pharmacology MH - Plasmids MH - Protein Kinases/*genetics/physiology MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Signal Transduction/*drug effects/genetics MH - Transcription Factors/*genetics/physiology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2784-90. PMID- 12183230 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Genetic and biochemical characterization of CGB-1, an Ambler class B carbapenem-hydrolyzing beta-lactamase from Chryseobacterium gleum. PG - 2791-6 AB - Chryseobacterium gleum (previously included in the Flavobacterium IIb species) is a gram-negative aerobe that is a source of nosocomial infections. An Ambler class B beta-lactamase gene was cloned and expressed in Escherichia coli from reference strain C. gleum CIP 103039 that had reduced susceptibility to expanded-spectrum cephalosporins and carbapenems. The purified beta-lactamase, CGB-1, with a pI value of 8.6 and a determined relative molecular mass of ca. 26 kDa, hydrolyzed penicillins; narrow- and expanded-spectrum cephalosporins; and carbapenems. CGB-1 was a novel member of the molecular subclass B1 of metallo-enzymes. It had 83 and 42% amino acid identity with IND-1 from Chryseobacterium indologenes and BlaB from C. meningosepticum, respectively. Thus, in addition to the previously characterized clavulanic acid-inhibited extended-spectrum beta-lactamase CGA-1 of Ambler class A, C. gleum produces a very likely chromosome-borne class B beta-lactamase. AD - Service de Bacteriologie-Virologie, Hopital de Bicetre, Assistance Publique/Hopitaux de Paris, Faculte de Medecine Paris-Sud, 94275 Le Kremlin-Bicetre Cedex, France. FAU - Bellais, Samuel AU - Bellais S FAU - Naas, Thierry AU - Naas T FAU - Nordmann, Patrice AU - Nordmann P LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Carbapenems) RN - 0 (DNA, Bacterial) RN - 0 (DNA, Recombinant) RN - 0 (Enzyme Inhibitors) RN - 0 (Plasmids) RN - 58001-44-8 (Clavulanic Acid) RN - EC 3.5.2.6 (beta-Lactamases) RN - EC 3.5.2.6 (beta-lactamase CGB-1, Chryseobacterium gleum) SB - IM MH - Amino Acid Sequence MH - Bacteria/drug effects/enzymology/*genetics MH - Base Sequence MH - Carbapenems/*pharmacology MH - Clavulanic Acid/pharmacology MH - Cloning, Molecular MH - Conjugation, Genetic MH - DNA, Bacterial/analysis MH - DNA, Recombinant/genetics MH - Drug Resistance MH - Enzyme Induction/drug effects MH - Enzyme Inhibitors/pharmacology MH - In Situ Hybridization MH - Isoelectric Focusing MH - Kinetics MH - Microbial Sensitivity Tests MH - Molecular Sequence Data MH - Plasmids/genetics MH - Research Support, Non-U.S. Gov't MH - beta-Lactamases/*genetics EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2791-6. PMID- 12183231 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Frequency of disinfectant resistance genes and genetic linkage with beta-lactamase transposon Tn552 among clinical staphylococci. PG - 2797-803 AB - A total of 61 strains of Staphylococcus aureus and 177 coagulase-negative staphylococcal strains were isolated from the blood of patients with bloodstream infections and from the skin of both children under cancer treatment and human immunodeficiency virus-positive patients. The MIC analyses revealed that 118 isolates (50%) were resistant to quaternary ammonium compound-based disinfectant benzalkonium chloride (BC). The frequencies of resistance to a range of antibiotics were significantly higher among BC-resistant staphylococci than among BC-sensitive staphylococci. Of 78 BC-resistant staphylococcal isolates, plasmid DNA from 65 (83%), 2 (3%), 43 (55%), and 15 (19%) isolates hybridized to qacA or -B (qacA/B), qacC, blaZ, and tetK probes, respectively. The qacA/B and blaZ probes hybridized to the same plasmid in 19 (24%) staphylococcal strains. The plasmids harboring both qacA/B and blaZ genes varied from approximately 20 to 40 kb. The Staphylococcus epidermidis Fol62 isolate, harboring multiresistance plasmid pMS62, contained qacA/B and blaZ together with tetK. Molecular and genetic studies indicated different structural arrangements of blaZ and qacA/B, including variable intergenic distances and transcriptional directions of the two genes on the same plasmid within the strains. The different organizations may be due to the presence of various genetic elements involved in cointegration, recombination, and rearrangements. These results indicate that qac resistance genes are common and that linkage between resistance to disinfectants and penicillin resistance occurs frequently in clinical isolates in Norway. Moreover, the higher frequency of antibiotic resistance among BC-resistant strains indicates that the presence of either resistance determinant selects for the other during antimicrobial therapy and disinfection in hospitals. AD - MATFORSK, Norwegian Food Research Institute, N-1430 As, Norway. FAU - Sidhu, Maan Singh AU - Sidhu MS FAU - Heir, Even AU - Heir E FAU - Leegaard, Truls AU - Leegaard T FAU - Wiger, Karianne AU - Wiger K FAU - Holck, Askild AU - Holck A LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Culture Media) RN - 0 (DNA Transposable Elements) RN - 0 (DNA, Bacterial) RN - 0 (Disinfectants) RN - 0 (Plasmids) SB - IM MH - Culture Media MH - DNA Transposable Elements/*genetics MH - DNA, Bacterial/biosynthesis/genetics MH - Disinfectants/*pharmacology MH - Drug Resistance, Microbial MH - Genes, Bacterial/genetics MH - Linkage (Genetics)/*genetics MH - Microbial Sensitivity Tests MH - Nucleic Acid Hybridization MH - Plasmids/genetics MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Staphylococcal Infections/microbiology MH - Staphylococcus/*drug effects/*genetics EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2797-803. PMID- 12183232 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Isoniazid-induced transient high-level resistance in Mycobacterium tuberculosis. PG - 2804-10 AB - An American Type Culture Collection reference strain and eight clinical strains of Mycobacterium tuberculosis, all of which were susceptible to isoniazid (INH) (mean MIC, 0.06 mg/liter) and negative for the Ser315Thr katG mutation, were left in their BACTEC 12B vials (for use with the BACTEC 460-TB method) containing 0.1 mg of INH per liter for periods of up to 28 days after the completion of the antibiotic susceptibility test. Each eventually grew to levels compatible with those of INH-resistant strains. Successive passages in INH-containing BACTEC 12B vials and onto solid media showed that the resistance noted above was maintained. Successive passages of these M. tuberculosis strains in which INH resistance had been induced into BACTEC 12B vials or solid media containing stepwise increases in INH concentrations eventually yielded organisms resistant to 20 mg of INH per liter. Transfer of cells in which INH resistance had been induced to drug-free medium followed by repeated passages in that medium eventually yielded organisms whose susceptibility to INH was identical to that of the original parent strains. The cycle of induced INH resistance could be repeated with these now INH-susceptible cells. The use of M. tuberculosis identification probes and IS6110-based restriction fragment length polymorphism analyses of cultures throughout the induction of INH resistance and the reversal of resistance in drug-free medium eliminated the possibility that the culture was contaminated or that the initial specimen had a mixed type of infection. Induced high-level resistance to INH (20 mg/liter) could be reduced 100-fold with a subinhibitory concentration of reserpine but not with verapamil. These results collectively suggest that high-level resistance to INH can be induced in INH-susceptible M. tuberculosis strains by the induction of a reserpine-sensitive efflux mechanism. AD - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, P-1349-008 Lisbon, Portugal. FAU - Viveiros, Miguel AU - Viveiros M FAU - Portugal, Isabel AU - Portugal I FAU - Bettencourt, Rosario AU - Bettencourt R FAU - Victor, Thomas C AU - Victor TC FAU - Jordaan, Annemarie M AU - Jordaan AM FAU - Leandro, Clara AU - Leandro C FAU - Ordway, Diane AU - Ordway D FAU - Amaral, Leonard AU - Amaral L LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antitubercular Agents) RN - 0 (Bacterial Proteins) RN - 0 (Calcium Channel Blockers) RN - 50-55-5 (Reserpine) RN - 52-53-9 (Verapamil) RN - 54-85-3 (Isoniazid) RN - EC 1.11.1. (Peroxidases) RN - EC 1.11.1.6 (catalase HPI) SB - IM MH - Antitubercular Agents/metabolism/*pharmacology MH - *Bacterial Proteins MH - Calcium Channel Blockers/pharmacology MH - Drug Resistance, Bacterial MH - Isoniazid/metabolism/*pharmacology MH - Microbial Sensitivity Tests MH - Mycobacterium tuberculosis/*drug effects/metabolism MH - Peroxidases/genetics MH - Polymorphism, Restriction Fragment Length MH - Research Support, Non-U.S. Gov't MH - Reserpine/pharmacology MH - Reverse Transcriptase Polymerase Chain Reaction MH - Verapamil/pharmacology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2804-10. PMID- 12183233 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Identification and analysis of amino acid mutations in porin IB that mediate intermediate-level resistance to penicillin and tetracycline in Neisseria gonorrhoeae. PG - 2811-20 AB - PenB is the third resistance determinant in the stepwise acquisition of multiple resistance genes in chromosomally mediated resistant Neisseria gonorrhoeae (CMRNG). Alterations in por(IB), one of two alleles at the por locus that encodes the outer membrane protein porin IB (PIB), were recently reported to be responsible for the increased resistance to penicillin and tetracycline conferred by penB, but the specific mutations conferring antibiotic resistance were not identified experimentally. To determine which amino acids in PIB confer increased resistance, we transformed a recipient strain with chimeras of the por(IB) genes from strains FA1090 and FA140 (penB2). These studies revealed that two amino acid changes, G120D and A121D, were both necessary and sufficient to confer increased resistance to penicillin and tetracycline. Site-saturation and site-directed mutagenesis of Gly-120 and Ala-121 revealed that both a single mutation, G120K, and the double mutations G120R A121H and G120P A121P also conferred antibiotic resistance to the recipient strain. The identical mutations in PIA increased penicillin and tetracycline resistance either moderately or not at all. Analysis of por(IB) genes present in the GenBank database from 51 clinical isolates demonstrated that lysine and aspartate mutations at positions 120 and/or 121 also occur in nature. These studies demonstrate that charged amino acids at positions 120 and 121 in PIB are highly preferential for conferring resistance to penicillin and tetracycline in N. gonorrhoeae. AD - Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365, USA. FAU - Olesky, Melanie AU - Olesky M FAU - Hobbs, Marcia AU - Hobbs M FAU - Nicholas, Robert A AU - Nicholas RA LA - eng GR - AI-36901/AI/NIAID PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Bacterial Outer Membrane Proteins) RN - 0 (Chimeric Proteins) RN - 0 (DNA, Bacterial) RN - 0 (Porins) RN - 0 (porin proteins, Neisseria) SB - IM MH - Amino Acid Sequence MH - Amino Acid Substitution MH - Bacterial Outer Membrane Proteins/drug effects/metabolism MH - Blotting, Western MH - Chimeric Proteins/chemistry/genetics MH - DNA, Bacterial/drug effects/genetics MH - Electrophoresis, Polyacrylamide Gel MH - Microbial Sensitivity Tests MH - Molecular Sequence Data MH - Mutation/*genetics MH - Neisseria gonorrhoeae/drug effects/*genetics/growth & development MH - Penicillin Resistance/*genetics MH - Porins/*genetics MH - Research Support, U.S. Gov't, P.H.S. MH - Reverse Transcriptase Polymerase Chain Reaction MH - Tetracycline Resistance/*genetics MH - Transformation, Bacterial EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2811-20. PMID- 12183234 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Antibiotic resistance genes and Salmonella genomic island 1 in Salmonella enterica serovar Typhimurium isolated in Italy. PG - 2821-8 AB - Fifty-four epidemiologically unrelated multidrug-resistant Salmonella enterica serovar Typhimurium isolates, collected between 1992 and 2000 in Italy, were analyzed for the presence of integrons. Strains were also tested for Salmonella genomic island 1 (SGI1), carrying antibiotic resistance genes in DT104 strains. A complete SGI1 was found in the majority of the DT104 strains. Two DT104 strains, showing resistance to streptomycin-spectinomycin and sulfonamides, carried a partially deleted SGI1 lacking the flo(st), tetR, and tetA genes, conferring chloramphenicol-florfenicol and tetracycline resistance, and the integron harboring the pse-1 gene cassette, conferring ampicillin resistance. The presence of SGI1 was also observed in serovar Typhimurium strains belonging to other phage types, suggesting either the potential mobility of this genomic island or changes in the phage-related phenotype of DT104 strains. AD - Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanita, Rome, Italy. alecara@iss.it FAU - Carattoli, Alessandra AU - Carattoli A FAU - Filetici, Emma AU - Filetici E FAU - Villa, Laura AU - Villa L FAU - Dionisi, Anna Maria AU - Dionisi AM FAU - Ricci, Antonia AU - Ricci A FAU - Luzzi, Ida AU - Luzzi I LA - eng SI - GENBANK/AF261825 PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (DNA, Bacterial) SB - IM MH - Blotting, Southern MH - Cloning, Molecular MH - DNA, Bacterial/genetics MH - Drug Resistance, Microbial MH - Electrophoresis, Gel, Pulsed-Field MH - Genes, Bacterial/*genetics MH - Genome, Bacterial MH - Humans MH - Italy MH - Molecular Sequence Data MH - Research Support, Non-U.S. Gov't MH - Salmonella Infections/microbiology MH - Salmonella enterica/*drug effects/*genetics EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2821-8. PMID- 12183235 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - The Candida dubliniensis CdCDR1 gene is not essential for fluconazole resistance. PG - 2829-41 AB - The present study investigated the role of the Candida dubliniensis CdCDR1 and CdCDR2 genes in the development of fluconazole resistance. The C. dubliniensis CdCDR1 gene was 92% identical at the nucleotide sequence level to the corresponding C. albicans gene. However, 58% (14 of 24) of C. dubliniensis genotype 1 isolates tested harbored a nonsense mutation in the CdCDR1 open reading frame that converted codon 756 (TAT) to a TAG translational stop codon. Analysis of five of these C. dubliniensis isolates by Western immunoblotting showed that they expressed a truncated 85-kDa CdCdr1p compared to the full-length 170-kDa CdCdr1p. Expression of CdCDR1 alleles from six C. dubliniensis isolates in a pdr5 Saccharomyces cerevisiae strain revealed that CdCDR1 alleles from three isolates that encoded truncated proteins were unable to confer resistance to drugs and antifungals. However, reassignment of the TAG sequence at codon 756 to TAT (encoding tyrosine) in an allele from strain CD36 conferred the ability to mediate resistance to multiple drugs. Fluconazole-resistant isolates of C. dubliniensis harboring functional alleles of CdCDR1 were found to exhibit two- to ninefold-higher levels of CdCDR1 mRNA than did matched fluconazole-susceptible isolates. By comparison, levels of CdMDR1 expression ranged from approximately 50- to 100-fold greater in resistant isolates. Fluconazole resistance was also identified in isolates harboring nonfunctional CdCDR1 alleles, but resistance in these isolates was only associated with increased CdMDR1 expression. Targeted disruption of two functional alleles of CdCDR1 in a fluconazole-resistant derivative of C. dubliniensis that overexpressed both CdCDR1 and CdMDR1 revealed that although CdCDR1 was important for mediating reduced susceptibility to itraconazole and ketoconazole, there was no affect on fluconazole susceptibility in the double mutant. Evidence presented in this study reveals that CdCDR1 is not essential for the development of fluconazole resistance in C. dubliniensis. AD - Microbiology Research Unit, Department of Oral Surgery, Oral Medicine and Pathology, School of Dental Science, Trinity College, University of Dublin, Republic of Ireland. FAU - Moran, Gary AU - Moran G FAU - Sullivan, Derek AU - Sullivan D FAU - Morschhauser, Joachim AU - Morschhauser J FAU - Coleman, David AU - Coleman D LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (ATP-Binding Cassette Transporters) RN - 0 (Antifungal Agents) RN - 0 (Azoles) RN - 0 (CDR1 protein, Candida albicans) RN - 0 (CDR2 protein, fungal) RN - 0 (DNA Primers) RN - 0 (DNA, Fungal) RN - 0 (Fungal Proteins) RN - 0 (Membrane Transport Proteins) RN - 0 (RNA, Messenger) RN - 86386-73-4 (Fluconazole) SB - IM MH - ATP-Binding Cassette Transporters/biosynthesis/genetics MH - Alleles MH - Antifungal Agents/*pharmacology MH - Azoles/pharmacology MH - Blotting, Southern MH - Blotting, Western MH - Candida/*drug effects/*genetics MH - DNA Primers/pharmacology MH - DNA, Fungal/drug effects/genetics MH - Drug Resistance, Fungal MH - Fluconazole/*pharmacology MH - Fungal Proteins/biosynthesis/genetics MH - Gene Targeting MH - Membrane Transport Proteins/biosynthesis/*genetics MH - Mutagenesis, Site-Directed MH - RNA, Messenger/biosynthesis MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Saccharomyces cerevisiae/drug effects/genetics MH - Transformation, Genetic/genetics EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2829-41. PMID- 12183236 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Effect of 5-iodo-2'-deoxyuridine on vaccinia virus (orthopoxvirus) infections in mice. PG - 2842-7 AB - There is a concern that there may be unregistered stocks of smallpox that can be used for bioterrorism or biological warfare. According to the WHO Advisory Committee on Variola Research, there is a need to develop strategies to treat smallpox infections should they reappear. It would also be important to have an effective drug at hand for the treatment of monkeypox disease in humans. We show here that 5-iodo-2'-deoxyuridine (IDU) is a potent inhibitor of vaccinia virus (VV) replication and that IDU inhibits VV DNA synthesis in a dose-dependent way. The in vivo protective effect of IDU was assessed in the VV tail lesion model in immunocompetent mice and in a lethal model for VV infection in SCID (severe combined immune deficiency) mice that had been infected either intranasally, intraperitoneally, or intravenously. Subcutaneous treatment with IDU at 150 and 100 mg/kg of body weight markedly reduced the number of tail lesions in immunocompetent NMRI mice. Untreated intranasally VV-infected SCID mice died at 20.8 +/- 3.1 days after infection (mean +/- standard deviation). Treatment with IDU (subcutaneously, 150 mg/kg/day [from day 0 to 4] and 75 mg/kg/day [from day 6 to 11]) delayed-virus induced mortality by 15 days (mean day of death +/- standard deviation, 35.8 +/- 6.7; P < 0.0001). This protective effect was associated with (i) an improvement of lung histology and (ii) a marked reduction in lung viral titers. IDU also delayed VV-induced mortality when mice had either been infected intraperitoneally or intravenously. Even when the start of treatment with IDU (in intraperitoneally VV-infected mice) was postponed until 2 or 4 days after infection, an important delay in virus-induced mortality was noted. AD - Rega Institute for Medical Research, Katholieke Universiteit Leuven, 3000 Leuven, Belgium. johan.neyts@rega.kuleuven.ac.be FAU - Neyts, Johan AU - Neyts J FAU - Verbeken, Erik AU - Verbeken E FAU - De Clercq, Erik AU - De Clercq E LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antiviral Agents) RN - 0 (DNA, Viral) RN - 54-42-2 (Idoxuridine) SB - IM MH - Animals MH - Antiviral Agents/*therapeutic use MH - Body Weight/physiology MH - Cells, Cultured MH - DNA, Viral/biosynthesis/genetics MH - Growth/physiology MH - Humans MH - Idoxuridine/*therapeutic use MH - Immunologic Deficiency Syndromes/immunology MH - Lung/virology MH - Mice MH - Mice, SCID MH - Plaque Assay MH - Research Support, Non-U.S. Gov't MH - Tail/pathology MH - Vaccinia/*drug therapy/pathology/virology MH - *Vaccinia virus EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2842-7. PMID- 12183237 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Penciclovir susceptibilities of herpes simplex virus isolates from patients using penciclovir cream for treatment of recurrent herpes labialis. PG - 2848-53 AB - The antiherpesvirus agent penciclovir (PCV) shares an identical activation pathway and a similar mode of action with acyclovir (ACV). However, since PCV represents a relatively recent treatment option, the clinical resistance profile to PCV is less well known. A susceptibility program was established to assess the resistance profile for serial herpes simplex virus isolates from immunocompetent patients with recurrent herpes labialis obtained throughout a 4-day period of treatment with topical PCV (1% cream) or a placebo. Two isolates (2 of 1,035 [0.19%]), representing 0.34% of the patients (2 of 585), were confirmed to be PCV-resistant (Pcv(r)) herpes simplex virus type 1 by a plaque reduction assay in MRC-5 cells. These two viruses were highly resistant to PCV (50% inhibitory concentrations [IC(50)s], >55 micro g/ml) and were isolated less than 17 h after the start of patient-initiated treatment. However, subsequent isolates on days 2 and 3 from these patients were completely susceptible to PCV (IC(50)s, <2.0 micro g/ml). Thus, it is not clear whether the resistance to PCV for these two early-treatment isolates was directly associated with the 17 h of PCV treatment; several possible explanations are discussed. In an analysis of the distribution of IC(50) differences between the first and last isolates, there were three patients with minor IC(50) increases in the PCV-treated population and one in the placebo-treated group, although statistically, only the latter was an outlier. No patients were found to have Pcv(r) virus at the end of acute treatment, regardless of treatment group. Overall, the prevalence of Pcv(r) was found to be similar to the 0.3% Acv(r) reported for immunocompetent, untreated populations. AD - Department of Host Defense, The Antimicrobial and Host Defense Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 19426-0989, USA. robert_t_sarisky@gsk.com FAU - Sarisky, Robert T AU - Sarisky RT FAU - Bacon, Teresa AU - Bacon T FAU - Boon, Ron AU - Boon R FAU - Locke, Leslie AU - Locke L FAU - Nguyen, Tammy T AU - Nguyen TT FAU - Leary, Jeffry AU - Leary J FAU - Esser, Klaus AU - Esser K FAU - Saltzman, Robin AU - Saltzman R LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antiviral Agents) RN - 39809-25-1 (penciclovir) RN - 59277-89-3 (Acyclovir) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) SB - IM MH - Acyclovir/*analogs & derivatives/*pharmacology MH - Antiviral Agents/*pharmacology MH - Autoradiography MH - Drug Resistance, Viral MH - Frameshift Mutation/genetics MH - Herpes Labialis/*drug therapy/*virology MH - Herpesvirus 1, Human/*drug effects/genetics/isolation & purification MH - Humans MH - Microbial Sensitivity Tests MH - Plaque Assay MH - Protein-Tyrosine Kinase/metabolism MH - Recurrence EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2848-53. PMID- 12183238 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Samarangenin B from Limonium sinense suppresses herpes simplex virus type 1 replication in Vero cells by regulation of viral macromolecular synthesis. PG - 2854-64 AB - Inhibitory effects of ethanolic extracts from 10 Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were investigated. By a bioassay-guided fractionation procedure, samarangenin B (Sam B) was isolated from Limonium sinense; Sam B significantly suppressed HSV-1 multiplication in Vero cells without apparent cytotoxicity. Time-of-addition experiments suggested that the inhibitory action of Sam B on HSV-1 replication was not due to the blocking of virus adsorption. In an attempt to further localize the point in the HSV-1 replication cycle where arrest occurred, a set of key regulatory events leading to viral multiplication was examined, including viral immediate-early (alpha), early (beta), and late (gamma) gene expression and DNA replication. Results indicated that levels of glycoprotein B (gB), gC, gD, gG, and infected-cell protein 5 (ICP5) expression and gB mRNA expression in Vero cells were impeded by Sam B. Data from PCR showed that replication of HSV-1 DNA in Vero cells was arrested by Sam B. Furthermore, Sam B decreased DNA polymerase, ICP0, and ICP4 gene expression in Vero cells. Results of an electrophoretic mobility shift assay demonstrated that Sam B interrupted the formation of an alpha-trans-induction factor/C1/Oct-1/GARAT multiprotein complex. The mechanisms of antiviral action of Sam B seem to be mediated, at least in part, by inhibiting HSV-1 alpha gene expression, including expression of the ICP0 and ICP4 genes, by blocking beta transcripts such as DNA polymerase mRNA, and by arresting HSV-1 DNA synthesis and structural protein expression in Vero cells. These results show that Sam B is an antiviral agent against HSV-1 replication. AD - National Research Institute of Chinese Medicine,Taipei, Taiwan, Republic of China. kuo9111@cma23.nricm.edu.tw FAU - Kuo, Yuh-Chi AU - Kuo YC FAU - Lin, Lie-Chwen AU - Lin LC FAU - Tsai, Wei-Jern AU - Tsai WJ FAU - Chou, Cheng-Jen AU - Chou CJ FAU - Kung, Szu-Hao AU - Kung SH FAU - Ho, Yen-Hui AU - Ho YH LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antiviral Agents) RN - 0 (Benzopyrans) RN - 0 (DNA, Complementary) RN - 0 (DNA, Viral) RN - 0 (Drugs, Chinese Herbal) RN - 0 (RNA, Viral) RN - 0 (samarangenin B) RN - EC 2.7.7.7 (DNA-Directed DNA Polymerase) SB - IM MH - Animals MH - Antiviral Agents/*pharmacology MH - Benzopyrans/isolation & purification/*pharmacology MH - Blotting, Northern MH - Blotting, Western MH - Cell Survival/drug effects MH - Cercopithecus aethiops MH - DNA Replication/drug effects MH - DNA, Complementary/biosynthesis/genetics MH - DNA, Viral/*biosynthesis MH - DNA-Directed DNA Polymerase/biosynthesis/genetics MH - Drugs, Chinese Herbal/pharmacology MH - Electrophoretic Mobility Shift Assay MH - Herpesvirus 1, Human/*drug effects/*metabolism MH - Plaque Assay MH - Plumbaginaceae/*chemistry MH - RNA, Viral/biosynthesis/genetics MH - Research Support, Non-U.S. Gov't MH - Vero Cells MH - Virus Replication/*drug effects EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2854-64. PMID- 12183239 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Immunomodulatory effect of zidovudine (ZDV) on cytotoxic T lymphocytes previously exposed to ZDV. PG - 2865-71 AB - In a previous study, zidovudine (ZDV) was shown to cause a concentration-dependent inhibition of antigen-specific cytotoxic T-lymphocyte (CTL) clonal expansion (S. Francke, C. G. Orosz, K. A. Hayes, and L. E. Mathes, Antimicrob. Agents Chemother. 44:1900-1905, 2000). However, this suppressive effect was lost if exposure to ZDV was delayed for 24 to 48 h during the antigen sensitization period, suggesting that antigen-primed CTL may be less susceptible than naive T lymphocytes to the suppressive effects of ZDV. The present study was undertaken to determine if naive T lymphocytes were more sensitive to the suppressive effects of ZDV than T lymphocytes previously exposed to antigen. The 50% inhibitory concentration (IC(50)) values of ZDV were determined on naive and antigen-primed T-cell responses in an alloantigen system. Lymphocyte cultures with continuous antigen exposure (double prime) were more resistant to ZDV suppression (IC(50) = 316 micro M) than were naive lymphocytes (IC(50) = 87.5 micro M). Interestingly, lymphocytes that were antigen primed but deprived of antigen during the final 7 days of culture (prime/hold) were exquisitely sensitive to ZDV suppression (IC(50) = 29.3 micro M). The addition of 80 micro M ZDV during the initial priming of the single-prime (prime/hold) and double-prime cultures did not select for a more drug-resistant cell population. The differences in ZDV sensitivities are likely a reflection of the physiological properties of the lymphocytes related to their activation state. AD - Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210, USA. FAU - Francke, Sabine AU - Francke S FAU - Orosz, Charles G AU - Orosz CG FAU - Hsu, Jason AU - Hsu J FAU - Mathes, Lawrence E AU - Mathes LE LA - eng GR - P30 CA16058/CA/NCI GR - R01 AI40855/AI/NIAID PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Adjuvants, Immunologic) RN - 0 (Anti-HIV Agents) RN - 0 (Antigens, Viral) RN - 0 (Chromium Radioisotopes) RN - 0 (Interleukin-2) RN - 30516-87-1 (Zidovudine) SB - IM MH - Adjuvants, Immunologic/*pharmacology MH - Animals MH - Anti-HIV Agents/*pharmacology MH - Antigens, Viral/immunology MH - Cell Separation MH - Chromium Radioisotopes/diagnostic use MH - Drug Resistance, Viral MH - Female MH - In Vitro MH - Interleukin-2/pharmacology MH - Lymphocyte Culture Test, Mixed MH - Mice MH - Mice, Inbred DBA MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - T-Lymphocytes, Cytotoxic/*drug effects/*immunology MH - Zidovudine/*pharmacology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2865-71. PMID- 12183240 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Antiviral activities of MCC-478, a novel and specific inhibitor of hepatitis B virus. PG - 2872-7 AB - MCC-478 is a newly synthesized 2-amino-6-arylthio-9-phosphonomethoxyethylpurine bis(2,2,2-trifluoroethyl) ester derivative. MCC-478 showed a substantially higher (ca. 80-fold) anti-hepatitis B virus (HBV) activity than that of lamivudine, despite no significant anti-human immunodeficiency virus activity. Since the bis(2,2,2-trifluoroethyl) ester group was used to improve the oral bioavailability of the phosphonomethoxyethylpurine derivatives, two monoester derivatives and one phosphonic acid derivative were also evaluated. It was suggested that these hydrolyzed derivatives, which appeared in animals given MCC-478, have enough anti-HBV activity to contribute to efficacy in vivo. Furthermore, no apparent cytotoxic effects or reductions of mitochondrial DNA content by MCC-478 and its derivatives were observed. These results indicated that MCC-478 may be a new promising anti-HBV agent. AD - Research Laboratory IV, Mitsubishi Pharma Corporation, Yokohama, Japan. Kamiya.Naohiro@mh.m-pharma.co.jp FAU - Kamiya, Naohiro AU - Kamiya N FAU - Kubota, Atsushi AU - Kubota A FAU - Iwase, Yumiko AU - Iwase Y FAU - Sekiya, Kouichi AU - Sekiya K FAU - Ubasawa, Masaru AU - Ubasawa M FAU - Yuasa, Satoshi AU - Yuasa S LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (2-amino-6-(4-methoxyphenylthio)-9-(2-(phosphonomethoxy)ethyl)purine bis(2,2,2-trifluoroethyl) ester) RN - 0 (Anti-HIV Agents) RN - 0 (Antiviral Agents) RN - 0 (DNA, Mitochondrial) RN - 0 (Purines) SB - IM MH - Animals MH - Anti-HIV Agents/pharmacology MH - Antiviral Agents/*pharmacology/toxicity MH - Cell Survival/drug effects MH - Cells, Cultured MH - DNA, Mitochondrial/drug effects MH - HIV-1/drug effects MH - Hepatitis B virus/*drug effects MH - Humans MH - Purines/*pharmacology MH - Tumor Cells, Cultured MH - Virus Replication/drug effects EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2872-7. PMID- 12183241 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Effects of neutrophils on cefazolin activity and penicillin-binding proteins in Staphylococcus aureus abscesses. PG - 2878-84 AB - Bacteria survive within abscesses despite antimicrobial therapy, usually necessitating drainage. Our previous work showed that bacterial killing is diminished within the neutrophils of animals with abscesses. To further assess the role of neutrophils in Staphylococcus aureus survival and the poor activities of beta-lactams in abscesses, tissue cage abscess-bearing rats were given polymorphonuclear leukocyte (PMN)-depleting antibody prior to and several times following inoculation of the tissue cages with S. aureus. Cefazolin (300 mg/kg of body weight/day) was administered to all animals in appropriately divided doses. After 7 days of antimicrobial therapy, the 17 animals that received anti-PMN serum had significantly fewer abscess neutrophils than the 18 controls and fewer abscess bacteria (5.55 versus 3.79 log(10) CFU/ml [P = 0.04]) than the 18 controls. The data were consistent with the premise that cefazolin is more effective in abscesses depleted of neutrophils. To investigate further, S. aureus was incubated with rat peritoneal neutrophils; and bacterial cell membrane proteins were isolated, labeled with biotinylated ampicillin, separated by electrophoresis, blotted onto nitrocellulose, and stained for biotin reactivity. PBP 2 expression was consistently and significantly decreased after a brief, nonkilling PMN exposure. These experiments showed that PMN depletion enhanced the activity of cefazolin in the abscess milieu. Furthermore, altered bacterial cell wall cefazolin targets may be the mechanism by which the PMN diminishes antimicrobial activity, suggesting the importance of the staphylococcus-PMN interaction in the outcome of established infections. AD - Section of Infectious Diseases, Department of Medicine, University of Missouri--Kansas City, Kansas City, Missouri 64108, USA. bambergerd@umkc.edu FAU - Bamberger, David M AU - Bamberger DM FAU - Herndon, Betty L AU - Herndon BL FAU - Fitch, Jeffrey AU - Fitch J FAU - Florkowski, Aaron AU - Florkowski A FAU - Parkhurst, Vera AU - Parkhurst V LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Bacterial Proteins) RN - 0 (Carrier Proteins) RN - 0 (Fluorescent Dyes) RN - 0 (Penicillin-Binding Proteins) RN - 25953-19-9 (Cefazolin) RN - 58-85-5 (Biotin) RN - 69-53-4 (Ampicillin) RN - EC 2.3.2.12 (Peptidyl Transferases) RN - EC 2.4.1.- (Hexosyltransferases) RN - EC 3.4.17.8 (Muramoylpentapeptide Carboxypeptidase) SB - IM MH - Abscess/immunology/*microbiology MH - Ampicillin/pharmacology MH - Animals MH - Anti-Bacterial Agents/pharmacology MH - *Bacterial Proteins MH - Biotin MH - Carrier Proteins/*metabolism MH - Cefazolin/*pharmacology MH - Fluorescent Dyes MH - *Hexosyltransferases MH - Lymphocyte Count MH - Male MH - Muramoylpentapeptide Carboxypeptidase/*metabolism MH - Neutropenia/microbiology MH - Neutrophils/drug effects/*physiology MH - Penicillin-Binding Proteins MH - *Peptidyl Transferases MH - Phagocytosis/drug effects MH - Rats MH - Research Support, Non-U.S. Gov't MH - Staphylococcal Infections/immunology/*microbiology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2878-84. PMID- 12183242 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20031114 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Efficacy of quinupristin-dalfopristin in preventing vascular graft infection due to Staphylococcus epidermidis with intermediate resistance to glycopeptides. PG - 2885-8 AB - A rat model was used to investigate the efficacy of quinupristin-dalfopristin (Q-D) in the prevention of vascular prosthetic graft infection due to methicillin-resistant Staphylococcus epidermidis with intermediate resistance to glycopeptides. The in vitro activity of the compound was compared to that of vancomycin by MIC determination and time-kill study. Moreover, the efficacy of collagen-sealed Q-D-soaked Dacron was evaluated in a rat model of graft infection. Graft infections were established in the subcutaneous tissue of the backs of 120 adult male Wistar rats. The in vivo study included a control group, one contaminated group that did not receive any antibiotic prophylaxis, two contaminated groups that received grafts soaked with 10 and 100 micro g of Q-D per ml, respectively, and two contaminated groups that received grafts soaked with 10 and 100 micro g of vancomycin per ml, respectively. Rats that received Dacron grafts soaked with 100 micro g of Q-D per ml showed no evidence of infection (<10 CFU/ml). In contrast, for rats that received Dacron grafts soaked with 10 micro g of Q-D per ml and Dacron grafts soaked with 10 or 100 micro g of vancomycin per ml, the quantitative graft cultures demonstrated 2.2 x 10(2) +/- 1.3 x 10(2), 2.2 x 10(6) +/- 1.9 x 10(5), and 5.6 x 10(2) +/- 0.3 x 10(2) CFU/ml, respectively. Taken together the results of the study demonstrate that the use of Dacron grafts soaked with Q-D can result in significant bacterial growth inhibition and show that this compound is potentially valuable for prevention of vascular prosthetic graft infection. AD - Institute of Infectious Diseases and Public Health, University of Ancona, Italy. anconacmi@interfree.it FAU - Giacometti, Andrea AU - Giacometti A FAU - Cirioni, Oscar AU - Cirioni O FAU - Ghiselli, Roberto AU - Ghiselli R FAU - Orlando, Fiorenza AU - Orlando F FAU - Mocchegiani, Federico AU - Mocchegiani F FAU - Riva, Alessandra AU - Riva A FAU - Del Prete, Maria Simona AU - Del Prete MS FAU - Saba, Vittorio AU - Saba V FAU - Scalise, Giorgio AU - Scalise G LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antibiotics, Glycopeptide) RN - 0 (Antibiotics, Peptide) RN - 11006-76-1 (Virginiamycin) RN - 126602-89-9 (quinupristin-dalfopristin) SB - IM MH - Animals MH - Antibiotics, Glycopeptide/*pharmacology MH - Antibiotics, Peptide/*therapeutic use MH - Blood Vessel Prosthesis/*adverse effects MH - Drug Resistance, Microbial MH - Male MH - Microbial Sensitivity Tests MH - Prosthesis-Related Infections/microbiology/*prevention & control MH - Rats MH - Rats, Wistar MH - Staphylococcal Infections/microbiology/*prevention & control MH - *Staphylococcus epidermidis MH - Vancomycin Resistance MH - Virginiamycin/*therapeutic use EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2885-8. PMID- 12183243 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - In vitro and in vivo synergy of fosmidomycin, a novel antimalarial drug, with clindamycin. PG - 2889-94 AB - Fosmidomycin acts through inhibition of 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, a key enzyme of the nonmevalonate pathway of isoprenoid biosynthesis. It possesses potent antimalarial activity in vitro and in murine malaria. In a recent clinical study, fosmidomycin was effective and well tolerated in the treatment of patients with acute uncomplicated Plasmodium falciparum malaria but resulted in an unacceptably high rate of recrudescence. In order to identify a potential combination partner, the interaction of fosmidomycin with a number of antimalarial drugs in current use was investigated in a series of in vitro experiments. Synergy was observed between fosmidomycin and the lincosamides, lincomycin and clindamycin. The efficacy of a combination of fosmidomycin and clindamycin was subsequently demonstrated in the Plasmodium vinckei mouse model. AD - Institute of Biochemistry, Academic Hospital Centre, Justus-Liebig-University, Giessen, Germany. Jochen.Wiesner@Jomaa.de FAU - Wiesner, Jochen AU - Wiesner J FAU - Henschker, Dajana AU - Henschker D FAU - Hutchinson, David B AU - Hutchinson DB FAU - Beck, Ewald AU - Beck E FAU - Jomaa, Hassan AU - Jomaa H LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Antimalarials) RN - 18323-44-9 (Clindamycin) RN - 23155-02-4 (Fosfomycin) RN - 66508-53-0 (fosmidomycin) SB - IM MH - Animals MH - Anti-Bacterial Agents/*pharmacology MH - Antimalarials/*pharmacology MH - Clindamycin/*pharmacology MH - Dose-Response Relationship, Drug MH - Drug Synergism MH - Fosfomycin/*analogs & derivatives/*pharmacology MH - Humans MH - Malaria/drug therapy/parasitology MH - Mice MH - Plasmodium falciparum/*drug effects EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2889-94. PMID- 12183244 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Ertapenem versus ceftriaxone followed by appropriate oral therapy for treatment of complicated urinary tract infections in adults: results of a prospective, randomized, double-blind multicenter study. PG - 2895-900 AB - The efficacy and safety of intravenous (i.v.) ertapenem (1 g once a day) with the option to switch to an oral agent for treatment of adults with complicated urinary tract infections (UTIs) were compared with that of i.v. ceftriaxone (1 g daily) with the same oral switch option in a multicenter, double-blind, prospective, randomized study. At entry, 592 patients were assigned to one of two strata: acute pyelonephritis or other complicated UTI without acute pyelonephritis. After a minimum of 3 days, patients could be switched to an oral antimicrobial agent. A total of 159 patients in the ertapenem group and 171 patients in the ceftriaxone group were microbiologically evaluable. Approximately 95% of patients in each treatment group were switched to oral therapy. The most common pathogens were Escherichia coli and Klebsiella pneumoniae. At the primary efficacy endpoint 5 to 9 days after treatment, 91.8% of patients who received ertapenem and 93.0% of those who received ceftriaxone had a favorable microbiological response (95% confidence interval for the difference, adjusting for strata, -7.6 to 5.1%), indicating that outcomes in the two treatment groups were equivalent. Microbiological success rates for the two treatment groups were similar when compared by stratum and also by severity of infection. The frequency and severity of drug-related adverse events were generally similar in both treatment groups. In this study, ertapenem was as effective as ceftriaxone for the initial treatment of complicated UTIs in adults, was generally well tolerated, and had a similar overall safety profile. AD - Alaska Clinical Research Center, Anchorage, Alaska, USA. FAU - Tomera, Kevin M AU - Tomera KM FAU - Burdmann, Emmanuel A AU - Burdmann EA FAU - Reyna, Oscar G Pamo AU - Reyna OG FAU - Jiang, Qi AU - Jiang Q FAU - Wimmer, Wendy M AU - Wimmer WM FAU - Woods, Gail L AU - Woods GL FAU - Gesser, Richard M AU - Gesser RM CN - Protocol 014 Study Group. LA - eng PT - Clinical Trial PT - Journal Article PT - Multicenter Study PT - Randomized Controlled Trial PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Anti-Infective Agents, Urinary) RN - 0 (Cephalosporins) RN - 0 (Lactams) RN - 0 (ertapenem) RN - 73384-59-5 (Ceftriaxone) SB - IM MH - Adult MH - Anti-Bacterial Agents/adverse effects/*therapeutic use MH - Anti-Infective Agents, Urinary/adverse effects/*therapeutic use MH - Ceftriaxone/adverse effects/*therapeutic use MH - Cephalosporins/adverse effects/*therapeutic use MH - Comparative Study MH - Double-Blind Method MH - Female MH - Humans MH - *Lactams MH - Male MH - Prospective Studies MH - Research Support, Non-U.S. Gov't MH - Urinary Tract Infections/*drug therapy/microbiology/urine EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2895-900. PMID- 12183245 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Method for estimation of low outer membrane permeability to beta-lactam antibiotics. PG - 2901-7 AB - The outer membrane of gram-negative bacteria plays a major role in beta-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with beta-lactamases and efflux systems. Up to now, the quantitative estimation of low outer membrane permeability by the method of Zimmermann and Rosselet was difficult because of the secreted and cell surface-associated beta-lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of beta-lactam penetration into the periplasm. The method is dedicated to measurement of low permeability and is only valid when the diffusion rate through the outer membrane is rate limiting. Cytoplasmic membrane associated PBPs do not interfere since they are acylated after the very sensitive BlaR-CTD. This method was used to measure the permeability of beta-lactamase-deficient strains of Enterobacter cloacae and Enterobacter aerogenes to benzylpenicillin, ampicillin, carbenicillin, cefotaxime, aztreonam, and cephacetrile. Except for that of cephacetrile, the permeability coefficients were equal to or below 10(-7) cm/s. For cephacetrile, carbenicillin, and benzylpenicillin, the outer membrane of E. cloacae was 20 to 60 times less permeable than that of Escherichia coli, whereas for cefotaxime, aztreonam, and ampicillin it was, respectively, 400, 1,000, and 700 times less permeable. The permeability coefficient for aztreonam is the lowest ever measured (P = 3.2 x 10(-9) cm/s). Using these values, the MICs for a beta-lactamase-overproducing strain of E. cloacae were successfully predicted, demonstrating the validity of the method. AD - Laboratoire d'Enzymologie and Centre d'Ingenierie des Proteines, Universite de Liege, Institut de Chimie, B-4000 Liege, Belgium. FAU - Lakaye, Bernard AU - Lakaye B FAU - Dubus, Alain AU - Dubus A FAU - Joris, Bernard AU - Joris B FAU - Frere, Jean-Marie AU - Frere JM LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Fluorescent Dyes) RN - 0 (Penicillins) RN - 0 (Plasmids) RN - 61-33-6 (Penicillin G) RN - EC 3.5.2.6 (beta-Lactamases) SB - IM MH - Acylation MH - Algorithms MH - Anti-Bacterial Agents/*metabolism MH - Bacteria/genetics/*metabolism MH - Electrophoresis, Polyacrylamide Gel MH - Enterobacter/drug effects MH - Fluorescent Dyes MH - Hydrolysis MH - Membranes/metabolism MH - Microbial Sensitivity Tests MH - Models, Biological MH - Penicillin G/metabolism MH - Penicillins/metabolism MH - Permeability MH - Plasmids MH - Research Support, Non-U.S. Gov't MH - beta-Lactamases/metabolism EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2901-7. PMID- 12183246 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20031114 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - High incidence of cefoxitin and clindamycin resistance among anaerobes in Taiwan. PG - 2908-13 AB - Susceptibilities to 16 antimicrobial agents were determined by measurement of MICs for 344 isolates of anaerobic bacteria recovered from patients with significant infections. Resistance rates varied among antimicrobial agents and the species tested. The beta-lactams were more active in gram-positive than in gram-negative anaerobes. Resistance to meropenem was low (<1%). For beta-lactam-beta-lactamase inhibitors, piperacillin-tazobactam was most active for all species (resistance, <6%). The rates of resistance to cefoxitin (31 to 65%) and clindamycin (50 to 70%) for non-Bacteroides fragilis species of the B. fragilis group were higher than those for B. fragilis (4% resistant to cefoxitin and 33% resistant to clindamycin). Among members of B. fragilis group, Bacteroides thetaiotaomicron was the most resistant to clindamycin (70%) and cefoxitin (65%). Rates of susceptibility to imipenem and metronidazole for B. fragilis continue to be high compared to those from a previous study 10 years ago. However, resistance to metronidazole was found recently in five strains of B. fragilis. We analyzed the genetic relationships among the metronidazole-resistant B. fragilis strains by pulsed-field gel electrophoresis. The metronidazole-resistant B. fragilis strains showed genotypic heterogeneity, excluding the dissemination of a single clone. AD - School of Medical Technology, National Taiwan University College of Medicine, Taipei, Taiwan. ljteng@ha.mc.ntu.edu.tw FAU - Teng, Lee-Jene AU - Teng LJ FAU - Hsueh, Po-Ren AU - Hsueh PR FAU - Tsai, Jui-Chang AU - Tsai JC FAU - Liaw, Shwu-Jen AU - Liaw SJ FAU - Ho, Shen-Wu AU - Ho SW FAU - Luh, Kwen-Tay AU - Luh KT LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Cephamycins) RN - 0 (DNA, Bacterial) RN - 18323-44-9 (Clindamycin) RN - 35607-66-0 (Cefoxitin) RN - 443-48-1 (Metronidazole) SB - IM MH - Anti-Bacterial Agents/*pharmacology MH - Bacteria, Anaerobic/*drug effects MH - Bacterial Infections/epidemiology/*microbiology MH - Bacteroides/drug effects MH - Bacteroides fragilis/drug effects MH - Cefoxitin/*pharmacology MH - Cephamycins/*pharmacology MH - Clindamycin/*pharmacology MH - DNA, Bacterial/drug effects/genetics MH - Drug Resistance, Microbial MH - Metronidazole/pharmacology MH - Microbial Sensitivity Tests MH - Taiwan/epidemiology MH - Time Factors EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2908-13. PMID- 12183247 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Characterization of sparsomycin resistance in Streptomyces sparsogenes. PG - 2914-9 AB - The antitumor antibiotic sparsomycin, produced by Streptomyces sparsogenes, is a universal translation inhibitor that blocks the peptide bond formation in ribosomes from all species. Sparsomycin-resistant strains were selected by transforming the sensitive Streptomyces lividans with an S. sparsogenes library. Resistance was linked to the presence of a plasmid containing an S. sparsogenes 5.9-kbp DNA insert. A restriction analysis of the insert traced down the resistance to a 3.6-kbp DNA fragment, which was sequenced. The analysis of the fragment nucleotide sequence together with the previous restriction data associate the resistance to srd, an open reading frame of 1,800 nucleotides. Ribosomes from S. sparsogenes and the S. lividans-resistant strains are equally sensitive to the inhibitor and bind the drug with similar affinity. Moreover, the drug was not modified by the resistant strains. However, resistant cells accumulated less antibiotic than the sensitive ones. In addition, membrane fractions from the resistant strains showed a higher capacity for binding the drug. The results indicate that resistance in the producer strain is not connected to either ribosome modification or drug inactivation, but it might be related to an alteration in the sparsomycin permeability barrier. AD - Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas y Universidad Autonoma de Madrid, Canto Blanco, 28049 Madrid, Spain. FAU - Lazaro, E AU - Lazaro E FAU - Sanz, E AU - Sanz E FAU - Remacha, M AU - Remacha M FAU - Ballesta, J P G AU - Ballesta JP LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antibiotics, Antineoplastic) RN - 0 (Culture Media) RN - 0 (DNA, Bacterial) RN - 1404-64-4 (Sparsomycin) RN - 63-91-2 (Phenylalanine) SB - IM MH - Antibiotics, Antineoplastic/metabolism/*pharmacology MH - Culture Media MH - DNA, Bacterial/genetics MH - Drug Resistance MH - Genomic Library MH - Kinetics MH - Microbial Sensitivity Tests MH - Phenylalanine/biosynthesis MH - Research Support, Non-U.S. Gov't MH - Ribosomes/genetics/metabolism MH - Sparsomycin/metabolism/*pharmacology MH - Streptomyces/*drug effects MH - Subcellular Fractions/drug effects/metabolism EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2914-9. PMID- 12183248 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Consumption of imipenem correlates with beta-lactam resistance in Pseudomonas aeruginosa. PG - 2920-5 AB - It is generally assumed that the antibiotic prescription policy of a hospital has a significant impact on bacterial resistance rates; however, few studies are available to support this concept with valid statistical data. During a 3-year period from 1997 to 2000, we monitored the consumption of beta-lactam and other antibiotics with known activity against Pseudomonas aeruginosa in a 600-bed community hospital. Monthly isolations of P. aeruginosa were assessed, and resistance rates were recorded. Partial correlation coefficients between consumption and resistance rates were determined, taking into account possible associations with other variables such as seasonal effects and transfers from other hospitals. A total of 30 +/- 7 novel P. aeruginosa strains per month were isolated without epidemic clustering. Prescriptions of imipenem varied significantly during the study period, while prescriptions of other antipseudomonal agents were stable, with the exception of an increase in piperacillin-tazobactam prescriptions. Rates of resistance of P. aeruginosa to the antimicrobial agents used showed a time course similar to figures for imipenem consumption. Monthly rates of resistance to imipenem (partial correlation coefficient [cc], 0.63), piperacillin-tazobactam (cc, 0.57), and ceftazidime (cc, 0.56) were significantly associated with imipenem prescription rates in the same or the preceding month, while consumption of ceftazidime or piperacillin-tazobactam had no apparent association with resistance. Among the variables investigated, imipenem consumption was identified as the major factor associated with both carbapenem and beta-lactam resistance in endemic P. aeruginosa. Periods of extensive imipenem use were associated with significant increases in resistance. Our data support the concept that a written antibiotic policy which balances the use of various antibiotic classes may help to avoid disturbances of a hospital's microbial sensitivity patterns. AD - Section of Hospital Hygiene, Department of Medical Microbiology and Hygiene, Ulm University Hospital, Germany. FAU - Lepper, Philipp M AU - Lepper PM FAU - Grusa, Eberhard AU - Grusa E FAU - Reichl, Helga AU - Reichl H FAU - Hogel, Josef AU - Hogel J FAU - Trautmann, Matthias AU - Trautmann M LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Thienamycins) RN - 64090-99-9 (thienamycin) RN - 74431-23-5 (Imipenem) SB - IM MH - Drug Utilization MH - Hospitals, Community/organization & administration MH - Humans MH - Hygiene MH - Imipenem/pharmacology/*therapeutic use MH - Prescriptions, Drug MH - Pseudomonas Infections/*epidemiology/*microbiology MH - Pseudomonas aeruginosa/*drug effects/*metabolism MH - Public Policy MH - Seasons MH - Thienamycins/pharmacology/*therapeutic use MH - *beta-Lactam Resistance EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2920-5. PMID- 12183249 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Human immunodeficiency virus type 1 genotypic and pharmacokinetic determinants of the virological response to lopinavir-ritonavir-containing therapy in protease inhibitor-experienced patients. PG - 2926-32 AB - The response to regimens including lopinavir-ritonavir (LPV/r) in patients who have received multiple protease (PR) inhibitors (PI) can be analyzed in terms of human immunodeficiency virus type 1 (HIV-1) genotypic and pharmacokinetic (pK) determinants. We studied these factors and the evolution of HIV-1 resistance in response to LPV/r in a prospective study of patients receiving LPV/r under a temporary authorization in Bordeaux, France. HIV-1 PR and reverse transcriptase sequences were determined at baseline LPV/r for all the patients and at month 3 (M3) and M6 in the absence of response to treatment. pK measurements were determined at M1 and M3. Virological failure (VF) was defined as a plasma viral load >or=400 copies/ml at M3. A multivariate analysis of the predictors of VF, including clinical and biological characteristics and the treatment history of the patients, was performed. The PR gene sequence at M0, including individual mutations or a previously defined LPV mutation score (D. J. Kempf, J. D. Isaacson, M. S. King, S. C. Brun, Y. Xu, K. Real, B. M. Bernstein, A. J. Japour, E. Sun, and R. A. Rode, J. Virol. 75:7262-7269, 2001), and the individual exposure to LPV were also included covariates. Sixty-eight patients were enrolled. Thirty-four percent had a virological response at M3. An LPV mutation score of >5 mutations, the presence of the PR I54V mutation at baseline, a high number of previous PIs, prior therapy with ritonavir or indinavir, absence of coprescription of efavirenz, and a lower exposure to LPV or lower LPV trough concentrations were independently associated with VF on LPV/r. Additional PI resistance mutations, including primary mutation I50V, could be selected in patients failing on LPV/r. Genotypic and pK parameters should be used to optimize the virological response to LPV/r in PI-experienced patients and to avoid further viral evolution. AD - Laboratoire de Virologie, Hopital Pellegrin, Bordeaux, France. bernard.masquelier@chu-bordeaux.fr FAU - Masquelier, Bernard AU - Masquelier B FAU - Breilh, Dominique AU - Breilh D FAU - Neau, Didier AU - Neau D FAU - Lawson-Ayayi, Sylvie AU - Lawson-Ayayi S FAU - Lavignolle, Valerie AU - Lavignolle V FAU - Ragnaud, Jean-Marie AU - Ragnaud JM FAU - Dupon, Michel AU - Dupon M FAU - Morlat, Philippe AU - Morlat P FAU - Dabis, F AU - Dabis F FAU - Fleury, H AU - Fleury H CN - Groupe d' Epidemiologie Clinique du SIDA en Aquitaine. LA - eng PT - Clinical Trial PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-HIV Agents) RN - 0 (Drug Combinations) RN - 0 (HIV Protease Inhibitors) RN - 0 (Pyrimidinones) RN - 0 (RNA, Viral) RN - 0 (Ritonavir) RN - 192725-17-0 (lopinavir) RN - EC 3.4.23.- (HIV Protease) SB - IM MH - Adult MH - Amino Acid Substitution MH - Anti-HIV Agents/*pharmacokinetics/*therapeutic use MH - Drug Combinations MH - Female MH - Genotype MH - HIV Infections/*drug therapy/*metabolism/virology MH - HIV Protease/genetics MH - HIV Protease Inhibitors/*pharmacokinetics/*therapeutic use MH - HIV-1/*drug effects/*genetics MH - Humans MH - Male MH - Models, Biological MH - Pyrimidinones/*pharmacokinetics/*therapeutic use MH - RNA, Viral/blood MH - Ritonavir/*pharmacokinetics/*therapeutic use MH - Salvage Therapy EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2926-32. PMID- 12183250 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20031114 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Comparative study of mechanisms of herpes simplex virus inactivation by sodium lauryl sulfate and n-lauroylsarcosine. PG - 2933-42 AB - The mechanisms of herpes simplex virus (HSV) inactivation by sodium lauryl sulfate (SLS) and n-lauroylsarcosine (LS), two anionic surfactants with protein denaturant potency, have been evaluated in cultured cells. Results showed that pretreatment of HSV type 1 (HSV-1) strain F and HSV-2 strain 333 with either surfactant inhibited, in a concentration- and time-dependent manner, their infectivities on Vero cells. SLS was a more potent inhibitor of HSV-2 strain 333 infectivity than LS with respect to the concentration (4.8-fold lower) and time (2.4-fold shorter) required to completely inactivate the virus. No inhibition of both herpesvirus strains infectivities was observed when Vero cells were pretreated with either surfactant. LS prevented the binding of HSV-2 strain 333 to cells without affecting the stable attachment and the rate of penetration into cells, whereas SLS exerted the opposite effect. Both SLS and LS inhibited, in a concentration-dependent manner, the HSV-2 strain 333-induced cytopathic effect, probably by affecting newly synthesized virions that come into contact with surfactant molecules present in culture medium. The pretreatment of HSV-2 strain 333 with specific combinations of SLS and LS concentrations inhibited the viral infectivity in a synergistic manner and resulted in only a small increase in their toxicities for exponentially growing Vero cells compared with that caused by each compound alone. Taken together, these results suggest that SLS and LS, alone or combined, could represent potent candidates as microbicides in topical vaginal formulations to prevent the transmission of herpes and possibly other pathogens that cause sexually transmitted diseases, including human immunodeficiency virus type 1. AD - Centre de Recherche en Infectiologie, Universite Laval, Quebec, Quebec, Canada. FAU - Piret, Jocelyne AU - Piret J FAU - Roy, Sylvie AU - Roy S FAU - Gagnon, Mylene AU - Gagnon M FAU - Landry, Sebastien AU - Landry S FAU - Desormeaux, Andre AU - Desormeaux A FAU - Omar, Rabeea F AU - Omar RF FAU - Bergeron, Michel G AU - Bergeron MG LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Receptors, Virus) RN - 0 (Surface-Active Agents) RN - 0 (Viral Proteins) RN - 107-97-1 (Sarcosine) RN - 151-21-3 (Sodium Dodecyl Sulfate) RN - 97-78-9 (sarkosyl) SB - IM MH - Animals MH - Cell Survival/drug effects MH - Cercopithecus aethiops MH - Comparative Study MH - Herpesvirus 1, Human/*drug effects MH - Herpesvirus 2, Human/*drug effects MH - Plaque Assay MH - Receptors, Virus/drug effects MH - Sarcosine/*analogs & derivatives/*pharmacology MH - Sodium Dodecyl Sulfate/*pharmacology MH - Surface-Active Agents/*pharmacology MH - Time Factors MH - Vero Cells MH - Viral Proteins/metabolism EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2933-42. PMID- 12183251 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Application of real-time PCR for determination of antiviral drug susceptibility of herpes simplex virus. PG - 2943-7 AB - A quantitative real-time PCR (TaqMan) assay was developed for determination of antiviral drug susceptibility of herpes simplex virus (HSV). After short-time culture of the virus, the antiviral drug susceptibility of HSV isolates for acyclovir (ACV) was determined by measuring the reduction of the HSV type 1 (HSV-1) DNA levels in culture supernatants using real-time PCR. The 50% inhibitory concentration was reported as the concentration of antiviral drug that reduced the number of HSV-1 DNA copies by 50%. A total of 15 well-characterized ACV-sensitive or -resistant strains and clinical isolates were used for assay evaluation. The new assay with real-time PCR readout permitted rapid (3 days), objective, and reproducible determination of HSV-1 drug susceptibilities with no need for stringent control of initial multiplicity of infection. Furthermore, the real-time PCR assay results showed good correlation (r = 0.86) with those for the plaque reduction assay. In conclusion, the real-time PCR assay described here is a suitable quantitative method for determination of antiviral susceptibility of HSV-1, amenable for use in the routine diagnostic virology laboratory. AD - Department of Virology, Eijkman-Winkler Center, University Medical Center Utrecht, The Netherlands. r.stranska@lab.azu.nl FAU - Stranska, Ruzena AU - Stranska R FAU - van Loon, Anton M AU - van Loon AM FAU - Polman, Merjo AU - Polman M FAU - Schuurman, Rob AU - Schuurman R LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antiviral Agents) RN - 0 (DNA, Viral) SB - IM MH - Animals MH - Antiviral Agents/*pharmacology MH - Cercopithecus aethiops MH - Computer Systems MH - DNA Replication/drug effects MH - DNA, Viral/analysis/biosynthesis MH - Herpesvirus 1, Human/drug effects MH - Kinetics MH - Plaque Assay MH - Reproducibility of Results MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction/*methods MH - Simplexvirus/*drug effects MH - Vero Cells EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2943-7. PMID- 12183253 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - In vitro selection of resistance in Haemophilus influenzae by amoxicillin-clavulanate, cefpodoxime, cefprozil, azithromycin, and clarithromycin. PG - 2956-62 AB - Abilities of amoxicillin-clavulanate, cefpodoxime, cefprozil, azithromycin, and clarithromycin to select resistant mutants of Haemophilus influenzae were tested by multistep and single-step methodologies. For multistep studies, 10 random strains were tested: 5 of these were beta-lactamase positive. After 50 daily subcultures in amoxicillin-clavulanate, MICs did not increase more than fourfold. However, cefprozil MICs increased eightfold for one strain. Clarithromycin and azithromycin gave a >4-fold increase in 8 and 10 strains after 14 to 46 and 20 to 50 days, respectively. Mutants selected by clarithromycin and azithromycin were associated with mutations in 23S rRNA and ribosomal proteins L4 and L22. Three mutants selected by clarithromycin or azithromycin had alterations in ribosomal protein L4, while five had alterations in ribosomal protein L22. Two mutants selected by azithromycin had mutations in the gene encoding 23S rRNA: one at position 2058 and the other at position 2059 (Escherichia coli numbering), with replacement of A by G. One clone selected by clarithromycin became hypersusceptible to macrolides. In single-step studies azithromycin and clarithromycin had the highest mutation rates, while amoxicillin-clavulanate had the lowest. All resistant clones were identical to parents as observed by pulsed-field gel electrophoresis. The MICs of azithromycin for azithromycin-resistant clones were 16 to >128 micro g/ml, and those of clarithromycin for clarithromycin-resistant clones were 32 to >128 micro g/ml in multistep studies. For strains selected by azithromycin, the MICs of clarithromycin were high and vice versa. After 50 daily subcultures in the presence of drugs, MICs of amoxicillin-clavulanate and cefpodoxime against H. influenzae did not rise more than fourfold, in contrast to cefprozil, azithromycin, and clarithromycin, whose MICs rose to variable degrees. AD - Department of Pathology, Hershey Medical Center, Hershey, Pennsylvania 17033, USA. FAU - Clark, Catherine AU - Clark C FAU - Bozdogan, Bulent AU - Bozdogan B FAU - Peric, Mihaela AU - Peric M FAU - Dewasse, Bonifacio AU - Dewasse B FAU - Jacobs, Michael R AU - Jacobs MR FAU - Appelbaum, Peter C AU - Appelbaum PC LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Antibiotics, Combined) RN - 0 (Cephalosporins) RN - 68401-81-0 (Ceftizoxime) RN - 74469-00-4 (Amoxicillin-Potassium Clavulanate Combination) RN - 81103-11-9 (Clarithromycin) RN - 82619-04-3 (cefpodoxime) RN - 83905-01-5 (Azithromycin) RN - 92665-29-7 (cefprozil) SB - IM MH - Amino Acid Sequence MH - Amoxicillin-Potassium Clavulanate Combination/*pharmacology MH - Anti-Bacterial Agents/*pharmacology MH - Antibiotics, Combined/*pharmacology MH - Azithromycin/*pharmacology MH - Ceftizoxime/*analogs & derivatives/*pharmacology MH - Cephalosporins/*pharmacology MH - Clarithromycin/*pharmacology MH - Drug Resistance MH - Genes, Bacterial/genetics MH - Haemophilus influenzae/*drug effects/genetics MH - Microbial Sensitivity Tests MH - Molecular Sequence Data MH - Mutation MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Transformation, Bacterial EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2956-62. PMID- 12183254 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Antistreptococcal activity of telithromycin compared with seven other drugs in relation to macrolide resistance mechanisms in Russia. PG - 2963-8 AB - The susceptibilities of 468 recent Russian clinical Streptococcus pneumoniae isolates and 600 Streptococcus pyogenes isolates, from 14 centers in Russia, to telithromycin, erythromycin, azithromycin, clarithromycin, clindamycin, levofloxacin, quinupristin-dalfopristin, and penicillin G were tested. Penicillin-nonsusceptible S. pneumoniae strains were rare except in Siberia, where their prevalence rate was 13.5%: most were penicillin intermediate, but for three strains (two from Smolensk and one from Novosibirsk) the MICs of penicillin G were 4 or 8 micro g/ml. Overall, 2.5% of S. pneumoniae isolates were resistant to erythromycin. Efflux was the prevalent resistance mechanism (five strains; 41.7%), followed by ribosomal methylation encoded by constitutive erm(B), which was found in four isolates. Ribosomal mutation was the mechanism of macrolide resistance in three isolates; one erythromycin-resistant S. pneumoniae isolate had an A2059G mutation in 23S rRNA, and two isolates had substitution of GTG by TPS at positions 69 to 71 in ribosomal protein L4. All S. pyogenes isolates were susceptible to penicillin, and 11% were erythromycin resistant. Ribosomal methylation was the most common resistance mechanism for S. pyogenes (89.4%). These methylases were encoded by erm(A) [subclass erm(TR)] genes, and their expression was inducible in 96.6% of isolates. The rest of the erythromycin-resistant Russian S. pyogenes isolates (7.6%) had an efflux resistance mechanism. Telithromycin was active against 100% of pneumococci and 99.2% of S. pyogenes, and levofloxacin and quinupristin-dalfopristin were active against all isolates of both species. AD - Institute of Antimicrobial Chemotherapy, Smolensk, Russia. FAU - Kozlov, Roman S AU - Kozlov RS FAU - Bogdanovitch, Tatiana M AU - Bogdanovitch TM FAU - Appelbaum, Peter C AU - Appelbaum PC FAU - Ednie, Lois AU - Ednie L FAU - Stratchounski, Leonid S AU - Stratchounski LS FAU - Jacobs, Michael R AU - Jacobs MR FAU - Bozdogan, Bulent AU - Bozdogan B LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Bacterial Proteins) RN - 0 (Ketolides) RN - 0 (Macrolides) RN - 0 (telithromycin) RN - EC 2.1.1. (Methyltransferases) RN - EC 2.1.1.48 (ErmA protein, Bacteria) SB - IM MH - Anti-Bacterial Agents/metabolism/*pharmacology MH - Bacterial Proteins/genetics MH - Comparative Study MH - Drug Resistance MH - Genes, Bacterial/genetics MH - *Ketolides MH - *Macrolides MH - Methylation MH - *Methyltransferases MH - Microbial Sensitivity Tests MH - Research Support, Non-U.S. Gov't MH - Ribosomes/metabolism MH - Russia/epidemiology MH - Streptococcal Infections/epidemiology/microbiology MH - Streptococcus/*drug effects/genetics/metabolism EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2963-8. PMID- 12183255 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Phase I dose escalation trial evaluating the pharmacokinetics, anti-human cytomegalovirus (HCMV) activity, and safety of 1263W94 in human immunodeficiency virus-infected men with asymptomatic HCMV shedding. PG - 2969-76 AB - 1263W94 [maribavir; 5,6-dichloro-2-(isopropylamino)-1,beta-L-ribofuranosyl-1-H-benzimidazole] is a novel benzimidazole compound for treatment of human cytomegalovirus (HCMV) infection and disease, with potent in vitro activity against HCMV and good oral bioavailability. A phase I study was conducted to determine the pharmacokinetics (PK), anti-HCMV activity, and safety of 1263W94 administered as multiple oral doses to human immunodeficiency virus type 1-infected adult male subjects with asymptomatic HCMV shedding. Subjects received one of six dosage regimens (100, 200, or 400 mg three times a day, or 600, 900, or 1,200 mg twice a day) or a placebo for 28 days. 1263W94 demonstrated linear PK, with steady-state plasma 1263W94 profiles predictable based on single-dose data. 1263W94 was rapidly absorbed following oral dosing, and values for the maximum concentration of the drug in plasma and the area under the concentration-time curve increased in proportion to the dose. 1263W94 demonstrated in vivo anti-HCMV activity in semen at all of the dosage regimens tested, with mean reductions in semen HCMV titers of 2.9 to 3.7 log(10) PFU/ml among the four regimens evaluated for anti-HCMV activity. 1263W94 was generally well tolerated; taste disturbance was the most frequently reported adverse event over the 28-day dosing period. AD - Quest Clinical Research, San Francisco, California, USA. FAU - Lalezari, Jacob P AU - Lalezari JP FAU - Aberg, Judith A AU - Aberg JA FAU - Wang, Laurene H AU - Wang LH FAU - Wire, Mary Beth AU - Wire MB FAU - Miner, Richard AU - Miner R FAU - Snowden, Wendy AU - Snowden W FAU - Talarico, Christine L AU - Talarico CL FAU - Shaw, Shuching AU - Shaw S FAU - Jacobson, Mark A AU - Jacobson MA FAU - Drew, W Lawrence AU - Drew WL LA - eng PT - Clinical Trial PT - Clinical Trial, Phase II PT - Journal Article PT - Randomized Controlled Trial PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Benzimidazoles) RN - 0 (Culture Media) RN - 0 (DNA, Viral) RN - 0 (Ribonucleosides) RN - 0 (maribavir) SB - IM MH - Adult MH - Area Under Curve MH - Benzimidazoles/adverse effects/*pharmacokinetics/*therapeutic use MH - Cells, Cultured MH - Culture Media MH - Cytomegalovirus Infections/*drug therapy/virology MH - DNA, Viral/biosynthesis/genetics MH - Dose-Response Relationship, Drug MH - HIV Infections/*drug therapy MH - Humans MH - Male MH - Plaque Assay MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Ribonucleosides/adverse effects/*pharmacokinetics/*therapeutic use MH - Semen/virology MH - Urine/virology MH - Virus Shedding EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2969-76. PMID- 12183256 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Characterization of a self-transferable plasmid from Salmonella enterica serotype typhimurium clinical isolates carrying two integron-borne gene cassettes together with virulence and drug resistance genes. PG - 2977-81 AB - An unusual self-transferable virulence-resistance plasmid (pUO-StVR2) was found in nine multidrug-resistant (ACSSuT phenotype) Salmonella enterica serotype Typhimurium clinical isolates that were assigned to four different phage types and a single and distinctive XbaI pulsed-field gel electrophoresis profile. pUO-StVR2 is an IncFII plasmid of about 140 kb in length carrying the spvA, spvB, and spvC (Salmonella plasmid virulence) and rck (resistance to complement killing) genes. It also carries the oxa1/aadA1a (ampicillin resistance and streptomycin-spectinomycin resistance) gene cassette configuration located within a class 1 integron with qacEDelta1/sul1 (ammonium antiseptics resistance and sulfadiazine resistance); the transposon genes merA, tnpA, and tnpR (mercury resistance, transposase, and resolvase of Tn21, respectively); and the catA1 (chloramphenicol resistance) and tet(B) (tetracycline resistance) genes. The insertion of resistance genes into a Salmonella virulence plasmid constitutes a new and interesting example of plasmid evolution and presents a serious public health problem. AD - Departamento de Biologia Funcional, Area de Microbiologia, Universidad de Oviedo, 33006 Oviedo, Principality of Asturias, Spain. FAU - Guerra, Beatriz AU - Guerra B FAU - Soto, Sara AU - Soto S FAU - Helmuth, Reiner AU - Helmuth R FAU - Mendoza, M Carmen AU - Mendoza MC LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (DNA, Bacterial) RN - 0 (Plasmids) RN - 0 (Virulence Factors) RN - EC 2.7.7.- (Tn21 resolvase) RN - EC 2.7.7.- (Transposon Resolvases) SB - IM MH - Bacteriophage Typing MH - DNA, Bacterial/biosynthesis/genetics/isolation & purification MH - Drug Resistance, Microbial MH - Genes, Bacterial/*genetics MH - Integrons/*genetics MH - Plasmids/*genetics MH - Research Support, Non-U.S. Gov't MH - Salmonella enterica/drug effects/*genetics/pathogenicity MH - Serotyping MH - *Transposon Resolvases MH - Virulence Factors EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2977-81. PMID- 12183257 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - In vitro interaction of flucytosine combined with amphotericin B or fluconazole against thirty-five yeast isolates determined by both the fractional inhibitory concentration index and the response surface approach. PG - 2982-9 AB - Combination therapy could be of benefit for the treatment of invasive yeast infections. However, in vitro interaction studies are relatively scarce and the interpretation of the fractional inhibitory concentration (FIC) index can be contradictory due to various definitions used; not all information on the interaction study is used in the index, and different MIC end points exist for different classes of drugs. Fitting an interaction model to the whole response surface and estimation of an interaction coefficient alpha (IC(alpha)) would overcome these objections and has the additional advantage that confidence intervals of the interaction are obtained. The efficacy of flucytosine (5FC) in combination with amphotericin B (AB) and fluconazole (FCZ) was studied against 35 yeast isolates in triplicate (Candida albicans [n = 9], Candida glabrata [n = 9], Candida krusei [n = 9], and Cryptococcus neoformans [n = 8]) using a broth microdilution checkerboard method and measuring growth after 48 h by a spectrophotometer. The FIC index and IC(alpha) were determined, the latter by estimation from the response surface approach described by Greco et al. (W. R. Greco, G. Bravo, and J. C. Parsons, Pharmacol. Rev. 47:331-385, 1995) by using a computer program developed for that purpose. For the 5FC-FCZ combination, the interactions determined by the IC(alpha) generally were in concordance with the interactions determined by the FIC index, but large discrepancies were found between both methods for the 5FC-AB combination. These could mainly be explained by shortcomings in the FIC approach. The in vitro interaction of 5FC-AB demonstrated variable results depending on the tested Candida isolate. In general, the 5FC-FCZ combination was antagonistic against Candida species, but for some Candida isolates synergism was found. For C. neoformans the interaction for both combinations was highly dependent on the tested isolate and the method used. Response surface approach is an alternative method for determining the interaction between antifungal agents. By using this approach, some of the problems encountered with the FIC were overcome. AD - Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands. FAU - Te Dorsthorst, D T A AU - Te Dorsthorst DT FAU - Verweij, P E AU - Verweij PE FAU - Meletiadis, J AU - Meletiadis J FAU - Bergervoet, M AU - Bergervoet M FAU - Punt, N C AU - Punt NC FAU - Meis, J F G M AU - Meis JF FAU - Mouton, J W AU - Mouton JW LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antibiotics, Antifungal) RN - 0 (Antifungal Agents) RN - 1397-89-3 (Amphotericin B) RN - 2022-85-7 (Flucytosine) RN - 86386-73-4 (Fluconazole) SB - IM MH - Algorithms MH - Amphotericin B/*pharmacology MH - Antibiotics, Antifungal/*pharmacology MH - Antifungal Agents/*pharmacology MH - Candida/*drug effects MH - Drug Interactions MH - Fluconazole/*pharmacology MH - Flucytosine/*pharmacology MH - Microbial Sensitivity Tests MH - Models, Biological EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2982-9. PMID- 12183258 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Pharmacodynamic assessment of ertapenem (MK-0826) against Streptococcus pneumoniae in a murine neutropenic thigh infection model. PG - 2990-5 AB - The objective of this study was to determine the susceptibility breakpoint of a new carbapenem, ertapenem (MK-0826), against Streptococcus pneumoniae strains based on bacterial density and survival studies in a murine thigh infection model. Sixteen S. pneumoniae isolates for which MICs ranged from 0.015 to 4.0 mg/liter were tested with neutropenic ICR mice. Animals were infected with bacteria at 10(5) to 10(6) CFU per thigh and were treated with ertapenem starting at 2 h postinfection for 4 days. Ertapenem was given subcutaneously at 50 mg/kg of body weight every 6 h, which simulates the human pharmacodynamic profile (in particular, the duration of time that the concentration of free drug remains above the MIC of 2 mg/liter). At 0 and 24 h postinfection, thighs were harvested for bacterial density determination. Survival was assessed during 4 days of therapy and 3 days after the therapy. A protein binding study was conducted with mice by use of the ultrafiltration method. Protein binding in mice was approximately 95%, which is comparable to that in humans. The average change in bacterial density ranged from -0.22 to -4.4 log CFU per thigh over 24 h compared to 0-h controls. The extent of microbial eradication was dependent on the MIC for the S. pneumoniae isolate. Substantial bactericidal activities (i.e., killing of approximately 2 log CFU per thigh) were consistently observed against isolates for which MICs were TTC, was present within this 361-bp fragment. This triple-base-pair substitution, present in both copies of the 16S rRNA gene of all our Tet(r) H. pylori transformants, resulted in an increased MIC of tetracycline that was identical to that for the Tet(r) strain 181. AD - Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center Rotterdam, The Netherlands. FAU - Gerrits, Monique M AU - Gerrits MM FAU - de Zoete, Marcel R AU - de Zoete MR FAU - Arents, Niek L A AU - Arents NL FAU - Kuipers, Ernst J AU - Kuipers EJ FAU - Kusters, Johannes G AU - Kusters JG LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (RNA, Ribosomal, 16S) RN - 0 (Repressor Proteins) RN - 0 (tetracycline resistance-encoding transposon repressor protein) SB - IM MH - Aged MH - Amino Acid Substitution/genetics MH - Binding Sites MH - Helicobacter Infections/microbiology MH - Helicobacter pylori/drug effects/*genetics MH - Humans MH - Male MH - Mutation/*genetics MH - RNA, Ribosomal, 16S/*genetics MH - Repressor Proteins/genetics MH - Reverse Transcriptase Polymerase Chain Reaction MH - Tetracycline Resistance/*genetics MH - Transformation, Bacterial EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):2996-3000. PMID- 12183260 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041209 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro. PG - 3001-12 AB - Caspofungin acetate is an antifungal antibiotic that inhibits synthesis of 1,3-beta-D-glucan, an essential component of the fungal cell wall. While caspofungin causes cell death in yeasts and dimorphic fungi such as Candida albicans, its effect on Aspergillus fumigatus is less well understood. We used the fluorescent dyes 5,(6)-carboxyfluorescein diacetate (CFDA) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC), which stain live and dead cells, respectively, to further characterize the antifungal activity of caspofungin. For comparison, compounds whose mode of action was either fungistatic (fluconazole, itraconazole) or fungicidal (amphotericin B) were also evaluated. A correlation between caspofungin-induced loss of viability, decreased CFDA staining, and increased DiBAC staining was established first with C. albicans. For A. fumigatus, caspofungin caused similar dye-staining changes, which were quantified by fluorimetric analysis of stained hyphae grown in a medium that promoted dispersed growth. The minimum concentration of caspofungin required to produce these changes also decreased the level of growth-dependent reduction of the indicator dye Alamar Blue. We observed a differential effect of caspofungin as a function of cell position: 88% of apical cells and 61% of subapical branching cells failed to stain with the viable dye CFDA, but only 24% of subapical cells were unstained. Complementary results were seen with germlings from DiBAC-stained, caspofungin-treated cultures. Extended incubation of A. fumigatus with a single dose of caspofungin affected the same proportion of apical and subapical branching cells for up to 72 h. The dye-staining patterns illustrate that the cells at the active centers for new cell wall synthesis within A. fumigatus hyphae are killed when they are exposed to caspofungin. AD - Department of Human and Animal Infectious Disease Research, Merck Research Laboratories, Rahway, New Jersey 07065-0900, USA. FAU - Bowman, J C AU - Bowman JC FAU - Hicks, P Scott AU - Hicks PS FAU - Kurtz, M B AU - Kurtz MB FAU - Rosen, H AU - Rosen H FAU - Schmatz, D M AU - Schmatz DM FAU - Liberator, P A AU - Liberator PA FAU - Douglas, C M AU - Douglas CM LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antibiotics, Antifungal) RN - 0 (Antibiotics, Peptide) RN - 0 (Fluorescent Dyes) RN - 0 (Peptides) RN - 0 (Peptides, Cyclic) RN - 0 (caspofungin) SB - IM MH - Antibiotics, Antifungal/*pharmacology MH - Antibiotics, Peptide/*pharmacology MH - Aspergillus fumigatus/*drug effects/growth & development MH - Cell Count MH - Dose-Response Relationship, Drug MH - Fluorescent Dyes MH - Fluorometry MH - Microbial Sensitivity Tests MH - *Peptides MH - *Peptides, Cyclic EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3001-12. PMID- 12183261 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Pharmacokinetic and pharmacodynamic profiles of danofloxacin administered by two dosing regimens in calves infected with Mannheimia (Pasteurella) haemolytica. PG - 3013-9 AB - The pharmacokinetics and pharmacodynamics of danofloxacin in calves with induced Mannheimia (Pasteurella) haemolytica pneumonia were evaluated. Calves received either saline as an intravenous (IV) bolus or danofloxacin (0.738 mg/kg of body weight) administered as either a single IV bolus or a 36-h continuous IV infusion. Blood samples and bronchial secretions were collected before and at predetermined times over 48 h following the start of treatment. Calves were assessed clinically throughout, and lung consolidation was assessed at necropsy. Bronchial secretions and lung tissue were cultured for M. haemolytica. Bolus administration of danofloxacin produced a high maximum drug concentration-to-MIC ratio (C(max):MIC) of 14.5 and a time period of 9.1 h when plasma danofloxacin concentrations exceeded the MIC (T>MIC). Following danofloxacin infusion, the C(max):MIC was low (2.3), with a long T>MIC (33.3 h). The area under the curve-to-MIC ratios were 43.3 and 49.1 for the bolus and infusion administrations, respectively. The single bolus of danofloxacin was more effective than the same dose administered by continuous infusion, as indicated by a significantly lower (P < 0.05) number of animals with M. haemolytica in bronchial secretions after treatment and lower rectal temperatures in the 24 h after the start of treatment. Thus, danofloxacin exhibited concentration-dependent antimicrobial activity in cattle with respiratory disease caused by M. haemolytica. AD - Ondax Scientific, 20280 Hondarribia, Gipuzkoa, Spain. FAU - Sarasola, Patxi AU - Sarasola P FAU - Lees, Peter AU - Lees P FAU - AliAbadi, Fariborz Shojaee AU - AliAbadi FS FAU - McKellar, Quintin A AU - McKellar QA FAU - Donachie, William AU - Donachie W FAU - Marr, Kate A AU - Marr KA FAU - Sunderland, Simon J AU - Sunderland SJ FAU - Rowan, Tim G AU - Rowan TG LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Infective Agents) RN - 0 (Fluoroquinolones) RN - 112398-08-0 (danofloxacin) SB - IM MH - Animals MH - Anti-Infective Agents/administration & dosage/*pharmacokinetics/*therapeutic use MH - Area Under Curve MH - Dogs MH - *Fluoroquinolones MH - Infusions, Intravenous MH - Injections, Intravenous MH - Lung/microbiology MH - Male MH - Mannheimia haemolytica/*drug effects MH - Pasteurella Infections/*drug therapy/microbiology/physiopathology MH - Pneumonia, Bacterial/drug therapy/microbiology/physiopathology MH - Respiratory Mechanics/drug effects EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3013-9. PMID- 12183262 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Mutation in 23S rRNA associated with macrolide resistance in Neisseria gonorrhoeae. PG - 3020-5 AB - Fifty-six azithromycin-resistant (MICs, 2.0 to 4.0 micro g/ml) Neisseria gonorrhoeae strains with cross-resistance to erythromycin (MICs, 2.0 to 64.0 micro g/ml), isolated in Canada between 1997 and 1999, were characterized, and their mechanisms of azithromycin resistance were determined. Most (58.9%) of them belonged to auxotype-serotype class NR/IB-03, with a 2.6-mDa plasmid. Based on resistance to crystal violet (MICs >or= 1 micro g/ml), 96.4% of these macrolide-resistant strains appeared to have increased efflux. Nine of the eleven strains selected for further characterization were found to have a promoter region mtrR mutation, a single-base-pair (A) deletion in the 13-bp inverted repeat, which is believed to cause overexpression of the mtrCDE-encoded efflux pump. The two remaining macrolide-resistant strains (erythromycin MIC, 64.0 micro g/ml; azithromycin MIC, 4.0 micro g/ml), which did not have the mutation in the mtrR promoter region, were found to have a C2611T mutation (Escherichia coli numbering) in the peptidyltransferase loop in domain V of the 23S rRNA alleles. Although mutations in domain V of 23S rRNA alleles had been reported in other bacteria, including E. coli, Streptococcus pneumoniae, and Helicobacter pylori, this is the first observation of these mutations associated with macrolide resistance in N. gonorrhoeae. AD - National Laboratory for Sexually Transmitted Diseases, National Microbiology Laboratory, Population and Public Health Branch, Health Canada, Winnipeg, Manitoba, Canada. Lai_King_Ng@hc-sc.gc.ca FAU - Ng, Lai-King AU - Ng LK FAU - Martin, Irene AU - Martin I FAU - Liu, Gary AU - Liu G FAU - Bryden, Louis AU - Bryden L LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (DNA, Bacterial) RN - 0 (Oligonucleotide Probes) RN - 0 (RNA, Ribosomal, 23S) RN - 114-07-8 (Erythromycin) RN - 83905-01-5 (Azithromycin) RN - EC 2.3.2.12 (Peptidyl Transferases) SB - IM MH - Anti-Bacterial Agents/*pharmacology MH - Azithromycin/pharmacology MH - Canada/epidemiology MH - DNA, Bacterial/drug effects/genetics MH - Drug Resistance MH - Erythromycin/pharmacology MH - Gonorrhea/epidemiology/microbiology MH - Humans MH - Microbial Sensitivity Tests MH - Mutation/genetics MH - Neisseria gonorrhoeae/*drug effects MH - Nucleic Acid Conformation MH - Oligonucleotide Probes MH - Peptidyl Transferases/genetics MH - RNA, Ribosomal, 23S/*genetics MH - Reverse Transcriptase Polymerase Chain Reaction MH - Transformation, Bacterial EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3020-5. PMID- 12183263 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Antibiotic pharmacodynamics in surgical prophylaxis: an association between intraoperative antibiotic concentrations and efficacy. PG - 3026-30 AB - The objective of this study was to characterize the relationship between gentamicin concentrations during surgery and the development of wound infection following colorectal operations. Despite decades of research in surgical prophylaxis, the relationship between intraoperative antibiotic concentrations and postoperative infection and the concentrations required for effective prophylaxis have not been established. A pharmacodynamic analysis was conducted using data from a previous prospective, randomized, double-blind clinical study which compared two dosage regimens of gentamicin plus metronidazole for prophylaxis in connection with elective colorectal surgery. Univariate and multivariate analyses of risk factors for postoperative wound infection were conducted, and the relationship between intraoperative gentamicin concentrations and surgical outcome was characterized. The gentamicin concentration at the time of surgical closure was one of the strongest independent risk factors for infection (P = 0.02), along with the presence of diabetes mellitus (P = 0.02), stoma (P = 0.04), and advanced age (P = 0.05). Gentamicin concentrations at closure of less than 0.5 mg/liter were associated with an infection rate of 80% (representing 8 of 10 patients with concentrations below that level) (P = 0.003). Receiver operating characteristic curve analysis identified a critical closure concentration of 1.6 mg/liter for effective surgical prophylaxis (P = 0.002; sensitivity, 70.8%; specificity, 65.9%). This study provides new and important information on antibiotic pharmacodynamics in surgical prophylaxis. It demonstrates the critical effect of the antibiotic concentration at closure on wound infection and suggests a significant association between the concentration and other well-established risk factors, like the timing of preoperative antibiotic administration and surgery duration. AD - Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada. zelenits@ms.umanitoba.ca FAU - Zelenitsky, Sheryl A AU - Zelenitsky SA FAU - Ariano, Robert E AU - Ariano RE FAU - Harding, Godfrey K M AU - Harding GK FAU - Silverman, Richard E AU - Silverman RE LA - eng PT - Clinical Trial PT - Journal Article PT - Randomized Controlled Trial PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Gentamicins) SB - IM MH - Algorithms MH - Anti-Bacterial Agents/blood/*pharmacokinetics/therapeutic use MH - *Antibiotic Prophylaxis MH - Area Under Curve MH - Colon/surgery MH - Double-Blind Method MH - Female MH - Gentamicins/blood/pharmacokinetics/therapeutic use MH - Humans MH - Intraoperative Period MH - Male MH - Middle Aged MH - Rectum/surgery MH - Risk Factors MH - Surgical Wound Infection/epidemiology/microbiology/prevention & control EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3026-30. PMID- 12183264 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Prospective survey of beta-lactamases produced by ceftazidime- resistant Pseudomonas aeruginosa isolated in a French hospital in 2000. PG - 3031-4 AB - In 2000, at the Universite d'Auvergne teaching hospital in Clermont-Ferrand, France, 44 (6.2%) strains of Pseudomonas aeruginosa were found to be resistant to ceftazidime. After genotyping, 34 strains were selected. Nine had an additional beta-lactamase: OXA-21 (n = 6), PSE-1 (CARB-2) (n = 2), or PER-1 (n = 1). Ceftazidime resistance was related solely to the overproduction of the cephalosporinase in 30 strains. Sequencing of five bla(AmpC) genes encoding cephalosporinases with different pIs showed 99% identity with the ampC gene of P. aeruginosa PAO1. AD - Service de Bacteriologie, Faculte de Medecine, Universite d'Auvergne, 63001 Clermont-Ferrand, France. christophe.dechamps@u-clermont1.fr FAU - De Champs, Christophe AU - De Champs C FAU - Poirel, Laurent AU - Poirel L FAU - Bonnet, Richard AU - Bonnet R FAU - Sirot, Danielle AU - Sirot D FAU - Chanal, Catherine AU - Chanal C FAU - Sirot, Jacques AU - Sirot J FAU - Nordmann, Patrice AU - Nordmann P LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Cephalosporins) RN - 78439-06-2 (Ceftazidime) RN - EC 3.5.2.- (Cephalosporinase) RN - EC 3.5.2.6 (beta-Lactamases) SB - IM MH - Ceftazidime/*pharmacology/therapeutic use MH - Cephalosporinase/antagonists & inhibitors/genetics/metabolism MH - Cephalosporins/*pharmacology/therapeutic use MH - Data Collection MH - Drug Resistance, Microbial MH - France/epidemiology MH - Genes, Bacterial MH - Genotype MH - Humans MH - Microbial Sensitivity Tests MH - Prospective Studies MH - Pseudomonas Infections/*epidemiology/*microbiology MH - Pseudomonas aeruginosa/*drug effects/*enzymology MH - Research Support, Non-U.S. Gov't MH - beta-Lactamases/antagonists & inhibitors/genetics/*metabolism EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3031-4. PMID- 12183265 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Predicting evolution by in vitro evolution requires determining evolutionary pathways. PG - 3035-8 AB - In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 beta-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele. AD - Biology Department, University of Rochester, Rochester, New York 14627-0211, USA. drbh@mail.rochester.edu FAU - Hall, Barry G AU - Hall BG LA - eng GR - GM60761/GM/NIGMS PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (DNA, Bacterial) RN - EC 3.5.2.6 (beta-Lactamases) SB - IM MH - Alleles MH - Amino Acid Substitution/genetics MH - *DNA Shuffling MH - DNA, Bacterial/genetics MH - Drug Resistance, Microbial/*genetics MH - *Evolution MH - Mutation/genetics MH - Research Support, U.S. Gov't, P.H.S. MH - beta-Lactamases/*genetics/metabolism EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3035-8. PMID- 12183266 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041209 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - In vitro interaction of caspofungin acetate with voriconazole against clinical isolates of Aspergillus spp. PG - 3039-41 AB - The interaction between caspofungin acetate and voriconazole was studied in vitro by using 48 clinical Aspergillus spp. isolates obtained from patients with invasive aspergillosis. MICs were determined by the NCCLS broth microdilution method. Synergy, defined as a fractional inhibitory concentration (FIC) index of <1, was detected in 87.5% of the interactions; an additive effect, defined as an FIC index of 1.0, was observed in 4.2% of the interactions; and a subadditive effect, defined as an FIC index of 1.0 to 2.0, was found in 8.3% of the interactions. No antagonism was observed. Animal models are required to validate the in vivo significance of these in vitro data presented for the combination of caspofungin and voriconazole. AD - Department of Medicine, Division of Infectious Diseases, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA. perea@uthscsa.edu FAU - Perea, Sofia AU - Perea S FAU - Gonzalez, Gloria AU - Gonzalez G FAU - Fothergill, Annette W AU - Fothergill AW FAU - Kirkpatrick, William R AU - Kirkpatrick WR FAU - Rinaldi, Michael G AU - Rinaldi MG FAU - Patterson, Thomas F AU - Patterson TF LA - eng GR - 5 R01 DE11381/DE/NIDCR GR - M01-RR-01346/RR/NCRR PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antibiotics, Peptide) RN - 0 (Antifungal Agents) RN - 0 (Peptides) RN - 0 (Peptides, Cyclic) RN - 0 (Pyrimidines) RN - 0 (Triazoles) RN - 0 (caspofungin) RN - 0 (voriconazole) SB - IM MH - Antibiotics, Peptide/*pharmacology MH - Antifungal Agents/*pharmacology MH - Aspergillosis/*microbiology MH - Aspergillus/*drug effects MH - Drug Interactions MH - Humans MH - Microbial Sensitivity Tests MH - *Peptides MH - *Peptides, Cyclic MH - Pyrimidines/*pharmacology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Triazoles/*pharmacology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3039-41. PMID- 12183267 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Surveillance for antiviral-agent-resistant herpes simplex virus in the general population with recurrent herpes labialis. PG - 3042-4 AB - In a general population survey in the United States, the prevalence of antiviral-agent-resistant herpes simplex virus was very low among more than 1,000 isolates from individuals with an episode of recurrent herpes labialis not treated with topical antiviral agents. Two isolates had borderline resistance to acyclovir (0.2%), and all were susceptible to penciclovir. AD - GlaxoSmithKline Consumer Healthcare, Weybridge, Surrey KT15 0DE, United Kingdom. teresa.h.bacon@gsk.com FAU - Bacon, Teresa H AU - Bacon TH FAU - Boon, Ron J AU - Boon RJ FAU - Schultz, Margaret AU - Schultz M FAU - Hodges-Savola, Cheryl AU - Hodges-Savola C LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antiviral Agents) RN - 39809-25-1 (penciclovir) RN - 59277-89-3 (Acyclovir) SB - IM MH - Acyclovir/administration & dosage/*analogs & derivatives/pharmacology/therapeutic use MH - Administration, Topical MH - Adult MH - Animals MH - Antiviral Agents/*pharmacology MH - Cells, Cultured MH - Drug Resistance, Viral MH - Female MH - Herpes Labialis/*drug therapy/epidemiology/virology MH - Herpesvirus 1, Human/drug effects MH - Humans MH - Male MH - Population MH - Rabbits MH - Recurrence MH - United States/epidemiology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3042-4. PMID- 12183268 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Beta-lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types. PG - 3045-9 AB - Kluyvera ascorbata produces a beta-lactamase that results in an atypical susceptibility pattern, including low-level resistance to penicillins, cephalothin, and cefuroxime, but this resistance is reversed by clavulanate. Ten nucleotide sequences of the corresponding gene, bla(KLUA), were obtained and were found to have minor variations (96 to 100%). Otherwise, bla(KLUA) was found to be similar (95 to 100%) to some plasmid-encoded CTX-M-type beta-lactamases. Finally, mobilization of bla(KLUA) on a plasmid was found to be mediated probably by a genetic mobile element like ISEcp1. AD - Service de Bacteriologie, CHU Cochin, Paris, France. FAU - Humeniuk, Christel AU - Humeniuk C FAU - Arlet, Guillaume AU - Arlet G FAU - Gautier, Valerie AU - Gautier V FAU - Grimont, Patrick AU - Grimont P FAU - Labia, Roger AU - Labia R FAU - Philippon, Alain AU - Philippon A LA - eng SI - GENBANK/AF252622 SI - GENBANK/AF252623 SI - GENBANK/AF311345 SI - GENBANK/AJ272538 PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Plasmids) RN - EC 3.5.2.6 (beta-Lactamases) RN - EC 3.5.2.6 (beta-lactamase bla(KLUA), Klyvera ascorbata) SB - IM MH - Enterobacteriaceae/drug effects/*enzymology/genetics MH - Genes, Bacterial/genetics MH - Genotype MH - Kinetics MH - Microbial Sensitivity Tests MH - Molecular Sequence Data MH - Plasmids/*genetics MH - Research Support, Non-U.S. Gov't MH - beta-Lactamases/*genetics/*metabolism EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3045-9. PMID- 12183269 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20031114 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Functional characterization of Brucella melitensis NorMI, an efflux pump belonging to the multidrug and toxic compound extrusion family. PG - 3050-3 AB - Two putative proteins (NorMI and NorMII) similar to the multidrug efflux protein NorM of Vibrio parahaemolyticus are encoded by the Brucella melitensis 16 M genome. We show that a drug-hypersusceptible Escherichia coli strain overexpressing NorMI displays increased resistance to norfloxacin, ciprofloxacin, gentamicin, tetraphenylphosphonium ion, acriflavine, and berberine. This elevated resistance was proven to be mediated by an energy-dependent efflux mechanism. NorMI belongs to the multidrug and toxic compound extrusion family and is the first multidrug efflux protein identified in Brucella spp. AD - Unite de Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France. FAU - Braibant, Martine AU - Braibant M FAU - Guilloteau, Laurence AU - Guilloteau L FAU - Zygmunt, Michel S AU - Zygmunt MS LA - eng SI - GENBANK/AE008917 SI - GENBANK/AE008918 PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Membrane Transport Proteins) SB - IM MH - Anti-Bacterial Agents/*metabolism/*pharmacology MH - Biological Transport, Active MH - Brucella melitensis/*genetics/metabolism MH - Drug Resistance, Multiple, Bacterial/genetics MH - Energy Metabolism/physiology MH - Escherichia coli/drug effects MH - Genome, Bacterial MH - Kinetics MH - Membrane Transport Proteins/*metabolism MH - Molecular Sequence Data EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3050-3. PMID- 12183270 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Clinical isolates of Staphylococcus aureus with ribosomal mutations conferring resistance to macrolides. PG - 3054-6 AB - Six strains of Staphylococcus aureus isolated from cystic fibrosis patients after treatment with azithromycin were cross-resistant to azithromycin and erythromycin. None of the isolates contained erm or msr(A) genes, but they all carried either A2058G/U or A2059G mutations within the rrl genes, with a majority of the rRNA copies bearing the mutation. One strain displayed an additional mutation in the rplV gene, encoding the L22 ribosomal protein. AD - Service de Microbiologie, CHU Cote de Nacre, Caen, France. FAU - Prunier, Anne-Laure AU - Prunier AL FAU - Malbruny, Brigitte AU - Malbruny B FAU - Tande, Didier AU - Tande D FAU - Picard, Bertrand AU - Picard B FAU - Leclercq, Roland AU - Leclercq R LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Bacterial Proteins) RN - 0 (DNA, Bacterial) RN - 0 (Membrane Transport Proteins) RN - 0 (Oligonucleotide Probes) RN - 0 (msrA protein, Staphylococcus epidermidis) RN - 114-07-8 (Erythromycin) RN - 83905-01-5 (Azithromycin) RN - EC 2.1.1. (Methyltransferases) RN - EC 2.1.1.48 (ErmA protein, Bacteria) SB - IM MH - Anti-Bacterial Agents/*pharmacology MH - Azithromycin/pharmacology MH - Bacterial Proteins/genetics MH - Cystic Fibrosis/microbiology MH - DNA, Bacterial/genetics MH - Drug Resistance MH - Erythromycin/pharmacology MH - Genes, Bacterial/genetics MH - Humans MH - *Membrane Transport Proteins MH - *Methyltransferases MH - Microbial Sensitivity Tests MH - Mutation/*genetics MH - Oligonucleotide Probes MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Ribosomes/*genetics MH - Staphylococcal Infections/*microbiology MH - Staphylococcus aureus/*genetics EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3054-6. PMID- 12183271 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Phenylpropenamide derivatives AT-61 and AT-130 inhibit replication of wild-type and lamivudine-resistant strains of hepatitis B virus in vitro. PG - 3057-60 AB - The phenylpropenamide derivatives AT-61 and AT-130 are nonnucleoside analogue inhibitors of hepatitis B virus (HBV) replication. They inhibited the replication of wild-type HBV with 50% inhibitory concentrations of 21.2 +/- 9.5 and 2.40 +/- 0.92 micro M, respectively, compared to 0.064 +/- 0.020 micro M lamivudine. There were no significant differences in sensitivity between wild-type and nucleoside analogue-resistant (rtL180M, rtM204I, and rtL180M + rtM204V) HBV. AD - Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria 3051, Australia. FAU - Delaney, William E 4th AU - Delaney WE 4th FAU - Edwards, Ros AU - Edwards R FAU - Colledge, Danni AU - Colledge D FAU - Shaw, Tim AU - Shaw T FAU - Furman, Phil AU - Furman P FAU - Painter, George AU - Painter G FAU - Locarnini, Stephen AU - Locarnini S LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (AT 130) RN - 0 (AT 61) RN - 0 (Amides) RN - 0 (Anti-HIV Agents) RN - 0 (Benzamides) RN - 0 (Benzene Derivatives) RN - 134678-17-4 (Lamivudine) SB - IM MH - Amides/*pharmacology MH - Anti-HIV Agents/*pharmacology MH - Benzamides/*pharmacology MH - Benzene Derivatives/*pharmacology MH - Drug Resistance, Viral MH - Hepatitis B virus/*drug effects MH - Lamivudine/*pharmacology MH - Plaque Assay MH - Research Support, Non-U.S. Gov't MH - Virus Replication/*drug effects EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3057-60. PMID- 12183272 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Efficacies of quinupristin-dalfopristin combined with vancomycin in vitro and in experimental endocarditis due to methicillin-resistant Staphylococcus aureus in relation to cross-resistance to macrolides, lincosamides, and streptogramin B- type antibiotics. PG - 3061-4 AB - A beneficial effect of the combination of quinupristin-dalfopristin and vancomycin was observed against two methicillin-resistant strains of Staphylococcus aureus harboring or not harboring the ermC gene, which codes for constitutive macrolide, lincosamide, and streptogramin B resistance. The beneficial effect was observed in time-kill studies, in which the drugs were used at inhibitory concentrations, and in a rabbit model of endocarditis, in which the combination was highly bactericidal and more active than monotherapies. AD - Institut National de la Sante et de la Recherche Medicale (EMI 9933), Faculte de Medecine Xavier Bichat, Paris. FAU - Pavie, Juliette AU - Pavie J FAU - Lefort, Agnes AU - Lefort A FAU - Zarrouk, Virginie AU - Zarrouk V FAU - Chau, Francoise AU - Chau F FAU - Garry, Louis AU - Garry L FAU - Leclercq, Roland AU - Leclercq R FAU - Fantin, Bruno AU - Fantin B LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Antibiotics, Combined) RN - 0 (Antibiotics, Glycopeptide) RN - 0 (Culture Media) RN - 0 (Macrolides) RN - 11006-76-1 (Virginiamycin) RN - 126602-89-9 (quinupristin-dalfopristin) RN - 1404-90-6 (Vancomycin) RN - 3131-03-1 (Streptogramin B) RN - 80738-43-8 (lincosamide) SB - IM MH - Animals MH - Anti-Bacterial Agents/pharmacology MH - Antibiotics, Combined/*pharmacology/*therapeutic use MH - Antibiotics, Glycopeptide/*pharmacology/*therapeutic use MH - Culture Media MH - Drug Resistance MH - Endocarditis, Bacterial/*drug therapy/microbiology MH - *Macrolides MH - Methicillin Resistance MH - Microbial Sensitivity Tests MH - Rabbits MH - Research Support, Non-U.S. Gov't MH - Staphylococcus aureus/*drug effects MH - Streptogramin B/pharmacology MH - Time Factors MH - Vancomycin/*pharmacology/*therapeutic use MH - Virginiamycin/*pharmacology/*therapeutic use EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3061-4. PMID- 12183273 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Antibiotic susceptibilities of Parachlamydia acanthamoeba in amoebae. PG - 3065-7 AB - Parachlamydia acanthamoeba are intracellular bacteria of amoebae and are considered potential etiological agents of human pneumonia. We have determined the in vitro antibiotic susceptibilities of two strains (strain Bn(9) and Hall's coccus) in Acanthamoeba polyphaga. The two strains were susceptible to tetracyclines, macrolides, and rifampin, but resistant to fluoroquinolones. AD - Unite des Rickettsies, CNRS UPRES A 6020, Faculte de Medecine, Universite de la Mediterranee, 13385 Marseille Cedex 05, France. FAU - Maurin, M AU - Maurin M FAU - Bryskier, A AU - Bryskier A FAU - Raoult, D AU - Raoult D LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Bacterial Agents) RN - 0 (Anti-Infective Agents) RN - 0 (Antibiotics, Antitubercular) RN - 0 (DNA, Bacterial) RN - 0 (Fluoroquinolones) RN - 0 (Macrolides) RN - 0 (Tetracyclines) RN - 13292-46-1 (Rifampin) SB - IM MH - Acanthamoeba/drug effects/*microbiology MH - Animals MH - Anti-Bacterial Agents/*pharmacology MH - Anti-Infective Agents/pharmacology MH - Antibiotics, Antitubercular/pharmacology MH - Bacteria/*drug effects MH - DNA, Bacterial/genetics MH - Drug Resistance, Bacterial MH - Fluoroquinolones MH - Humans MH - Macrolides MH - Microbial Sensitivity Tests MH - Pneumonia, Bacterial/microbiology MH - Rifampin/pharmacology MH - Tetracyclines/pharmacology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3065-7. PMID- 12183274 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - In vitro activities of garenoxacin (BMS-284756) against 170 clinical isolates of nine Pasteurella species. PG - 3068-70 AB - The in vitro susceptibilities of 170 clinical isolates plus 12 American Type Culture Collection strains of Pasteurella species comprising nine species and three Pasteurella multocida subspecies were studied by an agar dilution method. Garenoxacin (BMS-284756), a new des-fluoro(6) quinolone, was active at 90% of the strains susceptible to or=8-fold more active against QRSA than the other fluoroquinolones. And the 50% protective doses for DW286 were correspondent with the in vitro activities. AD - College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea. FAU - Yun, Hee-Jeong AU - Yun HJ FAU - Min, Yu-Hong AU - Min YH FAU - Lim, Jung-A AU - Lim JA FAU - Kang, Jin-Wook AU - Kang JW FAU - Kim, So-Young AU - Kim SY FAU - Kim, Mi-Jeong AU - Kim MJ FAU - Jeong, Jae-Hee AU - Jeong JH FAU - Choi, Yun-Jeong AU - Choi YJ FAU - Kwon, Hyun-Jin AU - Kwon HJ FAU - Jung, Yong-Ho AU - Jung YH FAU - Shim, Mi-Ja AU - Shim MJ FAU - Choi, Eung-Chil AU - Choi EC LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (7-(3-(aminomethyl)-4-(methoxyimino)-3-methyltetrahydro-1H-1-pyrrolyl)-1-c yclopropyl-6-fluoro-4-oxo-1,4-dihydro(1,8)naphthyridine-3-carboxylic acid) RN - 0 (Anti-Bacterial Agents) RN - 0 (Anti-Infective Agents) RN - 0 (Fluoroquinolones) RN - 0 (Naphthyridines) SB - IM MH - Animals MH - Anti-Bacterial Agents/*pharmacology/*therapeutic use MH - Anti-Infective Agents/pharmacology/therapeutic use MH - Bacteria/*drug effects MH - Bacterial Infections/*drug therapy/microbiology MH - Comparative Study MH - Dogs MH - Fluoroquinolones MH - Mice MH - Microbial Sensitivity Tests MH - Naphthyridines/*pharmacology MH - Rats MH - Research Support, Non-U.S. Gov't EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3071-4. PMID- 12183276 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Prevalence of protease and reverse transcriptase drug resistance mutations over time in drug-naive human immunodeficiency virus type 1-positive individuals in Rio de Janeiro, Brazil. PG - 3075-9 AB - The prevalence of mutations that confer resistance to protease inhibitors and to nucleoside and nonnucleoside reverse transcriptase inhibitors in 49 blood samples from drug-naive human immunodeficiency virus type 1-infected blood donors living in Rio de Janeiro state, Brazil, in 1998 was evaluated genotypically and phenotypically. AD - Unidade de Genetica, Departamento de Ciencias Morfologicas, Instituto Biomedico, Universidade do Rio de Janeiro, Brazil. FAU - Dumans, Ana T AU - Dumans AT FAU - Soares, Marcelo A AU - Soares MA FAU - Pieniazek, Danuta AU - Pieniazek D FAU - Kalish, Marcia L AU - Kalish ML FAU - De Vroey, Veronique AU - De Vroey V FAU - Hertogs, Kurt AU - Hertogs K FAU - Tanuri, Amilcar AU - Tanuri A LA - eng SI - GENBANK/AF413811 SI - GENBANK/AF413812 SI - GENBANK/AF413813 SI - GENBANK/AF413814 SI - GENBANK/AF413815 SI - GENBANK/AF413816 SI - GENBANK/AF413817 SI - GENBANK/AF413818 SI - GENBANK/AF413819 SI - GENBANK/AF413820 SI - GENBANK/AF413821 SI - GENBANK/AF413822 SI - GENBANK/AF413823 SI - GENBANK/AF413824 SI - GENBANK/AF413825 SI - GENBANK/AF413826 SI - GENBANK/AF413827 SI - GENBANK/AF413828 SI - GENBANK/AF413829 SI - GENBANK/AF413830 SI - GENBANK/AF413831 SI - GENBANK/AF413832 SI - GENBANK/AF413833 SI - GENBANK/AF413834 SI - GENBANK/AF413835 SI - GENBANK/AF413836 SI - GENBANK/AF413837 SI - GENBANK/AF413838 SI - GENBANK/AF413839 SI - GENBANK/AF413840 SI - GENBANK/AF413841 SI - GENBANK/AF413842 SI - GENBANK/AF413843 SI - GENBANK/AF413844 SI - GENBANK/AF413845 SI - GENBANK/AF413846 SI - GENBANK/AF413847 SI - GENBANK/AF413848 SI - GENBANK/AF413849 SI - GENBANK/AF413850 SI - GENBANK/AF413851 SI - GENBANK/AF413852 SI - GENBANK/AF413853 SI - GENBANK/AF413854 SI - GENBANK/AF413855 SI - GENBANK/AF413856 SI - GENBANK/AF413857 SI - GENBANK/AF413858 SI - GENBANK/AF413859 SI - GENBANK/AF413860 SI - GENBANK/AF413861 SI - GENBANK/AF413862 SI - GENBANK/AF413863 SI - GENBANK/AF413864 SI - GENBANK/AF413865 SI - GENBANK/AF413866 SI - GENBANK/AF413867 SI - GENBANK/AF413868 SI - GENBANK/AF413869 SI - GENBANK/AF413870 SI - GENBANK/AF413871 SI - GENBANK/AF413872 SI - GENBANK/AF413873 SI - GENBANK/AF413874 SI - GENBANK/AF413875 SI - GENBANK/AF413876 SI - GENBANK/AF413877 SI - GENBANK/AF413878 SI - GENBANK/AF413879 SI - GENBANK/AF413880 SI - GENBANK/AF413881 SI - GENBANK/AF413882 SI - GENBANK/AF413883 SI - GENBANK/AF413884 SI - GENBANK/AF413885 SI - GENBANK/AF413886 SI - GENBANK/AF413887 SI - GENBANK/AF413888 SI - GENBANK/AF413889 SI - GENBANK/AF413890 SI - GENBANK/AF413891 SI - GENBANK/AF413892 SI - GENBANK/AF413893 SI - GENBANK/AF413894 SI - GENBANK/AF413895 SI - GENBANK/AF413896 SI - GENBANK/AF413897 SI - GENBANK/AF413898 SI - GENBANK/AF413899 SI - GENBANK/AF413900 SI - GENBANK/AF413901 SI - GENBANK/AF413902 SI - GENBANK/AF413903 SI - GENBANK/AF413904 SI - GENBANK/AF413905 SI - GENBANK/AF413906 SI - GENBANK/AF413907 SI - GENBANK/AF413908 SI - GENBANK/AF413909 SI - GENBANK/AF413910 SI - GENBANK/AF413911 SI - GENBANK/AF413912 PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (HIV Protease Inhibitors) RN - 0 (Reverse Transcriptase Inhibitors) RN - EC 2.7.7.- (HIV-1 Reverse Transcriptase) RN - EC 3.4.23.- (HIV Protease) SB - IM MH - Amino Acid Substitution MH - Brazil/epidemiology MH - Drug Resistance, Viral MH - Genotype MH - HIV Infections/drug therapy/epidemiology MH - HIV Protease/metabolism MH - HIV Protease Inhibitors/*pharmacology MH - HIV Seropositivity/enzymology/metabolism MH - HIV-1/*drug effects/enzymology MH - HIV-1 Reverse Transcriptase/metabolism MH - Humans MH - Molecular Sequence Data MH - Mutation/genetics MH - Phenotype MH - Phylogeny MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Inhibitors/*pharmacology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3075-9. PMID- 12183277 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Effect of lamivudine on transmission of human T-cell lymphotropic virus type 1 to adult peripheral blood mononuclear cells in vitro. PG - 3080-3 AB - The effects of lamivudine (3TC) on in vitro infection of peripheral blood mononuclear cells (PBMC) from healthy donors with human T-cell lymphotropic virus type 1 (HTLV-1) were investigated. Direct measures of viral replication (viral DNA, RNA, and protein) all gave similar, very high 50% inhibitory concentrations in comparison with those previously reported for zidovudine. Nevertheless, 3TC inhibited HTLV-1-driven long-term growth of infected PBMC in vitro at concentrations (6.25 micro M) which had poor or no direct antiviral effects, suggesting that another mechanism may be playing a role. AD - Department of Microbiological, Genetic and Molecular Science, University of Messina, Messina, Italy. FAU - Balestrieri, Emanuela AU - Balestrieri E FAU - Forte, Giancarlo AU - Forte G FAU - Matteucci, Claudia AU - Matteucci C FAU - Mastino, Antonio AU - Mastino A FAU - Macchi, Beatrice AU - Macchi B LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-HIV Agents) RN - 0 (DNA, Viral) RN - 134678-17-4 (Lamivudine) SB - IM CIN - Antimicrob Agents Chemother. 2003 May;47(5):1774;author reply 1774-5. PMID: 12709359 MH - Anti-HIV Agents/*pharmacology MH - Coculture Techniques MH - DNA, Viral/biosynthesis/genetics MH - Deltaretrovirus Infections/prevention & control/*transmission/*virology MH - Human T-lymphotropic virus 1/*drug effects MH - Humans MH - Lamivudine/*pharmacology MH - Monocytes/*virology MH - Research Support, Non-U.S. Gov't MH - Virus Replication/drug effects EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3080-3. PMID- 12183278 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041209 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Comparative evaluation of disk diffusion with microdilution assay in susceptibility testing of caspofungin against Aspergillus and Fusarium isolates. PG - 3084-7 AB - We compared the disk diffusion and broth microdilution methods for susceptibility testing of caspofungin against Aspergillus (n = 78) and Fusarium (n = 22) isolates. Microdilution testing followed the NCCLS M-38P guidelines but was performed in antibiotic medium 3 supplemented to 2% glucose (AM3). Disk diffusion assays were performed on AM3 agar plates with a 2- micro g caspofungin disk. By both methods, caspofungin showed favorable activity against Aspergillus isolates and no activity against Fusarium isolates. In the disk-based format, intrazonal growth that was not influenced by the drug concentration gradient was consistently observed for all of the Aspergillus isolates tested. AD - Division of Infectious Diseases, Department of Internal Medicine, Center for the Study of Emerging and Reemerging Pathogens, University of Texas Medical School, Houston, Texas 77030, USA. sarikan@metu.edu.tr FAU - Arikan, Sevtap AU - Arikan S FAU - Paetznick, Victor AU - Paetznick V FAU - Rex, John H AU - Rex JH LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antibiotics, Peptide) RN - 0 (Peptides) RN - 0 (Peptides, Cyclic) RN - 0 (caspofungin) SB - IM MH - Antibiotics, Peptide/*pharmacology MH - Aspergillosis/microbiology MH - Aspergillus/*drug effects MH - Comparative Study MH - Fusarium/*drug effects MH - Microbial Sensitivity Tests MH - Mycoses/microbiology MH - *Peptides MH - *Peptides, Cyclic EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3084-7. PMID- 12183279 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Incidence of high-level evernimicin resistance in Enterococcus faecium among food animals and humans. PG - 3088-90 AB - Six high-level evernimicin-resistant Enterococcus faecium isolates were identified among 304 avilamycin-resistant E. faecium isolates from animals and 404 stool samples from humans with diarrhea. All four animal isolates, and one of the human isolates, were able to transfer resistance to a susceptible E. faecium strain. The resulting transconjugants all tested positive for the presence of emtA, a gene encoding a methyltransferase previously linked with high-level evernimicin resistance. The four transconjugants derived from animal isolates all carried the same plasmid, while a differently sized plasmid was found in the isolate from humans. This study demonstrated a low incidence of high-level evernimicin resistance mediated by the emtA gene in different E. faecium isolates of animal and human origin. AD - Danish Veterinary Institute, DK-1790 Copenhagen V, Denmark. faa@vetinst.dk FAU - Aarestrup, Frank Moller AU - Aarestrup FM FAU - McNicholas, Paul M AU - McNicholas PM LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Aminoglycosides) RN - 0 (Anti-Bacterial Agents) RN - 0 (Oligosaccharides) RN - 11051-71-1 (avilamycin) RN - 53024-98-9 (everninomicin) RN - EC 2.1.1. (Methyltransferases) SB - IM MH - *Aminoglycosides MH - Animal Feed/analysis MH - Animals MH - Anti-Bacterial Agents/*pharmacology MH - Cattle MH - Chickens/*microbiology MH - Conjugation, Genetic MH - Denmark/epidemiology MH - Diarrhea/microbiology MH - Drug Resistance MH - Enterococcus faecium/*drug effects MH - Humans MH - Methyltransferases/genetics MH - Oligosaccharides/pharmacology MH - Reverse Transcriptase Polymerase Chain Reaction MH - Swine/*microbiology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3088-90. PMID- 12183280 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Voriconazole inhibition of the metabolism of tacrolimus in a liver transplant recipient and in human liver microsomes. PG - 3091-3 AB - The purpose of this study was to assess the effect of voriconazole on the blood tacrolimus concentration in a liver transplant recipient and to examine the interaction between voriconazole and tacrolimus by using human liver microsomes. Two subjects were enrolled in the clinical study: one received voriconazole, and the other received a placebo. Tacrolimus metabolism was evaluated in human liver microsomes at various concentrations in the absence and presence of various concentrations of voriconazole. Coadministration of voriconazole and tacrolimus resulted in elevated (nearly 10-fold-higher) trough tacrolimus blood concentrations in the liver transplant patient. In the in vitro study, voriconazole at a concentration of 10.4 +/- 4.3 micro g/ml inhibited the metabolism of tacrolimus by 50%. Clinically relevant concentrations of voriconazole inhibited the metabolism of tacrolimus in human liver microsomes. Close monitoring of the blood concentration and adjustment in the dose of tacrolimus are warranted in transplant recipients treated with voriconazole. AD - School of Pharmacy, University of Pittsburgh, and Veterans Affairs Medical Center, Pittsburgh, Pennsylvania 15240, USA. FAU - Venkataramanan, Raman AU - Venkataramanan R FAU - Zang, Shimin AU - Zang S FAU - Gayowski, Timothy AU - Gayowski T FAU - Singh, Nina AU - Singh N LA - eng PT - Clinical Trial PT - Journal Article PT - Randomized Controlled Trial PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antifungal Agents) RN - 0 (Enzyme Inhibitors) RN - 0 (Immunosuppressive Agents) RN - 0 (Pyrimidines) RN - 0 (Triazoles) RN - 0 (voriconazole) RN - 109581-93-3 (Tacrolimus) RN - 9035-51-2 (Cytochrome P-450 Enzyme System) RN - EC 1.14.14.1 (CYP3A protein, human) SB - IM MH - Antifungal Agents/*pharmacology MH - Cytochrome P-450 Enzyme System/antagonists & inhibitors MH - Drug Interactions MH - Enzyme Inhibitors/pharmacology MH - Humans MH - Immunosuppressive Agents/*metabolism MH - In Vitro MH - Kinetics MH - Liver Transplantation/*physiology MH - Microsomes, Liver/drug effects/*metabolism MH - Pyrimidines/*pharmacology MH - Tacrolimus/*metabolism MH - Triazoles/*pharmacology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3091-3. PMID- 12183281 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Comparative activities of the oxazolidinone AZD2563 and linezolid against selected recent North American isolates of Streptococcus pneumoniae. PG - 3094-5 AB - The activity of AZD2563 against 250 highly resistant pneumococci and 267 drug-susceptible isolates was determined. The AZD2563 MICs for 50 and 90% of the strains tested were 1 and 2 micro g/ml and 0.5 and 1 micro g/ml, respectively, for the two isolate groups. These MICs were within 1 log(2) dilution of those of linezolid. AD - Infectious Disease Service of Brooke Army Medical Center, San Antonio, Texas, USA. FAU - Baum, Sue E AU - Baum SE FAU - Crawford, Sharon A AU - Crawford SA FAU - McElmeel, M L AU - McElmeel ML FAU - Whitney, Cynthia G AU - Whitney CG FAU - Jorgensen, James H AU - Jorgensen JH LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (AZD2563) RN - 0 (Acetamides) RN - 0 (Anti-Bacterial Agents) RN - 0 (Oxazolidinones) RN - 165800-03-3 (linezolid) SB - IM MH - Acetamides/*pharmacology MH - Anti-Bacterial Agents/*pharmacology MH - Comparative Study MH - Drug Resistance, Microbial MH - Humans MH - Microbial Sensitivity Tests MH - North America MH - Oxazolidinones/*pharmacology MH - Pneumococcal Infections/microbiology MH - Research Support, Non-U.S. Gov't MH - Streptococcus pneumoniae/*drug effects EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3094-5. PMID- 12183282 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041209 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Effect of the echinocandin caspofungin on expression of Candida albicans secretory aspartyl proteinases and phospholipase in vitro. PG - 3096-100 AB - Although the echinocandin caspofungin primarily inhibits the synthesis of cell wall 1,3-beta-D-glucan, its fungicidal activity could also potentially perturb the expression of virulence factors involved in the ability of Candida albicans to cause infection. Expression of the C. albicans secretory aspartyl proteinase (SAP) and phospholipase B (PLB) virulence genes was determined by reverse transcription-PCR after the addition of caspofungin to cells grown for 15 h in Sabouraud dextrose broth. In cells that remained viable, expression of SAP1 to SAP3, SAP7 to SAP9, and PLB1 was unaltered after exposure to fungicidal concentrations (4 to 16 micro g/ml) of caspofungin over a period of 7 h. However, expression of SAP5 increased steadily beginning 1 h after exposure to caspofungin. These results indicate that caspofungin is rapidly fungicidal against C. albicans, before any suppression of SAP or PLB1 gene expression can occur. AD - Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, Quebec, Canada. FAU - Ripeau, Jean-Sebastien AU - Ripeau JS FAU - Aumont, Francine AU - Aumont F FAU - Belhumeur, Pierre AU - Belhumeur P FAU - Ostrosky-Zeichner, Luis AU - Ostrosky-Zeichner L FAU - Rex, John H AU - Rex JH FAU - de Repentigny, Louis AU - de Repentigny L LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Antibiotics, Antifungal) RN - 0 (Antibiotics, Peptide) RN - 0 (Peptides) RN - 0 (Peptides, Cyclic) RN - 0 (RNA, Fungal) RN - 0 (RNA, Messenger) RN - 0 (caspofungin) RN - EC 3.1.- (Phospholipases) RN - EC 3.4.23 (Aspartic Endopeptidases) SB - IM MH - Antibiotics, Antifungal/*pharmacology MH - Antibiotics, Peptide/*pharmacology MH - Aspartic Endopeptidases/*biosynthesis/genetics MH - Candida albicans/drug effects/*enzymology/genetics MH - Gene Expression Regulation, Enzymologic/*drug effects MH - Genes, Fungal/genetics MH - Microbial Sensitivity Tests MH - *Peptides MH - *Peptides, Cyclic MH - Phospholipases/*biosynthesis/genetics MH - RNA, Fungal/biosynthesis/genetics MH - RNA, Messenger/biosynthesis/genetics MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Time Factors EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3096-100. PMID- 12183283 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Development of a yeast assay for rapid screening of inhibitors of human-derived Pneumocystis carinii dihydrofolate reductase. PG - 3101-3 AB - Human-derived Pneumocystis carinii dihydrofolate reductase (DHFR) was expressed in a Saccharomyces cerevisiae strain whose growth depends on complementation by this enzyme. We utilized a quantitative assay to measure the sensitivity of this yeast strain to DHFR inhibitors. This assay should be useful for identifying new inhibitors of human-derived P. carinii DHFR. AD - Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892-1662, USA. FAU - Ma, Liang AU - Ma L FAU - Jia, Qiuyao AU - Jia Q FAU - Kovacs, Joseph A AU - Kovacs JA LA - eng PT - Journal Article PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Folic Acid Antagonists) RN - 0 (Triazines) RN - 47326-86-3 (BRL 6231) RN - 52128-35-5 (Trimetrexate) RN - 58-14-0 (Pyrimethamine) RN - 738-70-5 (Trimethoprim) RN - EC 1.5.1.3 (Tetrahydrofolate Dehydrogenase) SB - IM MH - Animals MH - Drug Evaluation, Preclinical MH - Folic Acid Antagonists/*pharmacology MH - Humans MH - Pneumocystis/*enzymology MH - Pneumocystis Infections/enzymology/microbiology MH - Pyrimethamine/pharmacology MH - Rats MH - Saccharomyces cerevisiae/drug effects/*enzymology MH - Tetrahydrofolate Dehydrogenase/*metabolism MH - Triazines/pharmacology MH - Trimethoprim/pharmacology MH - Trimetrexate/pharmacology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3101-3. PMID- 12183284 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Prevalence of oxacillin resistance in Staphylococcus aureus among inpatients and outpatients in the United States during 2000. PG - 3104-5 FAU - Jones, Mark E AU - Jones ME FAU - Mayfield, David C AU - Mayfield DC FAU - Thornsberry, Clyde AU - Thornsberry C FAU - Karlowsky, James A AU - Karlowsky JA FAU - Sahm, Daniel F AU - Sahm DF FAU - Peterson, Dan AU - Peterson D LA - eng PT - Letter PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Penicillins) RN - 66-79-5 (Oxacillin) SB - IM MH - Databases, Factual MH - Humans MH - Methicillin Resistance MH - Oxacillin/*pharmacology MH - Penicillin Resistance MH - Penicillins/*pharmacology MH - Staphylococcal Infections/*epidemiology/*microbiology MH - Staphylococcus aureus/*drug effects MH - United States/epidemiology EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3104-5. PMID- 12183285 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Relationship between antibiotic resistance in Streptococcus pneumoniae and that in Haemophilus influenzae: evidence for common selective pressure. PG - 3106-7 FAU - Jones, Mark E AU - Jones ME FAU - Karlowsky, James A AU - Karlowsky JA FAU - Blosser-Middleton, Renee AU - Blosser-Middleton R FAU - Critchley, Ian AU - Critchley I FAU - Thornsberry, Clyde AU - Thornsberry C FAU - Sahm, Daniel F AU - Sahm DF LA - eng PT - Letter PL - United States TA - Antimicrob Agents Chemother JID - 0315061 SB - IM MH - *Drug Resistance MH - Drug Utilization MH - Haemophilus influenzae/*drug effects MH - Microbial Sensitivity Tests MH - Research Support, Non-U.S. Gov't MH - Selection (Genetics) MH - Species Specificity MH - Streptococcus pneumoniae/*drug effects EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3106-7. PMID- 12183286 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - gyrA Mutations associated with nalidixic acid-resistant salmonellae from wild birds. PG - 3108-9 FAU - Reche, M Paloma AU - Reche MP FAU - Garcia de los Rios, Jose E AU - Garcia de los Rios JE FAU - Jimenez, Pedro A AU - Jimenez PA FAU - Rojas, Ana M AU - Rojas AM FAU - Rotger, Rafael AU - Rotger R LA - eng SI - GENBANK/AF447053 SI - GENBANK/AF447054 SI - GENBANK/AF447055 SI - GENBANK/AF447056 SI - GENBANK/AF447057 SI - GENBANK/AF447058 SI - GENBANK/AF447059 SI - GENBANK/X78977 PT - Letter PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-Infective Agents) RN - 389-08-2 (Nalidixic Acid) RN - EC 5.99.1.- (DNA Gyrase) SB - IM MH - Animals MH - Anti-Infective Agents/*pharmacology MH - Birds/*microbiology MH - DNA Gyrase/*genetics MH - Drug Resistance, Bacterial MH - Feces/microbiology MH - Microbial Sensitivity Tests MH - Molecular Sequence Data MH - Mutation/*genetics MH - Nalidixic Acid/*pharmacology MH - Research Support, Non-U.S. Gov't MH - Salmonella/*drug effects EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3108-9. PMID- 12183287 OWN - NLM STAT- MEDLINE DA - 20020816 DCOM- 20030213 LR - 20041117 PUBM- Print IS - 0066-4804 VI - 46 IP - 9 DP - 2002 Sep TI - Amino acid substitutions at position 69 of the reverse transcriptase of human immunodeficiency virus type 1 are frequent in zalcitabine-naive antiretroviral-drug-experienced patients. PG - 3110-1 FAU - Montes, Brigitte AU - Montes B FAU - Segondy, Michel AU - Segondy M LA - eng PT - Comment PT - Letter PL - United States TA - Antimicrob Agents Chemother JID - 0315061 RN - 0 (Anti-HIV Agents) RN - 0 (Reverse Transcriptase Inhibitors) RN - 7481-89-2 (Zalcitabine) RN - EC 2.7.7.- (HIV-1 Reverse Transcriptase) SB - IM CON - Antimicrob Agents Chemother. 2001 Aug;45(8):2276-9. PMID: 11451685 MH - Amino Acid Substitution/*genetics MH - Anti-HIV Agents/*therapeutic use MH - *Antiretroviral Therapy, Highly Active MH - Drug Resistance, Viral MH - HIV-1/*enzymology/*genetics MH - HIV-1 Reverse Transcriptase/*genetics MH - Humans MH - Logistic Models MH - Reverse Transcriptase Inhibitors/*therapeutic use MH - Zalcitabine/*therapeutic use EDAT- 2002/08/17 10:00 MHDA- 2003/02/14 04:00 PST - ppublish SO - Antimicrob Agents Chemother 2002 Sep;46(9):3110-1. PMID- 12184817 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030512 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Aug 16 TI - Low agreement for assessing the risk of postoperative deep venous thrombosis when deciding prophylaxis strategies: a study using clinical vignettes. PG - 16 AB - BACKGROUND: Several clinical practice guidelines (CPG) on antithrombotic prophylaxis in surgical patients help to decide about the prophylaxis strategy based on the patient risk of deep venous thrombosis (DVT). However, the physician risk estimates of DVT could have little inter-observer reproducibility, which could lead to different individual prophylaxis practices. METHODS: Physicians were asked to evaluate DVT risk in eight clinical vignettes, describing actual patients cared for in our hospital. The vignettes included all possible levels of DVT risk. RESULTS: The degree of prophylaxis strategies accuracy was 63% (95% CI 523-75%). Overall agreement was 0.32 (z = 7.61, p < 0.001) and for each level of risk kappa was 0.38 (z = 6.50, p < 0.001); 0.1 (z = 1.65, p < 0.049) and 0.5 (z = 8.45, p < 0.001) for small, moderate and high risk group respectively CONCLUSIONS: Our results showed that there is poor agreement when physicians have to evaluate the risk for postoperative DVT, and in the cases of low and moderate risks of DVT there is the smallest agreement. In addition, the data also showed that the overall accuracy of DVT prophylaxis strategy was only moderate and the risk evaluation did not correlate to the selection of the strategy. The issue of inter-observers variability should be taken into account when CPG performance are analysed, especially when considering the risk-evaluation to choose the appropriate actions. AD - Departamento de Medicina, Hospital Universitario Austral. moflaherty@cas.austral.edu.ar FAU - O'Flaherty, Martin AU - O'Flaherty M FAU - Lerum, Kaja AU - Lerum K FAU - Martin, Paula AU - Martin P FAU - Grassi, Daniel AU - Grassi D LA - eng PT - Journal Article DEP - 20020816 PL - England TA - BMC Health Serv Res JID - 101088677 RN - 0 (Nadroparin) RN - 9005-49-6 (Heparin) SB - IM MH - Adult MH - Aged MH - Aged, 80 and over MH - Argentina/epidemiology MH - Bandages/utilization MH - Decision Making MH - Early Ambulation/utilization MH - Female MH - Heparin/therapeutic use MH - Hospitals, University/standards MH - Humans MH - Male MH - Medical Audit MH - Middle Aged MH - Nadroparin/therapeutic use MH - Observer Variation MH - Postoperative Complications/classification/*epidemiology/*prevention & control MH - *Practice Guidelines MH - Premedication/utilization MH - Risk Assessment/*statistics & numerical data MH - Venous Thrombosis/*epidemiology/etiology/*prevention & control EDAT- 2002/08/20 10:00 MHDA- 2003/05/13 05:00 PHST- 2002/04/19 [received] PHST- 2002/08/16 [accepted] PHST- 2002/08/16 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Aug 16;2(1):16. PMID- 12185243 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041203 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway. PG - 11975-80 AB - The genome of the Mastreviruses encodes a replication-associated protein (RepA) that interacts with members of the plant retinoblastoma-related protein family, which are putative cell cycle regulators. Expression of ZmRb1, a maize retinoblastoma-related gene, and RepA inhibited and stimulated, respectively, cell division in tobacco cell cultures. The effect of RepA was mitigated by over-expression of ZmRb1. RepA increased transformation frequency and callus growth rate of high type II maize germplasm. RepA-containing transgenic maize calli remained embryogenic, were readily regenerable, and produced fertile plants that transmitted transgene expression in a Mendelian fashion. In high type II, transformation frequency increased with the strength of the promoter driving RepA expression. When a construct in which RepA was expressed behind its native LIR promoter was used, primary transformation frequencies did not improve for two elite Pioneer maize inbreds. However, when LIR:RepA-containing transgenic embryos were used in subsequent rounds of transformation, frequencies were higher in the RepA+ embryos. These data demonstrate that RepA can stimulate cell division and callus growth in culture, and improve maize transformation. AD - Pioneer Hi-Bred, International, Inc., Johnston, IA 50131, USA. william.gordon-kamm@pioneer.com FAU - Gordon-Kamm, William AU - Gordon-Kamm W FAU - Dilkes, Brian P AU - Dilkes BP FAU - Lowe, Keith AU - Lowe K FAU - Hoerster, George AU - Hoerster G FAU - Sun, Xifan AU - Sun X FAU - Ross, Margit AU - Ross M FAU - Church, Laura AU - Church L FAU - Bunde, Chris AU - Bunde C FAU - Farrell, Jeff AU - Farrell J FAU - Hill, Patrea AU - Hill P FAU - Maddock, Sheila AU - Maddock S FAU - Snyder, Jane AU - Snyder J FAU - Sykes, Louisa AU - Sykes L FAU - Li, Zhongsen AU - Li Z FAU - Woo, Young-min AU - Woo YM FAU - Bidney, Dennis AU - Bidney D FAU - Larkins, Brian A AU - Larkins BA LA - eng SI - GENBANK/AY138520 SI - GENBANK/AY138521 PT - Journal Article DEP - 20020816 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (DNA-Binding Proteins) RN - 0 (Proteins) RN - 0 (Retinoblastoma Protein) RN - 0 (Trans-Activators) RN - 0 (replication initiator protein) RN - EC 5.99.- (DNA Helicases) SB - IM MH - *Cell Cycle MH - Cell Division MH - *DNA Helicases MH - *DNA-Binding Proteins MH - Molecular Sequence Data MH - Plants, Genetically Modified MH - Proteins/physiology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Retinoblastoma Protein/*metabolism MH - *Trans-Activators MH - Zea mays/*cytology/metabolism EDAT- 2002/08/20 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/16 [aheadofprint] AID - 10.1073/pnas.142409899 [doi] AID - 142409899 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11975-80. Epub 2002 Aug 16. PMID- 12185244 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041117 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - Formation of geometrically complex lipid nanotube-vesicle networks of higher-order topologies. PG - 11573-8 AB - We present a microelectrofusion method for construction of fluid-state lipid bilayer networks of high geometrical complexity up to fully connected networks with genus = 3 topology. Within networks, self-organizing branching nanotube architectures could be produced where intersections spontaneously arrange themselves into three-way junctions with an angle of 120 degrees between each nanotube. Formation of branching nanotube networks appears to follow a minimum-bending energy algorithm that solves for pathway minimization. It is also demonstrated that materials can be injected into specific containers within a network by nanotube-mediated transport of satellite vesicles having defined contents. Using a combination of microelectrofusion, spontaneous nanotube pattern formation, and satellite-vesicle injection, complex networks of containers and nanotubes can be produced for a range of applications in, for example, nanofluidics and artificial cell design. In addition, this electrofusion method allows integration of biological cells into lipid nanotube-vesicle networks. AD - Department of Physical Chemistry and Microtechnology Center, Chalmers University of Technology, SE-412 96 Goteborg, Sweden. FAU - Karlsson, Mattias AU - Karlsson M FAU - Sott, Kristin AU - Sott K FAU - Davidson, Maximillian AU - Davidson M FAU - Cans, Ann-Sofie AU - Cans AS FAU - Linderholm, Pontus AU - Linderholm P FAU - Chiu, Daniel AU - Chiu D FAU - Orwar, Owe AU - Orwar O LA - eng PT - Journal Article DEP - 20020816 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (Lipids) SB - IM MH - Lipids/*chemistry MH - Microscopy, Fluorescence MH - Molecular Structure MH - Research Support, Non-U.S. Gov't EDAT- 2002/08/20 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/16 [aheadofprint] AID - 10.1073/pnas.172183699 [doi] AID - 172183699 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11573-8. Epub 2002 Aug 16. PMID- 12185245 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041117 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - Loss of microsatellite diversity and low effective population size in an overexploited population of New Zealand snapper (Pagrus auratus). PG - 11742-7 AB - Although the effects of overfishing on species diversity and abundance are well documented, threats to the genetic diversity of marine fish populations have so far been largely neglected. Indeed, there seems to be little cause for concern, as even "collapsed" stocks usually consist of several million individuals, whereas population genetics theory suggests that only very small populations suffer significant loss of genetic diversity. On the other hand, in many marine species the genetically effective population size (N(e)), which determines the genetic properties of a population, may be orders of magnitude smaller than the census population size (N). Here, microsatellite analyses of a time series of archived scales demonstrated a significant decline in genetic diversity in a New Zealand snapper population during its exploitation history. Effective population sizes estimated both from the decline in heterozygosity and from temporal fluctuations in allele frequency were five orders of magnitude smaller than census population sizes from fishery data. If such low N(e)/N ratios are commonplace in marine species, many exploited marine fish stocks may be in danger of losing genetic variability, potentially resulting in reduced adaptability, population persistence, and productivity. AD - Molecular Ecology and Fisheries Genetics Laboratory, Department of Biological Sciences, University of Hull, Hull HU6 7RX, United Kingdom. lhauser@u.washington.edu FAU - Hauser, Lorenz AU - Hauser L FAU - Adcock, Greg J AU - Adcock GJ FAU - Smith, Peter J AU - Smith PJ FAU - Ramirez, Julio H Bernal AU - Ramirez JH FAU - Carvalho, Gary R AU - Carvalho GR LA - eng PT - Journal Article DEP - 20020816 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 SB - IM MH - Animals MH - *Evolution MH - Microsatellite Repeats/*genetics MH - Perciformes/*genetics MH - Population Density MH - Research Support, Non-U.S. Gov't MH - Species Specificity EDAT- 2002/08/20 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/16 [aheadofprint] AID - 10.1073/pnas.172242899 [doi] AID - 172242899 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11742-7. Epub 2002 Aug 16. PMID- 12186657 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021216 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Aug 19 TI - A approximately 35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP. PG - 23 AB - 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. AD - Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi-110067, India. skdhar2002@yahoo.co.in FAU - Dhar, Suman K AU - Dhar SK FAU - Mondal, Neelima AU - Mondal N FAU - Soni, Rajesh K AU - Soni RK FAU - Mukhopadhyay, Gauranga AU - Mukhopadhyay G LA - eng PT - Journal Article DEP - 20020819 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Cell Cycle Proteins) RN - 0 (DNA, Fungal) RN - 0 (DNA-Binding Proteins) RN - 0 (Peptides) RN - 0 (Proto-Oncogene Proteins c-myc) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 122544-18-7 (CDC6 protein, S cerevisiae) RN - 56-65-5 (Adenosine Triphosphate) RN - 60-00-4 (Edetic Acid) RN - 7647-14-5 (Sodium Chloride) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Adenosine Triphosphate/*pharmacology MH - Animals MH - Binding Sites/genetics MH - Cell Cycle Proteins/chemistry/genetics/metabolism MH - Cell Line MH - DNA, Fungal/genetics/metabolism MH - DNA-Binding Proteins/chemistry/genetics/*metabolism MH - Edetic Acid/pharmacology MH - Glutathione Transferase/genetics/metabolism MH - Humans MH - Molecular Weight MH - Mutation MH - Peptides/chemistry/genetics/*metabolism MH - Protein Binding/drug effects MH - Proto-Oncogene Proteins c-myc/genetics MH - Recombinant Fusion Proteins/genetics/metabolism MH - Replication Origin/*genetics MH - Research Support, Non-U.S. Gov't MH - Saccharomyces cerevisiae/genetics MH - *Saccharomyces cerevisiae Proteins MH - Sodium Chloride/pharmacology MH - Spodoptera MH - Temperature EDAT- 2002/08/21 10:00 MHDA- 2002/12/18 04:00 PHST- 2002/03/28 [received] PHST- 2002/08/19 [accepted] PHST- 2002/08/19 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Aug 19;3(1):23. PMID- 12186975 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041117 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - SALSA, a variant of yeast SAGA, contains truncated Spt7, which correlates with activated transcription. PG - 11622-7 AB - Spt-Ada-Gcn5 acetyltransferase (SAGA) is a previously described histone acetyltransferase/transcriptional coactivator complex in yeast. At promoters of certain genes (HIS3 and TRP3), SAGA has an inhibitory function involving a nonproductive TATA-binding protein interaction mediated by the Spt3 and Spt8 subunits. Related to this, Spt8-less SAGA is a major form of the complex under activating conditions for these genes. In the present study, we purify this activation-specific complex, called SALSA (SAGA altered, Spt8 absent). Besides lacking Spt8, SALSA contains Spt7 subunit that is truncated. Examining the role of this subunit, we find that C-terminally truncated SPT7 resulted in derepressed HIS3 transcription. Furthermore, when grown in rich media (repressing conditions), wild-type cells yielded predominantly SAGA, but Spt7 C-terminal truncations resulted primarily in a form of complex similar to SALSA. Thus, SALSA-like structure and activating function can be partially recapitulated in yeast by truncating the C terminus of Spt7. Overall, these results lead to a model that for a subset of promoters SAGA is inhibitory through Spt3, Spt8, and an Spt8-interacting subdomain of Spt7, whereas SALSA is a form of complex for positive transcriptional regulation. These data clarify a mechanism by which a transcriptional regulatory complex can switch between positive and negative modulation. AD - The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA. FAU - Sterner, David E AU - Sterner DE FAU - Belotserkovskaya, Rimma AU - Belotserkovskaya R FAU - Berger, Shelley L AU - Berger SL LA - eng PT - Journal Article DEP - 20020819 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (Fungal Proteins) RN - 0 (Plasmids) RN - 0 (SPT7 protein, S cerevisiae) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 0 (Transcription Factors) SB - IM MH - Amino Acid Sequence MH - Base Sequence MH - Blotting, Western MH - Fungal Proteins/*metabolism MH - Molecular Sequence Data MH - Plasmids MH - Precipitin Tests MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Saccharomyces cerevisiae/*metabolism MH - *Saccharomyces cerevisiae Proteins MH - Transcription Factors/chemistry/genetics/metabolism/*physiology MH - *Transcription, Genetic EDAT- 2002/08/21 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/19 [aheadofprint] AID - 10.1073/pnas.182021199 [doi] AID - 182021199 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11622-7. Epub 2002 Aug 19. PMID- 12186978 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041117 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - Essential myosin light chain as a target for caspase-3 in failing myocardium. PG - 11860-5 AB - Programmed cell death involves the activation of caspase proteases that can mediate the cleavage of vital cytoskeletal proteins. We have recently reported that, in failing cardiac myocytes, caspase-3 activation is associated with a reduction in contractile performance. In this study we used a modified yeast two-hybrid system to screen for caspase-3 interacting proteins of the cardiac cytoskeleton. We identified ventricular essential myosin light chain (vMLC1) as a target for caspase-3. By sequencing and site-directed mutagenesis, a noncanonical cleavage site for caspase-3 was mapped to the C-terminal DFVE(135)G motif. We demonstrated that vMLC1 cleavage in failing myocardium in vivo is associated with a morphological disruption of the organized vMLC1 staining of sarcomeres, and with a reduction in myocyte contractile performance. Adenoviral gene transfer of the caspase inhibitor p35 in vivo prevented caspase-3 activation and vMLC1 cleavage, with positive impact on contractility. These data suggest that direct cleavage of vMLC1 by activated caspase-3 may contribute to depression of myocyte function by altering cross-bridge interaction between myosin and actin molecules. Therefore, activation of apoptotic pathways in the heart may lead to contractile dysfunction before cell death. AD - I. Medizinische Klinik and Deutsches Herzzentrum, D-81675 Munich, Germany. FAU - Moretti, Alessandra AU - Moretti A FAU - Weig, Hans-Jorg AU - Weig HJ FAU - Ott, Thomas AU - Ott T FAU - Seyfarth, Melchior AU - Seyfarth M FAU - Holthoff, Hans-Peter AU - Holthoff HP FAU - Grewe, Diana AU - Grewe D FAU - Gillitzer, Angelika AU - Gillitzer A FAU - Bott-Flugel, Lorenz AU - Bott-Flugel L FAU - Schomig, Albert AU - Schomig A FAU - Ungerer, Martin AU - Ungerer M FAU - Laugwitz, Karl-Ludwig AU - Laugwitz KL LA - eng PT - Journal Article DEP - 20020819 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (Myosin Light Chains) RN - EC 3.4.22.- (Caspases) RN - EC 3.4.22.- (caspase-3) SB - IM MH - Animals MH - COS Cells MH - Caspases/*metabolism MH - Heart/*physiopathology MH - Hydrolysis MH - Kinetics MH - Mutagenesis, Site-Directed MH - Myocardium/enzymology/*metabolism MH - Myosin Light Chains/genetics/*metabolism MH - Precipitin Tests MH - Rabbits MH - Research Support, Non-U.S. Gov't MH - Sarcomeres/metabolism MH - Substrate Specificity EDAT- 2002/08/21 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/19 [aheadofprint] AID - 10.1073/pnas.182373099 [doi] AID - 182373099 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11860-5. Epub 2002 Aug 19. PMID- 12186979 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041117 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - Evidence for an ancient selective sweep in the MHC class I gene repertoire of chimpanzees. PG - 11748-53 AB - MHC class I molecules play an essential role in the immune defense against intracellular infections. The hallmark of the MHC is its extensive degree of polymorphism at the population level. However, the present comparison of MHC class I gene intron variation revealed that chimpanzees have experienced a severe repertoire reduction at the orthologues of the HLA-A, -B, and -C loci. The loss of variability predates the (sub)speciation of chimpanzees and did not effect other known gene systems. Therefore the selective sweep in the MHC class I gene may have resulted from a widespread viral infection. Based on the present results and the fact that chimpanzees have a natural resistance to the development of AIDS, we hypothesize that the selective sweep was caused by the chimpanzee-derived simian immunodeficiency virus (SIVcpz), the closest relative of HIV-1, or a closely related retrovirus. Hence, the contemporary chimpanzee populations represent the offspring of AIDS-resistant animals, the survivors of a HIV-like pandemic that took place in the distant past. AD - Department of Immunobiology, Biomedical Primate Research Centre, P.O. Box 3306, 2280 GH, Rijswijk, The Netherlands. FAU - de Groot, Natasja G AU - de Groot NG FAU - Otting, Nel AU - Otting N FAU - Doxiadis, Gaby G M AU - Doxiadis GG FAU - Balla-Jhagjhoorsingh, Sunita S AU - Balla-Jhagjhoorsingh SS FAU - Heeney, Jonathan L AU - Heeney JL FAU - van Rood, Jon J AU - van Rood JJ FAU - Gagneux, Pascal AU - Gagneux P FAU - Bontrop, Ronald E AU - Bontrop RE LA - eng SI - GENBANK/AJ313335 SI - GENBANK/AJ313336 SI - GENBANK/AJ313337 SI - GENBANK/AJ313338 SI - GENBANK/AJ313339 SI - GENBANK/AJ313340 SI - GENBANK/AJ313341 SI - GENBANK/AJ313342 SI - GENBANK/AJ313343 SI - GENBANK/AJ313344 SI - GENBANK/AJ313345 SI - GENBANK/AJ313346 SI - GENBANK/AJ313347 SI - GENBANK/AJ313348 SI - GENBANK/AJ313349 SI - GENBANK/AJ313350 SI - GENBANK/AJ313351 SI - GENBANK/AJ313352 SI - GENBANK/AJ313353 SI - GENBANK/AJ313354 SI - GENBANK/AJ313355 SI - GENBANK/AJ313356 SI - GENBANK/AJ313357 SI - GENBANK/AJ313358 SI - GENBANK/AJ313359 PT - Journal Article DEP - 20020819 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 9007-49-2 (DNA) SB - IM MH - Animals MH - Base Sequence MH - DNA MH - *Evolution, Molecular MH - *Genes, MHC Class I MH - Introns MH - Molecular Sequence Data MH - Pan troglodytes/*genetics MH - Phylogeny MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Nucleic Acid EDAT- 2002/08/21 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/19 [aheadofprint] AID - 10.1073/pnas.182420799 [doi] AID - 182420799 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11748-53. Epub 2002 Aug 19. PMID- 12192093 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041117 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - Functional plasticity of an antigen-specific memory CD4 T cell population. PG - 11802-7 AB - The protective nature of memory immune responses is attributed largely to terminally differentiated memory T cells that retain memory of the antigen via the antigen receptor and memory of the effector functions that initially cleared the pathogen. It is not known whether a given population of antigen-specific memory T cells is endowed with functional flexibility to provide protective responses against antigens reencountered in different immunological contexts. Here, we examine functional properties of influenza hemagglutinin (HA)-specific memory CD4 T cells recovered from adoptive hosts that received in vitro-activated HA-specific T cell receptor-transgenic CD4 T cells 2 months to 1 year previously. We demonstrate that this HA-specific memory CD4 T cell population bearing a clonal T cell receptor can produce predominantly T helper 1 or T helper 2 effector cytokines depending on the nature of the recall stimulus. Our findings reveal remarkable functional plasticity within an antigen-specific memory T cell population and have direct implications for modulating memory T cell function in vaccine design and treatments for autoimmune diseases. AD - Department of Surgery, University of Maryland School of Medicine, MSTF Building, Room 400, 685 West Baltimore Street, Baltimore, MD 21201, USA. FAU - Ahmadzadeh, Mojgan AU - Ahmadzadeh M FAU - Farber, Donna L AU - Farber DL LA - eng GR - AI42092/AI/NIAID PT - Journal Article DEP - 20020821 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (Antigens) RN - 0 (Cytokines) SB - IM MH - Adoptive Transfer MH - Animals MH - Antigens/*immunology MH - CD4-Positive T-Lymphocytes/cytology/*immunology MH - Cell Differentiation MH - Cytokines/biosynthesis MH - *Immunologic Memory MH - Mice MH - Mice, Inbred BALB C MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2002/08/23 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/21 [aheadofprint] AID - 10.1073/pnas.192263099 [doi] AID - 192263099 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11802-7. Epub 2002 Aug 21. PMID- 12193273 OWN - NLM STAT- Publisher DA - 20020823 PUBM- Print IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Aug 23 TI - Delineation of RAID1, the RACK1 interaction domain located within the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5. PG - 24 AB - Background The cyclic AMP specific phosphodiesterase, PDE4D5 interacts with the beta-propeller protein RACK1 to form a signaling scaffold complex in cells. Two-hybrid analysis of truncation and mutant constructs of the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5 was used to define a domain conferring interaction with the signaling scaffold protein, RACK1. Results Truncation and mutagenesis approaches showed that the RACK1-interacting domain on PDE4D5 comprised a cluster of residues provided by Asn-22/Pro-23/Trp-24/Asn-26 together with a series of hydrophobic amino acids, namely Leu-29, Val-30, Leu-33, Leu-37 and Leu-38 in a 'Leu-Xaa-Xaa-Xaa-Leu' repeat. This was done by 2-hybrid analyses and then confirmed in biochemical pull down analyses using GST-RACK1 and mutant PDE4D5 forms expressed in COS cells. Mutation of Arg-34, to alanine, in PDE4D5 attenuated its interaction with RACK1 both in 2-hybrid screens and in pull down analyses. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, bound to RACK1 with similar affinity to native PDE4D5 itself (Ka circa 6 nM). Conclusion The RACK1 Interaction Domain on PDE4D5, that we here call RAID1, is proposed to form an amphipathic helical structure that we suggest may interact with the C-terminal beta-propeller blades of RACK1 in a manner akin to the interaction of the helical G-gamma signal transducing protein with the beta-propeller protein, G-beta. AU - Bolger GB AU - McCahill A AU - Yarwood SJ AU - Steele MS AU - Warwicker J AU - Houslay MD LA - ENG PT - JOURNAL ARTICLE TA - BMC Biochem JID - 101084098 EDAT- 2002/08/24 10:00 MHDA- 2002/08/24 10:00 PHST- 2002/06/06 [received] PHST- 2002/08/23 [accepted] PHST- 2002/08/23 [aheadofprint] PST - aheadofprint SO - BMC Biochem 2002 Aug 23;3(1):24. PMID- 12193647 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041117 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase. PG - 11682-7 AB - Escherichia coli RNA polymerase translocates along the DNA discontinuously during the elongation phase of transcription, spending proportionally more time at some template positions, known as pause and arrest sites, than at others. Current models of elongation suggest that the enzyme backtracks at these locations, but the dynamics are unresolved. Here, we study the role of lateral displacement in pausing and arrest by applying force to individually transcribing molecules. We find that an assisting mechanical force does not alter the translocation rate of the enzyme, but does reduce the efficiency of both pausing and arrest. Moreover, arrested molecules cannot be rescued by force, suggesting that arrest occurs by a bipartite mechanism: the enzyme backtracks along the DNA followed by a conformational change of the ternary complex (RNA polymerase, DNA and transcript), which cannot be reversed mechanically. AD - The Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA. FAU - Forde, Nancy R AU - Forde NR FAU - Izhaky, David AU - Izhaky D FAU - Woodcock, Glenna R AU - Woodcock GR FAU - Wuite, Gijs J L AU - Wuite GJ FAU - Bustamante, Carlos AU - Bustamante C LA - eng GR - 5R37GM032543-21/GM/NIGMS PT - Journal Article DEP - 20020822 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - EC 2.7.7.6 (DNA-Directed RNA Polymerases) SB - IM MH - DNA-Directed RNA Polymerases/chemistry/*metabolism MH - Escherichia coli/*enzymology MH - Kinetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Transcription, Genetic EDAT- 2002/08/24 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/22 [aheadofprint] AID - 10.1073/pnas.142417799 [doi] AID - 142417799 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11682-7. Epub 2002 Aug 22. PMID- 12193650 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041117 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - Sleep forms memory for finger skills. PG - 11987-91 AB - Practicing a motor skill triggers a process of memory consolidation that continues for hours after practice has ended, and becomes manifest in an improved skill at later testing. We used a sequential motor task (finger-to-thumb opposition task) to show that, in humans, the formation of motor skill memories essentially benefits from sleep. Independent of whether placed during daytime or nighttime, sleep after practice enhanced speed of sequence performance on average by 33.5% and reduced error rate by 30.1% as compared with corresponding intervals of wakefulness. The effect of sleep after learning proved to be stable when retesting was postponed for another night, to exclude effects of sleep loss and to assure that all subjects had sufficient sleep before retrieval testing. Also, the consolidating effect of sleep was specific for the motor sequence learned. It did not generalize to a similar sequence containing identical movement segments in a different order. Retention periods of wakefulness improved performance only moderately and only if placed during daytime. The observations demonstrate a critical role of sleep for storing and optimizing motor skills. AD - Department of Neuroendocrinology, University of Lubeck, D-23538 Lubeck, Germany. FAU - Fischer, Stefan AU - Fischer S FAU - Hallschmid, Manfred AU - Hallschmid M FAU - Elsner, Anna Lisa AU - Elsner AL FAU - Born, Jan AU - Born J LA - eng PT - Journal Article DEP - 20020822 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 SB - IM MH - Adolescent MH - Adult MH - Fingers/*physiology MH - Humans MH - *Memory MH - *Motor Activity MH - Research Support, Non-U.S. Gov't MH - Sleep/*physiology EDAT- 2002/08/24 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/22 [aheadofprint] AID - 10.1073/pnas.182178199 [doi] AID - 182178199 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11987-91. Epub 2002 Aug 22. PMID- 12195021 OWN - NLM STAT- MEDLINE DA - 20020904 DCOM- 20020927 LR - 20041117 PUBM- Print-Electronic IS - 0027-8424 VI - 99 IP - 18 DP - 2002 Sep 3 TI - SAGE Genie: a suite with panoramic view of gene expression. PG - 11547-8 AD - Department of Cancer Biology, 658 MRB II, Vanderbilt-Ingram Cancer Center, Nashville, TN 37232, USA. peng.liang@vanderbilt.edu FAU - Liang, Peng AU - Liang P LA - eng PT - Comment PT - Journal Article DEP - 20020823 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (DNA, Complementary) RN - 0 (RNA, Messenger) SB - IM CON - Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11287-92. PMID: 12119410 MH - Brain/metabolism MH - Brain Neoplasms/genetics MH - DNA, Complementary MH - Expressed Sequence Tags MH - *Gene Expression Profiling MH - Humans MH - RNA, Messenger/genetics EDAT- 2002/08/27 10:00 MHDA- 2002/09/28 04:00 PHST- 2002/08/23 [aheadofprint] AID - 10.1073/pnas.192436299 [doi] AID - 192436299 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2002 Sep 3;99(18):11547-8. Epub 2002 Aug 23. PMID- 12202540 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Laboratory diagnosis of lower respiratory tract infections: controversy and conundrums. PG - 3115-20 AD - University of Utah School of Medicine and Diagnostic Infectious Diseases Laboratories, ARUP Laboratories, Inc., Salt Lake City, Utah, USA. kcarrol7@jhmi.edu FAU - Carroll, Karen C AU - Carroll KC LA - eng PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - J Clin Microbiol JID - 7505564 SB - IM MH - Humans MH - Laboratories MH - *Laboratory Techniques and Procedures MH - Microbiology MH - Respiratory Tract Infections/*diagnosis/*etiology RF - 26 EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3115-20. PMID- 12202541 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041118 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Emergence of Klebsiella pneumoniae isolates producing inducible DHA-1 beta-lactamase in a university hospital in Taiwan. PG - 3121-6 AB - Ten nonrepetitive clinical isolates of Klebsiella pneumoniae exhibiting an unusual inducible beta-lactam resistance phenotype were identified between January 1999 and September 2001 in a university hospital in Taiwan. In the presence of 2 micro g of clavulanic acid, the isolates showed a one to four twofold concentration increase in the MICs of ceftazidime, cefotaxime, and aztreonam but remained susceptible to cefepime (MICs, /=64 micro g/ml). C. dubliniensis was not detected at end of study in any of these patients. Of the remaining 30 patients, eight (27%) demonstrated a replacement of C. albicans by C. dubliniensis when a comparison of isolates obtained at baseline and those from the last culture was done. For the 22 of these 30 patients in whom no switch in species was detected, the fluconazole MICs for initial and end-of-study C. albicans isolates ranged from 0.125 to 2.0 micro g/ml. For the eight patients in whom a switch to C. dubliniensis was detected, the fluconazole MICs for C. dubliniensis isolates at end of study ranged from 0.25 to 64 micro g/ml: the fluconazole MICs for isolates from six patients were 0.25 to 2.0 micro g/ml and those for the other two were 32 and 64 micro g/ml, respectively. In conclusion, a considerable number of patients initially infected with C. albicans strains that failed to develop fluconazole resistance demonstrated a switch to C. dubliniensis. C. dubliniensis in this setting may be underestimated due to lack of identification and may occur due to the impact of fluconazole on the ecology of oral yeast species. AD - Department of Medicine, Division of Infectious Diseases, The University of Texas Health Science Center at San Antonio, 78245, USA. FAU - Martinez, Marcos AU - Martinez M FAU - Lopez-Ribot, Jose L AU - Lopez-Ribot JL FAU - Kirkpatrick, William R AU - Kirkpatrick WR FAU - Coco, Brent J AU - Coco BJ FAU - Bachmann, Stefano P AU - Bachmann SP FAU - Patterson, Thomas F AU - Patterson TF LA - eng GR - 5 R01 DE11381-04A2/DE/NIDCR GR - M01-RR-01346/RR/NCRR GR - R29 AI42401/AI/NIAID PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antifungal Agents) RN - 86386-73-4 (Fluconazole) SB - IM MH - AIDS-Related Opportunistic Infections/*drug therapy/microbiology MH - Antifungal Agents/pharmacology/*therapeutic use MH - Candida/*classification/drug effects/genetics/isolation & purification MH - Candida albicans/*classification/drug effects/genetics/isolation & purification MH - Candidiasis, Oral/*drug therapy/microbiology MH - DNA Fingerprinting MH - Drug Resistance, Fungal MH - Fluconazole/pharmacology/*therapeutic use MH - HIV Infections/complications MH - Humans MH - Karyotyping/methods MH - Microbial Sensitivity Tests MH - Mycological Typing Techniques MH - Oropharynx/microbiology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3135-9. PMID- 12202544 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Weak association between SEN virus viremia and liver disease. PG - 3140-5 AB - Recently, a novel DNA virus designated SEN virus (SEN-V), which is thought to be related to posttransfusion hepatitis, was discovered. The aim of the present study was to clarify the relationship between SEN-V infection and the development of liver disease. We examined SEN-V from the sera of 21 patients with non-B, non-C hepatocellular carcinoma (HCC) and 13 patients with non-B, non-C chronic liver disease (CLD) without HCC who were admitted to our hospital between 1995 and 1997. Thirty-two patients without liver disease served as controls and were also examined for SEN-V. SEN-V DNA was detected by the nested PCR method after extraction of DNA from serum. SEN-V DNA was detected in 74% (25 of 34) of patients with CLD with or without HCC who were negative for both hepatitis B virus surface antigen and anti-hepatitis C virus antibody. SEN-V DNA was detected in 69% (9 of 13) of CLD patients without HCC and in 76% (16 of 21) of HCC patients. The prevalence of SEN-V was no higher in patients with liver disease than in patients without liver disease (24 of 32; 75%). There were no significant differences in age, sex, liver function, history of blood transfusion, or amount of alcohol intake between SEN-V-positive and SEN-V-negative CLD and HCC patients. Genetic analysis suggested that SEN-V is closely related to the TT virus family. SEN-V was detected at almost the same frequency in patients with and without liver disease. SEN-V does not seem to contribute either to the pathogenesis of liver disease or to the development of HCC from chronic liver disease. AD - Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. ch2h-ysd@asahi-net.or.jp FAU - Yoshida, Hideo AU - Yoshida H FAU - Kato, Naoya AU - Kato N FAU - Shiratori, Yasushi AU - Shiratori Y FAU - Shao, Runxuan AU - Shao R FAU - Wang, Yue AU - Wang Y FAU - Shiina, Shuichiro AU - Shiina S FAU - Omata, Masao AU - Omata M LA - eng SI - GENBANK/AB059353 SI - GENBANK/AB059532 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Viral) SB - IM MH - Aged MH - Base Sequence MH - Carcinoma, Hepatocellular/*epidemiology/virology MH - DNA Virus Infections/*epidemiology/virology MH - DNA Viruses/genetics/*isolation & purification MH - DNA, Viral/blood MH - Female MH - Hepatitis, Viral, Human/virology MH - Humans MH - Incidence MH - Liver Diseases/*epidemiology/virology MH - Male MH - Middle Aged MH - Molecular Sequence Data MH - Polymerase Chain Reaction/methods MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - Viremia/*epidemiology/virology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3140-5. PMID- 12202545 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Cavitary pneumonia in an AIDS patient caused by an unusual Bordetella bronchiseptica variant producing reduced amounts of pertactin and other major antigens. PG - 3146-54 AB - Although Bordetella bronchiseptica can infect and colonize immunocompromised humans, its role as a primary pathogen in pneumonia and other respiratory processes affecting those patients remains controversial. A case of cavitary pneumonia caused by B. bronchiseptica in an AIDS patient is presented, and the basis of the seemingly enhanced pathogenic potential of this isolate (designated 814) is investigated. B. bronchiseptica was the only microorganism recovered from sputum, bronchoalveolar lavage fluid, and samples taken through the protected brush catheter. Unlike previous work reporting the involvement of B. bronchiseptica in cases of pneumonia, antibiotic treatment selected on the basis of in vitro antibacterial activity resulted in clearance of the infection and resolution of the pulmonary infiltrate. Although isolate 814 produced reduced amounts of several major antigens including at least one Bvg-activated factor (pertactin), the molecular basis of this deficiency was found to be BvgAS independent since the defect persisted after the bvgAS locus of isolate 814 was replaced with a wild-type bvgAS allele. Despite its prominent phenotype, isolate 814 displayed only a modest yet a significant deficiency in its ability to colonize the respiratory tracts of immunocompetent rats at an early time point. Interestingly, the antibody response elicited by isolate 814 in these animals was almost undetectable. We propose that isolate 814 may be more virulent in immunocompromised patients due, at least in part, to its innate ability to produce low amounts of immunogenic factors which may be required at only normal levels for the interaction of this pathogen with its immunocompetent natural hosts. AD - Departamento de Microbiologia y Parasitologia, Universidad de Navarra, 31080 Pamplona, Spain. FAU - Lorenzo-Pajuelo, Benito AU - Lorenzo-Pajuelo B FAU - Villanueva, Jose Luis AU - Villanueva JL FAU - Rodriguez-Cuesta, Juan AU - Rodriguez-Cuesta J FAU - Vergara-Irigaray, Nuria AU - Vergara-Irigaray N FAU - Bernabeu-Wittel, Maximo AU - Bernabeu-Wittel M FAU - Garcia-Curiel, Andres AU - Garcia-Curiel A FAU - Martinez de Tejada, Guillermo AU - Martinez de Tejada G LA - eng PT - Case Reports PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antigens, Bacterial) RN - 0 (Bacterial Outer Membrane Proteins) RN - 0 (Virulence Factors, Bordetella) RN - 0 (pertactin) SB - IM MH - AIDS-Related Opportunistic Infections/*microbiology/physiopathology/radiography MH - Adult MH - Animals MH - Antigens, Bacterial/genetics/metabolism MH - Bacterial Outer Membrane Proteins/genetics/metabolism MH - Bordetella Infections/*microbiology/physiopathology/radiography MH - Bordetella bronchiseptica/classification/genetics/*isolation & purification/pathogenicity MH - Female MH - Humans MH - Male MH - Phenotype MH - Pneumonia, Bacterial/*microbiology/physiopathology/radiography MH - Rats MH - Rats, Wistar MH - Research Support, Non-U.S. Gov't MH - *Variation (Genetics) MH - Virulence MH - Virulence Factors, Bordetella/genetics/metabolism EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3146-54. PMID- 12202546 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Detection of human papillomavirus DNA in urine specimens from human immunodeficiency virus-positive women. PG - 3155-61 AB - Human immunodeficiency virus (HIV)-positive women may represent one of the fastest-growing populations at risk for acquiring cervical cancer and thus require frequent screening. The purpose of the present studies was to validate a PCR-based urine assay by comparing detection and genotyping of human papillomavirus (HPV) DNA in urine samples and matching cervical swab specimens of HIV-positive women. Despite a difference in amplifiability, the prevalence of any HPV genotype (58% for the cervical swab specimens and 48% for the urine specimens) was not significantly different in this population. The levels of concordance were 70, 71, and 78% for detection of any HPV type, any high-risk HPV type, or any low-risk HPV type in the two specimen types, respectively. While instances of discordant detection were greater for the cervical swab specimens than for the urine specimens, this was not statistically significant. The distributions of HPV genotypes were similar in the cervix and the urine for the majority of types examined. Importantly, detection of HPV DNA in urine was associated with an abnormal Papanicolaou smear to the same extent that detection of HPV DNA in a cervical swab specimen was. These data provide preliminary support for the proposal to use urine testing as a primary or secondary screening tool for cervical cancer in HIV-positive women or as an epidemiological tool. Additional studies with larger sample sizes must be conducted in order to further verify these findings. AD - Department of Microbiology. Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112-2822, USA. jbrink@lsuhsc.edu FAU - Brinkman, Joeli A AU - Brinkman JA FAU - Jones, W Elizabeth AU - Jones WE FAU - Gaffga, Ann M AU - Gaffga AM FAU - Sanders, Jonathan A AU - Sanders JA FAU - Chaturvedi, Anil K AU - Chaturvedi AK FAU - Slavinsky III, Joseph AU - Slavinsky III J FAU - Clayton, John L AU - Clayton JL FAU - Dumestre, Jeanne AU - Dumestre J FAU - Hagensee, Michael E AU - Hagensee ME LA - eng GR - CA86378-01/CA/NCI PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Viral) SB - IM MH - Adult MH - Cervical Intraepithelial Neoplasia MH - Cervix Neoplasms/virology MH - Cervix Uteri/virology MH - Comparative Study MH - DNA, Viral/*urine MH - Female MH - *HIV Seropositivity MH - Humans MH - Papillomavirus, Human/*classification/genetics/*isolation & purification MH - Papovaviridae Infections/*virology MH - Polymerase Chain Reaction/methods MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Specimen Handling MH - Tumor Virus Infections/*virology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3155-61. PMID- 12202547 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Reactive nitrogen intermediates have a bacteriostatic effect on Mycobacterium tuberculosis in vitro. PG - 3162-6 AB - Susceptibility of six isolates of Mycobacterium tuberculosis (CB3.3, CDC1551, RJ2E, C.C.13, H37Rv, and H37Ra) and two isolates of Mycobacterium bovis (Ravenel and BCG) to reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) was determined by standard in vitro survival assays. After 21 days of incubation, the survival of most strains exposed to either acidified sodium nitrite (ASN) or hydrogen peroxide (H(2)O(2)) was significantly lower than the same strains unexposed to these RNI or ROI products. However, after 50 days of incubation, these differences in susceptibility became less apparent for strains exposed to ASN but not for strains exposed to H(2)O(2). The recovery of these strains after exposure to RNI suggests that the effect of RNI on M. tuberculosis is bacteriostatic. The in vitro concentrations of ROI and RNI used in these assays were higher than those expected in vivo. These observations suggest that, in vivo, RNI expression at physiologically achievable concentrations may keep M. tuberculosis from proliferating but that removal of RNI may allow the organisms to proliferate. Furthermore, the ability of some M. tuberculosis strains to cause rapidly progressive disease may relate to their intrinsic levels of RNI and ROI resistance. AD - School of Public Health, Division of Infectious Diseases, University of California at Berkeley, Berkeley, California 94720, USA. FAU - Firmani, Marcia A AU - Firmani MA FAU - Riley, Lee W AU - Riley LW LA - eng GR - F31 DA05874/DA/NIDA GR - HL51967/HL/NHLBI PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Reactive Nitrogen Species) RN - 0 (Reactive Oxygen Species) RN - 7632-00-0 (Sodium Nitrite) RN - 7722-84-1 (Hydrogen Peroxide) SB - IM MH - Humans MH - Hydrogen Peroxide/pharmacology MH - Microbial Sensitivity Tests/methods MH - Mycobacterium tuberculosis/*drug effects/growth & development MH - Reactive Nitrogen Species/*pharmacology MH - Reactive Oxygen Species/pharmacology MH - Research Support, U.S. Gov't, P.H.S. MH - Sodium Nitrite/*pharmacology MH - Tuberculosis, Pulmonary/microbiology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3162-6. PMID- 12202548 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Detection of simian immunodeficiency virus in diverse species and of human immunodeficiency virus Type 2 by using consensus primers within the pol region. PG - 3167-71 AB - Human immunodeficiency virus type 2 (HIV-2) is the result of cross-species transmission of simian immunodeficiency virus (SIV) from sooty mangabey monkeys to humans. Primer pairs (intHIV-2/SIV) based on a region of integrase that has considerable homology across HIV-2 and SIV lineages were designed to develop a broadly cross-reactive molecular assay to detect lentivirus infection in primates. The intHIV-2/SIV primers detect HIV-2 and simian viruses SIVcpz, SIVsmm, SIVsyk, SIVagm, and SIVmnd. The primers are also capable of amplifying some HIV-1 strains. Additionally, sequences from the integrase amplicons were of sufficient genetic diversity to permit not only phylogenetic clustering of all simian viruses to their respective lineages but also HIV type and group classification. Thus, the primers described here provide a method to detect primate lentiviruses from diverse species of nonhuman primates, as well as from persons infected with HIV-1 and HIV-2. AD - HIV Immunology and Diagnostics Branch, Division of AIDS, Sexually Transmitted Diseases, and Tuberculosis Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA. FAU - Masciotra, Silvina AU - Masciotra S FAU - Yang, Chunfu AU - Yang C FAU - Pieniazek, Danuta AU - Pieniazek D FAU - Thomas, Chanda AU - Thomas C FAU - Owen, Sherry M AU - Owen SM FAU - McClure, Harold M AU - McClure HM FAU - Lal, Renu B AU - Lal RB LA - eng SI - GENBANK/AF395546 SI - GENBANK/AF395547 SI - GENBANK/AF395548 SI - GENBANK/AF395549 SI - GENBANK/AF395550 SI - GENBANK/AF395551 SI - GENBANK/AF395552 SI - GENBANK/AF395553 SI - GENBANK/AF395554 SI - GENBANK/AF395555 SI - GENBANK/AF395556 SI - GENBANK/AF395557 SI - GENBANK/AF395558 SI - GENBANK/AF395559 SI - GENBANK/AF395560 SI - GENBANK/AF395561 SI - GENBANK/AF395562 SI - GENBANK/AF395563 SI - GENBANK/AF395564 SI - GENBANK/AF395565 SI - GENBANK/AF395566 SI - GENBANK/AF395567 SI - GENBANK/AF395568 SI - GENBANK/AF395569 SI - GENBANK/AF395570 SI - GENBANK/AF395571 GR - RR0016/RR/NCRR PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA Primers) RN - 0 (DNA, Viral) RN - 0 (Gene Products, pol) RN - EC 2.7.7.- (HIV Integrase) SB - IM MH - Animals MH - Cercopithecinae MH - DNA Primers MH - DNA, Viral/analysis MH - Gene Products, pol/*genetics MH - HIV Infections/virology MH - HIV Integrase/genetics MH - HIV-1/classification/genetics/isolation & purification MH - HIV-2/classification/genetics/*isolation & purification MH - Humans MH - Molecular Sequence Data MH - Monkey Diseases/virology MH - Phylogeny MH - Polymerase Chain Reaction MH - Research Support, U.S. Gov't, P.H.S. MH - SIV/classification/genetics/*isolation & purification MH - Sequence Analysis, DNA MH - Simian Acquired Immunodeficiency Syndrome/virology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3167-71. PMID- 12202549 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - groESL sequence determination, phylogenetic analysis, and species differentiation for viridans group streptococci. PG - 3172-8 AB - The full-length sequences of the groESL genes (also known as cpn10/60) of Streptococcus anginosus, Streptococcus constellatus, Streptococcus gordonii, and Streptococcus sanguis and the near full-length sequence of the groESL genes of Streptococcus intermedius, Streptococcus bovis, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, and Streptococcus salivarius were determined. The lengths of the groES genes from the 10 species listed above ranged from 282 to 288 bp, and the full-length sequences of groEL determined for 4 species (S. anginosus, S. constellatus, S. gordonii, and S. sanguis) revealed that each was 1,623 bp. The intergenic region (spacer) between the groES and groEL genes varies in size (15 to 111 bp) and sequence between species. The variation of the groES sequences among the species tested was greater (62.1 to 95.1% nucleotide sequence identities) than that of the groEL sequences (77.2 to 95.2% nucleotide sequence identities). Phylogenetic analysis of the groES and groEL genes yielded evolutionary trees similar to the tree constructed by use of the 16S rRNA gene. The intraspecies variation of the spacer was minimal for clinical isolates of some species. The groESL sequence data provide an additional parameter for identification of viridans group streptococcal species. AD - School of Medical Technology Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan. ljteng@ha.mc.ntu.edu.tw FAU - Teng, Lee-Jene AU - Teng LJ FAU - Hsueh, Po-Ren AU - Hsueh PR FAU - Tsai, Jui-Chang AU - Tsai JC FAU - Chen, Pin-Wun AU - Chen PW FAU - Hsu, Jia-Chuan AU - Hsu JC FAU - Lai, Hsin-Chih AU - Lai HC FAU - Lee, Chun-Nan AU - Lee CN FAU - Ho, Shen-Wu AU - Ho SW LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Bacterial Proteins) RN - 0 (Chaperonins) RN - 0 (GroESL protein, Bacteria) SB - IM MH - Bacterial Proteins/*genetics MH - Bacterial Typing Techniques MH - Base Sequence MH - Chaperonins/*genetics MH - Humans MH - Molecular Sequence Data MH - *Phylogeny MH - Polymerase Chain Reaction MH - Research Support, Non-U.S. Gov't MH - *Sequence Analysis, DNA MH - Streptococcal Infections/*microbiology MH - Streptococcus/*classification/genetics MH - Variation (Genetics) EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3172-8. PMID- 12202550 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Development and evaluation of rapid urinary antigen detection tests for diagnosis of penicilliosis marneffei. PG - 3179-83 AB - Penicilliosis, caused by the dimorphic fungus Penicillium marneffei, is an important opportunistic systemic fungal infection affecting immunocompromised individuals living in areas where penicilliosis is endemic. We have demonstrated previously that a urinary enzyme-linked immunosorbent assay (ELISA) with purified rabbit polyclonal antibody against killed whole-fission-form arthroconidia of P. marneffei was specific and highly sensitive for the diagnosis of penicilliosis. In this study, a dot blot ELISA and a latex agglutination (LA) test were developed with the same polyclonal antibody and compared with the ELISA for the detection of P. marneffei urinary antigen. Urine specimens from 37 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects and 248 hospitalized patients without penicilliosis) were tested. Antigen was detected in urine from all 37 (100%) penicilliosis patients by the LA test, 35 (94.6%) penicilliosis patients by the dot blot ELISA, and 36 (97.3%) penicilliosis patients by the ELISA. False-positive results were found by the three assays for 2 (0.7%), 8 (2.7%), and 6 (2%) of 300 controls, respectively. The overall sensitivities of the diagnostic tests were as follows: dot blot ELISA, 94.6%; ELISA, 97.3%; and LA test, 100% (specificities, 97.3, 98, and 99.3%, respectively). The LA test is simple, robust, rapid, and convenient and should prove to be an important addition to the existing diagnostic tests for penicilliosis. AD - Clinical Infectious Diseases Research Unit, Department of Clinical Tropical Medicine, Faculty of Tropical Medicine Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. FAU - Desakorn, Varunee AU - Desakorn V FAU - Simpson, Andrew J H AU - Simpson AJ FAU - Wuthiekanun, Vanaporn AU - Wuthiekanun V FAU - Sahassananda, Duangjai AU - Sahassananda D FAU - Rajanuwong, Adul AU - Rajanuwong A FAU - Pitisuttithum, Punnee AU - Pitisuttithum P FAU - Howe, Paul A AU - Howe PA FAU - Smith, Michael D AU - Smith MD FAU - White, Nicholas J AU - White NJ LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antigens, Fungal) SB - IM MH - AIDS-Related Opportunistic Infections/*diagnosis/microbiology MH - Antigens, Fungal/*urine MH - Enzyme-Linked Immunosorbent Assay/methods MH - Humans MH - Latex Fixation Tests MH - Mycoses/*diagnosis/microbiology MH - Penicillium/*isolation & purification MH - Research Support, Non-U.S. Gov't MH - Sensitivity and Specificity MH - Time Factors EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3179-83. PMID- 12202551 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20031114 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Molecular characterization of multiresistant d-tartrate-positive Salmonella enterica serovar paratyphi B isolates. PG - 3184-91 AB - Since 1996, the National Salmonella Reference Laboratory of Germany has received an increasing number of Salmonella enterica subsp. enterica serovar Paratyphi B isolates. Nearly all of these belonged to the dextrorotatory tartrate-positive variant (S. enterica subsp. enterica serovar Paratyphi B dT(+)), formerly called S. enterica subsp. enterica serovar Java. A total of 55 selected contemporary and older S. enterica subsp. enterica serovar Paratyphi B dT(+) isolates were analyzed by plasmid profiling, antimicrobial resistance testing, pulsed-field gel electrophoresis, IS200 profiling, and PCR-based detection of integrons. The results showed a high genetic heterogeneity among 10 old strains obtained from 1960 to 1993. In the following years, however, new distinct multiresistant S. enterica subsp. enterica serovar Paratyphi B dT(+) clones emerged, and one clonal lineage successfully displaced the older ones. Since 1994, 88% of the isolates investigated were multiple drug resistant. Today, a particular clone predominates in some German poultry production lines, poultry products, and various other sources. It was also detected in contemporary isolates from two neighboring countries as well. AD - National Salmonella Reference Laboratory, Federal Institute for Health Protection of Consumers and Veterinary Medicine, 12277 Berlin, Germany. FAU - Miko, Angelika AU - Miko A FAU - Guerra, Beatriz AU - Guerra B FAU - Schroeter, Andreas AU - Schroeter A FAU - Dorn, Christina AU - Dorn C FAU - Helmuth, Reiner AU - Helmuth R LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Anti-Bacterial Agents) RN - 0 (DNA Transposable Elements) RN - 0 (Plasmids) RN - 0 (Tartrates) RN - EC 2.7.7.- (Integrases) SB - IM MH - Animals MH - Anti-Bacterial Agents/pharmacology MH - *Bacterial Typing Techniques MH - DNA Transposable Elements MH - *Drug Resistance, Multiple, Bacterial MH - Electrophoresis, Gel, Pulsed-Field MH - Germany MH - Integrases/genetics MH - Microbial Sensitivity Tests MH - Plasmids/genetics MH - Polymerase Chain Reaction MH - Poultry MH - Poultry Diseases/microbiology MH - Poultry Products/microbiology MH - Salmonella Infections, Animal/microbiology MH - Salmonella enterica/*classification/drug effects/*genetics/isolation & purification MH - Serotyping MH - Tartrates/*metabolism EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3184-91. PMID- 12202552 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20031114 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Identification of Anaplasma phagocytophila (formerly Ehrlichia phagocytophila) variants in blood from sheep in Norway. PG - 3192-7 AB - A total of 41 blood samples were collected from 40 Anaplasma phagocytophila-infected sheep in 11 sheep flocks from four different counties of southern Norway. The presence and nature of the Anaplasma species were identified by microscopic detection of morulae, PCR, reverse line blot hybridization, and 16S rRNA gene sequencing. A. phagocytophila was identified in all of the samples, and sequencing of the 16S rRNA gene revealed the presence of four variants of A. phagocytophila. Two of these variants have been described before, but two were newly identified 16S rRNA variants of this species. A. phagocytophila variant 1 was found in nine flocks, A. phagocytophila variant 2 was found in four flocks, the A. phagocytophila prototype was found in two flocks, and A. phagocytophila variant 5 was found in one flock. In two flocks, some sheep were infected with A. phagocytophila variant 1, whereas others were infected with A. phagocytophila variant 2, and in three animals a double infection with two variants was registered. Analyses of the blood samples revealed that blood from sheep infected with A. phagocytophila variant 2 contained nearly twice as many neutrophils and eight times as many Anaplasma-infected neutrophils as blood from sheep infected with the A. phagocytophila variant 1. Furthermore, only 43% of the A. phagocytophila variant 2-infected sheep displayed antibody responses in an immune fluorescence assay, whereas 93% of the sheep with the A. phagocytophila variant 1-infected sheep were seropositive. AD - Department of Sheep and Goat Research, Norwegian School of Veterinary Science, Sandnes, Norway. snoore.Stuen@veths.no FAU - Stuen, Snorre AU - Stuen S FAU - Van De Pol, Ingrid AU - Van De Pol I FAU - Bergstrom, Karin AU - Bergstrom K FAU - Schouls, Leo M AU - Schouls LM LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Culture Media) RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal, 16S) SB - IM MH - Anaplasma/*classification/genetics/isolation & purification MH - Anaplasmosis/*microbiology MH - Animals MH - Bacterial Typing Techniques MH - Base Sequence MH - Blood/*microbiology MH - Culture Media MH - DNA, Ribosomal/analysis MH - Molecular Sequence Data MH - Norway MH - Nucleic Acid Hybridization/methods MH - Polymerase Chain Reaction MH - RNA, Ribosomal, 16S/genetics MH - Sequence Analysis, DNA MH - Sheep MH - Sheep Diseases/*microbiology MH - *Variation (Genetics) EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3192-7. PMID- 12202553 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Comparison of rapid, automated ribotyping and DNA macrorestriction analysis of Burkholderia pseudomallei. PG - 3198-203 AB - An automated ribotyping device (RiboPrinter) was used to determine the ribotypes of a collection of Burkholderia pseudomallei isolates. In a preliminary evaluation with the restriction enzymes BamHI and EcoRI, the protocol with EcoRI was more discriminating. The reproducibilities of the ribotypes obtained with EcoRI (EcoRI ribotypes) were determined by testing three levels of bacterial loads. The performance of the manufacturer's software was assessed by comparing the machine-optimized ribotypes with the type determined from the original gel image analyzed with Bionumerics software. The library of B. pseudomallei EcoRI ribotypes was then compared with the ribotypes obtained by DNA macrorestriction analysis of XbaI digests by pulsed-field gel electrophoresis. The typeability of B. pseudomallei by EcoRI ribotyping was 100%, and the discrimination index was 0.94. The slightly greater discrimination provided by DNA macrorestriction analysis (0.96) was achieved at the expense of a significantly longer processing time of 6 days, although the method was only half the cost of automated ribotyping. Typeability by macrorestriction analysis was lower (97%) unless a thiourea step was added to neutralize the action of Tris-dependent endonucleases. The digital record of B. pseudomallei isolates analyzed thus far provides a useful resource for future epidemiological studies and will help shorten the response time in the event of a further melioidosis outbreak or the deliberate release of B. pseudomallei as a biohazard. AD - Division of Microbiology and Infectious Diseases, Western Australian Centre for Pathology, Nedlands, Australia. tim.inglis@health.wa.gov.au FAU - Inglis, Timothy J J AU - Inglis TJ FAU - O'Reilly, Lyn AU - O'Reilly L FAU - Foster, Niki AU - Foster N FAU - Clair, Adele AU - Clair A FAU - Sampson, Judy AU - Sampson J LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - EC 3.1.21.- (Deoxyribonuclease BamHI) RN - EC 3.1.21.- (Deoxyribonuclease EcoRI) SB - IM MH - Automation MH - Bacterial Typing Techniques MH - Burkholderia pseudomallei/*classification/genetics MH - Comparative Study MH - Deoxyribonuclease BamHI MH - Deoxyribonuclease EcoRI MH - Electrophoresis, Gel, Pulsed-Field MH - Humans MH - Melioidosis/*microbiology MH - Research Support, Non-U.S. Gov't MH - Restriction Mapping/*methods MH - *Ribotyping EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3198-203. PMID- 12202554 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Testing conditions for determination of minimum fungicidal concentrations of new and established antifungal agents for Aspergillus spp.: NCCLS collaborative study. PG - 3204-8 AB - Standard conditions are not available for evaluating the minimum fungicidal concentrations (MFCs) of antifungal agents. This multicenter collaborative study investigated the reproducibility in three laboratories of itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B MFCs for 15 selected isolates of Aspergillus spp. After MIC determinations for the 15 isolates in each center by the NCCLS M38-A broth microdilution method with four media, standard RPMI 1640 (RPMI), RPMI with 2% dextrose, antibiotic medium 3 (M3), and M3 with 2% dextrose, MFCs were determined for each isolate-medium-drug combination. MFCs were defined as the lowest drug dilutions that yielded <3 colonies (approximately 99 to 99.5% killing activity). The highest reproducibility (96 to 100%) was for amphotericin B MFCs with the four media. Although reproducibility was more variable and medium dependent for the azoles (91 to 98%), agreement was good to excellent for itraconazole, ravuconazole, and voriconazole MFCs with RPMI and M3 (93 to 98%). For posaconazole, the agreement was higher with M3 media (91 to 96%) than with RPMI media (91%). These data extend the refinement of testing guidelines for susceptibility testing of Aspergillus spp. and warrant consideration for introduction into future versions of the M38 document. The role of the MFC under these standardized testing conditions as a predictor of clinical outcome needs to be established in clinical trials. AD - Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia 23298-0049, USA. avingrof@hsc.vcu.edu FAU - Espinel-Ingroff, A AU - Espinel-Ingroff A FAU - Fothergill, A AU - Fothergill A FAU - Peter, J AU - Peter J FAU - Rinaldi, M G AU - Rinaldi MG FAU - Walsh, T J AU - Walsh TJ LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antifungal Agents) RN - 0 (Culture Media) SB - IM MH - Antifungal Agents/*pharmacology MH - Aspergillus/*classification/*drug effects MH - Culture Media MH - Drug Resistance, Fungal MH - Humans MH - Microbial Sensitivity Tests/methods/*standards MH - Reference Standards MH - Reproducibility of Results MH - Research Support, Non-U.S. Gov't EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3204-8. PMID- 12202555 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Polyphyletic strains of hepatitis E virus are responsible for sporadic cases of acute hepatitis in Japan. PG - 3209-18 AB - Among 87 patients who were previously treated for acute hepatitis of unknown etiology between 1992 and 2001 at five hospitals in Japan, 11 (13%) patients were positive for immunoglobulin M-class antibodies to hepatitis E virus (HEV) by enzyme immunoassay and had detectable HEV RNA by reverse transcription-PCR with two independent sets of primers derived from well-conserved genomic areas in open reading frames 1 and 2. Clinical HEV infection was significantly associated with male sex (9 of 11 versus 29 of 76 patients [P < 0.01]) and older age (52 +/- 11 [mean +/- standard deviation] versus 41 +/- 17 years [P < 0.05]), and its prevalence differed by geographic region (6 to 25%), with a higher rate in the northern part of Japan. At admission, the 11 patients with HEV-associated hepatitis had elevated alanine aminotransferase levels of 914 to 4,850 IU/liter, and all but 1 had elevated bilirubin levels of 1.5 to 24.0 mg/dl. The 11 HEV isolates were of genotype III or IV and were segregated into three groups with intergroup nucleotide differences of 9.5 to 22.0%. Phylogenetic analysis revealed that four isolates of genotype III were closely related to a Japanese isolate, while the other four isolates of the same genotype were nearest those from the United States. The remaining three isolates were close to known isolates of genotype IV in China and Taiwan but shared less than 88% identity with them. These results indicate that multiple genotypes of HEV cocirculate in Japan and contribute to the development of sporadic acute hepatitis, with the prevalence differing by age, sex, and geographic region. AD - Department of Internal Medicine, Kin-ikyo Chuo Hospital, Hokkaido 007-0870, Japan. FAU - Mizuo, Hitoshi AU - Mizuo H FAU - Suzuki, Kazuyuki AU - Suzuki K FAU - Takikawa, Yasuhiro AU - Takikawa Y FAU - Sugai, Yoshiki AU - Sugai Y FAU - Tokita, Hajime AU - Tokita H FAU - Akahane, Yoshihiro AU - Akahane Y FAU - Itoh, Keiichi AU - Itoh K FAU - Gotanda, Yuhko AU - Gotanda Y FAU - Takahashi, Masaharu AU - Takahashi M FAU - Nishizawa, Tsutomu AU - Nishizawa T FAU - Okamoto, Hiroaki AU - Okamoto H LA - eng SI - GENBANK/AB082545 SI - GENBANK/AB082546 SI - GENBANK/AB082547 SI - GENBANK/AB082548 SI - GENBANK/AB082549 SI - GENBANK/AB082550 SI - GENBANK/AB082551 SI - GENBANK/AB082552 SI - GENBANK/AB082553 SI - GENBANK/AB082554 SI - GENBANK/AB082555 SI - GENBANK/AB082556 SI - GENBANK/AB082557 SI - GENBANK/AB082558 SI - GENBANK/AB082559 SI - GENBANK/AB082560 SI - GENBANK/AB082561 SI - GENBANK/AB082562 SI - GENBANK/AB082563 SI - GENBANK/AB082564 SI - GENBANK/AB082565 SI - GENBANK/AB082566 SI - GENBANK/AB082567 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Viral) RN - 0 (Hepatitis Antibodies) RN - 0 (Immunoglobulin G) RN - 0 (Immunoglobulin M) RN - 0 (RNA, Viral) SB - IM MH - Acute Disease MH - Adult MH - Aged MH - DNA, Viral/analysis MH - Female MH - Hepatitis Antibodies/blood MH - Hepatitis E/*epidemiology/*virology MH - Hepatitis E virus/*classification/genetics/*immunology/isolation & purification MH - Hepatitis, Viral, Human/epidemiology/virology MH - Humans MH - Immunoglobulin G/blood MH - Immunoglobulin M/blood MH - Japan/epidemiology MH - Male MH - Middle Aged MH - Molecular Sequence Data MH - Phylogeny MH - Prevalence MH - RNA, Viral/blood MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - *Variation (Genetics) EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3209-18. PMID- 12202556 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Comparison of C(18)-carboxypropylbetaine and standard N-acetyl-L-cysteine-NaOH processing of respiratory specimens for increasing tuberculosis smear sensitivity in Brazil. PG - 3219-22 AB - Techniques to improve the sensitivity of smear microscopy would facilitate early tuberculosis (TB) diagnosis and disease control, especially in low-income countries where the positive predictive value is high. C(18)-carboxypropylbetaine (CB-18) is a zwitterionic detergent that helps to compensate for the innate buoyancy of mycobacteria, potentially enhancing recovery by centrifugation. Previous data suggest that CB-18 may increase the sensitivity of smear, culture, and molecular amplification diagnostic testing. The goal of the present study was to evaluate if the sensitivity of the smear technique using light microscopy could be improved by treating respiratory samples with CB-18. In the first phase, respiratory specimens were collected consecutively from patients with suspected pulmonary tuberculosis in a tertiary-care hospital in Rio de Janeiro, Brazil (236 specimens were analyzed). After protocol modifications, another 120 respiratory specimens were evaluated. The standard technique was N-acetyl-L-cysteine with sodium hydroxide (NALC-NaOH) treatment, smear concentration with centrifugation, and Ziehl-Neelsen staining. Culture on Lowenstein-Jensen slants was performed on all specimens for use as the "gold standard." No specimens from patients undergoing active TB treatment were included. The initial protocol for CB-18 processing resulted in a sensitivity of 59.6% and specificity of 96.8% compared to standard processing with a sensitivity of 66.0% and specificity of 96.8%. Using the modified protocol, the sensitivity of CB-18 increased to 71.4% with a specificity of 97.0% versus standard processing with a sensitivity of 61.9% and a specificity of 99.0%. The diagnostic yield of acid-fast bacillus smear with CB-18 in the absence of fluorescence microscopy and PCR compared to standard processing with NALC-NaOH was not significantly different, although the power to detect a difference by the modified assay was low. AD - Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA. FAU - Scott, Cherise P AU - Scott CP FAU - Dos Anjos Filho, Luciano AU - Dos Anjos Filho L FAU - De Queiroz Mello, Fernanda Carvalho AU - De Queiroz Mello FC FAU - Thornton, Charles G AU - Thornton CG FAU - Bishai, William R AU - Bishai WR FAU - Fonseca, Leila S AU - Fonseca LS FAU - Kritski, AfrAnio L AU - Kritski AL FAU - Chaisson, Richard E AU - Chaisson RE FAU - Manabe, Yukari C AU - Manabe YC LA - eng GR - 2D43TW000010/TW/FIC GR - AI 45432/AI/NIAID GR - K24 AI 01637/AI/NIAID PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (C(18)-carboxypropylbetaine) RN - 0 (Culture Media) RN - 107-43-7 (Betaine) RN - 1310-73-2 (Sodium Hydroxide) RN - 616-91-1 (Acetylcysteine) SB - IM MH - *Acetylcysteine MH - Bacteriological Techniques MH - *Betaine/*analogs & derivatives MH - Brazil MH - Comparative Study MH - Culture Media MH - Humans MH - Mycobacterium tuberculosis/isolation & purification MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sensitivity and Specificity MH - Sodium Hydroxide MH - Specimen Handling/*methods MH - Sputum/*microbiology MH - Staining and Labeling/methods MH - Tuberculosis, Pulmonary/*diagnosis/microbiology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3219-22. PMID- 12202557 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - PCR-based identification of bacteria associated with endodontic infections. PG - 3223-31 AB - PCR primers that target the bacterial 16S rRNA genes (or the tuf gene for the genus Enterococcus) were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samples from the root canals of 24 teeth with necrotic pulp were included in the study. PCR with universal bacterial primers identified bacterial DNA in 22 specimens; the remaining 2 specimens were from intact teeth that had been traumatized 6 months prior to treatment. PCR with specific primers showed that preoperative symptoms were significantly associated with the presence of Streptococcus spp. (P < 0.001 by chi-square analysis). There was also a nonsignificant trend for symptoms to be associated with Fusobacterium nucleatum and Porphyromonas gingivalis (odds ratio, >2) and for diabetes mellitus to be associated with P. gingivalis and Porphyromonas endodontalis (odds ratio, >2). Cloning and sequencing of the universal PCR product in one specimen revealed the presence of an organism related to the genus Olsenella, which has not previously been described in endodontic infections. AD - Department of Endodontology, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030, U.S.A. fouad@nso.uchc.edu FAU - Fouad, Ashraf F AU - Fouad AF FAU - Barry, Jody AU - Barry J FAU - Caimano, Melissa AU - Caimano M FAU - Clawson, Michael AU - Clawson M FAU - Zhu, Qiang AU - Zhu Q FAU - Carver, Rachaele AU - Carver R FAU - Hazlett, Karsten AU - Hazlett K FAU - Radolf, Justin D AU - Radolf JD LA - eng SI - GENBANK/AF426827 GR - AI-26756/AI/NIAID GR - AI-29735/AI/NIAID GR - M01RR06192/RR/NCRR PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal, 16S) SB - IM MH - Bacteria/*classification/genetics/isolation & purification MH - Bacterial Infections/*microbiology MH - Bacterial Typing Techniques MH - DNA, Ribosomal/analysis MH - Dental Pulp Cavity/*microbiology MH - Dental Pulp Necrosis/*microbiology MH - Diabetes Mellitus/microbiology MH - Molecular Sequence Data MH - Periapical Periodontitis/*microbiology MH - Polymerase Chain Reaction/*methods MH - RNA, Ribosomal, 16S/genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3223-31. PMID- 12202558 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Sensitivity of three urinary antigen tests associated with clinical severity in a large outbreak of Legionnaires' disease in The Netherlands. PG - 3232-6 AB - In 1999 an outbreak involving 188 patients with Legionnaires' disease (LD) occurred among visitors to a flower show in the Netherlands. Two enzyme immunoassays (Binax and Biotest) and one immunochromatographic assay (Binax NOW) were tested, using urine samples from LD patients from the 1999 outbreak. Sensitivity was calculated using positive culture and/or seroconversion as the "gold standard" in outbreak-related patients with radiographically confirmed pneumonia who fulfilled the epidemiological critera. The Binax EIA, Biotest EIA, and Binax NOW assay showed overall sensitivities of 69, 71, and 72%, respectively. When the tests were performed with concentrated urine samples, the overall sensitivities increased to 79, 74, and 81%, respectively. Using multiple logistic regression analysis with backward elimination, a statistically significant association was found between clinical severity and test sensitivity for all tests. For patients with mild LD, the test sensitivities ranged from 40 to 53%, whereas for patients with severe LD who needed immediate special medical care, the sensitivities reached 88 to 100%. These findings have major implications for the diagnostic process in patients with mild pneumonia and suggest that patients with mild pneumonia may go underdiagnosed if urine antigen tests alone are used. AD - Regional Laboratory of Public Health Haarlem, The Netherlands. e.yzerman@streeklabhaarlem.nl FAU - Yzerman, Ed P F AU - Yzerman EP FAU - den Boer, Jeroen W AU - den Boer JW FAU - Lettinga, Kamilla D AU - Lettinga KD FAU - Schellekens, Joop AU - Schellekens J FAU - Dankert, Jacob AU - Dankert J FAU - Peeters, Marcel AU - Peeters M LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antigens, Bacterial) RN - 0 (Reagent Kits, Diagnostic) SB - IM MH - Aged MH - Antigens, Bacterial/*urine MH - Chromatography/methods MH - *Disease Outbreaks MH - Female MH - Humans MH - Immunoenzyme Techniques MH - Legionella pneumophila/*isolation & purification MH - Legionnaires' Disease/diagnosis/*epidemiology/microbiology/physiopathology MH - Male MH - Middle Aged MH - Netherlands/epidemiology MH - Reagent Kits, Diagnostic MH - Sensitivity and Specificity MH - *Severity of Illness Index EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3232-6. PMID- 12202559 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis. PG - 3237-44 AB - Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts. AD - Department of Pediatric Hematology-Oncology, School of Medicine, Cukurova University, 01330 Adana, Turkey. FAU - Tanriverdi, Sultan AU - Tanriverdi S FAU - Tanyeli, Atila AU - Tanyeli A FAU - Baslamisli, Fikri AU - Baslamisli F FAU - Koksal, Fatih AU - Koksal F FAU - Kilinc, Yurdanur AU - Kilinc Y FAU - Feng, Xiaochuan AU - Feng X FAU - Batzer, Glenda AU - Batzer G FAU - Tzipori, Saul AU - Tzipori S FAU - Widmer, Giovanni AU - Widmer G LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Fluorescent Dyes) RN - 0 (Organic Chemicals) RN - 0 (Tubulin) RN - 163795-75-3 (SYBR Green I) SB - IM MH - Animals MH - Cattle MH - Cryptosporidiosis/*parasitology MH - Cryptosporidium parvum/*classification/genetics/growth & development/*isolation & purification MH - Fluorescent Dyes MH - Genotype MH - Humans MH - *Organic Chemicals MH - Polymerase Chain Reaction/*methods MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Sensitivity and Specificity MH - *Temperature MH - Tubulin/genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3237-44. PMID- 12202560 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Dichotomy of glycoprotein g gene in herpes simplex virus type 1 isolates. PG - 3245-51 AB - Herpes simplex virus type 1 (HSV-1) encodes 11 envelope glycoproteins, of which glycoprotein G-1 (gG-1) induces a type-specific antibody response. Variability of the gG-1 gene among wild-type strains may be a factor of importance for a reliable serodiagnosis and typing of HSV-1 isolates. Here, we used a gG-1 type-specific monoclonal antibody (MAb) to screen for mutations in the immunodominant region of this protein in 108 clinical HSV-1 isolates. Of these, 42 isolates showed no reactivity to the anti-gG-1 MAb. One hundred five strains were further examined by DNA sequencing of the middle part of the gG-1 gene, encompassing 106 amino acids including the immunodominant region and epitope of the anti-gG-1 MAb. By phylogenetic comparisons based on the sequence data, we observed two (main) genetic variants of the gG-1 gene among the clinical isolates corresponding to reactivity or nonreactivity to the anti-gG-1 MAb. Furthermore, four strains appeared to be recombinants of the two gG-1 variants. In addition, one strain displayed a gG-1-negative phenotype due to a frameshift mutation, in the form of insertion of a cytosine nucleotide. When immunoglobulin G reactivity to HSV-1 in sera from patients infected with either of the two variants was investigated, no significant differences were found between the two groups, either in a type-common enzyme-linked immunosorbent assay (ELISA) or in a type-specific gG-1 antigen-based ELISA. Despite the here-documented existence of two variants of the gG-1 gene affecting the immunodominant region of the protein, other circumstances, such as early phase of infection, might be sought for explaining the seronegativity to gG-1 commonly found in a proportion of the HSV-1-infected patients. AD - Department of Clinical Virology, Goteborg University, Goteborg, Sweden. elham.rekabdar@microbio.gu.se FAU - Rekabdar, Elham AU - Rekabdar E FAU - Tunback, Petra AU - Tunback P FAU - Liljeqvist, Jan-Ake AU - Liljeqvist JA FAU - Lindh, Magnus AU - Lindh M FAU - Bergstrom, Tomas AU - Bergstrom T LA - eng SI - GENBANK/AF513114 SI - GENBANK/AF513115 SI - GENBANK/AF513116 SI - GENBANK/AF513117 SI - GENBANK/AF513118 SI - GENBANK/AF513119 SI - GENBANK/AF513120 SI - GENBANK/AF513121 SI - GENBANK/AF513122 SI - GENBANK/AF513123 SI - GENBANK/AF513124 SI - GENBANK/AF513125 SI - GENBANK/AF513126 SI - GENBANK/AF513127 SI - GENBANK/AF513128 SI - GENBANK/AF513129 SI - GENBANK/AF513130 SI - GENBANK/AF513131 SI - GENBANK/AF513132 SI - GENBANK/AF513133 SI - GENBANK/AF513134 SI - GENBANK/AF513135 SI - GENBANK/AF513136 SI - GENBANK/AF513137 SI - GENBANK/AF513138 SI - GENBANK/AF513139 SI - GENBANK/AF513140 SI - GENBANK/AF513141 SI - GENBANK/AF513142 SI - GENBANK/AF513143 SI - GENBANK/AF513144 SI - GENBANK/AF513145 SI - GENBANK/AF513146 SI - GENBANK/AF513147 SI - GENBANK/AF513148 SI - GENBANK/AF513149 SI - GENBANK/AF513150 SI - GENBANK/AF513151 SI - GENBANK/AF513152 SI - GENBANK/AF513153 SI - GENBANK/AF513154 SI - GENBANK/AF513155 SI - GENBANK/AF513156 SI - GENBANK/AF513157 SI - GENBANK/AF513158 SI - GENBANK/AF513159 SI - GENBANK/AF513160 SI - GENBANK/AF513161 SI - GENBANK/AF513162 SI - GENBANK/AF513163 SI - GENBANK/AF513164 SI - GENBANK/AF513165 SI - GENBANK/AF513166 SI - GENBANK/AF513167 SI - GENBANK/AF513168 SI - GENBANK/AF513169 SI - GENBANK/AF513170 SI - GENBANK/AF513171 SI - GENBANK/AF513172 SI - GENBANK/AF513173 SI - GENBANK/AF513174 SI - GENBANK/AF513175 SI - GENBANK/AF513176 SI - GENBANK/AF513177 SI - GENBANK/AF513178 SI - GENBANK/AF513179 SI - GENBANK/AF513180 SI - GENBANK/AF513181 SI - GENBANK/AF513182 SI - GENBANK/AF513183 SI - GENBANK/AF513184 SI - GENBANK/AF513185 SI - GENBANK/AF513186 SI - GENBANK/AF513187 SI - GENBANK/AF513188 SI - GENBANK/AF513189 SI - GENBANK/AF513190 SI - GENBANK/AF513191 SI - GENBANK/AF513192 SI - GENBANK/AF513193 SI - GENBANK/AF513194 SI - GENBANK/AF513195 SI - GENBANK/AF513196 SI - GENBANK/AF513197 SI - GENBANK/AF513198 SI - GENBANK/AF513199 SI - GENBANK/AF513200 SI - GENBANK/AF513201 SI - GENBANK/AF513202 SI - GENBANK/AF513203 SI - GENBANK/AF513204 SI - GENBANK/AF513205 SI - GENBANK/AF513206 SI - GENBANK/AF513207 SI - GENBANK/AF513208 SI - GENBANK/AF513209 SI - GENBANK/AF513210 SI - GENBANK/AF513211 SI - GENBANK/AF513212 SI - GENBANK/AF513213 SI - GENBANK/AF513214 SI - GENBANK/AF513215 SI - GENBANK/AF513216 SI - GENBANK/AF513217 SI - GENBANK/AF513218 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antibodies, Monoclonal) RN - 0 (Antibodies, Viral) RN - 0 (Immunodominant Epitopes) RN - 0 (Viral Envelope Proteins) RN - 0 (glycoprotein gG-1, herpes simplex virus type 1) SB - IM MH - Amino Acid Sequence MH - Antibodies, Monoclonal/*immunology MH - Antibodies, Viral/immunology MH - Cell Line MH - Enzyme-Linked Immunosorbent Assay MH - Frameshift Mutation MH - Herpes Simplex/*virology MH - Herpesvirus 1, Human/*classification/*genetics/immunology MH - Humans MH - Immunodominant Epitopes MH - Molecular Sequence Data MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - *Variation (Genetics) MH - Viral Envelope Proteins/chemistry/*genetics/immunology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3245-51. PMID- 12202561 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Detection and characterization of hepatitis C virus RNA in seminal plasma and spermatozoon fractions of semen from patients attempting medically assisted conception. PG - 3252-5 AB - To investigate the risk of transmission of hepatitis C virus (HCV) via semen in assisted reproduction techniques, semen samples from 32 men chronically infected with HCV attending a center for assisted procreation were tested for HCV RNA by a reverse transcription-PCR protocol by using a modified version of the Cobas AMPLICOR HCV assay (version 2.0; Roche Diagnostics). The sensitivity of the test was 40 copies/ml. Four of 32 seminal plasma samples (12.5%) were found to be positive for the presence of HCV RNA. The median HCV load in blood was significantly higher in patients who were found to be positive for the presence of HCV RNA in semen than in those who tested negative (P = 0.02). In one man, seven consecutive seminal plasma samples tested positive for HCV RNA, as did two consecutive motile spermatozoon fractions; the corresponding fractions obtained after migration of the spermatozoa remained negative. Despite the absence of the proven infectivity of virus in semen samples that test positive for HCV RNA, these findings highlight the fact that seminal fluid may exhibit prolonged HCV RNA excretion. The usefulness of HCV RNA detection in both seminal plasma and spermatozoon fractions before the start of a program of medically assisted reproduction in couples in whom the male partner is chronically infected with HCV would need to be evaluated prospectively with a larger population of subjects exhibiting HCV RNA in their semen. AD - Laboratoire de Bacteriologie-Virologie, GIMAP, Faculty of Medicine of Saint-Etienne, France. FAU - Bourlet, Thomas AU - Bourlet T FAU - Levy, Rachel AU - Levy R FAU - Maertens, Anne AU - Maertens A FAU - Tardy, Jean-Claude AU - Tardy JC FAU - Grattard, Florence AU - Grattard F FAU - Cordonier, Helene AU - Cordonier H FAU - Laurent, Jean-Louis AU - Laurent JL FAU - Guerin, Jean-Francois AU - Guerin JF FAU - Pozzetto, Bruno AU - Pozzetto B LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (RNA, Viral) SB - IM MH - Adult MH - Female MH - Genotype MH - Hepacivirus/*classification/genetics/*isolation & purification MH - Hepatitis C, Chronic/*virology MH - Humans MH - Male MH - Middle Aged MH - RNA, Viral/analysis/blood MH - *Reproductive Techniques, Assisted MH - Reverse Transcriptase Polymerase Chain Reaction MH - Semen/*virology MH - Sensitivity and Specificity MH - Spermatozoa/*virology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3252-5. PMID- 12202562 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. PG - 3256-60 AB - A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 10(3) to 10(4) gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI. AD - Southeast Poultry Research Laboratory, USDA Agricultural Research Service, Athens, Georgia 30605, USA. FAU - Spackman, Erica AU - Spackman E FAU - Senne, Dennis A AU - Senne DA FAU - Myers, T J AU - Myers TJ FAU - Bulaga, Leslie L AU - Bulaga LL FAU - Garber, Lindsey P AU - Garber LP FAU - Perdue, Michael L AU - Perdue ML FAU - Lohman, Kenton AU - Lohman K FAU - Daum, Luke T AU - Daum LT FAU - Suarez, David L AU - Suarez DL LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Fluorescent Dyes) RN - 0 (Hemagglutinin Glycoproteins, Influenza Virus) SB - IM MH - Animals MH - Chick Embryo MH - Fluorescent Dyes MH - Hemagglutination Inhibition Tests MH - Hemagglutinin Glycoproteins, Influenza Virus/*genetics MH - Influenza A Virus, Avian/classification/genetics/*isolation & purification MH - Influenza, Avian/*virology MH - Poultry MH - Poultry Diseases/*virology MH - Research Support, U.S. Gov't, Non-P.H.S. MH - *Reverse Transcriptase Polymerase Chain Reaction MH - Sensitivity and Specificity EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3256-60. PMID- 12202563 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Distribution of environmentally regulated genes of Streptococcus suis serotype 2 among S. suis serotypes and other organisms. PG - 3261-8 AB - The occurrence of 36 environmentally regulated genes of Streptococcus suis strain 10 among all 35 S. suis serotypes was determined by using hybridization with the amplified genes as probes. In addition, the distribution of these genes among the virulence phenotypes of serotypes 1 and 2 was assessed. Hybridization was also performed with various other streptococcal species and nonstreptococcal bacterial species which may be present in pigs. Interestingly, probe ivs-25/iri-1, similar to agrA and sapR, hybridized only with S. suis serotype 1 and 2 strains with virulent phenotypes and is therefore suitable as a diagnostic parameter. Only one probe was specific for S. suis. This probe's sequence was identical to the epf gene, a putative virulence factor of S. suis. Probe ivs-31 was similar to a virulence factor of S. suis, namely, a gene encoding a fibronectin- and fibrinogen-binding protein. This probe hybridized only with oral streptococci. Nearly half of the probes (45%) hybridized with the oral streptococci (S. oralis, S. milleri, S. sanguis, S. gordonii, and S. mitis) and with Streptococcus pneumoniae. This indicates a close relationship between S. suis, the oral streptococci, and S. pneumoniae with respect to the selected environmentally regulated genes. One probe only hybridized with gram-negative species and therefore seems to be obtained by S. suis from a gram-negative organism by horizontal transfer. AD - Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 1100 DD, The Netherlands. a.degreeff@id.wag-ur.nl FAU - De Greeff, Astrid AU - De Greeff A FAU - Buys, Herma AU - Buys H FAU - Verhaar, Robin AU - Verhaar R FAU - Van Alphen, Loek AU - Van Alphen L FAU - Smith, Hilde E AU - Smith HE LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Bacterial Proteins) RN - 0 (DNA Probes) SB - IM MH - Animals MH - Bacteria/genetics MH - Bacterial Proteins/*genetics MH - DNA Probes/genetics MH - *Environment MH - *Gene Expression Regulation, Bacterial MH - Humans MH - Serotyping MH - Streptococcal Infections/*microbiology/veterinary MH - Streptococcus suis/classification/genetics/*pathogenicity MH - Swine MH - Swine Diseases/*microbiology MH - Virulence/genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3261-8. PMID- 12202564 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Effects of various test media on the activities of 21 antimicrobial agents against Haemophilus influenzae. PG - 3269-76 AB - As considerable variation in the antimicrobial susceptibility of Haemophilus influenzae has been reported, the effects of various test media on the susceptibility of H. influenzae were studied. MICs were determined by three laboratories for 21 antimicrobial agents against a panel of 100 selected isolates. Testing was performed using a reference NCCLS frozen broth microdilution method with Haemophilus test medium (HTM) broth and dried commercial MIC trays rehydrated with the following media: in-house and commercially prepared HTM broth, Mueller-Hinton broth with 2% lysed horse blood and NAD, IsoSensitest broth with 2% lysed horse blood and NAD, and IsoSensitest broth-based HTM. Overall, all results were very reproducible, with the MIC at which 50% of the isolates tested are inhibited (MIC(50)), MIC(90), and geometric mean MIC being within one doubling dilution by all six methods and at all three testing centers for 15 of the 21 agents tested. Interlaboratory differences were more marked than intralaboratory differences or differences among media. Cefprozil, cefaclor, and trimethoprim-sulfamethoxazole results differed the most, while results for ampicillin, amoxicillin-clavulanic acid, cefdinir, cefixime, ceftriaxone, and clarithromycin were the most reproducible. However, these variations in results caused considerable differences in susceptibility rates for agents for which NCCLS susceptible breakpoints were close to the geometric mean MIC, particularly for cefaclor and cefprozil. This was much less of a problem when pharmacokinetic-pharmacodynamic breakpoints were used. Reproducible susceptibility results were obtained for a wide range of agents against H. influenzae in three laboratories using a variety of media that support the growth of this fastidious species. AD - Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA. mrj6@po.cwru.edu FAU - Jacobs, Michael R AU - Jacobs MR FAU - Bajaksouzian, Saralee AU - Bajaksouzian S FAU - Windau, Anne AU - Windau A FAU - Appelbaum, Peter C AU - Appelbaum PC FAU - Lin, Gengrong AU - Lin G FAU - Felmingham, David AU - Felmingham D FAU - Dencer, Christine AU - Dencer C FAU - Koeth, Laura AU - Koeth L FAU - Singer, Mendel E AU - Singer ME FAU - Good, Caryn E AU - Good CE LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Anti-Bacterial Agents) RN - 0 (Culture Media) SB - IM MH - Anti-Bacterial Agents/pharmacology MH - Comparative Study MH - Culture Media MH - Haemophilus influenzae/*drug effects/growth & development MH - Humans MH - Microbial Sensitivity Tests/methods/standards MH - Reference Standards MH - Reproducibility of Results MH - Research Support, Non-U.S. Gov't EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3269-76. PMID- 12202565 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Detection of Trichomonas vaginalis on modified Columbia agar in the routine laboratory. PG - 3277-80 AB - Broth culture of Trichomonas vaginalis is considered the "gold standard" for the diagnosis of trichomoniasis. Two studies were carried out to evaluate modified Columbia agar (MCA) for the isolation of T. vaginalis from clinical samples. Study I compared isolation on MCA to that on liquid medium with 889 vaginal samples. Out of 63 samples positive for T. vaginalis (7.1% of total), MCA identified 62 (98.4%) and broth identified 58 (92.1%). In study II, trichomoniasis was diagnosed within the scope of a screening program for a total of 39,585 men and women by culture on MCA and direct microscopy. Culture on MCA detected 199 (98.5%) and Gram staining detected 163 (80.7%) of 202 positive specimens. Wet-mount preparations used for symptomatic patients identified 103 (92.8%) of 111 cases. Culture of T. vaginalis from clinical samples on MCA is highly sensitive and reliable, as well as timesaving, and therefore suitable for screening of symptomatic and asymptomatic individuals. AD - Outpatients' Center for Diagnosis of Infectious Venerodermatological Diseases, A-1210 Vienna, Austria. angelika.stary@univie.ac.at FAU - Stary, Angelika AU - Stary A FAU - Kuchinka-Koch, Angelika AU - Kuchinka-Koch A FAU - Teodorowicz, Lilianna AU - Teodorowicz L LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Culture Media) RN - 0 (Gram's stain) RN - 0 (Phenazines) RN - 548-62-9 (Gentian Violet) RN - 9002-18-0 (Agar) SB - IM MH - *Agar MH - Animals MH - Culture Media MH - Female MH - Gentian Violet MH - Humans MH - Laboratories MH - Laboratory Techniques and Procedures MH - Male MH - Mass Screening MH - Phenazines MH - Sensitivity and Specificity MH - Specimen Handling/methods MH - Trichomonas Vaginitis/*diagnosis/parasitology MH - Trichomonas vaginalis/*growth & development/*isolation & purification MH - Urethra/parasitology MH - Vagina/parasitology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3277-80. PMID- 12202566 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Mycobacterium microti infection (vole tuberculosis) in wild rodent populations. PG - 3281-5 AB - Mycobacterium microti (vole tuberculosis) infections in small wild mammals were first described more than 60 years ago in several populations in Great Britain. Few studies of vole tuberculosis have been undertaken since then, and little is known about the relationship between M. microti isolates originating from different populations or at different times or of the prevalence of this infection in wild rodent populations, despite human cases of M. microti infections being increasingly reported. In this study, field voles (Microtus agrestis), bank voles (Clethrionomys glareolus), and wood mice (Apodemus sylvaticus) were found to be infected, with up to 8% having external tuberculous signs, in wild populations in Northumberland and Cheshire, England. Spoligotyping applied directly to the clinical material simultaneously detected and typed M. microti bacteria in skin lesions, lymph glands, and internal abcesses. IS6110 restriction fragment length polymorphism typing of cultured bacteria was used to compare these isolates with previously isolated strains from both animals and humans. This demonstrated that although the current rodent isolates were distinct from those isolated from voles in the 1930s in Great Britain, they had a high degree of similarity to these strains and were distinct from the M. microti isolates from humans, a pig, and a ferret from The Netherlands. Thus, M. microti infection seems to be widespread in wild rodent populations, but more studies are needed to understand how M. microti might be transmitted from animals to humans and to determine better the zoonotic risk posed. AD - Centre for Comparative Infectious Diseases, University of Liverpool, Liverpool, United Kingdom. rachel@naturebureau.co.uk FAU - Cavanagh, Rachel AU - Cavanagh R FAU - Begon, Michael AU - Begon M FAU - Bennett, Malcolm AU - Bennett M FAU - Ergon, Torbjorn AU - Ergon T FAU - Graham, Isla M AU - Graham IM FAU - De Haas, Petra E W AU - De Haas PE FAU - Hart, C A AU - Hart CA FAU - Koedam, Marianne AU - Koedam M FAU - Kremer, Kristin AU - Kremer K FAU - Lambin, Xavier AU - Lambin X FAU - Roholl, Paul AU - Roholl P FAU - Soolingen Dv, Dick van AU - Soolingen Dv D LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Culture Media) RN - 0 (DNA, Bacterial) RN - 0 (Oligonucleotides) SB - IM MH - Animals MH - *Animals, Wild MH - Culture Media MH - DNA, Bacterial/analysis MH - England/epidemiology MH - Microtinae MH - Muridae MH - Mycobacterium/*classification/genetics/isolation & purification MH - Mycobacterium Infections/epidemiology/microbiology/pathology/veterinary MH - Oligonucleotides/analysis MH - Polymorphism, Restriction Fragment Length MH - Research Support, Non-U.S. Gov't MH - Rodent Diseases/*epidemiology/*microbiology/pathology MH - Tuberculosis/epidemiology/microbiology/pathology/*veterinary EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3281-5. PMID- 12202567 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet. PG - 3286-90 AB - To identify ehrlichial agents in Boophilus microplus ticks, DNA samples of B. microplus collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of B. microplus from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5'-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of Anaplasma marginale, an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of Ehrlichia chaffeensis, an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the E. canis group of the genus EHRLICHIA: Sequence comparison of the 16S rRNA gene with the members of the genus Ehrlichia reveals that the novel ehrlichial agent detected in B. microplus ticks is a new species of the genus Ehrlichia and is most closely related to E. chaffeensis. AD - Department of Microbiology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China. bohaiwen@sohu.com FAU - Wen, Bohai AU - Wen B FAU - Jian, Rui AU - Jian R FAU - Zhang, Youzhi AU - Zhang Y FAU - Chen, Rong AU - Chen R LA - eng SI - GENBANK/AF414399 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal, 16S) SB - IM MH - Anaplasma/classification/genetics/*isolation & purification MH - Animals MH - Base Sequence MH - DNA, Ribosomal/analysis MH - Ehrlichia/*classification/genetics/*isolation & purification MH - Ehrlichia chaffeensis/*classification/genetics MH - Molecular Sequence Data MH - Phylogeny MH - Polymerase Chain Reaction/methods MH - RNA, Ribosomal, 16S/*genetics MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - Tibet MH - Ticks/*microbiology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3286-90. PMID- 12202568 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Rapid detection of methicillin resistance in coagulase-negative Staphylococci with the VITEK 2 system. PG - 3291-5 AB - The aim of the present study was to evaluate the accuracy of the new VITEK 2 system (bioMerieux, Marcy l' Etoile, France) for the detection of methicillin resistance in coagulase-negative staphylococci (CoNS) by using AST-P515 and AST-P523 test cards. Analyses of the VITEK 2 oxacillin MIC determination evaluated according to the actual breakpoint (>/=0.5 micro g/ml) of the National Committee for Clinical Laboratory Standards resulted in a high sensitivity of 99.2% but a moderate specificity of 80%. The newly included oxacillin resistance (OR) test of the VITEK 2 system displayed a high sensitivity and a high specificity of 97.5 and 98.7%, respectively. Concordance between the results of the mecA PCR and the VITEK 2 oxacillin MIC was observed for almost all Staphylococcus epidermidis strains, but the reduced specificity was attributable to higher oxacillin MICs for mecA-negative non-S. epidermidis strains, especially S. saprophyticus, S. lugdunensis, and S. cohnii. Evaluation of alternative oxacillin MIC breakpoints of 1, 2, or 4 micro g/ml resulted in improved degrees of specificity of 84, 90.7, and 97.3%, respectively. Only minor changes occurred in the corresponding sensitivity values, which were 98.4, 97.5, and 97.5%, respectively. Methicillin resistance in CoNS was detected after 7 and 8 h in 91.1 and 93.5% of the mecA-positive strains, respectively, by the VITEK 2 OR test and in 86.3 and 89.5% of the mecA-positive strains, respectively, by VITEK 2 oxacillin MIC determination. After 7 and 8 h the VITEK 2 OR test classified 59.2 and 78.9% of the mecA-negative strains, respectively, as susceptible to oxacillin, whereas comparable values were obtained 2 h later by VITEK 2 oxacillin MIC determination. The results of our study encourage the use of the VITEK 2 system, which proved to be a highly reliable and rapid phenotypic method for the detection of methicillin resistance in CoNS. AD - Institut fur Medizinische Mikrobiologie und Immunologie, Universitatsklinikum Hamburg-Eppendorf, D-20246 Hamburg, Germany. horstko@uke.uni-hamburg.de FAU - Horstkotte, Matthias A AU - Horstkotte MA FAU - Knobloch, Johannes K-M AU - Knobloch JK FAU - Rohde, Holger AU - Rohde H FAU - Dobinsky, Sabine AU - Dobinsky S FAU - Mack, Dietrich AU - Mack D LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Bacterial Proteins) RN - 0 (Carrier Proteins) RN - 0 (Coagulase) RN - 0 (Penicillin-Binding Proteins) RN - 0 (Penicillins) RN - 0 (Reagent Kits, Diagnostic) RN - 66-79-5 (Oxacillin) RN - EC 2.3.2.12 (Peptidyl Transferases) RN - EC 2.4.1.- (Hexosyltransferases) RN - EC 3.4.17.8 (Muramoylpentapeptide Carboxypeptidase) SB - IM MH - *Bacterial Proteins MH - Carrier Proteins MH - Coagulase/*metabolism MH - *Hexosyltransferases MH - Humans MH - *Methicillin Resistance MH - Microbial Sensitivity Tests/methods/standards MH - Muramoylpentapeptide Carboxypeptidase MH - Oxacillin/pharmacology MH - Penicillin-Binding Proteins MH - Penicillins/pharmacology MH - *Peptidyl Transferases MH - *Reagent Kits, Diagnostic MH - Research Support, Non-U.S. Gov't MH - Sensitivity and Specificity MH - Staphylococcal Infections MH - Staphylococcus/*drug effects MH - Time Factors EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3291-5. PMID- 12202569 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh. PG - 3296-9 AB - The sixth pandemic of cholera and, presumably, the earlier pandemics were caused by the classical biotype of Vibrio cholerae O1, which was progressively replaced by the El Tor biotype representing the seventh cholera pandemic. Although the classical biotype of V. cholerae O1 is extinct, even in southern Bangladesh, the last of the niches where this biotype prevailed, we have identified new varieties of V. cholerae O1, of the El Tor biotype with attributes of the classical biotype, from hospitalized patients with acute diarrhea in Bangladesh. Twenty-four strains of V. cholerae O1 isolated between 1991 and 1994 from hospitalized patients with acute diarrhea in Matlab, a rural area of Bangladesh, were examined for the phenotypic and genotypic traits that distinguish the two biotypes of V. cholerae O1. Standard reference strains of V. cholerae O1 belonging to the classical and El Tor biotypes were used as controls in all of the tests. The phenotypic traits commonly used to distinguish between the El Tor and classical biotypes, including polymyxin B sensitivity, chicken cell agglutination, type of tcpA and rstR genes, and restriction patterns of conserved rRNA genes (ribotypes), differentiated the 24 strains of toxigenic V. cholerae O1 into three types designated the Matlab types. Although all of the strains belonged to ribotypes that have been previously found among El Tor vibrios, type I strains had more traits of the classical biotype while type II and III strains appeared to be more like the El Tor biotype but had some classical biotype properties. These results suggest that, although the classical and El Tor biotypes have different lineages, there are possible naturally occurring genetic hybrids between the classical and El Tor biotypes that can cause cholera and thus provide new insight into the epidemiology of cholera in Bangladesh. Furthermore, the existence of such novel strains may have implications for the development of a cholera vaccine. AD - Laboratory Sciences Division, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka 1000, Bangladesh. FAU - Nair, G Balakrish AU - Nair GB FAU - Faruque, Shah M AU - Faruque SM FAU - Bhuiyan, N A AU - Bhuiyan NA FAU - Kamruzzaman, M AU - Kamruzzaman M FAU - Siddique, A K AU - Siddique AK FAU - Sack, David A AU - Sack DA LA - eng GR - R01 AI39129/AI/NIAID PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Bacterial Proteins) SB - IM MH - Acute Disease MH - Bacterial Proteins/genetics MH - Bacterial Typing Techniques MH - Bangladesh/epidemiology MH - Cholera/*epidemiology/microbiology MH - Diarrhea/*epidemiology/microbiology MH - Genotype MH - Hospitalization MH - Humans MH - Phenotype MH - Polymerase Chain Reaction MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - *Variation (Genetics) MH - Vibrio cholerae/*classification/*genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3296-9. PMID- 12202570 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Comparative assessment of genotyping methods for epidemiologic study of Burkholderia cepacia genomovar III. PG - 3300-7 AB - We analyzed a collection of 97 well-characterized Burkholderia cepacia genomovar III isolates to evaluate multiple genomic typing systems, including pulsed-field gel electrophoresis (PFGE), BOX-PCR fingerprinting and random amplified polymorphic DNA (RAPD) typing. The typeability, reproducibility, and discriminatory power of these techniques were evaluated, and the results were compared to each other and to data obtained in previous studies by using multilocus restriction typing (MLRT). All methods showed excellent typeability. PFGE with SpeI was more reproducible than RAPD and BOX-PCR fingerprinting. The discriminatory power of the methods was variable, with PFGE and RAPD typing having a higher index of discrimination than BOX-PCR fingerprinting. In general, the results obtained by PFGE, BOX-PCR fingerprinting, and MLRT were in good agreement. Our data indicate that different genomic-based methods can be used to type B. cepacia genomovar III isolates depending on the situation and the epidemiologic question being addressed. PFGE and RAPD fingerprinting are best suited to addressing small-scale studies (i.e., local epidemiology), whereas BOX-PCR fingerprinting is more appropriate for large-scale studies (i.e., global epidemiology). In this regard, BOX-PCR fingerprinting can be considered a rapid and easy alternative to MLRT. AD - Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan 48109-0646, USA. FAU - Coenye, Tom AU - Coenye T FAU - Spilker, Theodore AU - Spilker T FAU - Martin, Alissa AU - Martin A FAU - LiPuma, John J AU - LiPuma JJ LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 SB - IM MH - Bacterial Typing Techniques/*methods MH - Burkholderia Infections/*epidemiology/microbiology MH - Burkholderia cepacia/*classification/*genetics MH - Comparative Study MH - Cystic Fibrosis/*epidemiology/microbiology MH - Electrophoresis, Gel, Pulsed-Field MH - Epidemiology, Molecular MH - Genotype MH - Humans MH - Polymerase Chain Reaction/methods MH - Random Amplified Polymorphic DNA Technique MH - Reproducibility of Results MH - Research Support, Non-U.S. Gov't EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3300-7. PMID- 12202571 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Determination of novel Borrelia genospecies in Swedish Ixodes ricinus ticks. PG - 3308-12 AB - A total of 301 adult questing Ixodes ricinus ticks were collected at 15 different locations along the south and east coasts of Sweden to determine the Borrelia genospecies diversity. Thirty-two ticks (11%) were found to be positive by nested PCR with Borrelia burgdorferi sensu lato-specific primers. Species determination was based on partial sequencing of the 16S rRNA gene and the flagellin gene. Five different Borrelia species were found. The nucleotide sequence of the Borrelia DNA found in two ticks differed extensively from the nucleotide sequences of the Borrelia DNA found in the other ticks, and analysis revealed that they were closely related to the relapsing fever borrelia species Borrelia miyamotoi. This is the first report of a B. miyamotoi-like borrelia in I. ricinus and in Europe. Moreover, the Borrelia DNA of two ticks (6%) clustered within the B. valaisiana complex. B. valaisiana has not previously been reported in Sweden. B. afzelii DNA was found in 14 ticks (44%), and B. garinii DNA was found in 10 ticks (31%). B. burgdorferi sensu stricto DNA was found in four ticks (13%). We conclude that all of the known human-pathogenic species (B. garinii, B. afzelii, and B. burgdorferi sensu stricto) and B. valaisiana found elsewhere in Europe are also present in the Swedish host-seeking tick population and that a B. miyamotoi-like Borrelia species seems to be present in I. ricinus ticks in Europe. AD - Department of Infectious Diseases, Blekinge Hospital, S-371 85 Karlskrona, Sweden. carl-johan.fraenkel@ltblekinge.se FAU - Fraenkel, Carl-Johan AU - Fraenkel CJ FAU - Garpmo, Ulf AU - Garpmo U FAU - Berglund, Johan AU - Berglund J LA - eng SI - GENBANK/AY083470 SI - GENBANK/AY083471 SI - GENBANK/AY083472 SI - GENBANK/AY083473 SI - GENBANK/AY083474 SI - GENBANK/AY083475 SI - GENBANK/AY083476 SI - GENBANK/AY083477 SI - GENBANK/AY083478 SI - GENBANK/AY083479 SI - GENBANK/AY083480 SI - GENBANK/AY083481 SI - GENBANK/AY083482 SI - GENBANK/AY083483 SI - GENBANK/AY083484 SI - GENBANK/AY083485 SI - GENBANK/AY083486 SI - GENBANK/AY083487 SI - GENBANK/AY083488 SI - GENBANK/AY083489 SI - GENBANK/AY083490 SI - GENBANK/AY083491 SI - GENBANK/AY083492 SI - GENBANK/AY083493 SI - GENBANK/AY083494 SI - GENBANK/AY083495 SI - GENBANK/AY083496 SI - GENBANK/AY083497 SI - GENBANK/AY083498 SI - GENBANK/AY083499 SI - GENBANK/AY083500 SI - GENBANK/AY083501 SI - GENBANK/AY083502 SI - GENBANK/AY083503 SI - GENBANK/AY083504 SI - GENBANK/AY083505 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Bacterial) RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal, 16S) RN - 12777-81-0 (Flagellin) SB - IM MH - Animals MH - Borrelia/*classification/*genetics/isolation & purification MH - DNA, Bacterial/analysis MH - DNA, Ribosomal/analysis MH - Flagellin/genetics MH - Ixodes/*microbiology MH - Molecular Sequence Data MH - Phylogeny MH - RNA, Ribosomal, 16S/genetics MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - Sweden EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3308-12. PMID- 12202572 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Molecular epidemiology of erythromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites. PG - 3313-8 AB - Erythromycin-resistant isolates of Streptococcus pneumoniae from blood cultures and noninvasive sites were studied over a 3-year period. The prevalence of erythromycin resistance was 11.9% (19 of 160) in blood culture isolates but 4.2% (60 of 1,435) in noninvasive-site isolates. Sixty-two of the 79 resistant isolates were available for study. The M phenotype was responsible for 76% (47 of 62) of resistance, largely due to a serotype 14 clone, characterized by multilocus sequence typing as ST9, which accounted for 79% (37 of 47) of M phenotype resistance. The ST9 clone was 4.8 times more common in blood than in noninvasive sites. All M phenotype isolates were PCR positive for mef(A), but sequencing revealed that the ST9 clone possessed the mef(A) sequence commonly associated with Streptococcus pyogenes. All M phenotype isolates with this mef(A) sequence also had sequences consistent with the presence of the Tn1207.1 genetic element inserted in the celB gene. In contrast, isolates with the mef(E) sequence normally associated with S. pneumoniae contained sequences consistent with the presence of the mega insertion element. All MLS(B) isolates carried erm(B), and two isolates carried both erm(B) and mef(E). Fourteen of the 15 MLS(B) isolates were tetracycline resistant and contained tet(M). However, six M phenotype isolates of serotypes 19 (two isolates) and 23 (four isolates) were also tetracycline resistant and contained tet(M). MICs for isolates with the mef(A) sequence were significantly higher than MICs for isolates with the mef(E) sequence (P < 0.001). Thus, the ST9 clone of S. pneumoniae is a significant cause of invasive pneumococcal disease in northeast Scotland and is the single most important contributor to M phenotype erythromycin resistance. AD - Department of Medical Microbiology, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, United Kingdom. FAU - Amezaga, Maria Rosario AU - Amezaga MR FAU - Carter, Philip E AU - Carter PE FAU - Cash, Phillip AU - Cash P FAU - McKenzie, Hamish AU - McKenzie H LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Anti-Bacterial Agents) RN - 0 (Bacterial Proteins) RN - 0 (Culture Media) RN - 0 (DNA Transposable Elements) RN - 114-07-8 (Erythromycin) SB - IM MH - Anti-Bacterial Agents/*pharmacology MH - Bacteremia/epidemiology/microbiology MH - Bacterial Proteins/genetics MH - Blood/microbiology MH - Culture Media MH - DNA Transposable Elements MH - Drug Resistance, Bacterial/genetics MH - *Epidemiology, Molecular MH - Erythromycin/*pharmacology MH - Humans MH - Microbial Sensitivity Tests MH - Pneumococcal Infections/epidemiology/microbiology MH - Polymerase Chain Reaction MH - Prevalence MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - Streptococcus pneumoniae/*classification/*drug effects/genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3313-8. PMID- 12202573 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Rational design of DNA sequence-based strategies for subtyping Listeria monocytogenes. PG - 3319-25 AB - The ability to differentiate bacteria beyond the species level is essential for identifying and tracking infectious disease outbreaks and to improve our knowledge of the population genetics, epidemiology, and ecology of bacterial pathogens. Commonly used subtyping methods, such as serotyping, phage typing, ribotyping, and pulsed-field gel electrophoresis, can yield ambiguous results that are difficult to standardize and share among laboratories. DNA sequence-based subtyping strategies can reduce interpretation ambiguity. We report the development of a rational approach for designing sequence-based subtyping methods. Listeria monocytogenes was selected as the model organism for testing the efficacy of this approach. Two housekeeping genes (recA and prs), one stress response gene (sigB), two virulence genes (actA and inlA), and two intergenic regions (hly-mpl and plcA-hly) were sequenced for 15 L. monocytogenes isolates. Isolates were chosen from a representative collection of more than 1,000 L. monocytogenes isolates to reflect the genetic diversity of this species. DNA sequences were aligned, and sliding window analyses were performed for each gene to define 600-bp-long regions that were (i) most polymorphic (using ProSeq) or (ii) most discriminatory (using a new algorithm implemented in WINDOWMIN). Complete gene sequences for actA (1,929 bp) and inlA (2,235 bp) provided the highest discrimination (identifying 15 and 14 allelic types, respectively). WINDOWMIN allowed identification of 600-bp regions within these genes that provided similar discriminatory power (yielding 15 and 13 allelic types, respectively). The most discriminatory 600-bp fragments identified in the housekeeping and stress response genes differentiated the isolates into 8 to 10 subtypes; intergenic region sequences yielded 8 and 12 allelic types based on 335- and 242-bp sequences for hly-mpl and plcA-hly, respectively. Regions identified as most polymorphic were not necessarily most discriminatory; therefore, application of the WINDOWMIN algorithm provided a powerful tool for determining the best target regions for DNA sequence-based subtyping. Our specific results also show that inclusion of virulence gene target sequences in a DNA sequence-based subtyping scheme for L. monocytogenes is necessary to achieve maximum subtype differentiation. AD - Department of Food Science, Cornell University, Ithaca, New York 14853, USA. FAU - Cai, Steven AU - Cai S FAU - Kabuki, Dirce Yorika AU - Kabuki DY FAU - Kuaye, Arnaldo Yoshiteru AU - Kuaye AY FAU - Cargioli, Theresa Gina AU - Cargioli TG FAU - Chung, Michael S AU - Chung MS FAU - Nielsen, Rasmus AU - Nielsen R FAU - Wiedmann, Martin AU - Wiedmann M LA - eng SI - GENBANK/AF497139 SI - GENBANK/AF497140 SI - GENBANK/AF497141 SI - GENBANK/AF497142 SI - GENBANK/AF497143 SI - GENBANK/AF497144 SI - GENBANK/AF497145 SI - GENBANK/AF497146 SI - GENBANK/AF497147 SI - GENBANK/AF497148 SI - GENBANK/AF497149 SI - GENBANK/AF497150 SI - GENBANK/AF497151 SI - GENBANK/AF497152 SI - GENBANK/AF497153 SI - GENBANK/AF497154 SI - GENBANK/AF497155 SI - GENBANK/AF497156 SI - GENBANK/AF497157 SI - GENBANK/AF497158 SI - GENBANK/AF497159 SI - GENBANK/AF497160 SI - GENBANK/AF497161 SI - GENBANK/AF497162 SI - GENBANK/AF497163 SI - GENBANK/AF497164 SI - GENBANK/AF497165 SI - GENBANK/AF497166 SI - GENBANK/AF497167 SI - GENBANK/AF497168 SI - GENBANK/AF497169 SI - GENBANK/AF497170 SI - GENBANK/AF497171 SI - GENBANK/AF497172 SI - GENBANK/AF497173 SI - GENBANK/AF497174 SI - GENBANK/AF497175 SI - GENBANK/AF497176 SI - GENBANK/AF497177 SI - GENBANK/AF497178 SI - GENBANK/AF497179 SI - GENBANK/AF497180 SI - GENBANK/AF497181 SI - GENBANK/AF497182 SI - GENBANK/AF497183 SI - GENBANK/AF497184 SI - GENBANK/AF497185 SI - GENBANK/AF497186 SI - GENBANK/AF497187 SI - GENBANK/AF497188 SI - GENBANK/AF497189 SI - GENBANK/AF497190 SI - GENBANK/AF497191 SI - GENBANK/AF497192 SI - GENBANK/AF497193 SI - GENBANK/AF497194 SI - GENBANK/AF497195 SI - GENBANK/AF497196 SI - GENBANK/AF497197 SI - GENBANK/AF497198 SI - GENBANK/AF497199 SI - GENBANK/AF497200 SI - GENBANK/AF497201 SI - GENBANK/AF497202 SI - GENBANK/AF497203 SI - GENBANK/AF497204 SI - GENBANK/AF497205 SI - GENBANK/AF497206 SI - GENBANK/AF497207 SI - GENBANK/AF497208 SI - GENBANK/AF497209 SI - GENBANK/AF497210 SI - GENBANK/AF497211 SI - GENBANK/AF497212 SI - GENBANK/AF497213 SI - GENBANK/AF497214 SI - GENBANK/AF497215 SI - GENBANK/AF497216 SI - GENBANK/AF497217 SI - GENBANK/AF497218 SI - GENBANK/AF497219 SI - GENBANK/AF497220 SI - GENBANK/AF497221 SI - GENBANK/AF497222 SI - GENBANK/AF497223 SI - GENBANK/AF497224 SI - GENBANK/AF497225 SI - GENBANK/AF497226 SI - GENBANK/AF497227 SI - GENBANK/AF497228 SI - GENBANK/AF497229 SI - GENBANK/AF497230 SI - GENBANK/AF497231 SI - GENBANK/AF497232 SI - GENBANK/AF497233 SI - GENBANK/AF497234 SI - GENBANK/AF497235 SI - GENBANK/AF497236 SI - GENBANK/AF497237 SI - GENBANK/AF497238 SI - GENBANK/AF497239 SI - GENBANK/AF497240 SI - GENBANK/AF497241 SI - GENBANK/AF497242 SI - GENBANK/AF497243 GR - R01GM63259/GM/NIGMS PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Bacterial Proteins) RN - 0 (DNA, Bacterial) SB - IM MH - *Algorithms MH - Animals MH - Bacterial Proteins/*genetics MH - Bacterial Typing Techniques/*methods MH - DNA, Bacterial/analysis MH - Gene Deletion MH - Humans MH - Listeria Infections/microbiology MH - Listeria monocytogenes/*classification/*genetics/pathogenicity MH - Molecular Sequence Data MH - Polymorphism, Genetic MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Analysis, DNA/*methods EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3319-25. PMID- 12202574 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Possible connection between a widely disseminated conjugative gentamicin resistance (pMG1-like) plasmid and the emergence of vancomycin resistance in Enterococcus faecium. PG - 3326-33 AB - A total of 640 vancomycin-resistant Enterococcus faecium (VRE) isolates, which were obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were used in this study. Of the 640 strains, 611 and 29 were VanA and VanB VRE, respectively, based on PCR analysis. Four hundred ninety-two (77%) of the strains exhibited resistance to concentrations of gentamicin from 64 micro g/ml (MIC) to more than 1,024 micro g/ml (MIC). The gentamicin resistance of each of 261 (53%) of the 492 gentamicin-resistant strains was transferred to E. faecium at a frequency of about 10(-5) to 10(-6) per donor cell in broth mating. More than 90% of vancomycin resistances of the 261 strains cotransferred with the gentamicin resistances to E. faecium strains by filter mating. The conjugative gentamicin resistance plasmids were identified and were classified into five types (A through E) with respect to their EcoRI restriction profiles. The transfer frequencies of each type of plasmid between E. faecium strains or Enterococcus faecalis strains were around 10(-3) to 10(-5) per donor cell or around 10(-6) to 10(-7) per donor cell, respectively, in broth mating. Type A and type B were the most frequently isolated, at an isolation frequency of about 40% per VRE isolate harboring the gentamicin resistance conjugative plasmid. The plasmids did not show any homology in Southern hybridization with the pheromone-responsive plasmids and broad-host-range plasmids pAMbeta1 and pIP501. The EcoRI or NdeI restriction fragments of each type of plasmids hybridized to the conjugative gentamicin resistance plasmid pMG1 (65.1 kb), which was originally isolated from an E. faecium clinical isolate, and transfer efficiently in broth mating. AD - Department of Microbiology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan. FAU - Tomita, Haruyoshi AU - Tomita H FAU - Pierson, Carl AU - Pierson C FAU - Lim, Suk Kyung AU - Lim SK FAU - Clewell, Don B AU - Clewell DB FAU - Ike, Yasuyoshi AU - Ike Y LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Anti-Bacterial Agents) RN - 0 (Culture Media) RN - 0 (Gentamicins) RN - 0 (Plasmids) SB - IM MH - Anti-Bacterial Agents/*pharmacology MH - *Conjugation, Genetic MH - Culture Media MH - Drug Resistance, Bacterial/genetics MH - Enterococcus faecium/*drug effects/genetics MH - Gene Transfer, Horizontal MH - Gentamicins/*pharmacology MH - Nucleic Acid Hybridization MH - Plasmids/*genetics MH - Research Support, Non-U.S. Gov't MH - *Vancomycin Resistance EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3326-33. PMID- 12202575 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Detection and quantification of oral treponemes in subgingival plaque by real-time PCR. PG - 3334-40 AB - Oral treponemes have been associated with periodontal diseases. We developed a highly sensitive and specific method to detect and quantify cultivable oral treponemes (Treponema denticola, Treponema vincentii, and Treponema medium) in 50 subgingival plaque samples from 13 healthy subjects as well as 37 patients with periodontal diseases using real-time PCR assays with specific primers and a TaqMan probe for each 16S rRNA sequence. The specificity for each assay was examined by using DNA specimens from various treponemal and other bacterial species. The TaqMan real-time PCR was able to detect from 10(3) to 10(8) cells of the oral treponemes, with correlation coefficients as follows: T. denticola, 0.984; T. vincentii, 0.991; and T. medium, 0.984. The frequencies of occurrence of these three oral treponemes in subgingival plaque samples were as follows: T. denticola, 68.0%; T. vincentii, 36.0%; and T. medium, 48.0%. In addition, the number of T. denticola, T. vincentii, and T. medium cells in plaque samples detected by real-time PCR ranged from 3 to 15,184, 1 to 447, and 1 to 7,301 cells/pg of plaque DNA, respectively. Increased numbers of T. denticola cells were detected in plaque samples from deep periodontal pockets, and T. medium was also detected in deep pockets. On the other hand, T. vincentii was mainly found in shallow pockets. These results suggest that various oral treponemes are associated with the formation of each stage of periodontal disease. AD - Department of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi, Hozumi-cho, Motosu-gun, Gifu 501-0296, Japan. FAU - Asai, Yasuyuki AU - Asai Y FAU - Jinno, Takayoshi AU - Jinno T FAU - Igarashi, Hajime AU - Igarashi H FAU - Ohyama, Yoshinori AU - Ohyama Y FAU - Ogawa, Tomohiko AU - Ogawa T LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Bacterial) RN - EC 2.7.7.- (Taq Polymerase) SB - IM MH - Adolescent MH - Adult MH - Aged MH - Aged, 80 and over MH - DNA, Bacterial/analysis MH - Dental Plaque/*microbiology MH - Female MH - Humans MH - Male MH - Middle Aged MH - Mouth/*microbiology MH - Periodontal Diseases/*microbiology/physiopathology MH - Polymerase Chain Reaction/*methods MH - Research Support, Non-U.S. Gov't MH - Sensitivity and Specificity MH - Species Specificity MH - Taq Polymerase MH - Treponema/classification/genetics/*isolation & purification MH - Treponemal Infections/microbiology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3334-40. PMID- 12202576 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - High prevalence of human papillomavirus (HPV) infections and high frequency of multiple HPV genotypes in human immunodeficiency virus-infected women in Brazil. PG - 3341-5 AB - A group of 208 human immunodeficiency virus (HIV)-infected women in Brazil were studied for the presence of human papillomavirus with the general SPF(10) PCR primer set. Virtually all (98%) women were found positive for human papillomavirus (HPV) DNA. Genotyping by the reverse hybridization line probe assay (HPV-LiPA) revealed a high prevalence of multiple genotypes (78.9% of the cases), with an average of 3.1 genotypes per patient (range, 1 to 10 genotypes). HPV 6 was the most prevalent genotype and was observed in 80 (39.2%) patients, followed by types 51 (31.9%), 11 (26.0%), 18 (24.0%), and 16 (22.5%). Of the genotypes detected, 40.9% were low-risk genotypes. Twenty-two (10.5%) patients showed normal (Pap I) cytology, 149 (71.6%) patients had inflammation (Pap II), and 28 patients (13.4%) had a Pap III score. The prevalence of high-risk genotypes increased with the cytological classification. There were no significant associations between the number of HPV genotypes detected and the cytological classification, HIV viral load, and CD4 count in these patients. In conclusion, the highly sensitive SPF(10) LiPA system shows that a very high proportion of HIV-infected women in Brazil are infected with HPV and often carry multiple HPV genotypes. AD - Laboratorio de Virologia do Instituto de Medicina Tropical, Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, Brazil. FAU - Levi, Jose E AU - Levi JE FAU - Kleter, Bernhard AU - Kleter B FAU - Quint, Wim G V AU - Quint WG FAU - Fink, Maria C S AU - Fink MC FAU - Canto, Cynthia L M AU - Canto CL FAU - Matsubara, Regina AU - Matsubara R FAU - Linhares, Iara AU - Linhares I FAU - Segurado, Aluisio AU - Segurado A FAU - Vanderborght, Bart AU - Vanderborght B FAU - Neto, Jose Eluf AU - Neto JE FAU - Van Doorn, Leen-Jan AU - Van Doorn LJ LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Viral) SB - IM MH - Brazil/epidemiology MH - Cervical Intraepithelial Neoplasia/epidemiology/virology MH - Cervix Neoplasms/epidemiology/virology MH - DNA, Viral/analysis MH - Female MH - Genotype MH - HIV Infections/*complications/*epidemiology MH - HIV-1/isolation & purification/physiology MH - Humans MH - Papillomavirus, Human/*classification/genetics/isolation & purification MH - Papovaviridae Infections/*epidemiology/virology MH - Polymerase Chain Reaction MH - Prevalence MH - Research Support, Non-U.S. Gov't MH - Tumor Virus Infections/*epidemiology/virology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3341-5. PMID- 12202577 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Leptotrichia amnionii sp. nov., a novel bacterium isolated from the amniotic fluid of a woman after intrauterine fetal demise. PG - 3346-9 AB - A novel bacterium was isolated and characterized from the amniotic fluid of a woman who experienced intrauterine fetal demise in the second trimester of pregnancy. The bacterium was a slow-growing, gram-negative anaerobic coccobacillus belonging to the genus LEPTOTRICHIA: Unlike Leptotrichia sanguinegens, the isolate did not grow in chopped-meat glucose broth or on sheep blood agar upon subculturing. The isolate was characterized by sequencing and analyzing its 16S rRNA gene. The 1,493-bp 16S ribosomal DNA sequence had only 96% homology with L. sanguinegens. Several phylogenetic analyses indicated that L. amnionii is a distinct species and most closely related to L. sanguiegens. AD - Clinical Research Center, Marshfield Medical Research and Education Foundation, Wisconsin 54449, USA. shuklas@mmrf.mfldclin.edu FAU - Shukla, Sanjay K AU - Shukla SK FAU - Meier, Paul R AU - Meier PR FAU - Mitchell, Paul D AU - Mitchell PD FAU - Frank, Daniel N AU - Frank DN FAU - Reed, Kurt D AU - Reed KD LA - eng SI - GENBANK/AY078425 PT - Case Reports PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal, 16S) SB - IM MH - Adult MH - Amniotic Fluid/*microbiology MH - DNA, Ribosomal/analysis MH - Female MH - *Fetal Death MH - Gram-Negative Anaerobic Bacteria/*classification/genetics/*isolation & purification MH - Gram-Negative Bacterial Infections/microbiology MH - Humans MH - Molecular Sequence Data MH - Phylogeny MH - Polymerase Chain Reaction MH - Pregnancy MH - Pregnancy Complications, Infectious/*microbiology MH - Pregnancy Trimester, Second MH - RNA, Ribosomal, 16S/genetics MH - Research Support, Non-U.S. Gov't EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3346-9. PMID- 12202578 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Fast, noninvasive method for molecular detection and differentiation of Malassezia yeast species on human skin and application of the method to dandruff microbiology. PG - 3350-7 AB - Malassezia fungi have been the suspected cause of dandruff for more than a century. Previously referred to as Pityrosporum ovale, Pityrosporum orbiculare, or Malassezia, these fungi are now known to consist of at least seven Malassezia species. Each species has a specific ecological niche, as well as specific biochemical and genetic characteristics. Malassezia yeasts have fastidious culture conditions and exceedingly different growth rates. Therefore, the results of surveys of Malassezia based on culture methods can be difficult to interpret. We developed a molecular technique, terminal fragment length polymorphism analysis, to more accurately survey the ecology of Malassezia yeasts without bias from culture. This technique involves fluorescent nested PCR of the intergenic transcribed spacer (ITS) ITS I and ITS II region ribosomal gene clusters. All known Malassezia species can be differentiated by unique ITS fragment lengths. We have used this technique to directly analyze scalp samples from subjects enrolled in a demographic scalp health study. Results for subjects assigned composite adherent scalp flaking scores (ASFS) <10 were compared to those for subjects assigned composite ASFS >24. Malassezia restricta and M. globosa were found to be the predominant Malassezia species present in both groups. Importantly, we found no evidence of M. furfur in either group, indicating that M. furfur can be eliminated as the causal organism for dandruff. Both groups also showed the presence of non-Malassezia fungi. This method, particularly when it is used in combination with existing fungal ITS databases, is expected to be useful in the diagnosis of multiple other fungal infections. AD - The Procter & Gamble Company, Cincinnati, Ohio 45252. Yeast Division, Centraalbureau voor Schimmelcultures, 3584 CT Utrecht, The Netherlands. FAU - Gemmer, Christina M AU - Gemmer CM FAU - DeAngelis, Yvonne M AU - DeAngelis YM FAU - Theelen, Bart AU - Theelen B FAU - Boekhout, Teun AU - Boekhout T FAU - Dawson Jr, Thomas L Jr AU - Dawson Jr TL Jr LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Fungal) RN - 0 (DNA, Ribosomal Spacer) SB - IM MH - DNA, Fungal/analysis/isolation & purification MH - DNA, Ribosomal Spacer/analysis MH - Dermatitis, Seborrheic/*microbiology MH - Humans MH - Malassezia/*classification/genetics/isolation & purification MH - Polymerase Chain Reaction/*methods MH - Research Support, Non-U.S. Gov't MH - Scalp/*microbiology MH - Scalp Dermatoses/*microbiology MH - Sensitivity and Specificity MH - Specimen Handling MH - Time Factors EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3350-7. PMID- 12202579 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20031114 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Detection of Rickettsia prowazekii in body lice and their feces by using monoclonal antibodies. PG - 3358-63 AB - In order to identify Rickettsia prowazekii in lice, we developed a panel of 29 representative monoclonal antibodies selected from 187 positive hybridomas made by fusing splenocytes of immunized mice with SP2/0-Ag14 myeloma cells. Immunoblotting revealed that 15 monoclonal antibodies reacted with the lipopolysaccharide-like (LPS-L) antigen and 14 reacted with the epitopes of a 120-kDa protein. Only typhus group rickettsiae reacted with the monoclonal antibodies against LPS-L. R. felis, a recently identified rickettsial species, did not react with these monoclonal antibodies, confirming that it is not antigenically related to the typhus group. Monoclonal antibodies against the 120-kDa protein were highly specific for R. prowazekii. We successfully applied a selected monoclonal antibody against the 120-kDa protein to detect by immunofluorescence assay R. prowazekii in smears from 56 wild and laboratory lice, as well as in 10 samples of louse feces infected or not infected with the organism. We have developed a simple, practical, and specific diagnostic assay for clinical specimens and large-scale epidemiological surveys with a sensitivity of 91%. These monoclonal antibodies could be added to the rickettsial diagnostic panel and be used to differentiate R. prowazekii from other rickettsial species. AD - Unite des Rickettsies, CNRS UMR 6020, IFR 48, Faculty of Medicine, 13385 Marseille, France. FAU - Fang, Rong AU - Fang R FAU - Houhamdi, Linda AU - Houhamdi L FAU - Raoult, Didier AU - Raoult D LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antibodies, Bacterial) RN - 0 (Antibodies, Monoclonal) SB - IM MH - Animals MH - Antibodies, Bacterial/biosynthesis/*diagnostic use/immunology MH - Antibodies, Monoclonal/biosynthesis/*diagnostic use/immunology MH - *Antibody Specificity MH - Blotting, Western MH - Electrophoresis, Polyacrylamide Gel MH - Feces/*microbiology MH - Female MH - Mice MH - Mice, Inbred BALB C MH - Pediculus/*microbiology MH - Rickettsia prowazekii/immunology/*isolation & purification EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3358-63. PMID- 12202580 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Rapid-cycle PCR and fluorimetry for detection of mycobacteria. PG - 3364-73 AB - In this study we used LightCycler PCR amplification and product detection by fluorescence resonance energy transfer probes to identify mycobacteria and differentiate between Mycobacterium tuberculosis complex, Mycobacterium avium, and other nontuberculous mycobacteria. Targeting the 16S rRNA gene, three different probes specific for mycobacteria, M. tuberculosis complex, and M. avium were constructed. As few as five genome copies of target nucleic acid were detected by the probes, illustrating the high sensitivity of the system. All 33 mycobacterial species tested but none of the closely related actinomycetes and other bacteria produced a specific fluorescence signal. A specificity of 100% was also demonstrated for the M. tuberculosis complex-specific probe and the M. avium-specific probe. Within 45 min, the LightCycler method correctly detected mycobacteria and specifically identified M. tuberculosis complex and M. avium without any post-PCR sample manipulation. In view of future clinical studies, we also constructed and tested an internal control which could be used to assure successful amplification and detection of mycobacteria. Monitoring of PCR inhibition will be essential for evaluation of this system for direct detection of mycobacteria in clinical specimens. Finally, we tested our system on sputum seeded with mycobacteria and were able to detect as few as 10 organisms. At present, this system is the fastest available method for identification and differentiation of mycobacteria from culture-positive specimens and offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnostic mycobacterial laboratory. AD - Institute of Medical Microbiology, Medical School Hannover, 30625 Hannover, Germany. FAU - Lachnik, Jacqueline AU - Lachnik J FAU - Ackermann, Birgit AU - Ackermann B FAU - Bohrssen, Antje AU - Bohrssen A FAU - Maass, Silvia AU - Maass S FAU - Diephaus, Catharina AU - Diephaus C FAU - Puncken, Axel AU - Puncken A FAU - Stermann, Marion AU - Stermann M FAU - Bange, Franz-Christoph AU - Bange FC LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA Probes) RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal, 16S) SB - IM MH - *Bacterial Typing Techniques MH - DNA Probes MH - DNA, Ribosomal/analysis MH - Energy Transfer MH - Fluorometry MH - Humans MH - Mycobacterium/*classification/genetics/isolation & purification MH - Mycobacterium Infections/microbiology MH - Mycobacterium avium Complex/*classification MH - Mycobacterium tuberculosis/*classification MH - Polymerase Chain Reaction/*methods MH - RNA, Ribosomal, 16S/genetics MH - Research Support, Non-U.S. Gov't MH - Sensitivity and Specificity MH - Sputum/microbiology MH - Temperature MH - Time Factors EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3364-73. PMID- 12202581 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Clinical and epidemiological correlates of genotypes within the Mycobacterium avium complex defined by restriction and sequence analysis of hsp65. PG - 3374-80 AB - Species identification of isolates of the Mycobacterium avium complex (MAC) remains a difficult task. Although M. avium and Mycobacterium intracellulare can be identified with expensive, commercially available probes, many MAC isolates remain unresolved, including those representing Mycobacterium lentiflavum as well as other potentially undefined species. PCR restriction analysis (PRA) of the hsp65 gene has been proposed as a rapid and inexpensive approach. We applied PRA to 278 MAC isolates, including 126 from blood of human immunodeficiency virus (HIV)-infected patients, 59 from sputum of HIV-negative patients with chronic obstructive pulmonary disease, 88 from environmental sources, and 5 pulmonary isolates from a different study. A total of 15 different PRA patterns were observed. For 27 representative isolates, a 441-bp fragment of the hsp65 gene was sequenced; based on 54 polymorphic sites, 18 different alleles were defined, including 12 alleles not previously reported. Species and phylogenetic relationships were more accurately defined by sequencing than by PRA or commercial probe. The distribution of PRA types and, by implication, phylogenetic lineages among blood isolates was significantly different from that for pulmonary and environmental isolates, suggesting that particular lineages have appreciably greater virulence and invasive potential. AD - Department of Medicine, Boston University School of Medicine, VA Boston Healthcare System, Boston, Massachusetts 02130, USA. FAU - Smole, Sandra C AU - Smole SC FAU - McAleese, Fionnuala AU - McAleese F FAU - Ngampasutadol, Jutamas AU - Ngampasutadol J FAU - Von Reyn, C Fordham AU - Von Reyn CF FAU - Arbeit, Robert D AU - Arbeit RD LA - eng SI - GENBANK/AF241200 SI - GENBANK/AF241201 SI - GENBANK/AF241202 SI - GENBANK/AF241203 SI - GENBANK/AF241204 SI - GENBANK/AF241205 SI - GENBANK/AF241206 SI - GENBANK/AF241207 SI - GENBANK/AF241208 SI - GENBANK/AF241209 SI - GENBANK/AF241210 SI - GENBANK/AF241211 SI - GENBANK/AF241212 SI - GENBANK/AF241213 SI - GENBANK/AF241214 SI - GENBANK/AF241215 SI - GENBANK/AF241216 SI - GENBANK/AF354274 SI - GENBANK/AF354275 SI - GENBANK/AF354276 SI - GENBANK/AF354277 SI - GENBANK/AF354278 SI - GENBANK/AF354279 SI - GENBANK/AF354280 SI - GENBANK/AF354281 SI - GENBANK/AF354282 SI - GENBANK/AF354283 PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Bacterial Proteins) RN - 0 (Chaperonins) RN - 0 (DNA Transposable Elements) RN - 0 (heat-shock protein 65, Mycobacterium) SB - IM MH - *Bacterial Proteins MH - Bacterial Typing Techniques MH - Base Sequence MH - Blood/microbiology MH - Chaperonins/*genetics MH - DNA Transposable Elements MH - Genotype MH - Humans MH - Lung/microbiology MH - Molecular Sequence Data MH - Mycobacterium avium Complex/*classification/*genetics/isolation & purification MH - Mycobacterium avium-intracellulare Infection/*epidemiology/microbiology/*physiopathology MH - Phylogeny MH - Polymerase Chain Reaction/methods MH - Restriction Mapping MH - Sequence Analysis, DNA EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3374-80. PMID- 12202582 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Malignant catarrhal fever-like disease in Barbary red deer (Cervus elaphus barbarus) naturally infected with a virus resembling alcelaphine herpesvirus 2. PG - 3381-90 AB - Eight Barbary red deer (Cervus elaphus barbarus) developed clinical signs suggestive of malignant catarrhal fever (MCF) over a 28-day period. These animals were housed outdoors with four other species of ruminants. Affected red deer had lethargy, ocular signs, and nasal discharge and were euthanatized within 48 h. Lesions included ulcers of the muzzle, lips, and oral cavity associated with infiltrates of neutrophils and lymphocytes. Serologically, six of seven red deer tested during the outbreak were positive by competitive enzyme-linked immunosorbent assay for antibodies to a shared MCF virus antigen. PCR using oligonucleotide primers designed for a conserved protein of alcelaphine herpesviruses 1 (AlHV-1) and 2 (AlHV-2) and for conserved regions of a herpesvirus DNA polymerase gene was positive for tissues from all eight clinically affected animals and negative for eight out of eight red deer without clinical signs of MCF. DNA sequencing of PCR amplicons from the diseased red deer indicated that they were infected with a novel herpesvirus closely related to AlHV-2; immunohistochemistry using polyclonal anti-AlHV-2 serum and in situ hybridization demonstrated the presence of virus within salivary glands adjacent to oral lesions of affected animals. A survey of other ruminants near the outbreak subsequently showed that normal Jackson's hartebeest (Alcelaphus buselaphus jacksoni) that were cohoused with the diseased red deer were infected with the same virus and were shedding the virus in nasal excretions. These findings suggest that a herpesvirus closely related to AlHV-2 caused the MCF-like disease epizootic in Barbary red deer and that the virus may have originated from Jackson's hartebeest. AD - Molecular Diagnostics Laboratory, Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, California 92112-0551, USA. FAU - Klieforth, Robert AU - Klieforth R FAU - Maalouf, Gabriel AU - Maalouf G FAU - Stalis, Ilse AU - Stalis I FAU - Terio, Karen AU - Terio K FAU - Janssen, Donald AU - Janssen D FAU - Schrenzel, Mark AU - Schrenzel M LA - eng SI - GENBANK/AY092762 SI - GENBANK/AY092763 SI - GENBANK/AY125489 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antibodies, Viral) SB - IM MH - Amino Acid Sequence MH - Animals MH - Antibodies, Viral/blood MH - Base Sequence MH - *Deer MH - *Disease Outbreaks MH - Enzyme-Linked Immunosorbent Assay MH - Gammaherpesvirinae/*classification/genetics/*isolation & purification MH - Herpesviridae Infections/epidemiology/veterinary/virology MH - Malignant Catarrh/*epidemiology/virology MH - Molecular Sequence Data MH - Polymerase Chain Reaction MH - Research Support, Non-U.S. Gov't MH - Rhadinovirus/*classification/genetics MH - Sequence Analysis, DNA EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3381-90. PMID- 12202583 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Practical approach for typing strains of Leishmania infantum by microsatellite analysis. PG - 3391-7 AB - Currently the universally accepted standard procedure for characterizing and identifying strains of Leishmania is isoenzyme analysis. However, in the Mediterranean area, despite their very wide geographical distribution, most Leishmania infantum strains belong to zymodeme MON-1. In order to increase our understanding of polymorphism in strains of L. infantum, we developed PCR assays amplifying 10 microsatellites and sequenced PCR products. The discriminative power of microsatellite analysis was tested by using a panel of 50 L. infantum strains collected from patients and dogs from Spain, France, and Israel, including 32 strains belonging to zymodeme MON-1, 8 strains belonging to zymodemes MON-24, MON-29, MON-33, MON-34, or MON-80, and 10 untyped strains. Five of the microsatellites were polymorphic, revealing 22 genotypes, whereas the five remaining microsatellites were not variable. In particular, MON-1 strains could be separated into 13 different closely related genotypes. MON-33 and MON-34 strains also gave two additional genotypes closely related to MON-1, while MON-29, MON-24, and MON 80 strains exhibited more divergent genotypes. Among the foci examined, the Catalonian focus displayed a high polymorphism, probably reflecting isoenzyme polymorphism, while the Israeli focus exhibited a low polymorphism that could be consistent with the recent reemergence and rapid spread of canine leishmaniasis in northern and central Israel. The strains originating from the south of France and the Madrid, Spain, area displayed significant microsatellite polymorphism even though they were monomorphic by isoenzyme analysis. In conclusion, microsatellite polymorphism exhibits a high discriminative power and appears to be suitable for characterization of closely related strains of L. infantum in epidemiological studies. AD - Sante Environnement Rural, Universite de Franche-Comte, 25000 Besancon, France. FAU - Bulle, Beatrice AU - Bulle B FAU - Millon, Laurence AU - Millon L FAU - Bart, Jean-Mathieu AU - Bart JM FAU - Gallego, Montserrat AU - Gallego M FAU - Gambarelli, Francoise AU - Gambarelli F FAU - Portus, Montserrat AU - Portus M FAU - Schnur, Lee AU - Schnur L FAU - Jaffe, Charles L AU - Jaffe CL FAU - Fernandez-Barredo, Salceda AU - Fernandez-Barredo S FAU - Alunda, Jose Maria AU - Alunda JM FAU - Piarroux, Renaud AU - Piarroux R LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Ribosomal Spacer) RN - 0 (Isoenzymes) SB - IM MH - Animals MH - Base Sequence MH - DNA, Ribosomal Spacer/analysis MH - Dog Diseases/*parasitology MH - Dogs MH - France MH - Genotype MH - Humans MH - Isoenzymes/genetics MH - Israel MH - Leishmania infantum/*classification/genetics MH - Leishmaniasis, Visceral/*parasitology/veterinary MH - Microsatellite Repeats/*genetics MH - Molecular Sequence Data MH - Parasitology/methods MH - Polymerase Chain Reaction/*methods MH - Polymorphism, Genetic MH - Reproducibility of Results MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - Spain MH - Variation (Genetics) EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3391-7. PMID- 12202584 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Mycobacterium africanum subtype II is associated with two distinct genotypes and is a major cause of human tuberculosis in Kampala, Uganda. PG - 3398-405 AB - The population structure of 234 Mycobacterium tuberculosis complex strains obtained during 1995 and 1997 from tuberculosis patients living in Kampala, Uganda (East Africa), was analyzed by routine laboratory procedures, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. According to biochemical test results, 157 isolates (67%) were classified as M. africanum subtype II (resistant to thiophen-2-carboxylic acid hydrazide), 76 isolates (32%) were classified as M. tuberculosis, and 1 isolate was classified as classical M. bovis. Spoligotyping did not lead to clear differentiation of M. tuberculosis and M. africanum, but all M. africanum subtype II isolates lacked spacers 33 to 36, differentiating them from M. africanum subtype I. Moreover, spoligotyping was not sufficient for differentiation of isolates on the strain level, since 193 (82%) were grouped into clusters. In contrast, in the IS6110-based dendrogram, M. africanum strains were clustered into two closely related strain families (Uganda I and II) and clearly separated from the M. tuberculosis isolates. A further characteristic of both M. africanum subtype II families was the absence of spoligotype spacer 40. All strains of family I also lacked spacer 43. The clustering rate obtained by the combination of spoligotyping and RFLP IS6110 analysis was similar for M. africanum and M. tuberculosis, as 46% and 49% of the respective isolates were grouped into clusters. The results presented demonstrate that M. africanum subtype II isolates from Kampala, Uganda, belong to two closely related genotypes, which may represent unique phylogenetic branches within the M. tuberculosis complex. We conclude that M. africanum subtype II is the main cause of human tuberculosis in Kampala, Uganda. AD - National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany. sniemann@fz.borstel.de FAU - Niemann, S AU - Niemann S FAU - Rusch-Gerdes, S AU - Rusch-Gerdes S FAU - Joloba, M L AU - Joloba ML FAU - Whalen, C C AU - Whalen CC FAU - Guwatudde, D AU - Guwatudde D FAU - Ellner, J J AU - Ellner JJ FAU - Eisenach, K AU - Eisenach K FAU - Fumokong, N AU - Fumokong N FAU - Johnson, J L AU - Johnson JL FAU - Aisu, T AU - Aisu T FAU - Mugerwa, R D AU - Mugerwa RD FAU - Okwera, A AU - Okwera A FAU - Schwander, S K AU - Schwander SK LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA Transposable Elements) RN - 0 (Oligonucleotides) SB - IM CIN - J Clin Microbiol. 2003 Mar;41(3):1345-6; author reply 1346-8. PMID: 12624085 MH - *Bacterial Typing Techniques MH - DNA Fingerprinting MH - DNA Transposable Elements MH - Genotype MH - Humans MH - Mycobacterium/*classification/*genetics MH - Oligonucleotides/*analysis MH - Phylogeny MH - *Polymorphism, Restriction Fragment Length MH - Species Specificity MH - Tuberculosis, Pulmonary/*epidemiology/*microbiology MH - Uganda/epidemiology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3398-405. PMID- 12202585 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Fluorescent amplified fragment length polymorphism analysis of Salmonella enterica serovar typhimurium reveals phage-type- specific markers and potential for microarray typing. PG - 3406-15 AB - Fluorescent amplified fragment length polymorphism (AFLP) was applied to 46 Salmonella enterica serovar Typhimurium isolates of Australian origin comprising nine phage types, by using the restriction enzymes MseI and EcoRI and all 16 possible MseI +1-EcoRI +1 primer pair combinations. AFLP in the present study showed a very good discrimination power with a Simpson index of diversity of 0.98, and 35 different AFLP patterns were observed in the 46 isolates. AFLP grouped most serovar Typhimurium isolates by phage type and enabled differentiation of phage types. Furthermore, 84 phage-type-specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage type specificity) were observed in the 46 strains studied. Eighteen phage-type-specific AFLP fragments were cloned and sequenced. Fifteen are of known genes or have a homologue in the databases. Three sequences are plasmid related, eight are phage related, and four relate to chromosomal genes. Twelve of the 18 fragments are polymorphic because the DNA is present or absent as indicated by Southern hybridization, and we see good potential to use sequences of these fragments as the basis for multiplex PCR and development of a microarray-based molecular phage-typing method for serovar Typhimurium. AD - School of Molecular and Microbial Biosciences, The University of Sydney, Sydney, New South Wales 2006, Australia. FAU - Hu, Honghua AU - Hu H FAU - Lan, Ruiting AU - Lan R FAU - Reeves, Peter R AU - Reeves PR LA - eng SI - GENBANK/AF500153 SI - GENBANK/AF500154 SI - GENBANK/AF500155 SI - GENBANK/AF500156 SI - GENBANK/AF500157 SI - GENBANK/AF500158 SI - GENBANK/AF500159 SI - GENBANK/AF500160 SI - GENBANK/AF500161 SI - GENBANK/AF500162 SI - GENBANK/AF500163 SI - GENBANK/AF500164 SI - GENBANK/AF500165 SI - GENBANK/AF500166 SI - GENBANK/AF500167 SI - GENBANK/AF500168 SI - GENBANK/AF500169 SI - GENBANK/AF500170 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Bacterial Proteins) RN - 0 (Genetic Markers) RN - 0 (Plasmids) RN - EC 3.1.21.- (Deoxyribonuclease EcoRI) RN - EC 3.1.21.- (endodeoxyribonuclease MseI) RN - EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific) SB - IM MH - Animals MH - Bacterial Proteins/genetics MH - Bacterial Typing Techniques MH - *Bacteriophage Typing MH - Cattle MH - Deoxyribonuclease EcoRI MH - Deoxyribonucleases, Type II Site-Specific MH - *Genetic Markers MH - Humans MH - Molecular Sequence Data MH - *Oligonucleotide Array Sequence Analysis MH - Phylogeny MH - Plasmids/genetics MH - Polymerase Chain Reaction MH - *Polymorphism, Restriction Fragment Length MH - Research Support, Non-U.S. Gov't MH - Salmonella Phages/genetics MH - Salmonella typhimurium/*classification/genetics/virology MH - Variation (Genetics) EDAT- 2002/08/31 10:00 MHDA- 2003/01/09 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3406-15. PMID- 12202586 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Comparison of lysis filtration and an automated blood culture system (BACTEC) for detection, quantification, and identification of odontogenic bacteremia in children. PG - 3416-20 AB - Lysis filtration (LyF) was compared with BACTEC PAEDS PLUS in estimating the prevalence of, and sensitivity for, detection of odontogenic bacteremia. Both real bacteremia and simulated bacteremia (seeded blood or saline samples) were assessed to determine the validity of LyF in estimating bacteremia. The simulated bacteremia was also used to assess the reliability of LyF to estimate intensity of bacteremia in CFU per milliliter of blood. Reference organisms were used to assess the abilities of LyF and BACTEC to isolate known oral streptococci. There was no difference in the number of CFU per milliliter of seeded saline, seeded blood, and drop cultures of the organisms plated directly onto agar. Blood cell volume had a negligible effect on the yield of organisms for simulated bacteremia. When LyF and BACTEC were compared, the time to detection of bacteremia was always significantly shorter for BACTEC. For aerobic cultures, these times were 43.7 and 9.6 h, respectively (P < 0.01). For anaerobic cultures, these times were 45.1 and 9.9 h, respectively (P < 0.01). These differences occurred as well for bacteremia following the extraction of a single tooth, with LyF and BACTEC aerobic cultures taking 78 and 30.5 h, respectively (P < 0.0001). For anaerobic cultures, the times were 90.8 and 45 h, respectively (P < 0.0004). A preextraction bacteremia was detected on 2.1% of occasions with BACTEC compared to 31% of occasions with LyF (P < 0.05) The use of LyF was an effective and reliable means of estimating the intensity of pre- and postextraction bacteremia. The values were 3.6 and 5.9 CFU/ml, respectively (P < 0.4729), and the difference was not statistically significant. In summary, BACTEC is quicker than LyF, but less sensitive. LyF provides additional important information in estimating the intensity of bacteremia. AD - Department of Oral Medicine, The Eastman Dental Institute for Oral Healthcare Sciences, University College London, London WC1X 8LD, United Kingdom. v.lucas@eastman.ucl.ac.uk FAU - Lucas, Victoria S AU - Lucas VS FAU - Lytra, Vasiliki AU - Lytra V FAU - Hassan, Thoraya AU - Hassan T FAU - Tatham, Helen AU - Tatham H FAU - Wilson, M AU - Wilson M FAU - Roberts, Graham J AU - Roberts GJ LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Culture Media) RN - 0 (Reagent Kits, Diagnostic) SB - IM MH - Adolescent MH - Aerobiosis MH - Anaerobiosis MH - Bacteremia/diagnosis/*microbiology MH - Bacteria/classification/*isolation & purification MH - Blood/*microbiology MH - Child MH - Child, Preschool MH - Colony Count, Microbial MH - Comparative Study MH - Culture Media MH - Filtration/methods MH - Humans MH - *Reagent Kits, Diagnostic MH - Sensitivity and Specificity MH - Tooth Extraction/*adverse effects EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3416-20. PMID- 12202587 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Biological and biochemical characterization of sheep scrapie in Japan. PG - 3421-6 AB - Due to the apparent absence of an agent-specific nucleic acid genome, scrapie strains cannot be classified by genome characterization, which is commonly used for the classification of many viruses. However, scrapie strains can be distinguished to some extent by biological properties such as transmissibility to experimental animals and distribution of neuropathological lesions and by biochemical properties such as the molecular mass and relative protease-resistance of the disease-specific isoform of prion protein (PrP(Sc)). In order to preliminarily characterize the scrapie strains that are prevalent in Japan, we analyzed the transmissibility of sheep scrapie isolates to mice and the relative proteinase K (PK) resistance of the corresponding PrP(Sc). The results indicate that Japanese scrapie strains can be divided into at least three groups based on biological and biochemical properties. The first group includes isolates which cause disease in mice with an incubation period of approximately 400 days and possess PrP(Sc) with relatively high PK resistance. Isolates of the second group contain PrP(Sc) that is highly resistant to PK digestion but transmit poorly to mice. The final group consists of isolates that cause disease in mice with an incubation period of less than 300 days and are associated with PrP(Sc) with reduced PK resistance. Sheep scrapie has occurred sporadically in Japan since1982, with only approximately 60 officially reported cases so far. However, the diversity of scrapie strains in the field suggested by our data raises the concern that a scrapie strain similar to the parental agent of bovine spongiform encephalopathy could exist or emerge in Japan. Thus, continuous surveillance for scrapie will be required to prevent the further spread of scrapie, not only among the sheep population but also to other species, and to eliminate any potential risk of sheep scrapie to public health. AD - Department of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan. FAU - Horiuchi, Motohiro AU - Horiuchi M FAU - Nemoto, Takuya AU - Nemoto T FAU - Ishiguro, Naotaka AU - Ishiguro N FAU - Furuoka, Hidefumi AU - Furuoka H FAU - Mohri, Shirou AU - Mohri S FAU - Shinagawa, Morikazu AU - Shinagawa M LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (PrPSc Proteins) RN - EC 3.4.21.64 (Endopeptidase K) SB - IM MH - Animals MH - Brain/metabolism MH - Endopeptidase K/*metabolism MH - Female MH - Immunoblotting MH - Japan/epidemiology MH - Mice MH - Mice, Inbred ICR MH - PrPSc Proteins/*metabolism MH - Research Support, Non-U.S. Gov't MH - Scrapie/epidemiology/*transmission MH - Sheep EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3421-6. PMID- 12202588 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Actinomyces cardiffensis sp. nov. from human clinical sources. PG - 3427-31 AB - Eight strains of a previously undescribed catalase-negative Actinomyces-like bacterium were recovered from human clinical specimens. The morphological and biochemical characteristics of the isolates were consistent with their assignment to the genus Actinomyces, but they did not appear to correspond to any recognized species. 16S rRNA gene sequence analysis showed the organisms represent a hitherto unknown species within the genus Actinomyces related to, albeit distinct from, a group of species which includes Actinomyces turicensis and close relatives. Based on biochemical and molecular genetic evidence, it is proposed that the unknown isolates from human clinical sources be classified as a new species, Actinomyces cardiffensis sp. nov. The type strain of Actinomyces cardiffensis is CCUG 44997(T). AD - Anaerobe Reference Unit, PHLS, University Hospital of Wales, Cardiff, United Kingdom. hallv@cardiff.ac.uk FAU - Hall, Val AU - Hall V FAU - Collins, Mattew D AU - Collins MD FAU - Hutson, Roger AU - Hutson R FAU - Falsen, Enevold AU - Falsen E FAU - Duerden, Brian I AU - Duerden BI LA - eng SI - GENBANK/AJ421779 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal, 16S) SB - IM MH - Actinomyces/*classification/genetics/metabolism MH - Actinomycosis/*microbiology MH - Adult MH - Aged MH - Bacterial Typing Techniques MH - DNA, Ribosomal/analysis MH - Female MH - Humans MH - Male MH - Molecular Sequence Data MH - Phylogeny MH - RNA, Ribosomal, 16S/genetics MH - Sequence Analysis, DNA EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3427-31. PMID- 12202589 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Serodiagnosis of imported schistosomiasis by a combination of a commercial indirect hemagglutination test with Schistosoma mansoni adult worm antigens and an enzyme-linked immunosorbent assay with S. mansoni egg antigens. PG - 3432-7 AB - A commercial indirect hemagglutination (IHA) test using erythrocytes coated with Schistosoma mansoni adult worm antigens (WA) and an enzyme-linked immunosorbent assay (ELISA) with S. mansoni egg antigens (SEA) were assessed for their use in serodiagnosis of imported schistosomiasis (hereafter these tests are designated WA/IHA and SEA/ELISA, respectively). The sensitivity of the tests was evaluated with sera from 75 patients with proven S. mansoni infection, 25 with proven S. haematobium infection, and 10 with clinical Katayama fever. The specificity was assessed with sera from 283 patients with various parasitic, bacterial, viral, and fungal infections and sera containing autoimmune antibodies. Sensitivities of the WA/IHA with a cutoff titer of 1:160 (WA/IHA(160)) in detecting S. mansoni, S. haematobium, S. mansoni and S. haematobium combined, and clinical Katayama fever were 88.0, 80.0, 86.0, and 70.0%, respectively, with a specificity of 98.9%. The WA/IHA with a cutoff of 1:80 (WA/IHA(80)) showed sensitivities of 94.7, 92.0, 94.0, and 90.0%, respectively, with a specificity of 94.7%. The comparable values of SEA/ELISA were 93.3, 92.0, 93.0, and 50.0%, respectively, with a specificity of 98.2%. Combined use of ELISA and WA/IHA(80) gave sensitivities of 100% for S. mansoni, S. haematobium, and S. mansoni and S. haematobium combined and 90% for Katayama fever. The specificity of this combination in detecting schistosomiasis was 92.9%. Combination of SEA/ELISA with WA/IHA(160) gave sensitivities of 98.7, 96.0, 98.0, and 80% with a specificity of 97.2%. Our findings suggest that WA/IHA and SEA/ELISA are each sensitive and specific serological tests that are easy to use for the diagnosis of imported schistosomiasis. The combined use of these two tests enabled the serological diagnosis of schistosomiasis to be achieved with very high degrees of both sensitivity and specificity. AD - Department of Microbiology, Tropical Medicine and AIDS, Academic Medical Centre, University of Amsterdam, The Netherlands. T.vanGool@amc.uva.nl FAU - Van Gool, Tom AU - Van Gool T FAU - Vetter, Hans AU - Vetter H FAU - Vervoort, Tony AU - Vervoort T FAU - Doenhoff, Michael J AU - Doenhoff MJ FAU - Wetsteyn, Jose AU - Wetsteyn J FAU - Overbosch, David AU - Overbosch D LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antibodies, Helminth) RN - 0 (Antigens, Helminth) SB - IM MH - Animals MH - Antibodies, Helminth/*blood MH - Antigens, Helminth/*immunology MH - Enzyme-Linked Immunosorbent Assay MH - Hemagglutination Tests MH - Humans MH - Ovum/immunology MH - Parasite Egg Count MH - Schistosoma mansoni/*immunology MH - Schistosomiasis haematobia/*diagnosis/parasitology MH - Schistosomiasis mansoni/*diagnosis/parasitology MH - Sensitivity and Specificity MH - Serologic Tests MH - *Travel MH - Tropical Climate EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3432-7. PMID- 12202590 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Taxonomic subgroups of Pasteurella multocida correlate with clinical presentation. PG - 3438-41 AB - Pasteurella multocida is a small nonmotile gram-negative coccobacillus that is found in the nasopharynx and gastrointestinal tract of many wild and domesticated animals. In humans it most commonly causes cellulitis and localized superficial skin abscesses following an animal bite or scratch. The respiratory tract is the second most common site of infection for PASTEURELLA: Of the more than 17 species of Pasteurella known, Pasteurella multocida subsp. multocida and Pasteurella multocida subsp. septica are among the most common pathogens in humans. With the use of molecular techniques, distinction between different subspecies of P. multocida can be made more easily and accurately. We used the sequence of the 16S ribosomal DNA (rDNA) and repetitive extragenic palindromic sequence-PCR (REP-PCR) to characterize 20 strains (14 of P. multocida subsp. multocida and 6 of P. multocida subsp. septica; the 16S rDNA is identical for P. multocida subsp. multocida and Pasteurella multocida subsp. gallicida but differs from that of P. multocida subsp. septica) isolated from various anatomic sites. We found excellent correlation between the 16S rDNA sequence (a marker for a small conserved region of the genome), REP-PCR (a marker for a large portion of the genome), and biochemical tests (trehalose and sorbitol). We also found a correlation between the clinical presentation and the taxonomic group, with P. multocida subsp. septica more often associated with wounds than with respiratory infections (67 versus 17%, respectively) (P < 0.05, Z test) and P. multocida subsp. multocida more often associated with respiratory infections than with wounds (71 versus 14%, respectively) (P < 0.05, Z test). AD - Department of Pathology, Baylor College of Medicine Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, Houston, Texas USA. FAU - Chen, Henry I AU - Chen HI FAU - Hulten, Kristina AU - Hulten K FAU - Clarridge, Jill E 3rd AU - Clarridge JE 3rd LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal, 16S) RN - 50-70-4 (Sorbitol) RN - 99-20-7 (Trehalose) SB - IM MH - Bacterial Typing Techniques MH - DNA, Ribosomal/analysis MH - Humans MH - Pasteurella Infections/microbiology/*physiopathology MH - Pasteurella multocida/*classification/*genetics/metabolism MH - Polymerase Chain Reaction/methods MH - RNA, Ribosomal, 16S/genetics MH - Repetitive Sequences, Nucleic Acid/genetics MH - Respiratory Tract Infections/*microbiology MH - Sequence Analysis, DNA MH - Sorbitol/metabolism MH - Trehalose/metabolism MH - Wound Infection/*microbiology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3438-41. PMID- 12202591 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Genotypic diversity of clinical Actinomyces species: phenotype, source, and disease correlation among genospecies. PG - 3442-8 AB - We determined the frequency distribution of Actinomyces spp. recovered in a routine clinical laboratory and investigated the clinical significance of accurate identification to the species level. We identified 92 clinical strains of Actinomyces, including 13 strains in the related Arcanobacterium-Actinobaculum taxon, by 16S rRNA gene sequence analysis and recorded their biotypes, sources, and disease associations. The clinical isolates clustered into 21 genogroups. Twelve genogroups (74 strains) correlated with a known species, and nine genogroups (17 strains) did not. The individual species had source and disease correlates. Actinomyces turicensis was the most frequently isolated species and was associated with genitourinary tract specimens, often with other organisms and rarely with inflammatory cells. Actinomyces radingae was most often associated with serious, chronic soft tissue abscesses of the breast, chest, and back. Actinomyces europaeus was associated with skin abscesses of the neck and genital areas. Actinomyces lingnae, Actinomyces gravenitzii, Actinomyces odontolyticus, and Actinomyces meyeri were isolated from respiratory specimens, while A. odontolyticus-like strains were isolated from diverse sources. Several of the species were commonly coisolated with a particular bacterium: Actinomyces israelii was the only Actinomyces spp. coisolated with Actinobacillus (Haemophilus) actinomycetemcomitans; Actinomyces meyeri was coisolated with Peptostreptococcus micros and was the only species other than A. israelii associated with sulfur granules in histological specimens. Most genogroups had consistent biotypes (as determined with the RapID ANA II system); however, strains were misidentified, and many codes were not in the database. One biotype was common to several genogroups, with all of these isolates being identified as A. meyeri. Despite the recent description of new Actinomyces spp., 19% of the isolates recovered in our routine laboratory belonged to novel genospecies. One novel group with three strains, Actinomyces houstonensis sp. nov., was phenotypically similar to A. meyeri and A. turicensis but was genotypically closest to Actinomyces neuii. A. houstonensis sp. nov. was associated with abscesses. Our data documented consistent site and disease associations for 21 genogroups of Actinomyces spp. that provide greater insights into appropriate treatments. However, we also demonstrated a complexity within the Actinomyces genus that compromises the biochemical identification of Actinomyces that can be performed in most clinical laboratories. It is our hope that this large group of well-defined strains will be used to find a simple and accurate biochemical test for differentiation of the species in routine laboratories. AD - Department of Pathology, Baylor College of Medicine, Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, Houston, Texas, USA. jill.clarridge@med.va.gov FAU - Clarridge, Jill E 3rd AU - Clarridge JE 3rd FAU - Zhang, Qing AU - Zhang Q LA - eng SI - GENBANK/AF457638 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal, 16S) SB - IM MH - Actinomyces/*classification/*genetics/metabolism/pathogenicity MH - Actinomycosis/*microbiology/*physiopathology MH - Bacterial Typing Techniques MH - DNA, Ribosomal/analysis MH - Genotype MH - Humans MH - Molecular Sequence Data MH - Phenotype MH - Phylogeny MH - RNA, Ribosomal, 16S/genetics MH - Sequence Analysis, DNA MH - *Variation (Genetics) EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3442-8. PMID- 12202592 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens. PG - 3449-54 AB - We describe a novel adaptation of the TaqMan PCR assay which potentially allows for highly sensitive detection of any eubacterial species with simultaneous species identification. Our system relies on a unique multiprobe design in which a single set of highly conserved sequences encoded by the 16S rRNA gene serves as the primer pair and is used in combination with both an internal highly conserved sequence, the universal probe, and an internal variable region, the species-specific probe. A pre-PCR ultrafiltration step effectively decontaminates or removes background DNA. The TaqMan system described reliabAly detected 14 common bacterial species with a detection limit of 50 fg. Further, highly sensitive and specific pathogen detection was demonstrated with a prototype species-specific probe designed to detect Staphylococcus aureus. This assay has broad potential in the clinical arena for rapid and specific diagnosis of infectious diseases. AD - Department of Emergency Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. FAU - Yang, Samuel AU - Yang S FAU - Lin, Shin AU - Lin S FAU - Kelen, Gabor D AU - Kelen GD FAU - Quinn, Thomas C AU - Quinn TC FAU - Dick, James D AU - Dick JD FAU - Gaydos, Charlotte A AU - Gaydos CA FAU - Rothman, Richard E AU - Rothman RE LA - eng SI - GENBANK/AF179251 SI - GENBANK/AF179252 SI - GENBANK/AF179253 SI - GENBANK/AF179254 SI - GENBANK/AF179255 SI - GENBANK/AF179256 SI - GENBANK/AF179257 SI - GENBANK/AF179258 SI - GENBANK/AF179259 SI - GENBANK/AF179260 SI - GENBANK/AF179261 SI - GENBANK/AF179262 SI - GENBANK/AF179263 SI - GENBANK/AF179264 SI - GENBANK/AF179265 SI - GENBANK/AF179266 SI - GENBANK/AF209195 SI - GENBANK/AF378325 SI - GENBANK/AF378326 GR - M01RR00052-39-5(S1)/RR/NCRR PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA Probes) RN - 0 (DNA, Bacterial) RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal, 16S) RN - EC 2.7.7.- (Taq Polymerase) SB - IM MH - Bacteria/*classification/*genetics MH - Bacterial Infections/*microbiology MH - DNA Probes MH - DNA, Bacterial/analysis MH - DNA, Ribosomal/analysis MH - Filtration/methods MH - Humans MH - Molecular Sequence Data MH - Polymerase Chain Reaction/*methods MH - RNA, Ribosomal, 16S/*genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sensitivity and Specificity MH - Sequence Analysis, DNA MH - Species Specificity MH - Taq Polymerase EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3449-54. PMID- 12202593 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Characterization of hepatitis C virus quasispecies by matrix-assisted laser desorption ionization-time of flight (mass spectrometry) mutation detection. PG - 3455-62 AB - Hepatitis C virus (HCV), the causative agent of hepatitis C, frequently causes chronic infection. The mechanisms of viral persistence continue to be the object of investigation. An important aspect of HCV chronic infection is the quasispecies nature of the viral population, which has been particularly well documented in the hypervariable region 1 of the E2 glycoprotein. Recent studies show that characterization of the quasispecies diversity at the amino acid level can help to predict the outcome of HCV infection. Currently the accurate characterization of HCV quasispecies requires the cloning of PCR products, followed by the sequencing of many clones. In this study we present a new method to characterize HCV quasispecies, based on in vitro translation of the amplicons, followed by mass spectrometry analysis of the resulting peptide mix. The assay was used on reference HCV samples and on clinical samples. In principle, this method could be applied to other chronic viral infections in which quasispecies play a role. AD - Divisions of Microbiology. Gastroenterology and Nutrition, Hospital for Sick Children, Toronto, Ontario, Canada. FAU - Ayers, Melissa AU - Ayers M FAU - Siu, Karen AU - Siu K FAU - Roberts, Eve AU - Roberts E FAU - Garvin, Alex M AU - Garvin AM FAU - Tellier, Raymond AU - Tellier R LA - eng PT - Case Reports PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (HVR1 protein, Hepatitis C virus) RN - 0 (Peptides) RN - 0 (Viral Proteins) SB - IM MH - Child, Preschool MH - Female MH - Genotype MH - Hepacivirus/*classification/*genetics MH - Hepatitis C, Chronic/*virology MH - Humans MH - Infant MH - Male MH - *Mutation MH - Peptides/chemical synthesis/chemistry/genetics MH - Protein Biosynthesis MH - Research Support, Non-U.S. Gov't MH - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods MH - Viral Proteins/chemistry/genetics/metabolism EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3455-62. PMID- 12202594 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Molecular typing of papillomatous digital dermatitis-associated Treponema isolates based on analysis of 16S-23S ribosomal DNA intergenic spacer regions. PG - 3463-9 AB - Papillomatous digital dermatitis (PDD), an emerging infectious disease of cattle, is characterized by painful, ulcerative foot lesions. The detection of high numbers of invasive spirochetes in PDD lesions suggests an important role for these organisms in the pathogenesis of PDD. PDD-associated spirochetes have phenotypic characteristics consistent with members of the genus TREPONEMA: Partial 16S ribosomal DNA (rDNA) sequence analysis of clonal isolates from California cattle showed that they comprise three phylotypes which cluster closely with human-associated Treponema spp. of the oral cavity (T. denticola and T. medium/T. vincentii) or genital area (T. phagedenis). The goal of our study was to apply 16S-23S rDNA intergenic spacer region (ISR) sequence analysis to the molecular typing of U.S. PDD-associated Treponema isolates. This methodology has potentially greater discriminatory power for differentiation of closely related bacteria than 16S rDNA analysis. We PCR amplified, cloned, and sequenced the ISRs from six California PDD-associated Treponema isolates and, for comparative purposes, one strain each of T. denticola, T. medium, T. vincentii, and T. phagedenis. Two ISRs that varied in length and composition were present in all the PDD-associated Treponema isolates and in T. denticola, T. medium, and T. phagedenis. ISR1 contained a tRNA(Ala) gene, while ISR2 contained a tRNA(Ile) gene. Only a single ISR (ISR1) was identified in T. vincentii. Comparative analyses of the ISR1 and ISR2 sequences indicated that the California PDD-associated Treponema isolates comprised three phylotypes, in agreement with the results of 16S rDNA analysis. PCR amplification of the 16S-tRNA(Ile) region of ISR2 permitted rapid phylotyping of California and Iowa PDD-associated Treponema isolates based on product length polymorphisms. AD - Program in Infectious Diseases, Department of Epidemiology, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7435, USA. lstamm@email.unc.edu FAU - Stamm, L V AU - Stamm LV FAU - Bergen, H L AU - Bergen HL FAU - Walker, R L AU - Walker RL LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Ribosomal Spacer) RN - 0 (RNA, Ribosomal, 16S) RN - 0 (RNA, Ribosomal, 23S) SB - IM MH - Animals MH - Bacterial Typing Techniques MH - Base Sequence MH - California MH - Cattle MH - Cattle Diseases/*microbiology MH - DNA, Ribosomal Spacer/*analysis MH - Dermatitis/microbiology/*veterinary MH - Humans MH - Iowa MH - Molecular Sequence Data MH - RNA, Ribosomal, 16S/*genetics MH - RNA, Ribosomal, 23S/*genetics MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - Treponema/*classification/genetics MH - Treponemal Infections/microbiology/veterinary EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3463-9. PMID- 12202595 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. PG - 3470-5 AB - The pathogenicity locus (PaLoc) of Clostridium difficile contains toxin A and B genes and three accessory genes, including tcdD and tcdC, which are supposed to code for the positive and negative regulators of toxin expression, respectively. Different studies have described variations in C. difficile toxin A and B genes, but little is known about C. difficile variants for the accessory genes. The PaLoc of several C. difficile clinical isolates was investigated by three different PCR methods with the aim to identify variant strains. Of the toxinogenic C. difficile strains examined, 25% showed variations. No correlation between C. difficile variant strains and key patient groups was found. Interestingly, all of these strains showed a variant tcdC gene. Three different tcdC alleles were identified, and one of these had a nonsense mutation which reduced the TcdC protein from 232 to 61 amino acids. It is possible that different TcdC variants affect toxin production differently, a hypothesis with important implications for the pathogenic potential of variant C. difficile strains. AD - Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanita, Rome, Italy. FAU - Spigaglia, Patrizia AU - Spigaglia P FAU - Mastrantonio, Paola AU - Mastrantonio P LA - eng SI - GENBANK/AJ428941 SI - GENBANK/AJ428942 SI - GENBANK/AJ428943 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Bacterial Proteins) RN - 0 (Bacterial Toxins) RN - 0 (Enterotoxins) RN - 0 (Repressor Proteins) RN - 0 (TcdC protein, Clostridium difficile) RN - 0 (tcdA protein, Clostridium difficile) RN - 0 (toxB protein, Clostridium difficile) SB - IM MH - Amino Acid Sequence MH - Bacterial Proteins/*genetics/metabolism MH - Bacterial Toxins/genetics/metabolism MH - Base Sequence MH - Clostridium difficile/classification/genetics/*pathogenicity MH - Enterocolitis, Pseudomembranous/*microbiology MH - Enterotoxins/genetics/metabolism MH - *Gene Expression Regulation, Bacterial MH - Humans MH - Molecular Sequence Data MH - Polymerase Chain Reaction MH - *Polymorphism, Genetic MH - Repressor Proteins/*genetics/metabolism MH - Sequence Analysis, DNA MH - Variation (Genetics) MH - Virulence/genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3470-5. PMID- 12202596 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Labor and cost requirements of two commercial assays for qualitative molecular detection of hepatitis C virus. PG - 3476-7 AB - The Bayer transcription-mediated amplification (TMA) and the Roche PCR Amplicor version 2.0 molecular assays for the qualitative detection of hepatitis C virus were compared for cost, hands-on time, assay duration, and complexity. The TMA assay compares well to PCR and may be especially useful for laboratories with large numbers of test requests. AD - Department of Pathology, Stanford University School of Medicine, California, USA. FAU - Schrijver, Iris AU - Schrijver I FAU - Baron, Ellen Jo AU - Baron EJ LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (RNA, Viral) RN - 0 (Reagent Kits, Diagnostic) SB - IM MH - Costs and Cost Analysis MH - Health Manpower MH - Hepacivirus/*isolation & purification/physiology MH - Hepatitis C/*diagnosis/virology MH - Humans MH - Nucleic Acid Amplification Techniques/*economics/methods MH - Polymerase Chain Reaction/economics MH - RNA, Viral/*analysis MH - Reagent Kits, Diagnostic/*economics MH - Research Support, Non-U.S. Gov't MH - Time Factors MH - Transcription, Genetic MH - Viral Load EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3476-7. PMID- 12202597 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Evaluation of genotype and LiPA MYCOBACTERIA assays for identification of Finnish mycobacterial isolates. PG - 3478-81 AB - Two DNA strip assays, INNO-LiPA MYCOBACTERIA and GenoType Mykobakterien, were evaluated for identification of 81 Finnish mycobacterial isolates. The LiPA assay correctly identified 89.4% of the 66 isolates studied, and the GenoType assay identified 95.1% of 81 isolates. The GenoType assay had a wider selection of species and less stringent temperature requirements. AD - National Public Health Institute. Turku Graduate School of Biomedical Sciences Department of Medical Microbiology, University of Turku, Turku, Finland. FAU - Makinen, Johanna AU - Makinen J FAU - Sarkola, Aleksi AU - Sarkola A FAU - Marjamaki, Merja AU - Marjamaki M FAU - Viljanen, Matti K AU - Viljanen MK FAU - Soini, Hanna AU - Soini H LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA Probes) RN - 0 (DNA, Bacterial) RN - 0 (Reagent Kits, Diagnostic) RN - 0 (Reagent Strips) SB - IM MH - Bacterial Typing Techniques MH - *DNA Probes MH - DNA, Bacterial/analysis MH - Finland MH - Genotype MH - Humans MH - Mycobacterium/*classification/genetics MH - Mycobacterium Infections/*microbiology MH - Nucleic Acid Hybridization/*methods MH - Polymerase Chain Reaction/methods MH - *Reagent Kits, Diagnostic MH - *Reagent Strips EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3478-81. PMID- 12202598 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Are three sputum acid-fast bacillus smears necessary for discontinuing tuberculosis isolation? PG - 3482-4 AB - To evaluate the efficacy of three sputum acid-fast bacillus (AFB) smears to rule out pulmonary tuberculosis, sputum AFB smear and culture results were analyzed at two university-affiliated teaching hospitals. The negative predictive value of the smear increased by only 0.2% on days 2 and 3 each, indicating that in low-prevalence populations, there is limited value in requiring three negative sputum AFB smears before discontinuing tuberculosis isolation. AD - Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901, USA. phillip.mathew@mdsps.com FAU - Mathew, Philip AU - Mathew P FAU - Kuo, Yen-Hong AU - Kuo YH FAU - Vazirani, Bindu AU - Vazirani B FAU - Eng, Robert H K AU - Eng RH FAU - Weinstein, Melvin P AU - Weinstein MP LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Culture Media) SB - IM MH - Culture Media MH - Hospitals, Teaching/*standards MH - Humans MH - Mycobacterium tuberculosis/*isolation & purification MH - New Jersey MH - Predictive Value of Tests MH - Sensitivity and Specificity MH - Sputum/*microbiology MH - Staining and Labeling/*methods MH - Tuberculosis, Pulmonary/*diagnosis/microbiology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3482-4. PMID- 12202599 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Direct PCR detection of Burkholderia cepacia complex and identification of its genomovars by using sputum as source of DNA. PG - 3485-8 AB - We developed a nested PCR assay that detects the recA gene of the Burkholderia cepacia complex in sputum. The product of the first PCR round is also used to identify the genomovar of the pathogen. The protocol achieves high sensitivity and specificity with simple interpretation of genomovar status. AD - 2nd Department of Pediatrics, 2nd Medical School of Charles University, Prague, Czech Republic. pavel.drevinek@Lfmotol.cuni.cz FAU - Drevinek, Pavel AU - Drevinek P FAU - Hrbackova, Hana AU - Hrbackova H FAU - Cinek, Ondrej AU - Cinek O FAU - Bartosova, Jana AU - Bartosova J FAU - Nyc, Otakar AU - Nyc O FAU - Nemec, Alexandr AU - Nemec A FAU - Pohunek, Petr AU - Pohunek P LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Bacterial) RN - EC 2.7.7.- (Rec A Recombinases) SB - IM EIN - J Clin Microbiol 2002 Dec;40(12):4806 MH - Adolescent MH - Adult MH - Burkholderia Infections/diagnosis/epidemiology/microbiology MH - Burkholderia cepacia/*classification/genetics/*isolation & purification MH - Child MH - Child, Preschool MH - Cystic Fibrosis/epidemiology/*microbiology MH - Czech Republic/epidemiology MH - DNA, Bacterial/*analysis MH - Female MH - Humans MH - Infant MH - Infant, Newborn MH - Male MH - Polymerase Chain Reaction/*methods MH - Rec A Recombinases/genetics MH - Research Support, Non-U.S. Gov't MH - Sensitivity and Specificity MH - Sputum/*chemistry EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3485-8. PMID- 12202600 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Increasing incidence of candidemia: results from a 20-year nationwide study in Iceland. PG - 3489-92 AB - A nationwide study on candidemia was conducted in Iceland from 1980 to 1999. The annual incidence increased from 1.4 cases/100,000 inhabitants/year between 1980 and 1984 to 4.9 cases/100,000 inhabitants/year between 1995 and 1999 (P < 0.0001). Candidemia episodes at university hospitals increased from 0.15/1,000 admissions to 0.55/1,000 admissions (P < 0.0001). Candida albicans was the predominant species responsible (64.4%). The national import of fluconazole increased approximately fourfold during the second half of the study, but increased resistance to this agent was not observed. AD - University of Iceland, Reykjavik, Iceland. FAU - Asmundsdottir, Lena Ros AU - Asmundsdottir LR FAU - Erlendsdottir, Helga AU - Erlendsdottir H FAU - Gottfredsson, Magnus AU - Gottfredsson M LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antifungal Agents) RN - 0 (Culture Media) SB - IM MH - Adolescent MH - Adult MH - Age Distribution MH - Aged MH - Antifungal Agents/pharmacology MH - Blood/microbiology MH - Candida/*classification/drug effects/*isolation & purification MH - Candida albicans/drug effects/isolation & purification MH - Candidiasis/*epidemiology/microbiology MH - Child MH - Child, Preschool MH - Culture Media MH - Fungemia/*epidemiology/microbiology MH - Humans MH - Iceland/epidemiology MH - Incidence MH - Infant MH - Microbial Sensitivity Tests MH - Middle Aged MH - Research Support, Non-U.S. Gov't EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3489-92. PMID- 12202601 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Rate of hepatitis B virus infection in pregnant women determined by a monoclonal hepatitis B surface antigen immunoassay. PG - 3493-6 AB - The rate of HBsAg in 6,976 B-human chorionic gonadotropin (B-hCG)-positive specimens, as determined by the Auszyme Monoclonal assay (Abbott Laboratories, Abbott Park, Ill.), was 0.56% (39 of 6,986 repeatedly reactive [RR] and confirmed-positive specimens). All RR and confirmed specimens were hepatitis B virus positive by at least one additional test, yielding an assay specificity of 99.96%. The findings argue against unique attributes in the pregnant population that might produce inaccurate assay results. AD - Hepatitis Business Team, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064, USA. FAU - Gotstein, Melissa G AU - Gotstein MG FAU - Aide, Paula M AU - Aide PM FAU - Coleman, Paul F AU - Coleman PF FAU - Sanborn, Mark R AU - Sanborn MR LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antibodies, Monoclonal) RN - 0 (Hepatitis B Antibodies) RN - 0 (Hepatitis B Surface Antigens) RN - 0 (Reagent Kits, Diagnostic) SB - IM MH - Antibodies, Monoclonal/*diagnostic use/immunology MH - Female MH - Hepatitis B/diagnosis/*epidemiology MH - Hepatitis B Antibodies/diagnostic use/immunology MH - Hepatitis B Surface Antigens/*blood MH - Hepatitis B virus/*isolation & purification MH - Humans MH - Immunoassay MH - Pregnancy MH - Pregnancy Complications, Infectious/diagnosis/*epidemiology/virology MH - Prenatal Care MH - Reagent Kits, Diagnostic MH - Sensitivity and Specificity EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3493-6. PMID- 12202602 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Technical improvement to prevent DNA degradation of enteric pathogens in pulsed-field gel electrophoresis. PG - 3497-8 AB - This study used a modified pulsed-field gel electrophoresis (PFGE) method with HEPES as a running buffer to prevent electrophoresis-related DNA degradation of nine Salmonella enterica subsp. enterica serovar Ohio, seven Salmonella serovar Newport, and two enterohemorrhagic Escherichia coli (non-O157) strains. All strains yielded identifiable bands with this method in contrast to a commonly applied PFGE method using Tris buffer. AD - Laboratory of Enteric Pathogens, National Public Health Institute, Helsinki, Finland. FAU - Koort, Joanna M K AU - Koort JM FAU - Lukinmaa, Susanna AU - Lukinmaa S FAU - Rantala, Marjatta AU - Rantala M FAU - Unkila, Erja AU - Unkila E FAU - Siitonen, Anja AU - Siitonen A LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Buffers) RN - 0 (DNA, Bacterial) RN - 7365-45-9 (HEPES) SB - IM MH - Bacterial Typing Techniques/*methods MH - *Buffers MH - DNA, Bacterial/*chemistry MH - Electrophoresis, Gel, Pulsed-Field/methods MH - Escherichia coli/classification/*genetics MH - *HEPES MH - Humans MH - Salmonella enterica/classification/*genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3497-8. PMID- 12202603 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Early detection of negative BACTEC MGIT 960 cultures by PCR-reverse cross-blot hybridization assay. PG - 3499-501 AB - We evaluated the efficacy of a PCR-reverse cross-blot hybridization assay, a test which permits identification of mycobacteria by means of species-specific probes and a Mycobacterium-specific probe, for early detection of negative BACTEC MGIT 960 mycobacterial cultures. Aliquots of 549 cultures were collected 7 days after the culture media were inoculated with various clinical specimens and tested with the molecular assay. PCR results were compared to those obtained at the end times with the BACTEC MGIT 960 system. Of the 549 specimens analyzed, 484 were found to be negative and 64 were found positive by both methods; one specimen, found to be positive by the BACTEC MGIT 960 system, was identified as negative by the molecular assay. In view of its high negative predictive value (99.8%), the PCR-reverse cross-blot hybridization assay appears to be a valid tool for early detection of negative BACTEC MGIT 960 cultures. AD - Istituto di Microbiologia, Universita Cattolica del Sacro Cuore, Rome, Italy. FAU - Romano, Lucio AU - Romano L FAU - Sanguinetti, Maurizio AU - Sanguinetti M FAU - Posteraro, Brunella AU - Posteraro B FAU - Ardito, Fausta AU - Ardito F FAU - Gesu, Giovanni AU - Gesu G FAU - Schito, Anna Maria AU - Schito AM FAU - Fadda, Giovanni AU - Fadda G LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Culture Media) RN - 0 (DNA Probes) SB - IM MH - Bacteriological Techniques MH - Culture Media MH - DNA Probes MH - Humans MH - Mycobacterium/genetics/*growth & development/*isolation & purification MH - Mycobacterium Infections/*microbiology MH - Nucleic Acid Hybridization/*methods MH - Polymerase Chain Reaction/*methods MH - Predictive Value of Tests MH - Research Support, Non-U.S. Gov't MH - Sensitivity and Specificity MH - Species Specificity EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3499-501. PMID- 12202604 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Persistent endemicity of Salmonella bongori 48:z(35):--in Southern Italy: molecular characterization of human, animal, and environmental isolates. PG - 3502-5 AB - From 1984 to 1999, we collected 31 isolates of the rare serovar Salmonella bongori 48:z(35):- in southern Italy. Twenty-four of the isolates were from cases of acute enteritis in humans. Pulsed-field gel electrophoresis analysis showed that all but one of our isolates were at least 80% similar. Our findings suggest that genetically related S. bongori 48:z(35):- strains are endemically circulating in southern Italy. AD - Dipartimento di Igiene e Microbiologia G. D'Alessandro, Universita di Palermo, I-90127 Palermo, Italy. gmgiamm@libero.it FAU - Giammanco, Giovanni M AU - Giammanco GM FAU - Pignato, Sarina AU - Pignato S FAU - Mammina, Caterina AU - Mammina C FAU - Grimont, Francine AU - Grimont F FAU - Grimont, Patrick A D AU - Grimont PA FAU - Nastasi, Antonino AU - Nastasi A FAU - Giammanco, Giuseppe AU - Giammanco G LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Bacterial) SB - IM MH - Adult MH - Animals MH - Bacterial Typing Techniques MH - Child, Preschool MH - DNA, Bacterial/analysis MH - Electrophoresis, Gel, Pulsed-Field MH - *Endemic Diseases MH - Enteritis/*epidemiology/microbiology MH - Humans MH - Infant MH - Italy/epidemiology MH - Salmonella/classification/genetics/*growth & development/pathogenicity MH - Salmonella Infections/*epidemiology/microbiology MH - Salmonella Infections, Animal/epidemiology/microbiology MH - Water Microbiology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3502-5. PMID- 12202605 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Comparison of serological detection methods for diagnosis of Ehrlichia canis infections in dogs. PG - 3506-8 AB - We determined the value of four serological assays for the diagnosis of canine monocytic ehrlichiosis by comparing them to the indirect fluorescent-antibody assay "gold standard." The specificity of Dip-S-Ticks was significantly lower than that of all of the other tests evaluated. The sensitivity of Dip-S-Ticks was significantly higher than that of Snap3Dx or the Snap Canine Combo. The sensitivity of the rMAP2 enzyme-linked immunosorbent assay (ELISA) was significantly higher than that of the Snap Canine Combo. The accuracy levels of the rMAP2 ELISA, Snap3Dx, Dip-S-Ticks, and Snap Canine Combo were 97.0, 89.8, 85.1, and 82.9%, respectively. AD - Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610, USA. FAU - Belanger, Myriam AU - Belanger M FAU - Sorenson, Heather L AU - Sorenson HL FAU - France, Michelle K AU - France MK FAU - Bowie, Michael V AU - Bowie MV FAU - Barbet, Anthony F AU - Barbet AF FAU - Breitschwerdt, Edward B AU - Breitschwerdt EB FAU - Alleman, A Rick AU - Alleman AR LA - eng GR - AI45580-01S1/AI/NIAID PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antibodies, Bacterial) RN - 0 (Antigens, Bacterial) RN - 0 (Recombinant Proteins) SB - IM MH - Animals MH - Antibodies, Bacterial/*blood MH - Antigens, Bacterial/genetics/immunology MH - Comparative Study MH - Dog Diseases/*diagnosis/microbiology MH - Dogs MH - Ehrlichia/*immunology MH - Ehrlichiosis/diagnosis/*veterinary MH - Enzyme-Linked Immunosorbent Assay MH - Fluorescent Antibody Technique, Indirect MH - Recombinant Proteins/immunology MH - Reproducibility of Results MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sensitivity and Specificity MH - Serologic Tests EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3506-8. PMID- 12202606 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Evaluation of dipstick serologic tests for diagnosis of brucellosis and typhoid Fever in egypt. PG - 3509-11 AB - Two dipstick assays for the detection of Brucella- and typhoid-specific immunoglobulin M, recently developed by the Royal Tropical Institute of The Netherlands, were evaluated by use of 85 plasma samples from Egyptian patients. Both dipsticks were simple and accurate rapid diagnostic assays, and they can be useful adjuncts for the diagnosis of typhoid fever and brucellosis. AD - U.S. Medical Research Unit No. 3, Cairo, Egypt. IsmailT@namru3.med.navy.mil FAU - Ismail, Tharwat F AU - Ismail TF FAU - Smits, Henk AU - Smits H FAU - Wasfy, Momtaz O AU - Wasfy MO FAU - Malone, Joseph L AU - Malone JL FAU - Fadeel, Moustafa A AU - Fadeel MA FAU - Mahoney, Frank AU - Mahoney F LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antibodies, Bacterial) RN - 0 (Reagent Kits, Diagnostic) RN - 0 (Reagent Strips) SB - IM MH - Antibodies, Bacterial/*blood MH - Brucella/immunology MH - Brucellosis/*diagnosis MH - Egypt MH - Humans MH - *Reagent Kits, Diagnostic MH - *Reagent Strips MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Salmonella typhi/immunology MH - Sensitivity and Specificity MH - Serologic Tests MH - Typhoid Fever/*diagnosis EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3509-11. PMID- 12202607 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Simple and reliable method for detection and genotyping of hepatitis C virus RNA in dried blood spots stored at room temperature. PG - 3512-4 AB - We describe a simple, sensitive, and reproducible method for using whole blood collected onto filter paper (dried blood spots) for detection and genotyping of hepatitis C virus RNA that can be useful in large field studies, particularly in settings where collection, preparation, storage, and shipment of samples at controlled temperature can be difficult. AD - National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy. FAU - Solmone, Mariacarmela AU - Solmone M FAU - Girardi, Enrico AU - Girardi E FAU - Costa, Francesco AU - Costa F FAU - Pucillo, Leopoldo AU - Pucillo L FAU - Ippolito, Giuseppe AU - Ippolito G FAU - Capobianchi, Maria R AU - Capobianchi MR LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (RNA, Viral) SB - IM MH - Blood Specimen Collection/*methods MH - Genotype MH - Hepacivirus/*classification/genetics/*isolation & purification MH - Hepatitis C/virology MH - Humans MH - RNA, Viral/*blood MH - Reproducibility of Results MH - Research Support, Non-U.S. Gov't MH - Temperature EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3512-4. PMID- 12202608 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Evaluation of a new dot blot enzyme immunoassay (directigen flu A+B) for simultaneous and differential detection of influenza a and B virus antigens from respiratory samples. PG - 3515-7 AB - We report a prospective evaluation of a new dot blot enzyme immunoassay (EIA) method for the direct, rapid, qualitative, simultaneous, and differential detection of the influenza A (IA) and B (IB) virus antigen in different respiratory samples. The EIA method was compared with the shell vial culture system (MDCK cell line) used with the same samples. We studied 160 samples from 93 (58.1%) pediatric patients (hospital emergency room) and from 67 (41.9%) adult patients (sentinel network). Seventy-four(46.2%) samples were considered positive; of them, 46 (62.2%) were from pediatric patients and 28 (37.8%) were from an adult group (P < 0.05), with overall positive values of 49.9% and 41.7%, respectively. All 74 (100%) of the positive samples were isolated in cell culture versus the 68.9% that were detected as positive by the new EIA method (P < 0.05). Of the 41 samples positive for the IA virus, the EIA detected 34 (82.9%) positive samples; of the 33 samples positive for the IB virus, the EIA detected 17 (51.5%) positive samples (P < 0.05). No false-positive reaction was detected with the EIA method (specificity and positive predictive value of 100%). The overall results obtained in the comparison between the new EIA and the shell vial culture had a sensibility of 82.9% and predictive negative values of 92.4% for the IA virus and 51.5% and 84.3%, respectively, for the IB virus. This evaluation shows sensitivity and specificity percentages for the new EIA method that is acceptable for routine use in IA virus detection. The results obtained were worse for IB virus detection, but this new EIA method is actually the only one with the capacity to differentiate between the two influenza viruses. AD - Virology Unit, Clinical Microbiology Service, University Hospital Son Dureta (Universitat Illes Balears), 07014 Palma de Mallorca, Spain. jreina@hsd.es FAU - Reina, Jordi AU - Reina J FAU - Padilla, Emma AU - Padilla E FAU - Alonso, Fermin AU - Alonso F FAU - Ruiz De Gopegui, Enrique AU - Ruiz De Gopegui E FAU - Munar, Maria AU - Munar M FAU - Mari, Margarita AU - Mari M LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antigens, Viral) SB - IM MH - Adult MH - Antigens, Viral/*analysis MH - Child MH - Diagnosis, Differential MH - Humans MH - Immunoenzyme Techniques/*methods MH - Influenza/*virology MH - Influenza A Virus, Human/classification/*isolation & purification MH - Influenza B virus/classification/*isolation & purification MH - Prospective Studies MH - Research Support, Non-U.S. Gov't MH - Respiratory System/*virology MH - Virus Cultivation EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3515-7. PMID- 12202609 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20031114 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Molecular characterization confirms the presence of a divergent strain of canine coronavirus (UWSMN-1) in Australia. PG - 3518-22 AB - Canine coronavirus (CCV) UWSMN-1 was originally identified from an outbreak of fatal gastroenteritis in breeding colonies. In this report, we examined whether UWSMN-1 represents a novel divergent strain or is the result of recombination events between canine and feline coronavirus strains. Sequencing of various regions of the spike and polymerase genes confirms that UWSMN-1 is widely divergent from other CCV and feline coronavirus strains. These data raise the possibility that this strain is the first member of a novel third subtype of CCV. AD - School of Science, Food and Horticulture, University of Western Sydney, Penrith South DC, New South Wales, Australia. m.naylor@garvan.org.au FAU - Naylor, Matthew J AU - Naylor MJ FAU - Walia, Charanjiv S AU - Walia CS FAU - McOrist, Steven AU - McOrist S FAU - Lehrbach, Philip R AU - Lehrbach PR FAU - Deane, Elizabeth M AU - Deane EM FAU - Harrison, Gavan A AU - Harrison GA LA - eng SI - GENBANK/AF516906 SI - GENBANK/AF516907 PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Membrane Glycoproteins) RN - 0 (Viral Envelope Proteins) RN - 107476-75-5 (spike glycoprotein, coronavirus) SB - IM MH - Animals MH - Australia MH - Base Sequence MH - Coronavirus Infections/*veterinary/virology MH - Coronavirus, Canine/*classification/*genetics MH - Dog Diseases/*virology MH - Dogs MH - Genes, pol/*genetics MH - Membrane Glycoproteins/chemistry/*genetics MH - Molecular Sequence Data MH - Phylogeny MH - Sequence Alignment MH - Sequence Analysis, DNA MH - Viral Envelope Proteins/chemistry/*genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3518-22. PMID- 12202610 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Prevalence of blood-borne infectious diseases in blood donors in Ghana. PG - 3523-5 AB - Transfusion-transmissible infections among 808 blood donors in Ghana were investigated in 1999. Antibody seroprevalences of 3.8, 0.7, 8.4, and 13.5%, respectively, for human immunodeficiency virus, human T-cell lymphotrophic virus type 1, hepatitis C virus (HCV), and Treponema pallidum were obtained. The seroprevalence of HCV infection was confirmed to be 0.9% after supplementary testing, and the transfusion risk potential of these pathogens was demonstrated. AD - Noguchi Memorial Institute for Medical Research, University of Ghana, Legon. wampofo@noguchi.mimcom.net FAU - Ampofo, William AU - Ampofo W FAU - Nii-Trebi, Nicholas AU - Nii-Trebi N FAU - Ansah, Justina AU - Ansah J FAU - Abe, Kenji AU - Abe K FAU - Naito, Hideo AU - Naito H FAU - Aidoo, Simeon AU - Aidoo S FAU - Nuvor, Victor AU - Nuvor V FAU - Brandful, James AU - Brandful J FAU - Yamamoto, Naoki AU - Yamamoto N FAU - Ofori-Adjei, David AU - Ofori-Adjei D FAU - Ishikawa, Koichi AU - Ishikawa K LA - eng PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antibodies, Bacterial) RN - 0 (Antibodies, Viral) SB - IM MH - Adolescent MH - Adult MH - Aged MH - Antibodies, Bacterial/blood MH - Antibodies, Viral/blood MH - *Blood Donors MH - Blood Transfusion/*adverse effects MH - Ghana/epidemiology MH - HIV/immunology MH - Hepacivirus/immunology MH - Human T-lymphotropic virus 1/immunology MH - Humans MH - Middle Aged MH - Research Support, Non-U.S. Gov't MH - Syphilis/*epidemiology/microbiology MH - Treponema pallidum/immunology MH - Virus Diseases/*epidemiology/virology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3523-5. PMID- 12202611 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Evaluation of a novel commercial enzyme-linked immunosorbent assay detecting Coxiella burnetii-specific immunoglobulin G for Q fever prevaccination screening and diagnosis. PG - 3526-9 AB - A novel commercially available enzyme-linked immunosorbent assay (ELISA) for prevaccination screening and diagnosis of Q fever (PanBio Coxiella burnetii immunoglobulin G [IgG] ELISA) was compared to the complement fixation test (CFT), and the indirect fluorescent-antibody test (IFAT) was used to resolve discrepant results between the other two tests. A total of 214 serum samples was tested. The ELISA demonstrated a specificity of 96% (46 of 48 samples) and a sensitivity of 71% (95 of 134 samples). Of the six serum pairs showing CFT seroconversion, three pairs showed a corresponding ELISA seroconversion. No cross-reactivity was observed in the ELISA with serum samples from patients with mycoplasma, brucella, and chlamydia infections. One of the 13 patients with leptospirosis demonstrated a positive result in the ELISA but not in the CFT or the IFAT, and Legionella pneumophila serogroup 4 antibody was found in one of the two sera that were false-positive by ELISA. The results presented in this study suggest that the PanBio Q fever IgG ELISA is a specific alternative method for prevaccination testing and an aid for the diagnosis of Q fever. This test is suitable for use as a screening assay, with CFT and/or IFAT used to confirm negative results. AD - Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, Queensland, Australia. FAU - Field, Peter R AU - Field PR FAU - Santiago, Avelina AU - Santiago A FAU - Chan, Sau-Wan AU - Chan SW FAU - Patel, Dhara B AU - Patel DB FAU - Dickeson, David AU - Dickeson D FAU - Mitchell, Jody L AU - Mitchell JL FAU - Devine, Peter L AU - Devine PL FAU - Murphy, Alan M AU - Murphy AM LA - eng PT - Evaluation Studies PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Antibodies, Bacterial) RN - 0 (Immunoglobulin G) SB - IM MH - Antibodies, Bacterial/blood MH - *Antibody Specificity MH - Comparative Study MH - Complement Fixation Tests MH - Coxiella burnetii/*immunology MH - Enzyme-Linked Immunosorbent Assay MH - Fluorescent Antibody Technique, Indirect MH - Humans MH - Immunoglobulin G/*blood MH - *Mass Screening MH - Q Fever/*diagnosis MH - Sensitivity and Specificity MH - Vaccination EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3526-9. PMID- 12202612 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - A pilot with pain in his leg: thigh abscess caused by Salmonella enterica serotype Brandenburg. PG - 3530-1 AB - Salmonella enterica serotype Brandenburg is one of the more uncommon serotypes isolated from patients with gastroenteritis. Few cases of extraintestinal infections with serotype Brandenburg have been documented. The first case of a serotype Brandenburg-dependent thigh abscess originating from an atherosclerotic pseudoaneurysm of the femoral artery is reported. AD - Department of Infectious Diseases, University Hospital Malmo, Lund University, S-205 02 Malmo, Sweden. FAU - Bjorkman, Per AU - Bjorkman P FAU - Nilsson, Anna AU - Nilsson A FAU - Riesbeck, Kristian AU - Riesbeck K LA - eng PT - Case Reports PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 SB - IM MH - Abscess/*microbiology MH - Aircraft MH - Humans MH - Leg/pathology MH - Male MH - Middle Aged MH - Pain/*pathology MH - Salmonella Infections/*microbiology MH - Salmonella enterica/classification/*isolation & purification MH - Serotyping MH - *Thigh EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3530-1. PMID- 12202613 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Isolation of Nocardia paucivorans from the cerebrospinal fluid of a patient with relapse of cerebral nocardiosis. PG - 3532-4 AB - Nocardia paucivorans represents a new species of the genus Nocardia that has recently been isolated from bronchial secretions of a patient with chronic lung disease. Here, we report on the course of a disseminated infection caused by this species: i.e., cerebral and subsequent meningeal manifestations, isolation from the cerebrospinal fluid, and in vitro susceptibility to various antimicrobial agents. AD - Department of Medical Microbiology and Immunology of Infection, Benjamin Franklin Medical Center, Freie Universitat Berlin, 12203 Berlin, Germany. martin.eisenblaetter@ukbf.fu.berlin.de FAU - Eisenblatter, M AU - Eisenblatter M FAU - Disko, U AU - Disko U FAU - Stoltenburg-Didinger, G AU - Stoltenburg-Didinger G FAU - Scherubl, H AU - Scherubl H FAU - Schaal, K P AU - Schaal KP FAU - Roth, A AU - Roth A FAU - Ignatius, R AU - Ignatius R FAU - Zeitz, M AU - Zeitz M FAU - Hahn, H AU - Hahn H FAU - Wagner, J AU - Wagner J LA - eng PT - Case Reports PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 SB - IM MH - Brain Abscess/*microbiology MH - Cerebrospinal Fluid/*microbiology MH - Humans MH - Male MH - Middle Aged MH - Nocardia/classification/*isolation & purification MH - Nocardia Infections/*microbiology MH - Recurrence EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3532-4. PMID- 12202614 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20031114 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - First report on Schizophyllum commune from a dog. PG - 3535-7 AB - This report describes the first isolation of Schizophyllum commune from a granulomatous lesion on the neck of a dog. The biopsy specimen from the lesion disclosed granulomatous inflammation with branching fungal hyphae without clamp connections. The clinical isolate was identified as S. commune by mycological examination and analysis of ribosomal DNA sequences. AD - Department of Pathobiology, Nihon University School of Veterinary Medicine, 1866 Kameino, Fujisawa, Japan. kano@brs.nihon-u.ac.jp FAU - Kano, Rui AU - Kano R FAU - Oomae, Syougo AU - Oomae S FAU - Nakano, Yasuhiro AU - Nakano Y FAU - Minami, Takeo AU - Minami T FAU - Sukikara, Michio AU - Sukikara M FAU - Nakayama, Takao AU - Nakayama T FAU - Hasegawa, Atsuhiko AU - Hasegawa A LA - eng SI - GENBANK/AB080723 PT - Case Reports PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Ribosomal) RN - 0 (RNA, Ribosomal) RN - 130527-23-0 (RNA, ribosomal, 25S) SB - IM MH - Animals MH - DNA, Ribosomal/analysis MH - Dog Diseases/*microbiology MH - Dogs MH - Molecular Sequence Data MH - Mycological Typing Techniques MH - Mycoses/microbiology/*veterinary MH - RNA, Ribosomal/*genetics MH - Schizophyllum/classification/genetics/*isolation & purification MH - Sequence Analysis, DNA EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3535-7. PMID- 12202615 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Disseminated infection due to Blastobacter denitrificans following routine appendectomy in an adolescent. PG - 3538-9 AB - Until now, Blastobacter denitrificans has not been mentioned in the context of human infections. A case of severe complication caused by B. denitrificans after routine appendectomy in a young girl is described and confirms this organism to be an opportunistic human pathogen. AD - Institute of Medical Microbiology, Department of General Pediatrics and Neonatology, Otto von Guericke University, D-39120 Magdeburg, Germany. FAU - Trotha, Rene AU - Trotha R FAU - Guenther, Gudrun AU - Guenther G FAU - Konig, Wolfgang AU - Konig W FAU - Konig, Brigitte AU - Konig B LA - eng PT - Case Reports PT - Journal Article PL - United States TA - J Clin Microbiol JID - 7505564 SB - IM MH - Alphaproteobacteria/*isolation & purification MH - Appendectomy/*adverse effects MH - Child MH - Female MH - Gram-Negative Bacterial Infections/*microbiology MH - Humans MH - Opportunistic Infections/*microbiology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3538-9. PMID- 12202616 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Enteroaggregative Escherichia coli strains among classical enteropathogenic Escherichia coli O serogroups. PG - 3540-1 FAU - Elias, Waldir P AU - Elias WP FAU - Barros, Samar F AU - Barros SF FAU - Moreira, Cristiano G AU - Moreira CG FAU - Trabulsi, Luiz R AU - Trabulsi LR FAU - Gomes, Tania A T AU - Gomes TA LA - eng PT - Letter PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Bacterial Proteins) SB - IM MH - *Bacterial Adhesion MH - Bacterial Proteins/genetics/metabolism MH - Bacterial Typing Techniques MH - Cell Line MH - Epithelial Cells MH - Escherichia coli/*classification/genetics/*pathogenicity MH - Escherichia coli Infections/*microbiology MH - Humans MH - Serotyping MH - Virulence EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3540-1. PMID- 12202617 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Importance of testing stool specimens for Shiga toxins. PG - 3542-3 FAU - Park, C H AU - Park CH FAU - Kim, H J AU - Kim HJ FAU - Hixon, D L AU - Hixon DL LA - eng PT - Letter PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (Culture Media) RN - 0 (Shiga-Like Toxin I) RN - 0 (Shiga-Like Toxin II) SB - IM EIN - J Clin Microbiol. 2003 Jan;41(1):526. MH - Bacteriological Techniques MH - Culture Media MH - Escherichia coli/*classification/isolation & purification/metabolism MH - Escherichia coli Infections/*diagnosis/*epidemiology/microbiology MH - Escherichia coli O157/classification/*isolation & purification/metabolism MH - Feces/*microbiology MH - Humans MH - Incidence MH - Serotyping MH - Shiga-Like Toxin I/*biosynthesis MH - Shiga-Like Toxin II/*biosynthesis EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3542-3. PMID- 12202618 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Acrophialophora fusispora misidentified as Scedosporium prolificans. PG - 3544; author reply 3545 FAU - Guarro, Josep AU - Guarro J FAU - Gene, Josepa AU - Gene J LA - eng PT - Case Reports PT - Comment PT - Letter PL - United States TA - J Clin Microbiol JID - 7505564 SB - IM CON - J Clin Microbiol. 2001 Dec;39(12):4579-82. PMID: 11724890 MH - Contact Lenses/adverse effects MH - *Diagnostic Errors MH - Humans MH - Keratitis/*diagnosis/microbiology MH - Mitosporic Fungi/*classification/isolation & purification MH - Mycoses/microbiology MH - Scedosporium/*classification/isolation & purification MH - Uveitis/*diagnosis/microbiology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3544; author reply 3545. PMID- 12202619 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021114 LR - 20041117 PUBM- Print IS - 0095-1137 VI - 40 IP - 9 DP - 2002 Sep TI - Pulsed-field gel electrophoresis can yield DNA fingerprints of degradation-susceptible Clostridium difficile strains. PG - 3546-7; author reply 3547 FAU - Fawley, Warren N AU - Fawley WN FAU - Wilcox, Mark H AU - Wilcox MH LA - eng PT - Comment PT - Letter PL - United States TA - J Clin Microbiol JID - 7505564 RN - 0 (DNA, Bacterial) SB - IM CON - J Clin Microbiol. 2002 Jan;40(1):101-4. PMID: 11773100 MH - Bacterial Typing Techniques MH - Clostridium difficile/*classification/*genetics MH - DNA Fingerprinting/*methods MH - DNA, Bacterial/*metabolism MH - Electrophoresis, Gel, Pulsed-Field MH - Enterocolitis, Pseudomembranous/*microbiology MH - Humans EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - J Clin Microbiol 2002 Sep;40(9):3546-7; author reply 3547. PMID- 12202748 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Novel domains and orthologues of eukaryotic transcription elongation factors. PG - 3643-52 AB - The passage of RNA polymerase II across eukaryotic genes is impeded by the nucleosome, an octamer of histones H2A, H2B, H3 and H4 dimers. More than a dozen factors in the yeast Saccharomyces cerevisiae are known to facilitate transcription elongation through chromatin. In order to better understand the evolution and function of these factors, their sequences have been compared with known protein, EST and DNA sequences. Elongator subcomplex components Elp4p and Elp6p are shown to be homologues of ATPases, yet with substitutions of amino acids critical for ATP hydrolysis, and novel orthologues of Elp5p are detectable in human, and other animal, sequences. The yeast CP complex is shown to contain a likely inactive homologue of M24 family metalloproteases in Spt16p/Cdc68p and a 2-fold repeat in Pob3p, the orthologue of mammalian SSRP1. Archaeal DNA-directed RNA polymerase subunit E" is shown to be the orthologue of eukaryotic Spt4p, and Spt5p and prokaryotic NusG are shown to contain a novel 'NGN' domain. Spt6p is found to contain a domain homologous to the YqgF family of RNases, although this domain may also lack catalytic activity. These findings imply that much of the transcription elongation machinery of eukaryotes has been acquired subsequent to their divergence from prokaryotes. AD - MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK. chris.ponting@anat.ox.ac.uk FAU - Ponting, Chris P AU - Ponting CP LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Archaeal Proteins) RN - 0 (Bacterial Proteins) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 0 (Transcription Factors) SB - IM MH - Amino Acid Motifs/genetics MH - Amino Acid Sequence MH - Animals MH - Archaeal Proteins/genetics MH - Bacterial Proteins/genetics MH - Binding Sites/genetics MH - Databases, Protein MH - Eukaryotic Cells/metabolism MH - Evolution, Molecular MH - Humans MH - Molecular Sequence Data MH - Saccharomyces cerevisiae/*genetics MH - Saccharomyces cerevisiae Proteins/genetics MH - Sequence Homology, Amino Acid MH - Transcription Factors/*genetics MH - Transcription, Genetic/*genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3643-52. PMID- 12202749 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Displacement of DNA-PKcs from DNA ends by the Werner syndrome protein. PG - 3653-61 AB - The DNA-dependent protein kinase (DNA-PK) complex, which is composed of a DNA-dependent kinase subunit (DNA-PKcs) and the Ku70/80 heterodimer, is involved in DNA double-strand break repair by non-homologous end joining (NHEJ). Ku70/80 interacts with the Werner syndrome protein (WRN) and stimulates WRN exonuclease activity. To investigate a possible function of WRN in NHEJ, we have examined the relationship between DNA-PKcs, Ku and WRN. First, we showed that WRN forms a complex with DNA-PKcs and Ku in solution. Next, we determined whether this complex assembles on DNA ends. Interestingly, the addition of WRN to a Ku:DNA-PKcs:DNA complex results in the displacement of DNA-PKcs from the DNA, indicating that the triple complex WRN:Ku:DNA-PKcs cannot form on DNA ends. The displacement of DNA-PKcs from DNA requires the N- and C-terminal regions of WRN, both of which make direct contact with the Ku70/80 heterodimer. Moreover, exonuclease assays indicate that DNA-PKcs does not protect DNA from the nucleolytic action of WRN. These results suggest that WRN may influence the mechanism by which DNA ends are processed. AD - Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 509, Los Angeles, CA 90089, USA. FAU - Li, Baomin AU - Li B FAU - Comai, Lucio AU - Comai L LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Antigens, Nuclear) RN - 0 (DNA-Binding Proteins) RN - 0 (Ku autoantigen) RN - 0 (Nuclear Proteins) RN - 0 (XRCC5 protein, human) RN - 9007-49-2 (DNA) RN - EC 2.7.1.37 (DNA-activated protein kinase) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 3.1.- (Exonucleases) RN - EC 5.99.- (DNA Helicases) SB - IM MH - *Antigens, Nuclear MH - DNA/chemistry/genetics/metabolism MH - *DNA Helicases MH - DNA-Binding Proteins/chemistry/genetics/metabolism MH - Dimerization MH - Electrophoretic Mobility Shift Assay MH - Exonucleases/metabolism MH - Hela Cells MH - Humans MH - Mutation MH - Nuclear Proteins/chemistry/genetics/metabolism MH - Protein Binding MH - Protein-Serine-Threonine Kinases/chemistry/genetics/*metabolism MH - Research Support, Non-U.S. Gov't EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3653-61. PMID- 12202750 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Predicted structure and phyletic distribution of the RNA-binding protein Hfq. PG - 3662-71 AB - Hfq, a bacterial RNA-binding protein, was recently shown to contain the Sm1 motif, a characteristic of Sm and LSm proteins that function in RNA processing events in archaea and eukaryotes. In this report, comparative structural modeling was used to predict a three-dimensional structure of the Hfq core sequence. The predicted structure aligns with most major features of the Methanobacterium thermoautotrophicum LSm protein structure. Conserved residues in Hfq are positioned at the same structural locations responsible for subunit assembly and RNA interaction in Sm proteins. A highly conserved portion of Hfq assumes a structural fold similar to the Sm2 motif of Sm proteins. The evolution of the Hfq protein was explored by conducting a BLAST search of microbial genomes followed by phylogenetic analysis. Approximately half of the 140 complete or nearly complete genomes examined contain at least one gene coding for Hfq. The presence or absence of Hfq closely followed major bacterial clades. It is absent from high-level clades and present in the ancient Thermotogales-Aquificales clade and all proteobacteria except for those that have undergone major reduction in genome size. Residues at three positions in Hfq form signatures for the beta/gamma proteobacteria, alpha proteobacteria and low GC Gram-positive bacteria groups. AD - School of Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA. FAU - Sun, Xueguang AU - Sun X FAU - Zhulin, Igor AU - Zhulin I FAU - Wartell, Roger M AU - Wartell RM LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Host Factor 1 Protein) RN - 0 (RNA-Binding Proteins) RN - 0 (Ribonucleoproteins, Small Nuclear) RN - 0 (Sm antigen) SB - IM MH - Amino Acid Sequence MH - Bacteria/classification/*genetics MH - Databases, Protein MH - Escherichia coli/genetics MH - Genome, Bacterial MH - Host Factor 1 Protein/chemistry/*genetics MH - Models, Molecular MH - Molecular Sequence Data MH - *Phylogeny MH - Protein Conformation MH - Protein Structure, Secondary MH - RNA-Binding Proteins/chemistry/*genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Ribonucleoproteins, Small Nuclear/chemistry/genetics MH - Sequence Homology, Amino Acid EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3662-71. PMID- 12202751 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20050317 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Distinct domains in the CArG-box binding factor A destabilize tetraplex forms of the fragile X expanded sequence d(CGG)n. PG - 3672-81 AB - Formation of hairpin or tetraplex structures of the FMR1 gene d(CGG)n sequence triggers its expansion, setting off fragile X syndrome. In searching for proteins that destabilize d(CGG)n secondary structures we purified from rat liver quadruplex telomeric DNA binding protein 42 (qTBP42) that disrupts G'2 bimolecular tetraplex d(CGG)n while paradoxically stabilizing the G'2 structure of the telomeric sequence d(TTAGGG)n. Based on peptide sequence homology of qTBP42 and mouse CArG-box binding factor A (CBF-A), we provide direct evidence that recombinant CBF-A protein is physically and immunochemically indistinguishable from qTBP42 and that it too destabilizes G'2 d(CGG)n while stabilizing G'2 d(TTAGGG)n. We inquired whether CBF-A employs the same or different domains to differentially interact with G'2 d(CGG)n and G'2 d(TTAGGG)n. Mutant CBF-A proteins that lack each or combinations of its five conserved motifs: RNP1(1), RNP1(2), RNP2(1), RNP2(2) and ATP/GTP-binding box were tested for their G'2 d(CGG)n destabilization and G'2 d(TTAGGG)n stabilization activities. We find that either RNP1(1) or the ATP/GTP motifs are necessary and sufficient for G'2 d(CGG)n destabilization whereas RNP2(1) suppresses destabilization by either one of these two motifs. Neither RNP1(1) nor the ATP/GTP motif are required for G'2 d(TTAGGG)n stabilization. Hence, CBF-A employs different domains to destabilize G'2 d(CGG)n or stabilize G'2 d(TTAGGG)n. AD - Unit of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Haifa 31096, Israel. FAU - Weisman-Shomer, Pnina AU - Weisman-Shomer P FAU - Cohen, Esther AU - Cohen E FAU - Fry, Michael AU - Fry M LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (DNA-Binding Proteins) RN - 0 (Heterogeneous-Nuclear Ribonucleoprotein Group A-B) RN - 0 (Hnrpab protein, rat) RN - 0 (Immune Sera) RN - 0 (Nerve Tissue Proteins) RN - 0 (RNA-Binding Proteins) RN - 0 (Repressor Proteins) RN - 0 (quadruplex DNA) RN - 139135-51-6 (fragile X mental retardation protein) RN - 148710-92-3 (Hnrpab protein, mouse) RN - 9007-49-2 (DNA) SB - IM MH - Animals MH - Base Sequence MH - Binding Sites/genetics MH - DNA/chemistry/genetics/*metabolism MH - DNA-Binding Proteins/genetics/immunology/*metabolism MH - Heat MH - *Heterogeneous-Nuclear Ribonucleoprotein Group A-B MH - Immune Sera/immunology MH - Mice MH - Mutation MH - Nerve Tissue Proteins/*genetics MH - Nucleic Acid Conformation MH - Protein Binding MH - *RNA-Binding Proteins MH - Rats MH - Repressor Proteins/genetics/immunology/*metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Trinucleotide Repeats/*genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3672-81. PMID- 12202752 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding. PG - 3682-91 AB - The Drosophila melanogaster RECQ5/QE gene encodes a member of the DNA helicase family comprising the Escherichia coli RecQ protein and products of the human Bloom's, Werner's, and Rothmund-Thomson syndrome genes. The full-length product of RECQ5/QE was expressed in the baculovirus system and was purified. Gel filtration experiments indicated that RECQ5/QE was present in an oligomeric state. The RECQ5/QE protein hydrolyzed ATP and even more actively GTP in the presence of single-stranded DNA. ATP drove the DNA helicase activity of RECQ5/QE, whereas GTP had little effect. GTP exhibited a stimulatory effect on DNA unwinding when it was used together with ATP. This effect was more apparent with non-hydrolyzable GTP analogs, such as GTPgammaS and GMPPNP. These results indicate that GTP binding to RECQ5/QE triggers its DNA helicase activity. GTP binding increased the rate of strand separation without affecting the S(0.5) (K(m)) values for the substrates during the DNA helicase reaction. The data collectively suggest that the RECQ5/QE protein is activated upon GTP binding through the ATP-binding site. AD - Cellular and Molecular Biology Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. FAU - Kawasaki, Katsumi AU - Kawasaki K FAU - Maruyama, Sayako AU - Maruyama S FAU - Nakayama, Minoru AU - Nakayama M FAU - Matsumoto, Kohji AU - Matsumoto K FAU - Shibata, Takehiko AU - Shibata T LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (DNA, Single-Stranded) RN - 0 (Drosophila Proteins) RN - 0 (Recombinant Proteins) RN - 56-65-5 (Adenosine Triphosphate) RN - 86-01-1 (Guanosine Triphosphate) RN - EC 3.6.1.- (GTP Phosphohydrolases) RN - EC 3.6.1.3 (Adenosinetriphosphatase) RN - EC 5.99.- (DNA Helicases) RN - EC 5.99.- (RecQ5 helicase) SB - IM MH - Adenosine Triphosphate/metabolism MH - Adenosinetriphosphatase/metabolism MH - Animals MH - Cell Line MH - DNA Helicases/chemistry/genetics/*metabolism MH - DNA, Single-Stranded/metabolism MH - Dimerization MH - Drosophila Proteins/genetics/*metabolism MH - Enzyme Activation MH - GTP Phosphohydrolases/metabolism MH - Guanosine Triphosphate/metabolism MH - Protein Binding MH - Recombinant Proteins/genetics/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3682-91. PMID- 12202753 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - The switch region on Leishmania major chromosome 1 is not required for mitotic stability or gene expression, but appears to be essential. PG - 3692-7 AB - The Leishmania genome project reference strain, Leishmania major Friedlin, is trisomic for chromosome 1. The complete sequence of this chromosome has revealed that the genes are grouped into two large clusters of the polycistronic type, each borne by one DNA strand and located on each side of a 1.6-kb sequence often termed the switch region. Several hypotheses concerning the role of this switch region have been put forward (region of initiation of transcription for both gene clusters, origin of replication or centromeric sequence). In the present study, we have deleted this region on the three copies of chromosome 1 by sequential targeted replacements. The absence of the switch region did not alter the mitotic stability of the three deleted chromosomes. This region therefore does not appear necessary for chromosomal replication or segregation. However, during the third targeting round, which aimed at knocking out the last switch region, a fourth copy of chromosome 1 that retained this region appeared in all clones analysed. This suggests that the persistence of this switch region is necessary for parasite survival. We then showed that the presence/absence of the switch region did not act upon the expression of a resistance marker gene inserted beforehand into the left gene cluster of the same chromosomal molecule. This result suggests that the presence of this 1.6-kb sequence is not necessary for the expression of all genes on chromosome 1. AD - CNRS UMR5093 'Genome et Biologie Moleculaire des Protozoaires Parasites', Laboratoire de Parasitologie-Mycologie, Faculte de Medecine, 163 Rue Auguste Broussonet, F-34090 Montpellier, France. FAU - Dubessay, Pascal AU - Dubessay P FAU - Ravel, Christophe AU - Ravel C FAU - Bastien, Patrick AU - Bastien P FAU - Crobu, Lucien AU - Crobu L FAU - Dedet, Jean-Pierre AU - Dedet JP FAU - Pages, Michel AU - Pages M FAU - Blaineau, Christine AU - Blaineau C LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (DNA, Protozoan) RN - EC 3.1.21 (DNA Restriction Enzymes) SB - IM MH - Animals MH - Chromosome Deletion MH - Chromosomes/*genetics MH - DNA Restriction Enzymes/metabolism MH - DNA, Protozoan/genetics/metabolism MH - Drug Resistance/genetics MH - Gene Expression Regulation MH - Leishmania major/*genetics MH - Mitosis/*genetics MH - Mutation MH - Research Support, Non-U.S. Gov't MH - Transcription, Genetic/genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3692-7. PMID- 12202754 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - DNA double-strand break-induced phosphorylation of Drosophila histone variant H2Av helps prevent radiation-induced apoptosis. PG - 3698-705 AB - The response of eukaryotic cells to the formation of a double-strand break (DSB) in chromosomal DNA is highly conserved. One of the earliest responses to DSB formation is phosphorylation of the C-terminal tail of H2A histones located in nucleosomes near the break. Histone variant H2AX and core histone H2A are phosphorylated in mammals and budding yeast, respectively. We demonstrate the DSB-induced phosphorylation of histone variant H2Av in Drosophila melanogaster. H2Av is a member of the H2AZ family of histone variants. Ser137 within an SQ motif located near the C- terminus of H2Av was phosphorylated in response to gamma-irradiation in both tissue culture cells and larvae. Phosphorylation was detected within 1 min of irradiation and detectable after only 0.3 Gy of radiation exposure. Photochemically induced DSBs, but not general oxidative damage or UV-induced nicking of DNA, caused H2Av phosphorylation, suggesting that phosphorylation is DSB specific. Imaginal disc cells from Drosophila expressing a mutant allele of H2Av with its C-terminal tail deleted, and therefore unable to be phosphorylated, were more sensitive to radiation-induced apoptosis than were wildtype controls, suggesting that phosphorylation of H2Av is important for repair of radiation-induced DSBs. These observations suggest that in addition to providing the function of an H2AZ histone, H2Av is also the functional homolog in Drosophila of H2AX. AD - Wadsworth Center, New York State Department of Health, State University of New York, Albany, NY 12201-2002, USA. FAU - Madigan, James P AU - Madigan JP FAU - Chotkowski, Heather L AU - Chotkowski HL FAU - Glaser, Robert L AU - Glaser RL LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Histones) RN - 9007-49-2 (DNA) SB - IM MH - Amino Acid Sequence MH - Animals MH - Apoptosis/genetics/*radiation effects MH - Blotting, Western MH - Cell Line MH - DNA/genetics/metabolism MH - *DNA Damage MH - DNA Repair MH - Drosophila/*genetics/metabolism/radiation effects MH - Genotype MH - Histones/genetics/*metabolism MH - Larva/cytology/genetics/radiation effects MH - Molecular Sequence Data MH - Mutation MH - Phosphorylation MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Amino Acid EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3698-705. PMID- 12202755 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Partial reconstitution of human RNase P in HeLa cells between its RNA subunit with an affinity tag and the intact protein components. PG - 3706-11 AB - An RNA affinity tag was incorporated into the RNA subunit of human nuclear RNase P. The tagged RNA assembled with the protein components of RNase P inside HeLa cells to generate an active enzyme. Because of the specificity of the RNA tag to streptavidin, the reconstituted complex could be separated from the native enzyme and other ribonucleoproteins (particularly RNase MRP) by streptavidin agarose chromatography and could be recovered by the eluting agent, biotin. A mutant, tagged RNase P RNA, whose P3 domain was partially replaced, could not reconstitute with the proteins to yield an active enzyme. The P3 domain, therefore, is critical for the structure and function of RNase P. AD - Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, CT 06511, USA. FAU - Li, Yong AU - Li Y FAU - Altman, Sidney AU - Altman S LA - eng GR - GM19422/GM/NIGMS PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Affinity Labels) RN - 0 (Plasmids) RN - 0 (RNA, Catalytic) RN - 0 (Ribonucleoproteins) RN - 63231-63-0 (RNA) RN - 9013-20-1 (Streptavidin) RN - EC 3.1.- (Endoribonucleases) RN - EC 3.1.26.5 (RPP14 protein, human) RN - EC 3.1.26.5 (Ribonuclease P) SB - IM MH - Affinity Labels/*chemistry MH - Blotting, Northern MH - Blotting, Western MH - Chromatography, Agarose/methods MH - Cloning, Molecular MH - Endoribonucleases/chemistry/genetics/*metabolism MH - Hela Cells MH - Humans MH - Mutation MH - Nucleic Acid Conformation MH - Plasmids/genetics MH - RNA/chemistry/genetics/*metabolism MH - RNA, Catalytic/chemistry/genetics/*metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Ribonuclease P MH - Ribonucleoproteins/*metabolism MH - Streptavidin/chemistry MH - Transfection EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3706-11. PMID- 12202756 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Antiproliferative effect in chronic myeloid leukaemia cells by antisense peptide nucleic acids. PG - 3712-21 AB - Peptide nucleic acid (PNA) is a synthetic DNA analogue that is resistant to nucleases and proteases and binds with exceptional affinity to RNA. Because of these properties PNA has the potential to become a powerful therapeutic agent to be used in vivo. Until now, however, the use of PNA in vivo has not been much investigated. Here, we have attempted to reduce the expression of the bcr/abl oncogene in chronic myeloid leukaemia KYO-1 cells using a 13mer PNA sequence (asPNA) designed to hybridise to the b2a2 junction of bcr/abl mRNA. To enhance cellular uptake asPNA was covalently linked to the basic peptide VKRKKKP (NLS-asPNA). Moreover, to investigate the cellular uptake by confocal microscopy, both PNAs were linked by their N-terminus to fluorescein (FL). Studies of uptake, carried out at 4 and 37 degrees C on living KYO-1 cells stained with hexidium iodide, showed that both NLS-asPNA-FL and asPNA-FL were taken up by the cells, through a receptor-independent mechanism. The intracellular amount of NLS-asPNA-FL was about two to three times higher than that of asPNA-FL. Using a semi-quantitative RT- PCR technique we found that 10 micro M asPNA and NLS-asPNA reduced the level of b2a2 mRNA in KYO-1 cells to 20 +/- 5% and 60 +/- 10% of the control, respectively. Western blot analysis showed that asPNA promoted a significant inhibition of p210(BCR/ABL) protein: residual protein measured in cells exposed for 48 h to asPNA was approximately 35% of the control. Additionally, asPNA impaired cell growth to 50 +/- 5% of the control and inhibited completion of the cell cycle. In summary, these results demonstrate that a PNA 13mer is taken up by KYO-1 cells and is capable of producing a significant and specific down-regulation of the bcr/abl oncogene involved in leukaemogenesis. AD - Department of Biomedical Sciences and Technologies, School of Medicine, University of Udine, Piazzale Kolbe 4, 33100 Udine, Italy. FAU - Rapozzi, Valentina AU - Rapozzi V FAU - Burm, Brigitte E A AU - Burm BE FAU - Cogoi, Susanna AU - Cogoi S FAU - van der Marel, Gijs A AU - van der Marel GA FAU - van Boom, Jacques H AU - van Boom JH FAU - Quadrifoglio, Franco AU - Quadrifoglio F FAU - Xodo, Luigi E AU - Xodo LE LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Antineoplastic Agents) RN - 0 (DNA, Antisense) RN - 0 (Fusion Proteins, bcr-abl) RN - 0 (Nuclear Localization Signal) RN - 0 (Peptide Nucleic Acids) RN - 0 (RNA, Messenger) RN - 2321-07-5 (Fluorescein) SB - IM MH - Antineoplastic Agents/metabolism/*pharmacology MH - Apoptosis/drug effects MH - Base Sequence MH - Cell Cycle/drug effects MH - Cell Division/drug effects MH - DNA, Antisense/chemistry/genetics/*pharmacology MH - Down-Regulation MH - Flow Cytometry MH - Fluorescein/chemistry MH - Fusion Proteins, bcr-abl/genetics MH - Humans MH - K562 Cells MH - Leukemia, Myeloid, Chronic/*drug therapy/genetics/pathology MH - Microscopy, Confocal MH - Molecular Sequence Data MH - Nuclear Localization Signal/genetics MH - Peptide Nucleic Acids/*genetics MH - RNA, Messenger/drug effects/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Tumor Cells, Cultured/drug effects/metabolism EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3712-21. PMID- 12202757 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Polyamine structural effects on the induction and stabilization of liquid crystalline DNA: potential applications to DNA packaging, gene therapy and polyamine therapeutics. PG - 3722-31 AB - DNA undergoes condensation, conformational transitions, aggregation and resolubilization in the presence of polyamines, positively charged organic molecules present in all cells. Under carefully controlled environmental conditions, DNA can also transform to a liquid crystalline state in vitro. We undertook the present work to examine the ability of spermidine, N4-methylspermidine, spermine, N1-acetylspermine and a group of tetramine, pentamine and hexamine analogs of spermine to induce and stabilize liquid crystalline DNA. Liquid crystalline textures were identified under a polarizing microscope. In the absence of polyamines, calf thymus DNA assumed a diffused, planar cholesteric phase with entrapped bubbles when incubated on a glass slide at 37 degrees C. In the presence of spermidine and spermine, the characteristic fingerprint textures of the cholesteric phase, adopting a hexagonal order, were obtained. The helical pitch was 2.5 micro m. The final structures were dendrimeric and crystalline when DNA was treated with spermine homologs and bis(ethyl) derivatives. A cholesteric structure was observed when DNA was treated with a hexamine at 37 degrees C. This structure changed to a hexagonal dendrimer with fluidity on prolonged incubation. These data show a structural specificity effect of polyamines on liquid crystalline phase transitions of DNA and suggest a possible physiological function of natural polyamines. AD - Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA. FAU - Saminathan, M AU - Saminathan M FAU - Thomas, Thresia AU - Thomas T FAU - Shirahata, Akira AU - Shirahata A FAU - Pillai, C K S AU - Pillai CK FAU - Thomas, T J AU - Thomas TJ LA - eng GR - CA42439/CA/NCI GR - CA73058/CA/NCI GR - CA80163/CA/NCI PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Polyamines) RN - 100-97-0 (Methenamine) RN - 124-20-9 (Spermidine) RN - 137946-02-2 (1-methylspermidine) RN - 25593-72-0 (N'-acetylspermine) RN - 71-44-3 (Spermine) RN - 9007-49-2 (DNA) SB - IM MH - Animals MH - Cattle MH - Crystallization MH - DNA/*chemistry/metabolism MH - Gene Therapy/methods MH - Methenamine/chemistry/pharmacology MH - Microscopy, Polarization MH - Nucleic Acid Conformation/drug effects MH - Polyamines/*chemistry/pharmacology/therapeutic use MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Spermidine/*analogs & derivatives/chemistry/pharmacology MH - Spermine/*analogs & derivatives/chemistry/pharmacology MH - Structure-Activity Relationship MH - Thymus Gland/metabolism EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3722-31. PMID- 12202758 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Transcriptome analysis of Escherichia coli using high-density oligonucleotide probe arrays. PG - 3732-8 AB - Microarrays traditionally have been used to analyze the expression behavior of large numbers of coding transcripts. Here we present a comprehensive approach for high-throughput transcript discovery in Escherichia coli focused mainly on intergenic regions which, together with analysis of coding transcripts, provides us with a more complete insight into the organism's transcriptome. Using a whole genome array, we detected expression for 4052 coding transcripts and identified 1102 additional transcripts in the intergenic regions of the E.coli genome. Further classification reveals 317 novel transcripts with unknown function. Our results show that, despite sophisticated approaches to genome annotation, many cellular transcripts remain unidentified. Through the experimental identification of all RNAs expressed under a specific condition, we gain a more thorough understanding of all cellular processes. AD - Department of Computer Science, University of Washington, Seattle, WA 98195, USA. FAU - Tjaden, Brian AU - Tjaden B FAU - Saxena, Rini Mukherjee AU - Saxena RM FAU - Stolyar, Sergey AU - Stolyar S FAU - Haynor, David R AU - Haynor DR FAU - Kolker, Eugene AU - Kolker E FAU - Rosenow, Carsten AU - Rosenow C LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (3' Untranslated Regions) RN - 0 (5' Untranslated Regions) RN - 0 (RNA, Bacterial) SB - IM MH - 3' Untranslated Regions/genetics MH - 5' Untranslated Regions/genetics MH - Escherichia coli/*genetics MH - Gene Expression Regulation, Bacterial MH - Oligonucleotide Array Sequence Analysis/*methods MH - Operon/genetics MH - RNA, Bacterial/genetics/metabolism MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Reverse Transcriptase Polymerase Chain Reaction MH - Transcription, Genetic/*genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3732-8. PMID- 12202759 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides. PG - 3739-47 AB - Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5'-amino-5'-deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully explored, which may possibly have limited the scope of their utility. Here we describe the synthesis of the 5'-amino-2',5'-dideoxy analogs of adenosine, cytidine, guanosine, inosine and uridine from their respective naturally occurring nucleosides via the reduction of 5'-azido-2',5'-dideoxy intermediates using the Staudinger reaction, and the high yield conversion of these modified nucleosides and 5'-amino-5'-deoxythymidine to the corresponding 5'-N-triphosphates through reaction with trisodium trimetaphosphate in the presence of tris(hydroxymethyl)aminomethane (Tris). We also show that each of these nucleotide analogs can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I when individually substituted for its naturally occurring counterpart. Mild acid treatment of the resulting DNA generates polynucleotide fragments that arise from specific cleavage at each modified nucleotide, providing a sequence ladder for each base. Because the ladders are generated after the extension, the corresponding products may be manipulated by enzymatic and/or purification processes. The potential utility of this extension-cleavage procedure in genomic sequence analysis is discussed. AD - Variagenics Inc., 60 Hampshire Street, Cambridge, MA 02139, USA. jwolfe@variagenics.com FAU - Wolfe, Jia Liu AU - Wolfe JL FAU - Kawate, Tomohiko AU - Kawate T FAU - Belenky, Alexei AU - Belenky A FAU - Stanton, Vincent Jr AU - Stanton V Jr LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Dideoxynucleosides) RN - 118-00-3 (Guanosine) RN - 50-89-5 (Thymidine) RN - 58-61-7 (Adenosine) RN - 58-63-9 (Inosine) RN - 58-96-8 (Uridine) RN - 65-46-3 (Cytidine) RN - 9007-49-2 (DNA) RN - EC 2.7.7.- (DNA Polymerase I) SB - IM MH - Adenosine/analogs & derivatives/metabolism MH - Base Sequence MH - Chromatography, High Pressure Liquid MH - Cytidine/analogs & derivatives/metabolism MH - DNA/chemistry/genetics/metabolism MH - DNA Polymerase I/*metabolism MH - Dideoxynucleosides/chemical synthesis/*metabolism MH - Guanosine/analogs & derivatives/metabolism MH - Inosine/analogs & derivatives/metabolism MH - Magnetic Resonance Spectroscopy MH - Molecular Sequence Data MH - Sequence Analysis, DNA MH - Thymidine/analogs & derivatives/metabolism MH - Uridine/analogs & derivatives/metabolism EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3739-47. PMID- 12202760 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole-imidazole-cyclopropapyrroloindole conjugates. PG - 3748-53 AB - The pyrrole-imidazole (Py-Im) triamide-cyclopropa pyrroloindole (CPI) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 specifically alkylated DNA at 5'-CGCGCG-3' and 5'-PyGGCCPu-3', respectively. Agarose gel electrophoresis indicated that incubation of a supercoiled plasmid, pSPORT I (4109 bp), with conjugate 7 effectively inhibited its hydrolysis by BssHII (5'-G_CGCGC-3'), whereas conjugate 14 had no effect on this hydrolysis. These results suggest that conjugate 7 sequence-specifically inhibits the hydrolysis of DNA by BssHII. Sequence-specific alkylation by the Py-Im triamide-CPI conjugates was further confirmed by inhibition of the Eco52I (5'-C_GGCCG-3') hydrolysis of conjugate 14-treated pQBI PGK (5387 bp). In clear contrast, hydrolysis of pQB1 PGK by DraI (3'-TTT_AAA-3') was not inhibited by 5 micro M conjugate 14. That ImImPy did not inhibit the hydrolysis of pQB1 PGK indicates that covalent bond formation is necessary for inhibition. A similar experiment, using linear pQBI PGK, achieved the same extent of protection of the DNA with approximately half the concentration of conjugate 14 as was required to protect supercoiled DNA from hydrolysis. AD - Division of Biofunctional Molecules, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Surugadai, Kanda, Chiyoda, Tokyo 101-0062, Japan. FAU - Fujimoto, Kazuhisa AU - Fujimoto K FAU - Iida, Hirokazu AU - Iida H FAU - Kawakami, Masako AU - Kawakami M FAU - Bando, Toshikazu AU - Bando T FAU - Tao, Zhi-Fu AU - Tao ZF FAU - Sugiyama, Hiroshi AU - Sugiyama H LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (DNA, Superhelical) RN - 0 (Imidazoles) RN - 0 (Plasmids) RN - 0 (Pyrroles) RN - 9007-49-2 (DNA) RN - EC 3.1.21 (DNA Restriction Enzymes) RN - EC 3.1.21.- (endodeoxyribonuclease AhaIII) RN - EC 3.1.21.- (endodeoxyribonuclease BSSHII) RN - EC 3.1.21.- (endodeoxyribonuclease XmaIII) RN - EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific) SB - IM MH - Alkylation MH - Base Sequence MH - DNA/chemistry/*metabolism MH - DNA Restriction Enzymes/*metabolism MH - DNA, Superhelical/chemistry/metabolism MH - Deoxyribonucleases, Type II Site-Specific/metabolism MH - Electrophoresis, Polyacrylamide Gel MH - Imidazoles/*chemistry MH - Plasmids/chemistry/genetics/metabolism MH - Pyrroles/*chemistry MH - Sequence Analysis, DNA EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3748-53. PMID- 12202761 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Genome-wide detection of tissue-specific alternative splicing in the human transcriptome. PG - 3754-66 AB - We have developed an automated method for discovering tissue-specific regulation of alternative splicing through a genome-wide analysis of expressed sequence tags (ESTs). Using this approach, we have identified 667 tissue-specific alternative splice forms of human genes. We validated our muscle-specific and brain-specific splice forms for known genes. A high fraction (8/10) were reported to have a matching tissue specificity by independent studies in the published literature. The number of tissue-specific alternative splice forms is highest in brain, while eye-retina, muscle, skin, testis and lymph have the greatest enrichment of tissue-specific splicing. Overall, 10-30% of human alternatively spliced genes in our data show evidence of tissue-specific splice forms. Seventy-eight percent of our tissue-specific alternative splices appear to be novel discoveries. We present bioinformatics analysis of several tissue-specific splice forms, including automated protein isoform sequence and domain prediction, showing how our data can provide valuable insights into gene function in different tissues. For example, we have discovered a novel kidney-specific alternative splice form of the WNK1 gene, which appears to specifically disrupt its N-terminal kinase domain and may play a role in PHAII hypertension. Our database greatly expands knowledge of tissue-specific alternative splicing and provides a comprehensive dataset for investigating its functional roles and regulation in different human tissues. AD - Molecular Biology Institute and Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, CA 90095-1570, USA. FAU - Xu, Qiang AU - Xu Q FAU - Modrek, Barmak AU - Modrek B FAU - Lee, Christopher AU - Lee C LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (DNA-Binding Proteins) RN - 0 (Protein Isoforms) RN - 0 (Transcription Factors) RN - 0 (interferon regulatory factor-3) RN - EC 3.6.1.- (cdc42 GTP-Binding Protein) SB - IM MH - Alternative Splicing/*genetics MH - Amino Acid Sequence MH - Brain/metabolism MH - Computational Biology/statistics & numerical data MH - DNA-Binding Proteins/genetics MH - Expressed Sequence Tags MH - Female MH - Gene Expression Profiling MH - Gene Library MH - *Genome, Human MH - Humans MH - Male MH - Molecular Sequence Data MH - Protein Isoforms/genetics MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Sequence Homology, Amino Acid MH - Transcription Factors/genetics MH - Transcription, Genetic/*genetics MH - cdc42 GTP-Binding Protein/genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3754-66. PMID- 12202762 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA. PG - 3767-77 AB - In previous papers of this series the temperature-dependent Raman spectra of poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A.T and alternating A.T/T.A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA).poly(dT) and poly(dA-dT). poly(dA-dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van't Hoff premelting enthalpies of poly(dA).poly(dT) [DeltaH(vH)(pm) = 18.0 +/- 1.6 kcal x mol(-1) (kilocalories per mole cooperative unit)] and poly(dA-dT).poly(dA-dT) (DeltaH(vH)(pm) = 13.4 +/- 2.5 kcal x mol(-1)) differ by an amount (approximately 4.6 kcal x mol(-1)) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)(n).(dT)(n) tracts. (iii) The overall stacking free energy of poly(dA). poly(dT) [-6.88 kcal x mol(bp)(-1) (kilocalories per mole base pair)] is greater than that of poly(dA-dT). poly(dA-dT) (-6.31 kcal x mol(bp)(-1)). (iv) The difference between stacking free energies of A and T is significant in poly(dA).poly(dT) (DeltaDeltaG(st) = 0.8 +/- 0.3 kcal. mol(bp)(-1)), but marginal in poly(dA-dT).poly(dA-dT) (DeltaDeltaG(st) = 0.3 +/- 0.3 kcal x mol(bp)(-1)). (v) In poly(dA). poly(dT), the van't Hoff parameters for melting of A (DeltaH(vH)(A) = 407 +/- 23 kcal.mol(-1), DeltaS(vH)(A) = 1166 +/- 67 cal. degrees K(-1) x mol(-1), DeltaG(vH(25 degrees C))(A) = 60.0 +/- 3.2 kcal x mol(-1)) are clearly distinguished from those of T (DeltaH(vH)(T) = 185 +/- 38 kcal x mol(-1), DeltaS(vH)(T) = 516 +/- 109 cal. degrees K(-1) x mol(-1), DeltaG(vH(25 degrees C))(T) = 27.1 +/- 5.5 kcal x mol(-1)). (vi) Similar relative differences are observed in poly(dA-dT). poly(dA-dT) (DeltaH(vH)(A) = 333 +/- 54 kcal x mol(-1), DeltaS(vH)(A) = 961 +/- 157 cal. degrees K(-1) x mol(-1), DeltaG(vH(25 degrees C))(A) = 45.0 +/- 7.6 kcal x mol(-1); DeltaH(vH)(T) = 213 +/- 30 kcal x mol(-1), DeltaS(vH)(T) = 617 +/- 86 cal. degrees K(-1) x mol(-1), DeltaG(vH(25 degrees C))(T) = 29.3 +/- 4.9 kcal x mol(-1)). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation. AD - Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499, USA. FAU - Movileanu, Liviu AU - Movileanu L FAU - Benevides, James M AU - Benevides JM FAU - Thomas, George J Jr AU - Thomas GJ Jr LA - eng GR - GM54378/GM/NIGMS PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 26966-61-0 (Poly dA-dT) RN - 9007-49-2 (DNA) SB - IM MH - Base Composition MH - Base Sequence MH - DNA/*chemistry MH - Entropy MH - Hydrogen Bonding MH - Nucleic Acid Conformation MH - Nucleic Acid Denaturation MH - Poly dA-dT/chemistry MH - Reproducibility of Results MH - Research Support, U.S. Gov't, P.H.S. MH - Spectrum Analysis, Raman/methods MH - Temperature MH - *Thermodynamics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3767-77. PMID- 12202763 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Structural insights by molecular dynamics simulations into differential repair efficiency for ethano-A versus etheno-A adducts by the human alkylpurine-DNA N-glycosylase. PG - 3778-87 AB - 1,N6-ethenoadenine adducts (epsilonA) are formed by known environmental carcinogens and found to be removed by human alkylpurine-DNA N-glycosylase (APNG). 1,N6-ethanoadenine (EA) adducts differ from epsilonA by change of a double bond to a single bond in the 5-member exocyclic ring and are formed by chloroethyl nitrosoureas, which are used in cancer therapy. In this work, using purified recombinant human APNG, we show that EA is a substrate for the enzyme. However, the excision efficiency of EA was 65-fold lower than that of epsilonA. Molecular dynamics simulation produced similar structural motifs for epsilonA and EA when incorporated into a DNA duplex, suggesting that there are no specific conformational features in the DNA duplex which can account for the differences in repair efficiency. However, when EA was modeled into the APNG active site, based on the APNG/epsilonA-DNA crystallographic coordinates, in structures produced by 2 ns molecular dynamics simulation, we observed weakening in the stacking interaction between EA and aromatic side chains of the key amino acids in the active site. In contrast, the planar epsilonA is better stacked at the enzyme active site. We propose that the observed destabilization of the EA adduct at the active site, such as reduced stacking interactions, could account for the biochemically observed weaker recognition of EA by APNG as compared to epsilonA. AD - Donner Laboratory, Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA. FAU - Guliaev, Anton B AU - Guliaev AB FAU - Hang, Bo AU - Hang B FAU - Singer, B AU - Singer B LA - eng GR - CA 47723/CA/NCI GR - CA 72079/CA/NCI PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (DNA Adducts) RN - 0 (Oligonucleotides) RN - 13875-63-3 (1,N(6)-ethenoadenine) RN - 73-24-5 (Adenine) RN - EC 3.2.2.- (DNA Glycosylases) RN - EC 3.2.2.- (N-Glycosyl Hydrolases) RN - EC 3.2.2.21 (DNA-3-methyladenine glycosidase II) SB - IM MH - Adenine/*analogs & derivatives/chemistry/*metabolism MH - DNA Adducts/chemistry/*metabolism MH - *DNA Glycosylases MH - *DNA Repair MH - Humans MH - Molecular Structure MH - N-Glycosyl Hydrolases/chemistry/*metabolism MH - Oligonucleotides/chemistry/metabolism MH - Protein Structure, Tertiary MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Substrate Specificity EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3778-87. PMID- 12202764 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Gene expression profiling of the aging mouse cardiac myocytes. PG - 3788-94 AB - Heart disease remains the most frequent cause of death in the general population with increasing incidence in the elderly population. The pathologic failure of the aging heart may be related to structural and functional alterations in cardiac muscle cells. However, the molecular mechanisms underlying the aging-related decline in cardiac muscle function are largely unknown. To provide the first analysis of cardiac aging at the level of gene expression, we established and compared cDNA libraries from apparently healthy young and aged mouse ventricular cardiac muscle cells. We report the identification of genes that exhibit aging-related changes of mRNA levels. Aging expression profiles in aged hearts indicate decreased cellular adaptation and protection against stress-induced injury together with the development of contractile dysfunction. The data suggest reduced activity of the mitochondrial electron transport system and reduced levels of cardiac-specific transcription regulators. The cardiomyocyte aging profile of gene expression displays similarities with known heart disorders. Genes whose mRNA levels change with aging in cardiomyocytes might profoundly affect pathological changes in the heart. AD - Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. FAU - Bodyak, Natalya AU - Bodyak N FAU - Kang, Peter M AU - Kang PM FAU - Hiromura, Makoto AU - Hiromura M FAU - Sulijoadikusumo, Indra AU - Sulijoadikusumo I FAU - Horikoshi, Nobuo AU - Horikoshi N FAU - Khrapko, Konstantin AU - Khrapko K FAU - Usheva, Anny AU - Usheva A LA - eng GR - AG18536/AG/NIA GR - DC03299/DC/NIDCD GR - HL62458/HL/NHLBI PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (RNA, Messenger) RN - 11003-00-2 (Actinin) RN - 3326-32-7 (Fluorescein-5-isothiocyanate) RN - 63231-63-0 (RNA) SB - IM MH - Actinin/chemistry MH - Aging/*physiology MH - Animals MH - Blotting, Northern MH - Fluorescein-5-isothiocyanate/chemistry MH - *Gene Expression Profiling MH - Gene Library MH - Heart Ventricles/chemistry/cytology/*metabolism MH - Male MH - Mice MH - Mice, Inbred C57BL MH - Microscopy, Confocal MH - Oligonucleotide Array Sequence Analysis/methods MH - RNA/genetics/metabolism MH - RNA, Messenger/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Time Factors EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3788-94. PMID- 12202765 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041118 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Drosophila damage-specific DNA-binding protein 1 (D-DDB1) is controlled by the DRE/DREF system. PG - 3795-808 AB - We succeeded in cloning the gene, termed d-ddb1, for a Drosophila homolog of the p127 subunit of the human damage-specific DNA-binding protein, thought to recognize (6-4) photoproducts and related structures. In Drosophila, the gene product (D-DDB1) also appeared to play a role as a repair factor, d-ddb1 knockout Kc cells generated with a RNAi method being sensitive to UV. In addition, UV or methyl methanesulfonate treatment increased d-ddb1 transcripts. However, we found that the gene is controlled by the DRE/DREF system, which is generally responsible for activating the promoters of proliferation-related genes. Moreover, during Drosophila development, the transcription of d-ddb1 changed greatly, with the highest levels in unfertilized eggs, indicating that external injury to DNA is not essential to D-DDB1 function. Interestingly, as with UV irradiation-induced transfer of D-DDB1 to the nucleus from the cytoplasm, during spermatogenesis the protein transiently shifted from one cell compartment to the other. The results indicate that D-DDB1 not only contributes to the DNA repair system, but also has a role in cell proliferation and development. AD - Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda-shi, Chiba-ken 278-8510, Japan. FAU - Takata, Kei-ichi AU - Takata K FAU - Ishikawa, Gen AU - Ishikawa G FAU - Hirose, Fumiko AU - Hirose F FAU - Sakaguchi, Kengo AU - Sakaguchi K LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (DNA-Binding Proteins) RN - 0 (Dref protein, Drosophila) RN - 0 (Drosophila Proteins) RN - 0 (RNA I) RN - 0 (RNA, Bacterial) RN - 0 (RNA, Double-Stranded) RN - 0 (RNA, Messenger) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Transcription Factors) RN - 66-27-3 (Methyl Methanesulfonate) RN - EC 1.13.12.- (Luciferases) SB - IM MH - 5' Flanking Region/genetics MH - Animals MH - Base Sequence MH - Binding Sites/genetics MH - Cell Line MH - DNA Replication/genetics MH - DNA-Binding Proteins/*genetics/metabolism MH - *Drosophila Proteins MH - Drosophila melanogaster/cytology/*genetics MH - Embryo, Nonmammalian/metabolism MH - Embryonic Development MH - Female MH - Gene Expression Regulation/drug effects/radiation effects MH - Gene Expression Regulation, Developmental MH - Gene Silencing MH - Luciferases/genetics/metabolism MH - Male MH - Methyl Methanesulfonate/pharmacology MH - Molecular Sequence Data MH - Mutation MH - RNA, Bacterial/genetics/physiology MH - RNA, Double-Stranded/genetics/physiology MH - RNA, Messenger/genetics/metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Response Elements/genetics/*physiology MH - Sequence Deletion MH - Time Factors MH - Transcription Factors/genetics/*physiology MH - Ultraviolet Rays EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3795-808. PMID- 12202766 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Definition and prediction of the full range of transcription factor binding sites--the hepatocyte nuclear factor 1 dimeric site. PG - 3809-17 AB - In animals, transcription factor binding sites are hard to recognize because of their extensive variation. We therefore characterized the general relationship between a specific protein-binding site and its DNA sequence and used this relationship to generate a predictive algorithm for searching other DNA sequences. The experimental process was defined by studying hepatocyte nuclear factor 1 (HNF1), which binds DNA as a dimer on two inverted-repeat 7-bp half sites separated by one base. The binding model was based on the equivalence of the two half sites, which was confirmed in examples where specific modified sites were compared. Binding competition analysis was used to determine the effects of substitution of all four bases at each position in the half site. From these data, a weighted half-site matrix was generated and the full site was evaluated as the sum of two half-site scores. This process accurately predicted even weak binding sites that were significantly different from the consensus sequence. The predictions also showed a direct correlation with measured protein binding. AD - Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. locker@aecom.yu.edu FAU - Locker, Joseph AU - Locker J FAU - Ghosh, David AU - Ghosh D FAU - Luc, Phuong-Van AU - Luc PV FAU - Zheng, Jianhua AU - Zheng J LA - eng GR - CA68440/CA/NCI GR - CA76354/CA/NCI PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (DNA-Binding Proteins) RN - 0 (Nuclear Proteins) RN - 0 (Oligonucleotides) RN - 0 (Transcription Factors) RN - 126548-29-6 (hepatocyte nuclear factor 1) RN - 138674-15-4 (hepatocyte nuclear factor 1-beta) SB - IM MH - Algorithms MH - Base Sequence MH - Binding Sites/genetics MH - Binding, Competitive MH - *DNA-Binding Proteins MH - Electrophoretic Mobility Shift Assay MH - *Nuclear Proteins MH - Oligonucleotides/genetics/metabolism MH - Protein Binding MH - Research Support, U.S. Gov't, P.H.S. MH - Transcription Factors/*genetics/metabolism EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3809-17. PMID- 12202767 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships. PG - 3818-30 AB - DNA methylation is now seen as a primary signal in the cell for mediating transcriptional repression through chromatin formation. The construction and evaluation of enzymes capable of influencing this process in vivo is therefore of significant interest. We have fused the C5-cytosine DNA methyltransferases, M.HhaI and M.HpaII, which both methylate 4 bp sequences containing a CpG dinucleotide, to a three zinc finger protein recognising a 9 bp DNA sequence. DNA methylation analyses demonstrate specific DNA methylation by both enzymes at target sites comprising adjacent methyltransferase and zinc finger subsites, targeted M.HpaII being the most specific. Binding analysis of the targeted M.HpaII enzyme reveals an 8-fold preference for binding to its target site, compared to binding to a zinc finger site alone, and an 18-fold preference over binding to a methyltransferase site alone, thereby demonstrating enhanced binding by the fusion protein, compared to its component proteins. Both DNA binding and methylation are specific for the target site up to separations of approximately 40 bp between the zinc finger and methyltransferase subsites. Ex vivo plasmid methylation experiments are also described that demonstrate targeted methylation. These targeted enzymes, however, are shown to be not fully mono-functional, retaining a significant non-targeted activity most evident at elevated protein concentrations. AD - Department of Molecular Medicine, Guy's, King's and St Thomas' School of Medicine, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK. FAU - McNamara, Andrew R AU - McNamara AR FAU - Hurd, Paul J AU - Hurd PJ FAU - Smith, Alexander E F AU - Smith AE FAU - Ford, Kevin G AU - Ford KG LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Oligonucleotides) RN - 0 (Plasmids) RN - 9007-49-2 (DNA) RN - EC 2.1.1.73 (Site-Specific DNA Methyltransferase (Cytosine-Specific)) RN - EC 3.1.21.- (Deoxyribonuclease HpaII) RN - EC 3.1.21.- (endodeoxyribonuclease HhaI) RN - EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific) SB - IM MH - Binding Sites/genetics MH - Binding, Competitive MH - DNA/genetics/metabolism MH - DNA Methylation MH - Deoxyribonuclease HpaII/metabolism MH - Deoxyribonucleases, Type II Site-Specific/metabolism MH - Kinetics MH - Oligonucleotides/genetics/metabolism MH - Plasmids/genetics MH - Protein Binding MH - Research Support, Non-U.S. Gov't MH - Site-Specific DNA Methyltransferase (Cytosine-Specific)/genetics/*metabolism MH - Substrate Specificity MH - Time Factors MH - Zinc Fingers/genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3818-30. PMID- 12202768 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Dnmt3L is a transcriptional repressor that recruits histone deacetylase. PG - 3831-8 AB - The Dnmt3L protein belongs to the Dnmt3 family of DNA methyltransferases by virtue of its sequence homology in the plant homeodomain (PHD)-like motif. Dnmt3L is essential for the establishment of maternal genomic imprints and, given its lack of key methyltransferase motifs, is more likely to act as a regulator of methylation rather than as an enzyme that methylates DNA. Here, we show that Dnmt3L, like Dnmt3a and Dnmt3b, interacts both in vitro and in vivo with the histone deacetylase HDAC1. Consistent with the binding to a deacetylase, Dnmt3L purifies histone deacetylase activity from nuclear extracts. We find that Dnmt3L can repress transcription and that this repression is dependent on HDAC1 and is relieved by treatment with the HDAC inhibitor trichostatin A. Binding of Dnmt3L to HDAC1 as well as its repressive function require the PHD-like motif. Our results indicate that Dnmt3L plays a role in transcriptional regulation and that recruitment of the HDAC repressive machinery is a shared and conserved feature of the Dnmt3 family. The fact that, despite the absence of a methyltransferase domain, Dnmt3L retains the capacity to contact deacetylase further substantiates the notion that the Dnmts can repress transcription independently of their methylating activities. AD - Free University of Brussels, Faculty of Medicine, Laboratory of Molecular Virology, 808 route de Lennik, 1070 Brussels, Belgium. FAU - Deplus, Rachel AU - Deplus R FAU - Brenner, Carmen AU - Brenner C FAU - Burgers, Wendy A AU - Burgers WA FAU - Putmans, Pascale AU - Putmans P FAU - Kouzarides, Tony AU - Kouzarides T FAU - de Launoit, Yvan AU - de Launoit Y FAU - Fuks, Francois AU - Fuks F LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Recombinant Fusion Proteins) RN - 0 (Repressor Proteins) RN - EC 2.1.1.- (DNMT3L protein, human) RN - EC 2.1.1.37 (DNA (Cytosine-5-)-Methyltransferase) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.5.1.- (HDAC1 protein, human) RN - EC 3.5.1.- (Histone Deacetylases) SB - IM MH - Cell Line MH - DNA (Cytosine-5-)-Methyltransferase/genetics/*metabolism MH - Glutathione Transferase/genetics/metabolism MH - Hela Cells MH - Histone Deacetylases/genetics/*metabolism MH - Humans MH - Precipitin Tests MH - Protein Binding MH - Recombinant Fusion Proteins/genetics/metabolism MH - Repressor Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Transcription, Genetic MH - Tumor Cells, Cultured MH - Zinc Fingers/genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3831-8. PMID- 12202769 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Functional analysis of iceA1, a CATG-recognizing restriction endonuclease gene in Helicobacter pylori. PG - 3839-47 AB - iceA1 in Helicobacter pylori is a homolog of nlaIIIR, which encodes the CATG-specific restriction endonuclease NlaIII in Neisseria lactamica. Analysis of iceA1 sequences from 49 H.pylori strains shows that a full-length NlaIII-like ORF is present in 10 strains, including CH4, but in other strains, including strain 60190, the ORFs are truncated due to a variety of mutations. Our goal was to determine whether iceA1 can encode a NlaIII-like endonuclease. Overexpression in Escherichia coli of iceA1 from CH4, but not from 60190, yielded NlaIII-like activity, indicating that the full-length iceA1 is a functional endonuclease gene. Repair of the iceA1 frameshift mutation in strain 60190 and its expression in E.coli yielded functional NlaIII-like activity. We conclude that iceA1 in CH4 is a functional restriction endonuclease gene, while iceA1 in 60190 is not, due to a frameshift mutation, but that its repair restores its restriction endonuclease activity. AD - Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. FAU - Xu, Qing AU - Xu Q FAU - Morgan, R D AU - Morgan RD FAU - Roberts, R J AU - Roberts RJ FAU - Xu, S Y AU - Xu SY FAU - van Doorn, L J AU - van Doorn LJ FAU - Donahue, J P AU - Donahue JP FAU - Miller, G G AU - Miller GG FAU - Blaser, Martin J AU - Blaser MJ LA - eng SI - GENBANK/AF459446 GR - DK53707/DK/NIDDK GR - GM56534/GM/NIGMS GR - GM63270/GM/NIGMS PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Bacterial Proteins) RN - 0 (DNA, Bacterial) RN - 0 (Recombinant Proteins) RN - 0 (iceA1 protein, Helicobacter pylori) RN - EC 3.1.21 (DNA Restriction Enzymes) RN - EC 3.1.21.- (endodeoxyribonuclease NlaIII) RN - EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific) SB - IM MH - Amino Acid Sequence MH - Bacterial Proteins/genetics/*metabolism MH - Base Sequence MH - Comparative Study MH - DNA Restriction Enzymes/genetics/*metabolism MH - DNA, Bacterial/chemistry/genetics MH - Deoxyribonucleases, Type II Site-Specific/genetics/metabolism MH - Escherichia/genetics MH - Frameshift Mutation/genetics MH - Helicobacter pylori/*enzymology/genetics MH - Molecular Sequence Data MH - Neisseria/enzymology MH - Recombinant Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Analysis, DNA MH - Sequence Homology, Nucleic Acid EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3839-47. PMID- 12202770 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin. PG - 3848-56 AB - The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link. AD - Cancer Research UK Drug-DNA Interactions Research Group, Department of Oncology, Royal Free and University College Medical School, 91 Riding House Street, London W1W 7BS, UK. FAU - De Silva, Inusha U AU - De Silva IU FAU - McHugh, Peter J AU - McHugh PJ FAU - Clingen, Peter H AU - Clingen PH FAU - Hartley, John A AU - Hartley JA LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Antineoplastic Agents) RN - 0 (DNA-Binding Proteins) RN - 0 (ERCC4 protein) RN - 0 (Proteins) RN - 15663-27-1 (Cisplatin) RN - 9007-49-2 (DNA) RN - EC 3.1.- (ERCC-1 protein, human) RN - EC 3.1.- (Endonucleases) SB - IM MH - Animals MH - Antineoplastic Agents/*pharmacology MH - CHO Cells MH - Cell Division/drug effects/genetics MH - Cell Line MH - Cisplatin/*pharmacology MH - DNA/drug effects/genetics MH - DNA Damage MH - DNA Repair/*genetics MH - DNA-Binding Proteins/*genetics MH - Dose-Response Relationship, Drug MH - Electrophoresis, Gel, Pulsed-Field MH - *Endonucleases MH - Hamsters MH - Mutation MH - Proteins/*genetics MH - Research Support, Non-U.S. Gov't EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3848-56. PMID- 12202771 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Challenging artificial genetic systems: thymidine analogs with 5-position sulfur functionality. PG - 3857-69 AB - Eight different polymerases, chosen from evolutionary families A (Taq, Tfl, HotTub and Tth) and B (Pfu, Pwo, Vent and Deep Vent), were examined for their ability to incorporate 5-position modified 2'-deoxyuridine derivatives that carry a protected thiol group appended via different linkers containing either three or four carbon atoms. This represents the first attempt to incorporate the thiol functionality into DNA via enzymatic synthesis. Each polymerase-substrate combination was evaluated using a hierarchy of increasingly more difficult challenges, starting with incorporation of a single derivative, proceeding to incorporation of two derivatives at adjacent sites and non-adjacent sites, then examining the ability of the polymerase to accept the derivative within the template, and concluding with a challenge involving PCR. The evaluation of thiol-bearing 2'-deoxyuridine derivatives was then extended to consider their chemical stabilities. Stability was found to be less than satisfactory when the thiol functionality has a 'propargylic' relationship to the unsaturation in the linker. The best polymerase-appendage combination used the polymerase from Pyrococcus woesei (Pwo) and the 5'-tBu-SS-CH2-CH2-C [triple bond] C-linker. This pair supported PCR amplification and therefore should have value in artificial in vitro selection experiments. Indeed, we discovered that Pwo and Pfu preferred the derivative triphosphate over TTP, the natural substrate, in competition studies. These studies confirm an earlier suggestion that membership of an evolutionary family of polymerases is a partial predictor of the ability of the polymerase to accept 5-modified 2'-deoxyuridines. Considerable differences are displayed by different members within a polymerase family, however. This remains curious, as the ability of the polymerase to replicate natural DNA with high fidelity and its propensity to exclude unnatural analogs are presumed to be correlated. AD - Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA. FAU - Held, Heike A AU - Held HA FAU - Benner, Steven A AU - Benner SA LA - eng GR - GM 54048/GM/NIGMS PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (DNA Primers) RN - 0 (Deoxyuracil Nucleotides) RN - 0 (Oligonucleotides) RN - 50-89-5 (Thymidine) RN - 7704-34-9 (Sulfur) RN - EC 2.7.7.7 (DNA-Directed DNA Polymerase) SB - IM MH - DNA Primers/genetics MH - DNA-Directed DNA Polymerase/*metabolism MH - Deoxyuracil Nucleotides/chemistry/metabolism MH - Oligonucleotides/chemistry/genetics/metabolism MH - Polymerase Chain Reaction/methods MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Sulfur/chemistry MH - Templates, Genetic MH - Thymidine/chemistry/metabolism EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3857-69. PMID- 12202772 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Mutations altering the cleavage specificity of a homing endonuclease. PG - 3870-9 AB - The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein-DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences. AD - Department of Biology and Program in Molecular Biology, Pomona College, 609 North College Avenue, Claremont, CA 91711, USA. lms14747@pomona.edu FAU - Seligman, Lenny M AU - Seligman LM FAU - Chisholm, Karen M AU - Chisholm KM FAU - Chevalier, Brett S AU - Chevalier BS FAU - Chadsey, Meggen S AU - Chadsey MS FAU - Edwards, Samuel T AU - Edwards ST FAU - Savage, Jeremiah H AU - Savage JH FAU - Veillet, Adeline L AU - Veillet AL LA - eng GR - CA88942/CA/NCI GR - T32 CA 09437/CA/NCI GR - T32 CA80416/CA/NCI PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Plasmids) RN - 9007-49-2 (DNA) RN - EC 3.1.21 (DNA Restriction Enzymes) RN - EC 3.1.21.- (endodeoxyribonuclease CreI) SB - IM MH - Amino Acid Substitution MH - Base Sequence MH - Binding Sites/genetics MH - Binding, Competitive MH - DNA/genetics/*metabolism MH - DNA Restriction Enzymes/genetics/*metabolism MH - Escherichia coli/genetics MH - Kinetics MH - Mutation MH - Plasmids/genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Nucleic Acid MH - Substrate Specificity EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3870-9. PMID- 12202773 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20040506 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Promiscuous methylation of non-canonical DNA sites by HaeIII methyltransferase. PG - 3880-5 AB - The cytosine C5 methyltransferase M.HaeIII recognises and methylates the central cytosine of its canonical site GGCC. Here we report that M.HaeIII can also, with lower efficiency, methylate cytosines located in a wide range of non-canonical sequences. Using bisulphite sequencing we mapped the methyl- cytosine residues in DNA methylated in vitro and in vivo by M.HaeIII. Methyl-cytosine residues were observed in multiple sequence contexts, most commonly, but not exclusively, at star sites (sites differing by a single base from the canonical sequence). The most frequently used star sites had changes at positions 1 and 4, but there is little or no methylation at star sites changed at position 2. The rate of methylation of non-canonical sites can be quite significant: a DNA substrate lacking a canonical site was methylated by M.HaeIII in vitro at a rate only an order of magnitude slower than an otherwise identical substrate containing the canonical site. In vivo methylation of non-canonical sites may therefore be significant and may have provided the starting point for the evolution of restriction-modification systems with novel sequence specificities. AD - MRC Centre for Protein Engineering and MRC Laboratory for Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK and. Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76 100, Israel. FAU - Cohen, Helen M AU - Cohen HM FAU - Tawfik, Dan S AU - Tawfik DS FAU - Griffiths, Andrew D AU - Griffiths AD LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Oligonucleotides) RN - 10028-17-8 (Tritium) RN - 29908-03-0 (S-Adenosylmethionine) RN - 9007-49-2 (DNA) RN - EC 2.1.1.37 (GGCC-specific DNA methyltransferase) RN - EC 2.1.1.73 (Site-Specific DNA Methyltransferase (Cytosine-Specific)) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Base Sequence MH - Binding Sites/genetics MH - DNA/genetics/*metabolism MH - *DNA Methylation MH - Glutathione Transferase/genetics/metabolism MH - Molecular Sequence Data MH - Oligonucleotides/genetics/metabolism MH - S-Adenosylmethionine/metabolism MH - Sequence Homology, Nucleic Acid MH - Site-Specific DNA Methyltransferase (Cytosine-Specific)/*metabolism MH - Substrate Specificity MH - Tritium EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3880-5. PMID- 12202774 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Prediction of consensus structural motifs in a family of coregulated RNA sequences. PG - 3886-93 AB - Given a set of homologous or functionally related RNA sequences, the consensus motifs may represent the binding sites of RNA regulatory proteins. Unlike DNA motifs, RNA motifs are more conserved in structures than in sequences. Knowing the structural motifs can help us gain a deeper insight of the regulation activities. There have been various studies of RNA secondary structure prediction, but most of them are not focused on finding motifs from sets of functionally related sequences. Although recent research shows some new approaches to RNA motif finding, they are limited to finding relatively simple structures, e.g. stem-loops. In this paper, we propose a novel genetic programming approach to RNA secondary structure prediction. It is capable of finding more complex structures than stem-loops. To demonstrate the performance of our new approach as well as to keep the consistency of our comparative study, we first tested it on the same data sets previously used to verify the current prediction systems. To show the flexibility of our new approach, we also tested it on a data set that contains pseudoknot motifs which most current systems cannot identify. A web-based user interface of the prediction system is set up at http://bioinfo. cis.nctu.edu.tw/service/gprm/. AD - Computer and Information Science Department, National Chiao Tung University, 1001 Ta Hsueh Road, Hsinchu, Taiwan. yhu@cis.nctu.edu.tw FAU - Hu, Yuh-Jyh AU - Hu YJ LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 63231-63-0 (RNA) SB - IM MH - Algorithms MH - Base Sequence MH - Computational Biology/methods MH - Mutation MH - *Nucleic Acid Conformation MH - RNA/*chemistry/genetics MH - Reproducibility of Results MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Nucleic Acid MH - *Software EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3886-93. PMID- 12202775 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Human non-synonymous SNPs: server and survey. PG - 3894-900 AB - Human single nucleotide polymorphisms (SNPs) represent the most frequent type of human population DNA variation. One of the main goals of SNP research is to understand the genetics of the human phenotype variation and especially the genetic basis of human complex diseases. Non-synonymous coding SNPs (nsSNPs) comprise a group of SNPs that, together with SNPs in regulatory regions, are believed to have the highest impact on phenotype. Here we present a World Wide Web server to predict the effect of an nsSNP on protein structure and function. The prediction method enabled analysis of the publicly available SNP database HGVbase, which gave rise to a dataset of nsSNPs with predicted functionality. The dataset was further used to compare the effect of various structural and functional characteristics of amino acid substitutions responsible for phenotypic display of nsSNPs. We also studied the dependence of selective pressure on the structural and functional properties of proteins. We found that in our dataset the selection pressure against deleterious SNPs depends on the molecular function of the protein, although it is insensitive to several other protein features considered. The strongest selective pressure was detected for proteins involved in transcription regulation. AD - European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany. FAU - Ramensky, Vasily AU - Ramensky V FAU - Bork, Peer AU - Bork P FAU - Sunyaev, Shamil AU - Sunyaev S LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Proteins) SB - IM MH - *Databases, Genetic MH - Humans MH - Information Storage and Retrieval/methods MH - *Internet MH - Mutation MH - Polymorphism, Single Nucleotide/*genetics MH - Proteins/genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3894-900. PMID- 12202776 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Dictionary-driven protein annotation. PG - 3901-16 AB - Computational methods seeking to automatically determine the properties (functional, structural, physicochemical, etc.) of a protein directly from the sequence have long been the focus of numerous research groups. With the advent of advanced sequencing methods and systems, the number of amino acid sequences that are being deposited in the public databases has been increasing steadily. This has in turn generated a renewed demand for automated approaches that can annotate individual sequences and complete genomes quickly, exhaustively and objectively. In this paper, we present one such approach that is centered around and exploits the Bio-Dictionary, a collection of amino acid patterns that completely covers the natural sequence space and can capture functional and structural signals that have been reused during evolution, within and across protein families. Our annotation approach also makes use of a weighted, position-specific scoring scheme that is unaffected by the over-representation of well-conserved proteins and protein fragments in the databases used. For a given query sequence, the method permits one to determine, in a single pass, the following: local and global similarities between the query and any protein already present in a public database; the likeness of the query to all available archaeal/ bacterial/eukaryotic/viral sequences in the database as a function of amino acid position within the query; the character of secondary structure of the query as a function of amino acid position within the query; the cytoplasmic, transmembrane or extracellular behavior of the query; the nature and position of binding domains, active sites, post-translationally modified sites, signal peptides, etc. In terms of performance, the proposed method is exhaustive, objective and allows for the rapid annotation of individual sequences and full genomes. Annotation examples are presented and discussed in Results, including individual queries and complete genomes that were released publicly after we built the Bio-Dictionary that is used in our experiments. Finally, we have computed the annotations of more than 70 complete genomes and made them available on the World Wide Web at http://cbcsrv.watson.ibm.com/Annotations/. AD - Bioinformatics and Pattern Discovery Group, IBM TJ Watson Research Center, Yorktown Heights, NY 10598, USA. rigoutso@us.ibm.com FAU - Rigoutsos, Isidore AU - Rigoutsos I FAU - Huynh, Tien AU - Huynh T FAU - Floratos, Aris AU - Floratos A FAU - Parida, Laxmi AU - Parida L FAU - Platt, Daniel AU - Platt D LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Proteins) RN - 0 (Proteome) RN - 0 (Ubiquitin) SB - IM MH - Algorithms MH - Amino Acid Sequence MH - Comparative Study MH - Computational Biology/methods MH - *Databases, Protein MH - *Dictionaries, Chemical MH - Genomics MH - Humans MH - Internet MH - Molecular Sequence Data MH - Proteins/*genetics MH - Proteome/genetics MH - Sequence Alignment/methods MH - Sequence Homology, Amino Acid MH - Ubiquitin/genetics EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):3901-16. PMID- 12202777 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Improved quantitative real-time RT-PCR for expression profiling of individual cells. PG - e89 AB - The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single-cell resolution is highly desirable, in particular for complex tissues like the brain that contain a large variety of different cell types in close proximity. The patch-clamp technique allows selective harvesting of single-cell cytoplasm after recording of cellular activity. However, components of the cDNA reaction, in particular the reverse transcriptase itself, significantly inhibit subsequent rtqPCR amplification. Using undiluted single-cell cDNA reaction mix directly as template for rtqPCR, I observed that the amplification kinetics of rtqPCRs were dramatically altered in a non-systematic fashion. Here, I describe a simple and robust precipitation protocol suitable for purification of single-cell cDNA that completely removes inhibitory RT components without detectable loss of cDNA. This improved single-cell real-time RT-PCR protocol provides a powerful tool to quantify differential gene expression of individual cells and thus could complement global microarray-based expression profiling strategies. AD - University Laboratory of Physiology and MRC Anatomical Neuropharmacology Unit, Department of Pharmacology, Oxford University, Parks Road, Oxford OX1 3PT, UK. birgit.liss@physiol.ox.ac.uk FAU - Liss, Birgit AU - Liss B LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (DNA, Complementary) RN - 0 (RNA, Messenger) SB - IM MH - Animals MH - Brain/cytology/*metabolism MH - DNA, Complementary/metabolism MH - Gene Expression Profiling/*methods MH - Mice MH - Mice, Inbred C57BL MH - Neurons/cytology/metabolism MH - RNA, Messenger/genetics MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Time Factors EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):e89. PMID- 12202778 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein. PG - e90 AB - CREB-binding protein (CBP) is a multifunctional cofactor implicated in many intracellular signal transduction pathways. We aimed to investigate the involvement of CBP in the cAMP response element-binding protein (CREB)-mediated pathway. The point mutation Tyr658Ala in the CREB-binding domain (CBD) was shown to abolish the binding activity of CBP to phospho-CREB, the activated form of CREB. By using a mutant Cre/loxP recombination system, this point mutation was aimed to be generated in the mouse genome in a tissue- and time-specific manner. A targeting construct in which CBD exon 5 and inverted exon 5* containing the point mutation flanked by two mutant loxP sites (lox66 and lox71) oriented in a head-to-head position was generated. When Cre recombinase is present, the DNA flanked by the two mutant loxP sites is inverted, forming one loxP and one double mutated loxP site. As the double mutated loxP site shows low affinity for Cre recombinase, the favorable reaction leads to a product where the mutated exon 5* is placed into the position to be correctly transcribed and spliced. Inversion was observed to be complete in both bacteria and mouse embryonic stem cells. Our results indicate that this Cre- mediated inversion method is a valuable tool to introduce point mutations in the mouse genome in a regulatable manner. AD - Research Group Molecular Genetics of Behavior, Max-Planck-Institute of Psychiatry, Kraepelinstrasse 2-10, D-80804 Munich, Germany. FAU - Zhang, Zuwen AU - Zhang Z FAU - Lutz, Beat AU - Lutz B LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (CREB-binding protein) RN - 0 (Nuclear Proteins) RN - 0 (Plasmids) RN - 0 (Trans-Activators) RN - 0 (Viral Proteins) RN - 9007-49-2 (DNA) RN - EC 2.7.7.- (Cre recombinase, virus) RN - EC 2.7.7.- (Integrases) SB - IM MH - Animals MH - Attachment Sites (Microbiology)/*genetics MH - Bacteria/genetics MH - Bacteriophage P1/genetics MH - Base Sequence MH - Blotting, Southern MH - Cell Line MH - Cells, Cultured MH - DNA/genetics/metabolism MH - Gene Targeting/methods MH - Integrases/genetics/*metabolism MH - *Inversion, Chromosome MH - Mice MH - Mutation, Missense MH - Nuclear Proteins/genetics MH - Plasmids/genetics MH - Point Mutation MH - Research Support, Non-U.S. Gov't MH - Trans-Activators/genetics MH - Viral Proteins/genetics/*metabolism EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):e90. PMID- 12202779 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection. PG - e91 AB - Locked nucleic acids (LNAs) are synthetic nucleic acid analogs that bind to complementary target molecules (DNA, RNA or LNA) with very high affinity. At the same time, this binding affinity is decreased substantially when the hybrids thus formed contain even a single mismatched base pair. We have exploited these properties of LNA probes to develop a new method for single nucleotide polymorphism genotyping. In this method, very short (hexamer or heptamer) LNA probes are labeled with either rhodamine or hexachlorofluorescein (HEX), and their hybridization to target DNAs is followed by measuring the fluorescence polarization (FP) of the dyes. The formation of perfectly complementary double-stranded hybrids gives rise to significant FP increases, whereas the presence of single mismatches results in very small or no changes of this parameter. Multiplexing of the assay can be achieved by using differentially labeled wild-type and mutant specific probes in the same solution. The method is homogeneous, and because of the use of extremely short LNA probes, the generation of a universal set of genotyping reagents is possible. AD - Caliper Technologies Corporation, Mountain View, CA 94043, USA. FAU - Simeonov, Anton AU - Simeonov A FAU - Nikiforov, Theo T AU - Nikiforov TT LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Fluorescent Dyes) RN - 0 (Oligonucleotide Probes) RN - 0 (Rhodamines) SB - IM MH - Base Composition MH - Fluorescence Polarization/*methods MH - Fluorescent Dyes/chemistry MH - Genotype MH - Nucleic Acid Denaturation MH - Nucleic Acid Hybridization/methods MH - Oligonucleotide Probes/chemistry/*genetics MH - Polymerase Chain Reaction MH - Polymorphism, Single Nucleotide/*genetics MH - Rhodamines/chemistry MH - Temperature EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):e91. PMID- 12202780 OWN - NLM STAT- MEDLINE DA - 20020830 DCOM- 20021112 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 30 IP - 17 DP - 2002 Sep 1 TI - Multivariate curve resolution: a powerful tool for the analysis of conformational transitions in nucleic acids. PG - e92 AB - A successful application is reported of the multivariate curve resolution alternating least-squares method (MCR-ALS) for the analysis of nucleic acid melting and salt-induced transitions. Under conditions where several structures co-exist in a conformational equilibrium, MCR-ALS analysis of the UV and circular dichroism (CD) spectra at different temperatures, ionic strength and oligonucleotide concentration allows for the resolution of concentration profiles and pure spectra of the different species. The methodology is illustrated by the case of the cyclic oligonucleotide d. The melting transition of this molecule at different oligonucleotide concentrations was studied at 0, 2 and 10 mM MgCl2 by UV and CD spectroscopy. In addition, salt titration experiments were carried out at 21.0 and 54.0 degrees C. The MCR-ALS analysis indicates that three different conformations of this molecule co-exist in solution. In agreement with previous NMR studies, these conformations were assigned to a monomeric dumbbell-like structure, a dimeric four-stranded conformation and a disordered (random coil) structure. The MCR-ALS methodology allows for a detailed analysis of how this equilibrium is affected by temperature, salt and oligonucleotide concentration. AD - Departament de Quimica Analitica, Universitat de Barcelona, Diagonal 647, E-08028, Barcelona, Spain. FAU - Jaumot, Joaquim AU - Jaumot J FAU - Escaja, Nuria AU - Escaja N FAU - Gargallo, Raimundo AU - Gargallo R FAU - Gonzalez, Carlos AU - Gonzalez C FAU - Pedroso, Enrique AU - Pedroso E FAU - Tauler, Roma AU - Tauler R LA - eng PT - Journal Article PL - England TA - Nucleic Acids Res JID - 0411011 RN - 0 (Nucleic Acids) RN - 7647-14-5 (Sodium Chloride) RN - 7732-18-5 (Water) RN - 7786-30-3 (Magnesium Chloride) SB - IM MH - *Algorithms MH - Base Sequence MH - Circular Dichroism MH - Least-Squares Analysis MH - Magnesium Chloride/pharmacology MH - Multivariate Analysis MH - *Nucleic Acid Conformation MH - Nucleic Acid Denaturation MH - Nucleic Acids/*chemistry/drug effects MH - Research Support, Non-U.S. Gov't MH - Sodium Chloride/pharmacology MH - Spectrophotometry, Ultraviolet MH - Temperature MH - Water/pharmacology EDAT- 2002/08/31 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Nucleic Acids Res 2002 Sep 1;30(17):e92. PMID- 12204094 OWN - NLM STAT- Publisher DA - 20020902 PUBM- Print IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Aug 31 TI - Pro-domain removal in ASP-2 and the cleavage of the amyloid precursor are influenced by pH. PG - 25 AB - Background One of the signatures of Alzheimer's disease is the accumulation of aggregated amyloid protein, A-beta, in the brain. A-beta arises from cleavage of the Amyloid Precursor protein by alpha and gamma secretases, which present attractive candidates for therapeutic targeting. Two beta-secretase candidates, ASP-1 and ASP-2, were identified as aspartic proteases, both of which cleave the amyloid precursor at the beta-site. These are produced as immature transmembrane proteins containing a pro-segment. Results ASP-2 expressed in HEK293-cells cleaved the Swedish mutant amyloid precursor at different BETA-sites at different pHs in vitro. Recent reports show that furin cleaves the pro-peptide of ASP-2, whereas ASP-1 undergoes auto-catalysis. We show that purified recombinant ASP-2 cleaves its own pro-peptide at pH5 but not pH8.5 as seen by mass spectrometry, electrophoresis and N-terminal sequencing. Conclusion We suggest that ASP-2 processing as well as activity are influenced by pH, and hence the cellular localisation of the protein may have profound effects on the production of A-beta. These factors should be taken into consideration in the design of potential inhibitors for these enzymes. AU - Sidera C AU - Liu C AU - Austen BM LA - ENG PT - JOURNAL ARTICLE TA - BMC Biochem JID - 101084098 EDAT- 2002/09/03 10:00 MHDA- 2002/09/03 10:00 PHST- 2002/05/16 [received] PHST- 2002/08/31 [accepted] PHST- 2002/08/31 [aheadofprint] PST - aheadofprint SO - BMC Biochem 2002 Aug 31;3(1):25. PMID- 12204942 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Biological and clinical significance of lipids as modulators of immune system functions. PG - 945-50 AD - Unit of Microbiology, Department of Health Sciences, Faculty of Experimental Sciences, University of Jaen, E-23071, Jaen, Spain. mapablo@ujaen.es FAU - de Pablo, Manuel A AU - de Pablo MA FAU - Puertollano, Maria A AU - Puertollano MA FAU - Alvarez de Cienfuegos, Gerardo AU - Alvarez de Cienfuegos G LA - eng PT - Journal Article PT - Review PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Dietary Fats) RN - 0 (Fatty Acids) SB - IM MH - Dietary Fats/*immunology MH - Fatty Acids/*immunology MH - Humans MH - Immune System/*immunology MH - Nutritional Status RF - 83 EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):945-50. PMID- 12204943 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Laboratory diagnosis of visceral leishmaniasis. PG - 951-8 AD - Kala-Azar Medical Research Center, Department of Medicine, Banaras Hindu University, Institute of Medical Sciences, Varanasi 221 005, India. shyam_vns@satyam.net.in FAU - Sundar, Shyam AU - Sundar S FAU - Rai, M AU - Rai M LA - eng PT - Journal Article PT - Review PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 SB - IM MH - Animals MH - Humans MH - Leishmania donovani/*isolation & purification MH - Leishmaniasis, Visceral/*diagnosis MH - Research Support, Non-U.S. Gov't RF - 86 EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):951-8. PMID- 12204944 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Peripheral blood lymphocyte subsets in adolescents: a longitudinal analysis from the REACH project. PG - 959-65 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. AD - Children's Hospital of Philadelphia, Department of Pediatrics, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, 19104, USA. rudy@email.chop.edu FAU - Rudy, Bret J AU - Rudy BJ FAU - Wilson, Craig M AU - Wilson CM FAU - Durako, Stephen AU - Durako S FAU - Moscicki, Anna-Barbara AU - Moscicki AB FAU - Muenz, Larry AU - Muenz L FAU - Douglas, Steven D AU - Douglas SD LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antigens, CD19) RN - 0 (Receptors, IgG) SB - IM MH - Adolescent MH - Adult MH - Antigens, CD19/analysis MH - CD4-Positive T-Lymphocytes/chemistry/*cytology MH - CD8-Positive T-Lymphocytes/chemistry/*cytology MH - Child MH - Cohort Studies MH - Female MH - Flow Cytometry/*standards MH - Humans MH - Immunologic Memory MH - Longitudinal Studies MH - Male MH - Receptors, IgG/analysis MH - Reference Values MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):959-65. PMID- 12204945 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Active surveillance for scrapie by third eyelid biopsy and genetic susceptibility testing of flocks of sheep in Wyoming. PG - 966-71 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 PrP(Sc), the protein marker for this group of disorders. For live, asymptomatic sheep, diagnosis using tonsil or third-eyelid lymphoid tissue biopsy and PrP(Sc) 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 PrP(Sc)-positive sheep were identified through the passive surveillance program, and an additional three PrP(Sc)-positive sheep, including two from flocks with no history of scrapie, were identified through the active surveillance program. All PrP(Sc)-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. AD - Animal Disease Research Unit, Agricultural Research Service, Pullman, Washington 99164, USA. korouke@vetmed.wsu.edu FAU - O'Rourke, Katherine I AU - O'Rourke KI FAU - Duncan, John V AU - Duncan JV FAU - Logan, James R AU - Logan JR FAU - Anderson, Anne K AU - Anderson AK FAU - Norden, Dianne K AU - Norden DK FAU - Williams, Elizabeth S AU - Williams ES FAU - Combs, Bret A AU - Combs BA FAU - Stobart, Robert H AU - Stobart RH FAU - Moss, Gary E AU - Moss GE FAU - Sutton, Diane L AU - Sutton DL LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (PrPSc Proteins) SB - IM MH - Animals MH - Biopsy MH - Genetic Predisposition to Disease MH - Nictitating Membrane/chemistry/*pathology MH - PrPSc Proteins/analysis MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Scrapie/*genetics/*pathology MH - Sheep MH - Sheep Diseases/*genetics/*pathology MH - Wyoming EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):966-71. PMID- 12204946 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041124 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep 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. PG - 972-82 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 (L(1) to S(37)) 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 (K(15) to V(32)) 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 E(16) and K(25) with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q(22), D(26), and N(30) 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. AD - Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan. nagaoki@med.juntendo.ac.jp FAU - Nagaoka, Isao AU - Nagaoka I FAU - Hirota, Satoko AU - Hirota S FAU - Niyonsaba, Francois AU - Niyonsaba F FAU - Hirata, Michimasa AU - Hirata M FAU - Adachi, Yoshiyuki AU - Adachi Y FAU - Tamura, Hiroshi AU - Tamura H FAU - Tanaka, Shigenori AU - Tanaka S FAU - Heumann, Didier AU - Heumann D LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Amino Acids) RN - 0 (Antigens, CD14) RN - 0 (Antimicrobial Cationic Peptides) RN - 0 (Ion Channels) RN - 0 (Lipopolysaccharides) RN - 0 (Peptide Fragments) RN - 0 (cathelicidin antimicrobial peptide) RN - 0 (non-selective cation channel protein, human) RN - 143108-26-3 (CAP18 lipopolysaccharide-binding protein) SB - IM MH - Amino Acid Sequence MH - Amino Acids/*chemistry MH - Animals MH - Antigens, CD14/analysis MH - Antimicrobial Cationic Peptides/*chemistry MH - Cell Line MH - Disease Models, Animal MH - Flow Cytometry MH - Humans MH - Hydrophobicity MH - Ion Channels/chemistry MH - Lipopolysaccharides/*chemistry/pharmacology MH - Macrophages/chemistry/cytology/metabolism MH - Molecular Sequence Data MH - Peptide Fragments/chemistry/metabolism/pharmacology MH - Research Support, Non-U.S. Gov't MH - Shock, Septic/chemically induced/metabolism EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):972-82. PMID- 12204947 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Impaired macrophage phagocytosis of apoptotic neutrophils in patients with human immunodeficiency virus type 1 infection. PG - 983-6 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/mm(3) and in particular in those with <200 CD4(+) T lymphocyte cells/mm(3). 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. AD - Section of Pediatric Infectious Diseases, Regional Hospital, Varese, Italy. eiwlw@tin.it FAU - Torre, Donato AU - Torre D FAU - Gennero, Luisa AU - Gennero L FAU - Baccino, F M AU - Baccino FM FAU - Speranza, Filippo AU - Speranza F FAU - Biondi, Gilberto AU - Biondi G FAU - Pugliese, Agostino AU - Pugliese A LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Gene Products, nef) SB - IM MH - Adult MH - Apoptosis/*immunology MH - Gene Products, nef/immunology/pharmacology MH - HIV Infections/*immunology MH - *HIV-1 MH - Humans MH - Macrophages/*immunology/metabolism/virology MH - Neutrophils/*immunology/virology MH - Oxidation-Reduction MH - Phagocytosis/drug effects/*immunology EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):983-6. PMID- 12204948 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Evaluation of an in-house-developed radioassay kit for antibody detection in cases of pulmonary tuberculosis and tuberculous meningitis. PG - 987-93 AB - A radioassay for the detection of antitubercular antibody has been developed. The technique involves the addition of (125)I-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. AD - Laboratory Nuclear Medicine Section, Bhabha Atomic Research Centre, c/o Tata Memorial Centre Annexe. Department of Microbiology. Department of Pediatrics, K.E.M. Hospital, Mumbai-400012, India. FAU - Kameswaran, M AU - Kameswaran M FAU - Shetty, K AU - Shetty K FAU - Ray, M K AU - Ray MK FAU - Jaleel, M A AU - Jaleel MA FAU - Kadival, G V AU - Kadival GV LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antibodies, Bacterial) RN - 0 (Reagent Kits, Diagnostic) SB - IM MH - Antibodies, Bacterial/*analysis MH - Humans MH - Mycobacterium tuberculosis/*immunology MH - Radioligand Assay/*methods MH - Reagent Kits, Diagnostic MH - Serologic Tests/methods MH - Tuberculosis, Meningeal/*diagnosis MH - Tuberculosis, Pulmonary/*diagnosis EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):987-93. PMID- 12204949 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Functional and phenotypic changes in circulating lymphocytes from hospitalized zambian children with measles. PG - 994-1003 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 >38.5 degrees C, 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 beta(2) 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. AD - W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA. FAU - Ryon, Judith J AU - Ryon JJ FAU - Moss, William J AU - Moss WJ FAU - Monze, Mwaka AU - Monze M FAU - Griffin, Diane E AU - Griffin DE LA - eng GR - AI 23047/AI/NIAID PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Annexin A5) RN - 0 (Antigens, CD95) RN - 0 (Antigens, Surface) SB - IM MH - Annexin A5/metabolism MH - Antigens, CD95/blood MH - Antigens, Surface/blood MH - Apoptosis/immunology MH - Cell Survival/immunology MH - Cells, Cultured MH - Child MH - Child, Hospitalized MH - Child, Preschool MH - Female MH - Follow-Up Studies MH - Humans MH - *Immunophenotyping MH - Infant MH - Lymphocytes/cytology/immunology/metabolism MH - Lymphopenia/immunology/virology MH - Male MH - Measles/*immunology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Zambia EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):994-1003. PMID- 12204950 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Comparison of in-house and commercial slides for detection by immunofluorescence of immunoglobulins G and M against Bartonella henselae and Bartonella quintana. PG - 1004-9 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. AD - Unite des Rickettsies, CNRS UMR 6020, IFR 48, Faculte de Medecine, Universite de la Mediterranee, 13385 Marseille Cedex 05, France. FAU - Maurin, M AU - Maurin M FAU - Rolain, J M AU - Rolain JM FAU - Raoult, D AU - Raoult D LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antibodies, Bacterial) RN - 0 (Immunoglobulin G) RN - 0 (Immunoglobulin M) SB - IM MH - Antibodies, Bacterial/*analysis MH - Bacteremia/diagnosis MH - Bartonella henselae/*immunology MH - Bartonella quintana/*immunology MH - Cat-Scratch Disease/*diagnosis MH - Chronic Disease MH - Comparative Study MH - Endocarditis, Bacterial/diagnosis MH - Fluorescent Antibody Technique, Indirect/*methods MH - Humans MH - Immunoglobulin G/analysis MH - Immunoglobulin M/analysis MH - Sensitivity and Specificity MH - Trench Fever/*diagnosis EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1004-9. PMID- 12204951 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep 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. PG - 1010-3 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. AD - Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803, USA. FAU - Lin, Xiao-Qing AU - Lin XQ FAU - O'Reilly, Kathy L AU - O'Reilly KL FAU - Storz, Johannes AU - Storz J LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antibodies, Viral) RN - 0 (Antigens, Viral) SB - IM MH - Animals MH - Antibodies, Viral/*blood MH - Antigens, Viral/blood MH - Cattle MH - Comparative Study MH - Coronavirus Infections/*immunology/*veterinary MH - Coronavirus, Bovine/classification/*immunology MH - Intestines/virology MH - Lung/virology MH - Neutralization Tests MH - Pasteurellosis, Pneumonic/etiology/*immunology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Serotyping/veterinary EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1010-3. PMID- 12204952 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep 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. PG - 1014-20 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. AD - Microbiology and Tumorbiology Center, Karolinska Institute and Swedish Institute for Infectious Disease Control, S-171 77 Stockholm, Sweden. FAU - Guzman, G E AU - Guzman GE FAU - Awadzi, K AU - Awadzi K FAU - Opoku, N AU - Opoku N FAU - Narayanan, R B AU - Narayanan RB FAU - Akuffo, H O AU - Akuffo HO LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antigens, Helminth) SB - IM MH - Animals MH - Antigens, Helminth/analysis/immunology MH - Biopsy/*methods/standards MH - Comparative Study MH - Cross Reactions MH - Ghana MH - Humans MH - Immunoblotting/*methods/standards MH - Mass Screening/methods MH - Onchocerca volvulus/*isolation & purification MH - Onchocerciasis/*pathology MH - Reproducibility of Results MH - Research Support, Non-U.S. Gov't MH - Skin/parasitology/*pathology EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1014-20. PMID- 12204953 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Calcium phosphate nanoparticles induce mucosal immunity and protection against herpes simplex virus type 2. PG - 1021-4 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. AD - BioSante Pharmaceuticals, Inc., Smyrna, Georgia 30082, USA. qinghe@bellsouth.net FAU - He, Qing AU - He Q FAU - Mitchell, Alaina AU - Mitchell A FAU - Morcol, Tulin AU - Morcol T FAU - Bell, Steve J D AU - Bell SJ LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Adjuvants, Immunologic) RN - 0 (Calcium Phosphates) RN - 0 (alpha-tricalcium phosphate) RN - 0 (dicalcium phosphate anhydrous) RN - 0 (monocalcium phosphate) RN - 0 (tetracalcium phosphate) RN - 10103-46-5 (calcium phosphate) SB - IM MH - Adjuvants, Immunologic/*pharmacology MH - Animals MH - Calcium Phosphates/*pharmacology MH - Female MH - Herpes Genitalis/immunology/mortality/*prevention & control MH - Herpesvirus 2, Human/*immunology MH - *Immunization MH - Mice MH - Mice, Inbred BALB C MH - Nasal Mucosa MH - Particle Size MH - Research Support, Non-U.S. Gov't MH - Vagina EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1021-4. PMID- 12204954 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20031114 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Serodiagnosis of mice minute virus and mouse parvovirus infections in mice by enzyme-linked immunosorbent assay with baculovirus-expressed recombinant VP2 proteins. PG - 1025-31 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. AD - Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211, USA. LivingstonR@missouri.edu FAU - Livingston, Robert S AU - Livingston RS FAU - Besselsen, David G AU - Besselsen DG FAU - Steffen, Earl K AU - Steffen EK FAU - Besch-Williford, Cynthia L AU - Besch-Williford CL FAU - Franklin, Craig L AU - Franklin CL FAU - Riley, Lela K AU - Riley LK LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Capsid Proteins) RN - 0 (Recombinant Proteins) SB - IM MH - Animals MH - Baculoviridae/genetics MH - Blotting, Western MH - Capsid Proteins/*diagnostic use/*genetics/immunology MH - Enzyme-Linked Immunosorbent Assay/*methods MH - Gene Expression Regulation, Viral MH - Male MH - Mice MH - *Mice Minute Virus MH - Mice, Inbred BALB C MH - Mice, Inbred C3H MH - Mice, Inbred C57BL MH - Mice, Inbred DBA MH - Parvoviridae Infections/*diagnosis/immunology MH - Prevalence MH - Recombinant Proteins/diagnostic use/genetics/immunology MH - Sensitivity and Specificity MH - Serologic Tests MH - Virion EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1025-31. PMID- 12204955 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Specificities and opsonophagocytic activities of antibodies to pneumococcal capsular polysaccharides in sera of unimmunized young children. PG - 1032-8 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. AD - Department of Vaccines, National Public Health Institute, Helsinki, Finland. anu.soininen@kyl.fi FAU - Soininen, Anu AU - Soininen A FAU - Karpala, Maijastiina AU - Karpala M FAU - Wahlman, Sirkka-Liisa AU - Wahlman SL FAU - Lehtonen, Hannele AU - Lehtonen H FAU - Kayhty, Helena AU - Kayhty H LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antibodies, Bacterial) RN - 0 (Bacterial Capsules) SB - IM MH - Absorption MH - Antibodies, Bacterial/blood/immunology MH - *Antibody Specificity MH - Bacterial Capsules/*immunology/metabolism MH - Child, Preschool MH - Cross Reactions MH - Humans MH - Immunoenzyme Techniques MH - Infant MH - Phagocytosis/*immunology MH - Pneumococcal Infections/*immunology MH - Prospective Studies MH - Research Support, Non-U.S. Gov't MH - Streptococcus pneumoniae/*immunology EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1032-8. PMID- 12204956 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - In vitro whole-blood analysis of cellular immunity in patients with active coccidioidomycosis by using the antigen preparation T27K. PG - 1039-43 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. AD - Medicine and Primary Care Service, Southern Arizona Veterans Affairs Health Care System, Tucson, Arizona 85723, USA. nampel@u.arizona.edu FAU - Ampel, Neil M AU - Ampel NM FAU - Kramer, Larissa A AU - Kramer LA FAU - Li, Lijin AU - Li L FAU - Carroll, Deborah S AU - Carroll DS FAU - Kerekes, Kathleen M AU - Kerekes KM FAU - Johnson, Suzanne M AU - Johnson SM FAU - Pappagianis, Demosthenes AU - Pappagianis D LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antigens, CD) RN - 0 (Antigens, Differentiation, T-Lymphocyte) RN - 0 (Antigens, Fungal) RN - 0 (CD69 antigen) RN - 0 (Cytokines) SB - IM MH - Adult MH - Aged MH - Aged, 80 and over MH - Antigens, CD/analysis MH - Antigens, Differentiation, T-Lymphocyte/analysis MH - Antigens, Fungal/diagnostic use MH - Coccidioidomycosis/*diagnosis/*immunology MH - Cytokines/biosynthesis MH - Female MH - Humans MH - *Immunity, Cellular MH - In Vitro MH - Male MH - Middle Aged MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - T-Lymphocytes/chemistry/*immunology/metabolism EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1039-43. PMID- 12204957 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Immunoglobulin G antibody against Helicobacter pylori: clinical implications of levels found in serum. PG - 1044-8 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. AD - Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital. National Yang-Ming University, Taipei, Taiwan, Republic of China. tschen@vghtpe.gov.tw FAU - Chen, Tseng-Shing AU - Chen TS FAU - Li, Fen-Yau AU - Li FY FAU - Chang, Full-Young AU - Chang FY FAU - Lee, Shou-Dong AU - Lee SD LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antibodies, Bacterial) RN - 0 (Immunoglobulin G) SB - IM MH - Adult MH - Antibodies, Bacterial/*blood MH - Atrophy MH - Chronic Disease MH - Enzyme-Linked Immunosorbent Assay MH - Female MH - Gastric Mucosa/microbiology/pathology MH - Gastritis/*diagnosis/microbiology/pathology MH - Helicobacter Infections/*diagnosis/immunology/pathology MH - Helicobacter pylori/*immunology MH - Humans MH - Immunoglobulin G/blood MH - Male MH - Middle Aged MH - Multivariate Analysis MH - Predictive Value of Tests MH - Research Support, Non-U.S. Gov't MH - Severity of Illness Index EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1044-8. PMID- 12204958 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Spontaneous cytokine production and its effect on induced production. PG - 1049-56 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. AD - Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia 30333, USA. ziq4@cdc.gov FAU - Walker, Derrick AU - Walker D FAU - Jason, Janine AU - Jason J FAU - Wallace, Kelly AU - Wallace K FAU - Slaughter, Justin AU - Slaughter J FAU - Whatley, Virginia AU - Whatley V FAU - Han, Alison AU - Han A FAU - Nwanyanwu, Okey C AU - Nwanyanwu OC FAU - Kazembe, Peter N AU - Kazembe PN FAU - Dobbie, Hamish AU - Dobbie H FAU - Archibald, Lennox AU - Archibald L FAU - Jarvis, William R AU - Jarvis WR LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Cytokines) RN - 0 (Interleukin-2) RN - 0 (Interleukin-6) RN - 0 (Interleukin-8) RN - 0 (Tumor Necrosis Factor-alpha) RN - 130068-27-8 (Interleukin-10) RN - 207137-56-2 (Interleukin-4) RN - 82115-62-6 (Interferon Type II) SB - IM MH - Acute Disease MH - Adolescent MH - Adult MH - Child MH - Cytokines/*biosynthesis/immunology MH - Flow Cytometry MH - Humans MH - Immunity, Cellular/*immunology MH - Interferon Type II/biosynthesis/immunology MH - Interleukin-10/biosynthesis/immunology MH - Interleukin-2/biosynthesis/immunology MH - Interleukin-4/biosynthesis/immunology MH - Interleukin-6/biosynthesis/immunology MH - Interleukin-8/biosynthesis/immunology MH - Lymphocytes/immunology/*metabolism MH - Monocytes/immunology/*metabolism MH - Tumor Necrosis Factor-alpha/biosynthesis/immunology EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1049-56. PMID- 12204959 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Bacillus species are present in chewing tobacco sold in the United States and evoke plasma exudation from the oral mucosa. PG - 1057-60 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 10(6) 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. AD - Department of Medicine, University of Illinois at Chicago,Chicago, Illinois 60612, USA. IRubinst@uic.edu FAU - Rubinstein, Israel AU - Rubinstein I FAU - Pedersen, Gerald W AU - Pedersen GW LA - eng GR - DE00386/DE/NIDCR GR - DE10347/DE/NIDCR PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 SB - IM MH - Bacillus megaterium/growth & development/*isolation & purification MH - Exudates and Transudates/metabolism/microbiology MH - Humans MH - Keratinocytes/metabolism/microbiology MH - Mouth Mucosa/cytology/metabolism/*microbiology MH - Plasma/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Spores, Bacterial MH - *Tobacco, Smokeless MH - United States EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1057-60. PMID- 12204960 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection. PG - 1061-6 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 (10(8) 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 60 degrees C 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. AD - Research Center for Gastroenterology, Dankook University College of Medicine, Cheonan, Korea. FAU - Shin, Ji-Hyun AU - Shin JH FAU - Yang, Mierha AU - Yang M FAU - Nam, Seung Woo AU - Nam SW FAU - Kim, Jung Taik AU - Kim JT FAU - Myung, Na Hye AU - Myung NH FAU - Bang, Won-Gi AU - Bang WG FAU - Roe, Im Hwan AU - Roe IH LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Anti-Bacterial Agents) RN - 0 (IgY) RN - 0 (Immunoglobulins) RN - EC 3.5.1.5 (Urease) SB - IM MH - Animals MH - Anti-Bacterial Agents MH - Bacterial Adhesion MH - Chickens MH - Disease Models, Animal MH - Egg Yolk/immunology MH - Gastritis/immunology/therapy MH - Gerbillinae MH - Helicobacter Infections/immunology/*therapy MH - Helicobacter pylori/enzymology/growth & development/*immunology MH - Humans MH - Immunization MH - Immunoglobulins/isolation & purification/*therapeutic use MH - *Immunotherapy MH - In Vitro MH - Research Support, Non-U.S. Gov't MH - Stomach Neoplasms MH - Tumor Cells, Cultured MH - Urease/metabolism EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1061-6. PMID- 12204961 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Immunoglobulin G antibody response to infection with coccoid forms of Helicobacter pylori. PG - 1067-71 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. AD - Laboratory of Microbiology, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile. gfiguero@uchile.cl FAU - Figueroa, G AU - Figueroa G FAU - Faundez, G AU - Faundez G FAU - Troncoso, M AU - Troncoso M FAU - Navarrete, P AU - Navarrete P FAU - Toledo, M S AU - Toledo MS LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antibodies, Bacterial) RN - 0 (Antigens, Bacterial) RN - 0 (Immunoglobulin G) SB - IM MH - Adult MH - Antibodies, Bacterial/*blood MH - Antigens, Bacterial/analysis MH - Blotting, Western MH - Electrophoresis, Gel, Two-Dimensional MH - Enzyme-Linked Immunosorbent Assay MH - Helicobacter Infections/*immunology/microbiology MH - Helicobacter pylori/*immunology/pathogenicity/ultrastructure MH - Humans MH - Immunoglobulin G/blood MH - Research Support, Non-U.S. Gov't MH - Virulence EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1067-71. PMID- 12204962 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Clinical and epidemiological relevance of quantitating hepatitis E virus-specific immunoglobulin M. PG - 1072-8 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. AD - Department of Virus Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, USA. FAU - Seriwatana, Jitvimol AU - Seriwatana J FAU - Shrestha, Mrigendra P AU - Shrestha MP FAU - Scott, Robert M AU - Scott RM FAU - Tsarev, Sergei A AU - Tsarev SA FAU - Vaughn, David W AU - Vaughn DW FAU - Myint, Khin Saw Aye AU - Myint KS FAU - Innis, Bruce L AU - Innis BL LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (DNA, Viral) RN - 0 (Hepatitis Antibodies) RN - 0 (Immunoglobulin G) RN - 0 (Immunoglobulin M) SB - IM MH - Comparative Study MH - DNA, Viral/analysis MH - Hepatitis Antibodies/blood/*diagnostic use MH - Hepatitis E/*diagnosis/*epidemiology/immunology MH - Hepatitis E virus/genetics/*isolation & purification MH - Humans MH - Immunoenzyme Techniques/methods/standards MH - Immunoglobulin G/blood/diagnostic use MH - Immunoglobulin M/blood/*diagnostic use MH - Kinetics MH - Recurrence MH - Reference Standards MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Sensitivity and Specificity MH - Seroepidemiologic Studies EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1072-8. PMID- 12204963 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Cytokine gene expression by peripheral blood leukocytes in horses experimentally infected with Anaplasma phagocytophila. PG - 1079-84 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. AD - Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210-1093, USA. FAU - Kim, Hyung-Yong AU - Kim HY FAU - Mott, Jason AU - Mott J FAU - Zhi, Ning AU - Zhi N FAU - Tajima, Tomoko AU - Tajima T FAU - Rikihisa, Yasuko AU - Rikihisa Y LA - eng GR - AI30010/AI/NIAID GR - AI40934/AI/NIAID PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Bacterial Outer Membrane Proteins) RN - 0 (Cytokines) RN - 0 (RNA, Messenger) RN - 0 (p44 protein, Anaplasma phagocytophila) SB - IM MH - Anaplasma phagocytophilum/*immunology MH - Animals MH - Bacterial Outer Membrane Proteins/genetics MH - Cytokines/*genetics MH - Disease Models, Animal MH - Ehrlichiosis/*immunology MH - Female MH - Fluorescent Antibody Technique, Indirect MH - Gene Expression/immunology MH - Horses MH - Leukocytes/*microbiology/*physiology MH - Male MH - RNA, Messenger/analysis MH - Research Support, U.S. Gov't, P.H.S. MH - Tick-Borne Diseases/immunology EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1079-84. PMID- 12204964 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Affordable CD4(+)-T-cell counting by flow cytometry: CD45 gating for volumetric analysis. PG - 1085-94 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/mm(3) with very little bias for CD4 counts (in favor of the Trio method: +8 CD4(+) lymphocytes/mm(3); 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/mm(3), again with a negligible bias (-10 CD4(+) lymphocytes/mm(3)). 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/mm(3); 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. AD - HIV Immunology, Department of Immunology and Molecular Pathology, Royal Free and University College Medical School, London, United Kingdom. janossy@rfhsm.u-net.com FAU - Janossy, George AU - Janossy G FAU - Jani, Ilesh V AU - Jani IV FAU - Bradley, Nicholas J AU - Bradley NJ FAU - Bikoue, Arsene AU - Bikoue A FAU - Pitfield, Tim AU - Pitfield T FAU - Glencross, Debbie K AU - Glencross DK LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antigens, CD4) RN - 0 (Antigens, CD45) RN - 0 (Antigens, CD8) SB - IM MH - Acquired Immunodeficiency Syndrome/*diagnosis/immunology MH - Adult MH - Antigens, CD4/analysis MH - Antigens, CD45/*analysis MH - Antigens, CD8/analysis MH - CD4-Positive T-Lymphocytes/*chemistry/*cytology MH - Cell Count/methods MH - Female MH - Flow Cytometry/*methods MH - Humans MH - Male MH - Middle Aged MH - Research Support, Non-U.S. Gov't EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1085-94. PMID- 12204965 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Gamma interferon inhibits production of Anti-OspA borreliacidal antibody in vitro. PG - 1095-101 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. AD - Wisconsin State Laboratory of Hygiene, University of Wisconsin, Madison, Wisconsin 53706, USA. FAU - Munson, Erik L AU - Munson EL FAU - Du Chateau, Brian K AU - Du Chateau BK FAU - Jensen, Jani R AU - Jensen JR FAU - Callister, Steven M AU - Callister SM FAU - DeCoster, David J AU - DeCoster DJ FAU - Schell, Ronald F AU - Schell RF LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antibodies, Bacterial) RN - 0 (Antigens, Surface) RN - 0 (Bacterial Outer Membrane Proteins) RN - 0 (Lipoproteins) RN - 0 (Lyme Disease Vaccines) RN - 0 (OspA protein) RN - 0 (Recombinant Proteins) RN - 82115-62-6 (Interferon Type II) SB - IM MH - Animals MH - Antibodies, Bacterial/*biosynthesis MH - Antigens, Surface/*immunology MH - Bacterial Outer Membrane Proteins/*immunology MH - Borrelia burgdorferi/*immunology MH - Cells, Cultured MH - Flow Cytometry MH - In Vitro MH - Interferon Type II/immunology/*pharmacology MH - *Lipoproteins MH - Lyme Disease/*immunology/prevention & control MH - Lyme Disease Vaccines MH - Lymph Nodes/cytology/metabolism MH - Macrophages/cytology MH - Mice MH - Mice, Inbred C3H MH - Recombinant Proteins/immunology/pharmacology EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1095-101. PMID- 12204966 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041217 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Suppression of growth by all-trans retinoic acid requires prolonged induction of interferon regulatory factor 1 in cervical squamous carcinoma (SiHa) cells. PG - 1102-6 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. AD - Department of Microbiology, The University of Texas Medical Branch, Galveston, Texas 77555-1070, USA. AranyIstvan@uams.edu FAU - Arany, Istvan AU - Arany I FAU - Whitehead, William E AU - Whitehead WE FAU - Grattendick, Kenneth J AU - Grattendick KJ FAU - Ember, Istvan A AU - Ember IA FAU - Tyring, Stephen K AU - Tyring SK LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antineoplastic Agents) RN - 0 (DNA-Binding Proteins) RN - 0 (NF-kappa B) RN - 0 (Oligonucleotides) RN - 0 (Phosphoproteins) RN - 0 (Stat1 protein) RN - 0 (Trans-Activators) RN - 0 (interferon regulatory factor-1) RN - 302-79-4 (Tretinoin) SB - IM MH - Antineoplastic Agents/*pharmacology MH - *Carcinoma, Squamous Cell MH - Cell Division/drug effects MH - *Cervix Neoplasms MH - DNA-Binding Proteins/*genetics/metabolism MH - Dose-Response Relationship, Drug MH - Female MH - Gene Expression Regulation, Neoplastic/drug effects MH - Humans MH - NF-kappa B/metabolism MH - Oligonucleotides/metabolism MH - Phosphoproteins/*genetics MH - Phosphorylation/drug effects MH - Promoter Regions (Genetics)/physiology MH - Trans-Activators/metabolism MH - Tretinoin/*pharmacology MH - Tumor Cells, Cultured EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1102-6. PMID- 12204967 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Double-blind study to evaluate flow cytometry analysis of anti-live trypomastigote antibodies for monitoring treatment efficacy in cases of human Chagas' disease. PG - 1107-13 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 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. AD - Laboratorio de Doenca de Chagas, Centro de Pesquisas Rene Rachou, FIOCRUZ, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. oamfilho@cpqrr.fiocruz.br FAU - Martins-Filho, Olindo Assis AU - Martins-Filho OA FAU - Eloi-Santos, Silvana Maria AU - Eloi-Santos SM FAU - Carvalho, Andrea Teixeira AU - Carvalho AT FAU - Oliveira, Rodrigo Correa AU - Oliveira RC FAU - Rassi, Anis AU - Rassi A FAU - Luquetti, Alejandro Ostemayer AU - Luquetti AO FAU - Rassi, Gustavo Gabriel AU - Rassi GG FAU - Brener, Zigman AU - Brener Z LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antibodies, Protozoan) RN - 0 (Cryoprotective Agents) RN - 56-81-5 (Glycerol) SB - IM MH - Adolescent MH - Adult MH - Aged MH - Animals MH - Antibodies, Protozoan/*analysis MH - Chagas Disease/*diagnosis/immunology/*therapy MH - Child MH - Child, Preschool MH - Cryoprotective Agents MH - Double-Blind Method MH - Evaluation Studies MH - Female MH - Flow Cytometry/*methods/standards MH - Glycerol MH - Humans MH - Infant MH - Male MH - Middle Aged MH - Research Support, Non-U.S. Gov't MH - Temperature MH - Trypanosoma cruzi/*immunology EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1107-13. PMID- 12204968 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep 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. PG - 1114-8 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/ micro l; >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. AD - Cattedra di Immunologia, Universita di Milano, DISP, LITA Vialba, Milan, Italy. mago@mailserver.unimi.it FAU - Trabattoni, Daria AU - Trabattoni D FAU - Lo Caputo, Sergio AU - Lo Caputo S FAU - Biasin, Mara AU - Biasin M FAU - Seminari, Elena AU - Seminari E FAU - Di Pietro, Massimo AU - Di Pietro M FAU - Ravasi, Giovanni AU - Ravasi G FAU - Mazzotta, Francesco AU - Mazzotta F FAU - Maserati, Renato AU - Maserati R FAU - Clerici, Mario AU - Clerici M LA - eng PT - Clinical Trial PT - Journal Article PT - Randomized Controlled Trial PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Anti-HIV Agents) RN - 0 (HIV Protease Inhibitors) RN - 0 (Interleukin-2) RN - 0 (Oxazines) RN - 0 (RNA, Messenger) RN - 130068-27-8 (Interleukin-10) RN - 154635-17-3 (efavirenz) RN - 159989-64-7 (Nelfinavir) RN - 3056-17-5 (Stavudine) RN - 82115-62-6 (Interferon Type II) SB - IM MH - Adult MH - Anti-HIV Agents/*pharmacology MH - CD4-Positive T-Lymphocytes/cytology/immunology MH - Cohort Studies MH - Drug Therapy, Combination MH - Female MH - HIV/*immunology MH - HIV Infections/*drug therapy/*immunology MH - HIV Protease Inhibitors/pharmacology MH - Humans MH - Interferon Type II/genetics/metabolism MH - Interleukin-10/genetics/metabolism MH - Interleukin-2/genetics/metabolism MH - Male MH - Nelfinavir/pharmacology MH - Oxazines/*pharmacology MH - RNA, Messenger/analysis MH - Random Allocation MH - Research Support, Non-U.S. Gov't MH - Stavudine/pharmacology MH - Treatment Failure EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1114-8. PMID- 12204969 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Immunoblot analysis of the humoral immune response to Leishmania donovani polypeptides in cases of human visceral leishmaniasis: its usefulness in prognosis. PG - 1119-23 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. AD - Department of Biochemistry, Faculty of Science, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221 005, India. FAU - Kumar, Promod AU - Kumar P FAU - Pai, Kalpana AU - Pai K FAU - Tripathi, Kiran AU - Tripathi K FAU - Pandey, H P AU - Pandey HP FAU - Sundar, Shyam AU - Sundar S LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antibodies, Protozoan) RN - 0 (Antigens, Protozoan) RN - 0 (Antimony Sodium Gluconate) RN - 0 (Antiprotozoal Agents) RN - 0 (Peptides) RN - 107-73-3 (Phosphorylcholine) RN - 58066-85-6 (miltefosine) SB - IM MH - Adult MH - Animals MH - Antibodies, Protozoan/analysis MH - Antibody Formation MH - Antigens, Protozoan/diagnostic use/immunology MH - Antimony Sodium Gluconate/administration & dosage MH - Antiprotozoal Agents/administration & dosage MH - Humans MH - *Immunoblotting MH - Leishmania donovani/*immunology MH - Leishmaniasis, Visceral/*diagnosis/drug therapy/*immunology MH - Peptides/immunology MH - Phosphorylcholine/administration & dosage/*analogs & derivatives MH - Predictive Value of Tests MH - Prognosis MH - Research Support, Non-U.S. Gov't EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1119-23. PMID- 12204970 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep 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. PG - 1124-5 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. AD - Department of Pediatrics, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA. leachc@uthscsa.edu FAU - Leach, Charles T AU - Leach CT FAU - Ashley, Rhoda L AU - Ashley RL FAU - Baillargeon, Jacques AU - Baillargeon J FAU - Jenson, Hal B AU - Jenson HB LA - eng GR - P01 AI 30731/AI/NIAID PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Reagent Kits, Diagnostic) RN - 0 (Viral Envelope Proteins) RN - 0 (glycoprotein G, herpes simplex virus type 2) SB - IM MH - Adolescent MH - Child MH - Child, Preschool MH - Herpes Simplex/*diagnosis/immunology MH - *Herpesvirus 1, Human MH - *Herpesvirus 2, Human MH - Humans MH - *Immunoenzyme Techniques MH - Infant MH - Reagent Kits, Diagnostic MH - Research Support, U.S. Gov't, P.H.S. MH - Sensitivity and Specificity MH - Viral Envelope Proteins/*analysis/immunology EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1124-5. PMID- 12204971 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Helicobacter pylori-specific immune responses of children: implications for future vaccination strategy. PG - 1126-8 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). AD - Department of Epidemiology, University of Ulm, Germany. FAU - Bode, Gunter AU - Bode G FAU - Piechotowski, Isolde AU - Piechotowski I FAU - Rothenbacher, Dietrich AU - Rothenbacher D FAU - Brenner, Hermann AU - Brenner H LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antibodies, Bacterial) RN - 0 (Bacterial Vaccines) RN - 0 (Immunoglobulin G) SB - IM MH - Adolescent MH - Antibodies, Bacterial/blood MH - *Bacterial Vaccines MH - Child MH - Female MH - Helicobacter Infections/epidemiology/*immunology/*prevention & control MH - Helicobacter pylori/*immunology MH - Humans MH - Immunoglobulin G/blood MH - Male MH - Seroepidemiologic Studies EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1126-8. PMID- 12204972 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - CD4(+) CD25(+) T-cell production in healthy humans and in patients with thymic hypoplasia. PG - 1129-31 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. AD - Division of Immunologic and Infectious Diseases, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA. sullivak@mail.med.upenn.edu FAU - Sullivan, Kathleen E AU - Sullivan KE FAU - McDonald-McGinn, Donna AU - McDonald-McGinn D FAU - Zackai, Elaine H AU - Zackai EH LA - eng GR - M01-RR00240/RR/NCRR PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Antigens, CD4) RN - 0 (Receptors, Interleukin-2) SB - IM MH - Antigens, CD4/analysis MH - Autoimmune Diseases/immunology/*pathology MH - CD4 Lymphocyte Count MH - CD4-Positive T-Lymphocytes/*chemistry/*cytology MH - Humans MH - Receptors, Interleukin-2/*analysis MH - Research Support, U.S. Gov't, P.H.S. MH - Thymus Gland/immunology/*pathology EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1129-31. PMID- 12204973 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep 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. PG - 1132-6 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. AD - Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Yamaguchi University, 753-8515 Yamaguchi, Japan. InokumaHisashi@aol.com FAU - Inokuma, Hisashi AU - Inokuma H FAU - Fujii, Kaori AU - Fujii K FAU - Okuda, Masaru AU - Okuda M FAU - Onishi, Takafumi AU - Onishi T FAU - Beaufils, Jean-Pierre AU - Beaufils JP FAU - Raoult, Didier AU - Raoult D FAU - Brouqui, Philippe AU - Brouqui P LA - eng SI - GENBANK/AB058782 SI - GENBANK/AY044161 PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Bacterial Proteins) RN - 0 (Chaperonins) RN - 0 (GroESL protein, Bacteria) RN - 0 (RNA, Ribosomal, 16S) RN - EC 4.1.3.7 (Citrate (si)-Synthase) SB - IM MH - Anaplasma/*genetics MH - Animals MH - Bacterial Proteins/*genetics MH - Chaperonins/*genetics MH - Citrate (si)-Synthase/*genetics MH - Dogs MH - Ehrlichiosis/*diagnosis MH - Molecular Sequence Data MH - *Phylogeny MH - Polymerase Chain Reaction MH - RNA, Ribosomal, 16S/genetics MH - Research Support, Non-U.S. Gov't EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1132-6. PMID- 12204974 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Diagnostic techniques to detect cryptic leishmaniasis in dogs. PG - 1137-41 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. AD - Laboratori de Parasitologia, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Spain. FAU - Iniesta, Laura AU - Iniesta L FAU - Fernandez-Barredo, Salceda AU - Fernandez-Barredo S FAU - Bulle, Beatrice AU - Bulle B FAU - Gomez, M Teresa AU - Gomez MT FAU - Piarroux, Renaud AU - Piarroux R FAU - Gallego, Montserrat AU - Gallego M FAU - Alunda, Jose M AU - Alunda JM FAU - Portus, Montserrat AU - Portus M LA - eng PT - Journal Article PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 SB - IM MH - Animals MH - Blotting, Western MH - Carrier State MH - Dog Diseases/*diagnosis/immunology/parasitology MH - Dogs MH - *Enzyme-Linked Immunosorbent Assay MH - Fluorescent Antibody Technique MH - Hypersensitivity, Delayed/diagnosis/parasitology MH - Leishmania infantum/*isolation & purification MH - Leishmaniasis/*diagnosis/immunology MH - Research Support, Non-U.S. Gov't EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1137-41. PMID- 12204975 OWN - NLM STAT- MEDLINE DA - 20020902 DCOM- 20030211 LR - 20041117 PUBM- Print IS - 1071-412X VI - 9 IP - 5 DP - 2002 Sep TI - Centrifugation of human lung epithelial carcinoma a549 cells up-regulates interleukin-1beta gene expression. PG - 1142-3 FAU - Yang, Jun AU - Yang J FAU - Hooper, W Craig AU - Hooper WC FAU - Phillips, Donald J AU - Phillips DJ FAU - Tondella, M Lucia AU - Tondella ML FAU - Talkington, Deborah F AU - Talkington DF LA - eng PT - Letter PL - United States TA - Clin Diagn Lab Immunol JID - 9421292 RN - 0 (Interleukin-1) SB - IM MH - *Centrifugation MH - Epithelial Cells MH - Gene Expression Regulation, Neoplastic/*immunology MH - Humans MH - Interleukin-1/*genetics MH - *Lung Neoplasms MH - Tumor Cells, Cultured MH - Up-Regulation EDAT- 2002/09/03 10:00 MHDA- 2003/02/13 04:00 PST - ppublish SO - Clin Diagn Lab Immunol 2002 Sep;9(5):1142-3. PMID- 12207826 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030423 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Sep 3 TI - Optimizing antibiotics in residents of nursing homes: protocol of a randomized trial. PG - 17 AB - BACKGROUND: Antibiotics are frequently prescribed for older adults who reside in long-term care facilities. A substantial proportion of antibiotic use in this setting is inappropriate. Antibiotics are often prescribed for asymptomatic bacteriuria, a condition for which randomized trials of antibiotic therapy indicate no benefit and in fact harm. This proposal describes a randomized trial of diagnostic and therapeutic algorithms to reduce the use of antibiotics in residents of long-term care facilities. METHODS: In this on-going study, 22 nursing homes have been randomized to either use of algorithms (11 nursing homes) or to usual practise (11 nursing homes). The algorithms describe signs and symptoms for which it would be appropriate to send urine cultures or to prescribe antibiotics. The algorithms are introduced by inservicing nursing staff and by conducting one-on-one sessions for physicians using case-scenarios. The primary outcome of the study is courses of antibiotics per 1000 resident days. Secondary outcomes include urine cultures sent and antibiotic courses for urinary indications. Focus groups and semi-structured interviews with key informants will be used to assess the process of implementation and to identify key factors for sustainability. AD - Department of Pathology and Molecular Medicine, McMaster University Hamilton, ON, Canada. loebm@mcmaster.ca FAU - Loeb, Mark AU - Loeb M FAU - Brazil, Kevin AU - Brazil K FAU - Lohfeld, Lynne AU - Lohfeld L FAU - McGeer, Allison AU - McGeer A FAU - Simor, Andrew AU - Simor A FAU - Stevenson, Kurt AU - Stevenson K FAU - Walter, Stephen AU - Walter S FAU - Zoutman, Dick AU - Zoutman D LA - eng PT - Clinical Trial PT - Journal Article PT - Randomized Controlled Trial DEP - 20020903 PL - England TA - BMC Health Serv Res JID - 101088677 RN - 0 (Anti-Bacterial Agents) SB - IM MH - Aged MH - *Algorithms MH - Anti-Bacterial Agents/adverse effects/*therapeutic use MH - Bacteriuria/*drug therapy/*urine MH - *Clinical Protocols MH - Drug Resistance, Microbial MH - Drug Utilization/*standards/statistics & numerical data MH - Female MH - Humans MH - Male MH - Medical Staff/education/standards MH - Middle Aged MH - Nursing Homes/*standards MH - Nursing Staff/education/standards MH - Ontario MH - Randomized Controlled Trials/*methods MH - Research Support, U.S. Gov't, P.H.S. MH - Treatment Outcome EDAT- 2002/09/05 10:00 MHDA- 2003/04/24 05:00 PHST- 2002/07/16 [received] PHST- 2002/09/03 [accepted] PHST- 2002/09/03 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Sep 3;2(1):17. PMID- 12213183 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030423 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Sep 4 TI - Funding source, trial outcome and reporting quality: are they related? Results of a pilot study. PG - 18 AB - BACKGROUND: There has been increasing concern regarding the potential effects of the commercialization of research. METHODS: In order to examine the relationships between funding source, trial outcome and reporting quality, recent issues of five peer-reviewed, high impact factor, general medical journals were hand-searched to identify a sample of 100 randomized controlled trials (20 trials/journal). Relevant data, including funding source (industry/not-for-profit/mixed/not reported) and statistical significance of primary outcome (favouring new treatment/favouring conventional treatment/neutral/unclear), were abstracted. Quality scores were assigned using the Jadad scale and the adequacy of allocation concealment. RESULTS: Sixty-six percent of trials received some industry funding. Trial outcome was not associated with funding source (p=.461). There was a preponderance of favourable statistical conclusions among published trials with 67% reporting results that favored a new treatment whereas 6% favoured the conventional treatment. Quality scores were not associated with funding source or trial outcome. CONCLUSIONS: It is not known whether the absence of significant associations between funding source, trial outcome and reporting quality reflects a true absence of an association or is an artefact of inadequate statistical power, reliance on voluntary disclosure of funding information, a focus on trials recently published in the top medical journals, or some combination thereof. Continued and expanded monitoring of potential conflicts is recommended, particularly in light of new guidelines for disclosure that have been endorsed by the ICMJE. AD - Chalmers Research Group, Ottawa, Canada. tclifford@cheo.on.ca FAU - Clifford, Tammy J AU - Clifford TJ FAU - Barrowman, Nicholas J AU - Barrowman NJ FAU - Moher, David AU - Moher D LA - eng PT - Journal Article DEP - 20020904 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Bibliometrics MH - Conflict of Interest MH - Disclosure MH - Drug Evaluation/*economics/standards MH - Drug Industry MH - Humans MH - Organizations, Nonprofit MH - Periodicals/*standards/statistics & numerical data MH - Pilot Projects MH - Publication Bias MH - Quality Control MH - Randomized Controlled Trials/*economics/standards MH - Research Design/*standards MH - Research Support/classification/*statistics & numerical data MH - *Treatment Outcome EDAT- 2002/09/06 10:00 MHDA- 2003/04/24 05:00 PHST- 2002/04/23 [received] PHST- 2002/09/04 [accepted] PHST- 2002/09/04 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Sep 4;2(1):18. PMID- 12221110 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Role of fission yeast Tup1-like repressors and Prr1 transcription factor in response to salt stress. PG - 2977-89 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 Ca(2+)) 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. AD - School of Biochemistry and Genetics, University of Newcastle upon Tyne, United Kingdom. FAU - Greenall, Amanda AU - Greenall A FAU - Hadcroft, Andrew P AU - Hadcroft AP FAU - Malakasi, Panagiota AU - Malakasi P FAU - Jones, Nic AU - Jones N FAU - Morgan, Brian A AU - Morgan BA FAU - Hoffman, Charles S AU - Hoffman CS FAU - Whitehall, Simon K AU - Whitehall SK LA - eng GR - GM-46226/GM/NIGMS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Cations) RN - 0 (Ions) RN - 0 (Nuclear Proteins) RN - 0 (Plasmids) RN - 0 (Repressor Proteins) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 0 (Salts) RN - 0 (Schizosaccharomyces pombe Proteins) RN - 0 (TUP1 protein, S pombe) RN - 0 (Transcription Factors) RN - 0 (Tup11 protein, S pombe) RN - 0 (Tup12 protein, S pombe) RN - 133135-32-7 (TUP1 protein, S cerevisiae) RN - 63231-63-0 (RNA) RN - 7440-09-7 (Potassium) RN - EC 3.2.1.23 (beta-Galactosidase) SB - IM MH - Biological Transport MH - Cations MH - Dose-Response Relationship, Drug MH - Gene Expression Regulation, Fungal MH - Ions MH - Models, Biological MH - Nuclear Proteins/*metabolism MH - Phenotype MH - Plasmids/metabolism MH - Potassium/metabolism MH - Precipitin Tests MH - Promoter Regions (Genetics) MH - Protein Binding MH - RNA/metabolism MH - Repressor Proteins/*metabolism/*physiology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Saccharomyces cerevisiae Proteins/*metabolism MH - Salts/*pharmacology MH - Schizosaccharomyces/*metabolism MH - Schizosaccharomyces pombe Proteins/metabolism/*physiology MH - Time Factors MH - Transcription Factors/metabolism MH - beta-Galactosidase/metabolism EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.01-12-0568 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):2977-89. PMID- 12221111 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041215 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - The roles of bud-site-selection proteins during haploid invasive growth in yeast. PG - 2990-3004 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. AD - Institute of Molecular Biology, University of Oregon, Eugene 97403-1229, USA. FAU - Cullen, Paul J AU - Cullen PJ FAU - Sprague, George F Jr AU - Sprague GF Jr LA - eng GR - GM-19188/GM/NIGMS GR - GM-30027/GM/NIGMS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (AXL1 protein, S cerevisiae) RN - 0 (Actins) RN - 0 (Axl2 protein, S cerevisiae) RN - 0 (BUD2 protein, S cerevisiae) RN - 0 (BUD3 protein, S cerevisiae) RN - 0 (BUD4 protein, S cerevisiae) RN - 0 (Cell Cycle Proteins) RN - 0 (GTP Phosphohydrolase Activators) RN - 0 (Luminescent Proteins) RN - 0 (Membrane Glycoproteins) RN - 0 (Plasmids) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 147336-22-9 (Green Fluorescent Proteins) RN - 50-99-7 (Glucose) RN - 9002-18-0 (Agar) RN - EC 2.7.1.- (GLC2 protein) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 3.6.1.- (GTP-Binding Proteins) SB - IM MH - Actins/metabolism MH - Agar/metabolism MH - Blotting, Western MH - Cell Cycle Proteins/metabolism MH - Cell Division MH - GTP Phosphohydrolase Activators/metabolism MH - GTP-Binding Proteins/metabolism MH - Glucose/pharmacology MH - Green Fluorescent Proteins MH - Luminescent Proteins/metabolism MH - Membrane Glycoproteins/metabolism MH - Microscopy, Fluorescence MH - Plasmids/metabolism MH - Ploidies MH - Protein-Serine-Threonine Kinases/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Saccharomyces cerevisiae/metabolism/*physiology MH - Saccharomyces cerevisiae Proteins/metabolism MH - Temperature MH - Time Factors EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-03-0151 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):2990-3004. PMID- 12221112 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041215 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Pkh1 and pkh2 differentially phosphorylate and activate ypk1 and ykr2 and define protein kinase modules required for maintenance of cell wall integrity. PG - 3005-28 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, ypk1-1(ts) ykr2Delta 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 ypk1-1(ts) ykr2Delta 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. AD - Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720-3202, USA. FAU - Roelants, Francoise M AU - Roelants FM FAU - Torrance, Pamela D AU - Torrance PD FAU - Bezman, Natalie AU - Bezman N FAU - Thorner, Jeremy AU - Thorner J LA - eng GR - CA-09041/CA/NCI GR - GM-07232/GM/NIGMS GR - GM-21841/GM/NIGMS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (DNA Transposable Elements) RN - 0 (Luminescent Proteins) RN - 0 (Plasmids) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 147336-22-9 (Green Fluorescent Proteins) RN - EC 2.7.1.- (MCK1 protein, S cerevisiae) RN - EC 2.7.1.- (PKH1 protein, S cerevisiae) RN - EC 2.7.1.- (PKH2 protein, S cerevisiae) RN - EC 2.7.1.- (YKR2 protein kinase) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.37 (Protein Kinases) SB - IM MH - Catalysis MH - Cell Wall/*metabolism MH - DNA Transposable Elements MH - Escherichia coli/metabolism MH - Green Fluorescent Proteins MH - Immunoblotting MH - Luminescent Proteins/metabolism MH - Models, Biological MH - Mutation MH - Phenotype MH - Phosphorylation MH - Plasmids/metabolism MH - Precipitin Tests MH - Protein Kinases/genetics/*physiology MH - Protein-Tyrosine Kinase/genetics/*physiology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Saccharomyces cerevisiae/*metabolism MH - *Saccharomyces cerevisiae Proteins MH - Suppression, Genetic MH - Temperature EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-04-0201 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3005-28. PMID- 12221113 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Distinct chromosome segregation roles for spindle checkpoint proteins. PG - 3029-41 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. AD - McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. FAU - Warren, Cheryl D AU - Warren CD FAU - Brady, D Michelle AU - Brady DM FAU - Johnston, Raymond C AU - Johnston RC FAU - Hanna, Joseph S AU - Hanna JS FAU - Hardwick, Kevin G AU - Hardwick KG FAU - Spencer, Forrest A AU - Spencer FA LA - eng GR - GM50842/GM/NIGMS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Cell Cycle Proteins) RN - 0 (Plasmids) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 0 (mitotic arrest defective protein 3) RN - 142008-15-9 (BUB3 protein, S cerevisiae) RN - EC 2.7.1.- (Bub1 protein) RN - EC 2.7.1.37 (Protein Kinases) SB - IM MH - Alleles MH - Cell Cycle Proteins/metabolism/physiology MH - Chromosome Segregation MH - Chromosomes/*physiology/ultrastructure MH - Immunoblotting MH - Mitotic Spindle Apparatus/*physiology/ultrastructure MH - Mutation MH - Phenotype MH - Plasmids/metabolism MH - Precipitin Tests MH - Protein Binding MH - Protein Kinases/physiology MH - Protein Structure, Tertiary MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Saccharomyces cerevisiae/*genetics/*physiology MH - *Saccharomyces cerevisiae Proteins MH - Two-Hybrid System Techniques EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-04-0203 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3029-41. PMID- 12221114 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Activation of mitogen-activated protein kinase (mitogen-activated protein kinase/extracellular signal-regulated kinase) cascade by aldosterone. PG - 3042-54 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. AD - Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio 78229-3900, USA. FAU - Hendron, Eunan AU - Hendron E FAU - Stockand, James D AU - Stockand JD LA - eng GR - R01-DK59594/DK/NIDDK PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (MAP Kinase Signaling System) RN - 0 (Plasmids) RN - 0 (RNA, Messenger) RN - 52-39-1 (Aldosterone) RN - 86-01-1 (Guanosine Triphosphate) RN - EC 1.13.12.- (Luciferases) RN - EC 3.6.1.- (Proto-Oncogene Protein p21(ras)) SB - IM MH - Aldosterone/metabolism/*pharmacology MH - Animals MH - Blotting, Western MH - Cell Line MH - Cell Nucleus/metabolism MH - Electrophysiology MH - Enzyme Activation MH - Exons MH - Genes, Reporter MH - Guanosine Triphosphate/metabolism MH - Introns MH - Kidney/enzymology/*metabolism MH - Luciferases/metabolism MH - *MAP Kinase Signaling System MH - Mice MH - Models, Biological MH - Plasmids/metabolism MH - Promoter Regions (Genetics) MH - Proto-Oncogene Protein p21(ras)/metabolism MH - RNA, Messenger/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction MH - Time Factors EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-05-0260 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3042-54. PMID- 12221115 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Functional heterogeneity of bone morphogenetic protein receptor-II mutants found in patients with primary pulmonary hypertension. PG - 3055-63 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. AD - Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Japan. FAU - Nishihara, Ayako AU - Nishihara A FAU - Watabe, Tetsuro AU - Watabe T FAU - Imamura, Takeshi AU - Imamura T FAU - Miyazono, Kohei AU - Miyazono K LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (DNA, Complementary) RN - 0 (DNA-Binding Proteins) RN - 0 (Ligands) RN - 0 (Phosphoproteins) RN - 0 (Plasmids) RN - 0 (Smad5 protein) RN - 0 (Trans-Activators) RN - 0 (Transcription Factors) RN - 0 (Transforming Growth Factor beta) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 2.7.1.37 (bone morphogenetic protein receptor type II) SB - IM MH - Animals MH - COS Cells MH - Cytoplasm/metabolism MH - DNA, Complementary/metabolism MH - DNA-Binding Proteins/metabolism MH - Germ-Line Mutation MH - Humans MH - Hypertension, Pulmonary/*genetics/metabolism MH - Ligands MH - Lung/cytology MH - Microscopy, Fluorescence MH - *Mutation MH - Mutation, Missense MH - Phosphoproteins/metabolism MH - Phosphorylation MH - Plasmids/metabolism MH - Precipitin Tests MH - Protein Binding MH - Protein Structure, Tertiary MH - Protein-Serine-Threonine Kinases/*genetics MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Signal Transduction MH - Trans-Activators/metabolism MH - Transcription Factors/metabolism MH - Transcription, Genetic MH - Transforming Growth Factor beta/metabolism EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-02-0063 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3055-63. PMID- 12221116 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20050131 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin binding and phosphorylation. PG - 3064-77 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. AD - Department of Biochemistry and Molecular Genetics, University of Virginia Medical Center, Charlottesville 22908, USA. FAU - Bolton, Margaret A AU - Bolton MA FAU - Lan, Weijie AU - Lan W FAU - Powers, Shannon E AU - Powers SE FAU - McCleland, Mark L AU - McCleland ML FAU - Kuang, Jian AU - Kuang J FAU - Stukenberg, P Todd AU - Stukenberg PT LA - eng GR - GM63045-01/GM/NIGMS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Antibodies) RN - 0 (Chromosomal Proteins, Non-Histone) RN - 0 (Microtubule-Associated Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (inner centromere protein) RN - 0 (survivin protein, human) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.- (aurora B kinase) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Amino Acid Sequence MH - Animals MH - Antibodies/metabolism MH - Cell Cycle MH - Cells, Cultured MH - Chromatography, Gel MH - Chromosomal Proteins, Non-Histone/*metabolism MH - Cloning, Molecular MH - Dose-Response Relationship, Drug MH - Glutathione Transferase/metabolism MH - Humans MH - Immunoblotting MH - Microscopy, Fluorescence MH - Microtubule-Associated Proteins/chemistry/genetics/*metabolism MH - Mitosis MH - Models, Molecular MH - Molecular Sequence Data MH - Phosphorylation MH - Protein Binding MH - Protein Structure, Tertiary MH - Protein-Serine-Threonine Kinases/chemistry/*metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Amino Acid MH - Xenopus MH - Xenopus laevis EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-02-0092 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3064-77. PMID- 12221117 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041215 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization. PG - 3078-95 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 ~25% 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. AD - Department of Biochemistry and Molecular Biology, University of Minnesota Duluth School of Medicine, Duluth 55812, USA. aboman@d.umn.edu FAU - Boman, Annette L AU - Boman AL FAU - Salo, Paul D AU - Salo PD FAU - Hauglund, Melissa J AU - Hauglund MJ FAU - Strand, Nicole L AU - Strand NL FAU - Rensink, Shelly J AU - Rensink SJ FAU - Zhdankina, Olga AU - Zhdankina O LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Adaptor Proteins, Vesicular Transport) RN - 0 (Carrier Proteins) RN - 0 (Luminescent Proteins) RN - 0 (Plasmids) RN - 0 (Recombinant Fusion Proteins) RN - 0 (golgi associated, gamma adaptin homologous, ADP-ribosylation factor interacting protein) RN - 147336-22-9 (Green Fluorescent Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.6.1.47 (ADP-Ribosylation Factors) SB - IM MH - ADP-Ribosylation Factors/genetics/*metabolism MH - *Adaptor Proteins, Vesicular Transport MH - Amino Acid Sequence MH - Animals MH - Carrier Proteins/genetics/*metabolism MH - Chromatography, Affinity MH - Genotype MH - Glutathione Transferase/metabolism MH - Green Fluorescent Proteins MH - Humans MH - Immunoblotting MH - Luminescent Proteins/metabolism MH - Microscopy, Fluorescence MH - Molecular Sequence Data MH - Mutation MH - Phenotype MH - Plasmids/metabolism MH - Protein Binding MH - Protein Structure, Tertiary MH - Rats MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid MH - Subcellular Fractions MH - Temperature MH - Transfection MH - Two-Hybrid System Techniques EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-02-0078 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3078-95. PMID- 12221118 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041118 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Inversin forms a complex with catenins and N-cadherin in polarized epithelial cells. PG - 3096-106 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. AD - Indiana University School of Medicine Department of Medicine, Division of Nephrology, Indianapolis 46202-5116, USA. FAU - Nurnberger, Jens AU - Nurnberger J FAU - Bacallao, Robert L AU - Bacallao RL FAU - Phillips, Carrie L AU - Phillips CL LA - eng GR - R01 DK50141/DK/NIDDK PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Cadherins) RN - 0 (Cytoskeletal Proteins) RN - 0 (INVS protein, human) RN - 0 (Invs protein, mouse) RN - 0 (Protein Isoforms) RN - 0 (Proteins) RN - 0 (Trans-Activators) RN - 0 (Transcription Factors) RN - 0 (Triiodobenzoic Acids) RN - 146409-33-8 (beta catenin) RN - 7440-70-2 (Calcium) RN - 92339-11-2 (iodixanol) SB - IM MH - Animals MH - Body Patterning MH - Cadherins/*metabolism MH - Calcium/metabolism/pharmacology MH - Cell Adhesion MH - Cell Membrane/metabolism MH - Cell Nucleus/metabolism MH - Cells, Cultured MH - Cytoskeletal Proteins/*metabolism MH - Electrophoresis, Polyacrylamide Gel MH - Epithelial Cells/*metabolism MH - Homozygote MH - Immunoblotting MH - Immunohistochemistry MH - Mice MH - Microscopy, Confocal MH - Mutation MH - Phenotype MH - Precipitin Tests MH - Protein Binding MH - Protein Isoforms MH - Protein Structure, Tertiary MH - Proteins/*metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Spectrum Analysis, Mass MH - Trans-Activators/*metabolism MH - *Transcription Factors MH - Transcription, Genetic MH - Triiodobenzoic Acids/pharmacology EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-04-0195 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3096-106. PMID- 12221119 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041215 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Modulation of cellular cholesterol transport and homeostasis by Rab11. PG - 3107-22 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-labele d 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. AD - Department of Molecular Medicine, National Public Health Institute, Biomedicum Helsinki, Finland. FAU - Holtta-Vuori, Maarit AU - Holtta-Vuori M FAU - Tanhuanpaa, Kimmo AU - Tanhuanpaa K FAU - Mobius, Wiebke AU - Mobius W FAU - Somerharju, Pentti AU - Somerharju P FAU - Ikonen, Elina AU - Ikonen E LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Carrier Proteins) RN - 0 (Cyclodextrins) RN - 0 (Lipoproteins, LDL) RN - 0 (Luminescent Proteins) RN - 0 (Membrane Glycoproteins) RN - 0 (NPC1 protein, human) RN - 0 (Receptors, Transferrin) RN - 0 (Xanthenes) RN - 0 (rab11 protein) RN - 11096-37-0 (Transferrin) RN - 147336-22-9 (Green Fluorescent Proteins) RN - 56-65-5 (Adenosine Triphosphate) RN - 57-88-5 (Cholesterol) RN - 58-85-5 (Biotin) RN - 82354-19-6 (Texas red) RN - EC 3.6.1.- (GTP Phosphohydrolases) RN - EC 3.6.1.- (rab GTP-Binding Proteins) SB - IM MH - Adenosine Triphosphate/metabolism MH - Animals MH - Biological Transport MH - Biotin/metabolism MH - Blotting, Western MH - COS Cells MH - Carrier Proteins/metabolism MH - Cholesterol/*metabolism MH - Cyclodextrins/metabolism MH - Endocytosis MH - Endosomes/metabolism MH - GTP Phosphohydrolases/metabolism MH - Genes, Dominant MH - Green Fluorescent Proteins MH - Immunohistochemistry MH - Lipoproteins, LDL/metabolism MH - Luminescent Proteins/metabolism MH - Membrane Glycoproteins/metabolism MH - Microscopy, Fluorescence MH - Mutation MH - Phenotype MH - Receptors, Transferrin/metabolism MH - Research Support, Non-U.S. Gov't MH - Time Factors MH - Transfection MH - Transferrin/metabolism MH - Xanthenes/pharmacology MH - rab GTP-Binding Proteins/*metabolism EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-01-0025 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3107-22. PMID- 12221120 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - All small nuclear RNAs (snRNAs) of the [U4/U6.U5] Tri-snRNP localize to nucleoli; Identification of the nucleolar localization element of U6 snRNA. PG - 3123-37 AB - Previously, we showed that spliceosomal U6 small nuclear RNA (snRNA) transiently passes through the nucleolus. Herein, we report that all individual snRNAs of the [U4/U6.U5] tri-snRNP localize to nucleoli, demonstrated by fluorescence microscopy of nucleolar preparations after injection of fluorescein-labeled snRNA into Xenopus oocyte nuclei. Nucleolar localization of U6 is independent from [U4/U6] snRNP formation since sites of direct interaction of U6 snRNA with U4 snRNA are not nucleolar localization elements. Among all regions in U6, the only one required for nucleolar localization is its 3' end, which associates with the La protein and subsequently during maturation of U6 is bound by Lsm proteins. This 3'-nucleolar localization element of U6 is both essential and sufficient for nucleolar localization and also required for localization to Cajal bodies. Conversion of the 3' hydroxyl of U6 snRNA to a 3' phosphate prevents association with the La protein but does not affect U6 localization to nucleoli or Cajal bodies. AD - Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA. FAU - Gerbi, Susan A AU - Gerbi SA FAU - Lange, Thilo Sascha AU - Lange TS LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (RNA, Small Nuclear) RN - 0 (Ribonucleoproteins, Small Nuclear) RN - 0 (U6 small nuclear RNA) RN - 9007-49-2 (DNA) SB - IM MH - Animals MH - Base Sequence MH - Cell Nucleolus/*metabolism MH - Cell Nucleus/metabolism MH - Coiled Bodies/metabolism MH - DNA/metabolism MH - Microscopy, Fluorescence MH - Molecular Sequence Data MH - Mutation MH - Nucleic Acid Conformation MH - Oocytes/metabolism MH - Precipitin Tests MH - RNA, Small Nuclear/*metabolism MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Ribonucleoproteins, Small Nuclear/*metabolism MH - Sequence Homology, Nucleic Acid MH - Time Factors MH - Xenopus/embryology EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.01-12-0596 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3123-37. PMID- 12221121 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - A role for Caenorhabditis elegans importin IMA-2 in germ line and embryonic mitosis. PG - 3138-47 AB - The importin alpha family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin alpha proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin alpha proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis. AD - Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. FAU - Geles, Kenneth G AU - Geles KG FAU - Johnson, Jeffrey J AU - Johnson JJ FAU - Jong, Sena AU - Jong S FAU - Adam, Stephen A AU - Adam SA LA - eng GR - GM-47866/GM/NIGMS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Caenorhabditis elegans Proteins) RN - 0 (IMA-2 protein, C elegans) RN - 0 (Karyopherins) RN - 0 (alpha Karyopherins) SB - IM MH - Animals MH - Caenorhabditis elegans/*metabolism MH - Caenorhabditis elegans Proteins/*metabolism/*physiology MH - Cell Nucleus/metabolism MH - Crosses, Genetic MH - Cytoplasm/metabolism MH - Fluorescent Antibody Technique, Indirect MH - Karyopherins/*metabolism/*physiology MH - *Mitosis MH - Nuclear Envelope/metabolism MH - Phenotype MH - RNA Interference MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Time Factors MH - alpha Karyopherins/*metabolism/*physiology EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-02-0069 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3138-47. PMID- 12221122 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - The major sites of cellular phospholipid synthesis and molecular determinants of Fatty Acid and lipid head group specificity. PG - 3148-61 AB - Phosphatidylcholine and phosphatidylethanolamine are the two main phospholipids in eukaryotic cells comprising ~50 and 25% of phospholipid mass, respectively. Phosphatidylcholine is synthesized almost exclusively through the CDP-choline pathway in essentially all mammalian cells. Phosphatidylethanolamine is synthesized through either the CDP-ethanolamine pathway or by the decarboxylation of phosphatidylserine, with the contribution of each pathway being cell type dependent. Two human genes, CEPT1 and CPT1, code for the total compliment of activities that directly synthesize phosphatidylcholine and phosphatidylethanolamine through the CDP-alcohol pathways. CEPT1 transfers a phosphobase from either CDP-choline or CDP-ethanolamine to diacylglycerol to synthesize both phosphatidylcholine and phosphatidylethanolamine, whereas CPT1 synthesizes phosphatidylcholine exclusively. We show through immunofluorescence that brefeldin A treatment relocalizes CPT1, but not CEPT1, implying CPT1 is found in the Golgi. A combination of coimmunofluorescence and subcellular fractionation experiments with various endoplasmic reticulum, Golgi, and nuclear markers confirmed that CPT1 was found in the Golgi and CEPT1 was found in both the endoplasmic reticulum and nuclear membranes. The rate-limiting step for phosphatidylcholine synthesis is catalyzed by the amphitropic CTP:phosphocholine cytidylyltransferase alpha, which is found in the nucleus in most cell types. CTP:phosphocholine cytidylyltransferase alpha is found immediately upstream cholinephosphotransferase, and it translocates from a soluble nuclear location to the nuclear membrane in response to activators of the CDP-choline pathway. Thus, substrate channeling of the CDP-choline produced by CTP:phosphocholine cytidylyltransferase alpha to nuclear located CEPT1 is the mechanism by which upregulation of the CDP-choline pathway increases de novo phosphatidylcholine biosynthesis. In addition, a series of CEPT1 site-directed mutants was generated that allowed for the assignment of specific amino acid residues as structural requirements that directly alter either phospholipid head group or fatty acyl composition. This pinpointed glycine 156 within the catalytic motif as being responsible for the dual CDP-alcohol specificity of CEPT1, whereas mutations within helix 214-228 allowed for the orientation of transmembrane helices surrounding the catalytic site to be definitively positioned. AD - The Atlantic Research Centre, Department of Pediatrics, IWK Health Centre, Dalhousie University, Halifax, Nova Scotia, B3H 4H7 Canada. FAU - Henneberry, Annette L AU - Henneberry AL FAU - Wright, Marcia M AU - Wright MM FAU - McMaster, Christopher R AU - McMaster CR LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (DNA, Complementary) RN - 0 (Diglycerides) RN - 0 (Fatty Acids) RN - 0 (Lipids) RN - 0 (Phospholipids) RN - 56-40-6 (Glycine) SB - IM MH - Amino Acid Sequence MH - Animals MH - Blotting, Western MH - CHO Cells MH - Catalytic Domain MH - Cell Membrane/*metabolism MH - Cell Nucleus/metabolism MH - DNA, Complementary/metabolism MH - Diglycerides/metabolism MH - Fatty Acids/metabolism MH - Glycine/chemistry MH - Hamsters MH - Humans MH - Lipids/*metabolism MH - Microscopy, Fluorescence MH - Models, Biological MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - Mutation MH - Open Reading Frames MH - Phospholipids/*metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid MH - Structure-Activity Relationship EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.01-11-0540 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3148-61. PMID- 12221123 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041215 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - An essential subfamily of Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex and endosomal/vacuolar system. PG - 3162-77 AB - The Saccharomyces cerevisiae genome contains five genes encoding P-type ATPases that are potential aminophospholipid translocases (APTs): DRS2, NEO1, and three uncharacterized open reading frames that we have named DNF1, DNF2, and DNF3 for DRS2/NEO1 family. NEO1 is the only essential gene in APT family and seems to be functionally distinct from the DRS2/DNF genes. The drs2Delta dnf1Delta dnf2Delta dnf3Delta quadruple mutant is inviable, although any one member of this group can maintain viability, indicating that there is a substantial functional overlap between the encoded proteins. We have previously implicated Drs2p in clathrin function at the trans-Golgi network. In this study, we constructed strains carrying all possible viable combinations of null alleles from this group and analyzed them for defects in protein transport. The drs2Delta dnf1Delta mutant grows slowly, massively accumulates intracellular membranes, and exhibits a substantial defect in the transport of alkaline phosphatase to the vacuole. Transport of carboxypeptidase Y to the vacuole is also perturbed, but to a lesser extent. In addition, the dnf1Delta dnf2Delta dnf3Delta mutant exhibits a defect in recycling of GFP-Snc1p in the early endocytic-late secretory pathways. Drs2p and Dnf3p colocalize with the trans-Golgi network marker Kex2p, whereas Dnf1p and Dnf2p seem to localize to the plasma membrane and late exocytic or early endocytic membranes. We propose that eukaryotes express multiple APT subfamily members to facilitate protein transport in multiple pathways. AD - Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235-1634, USA. FAU - Hua, Zhaolin AU - Hua Z FAU - Fatheddin, Parvin AU - Fatheddin P FAU - Graham, Todd R AU - Graham TR LA - eng GR - GM-62367/GM/NIGMS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Luminescent Proteins) RN - 0 (Membrane Proteins) RN - 0 (Plasmids) RN - 0 (SNC1 protein, S cerevisiae) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 147336-22-9 (Green Fluorescent Proteins) RN - EC 3.1.3.1 (Alkaline Phosphatase) RN - EC 3.2.1. (Glycoside Hydrolases) RN - EC 3.2.1.26 (beta-Fructofuranosidase) RN - EC 3.4.- (Carboxypeptidases) RN - EC 3.4.16.5 (Carboxypeptidase C) RN - EC 3.6.1.- (DRS2 protein, S cerevisiae) RN - EC 3.6.1.3 (Adenosinetriphosphatase) RN - EC 3.6.1.38 (Ca(2+)-Transporting ATPase) SB - IM MH - Adenosinetriphosphatase/*chemistry/metabolism MH - Alkaline Phosphatase/metabolism MH - Alleles MH - Biological Transport MH - Ca(2+)-Transporting ATPase/chemistry/*metabolism/physiology MH - Carboxypeptidase C MH - Carboxypeptidases/metabolism MH - Endosomes/*metabolism MH - Glycoside Hydrolases/metabolism MH - Golgi Apparatus/*metabolism MH - Green Fluorescent Proteins MH - Luminescent Proteins/metabolism MH - Membrane Proteins/metabolism MH - Models, Biological MH - Mutation MH - Open Reading Frames MH - Phenotype MH - Plasmids/metabolism MH - Precipitin Tests MH - Protein Transport MH - Research Support, U.S. Gov't, P.H.S. MH - Saccharomyces cerevisiae Proteins/metabolism MH - Signal Transduction MH - Temperature MH - Time Factors MH - Vacuoles/metabolism MH - beta-Fructofuranosidase MH - trans-Golgi Network/metabolism EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-03-0172 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3162-77. PMID- 12221124 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20050209 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Regulation of focal adhesion kinase by a novel protein inhibitor FIP200. PG - 3178-91 AB - Focal adhesion kinase (FAK) is a major mediator of integrin signaling pathways. The mechanisms of regulation of FAK activity and its associated cellular functions are not very well understood. Here, we present data suggesting that a novel protein FIP200 functions as an inhibitor for FAK. We show the association of endogenous FIP200 with FAK, which is decreased upon integrin-mediated cell adhesion concomitant with FAK activation. In vitro- and in vivo-binding studies indicate that FIP200 interacts with FAK through multiple domains directly. FIP200 bound to the kinase domain of FAK inhibited its kinase activity in vitro and its autophosphorylation in vivo. Overexpression of FIP200 or its segments inhibited cell spreading, cell migration, and cell cycle progression, which correlated with their inhibition of FAK activity in vivo. The inhibition of these cellular functions by FIP200 could be rescued by coexpression of FAK. Last, we show that disruption of the functional interaction between endogenous FIP200 with FAK leads to increased FAK phosphorylation and partial restoration of cell cycle progression in cells plated on poly-L-lysine, providing further support for FIP200 as a negative regulator of FAK. Together, these results identify FIP200 as a novel protein inhibitor for FAK. AD - Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA. FAU - Abbi, Smita AU - Abbi S FAU - Ueda, Hiroki AU - Ueda H FAU - Zheng, Chuanhai AU - Zheng C FAU - Cooper, Lee Ann AU - Cooper LA FAU - Zhao, Jihe AU - Zhao J FAU - Christopher, Renee AU - Christopher R FAU - Guan, Jun-Lin AU - Guan JL LA - eng GR - GM48050/GM/NIGMS GR - GM52890/GM/NIGMS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Genetic Vectors) RN - 0 (Recombinant Fusion Proteins) RN - 59-14-3 (Bromodeoxyuridine) RN - 70-18-8 (Glutathione) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.- (RB1CC1 protein, human) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.112 (focal adhesion kinase) SB - IM MH - 3T3 Cells MH - Animals MH - Blotting, Western MH - Bromodeoxyuridine/pharmacology MH - CHO Cells MH - Cell Adhesion MH - Cell Cycle MH - Cell Movement MH - Dose-Response Relationship, Drug MH - Escherichia coli/metabolism MH - Genetic Vectors MH - Glutathione/metabolism MH - Glutathione Transferase/metabolism MH - Hamsters MH - Mice MH - Microscopy, Fluorescence MH - Models, Biological MH - Phosphorylation MH - Precipitin Tests MH - Protein Binding MH - Protein Structure, Tertiary MH - Protein-Tyrosine Kinase/*antagonists & inhibitors/*metabolism/*pharmacology MH - Recombinant Fusion Proteins/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-05-0295 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3178-91. PMID- 12221125 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - The product of the survival of motor neuron (SMN) gene is a human telomerase-associated protein. PG - 3192-202 AB - Telomerase is a ribonucleoprotein (RNP) complex that is minimally composed of a protein catalytic subunit, the telomerase reverse transcriptase (TERT), and an RNA component, the telomerase RNA. The survival of motor neuron (SMN) gene codes for a protein involved in the biogenesis of certain RNPs. Here, we report that SMN is a telomerase-associated protein. Using in vitro binding assays and immunoprecipitation experiments, we demonstrate an association between SMN and the telomerase RNP in vitro and in human cells. The specific immunopurification of SMN from human 293 cells copurified telomerase activity, suggesting that SMN associates with a subset of the functional telomerase holoenzyme. Our results also indicate that the human telomerase RNA and the human (h) TERT are not associated with Sm proteins, in contrast to Saccharomyces cerevisiae telomerase. Immunofluorescence analysis showed that hTERT does not specifically colocalize with wild-type SMN in gems or Cajal bodies. However, a dominant-negative mutant of SMN (SMNDeltaN27) previously characterized to elicit the cellular reorganization of small nuclear RNPs caused the accumulation of hTERT in specific SMNDeltaN27-induced cellular bodies. Furthermore, coexpression of SMNDeltaN27 and hTERT in rabbit reticulocyte lysates decreased the efficiency of human telomerase reconstitution in vitro. Our results establish SMN as a novel telomerase-associated protein that is likely to function in human telomerase biogenesis. AD - Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, H3T 1E2, Canada. FAU - Bachand, Francois AU - Bachand F FAU - Boisvert, Francois-Michel AU - Boisvert FM FAU - Cote, Jocelyn AU - Cote J FAU - Richard, Stephane AU - Richard S FAU - Autexier, Chantal AU - Autexier C LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (DNA, Complementary) RN - 0 (Nerve Tissue Proteins) RN - 0 (Plasmids) RN - 0 (SMN protein (spinal muscular atrophy)) RN - EC 2.7.7.- (Telomerase) SB - IM MH - Catalysis MH - Cell Line MH - Cell Nucleus/metabolism MH - DNA, Complementary/metabolism MH - Electrophoresis, Polyacrylamide Gel MH - Genes, Dominant MH - Hela Cells MH - Humans MH - Microscopy, Fluorescence MH - Mutation MH - Nerve Tissue Proteins/*metabolism/*physiology MH - Plasmids/metabolism MH - Precipitin Tests MH - Protein Binding MH - Research Support, Non-U.S. Gov't MH - Telomerase/*metabolism MH - Time Factors EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-04-0216 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3192-202. PMID- 12221126 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041215 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - alpha4beta1 integrin regulates lamellipodia protrusion via a focal complex/focal adhesion-independent mechanism. PG - 3203-17 AB - alpha4beta1 integrin plays an important role in cell migration. We show that when ectopically expressed in Chinese hamster ovary cells, alpha4beta1 is sufficient and required for promoting protrusion of broad lamellipodia in response to scratch-wounding, whereas alpha5beta1 does not have this effect. By time-lapse microscopy of cells expressing an alpha4/green fluorescent protein fusion protein, we show that alpha4beta1 forms transient puncta at the leading edge of cells that begin to protrude lamellipodia in response to scratch-wounding. The cells expressing a mutant alpha4/green fluorescent protein that binds paxillin at a reduced level had a faster response to scratch-wounding, forming alpha4-positive puncta and protruding lamellipodia much earlier. While enhancing lamellipodia protrusion, this mutation reduces random motility of the cells in Transwell assays, indicating that lamellipodia protrusion and random motility are distinct types of motile activities that are differentially regulated by interactions between alpha4beta1 and paxillin. Finally, we show that, at the leading edge, alpha4-positive puncta and paxillin-positive focal complexes/adhesions do not colocalize, but alpha4beta1 and paxillin colocalize partially in ruffles. These findings provide evidence for a specific role of alpha4beta1 in lamellipodia protrusion that is distinct from the motility-promoting functions of alpha5beta1 and other integrins that mediate cell adhesion and signaling events through focal complexes and focal adhesions. AD - Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. FAU - Pinco, Karen A AU - Pinco KA FAU - He, Wei AU - He W FAU - Yang, Joy T AU - Yang JT LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Cytoskeletal Proteins) RN - 0 (Integrin alpha4beta1) RN - 0 (Integrin alpha5beta1) RN - 0 (Ligands) RN - 0 (Luminescent Proteins) RN - 0 (Phosphoproteins) RN - 0 (Plasmids) RN - 0 (Recombinant Fusion Proteins) RN - 0 (paxillin) RN - 147336-22-9 (Green Fluorescent Proteins) SB - IM MH - Animals MH - Biotinylation MH - CHO Cells MH - Cell Adhesion MH - Cell Division MH - Cell Movement MH - Cytoskeletal Proteins/metabolism MH - Flow Cytometry MH - Focal Adhesions/metabolism MH - Green Fluorescent Proteins MH - Hamsters MH - Integrin alpha4beta1/metabolism/*physiology MH - Integrin alpha5beta1/metabolism MH - Ligands MH - Luminescent Proteins/metabolism MH - Microscopy, Confocal MH - Mutation MH - Phosphoproteins/metabolism MH - Plasmids/metabolism MH - Precipitin Tests MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Signal Transduction MH - Time Factors MH - Transfection MH - Wound Healing EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.02-05-0086 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3203-17. PMID- 12221127 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Regulation of airway tight junctions by proinflammatory cytokines. PG - 3218-34 AB - Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions. AD - Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, 27599, USA. FAU - Coyne, Carolyn B AU - Coyne CB FAU - Vanhook, Miriam K AU - Vanhook MK FAU - Gambling, Todd M AU - Gambling TM FAU - Carson, Johnny L AU - Carson JL FAU - Boucher, Richard C AU - Boucher RC FAU - Johnson, Larry G AU - Johnson LG LA - eng GR - HL-54832/HL/NHLBI PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Cytokines) RN - 0 (Interleukin-1) RN - 0 (Membrane Proteins) RN - 0 (Phosphoproteins) RN - 0 (RNA, Messenger) RN - 0 (Tumor Necrosis Factor-alpha) RN - 0 (zonula occludens-1 protein) RN - 7440-23-5 (Sodium) RN - 7782-50-5 (Chlorine) RN - 82115-62-6 (Interferon Type II) RN - EC 2.7.1.37 (Protein Kinase C) SB - IM MH - Blotting, Western MH - Cell Movement MH - Cells, Cultured MH - Chlorine/metabolism MH - Cystic Fibrosis/*metabolism MH - Cytokines/biosynthesis/*metabolism MH - Electrophysiology MH - Enzyme-Linked Immunosorbent Assay MH - Epithelial Cells/*metabolism MH - Humans MH - Interferon Type II/metabolism MH - Interleukin-1/metabolism MH - Membrane Proteins/metabolism MH - Microscopy, Confocal MH - Microscopy, Electron MH - Microscopy, Fluorescence MH - Permeability MH - Phosphoproteins/metabolism MH - Protein Kinase C/metabolism MH - RNA, Messenger/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Reverse Transcriptase Polymerase Chain Reaction MH - Sodium/pharmacology MH - Tight Junctions/*metabolism MH - Time Factors MH - Tumor Necrosis Factor-alpha/metabolism EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-03-0134 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3218-34. PMID- 12221128 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex. PG - 3235-45 AB - Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC. AD - Biosignal Research Center, Kobe University, Japan. FAU - Takahashi, Mikiko AU - Takahashi M FAU - Yamagiwa, Akiko AU - Yamagiwa A FAU - Nishimura, Tamako AU - Nishimura T FAU - Mukai, Hideyuki AU - Mukai H FAU - Ono, Yoshitaka AU - Ono Y LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (AKAP9 protein, human) RN - 0 (Adaptor Proteins, Signal Transducing) RN - 0 (Calmodulin-Binding Proteins) RN - 0 (Carrier Proteins) RN - 0 (Cytoskeletal Proteins) RN - 0 (DNA, Complementary) RN - 0 (Microtubule-Associated Proteins) RN - 0 (TUBGCP2 protein, human) RN - 0 (TUBGCP3 protein, human) RN - 0 (Tubulin) RN - 0 (kendrin) SB - IM MH - *Adaptor Proteins, Signal Transducing MH - Amino Acid Sequence MH - Animals MH - CHO Cells MH - COS Cells MH - Calmodulin-Binding Proteins/*metabolism MH - Carrier Proteins/*metabolism MH - Cell Line MH - Cell Nucleus/*metabolism MH - Centrosome/metabolism MH - *Cytoskeletal Proteins MH - DNA, Complementary/metabolism MH - Hamsters MH - Hela Cells MH - Humans MH - Immunoblotting MH - Microscopy, Fluorescence MH - Microtubule-Associated Proteins/metabolism MH - Microtubules/*metabolism/ultrastructure MH - Models, Genetic MH - Molecular Sequence Data MH - Precipitin Tests MH - Protein Binding MH - Protein Structure, Tertiary MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid MH - Transfection MH - Tubulin/*metabolism MH - Two-Hybrid System Techniques EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-02-0112 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3235-45. PMID- 12221129 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041203 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - DNA repair and transcriptional effects of mutations in TFIIH in Drosophila development. PG - 3246-56 AB - Mutations in XPB and XPD TFIIH helicases have been related with three hereditary human disorders: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. The dual role of TFIIH in DNA repair and transcription makes it difficult to discern which of the mutant TFIIH phenotypes is due to defects in any of these different processes. We used haywire (hay), the Drosophila XPB homolog, to dissect this problem. Our results show that when hay dosage is affected, the fly shows defects in structures that require high levels of transcription. We found a genetic interaction between hay and cdk7, and we propose that some of these phenotypes are due to transcriptional deficiencies. We also found more apoptotic cells in imaginal discs and in the CNS of hay mutant flies than in wild-type flies. Because this abnormal level of apoptosis was not detected in cdk7 flies, this phenotype could be related to defects in DNA repair. In addition the apoptosis induced by p53 Drosophila homolog (Dmp53) is suppressed in heterozygous hay flies. AD - Department of Genetics and Molecular Physiology, Institute of Biotechnology, Universidad Nacional Autonoma de Mexico, Morelos 62250, Mexico. FAU - Merino, Carlos AU - Merino C FAU - Reynaud, Enrique AU - Reynaud E FAU - Vazquez, Martha AU - Vazquez M FAU - Zurita, Mario AU - Zurita M LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (DNA-Binding Proteins) RN - 0 (Drosophila Proteins) RN - 0 (Protein p53) RN - 0 (TATA-Binding Protein Associated Factors) RN - 0 (Taf6 protein, Drosophila) RN - 0 (Transcription Factor TFIID) RN - 0 (Transcription Factors, TFII) RN - 148710-81-0 (transcription factor TFIIH) RN - 148998-67-8 (haywire protein, Drosophila) RN - 63231-63-0 (RNA) RN - EC 2.7.1.37 (Cyclin-Dependent Kinases) RN - EC 2.7.1.37 (cyclin-dependent kinase-activating kinase) SB - IM MH - Alleles MH - Animals MH - Animals, Genetically Modified MH - Blotting, Western MH - Cyclin-Dependent Kinases/genetics/metabolism MH - *DNA Repair MH - DNA-Binding Proteins/genetics/metabolism MH - Drosophila/*embryology/genetics MH - Drosophila Proteins/genetics/metabolism MH - Gene Expression Regulation, Developmental MH - Heterozygote MH - Homozygote MH - Humans MH - Mutation MH - Nucleic Acid Hybridization MH - Phenotype MH - Protein p53/genetics MH - RNA/metabolism MH - Research Support, Non-U.S. Gov't MH - *TATA-Binding Protein Associated Factors MH - *Transcription Factor TFIID MH - Transcription Factors, TFII/*genetics/metabolism MH - *Transcription, Genetic MH - Ultraviolet Rays MH - Wing/embryology EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-02-0087 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3246-56. PMID- 12221130 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041215 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Agonist-induced PIP(2) hydrolysis inhibits cortical actin dynamics: regulation at a global but not at a micrometer scale. PG - 3257-67 AB - Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) at the inner leaflet of the plasma membrane has been proposed to locally regulate the actin cytoskeleton. Indeed, recent studies that use GFP-tagged pleckstrin homology domains (GFP-PH) as fluorescent PIP(2) sensors suggest that this lipid is enriched in membrane microdomains. Here we report that this concept needs revision. Using three distinct fluorescent GFP-tagged pleckstrin homology domains, we show that highly mobile GFP-PH patches colocalize perfectly with various lipophilic membrane dyes and, hence, represent increased lipid content rather than PIP(2)-enriched microdomains. We show that bright patches are caused by submicroscopical folds and ruffles in the membrane that can be directly visualized at approximately 15 nm axial resolution with a novel numerically enhanced imaging method. F-actin motility is inhibited significantly by agonist-induced PIP(2) breakdown, and it resumes as soon as PIP(2) levels are back to normal. Thus, our data support a role for PIP(2) in the regulation of cortical actin, but they challenge a model in which spatial differences in PIP(2) regulation of the cytoskeleton exist at a micrometer scale. AD - Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands. FAU - van Rheenen, Jacco AU - van Rheenen J FAU - Jalink, Kees AU - Jalink K LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Actins) RN - 0 (Luminescent Proteins) RN - 0 (Phosphatidylinositol 4,5-Diphosphate) RN - 147336-22-9 (Green Fluorescent Proteins) SB - IM MH - 3T3 Cells MH - Actins/*metabolism MH - Animals MH - Cell Membrane/metabolism MH - Cell Movement MH - Cytoskeleton/metabolism MH - Green Fluorescent Proteins MH - Humans MH - Hydrolysis MH - Luminescent Proteins/metabolism MH - Mice MH - Microscopy, Confocal MH - Microscopy, Fluorescence MH - Models, Statistical MH - Phosphatidylinositol 4,5-Diphosphate/agonists/*metabolism MH - Photobleaching MH - Research Support, Non-U.S. Gov't MH - Time Factors MH - Transfection MH - Tumor Cells, Cultured EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-04-0231 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3257-67. PMID- 12221131 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041203 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport. PG - 3268-80 AB - The mechanisms mediating polarized delivery of vesicles to cell surface domains are poorly understood in animal cells. We have previously shown that expression of Rab8 promotes the formation of new cell surface domains through reorganization of actin and microtubules. To unravel the function of Rab8, we used the yeast two-hybrid system to search for potential Rab8-specific activators. We identified a coil-coiled protein (Rabin8), homologous to the rat Rabin3 that stimulated nucleotide exchange on Rab8 but not on Rab3A and Rab5. Furthermore, we show that rat Rabin3 has exchange activity on Rab8 but not on Rab3A, supporting the view that rat Rabin3 is the rat equivalent of human Rabin8. Rabin8 localized to the cortical actin and expression of Rabin8 resulted in remodeling of actin and the formation of polarized cell surface domains. Activation of PKC by phorbol esters enhanced translocation of both Rabin8 and Rab8-specific vesicles to the outer edge of lamellipodial structures. Moreover, coexpression of Rabin8 with dominant negative Rab8 (T22N) redistributes Rabin8 from cortical actin to Rab8-specific vesicles and promotes their polarized transport to cell protrusions. The C-terminal region of Rabin8 plays an essential role in this transport. We propose that Rabin8 is a Rab8-specific activator that is connected to processes that mediate polarized membrane traffic to dynamic cell surface structures. AD - Institute of Biotechnology, Program in Cellular Biotechnology, FIN-00014 University of Helsinki, Finland. FAU - Hattula, Katarina AU - Hattula K FAU - Furuhjelm, Johanna AU - Furuhjelm J FAU - Arffman, Airi AU - Arffman A FAU - Peranen, Johan AU - Peranen J LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Actins) RN - 0 (Guanine Nucleotide Exchange Factors) RN - 0 (Rab8 protein) RN - 0 (Recombinant Proteins) RN - 146-91-8 (Guanosine Diphosphate) RN - 86-01-1 (Guanosine Triphosphate) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.- (germinal center kinases) RN - EC 2.7.1.37 (Protein Kinase C) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 3.6.1.- (rab GTP-Binding Proteins) RN - EC 3.6.1.- (rab3A GTP-Binding Protein) RN - EC 3.6.1.- (rab5 GTP-Binding Proteins) SB - IM MH - Actins/*metabolism MH - Amino Acid Sequence MH - Biological Transport MH - Blotting, Northern MH - Cell Membrane/*metabolism MH - Cloning, Molecular MH - Glutathione Transferase/metabolism MH - Guanine Nucleotide Exchange Factors/*genetics/*metabolism MH - Guanosine Diphosphate/*metabolism MH - Guanosine Triphosphate/*metabolism MH - Hela Cells MH - Humans MH - Microscopy, Confocal MH - Molecular Sequence Data MH - Polymerase Chain Reaction MH - Protein Binding MH - Protein Kinase C/metabolism MH - Protein Structure, Tertiary MH - *Protein-Serine-Threonine Kinases MH - Recombinant Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid MH - Time Factors MH - Tissue Distribution MH - Transfection MH - Two-Hybrid System Techniques MH - rab GTP-Binding Proteins/*metabolism MH - rab3A GTP-Binding Protein/metabolism MH - rab5 GTP-Binding Proteins/metabolism EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-03-0143 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3268-80. PMID- 12221132 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041215 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Calcineurin, a calcium/calmodulin-dependent protein phosphatase, is involved in movement, fertility, egg laying, and growth in Caenorhabditis elegans. PG - 3281-93 AB - Calcineurin is a Ca(2+)-calmodulin-dependent serine/threonine protein phosphatase that has been implicated in various signaling pathways. Here we report the identification and characterization of calcineurin genes in Caenorhabditis elegans (cna-1 and cnb-1), which share high homology with Drosophila and mammalian calcineurin genes. C. elegans calcineurin binds calcium and functions as a heterodimeric protein phosphatase establishing its biochemical conservation in the nematode. Calcineurin is expressed in hypodermal seam cells, body-wall muscle, vulva muscle, neuronal cells, and in sperm and the spermatheca. cnb-1 mutants showed pleiotropic defects including lethargic movement and delayed egg-laying. Interestingly, these characteristic defects resembled phenotypes observed in gain-of-function mutants of unc-43/Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and goa-1/G(o)-protein alpha-subunit. Double mutants of cnb-1 and unc-43(gf) displayed an apparent synergistic severity of movement and egg-laying defects, suggesting that calcineurin may have an antagonistic role in CaMKII-regulated phosphorylation signaling pathways in C. elegans. AD - Department of Life Science, Kwangju Institute of Science and Technology, Korea. FAU - Bandyopadhyay, Jaya AU - Bandyopadhyay J FAU - Lee, Jiyeon AU - Lee J FAU - Lee, Jungsoo AU - Lee J FAU - Lee, Jin Il AU - Lee JI FAU - Yu, Jae-Ran AU - Yu JR FAU - Jee, Changhoon AU - Jee C FAU - Cho, Jeong-Hoon AU - Cho JH FAU - Jung, Sunki AU - Jung S FAU - Lee, Myon Hee AU - Lee MH FAU - Zannoni, Sonia AU - Zannoni S FAU - Singson, Andrew AU - Singson A FAU - Kim, Do Han AU - Kim do H FAU - Koo, Hyeon-Sook AU - Koo HS FAU - Ahnn, Joohong AU - Ahnn J LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (DNA, Complementary) RN - 0 (Luminescent Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 147336-22-9 (Green Fluorescent Proteins) RN - EC 3.1.3 (Phosphoric Monoester Hydrolases) RN - EC 3.1.3.- (Calcineurin) SB - IM MH - Amino Acid Sequence MH - Animals MH - Blotting, Northern MH - Caenorhabditis elegans/*metabolism MH - Calcineurin/*genetics/*metabolism MH - Cell Division MH - Cell Movement MH - Cloning, Molecular MH - DNA, Complementary/metabolism MH - Dose-Response Relationship, Drug MH - Gene Deletion MH - Gene Library MH - Green Fluorescent Proteins MH - Immunohistochemistry MH - Luminescent Proteins/metabolism MH - Microscopy, Fluorescence MH - Models, Genetic MH - Molecular Sequence Data MH - Mutation MH - Phenotype MH - Phosphoric Monoester Hydrolases/metabolism MH - Phosphorylation MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid MH - Signal Transduction MH - Two-Hybrid System Techniques EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-01-0005 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3281-93. PMID- 12221133 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041215 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Membrane trafficking of heterotrimeric G proteins via the endoplasmic reticulum and Golgi. PG - 3294-302 AB - Membrane targeting of G-protein alphabetagamma heterotrimers was investigated in live cells by use of Galpha and Ggamma subunits tagged with spectral mutants of green fluorescent protein. Unlike Ras proteins, Gbetagamma contains a single targeting signal, the CAAX motif, which directed the dimer to the endoplasmic reticulum. Endomembrane localization of farnesylated Ggamma(1), but not geranylgeranylated Ggamma(2), required carboxyl methylation. Targeting of the heterotrimer to the plasma membrane (PM) required coexpression of all three subunits, combining the CAAX motif of Ggamma with the fatty acyl modifications of Galpha. Galpha associated with Gbetagamma on the Golgi and palmitoylation of Galpha was required for translocation of the heterotrimer to the PM. Thus, two separate signals, analogous to the dual-signal targeting mechanism of Ras proteins, cooperate to target heterotrimeric G proteins to the PM via the endomembrane. AD - Department of Medicine, Cell Biology and Pharmacology, NYU School of Medicine, 10016, USA. FAU - Michaelson, David AU - Michaelson D FAU - Ahearn, Ian AU - Ahearn I FAU - Bergo, Martin AU - Bergo M FAU - Young, Stephen AU - Young S FAU - Philips, Mark AU - Philips M LA - eng GR - AI-36224/AI/NIAID GR - CA-09161/CA/NCI GR - GM-55279/GM/NIGMS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Luminescent Proteins) RN - 0 (Plasmids) RN - 0 (Recombinant Fusion Proteins) RN - 147336-22-9 (Green Fluorescent Proteins) RN - EC 3.6.1.- (GTP-Binding Proteins) SB - IM MH - Amino Acid Motifs MH - Animals MH - Biological Transport MH - COS Cells MH - Cell Line MH - Cell Membrane/metabolism MH - Dogs MH - Endoplasmic Reticulum/*metabolism MH - GTP-Binding Proteins/*metabolism MH - Golgi Apparatus/*metabolism MH - Green Fluorescent Proteins MH - Humans MH - Luminescent Proteins/metabolism MH - Microscopy, Fluorescence MH - Models, Biological MH - Plasmids/metabolism MH - Protein Binding MH - Protein Isoprenylation MH - Protein Structure, Tertiary MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-02-0095 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3294-302. PMID- 12221134 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Regulation of flagellar dynein by calcium and a role for an axonemal calmodulin and calmodulin-dependent kinase. PG - 3303-13 AB - Ciliary and flagellar motility is regulated by changes in intraflagellar calcium. However, the molecular mechanism by which calcium controls motility is unknown. We tested the hypothesis that calcium regulates motility by controlling dynein-driven microtubule sliding and that the central pair and radial spokes are involved in this regulation. We isolated axonemes from Chlamydomonas mutants and measured microtubule sliding velocity in buffers containing 1 mM ATP and various concentrations of calcium. In buffers with pCa > 8, microtubule sliding velocity in axonemes lacking the central apparatus (pf18 and pf15) was reduced compared with that of wild-type axonemes. In contrast, at pCa4, dynein activity in pf18 and pf15 axonemes was restored to wild-type level. The calcium-induced increase in dynein activity in pf18 axonemes was inhibited by antagonists of calmodulin and calmodulin-dependent kinase II. Axonemes lacking the C1 central tubule (pf16) or lacking radial spoke components (pf14 and pf17) do not exhibit calcium-induced increase in dynein activity in pCa4 buffer. We conclude that calcium regulation of flagellar motility involves regulation of dynein-driven microtubule sliding, that calmodulin and calmodulin-dependent kinase II may mediate the calcium signal, and that the central apparatus and radial spokes are key components of the calcium signaling pathway. AD - Dartmouth College, Department of Biological Sciences, Hanover, New Hampshire 03755, USA. elizabeth.f.smith@dartmouth.edu FAU - Smith, Elizabeth F AU - Smith EF LA - eng GR - GM51379/GM/NIGMS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Calmodulin) RN - 7440-70-2 (Calcium) RN - EC 2.7.1.123 (Ca(2+)-Calmodulin Dependent Protein Kinase) RN - EC 2.7.1.37 (Casein Kinases) RN - EC 2.7.1.37 (Protein Kinases) RN - EC 3.1.3 (Phosphoric Monoester Hydrolases) RN - EC 3.1.3.16 (Phosphoprotein Phosphatase) RN - EC 3.6.1.33 (Dynein ATPase) SB - IM MH - Animals MH - Axons/*metabolism MH - Ca(2+)-Calmodulin Dependent Protein Kinase/*metabolism MH - Calcium/*metabolism MH - Calmodulin/*metabolism MH - Casein Kinases MH - Chlamydomonas/*cytology/genetics MH - Dynein ATPase/*metabolism MH - Flagella/*metabolism MH - Microtubules/metabolism MH - Models, Biological MH - Phosphoprotein Phosphatase/metabolism MH - Phosphoric Monoester Hydrolases/metabolism MH - Protein Kinases/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-04-0185 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3303-13. PMID- 12221135 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Analysis of Sec22p in endoplasmic reticulum/Golgi transport reveals cellular redundancy in SNARE protein function. PG - 3314-24 AB - Membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form heteromeric complexes that are required for intracellular membrane fusion and are proposed to encode compartmental specificity. In yeast, the R-SNARE protein Sec22p acts in transport between the endoplasmic reticulum (ER) and Golgi compartments but is not essential for cell growth. Other SNARE proteins that function in association with Sec22p (i.e., Sed5p, Bos1p, and Bet1p) are essential, leading us to question how transport through the early secretory pathway is sustained in the absence of Sec22p. In wild-type strains, we show that Sec22p is directly required for fusion of ER-derived vesicles with Golgi acceptor membranes. In sec22Delta strains, Ykt6p, a related R-SNARE protein that operates in later stages of the secretory pathway, is up-regulated and functionally substitutes for Sec22p. In vivo combination of the sec22Delta mutation with a conditional ykt6-1 allele results in lethality, consistent with a redundant mechanism. Our data indicate that the requirements for specific SNARE proteins in intracellular membrane fusion are less stringent than appreciated and suggest that combinatorial mechanisms using both upstream-targeting elements and SNARE proteins are required to maintain an essential level of compartmental organization. AD - Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA. FAU - Liu, Yiting AU - Liu Y FAU - Barlowe, Charles AU - Barlowe C LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Membrane Proteins) RN - 0 (Plasmids) RN - 0 (Receptors, Cell Surface) RN - 0 (SNAP receptor) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 0 (Sec22 protein, S cerevisiae) RN - 0 (Vesicular Transport Proteins) RN - 0 (YKT6 protein, S cerevisiae) SB - IM MH - Biological Transport MH - Cell-Free System MH - Endoplasmic Reticulum/*metabolism MH - Golgi Apparatus/*metabolism MH - Immunoblotting MH - Membrane Proteins/*biosynthesis/*metabolism/physiology MH - Plasmids/metabolism MH - Precipitin Tests MH - Protein Binding MH - Protein Transport MH - Receptors, Cell Surface/*biosynthesis/physiology MH - Research Support, U.S. Gov't, P.H.S. MH - Saccharomyces cerevisiae/*metabolism/physiology MH - Saccharomyces cerevisiae Proteins/*biosynthesis/physiology MH - *Vesicular Transport Proteins EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-04-0204 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3314-24. PMID- 12221136 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Proteasome regulates the delivery of LDL receptor-related protein into the degradation pathway. PG - 3325-35 AB - The low-density lipoprotein receptor (LDLR)-related protein (LRP) is a multiligand endocytic receptor that has broad cellular and physiological functions. Previous studies have shown that both tyrosine-based and di-leucine motifs within the LRP cytoplasmic tail are responsible for mediating its rapid endocytosis. Little is known, however, about the mechanism by which LRP is targeted for degradation. By examining both endogenous full-length and a minireceptor form of LRP, we found that proteasomal inhibitors, MG132 and lactacystin, prolong the cellular half-life of LRP. The presence of proteasomal inhibitors also significantly increased the level of LRP at the cell surface, suggesting that the delivery of LRP to the degradation pathway was blocked at a compartment from which recycling of the receptor to the cell surface still occurred. Immunoelectron microscopy analyses demonstrated a proteasomal inhibitor-dependent reduction in LRP minireceptor within both limiting membrane and internal vesicles of the multivesicular bodies, which are compartments that lead to receptor degradation. In contrast to the growth hormone receptor, we found that the initial endocytosis of LRP minireceptor does not require a functional ubiquitin-proteasome system. Finally, using truncated cytoplasmic mutants of LRP minireceptors, we found that a region of 19 amino acids within the LRP tail is required for proteasomal regulation. Taken together our results provide strong evidence that the cellular turnover of a cargo receptor, i.e., LRP, is regulated by the proteasomal system, suggesting a broader function of the proteasome in regulating the trafficking of receptors into the degradation pathway. AD - Department of Pediatrics, Washington University School of Medicine, CB 8208, St. Louis Children's Hospital, Missouri 63110, USA. FAU - Melman, Lora AU - Melman L FAU - Geuze, Hans J AU - Geuze HJ FAU - Li, Yonghe AU - Li Y FAU - McCormick, Lynn M AU - McCormick LM FAU - Van Kerkhof, Peter AU - Van Kerkhof P FAU - Strous, Ger J AU - Strous GJ FAU - Schwartz, Alan L AU - Schwartz AL FAU - Bu, Guojun AU - Bu G LA - eng GR - AG05681/AG/NIA GR - DK61761/DK/NIDDK GR - HL53280/HL/NHLBI GR - NS37525/NS/NINDS PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Cysteine Proteinase Inhibitors) RN - 0 (LDL-Receptor Related Protein 1) RN - 0 (Leupeptins) RN - 0 (Ligands) RN - 0 (Lipoproteins, LDL) RN - 0 (Multienzyme Complexes) RN - 0 (Recombinant Proteins) RN - 133407-82-6 (benzyloxycarbonylleucyl-leucyl-leucine aldehyde) RN - EC 3.4.22 (Cysteine Endopeptidases) RN - EC 3.4.25.1 (Proteasome Endopeptidase Complex) SB - IM MH - Amino Acid Motifs MH - Animals MH - Blotting, Western MH - CHO Cells MH - Cysteine Endopeptidases/*metabolism MH - Cysteine Proteinase Inhibitors/pharmacology MH - Endocytosis MH - Flow Cytometry MH - Hamsters MH - Humans MH - Kinetics MH - LDL-Receptor Related Protein 1/chemistry/*metabolism MH - Leupeptins/pharmacology MH - Ligands MH - Lipoproteins, LDL/metabolism MH - Microscopy, Fluorescence MH - Microscopy, Immunoelectron MH - Multienzyme Complexes/*metabolism MH - Precipitin Tests MH - Proteasome Endopeptidase Complex MH - Protein Binding MH - Protein Transport MH - Recombinant Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Time Factors MH - Tumor Cells, Cultured EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-03-0152 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3325-35. PMID- 12221137 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - A Ypt32p exchange factor is a putative effector of Ypt1p. PG - 3336-43 AB - Ypt1p regulates vesicle tethering and fusion events from the ER to the Golgi and through the early Golgi. Genetic studies have suggested a functional relationship between Ypt1p and Ypt31p/Ypt32p. Ypt31p and Ypt32p are a pair of functionally redundant GTPases that act after Ypt1p to mediate intra-Golgi traffic or the budding of post-Golgi vesicles from the trans-Golgi. Here we report that a novel Ypt32p exchange factor is a putative effector of Ypt1p. These findings implicate small GTP-binding proteins of the Ypt/Rab family in a signal cascade that directs membrane traffic through the secretory pathway. AD - Howard Hughes Medical Institute and the Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06519-1418, USA. FAU - Wang, Wei AU - Wang W FAU - Ferro-Novick, Susan AU - Ferro-Novick S LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Carrier Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 0 (USO1 protein, S cerevisiae) RN - 0 (Vesicular Transport Proteins) RN - 0 (YPT1 protein, S cerevisiae) RN - 0 (YPT32 protein, S cerevisiae) RN - 86-01-1 (Guanosine Triphosphate) RN - EC 3.6.1.- (rab GTP-Binding Proteins) SB - IM MH - Carrier Proteins/metabolism MH - Cell Membrane/metabolism MH - Cytosol/metabolism MH - Golgi Apparatus/metabolism MH - Guanosine Triphosphate/metabolism MH - Protein Binding MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Saccharomyces cerevisiae/*metabolism MH - Saccharomyces cerevisiae Proteins/metabolism MH - Signal Transduction MH - Subcellular Fractions/metabolism MH - Time Factors MH - *Vesicular Transport Proteins MH - rab GTP-Binding Proteins/*metabolism EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.01-12-0577 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3336-43. PMID- 12221138 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20041117 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Purification and identification of secernin, a novel cytosolic protein that regulates exocytosis in mast cells. PG - 3344-54 AB - After permeabilization with the pore-forming toxin streptolysin-O mast cells can be triggered to secrete by addition of both calcium and a GTP analogue. If stimulation is delayed after permeabilization, there is a progressive decrease in the extent of secretion upon stimulation, eventually leading to a complete loss of the secretory response. This loss of secretory response can be retarded by the addition of cytosol from other secretory tissues, demonstrating that the response is dependent on a number of cytosolic proteins. We have used this as the basis of a bioassay to purify Secernin 1, a novel 50-kDa cytosolic protein that appears to be involved in the regulation of exocytosis from peritoneal mast cells. Secernin 1 increases both the extent of secretion and increases the sensitivity of mast cells to stimulation with calcium. AD - Department of Biological and Biomedical Sciences, University of Durham, United Kingdom. FAU - Way, Gemma AU - Way G FAU - Morrice, Nicholas AU - Morrice N FAU - Smythe, Carl AU - Smythe C FAU - O'Sullivan, Antony J AU - O'Sullivan AJ LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Nerve Tissue Proteins) RN - 0 (Plasmids) RN - 0 (Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (phenyl-superose) RN - 0 (secernin 1 protein, human) RN - 0 (secernin 1 protein, mouse) RN - 1306-06-5 (Durapatite) RN - 7440-70-2 (Calcium) RN - 9012-36-6 (Sepharose) RN - 97599-42-3 (Superose 12) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Calcium/pharmacology MH - Cattle MH - Chromatography MH - Chromatography, Gel/*methods MH - Cytosol/*metabolism MH - Dose-Response Relationship, Drug MH - Durapatite/pharmacology MH - Exocytosis MH - Glutathione Transferase/metabolism MH - Humans MH - Male MH - Mast Cells/*metabolism MH - Molecular Sequence Data MH - Nerve Tissue Proteins/*chemistry/*isolation & purification MH - Plasmids/metabolism MH - Proteins/*chemistry/*isolation & purification MH - Rats MH - Rats, Sprague-Dawley MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Sepharose/*analogs & derivatives/pharmacology MH - Sequence Homology, Amino Acid MH - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MH - Spectrum Analysis, Mass MH - Time Factors EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E01-10-0094 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3344-54. PMID- 12221139 OWN - NLM STAT- MEDLINE DA - 20020910 DCOM- 20030312 LR - 20050319 PUBM- Print IS - 1059-1524 VI - 13 IP - 9 DP - 2002 Sep TI - Role of LAMP-2 in lysosome biogenesis and autophagy. PG - 3355-68 AB - In LAMP-2-deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2-deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2-deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation. AD - Centre for High Resolution Imaging and Processing, School of Life Sciences, University of Dundee, Scotland, UK. FAU - Eskelinen, Eeva-Liisa AU - Eskelinen EL FAU - Illert, Anna Lena AU - Illert AL FAU - Tanaka, Yoshitaka AU - Tanaka Y FAU - Schwarzmann, Gunter AU - Schwarzmann G FAU - Blanz, Judith AU - Blanz J FAU - Von Figura, Kurt AU - Von Figura K FAU - Saftig, Paul AU - Saftig P LA - eng PT - Journal Article PL - United States TA - Mol Biol Cell JID - 9201390 RN - 0 (Antigens, CD) RN - 0 (Lipids) RN - 0 (lysosome-associated membrane glycoproteins) RN - EC 3.4.23.5 (Cathepsin D) SB - IM MH - Animals MH - Antigens, CD/metabolism/*physiology MH - Blotting, Western MH - Cathepsin D/metabolism MH - Cell Membrane MH - Cells, Cultured MH - Endocytosis MH - Endosomes MH - Genotype MH - Hepatocytes/*metabolism MH - Immunohistochemistry MH - Lipids/metabolism MH - Lysosomes/*metabolism MH - Mice MH - Microscopy, Electron MH - Microscopy, Fluorescence MH - Phenotype MH - Precipitin Tests MH - Protein Binding MH - Research Support, Non-U.S. Gov't MH - Time Factors EDAT- 2002/09/11 10:00 MHDA- 2003/03/13 04:00 AID - 10.1091/mbc.E02-02-0114 [doi] PST - ppublish SO - Mol Biol Cell 2002 Sep;13(9):3355-68. PMID- 12223118 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - IkB kinase alpha: a link in the chain of the mammary cycle. PG - 173-5 AB - The transcription factor NF-kappaB exhibits altered activity in some breast cancers but the relevance of this association has not been established. Cao et al.'s elegant study recently published in Cell reveals a NF-kappaB-dependent signalling pathway responsible for epithelial proliferation in the mouse mammary gland. Could this mechanism, rather than prevention of apoptosis, be responsible for the reported association between NF-kappaB and breast cancer? Could the specificity of NF-kappaB modulators of the IkB kinase complex determine the fate of epithelial cells at different stages of mammary development? AD - Department of Pathology, University of Cambridge, UK. rwec2@mole.bio.ac.uk FAU - Clarkson, Richard AU - Clarkson R LA - eng PT - Editorial DEP - 20020705 PL - England TA - Breast Cancer Res JID - 100927353 RN - 0 (NF-kappa B) RN - EC 2.7.1.- (I kappa B kinase) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Animals MH - Apoptosis MH - Breast/*enzymology MH - Breast Neoplasms/metabolism/pathology MH - Cell Division MH - Epithelial Cells/enzymology MH - Female MH - Humans MH - NF-kappa B/*metabolism MH - Protein-Serine-Threonine Kinases/*metabolism MH - Signal Transduction EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/05/20 [received] PHST- 2002/06/17 [revised] PHST- 2002/06/18 [accepted] PHST- 2002/07/05 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):173-5. Epub 2002 Jul 5. PMID- 12223119 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - A new model for ductal carcinoma in situ suggests strategies for treatment. PG - 176-8 AB - Human ductal carcinoma in situ (DCIS) of the breast is now diagnosed quite frequently, due largely to the introduction of mammographic screening. It has been shown in a cell culture system that activation of c-erbB-2, but not the epidermal growth factor receptor, results in a DCIS-like phenotype. Since overexpression of c-erbB-2 occurs in 60% of DCIS, this suggests that it could be a target for treatment in this disease. AD - Department of Biosciences, University of Kent at Canterbury, UK. W.J.Gullick@ukc.ac.uk FAU - Gullick, William John AU - Gullick WJ LA - eng PT - Editorial PT - Review PT - Review, Tutorial DEP - 20020719 PL - England TA - Breast Cancer Res JID - 100927353 RN - 0 (Antineoplastic Agents) RN - 0 (Tumor Markers, Biological) RN - EC 2.7.1.112 (Receptor, Epidermal Growth Factor) RN - EC 2.7.1.112 (Receptor, erbB-2) SB - IM MH - Animals MH - Antineoplastic Agents/therapeutic use MH - Breast Neoplasms/drug therapy/*metabolism/pathology MH - Carcinoma, Intraductal, Noninfiltrating/drug therapy/*metabolism/pathology MH - Female MH - Humans MH - Mammography MH - Models, Biological MH - Receptor, Epidermal Growth Factor/metabolism MH - Receptor, erbB-2/antagonists & inhibitors/*metabolism MH - Tumor Markers, Biological/*metabolism RF - 15 EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/06/05 [received] PHST- 2002/07/04 [revised] PHST- 2002/07/08 [accepted] PHST- 2002/07/19 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):176-8. Epub 2002 Jul 19. PMID- 12223120 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - Familial risks of breast cancer. PG - 179-81 AB - A recent analysis by the Collaborative Group on Hormonal Factors in Breast Cancer has provided the most precise quantification to date of the familial risks of breast cancer. The familial relative risks are shown to decrease from more than fivefold in women younger than age 40 years with a first-degree relative aged younger than 40 years at diagnosis, to 1.4-fold in women older than 60 years with a relative diagnosed over age 60 years. These risks increase progressively with the number of affected relatives. The risks associated with an affected mother and an affected sister are similar, and the relative (but not absolute) risks are similar in subgroups defined by other established breast cancer risk factors. These results provide a useful basis for counselling of women with a family history of breast cancer, and they have implications for the genetic basis of the disease. AD - Cancer Research UK, Genetic Epidemiology Research Group, University of Cambridge, UK. douglas@srl.cam.ac.uk FAU - Easton, Douglas F AU - Easton DF LA - eng PT - Editorial DEP - 20020802 PL - England TA - Breast Cancer Res JID - 100927353 SB - IM MH - Adult MH - Breast Neoplasms/*genetics/prevention & control MH - Family Health MH - Female MH - Genes, BRCA1 MH - Genes, BRCA2 MH - *Genetic Predisposition to Disease MH - Humans MH - Incidence MH - Middle Aged MH - Probability MH - Risk Factors EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/04/26 [received] PHST- 2002/07/11 [revised] PHST- 2002/07/11 [accepted] PHST- 2002/08/02 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):179-81. Epub 2002 Aug 2. PMID- 12223121 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - Progesterone receptors - animal models and cell signaling in breast cancer: Role of steroid receptor coactivators and corepressors of progesterone receptors in breast cancer. PG - 182-6 AB - Progesterone, an ovarian steroid hormone, plays a key role in the development and function of the mammary gland, as it also does in the uterus and the ovary. The action of progesterone is mediated through its intracellular cognate receptor, the progesterone receptor (PR), which functions as a transcription factor that regulates gene expression. As with other nuclear receptors, coregulators (coactivators and corepressors) recruited by the liganded or unliganded PR, either to enhance or to suppress transcription activity, modulate the function of the PR. Mutation or aberrant expression of the coregulators might thus affect the normal function of the PR and hence disrupt the normal development of the mammary gland, which may lead to breast cancer. AD - Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA. FAU - Gao, Xiuhua AU - Gao X FAU - Nawaz, Zafar AU - Nawaz Z LA - eng GR - DK56833/DK/NIDDK PT - Journal Article PT - Review PT - Review, Tutorial DEP - 20020628 PL - England TA - Breast Cancer Res JID - 100927353 RN - 0 (Nuclear Proteins) RN - 0 (Receptors, Progesterone) RN - 0 (Repressor Proteins) RN - 0 (Trans-Activators) RN - 0 (nuclear receptor co-repressor) SB - IM MH - Animals MH - Breast Neoplasms/*metabolism/pathology MH - Cell Division MH - Disease Models, Animal MH - Female MH - Humans MH - Mice MH - Nuclear Proteins/metabolism MH - Receptor Cross-Talk/*physiology MH - Receptors, Progesterone/*physiology MH - Repressor Proteins/metabolism MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction MH - Trans-Activators/metabolism MH - Transcription, Genetic RF - 37 EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/04/15 [received] PHST- 2002/05/21 [revised] PHST- 2002/05/30 [accepted] PHST- 2002/06/28 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):182-6. Epub 2002 Jun 28. PMID- 12223122 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - Expression and transcriptional activity of progesterone receptor A and progesterone receptor B in mammalian cells. PG - 187-90 AB - Progesterone is an essential regulator of normal female reproductive function. Its effects are mediated by two nuclear progesterone receptor (PR) proteins, PRA and PRB, which are identical except for an additional 164 amino acids at the N-terminal end of PRB. Transcriptional analyses of the two receptor forms have assigned strikingly distinct functional signatures to the two PRs, despite their apparent physical similarity. The basis of these differences is yet to be fully understood. Furthermore, these differences are strongly influenced by the cell type and the promoter used. We review the mammalian transcriptional studies of PRA and PRB, and compare them with what is known about their expression and function in target tissues. AD - Westmead Institute for Cancer Research, University of Sydney Westmead Hospital, New South Wales, Australia. Dinny_Graham@wmi.usyd.edu.au FAU - Graham, J Dinny AU - Graham JD FAU - Clarke, Christine L AU - Clarke CL LA - eng PT - Journal Article PT - Review PT - Review, Tutorial DEP - 20020702 PL - England TA - Breast Cancer Res JID - 100927353 RN - 0 (Receptors, Progesterone) RN - 0 (Tumor Markers, Biological) RN - 0 (progesterone receptor A) RN - 0 (progesterone receptor B) SB - IM MH - Breast Neoplasms/*genetics/metabolism MH - Cell Transformation, Neoplastic/genetics/metabolism MH - Female MH - Humans MH - Receptors, Progesterone/*genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Signal Transduction MH - *Trans-Activation (Genetics) MH - *Transcription, Genetic MH - Tumor Markers, Biological/metabolism RF - 27 EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/05/28 [received] PHST- 2002/06/07 [accepted] PHST- 2002/07/02 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):187-90. Epub 2002 Jul 2. PMID- 12223123 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - Progesterone's role in mammary gland development and tumorigenesis as disclosed by experimental mouse genetics. PG - 191-6 AB - The progesterone receptor knockout mouse demonstrated progesterone's importance to parity-induced mammary tertiary branching and lobuloalveologenesis. Because early parity provides significant protection against breast cancer whereas prolonged exposure to premenopausal ovarian progesterone (or to postmenopausal supplementations thereof) has been linked to breast cancer risk, this steroid can be considered to exhibit contrasting roles in breast cancer etiology. This review describes the important mouse models that have contributed to our understanding of progesterone's role in mammary gland development and neoplasia. We conclude by emphasising the urgent need to identify the molecular targets of the progesterone receptor, and to determine whether these targets are modulated differently by the progesterone receptor isoforms (A and B) during mammary morphogenesis and tumorigenesis. AD - Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA. FAU - Soyal, Selma AU - Soyal S FAU - Ismail, Preeti M AU - Ismail PM FAU - Li, Jie AU - Li J FAU - Mulac-Jericevic, Biserka AU - Mulac-Jericevic B FAU - Conneely, Orla M AU - Conneely OM FAU - Lydon, John P AU - Lydon JP LA - eng GR - CA-77530/CA/NCI PT - Journal Article PT - Review PT - Review, Tutorial DEP - 20020705 PL - England TA - Breast Cancer Res JID - 100927353 RN - 0 (Receptors, Progesterone) RN - 57-83-0 (Progesterone) SB - IM MH - Animals MH - Cell Transformation, Neoplastic MH - Disease Progression MH - Female MH - Mammary Neoplasms, Experimental/*metabolism/*pathology MH - Mice MH - Mice, Knockout MH - Parity/physiology MH - Progesterone/*physiology MH - Receptors, Progesterone/deficiency/physiology MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. RF - 29 EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/04/17 [received] PHST- 2002/05/24 [revised] PHST- 2002/05/28 [accepted] PHST- 2002/07/05 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):191-6. Epub 2002 Jul 5. PMID- 12223124 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - The role of oestrogen and progesterone receptors in human mammary development and tumorigenesis. PG - 197-201 AB - A relatively small number of cells in the normal human mammary gland express receptors for oestrogen and progesterone (ER and PR), and there is almost complete dissociation between steroid receptor expression and proliferation. Increased expression of the ER alpha (ERalpha) and loss of the inverse relationship between receptor expression and proliferation occur at the very earliest stages of tumorigenesis, implying that dysregulation of ERalpha expression contributes to breast tumour formation. There is evidence also for alterations in the ratio between the two PR isoforms in premalignant breast lesions. Elucidation of the factors mediating the effects of oestradiol and progesterone on development of the normal breast and of the mechanisms by which expression of the ERalpha and the PR isoforms is controlled could identify new targets for breast cancer prevention and improved prediction of breast cancer risk. AD - Tumour Biochemistry Laboratory, Christie Hospital NHS Trust, Manchester, UK. eanderson@picr.man.ac.uk FAU - Anderson, Elizabeth AU - Anderson E LA - eng PT - Journal Article PT - Review PT - Review, Tutorial DEP - 20020724 PL - England TA - Breast Cancer Res JID - 100927353 RN - 0 (Receptors, Estrogen) RN - 0 (Receptors, Progesterone) SB - IM MH - Breast/*growth & development/metabolism MH - Breast Neoplasms/*metabolism/pathology MH - Epithelial Cells/metabolism MH - Female MH - Humans MH - Receptors, Estrogen/*physiology MH - Receptors, Progesterone/*physiology MH - Signal Transduction RF - 29 EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/05/17 [received] PHST- 2002/06/27 [revised] PHST- 2002/07/02 [accepted] PHST- 2002/07/24 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):197-201. Epub 2002 Jul 24. PMID- 12223125 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - British Cancer Research Meeting, 30 June-3 July 2002, Glasgow. PG - 202-4 AB - The 2002 British Cancer Research Meeting was held from 30th June to 3rd July in Glasgow, UK. The meeting was structured to include educational workshops, plenary lectures, symposia, and poster sessions, which brought together scientists and clinicians. Presentations ranged from the impact that modifications to basic chromatin structure can have on diagnosis and targeted gene therapy, to the outcome of novel therapeutics through clinical trials. The emphasis was clear: patient survival is the main priority and treatment of organ-specific cancer must inevitably be replaced by individualised tumour-specific therapy. AD - Molecular Medicine Unit, University of Leeds, UK. FAU - Parkes, Alicia T AU - Parkes AT FAU - Speirs, Valerie AU - Speirs V LA - eng PT - Congresses DEP - 20020729 PL - England TA - Breast Cancer Res JID - 100927353 RN - 0 (Antineoplastic Agents) SB - IM MH - Antineoplastic Agents/therapeutic use MH - Biomedical Research MH - Clinical Trials MH - England MH - Gene Therapy MH - Humans MH - Neoplasms/*diagnosis/*therapy EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/07/12 [received] PHST- 2002/07/23 [accepted] PHST- 2002/07/29 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):202-4. Epub 2002 Jul 29. PMID- 12223126 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - Tumour Fas ligand:Fas ratio greater than 1 is an independent marker of relative resistance to tamoxifen therapy in hormone receptor positive breast cancer. PG - R9 AB - BACKGROUND: The objective of the present study was to examine the prognostic and predictive significance of the apoptosis-related marker Fas ligand (FasL):Fas ratio in breast cancer. METHODS: Tumour biopsies from 215 primary invasive breast cancer patients were examined for the expression of FasL and Fas mRNA transcripts by quantitative real-time RT-PCR. Their prognostic and predictive impact on patient survival was determined in univariate and multivariate survival analyses. RESULTS: Using a cutoff value of 1, a FasL:Fas ratio greater than 1 was found to have significant prognostic value for disease-free survival among the total population (median follow up 54 months). It was associated with a significantly decreased disease-free survival (P = 0.022) and with a tendency toward increased mortality (P = 0.14) in univariate analysis. Hormone receptor positive women exclusively treated with tamoxifen (n = 86) and with a FasL:Fas ratio greater than 1 had a significantly decreased disease-free survival (P = 0.008) and overall survival (P = 0.03) in univariate Kaplan-Meier analysis. Furthermore, tumour size and FasL:Fas ratio were of independent predictive significance in the multivariate model for disease-free and overall survival in that subgroup. Among postmenopausal patients (n = 148) both of those factors retained independent prognostic significance in the multivariate model for disease-free survival. In contrast, FasL:Fas ratio had no significant predictive value in patients exclusively treated with chemotherapy. CONCLUSION: The data presented indicate that FasL:Fas ratio may be useful not only as a prognostic factor but also as a predictive factor for projecting response to the antioestrogen tamoxifen. The results strongly support a correlation between FasL:Fas ratio greater than 1 and lack of efficacy of tamoxifen in hormone receptor positive patients. AD - Department of Obstetrics & Gynaecology, University of Rostock, Faculty of Medicine, Germany. toralf.reimer@med.uni-rostock.de FAU - Reimer, Toralf AU - Reimer T FAU - Koczan, Dirk AU - Koczan D FAU - Muller, Heiner AU - Muller H FAU - Friese, Klaus AU - Friese K FAU - Thiesen, Hans-Jurgen AU - Thiesen HJ FAU - Gerber, Bernd AU - Gerber B LA - eng PT - Journal Article DEP - 20020621 PL - England TA - Breast Cancer Res JID - 100927353 RN - 0 (Antigens, CD95) RN - 0 (Estrogen Antagonists) RN - 0 (FasL protein) RN - 0 (Ligands) RN - 0 (Membrane Glycoproteins) RN - 0 (RNA, Messenger) RN - 0 (Receptors, Estrogen) RN - 0 (Receptors, Progesterone) RN - 0 (Tumor Markers, Biological) RN - 10540-29-1 (Tamoxifen) SB - IM MH - Antigens, CD95/*genetics/metabolism MH - Biopsy MH - Breast Neoplasms/drug therapy/*genetics/mortality MH - Disease-Free Survival MH - Drug Resistance, Neoplasm/*genetics MH - Estrogen Antagonists/*therapeutic use MH - Female MH - Humans MH - Ligands MH - Membrane Glycoproteins/*genetics/metabolism MH - Neoplasm Invasiveness/pathology MH - Neoplasms, Hormone-Dependent/drug therapy/*genetics/mortality MH - Prognosis MH - RNA, Messenger/metabolism MH - Receptors, Estrogen/metabolism MH - Receptors, Progesterone/metabolism MH - Reverse Transcriptase Polymerase Chain Reaction MH - Survival Rate MH - Tamoxifen/*therapeutic use MH - Tumor Markers, Biological/*genetics EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/04/14 [received] PHST- 2002/05/24 [revised] PHST- 2002/05/28 [accepted] PHST- 2002/06/21 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):R9. Epub 2002 Jun 21. PMID- 12223127 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - Risk factors for breast cancer in Iran: a case-control study. PG - R10 AB - BACKGROUND: Iranian breast cancer patients are relatively younger than their Western counterparts. The objective of the present study was to investigate risk factors for breast cancer in Iranian women. METHOD: A case-control study was conducted from April 1997 to April 1998 in Tehran, Iran. Demographical data and risk factor related information were collected using a short structured questionnaire. Odds ratios (ORs) and 95% confidence intervals (CIs) were derived from logistic regression analysis. RESULTS: In all, 286 women with breast cancer and 249 control women were interviewed. In multivariate analysis, only marital status (never married: OR 4.24, 95% CI 1.70-10.57 [P = 0.002]; widowed/divorced: OR 1.71, 95% CI 1.05-2.68 [P = 0.03]) and family history (positive family history of breast cancer: OR 2.95, 95% CI 1.15-7.59 [P = 0.02]) were associated with significantly increased risk for breast cancer. CONCLUSION: The findings of the present study suggest that family history and marital status may have an impact on the incidence of breast cancer in Iranian women. AD - Iranian Centre for Breast Cancer, Tehran, Iran. FAU - Ebrahimi, Mandana AU - Ebrahimi M FAU - Vahdaninia, Mariam AU - Vahdaninia M FAU - Montazeri, Ali AU - Montazeri A LA - eng PT - Journal Article DEP - 20020709 PL - England TA - Breast Cancer Res JID - 100927353 SB - IM MH - Age Distribution MH - Breast Neoplasms/*epidemiology/genetics MH - Case-Control Studies MH - Family MH - Female MH - Humans MH - Incidence MH - Iran/epidemiology MH - Marital Status MH - Odds Ratio MH - Questionnaires MH - Risk Factors EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/01/12 [received] PHST- 2002/05/07 [revised] PHST- 2002/06/20 [accepted] PHST- 2002/07/09 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):R10. Epub 2002 Jul 9. PMID- 12223128 OWN - NLM STAT- MEDLINE DA - 20020911 DCOM- 20030318 LR - 20041117 PUBM- Print-Electronic IS - 1465-5411 VI - 4 IP - 5 DP - 2002 TI - Impact of false-positive mammography on subsequent screening attendance and risk of cancer. PG - R11 AB - BACKGROUND: One area of concern within the largely successful UK National Health Service breast screening programme is the relatively high proportion of women showing mammographic abnormalities who undergo further diagnostic tests that prove negative. Previous studies suggest that, in addition to increasing anxiety, such false-positive mammography is associated with increased risk of subsequent interval cancer. In the present article, we quantify this increased risk, investigate whether it extends to cancers detected at rescreening, and determine whether cancers differ between women who have, and have not, experienced false-positive mammography. METHODS: This was a retrospective cohort study of 140,387 women aged 49-63 years routinely invited for first screening by the East Anglian National Health Service breast screening programme. Proportions reattending, and subsequent risk and pathological attributes of cancer were compared between women who underwent further (negative) assessment following false-positive mammography and women mammographically normal at first screen. RESULTS: At first screen, 108,617 (91.9%) of the screened women were mammographically normal, 4278 (3.6%) were assessed and then judged normal, and 514 (0.4%) underwent benign biopsy. Compared with nonassessed normal women, reattendance was lower among assessed women: 83.1% (95% confidence interval [CI], 82.0-84.1) versus 85.7% (95% CI, 85.5-85.9) (odds ratio [OR], 0.82; 95% CI, 0.76-0.89). Assessed women were at greater risk of interval cancer (rate per 1000 women screened, 9.6 [95% CI, 6.8-12.4] versus 3.0 [95% CI, 2.7-3.4]; OR, 3.19 [95% CI, 2.34-4.35]), and also of cancer detected at second screen (rate per 1000, 8.4 [95% CI, 5.8-10.9] versus 3.9 [95% CI, 3.5-4.3]; OR, 2.15 [95% CI, 1.55-2.98]). More cancers in assessed women measured >or = 20 mm (OR, 1.59; 95% CI, 0.99-2.55). CONCLUSIONS: Women undergoing false-positive mammography at first screen were less likely to reattend for subsequent screens than were nonassessed women, yet they were more likely to develop interval cancers or cancers at second screen, and their cancers were larger. Factors predisposing for false-positive mammography require investigation. Women should be encouraged to continue with screening. AD - Cancer Intelligence Unit, Strangeways Research Laboratory, Cambridge, UK. jenny.mccann@srl.cam.ac.uk FAU - McCann, Jenny AU - McCann J FAU - Stockton, Diane AU - Stockton D FAU - Godward, Sara AU - Godward S LA - eng PT - Journal Article DEP - 20020717 PL - England TA - Breast Cancer Res JID - 100927353 SB - IM MH - Breast/*pathology MH - Breast Neoplasms/pathology/prevention & control/*radiography MH - Cohort Studies MH - Comparative Study MH - False Positive Reactions MH - Female MH - Great Britain MH - Humans MH - *Mammography MH - Mass Screening/*methods MH - Middle Aged MH - *Patient Participation MH - Predictive Value of Tests MH - Retrospective Studies MH - Risk Factors MH - Time Factors EDAT- 2002/09/12 10:00 MHDA- 2003/03/19 04:00 PHST- 2002/03/28 [received] PHST- 2002/06/04 [revised] PHST- 2002/06/12 [accepted] PHST- 2002/07/17 [aheadofprint] PST - ppublish SO - Breast Cancer Res 2002;4(5):R11. Epub 2002 Jul 17. PMID- 12225617 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030423 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Sep 11 TI - Patient attitudes toward using computers to improve health services delivery. PG - 19 AB - BACKGROUND: The aim of this study was to examine the acceptability of point of care computerized prompts to improve health services delivery among a sample of primary care patients. METHODS: Primary data collection. Cross-sectional survey. Patients were surveyed after their visit with a primary care provider. Data were obtained from patients of ten community-based primary care practices in the spring of 2001. RESULTS: Almost all patients reported that they would support using a computer before each visit to prompt their doctor to: "do health screening tests" (92%), "counsel about health behaviors (like diet and exercise)" (92%) and "change treatments for health conditions" (86%). In multivariate testing, the only variable that was associated with acceptability of the point of care computerized prompts was patient's confidence in their ability to answer questions about their health using a computer (beta = 0.39, p =.001). Concerns about data security were expressed by 36.3% of subjects, but were not related to acceptability of the prompts. CONCLUSIONS: Support for using computers to generate point of care prompts to improve quality-oriented processes of care was high in our sample, but may be contingent on patients feeling familiar with their personal medical history. AD - Brown University School of Medicine Centers for Behavioral and Preventive Medicine at The Miriam Hospital, USA. csciamanna@lifespan.org FAU - Sciamanna, Christopher N AU - Sciamanna CN FAU - Diaz, Joseph AU - Diaz J FAU - Myne, Puja AU - Myne P LA - eng PT - Journal Article DEP - 20020911 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Adolescent MH - Adult MH - Aged MH - Aged, 80 and over MH - *Attitude to Computers MH - Cross-Sectional Studies MH - Female MH - Focus Groups MH - Health Care Surveys MH - Humans MH - Male MH - Middle Aged MH - Multivariate Analysis MH - Patient Acceptance of Health Care/*statistics & numerical data MH - Point-of-Care Systems/*utilization MH - Preventive Health Services/methods MH - Primary Health Care/*methods/organization & administration MH - *Process Assessment (Health Care) MH - Quality Assurance, Health Care/methods MH - Reminder Systems MH - Research Support, U.S. Gov't, P.H.S. MH - Rhode Island EDAT- 2002/09/13 10:00 MHDA- 2003/04/24 05:00 PHST- 2002/06/05 [received] PHST- 2002/09/11 [accepted] PHST- 2002/09/11 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Sep 11;2(1):19. PMID- 12226484 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Inositol phospholipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C. PG - 22-46 AB - Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells. They fulfill many important functions through interaction with a wide range of cellular proteins. Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol. The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C. Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development. In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs. The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom. AD - Universitat Potsdam, Institut fur Biochemie und Biologie, Abteilung Molekularbiologie, Karl-Liebknecht-Strasse 25, Haus 20, D-14476 Golm/Potsdam, Germany. FAU - Mueller-Roeber, Bernd AU - Mueller-Roeber B FAU - Pical, Christophe AU - Pical C LA - eng PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Isoenzymes) RN - 0 (Phosphatidylinositols) RN - EC 2.7.1 (Phosphotransferases (Alcohol Group Acceptor)) RN - EC 2.7.1.137 (1-Phosphatidylinositol 3-Kinase) RN - EC 2.7.1.67 (1-Phosphatidylinositol 4-Kinase) RN - EC 3.1.4.3 (Phospholipase C) RN - EC 4.6.1.13 (Phosphatidylinositol Diacylglycerol-Lyase) SB - IM MH - 1-Phosphatidylinositol 3-Kinase/genetics/metabolism MH - 1-Phosphatidylinositol 4-Kinase/genetics/metabolism MH - Amino Acid Sequence MH - Animals MH - Arabidopsis/enzymology/genetics/*metabolism MH - Comparative Study MH - Humans MH - Isoenzymes/genetics/metabolism MH - Molecular Sequence Data MH - Phosphatidylinositol Diacylglycerol-Lyase MH - Phosphatidylinositols/*metabolism MH - Phospholipase C/genetics/*metabolism MH - Phosphotransferases (Alcohol Group Acceptor)/genetics/*metabolism MH - Phylogeny MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid MH - Yeasts/enzymology/genetics RF - 144 EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004770 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):22-46. PMID- 12226485 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Cell-specific expression of homospermidine synthase, the entry enzyme of the pyrrolizidine alkaloid pathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase. PG - 47-57 AB - Pyrrolizidine alkaloids (PAs) are constitutive plant defense compounds with a sporadic taxonomic occurrence. The first committed step in PA biosynthesis is catalyzed by homospermidine synthase (HSS). Recent evidence confirmed that HSS evolved by gene duplication from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of the eukaryotic translation initiation factor 5A. To better understand the evolutionary relationship between these two enzymes, which are involved in completely different biological processes, we studied their tissue-specific expression. RNA-blot analysis, reverse transcriptase-PCR, and immunolocalization techniques demonstrated that DHS is constitutively expressed in shoots and roots of Senecio vernalis (Asteraceae), whereas HSS expression is root specific and restricted to distinct groups of endodermis and neighboring cortex cells located opposite to the phloem. All efforts to detect DHS by immunolocalization failed, but studies with promoter-beta-glucuronidase fusions confirmed a general expression pattern, at least in young seedlings of tobacco (Nicotiana tabacum). The expression pattern for HSS differs completely from its ancestor DHS due to the adaptation of HSS to the specific requirements of PA biosynthesis. AD - Institut fur Pharmazeutische Biologie der Technischen Universitat, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany. FAU - Moll, Stefanie AU - Moll S FAU - Anke, Sven AU - Anke S FAU - Kahmann, Uwe AU - Kahmann U FAU - Hansch, Robert AU - Hansch R FAU - Hartmann, Thomas AU - Hartmann T FAU - Ober, Dietrich AU - Ober D LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Pyrrolizidine Alkaloids) RN - 0 (Recombinant Fusion Proteins) RN - EC 1.5. (Amine Oxidoreductases) RN - EC 1.5.1.- (deoxyhypusine synthase) RN - EC 2.5 (Alkyl and Aryl Transferases) RN - EC 2.5.1.- (homospermidine synthetase) RN - EC 3.2.1.31 (Glucuronidase) SB - IM MH - Alkyl and Aryl Transferases/genetics/*metabolism MH - Amine Oxidoreductases/genetics/*metabolism MH - Comparative Study MH - Gene Expression Regulation, Enzymologic MH - Gene Expression Regulation, Plant MH - Glucuronidase/genetics/metabolism MH - Microscopy, Confocal MH - Microscopy, Immunoelectron MH - Microscopy, Ultraviolet MH - Plant Roots/enzymology/ultrastructure MH - Plants, Genetically Modified MH - Pyrrolizidine Alkaloids/*metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Senecio/*enzymology/genetics/ultrastructure MH - Substrate Specificity MH - Tobacco/enzymology/genetics EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004259 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):47-57. PMID- 12226486 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Differential expression of a metallothionein gene during the presymbiotic versus the symbiotic phase of an arbuscular mycorrhizal fungus. PG - 58-67 AB - A full-length cDNA encoding a metallothionein (MT)-like polypeptide, designated GmarMT1, was identified in an expressed sequence tag collection from germinated spores of the arbuscular mycorrhizal fungus Gigaspora margarita (BEG34). The GmarMT1 gene is composed of two exons separated by an 81-bp intron. It codes for a 65-amino acid polypeptide comprising a plant type 1 MT-like N-terminal domain and a C-terminal domain that is most closely related to an as-yet-uncharacterized fungal MT. As revealed by heterologous complementation assays in yeast, GmarMT1 encodes a functional polypeptide capable of conferring increased tolerance against Cd and Cu. The GmarMT1 RNA is expressed in both presymbiotic spores and symbiotic mycelia, even in the absence of metal exposure, but is significantly less abundant in the latter stage. An opposite pattern was observed upon Cu exposure, which up-regulated GmarMT1 expression in symbiotic mycelia but not in germinated spores. Together, these data provide the first evidence, to our knowledge, for the occurrence in an arbuscular mycorrhizal fungus of a structurally novel MT that is modulated in a metal and life cycle stage-dependent manner and may afford protection against heavy metals (and other types of stress) to both partners of the endomycorrhizal symbiosis. AD - Dipartimento di Biologia Vegetale, Universita di Torino and Istituto per la Protezione delle Piante-Sezione di Torino, Consiglio Nazionale delle Ricerche, Viale Mattioli 25, 10125 Torino, Italy. FAU - Lanfranco, Luisa AU - Lanfranco L FAU - Bolchi, Angelo AU - Bolchi A FAU - Ros, Emanuele Cesale AU - Ros EC FAU - Ottonello, Simone AU - Ottonello S FAU - Bonfante, Paola AU - Bonfante P LA - eng SI - GENBANK/AJ421527 PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Fungal Proteins) RN - 0 (RNA, Messenger) RN - 7440-43-9 (Cadmium) RN - 7440-50-8 (Copper) RN - 9038-94-2 (Metallothionein) SB - IM MH - Adaptation, Physiological/genetics/physiology MH - Amino Acid Sequence MH - Base Sequence MH - Cadmium/pharmacology MH - Cloning, Molecular MH - Copper/pharmacology MH - Fungal Proteins/*genetics/metabolism MH - Gene Expression Regulation, Fungal/drug effects MH - Genetic Complementation Test MH - Metallothionein/*genetics/metabolism MH - Molecular Sequence Data MH - Mycelium/drug effects/genetics/growth & development MH - RNA, Messenger/drug effects/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid MH - Spores, Fungal/drug effects/genetics/growth & development MH - *Symbiosis/genetics MH - Zygomycota/drug effects/*genetics/growth & development EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.003525 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):58-67. PMID- 12226487 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - VFL, the grapevine FLORICAULA/LEAFY ortholog, is expressed in meristematic regions independently of their fate. PG - 68-77 AB - The flowering process in grapevine (Vitis vinifera) takes place in buds and extends for two consecutive growing seasons. To understand the genetic and molecular mechanisms underlying this process, we have characterized grapevine bud development, cloned the grapevine FLORICAULA/LEAFY (FLO/LFY) ortholog, VFL, and analyzed its expression patterns during vegetative and reproductive development. Flowering induction takes place during the first season. Upon induction, the shoot apical meristem begins to produce lateral meristems that will give rise to either inflorescences or tendrils. During the second season, after a winter dormancy period, buds reactivate and inflorescence meristems give rise to flower meristems. VFL is expressed in lateral meristems that give rise to inflorescence and flower meristems, consistent with a role in reproductive development. Furthermore, VFL is also detected in other meristematic regions such as the vegetative shoot apical meristem and the lateral meristems that will give rise to tendrils. VFL is also expressed in leaf primordia and in growing leaf margins until later stages of development. Accumulation of VFL transcripts in cell-proliferating regions suggests a role for VFL not only in flower meristem specification, but also in the maintenance of indeterminacy before the differentiation of derivatives of the apical meristem: flowers, leaves, or tendrils. AD - Departmento de Biotecnologia, Escuela Tecnics Suoerior Ingenieros Agronomos, Universidad Politecnica de Madrid, Spain. FAU - Carmona, Maria Jose AU - Carmona MJ FAU - Cubas, Pilar AU - Cubas P FAU - Martinez-Zapater, Jose M AU - Martinez-Zapater JM LA - eng SI - GENBANK/AF450278 PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Plant Proteins) SB - IM MH - Amino Acid Sequence MH - Cloning, Molecular MH - Gene Expression Regulation, Developmental MH - Gene Expression Regulation, Plant MH - In Situ Hybridization MH - Meristem/*genetics/growth & development/metabolism MH - Molecular Sequence Data MH - Phylogeny MH - Plant Proteins/*genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid MH - Vitis/*genetics/growth & development/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.002428 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):68-77. PMID- 12226488 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Effect of regulated overexpression of the MADS domain factor AGL15 on flower senescence and fruit maturation. PG - 78-89 AB - We have examined the effect of regulated overexpression of AGL15, a member of the MADS domain family of regulatory factors, on reproductive tissues. Using molecular and physiological markers, we show that constitutive overexpression of AGL15 in Arabidopsis leads to delay and down-regulation of senescence programs in perianth organs and developing fruits and alters the process of seed desiccation. Through genetic crosses, we show that the rate of water loss in the maturing seeds is dictated by the genetic composition and physiological state of the maternal tissue, rather than the embryo. To define the developmental time and/or place when senescence programs are most affected by elevated AGL15 levels, we expressed AGL15 under the control of various promoters. Expression during senescence or in abscission zone cells did not produce delays in floral organ senescence or abscission. Using a glucocorticoid-inducible expression system, we show that an increase in AGL15 levels around the time of flower opening is necessary to delay senescence and increase floral organ longevity. AD - Department of Botany, University of Wisconsin, 430 Lincoln Drive, Madison, Wisconsin 53706-1381, USA. FAU - Fang, Su-Chiung AU - Fang SC FAU - Fernandez, Donna E AU - Fernandez DE LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (AGAMOUS Protein, Arabidopsis) SB - IM MH - AGAMOUS Protein, Arabidopsis/genetics/*metabolism MH - Arabidopsis/genetics/*growth & development/metabolism MH - Cell Wall/genetics/physiology MH - Desiccation MH - Fruit/genetics/growth & development/metabolism MH - Gene Expression Regulation, Developmental MH - Gene Expression Regulation, Plant MH - Immunohistochemistry MH - Plant Stems/genetics/growth & development/metabolism MH - Plants, Genetically Modified MH - Research Support, Non-U.S. Gov't MH - Seeds/genetics/growth & development/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004721 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):78-89. PMID- 12226489 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041215 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Molecular identification of cytosolic, patatin-related phospholipases A from Arabidopsis with potential functions in plant signal transduction. PG - 90-101 AB - Rapid activation of phospholipase A (PLA) by auxin or plant-pathogen interaction suggests a function in signal transduction for this enzyme, but the molecular identification of a cytosolic PLA carrying out this function remains open. We isolated four cDNA sequences from Arabidopsis (ecotype Columbia), AtPLA I, AtPLA IIA, AtPLA IVA, and AtPLA IVC, which are members of the patatin-related PLA gene family in plants and which are homologous to the animal Ca(2+)-independent PLA(2) gene family. Expression was measured by reverse transcriptase-polymerase chain reaction, and AtPLA I transcripts were found preferentially in shoots, AtPLA IIA and AtPLA IVA in roots, and AtPLA IVC in flowers. Transient expression of the four PLA-green fluorescent protein fusion proteins in tobacco (Nicotiana tabacum) leaves showed they were located in the cytosol and not in the vacuoles. Surprisingly, AtPLA::green fluorescent protein was also localized to chloroplasts. The enzymatic activity of the purified recombinant AtPLA IVA toward phosphatidylcholine was dependent on Ca(2+), saturated at 0.5 mM, and had a pH optimum of about 7.0. It had both PLA(1) and PLA(2) specificity. The enzyme showed in vitro highest sensitivity toward the PLA(2) inhibitors palmitoyltrifluoromethyl ketone (PACOCF(3), K(i) approximately 30 nM), arachidonyltrifluoromethyl ketone (AACOCF(3), K(i) approximately 25 microM), and tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (K(i) approximately 200 nM) and was also sensitive to other previously used inhibitors 5,8,11,14-eicosatetraynoic acid (K(i) approximately 3 microM) and nordihydroguajaretic acid (K(i) approximately 15 microM). The influence of these PLA(2) inhibitors on elongation in etiolated Arabidopsis seedlings was tested, and tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one and 5,8,11,14-eicosatetraynoic acid inhibited hypocotyl elongation maximally at concentrations close to their K(i) in vitro. AD - Universitat Hannover, Institut fur Zierpflanzenbau, Baumschule und Pflanzenzuchtung, Herrenhauser Strasse 2, D-30419 Hannover. holk@zier.uni-hannover.de FAU - Holk, Andre AU - Holk A FAU - Rietz, Steffen AU - Rietz S FAU - Zahn, Marc AU - Zahn M FAU - Quader, Hartmut AU - Quader H FAU - Scherer, Gunther F E AU - Scherer GF LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Arabidopsis Proteins) RN - 0 (DNA, Complementary) RN - 0 (Enzyme Inhibitors) RN - 0 (Luminescent Proteins) RN - 0 (Plant Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (patatin protein, Solanum tuberosum) RN - 147336-22-9 (Green Fluorescent Proteins) RN - 7440-70-2 (Calcium) RN - EC 3.1.- (Phospholipases) RN - EC 3.1.- (phospholipase A I, Arabidopsis) RN - EC 3.1.- (phospholipase IIA, Arabidopsis) RN - EC 3.1.- (phospholipase IVA, Arabidopsis) RN - EC 3.1.- (phospholipase IVC, Arabidopsis) RN - EC 3.1.1 (Carboxylic Ester Hydrolases) RN - EC 3.1.1.- (Phospholipases A) SB - IM MH - Amino Acid Sequence MH - Arabidopsis/enzymology/*genetics MH - Arabidopsis Proteins/drug effects/*genetics/metabolism MH - Calcium/pharmacology MH - Carboxylic Ester Hydrolases/genetics/metabolism MH - Cytosol/enzymology MH - DNA, Complementary/chemistry/genetics MH - Enzyme Inhibitors/pharmacology MH - Gene Expression Regulation, Enzymologic MH - Gene Expression Regulation, Plant MH - Green Fluorescent Proteins MH - Hydrogen-Ion Concentration MH - Hypocotyl/drug effects/growth & development MH - Luminescent Proteins/genetics/metabolism MH - Molecular Sequence Data MH - Phospholipases/*genetics/metabolism MH - Phospholipases A/antagonists & inhibitors/*genetics/metabolism MH - Phylogeny MH - Plant Leaves/genetics/metabolism MH - Plant Proteins/genetics/metabolism MH - Plant Roots/genetics/metabolism MH - Plant Stems/genetics/metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - Sequence Homology, Amino Acid MH - Signal Transduction/genetics/*physiology MH - Substrate Specificity MH - Tobacco/genetics EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.006288 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):90-101. PMID- 12226490 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - The circadian clock that controls gene expression in Arabidopsis is tissue specific. PG - 102-10 AB - The expression of CHALCONE SYNTHASE (CHS) expression is an important control step in the biosynthesis of flavonoids, which are major photoprotectants in plants. CHS transcription is regulated by endogenous programs and in response to environmental signals. Luciferase reporter gene fusions showed that the CHS promoter is controlled by the circadian clock both in roots and in aerial organs of transgenic Arabidopsis plants. The period of rhythmic CHS expression differs from the previously described rhythm of chlorophyll a/b-binding protein (CAB) gene expression, indicating that CHS is controlled by a distinct circadian clock. The difference in period is maintained in the wild-type Arabidopsis accessions tested and in the de-etiolated 1 and timing of CAB expression 1 mutants. These clock-affecting mutations alter the rhythms of both CAB and CHS markers, indicating that a similar (if not identical) circadian clock mechanism controls these rhythms. The distinct tissue distribution of CAB and CHS expression suggests that the properties of the circadian clock differ among plant tissues. Several animal organs also exhibit heterogeneous circadian properties in culture but are believed to be synchronized in vivo. The fact that differing periods are manifest in intact plants supports our proposal that spatially separated copies of the plant circadian clock are at most weakly coupled, if not functionally independent. This autonomy has apparently permitted tissue-specific specialization of circadian timing. AD - Department of Biological Sciences, University of Warwick, Coventry, United Kingdom. FAU - Thain, Simon C AU - Thain SC FAU - Murtas, Giovanni AU - Murtas G FAU - Lynn, James R AU - Lynn JR FAU - McGrath, Robert B AU - McGrath RB FAU - Millar, Andrew J AU - Millar AJ LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Arabidopsis Proteins) RN - 0 (CAB2 protein, Arabidopsis) RN - 0 (Carrier Proteins) RN - 0 (Recombinant Fusion Proteins) RN - EC 1.13.12.- (Luciferases) RN - EC 2.3. (Acyltransferases) RN - EC 2.3.1.74 (flavanone synthetase) SB - IM MH - Acyltransferases/genetics/metabolism MH - Arabidopsis/genetics/*physiology MH - *Arabidopsis Proteins MH - Biological Transport MH - Carrier Proteins/genetics/metabolism MH - Circadian Rhythm/*physiology MH - Comparative Study MH - Gene Expression Regulation, Plant MH - Luciferases/genetics/metabolism MH - Mutation MH - Plant Leaves/enzymology/genetics MH - Plant Roots/enzymology/genetics MH - Plants, Genetically Modified MH - Promoter Regions (Genetics) MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005405 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):102-10. PMID- 12226491 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - A role for the DOF transcription factor BPBF in the regulation of gibberellin-responsive genes in barley aleurone. PG - 111-9 AB - Functional analyses of a number of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells following germination, have identified a GA-responsive complex as a tripartite element containing a pyrimidine box motif 5'-CCTTTT-3'. We describe here that BPBF, a barley (Hordeum vulgare) transcription factor of the DOF (DNA-Binding with One Finger) class, previously shown to be an activator of reserve protein encoding genes during development, also has a role in the control of hydrolase genes following seed germination. Northern-blot, reverse transcriptase-polymerase chain reaction, and in situ hybridization analyses evidenced that the transcripts of the BPBF-encoding gene (Pbf), besides being present during endosperm development, are also expressed in aleurone cells of germinated seeds where they are induced by GA, an effect counteracted by abscisic acid. Electrophoretic mobility shift assays have shown that the BPBF protein binds specifically to the pyrimidine box motif in vitro within the different sequence contexts that naturally occur in the promoters of genes encoding a cathepsin B-like protease (Al21) and a low-isoelectric point alpha-amylase (Amy2/32b), both induced in the aleurone layers in response to GA. In transient expression experiments, BPBF repressed transcription of the Al21 promoter in GA-treated barley aleurone layers and reverted the GAMYB-mediated activation of this protease promoter. AD - Laboratorio de Bioquimica y Biologia Molecular, Departmento de Biotecnologia-Universidad Politecnica de Madrid, Spain. FAU - Mena, Montana AU - Mena M FAU - Cejudo, Francisco Javier AU - Cejudo FJ FAU - Isabel-Lamoneda, Ines AU - Isabel-Lamoneda I FAU - Carbonero, Pilar AU - Carbonero P LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (DNA-Binding Proteins) RN - 0 (Gibberellins) RN - 0 (PBF protein, plant) RN - 0 (Plant Growth Regulators) RN - 0 (Plant Proteins) RN - 0 (Proto-Oncogene Proteins c-myb) RN - 0 (Transcription Factors) RN - 137950-93-7 (Myb protein, plant) RN - 21293-29-8 (Abscisic Acid) RN - 77-06-5 (gibberellic acid) RN - EC 3. (Hydrolases) RN - EC 3.4.22 (Cysteine Endopeptidases) SB - IM MH - Abscisic Acid/pharmacology MH - Cysteine Endopeptidases/genetics/metabolism MH - DNA-Binding Proteins/genetics/*physiology MH - Gene Expression Regulation, Developmental/drug effects MH - Gene Expression Regulation, Plant/drug effects MH - Germination/drug effects/physiology MH - Gibberellins/*pharmacology MH - Hordeum/drug effects/*genetics/growth & development MH - Hydrolases/genetics/metabolism MH - In Situ Hybridization MH - Plant Growth Regulators/pharmacology MH - Plant Proteins/*genetics/metabolism MH - Promoter Regions (Genetics)/genetics MH - Protein Binding MH - Proto-Oncogene Proteins c-myb/*genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Seeds/drug effects/genetics/growth & development MH - Transcription Factors/genetics/*physiology EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005561 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):111-9. PMID- 12226492 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - A strobilurin fungicide enhances the resistance of tobacco against tobacco mosaic virus and Pseudomonas syringae pv tabaci. PG - 120-7 AB - The strobilurin class of fungicides comprises a variety of synthetic plant-protecting compounds with broad-spectrum antifungal activity. In the present study, we demonstrate that a strobilurin fungicide, F 500 (Pyraclostrobin), enhances the resistance of tobacco (Nicotiana tabacum cv Xanthi nc) against infection by either tobacco mosaic virus (TMV) or the wildfire pathogen Pseudomonas syringae pv tabaci. F 500 was also active at enhancing TMV resistance in NahG transgenic tobacco plants unable to accumulate significant amounts of the endogenous inducer of enhanced disease resistance, salicylic acid (SA). This finding suggests that F 500 enhances TMV resistance in tobacco either by acting downstream of SA in the SA signaling mechanism or by functioning independently of SA. The latter assumption is the more likely because in infiltrated leaves, F 500 did not cause the accumulation of SA-inducible pathogenesis-related (PR)-1 proteins that often are used as conventional molecular markers for SA-induced disease resistance. However, accumulation of PR-1 proteins and the associated activation of the PR-1 genes were elicited upon TMV infection of tobacco leaves and both these responses were induced more rapidly in F 500-pretreated plants than in the water-pretreated controls. Taken together, our results suggest that F 500, in addition to exerting direct antifungal activity, may also protect plants by priming them for potentiated activation of subsequently pathogen-induced cellular defense responses. AD - Department of Biology, University of Kaiserslautern, P.O. Box 3049, D-67653 Kaiserslautern, Germany. FAU - Herms, Stefan AU - Herms S FAU - Seehaus, Kai AU - Seehaus K FAU - Koehle, Harald AU - Koehle H FAU - Conrath, Uwe AU - Conrath U LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Antibiotics, Antifungal) RN - 0 (Fatty Acids, Unsaturated) RN - 0 (Plant Proteins) RN - 0 (pathogenesis-related proteins, plant) RN - 52110-55-1 (mucidin) RN - 69-72-7 (Salicylic Acid) SB - IM MH - Antibiotics, Antifungal/*pharmacology MH - Fatty Acids, Unsaturated/*pharmacology MH - Immunity, Natural/drug effects MH - Microbial Sensitivity Tests MH - Plant Diseases/microbiology/virology MH - Plant Leaves/drug effects/microbiology/virology MH - Plant Proteins/biosynthesis MH - Pseudomonas/*drug effects/growth & development MH - Research Support, Non-U.S. Gov't MH - Salicylic Acid/metabolism MH - Signal Transduction/drug effects MH - Tobacco/microbiology/*physiology/virology MH - Tobacco Mosaic Virus/*drug effects/growth & development EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004432 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):120-7. PMID- 12226493 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - An endoplasmic reticulum-bound Ca(2+)/Mn(2+) pump, ECA1, supports plant growth and confers tolerance to Mn(2+) stress. PG - 128-37 AB - Plants can grow in soils containing highly variable amounts of mineral nutrients, like Ca(2+) and Mn(2+), though the mechanisms of adaptation are poorly understood. Here, we report the first genetic study to determine in vivo functions of a Ca(2+) pump in plants. Homozygous mutants of Arabidopsis harboring a T-DNA disruption in ECA1 showed a 4-fold reduction in endoplasmic reticulum-type calcium pump activity. Surprisingly, the phenotype of mutant plants was indistinguishable from wild type when grown on standard nutrient medium containing 1.5 mM Ca(2+) and 50 microM Mn(2+). However, mutants grew poorly on medium with low Ca(2+) (0.2 mM) or high Mn(2+) (0.5 mM). On high Mn(2+), the mutants failed to elongate their root hairs, suggesting impairment in tip growth processes. Expression of the wild-type gene (CAMV35S::ECA1) reversed these conditional phenotypes. The activity of ECA1 was examined by expression in a yeast (Saccharomyces cerevisiae) mutant, K616, which harbors a deletion of its endogenous calcium pumps. In vitro assays demonstrated that Ca(2+), Mn(2+), and Zn(2+) stimulated formation of a phosphoenzyme intermediate, consistent with the translocation of these ions by the pump. ECA1 provided increased tolerance of yeast mutant to toxic levels of Mn(2+) (1 mM) and Zn(2+)(3 mM), consistent with removal of these ions from the cytoplasm. These results show that despite the potential redundancy of multiple Ca(2+) pumps and Ca(2+)/H(+) antiporters in Arabidopsis, pumping of Ca(2+) and Mn(2+) by ECA1 into the endoplasmic reticulum is required to support plant growth under conditions of Ca(2+) deficiency or Mn(2+) toxicity. AD - Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA. FAU - Wu, Zhongyi AU - Wu Z FAU - Liang, Feng AU - Liang F FAU - Hong, Bimei AU - Hong B FAU - Young, Jeff C AU - Young JC FAU - Sussman, Michael R AU - Sussman MR FAU - Harper, Jeffrey F AU - Harper JF FAU - Sze, Heven AU - Sze H LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Carrier Proteins) RN - 0 (Plant Proteins) RN - 7439-96-5 (Manganese) RN - 7440-66-6 (Zinc) RN - 7440-70-2 (Calcium) RN - EC 3.6.1.38 (Ca(2+)-Transporting ATPase) SB - IM SB - S MH - Adaptation, Physiological/*drug effects MH - Amino Acid Sequence MH - Arabidopsis/drug effects/genetics/*growth & development MH - Base Sequence MH - Ca(2+)-Transporting ATPase/antagonists & inhibitors/genetics/*metabolism MH - Calcium/*metabolism/pharmacology MH - Carrier Proteins/metabolism MH - Comparative Study MH - Endoplasmic Reticulum/drug effects/enzymology/*metabolism MH - Gene Expression Regulation, Enzymologic MH - Gene Expression Regulation, Plant MH - Genetic Complementation Test MH - Manganese/*metabolism/pharmacology MH - Molecular Sequence Data MH - Mutation MH - Plant Proteins/genetics/metabolism MH - Plant Roots/drug effects/growth & development MH - Plants, Genetically Modified MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Saccharomyces cerevisiae/genetics MH - Zinc/pharmacology OTO - NASA OT - Non-programmatic EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004440 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):128-37. PMID- 12226494 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Movement of potato spindle tuber viroid reveals regulatory points of phloem-mediated RNA traffic. PG - 138-46 AB - Increasing evidence indicates that the phloem mediates traffic of selective RNAs within a plant. How an RNA enters, moves in, and exits the phloem is poorly understood. Potato spindle tuber viroid (PSTVd) is a pathogenic RNA that does not encode proteins and is not encapsidated, and yet it replicates autonomously and traffics systemically within an infected plant. The viroid RNA genome must interact directly with cellular factors to accomplish these functions and is, therefore, an excellent probe to study mechanisms that regulate RNA traffic. Our analyses of PSTVd traffic in Nicotiana benthamiana yielded evidence that PSTVd movement within sieve tubes does not simply follow mass flow from source to sink organs. Rather, this RNA is transported into selective sink organs. Furthermore, two PSTVd mutants can enter the phloem to spread systemically but cannot exit the phloem in systemic leaves of tobacco (Nicotiana tabacum). A viroid most likely has evolved structural motifs that mimic endogenous plant RNA motifs so that they are recognized by cellular factors for traffic. Thus, analysis of PSTVd traffic functions may provide insights about endogenous mechanisms that control phloem entry, transport, and exit of RNAs. AD - Department of Plant Biology and Plant Biotechnology Center, Ohio State University, Columbus, Ohio 43210, USA. FAU - Zhu, Yali AU - Zhu Y FAU - Qi, Yijun AU - Qi Y FAU - Xun, Yan AU - Xun Y FAU - Owens, Robert AU - Owens R FAU - Ding, Biao AU - Ding B LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (RNA, Plant) RN - 0 (RNA, Viral) SB - IM MH - Biological Transport/physiology MH - Cloning, Molecular MH - Gene Expression Regulation, Plant MH - Gene Expression Regulation, Viral MH - In Situ Hybridization MH - Plant Diseases/virology MH - Plant Leaves/metabolism/virology MH - Plant Stems/metabolism/virology MH - Plant Viruses/genetics/*physiology MH - Plants, Genetically Modified MH - Point Mutation MH - Potatoes/virology MH - RNA, Plant/*metabolism MH - RNA, Viral/metabolism MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Tobacco/genetics/*metabolism/virology MH - Viroids/genetics/*physiology MH - Virus Replication EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.006403 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):138-46. PMID- 12226495 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - The bifunctional LKR/SDH locus of plants also encodes a highly active monofunctional lysine-ketoglutarate reductase using a polyadenylation signal located within an intron. PG - 147-54 AB - Both plants and animals catabolize lysine (Lys) via two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide encoded by a single LKR/SDH gene. We have previously shown that the Arabidopsis LKR/SDH gene also encodes a monofunctional SDH that is transcribed from an internal promoter. In the present report, we have identified two cDNAs derived from cotton (Gossypium hirsutum) boll abscission zone that encode a novel enzymatic form of Lys catabolism, i.e. a catabolic monofunctional LKR. The monofunctional LKR mRNA is also encoded by the LKR/SDH gene, using two weak polyadenylation sites located within an intron. In situ mRNA hybridization and quantitative reverse transcriptase-polymerase chain reaction analyses also suggest that the cotton monofunctional LKR is relatively abundantly expressed in parenchyma cells of the abscission zone. DNA sequence analysis of the LKR/SDH genes of Arabidopsis, maize (Zea mays), and tomato (Lycopersicon esculentum) suggests that these genes can also encode a monofunctional LKR mRNA by a similar mechanism. To test whether the LKR/SDH and monofunctional LKR enzymes possess different biochemical properties, we used recombinant Arabidopsis LKR/SDH and monofunctional LKR enzymes expressed in yeast (Saccharomyces cerevisiae) cells. The K(m) of the monofunctional LKR to Lys was nearly 10-fold lower than its counterpart that is linked to SDH. Taken together, our results suggest that the LKR/SDH locus of plants is a super-composite locus that can encode three related but distinct enzymes of Lys catabolism. These three enzymes apparently operate in concert to finely regulate Lys catabolism during plant development. AD - Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100 Israel. FAU - Tang, Guiliang AU - Tang G FAU - Zhu, Xiaohong AU - Zhu X FAU - Gakiere, Bertrand AU - Gakiere B FAU - Levanony, Hanna AU - Levanony H FAU - Kahana, Anat AU - Kahana A FAU - Galili, Gad AU - Galili G LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (RNA, Messenger) RN - 56-87-1 (Lysine) RN - EC 1.5.1.- (Saccharopine Dehydrogenases) SB - IM MH - Arabidopsis/enzymology/genetics MH - Base Sequence MH - Gossypium/*enzymology/genetics MH - In Situ Hybridization MH - Introns/*genetics MH - Lycopersicon esculentum/enzymology/genetics MH - Lysine/metabolism MH - Molecular Sequence Data MH - Polyadenylation/*genetics MH - RNA, Messenger/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Saccharopine Dehydrogenases/*genetics MH - Sequence Homology, Nucleic Acid MH - Signal Transduction/genetics MH - Zea mays/enzymology/genetics EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005660 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):147-54. PMID- 12226496 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Elongated mesocotyl1, a phytochrome-deficient mutant of maize. PG - 155-63 AB - To begin the functional dissection of light signal transduction pathways of maize (Zea mays), we have identified and characterized the light-sensing mutant elm1 (elongated mesocotyl1). Seedlings homozygous for elm1 are pale green, show pronounced elongation of the mesocotyl, and fail to de-etiolate under red or far-red light. Etiolated elm1 mutants contain no spectrally active phytochrome and do not deplete levels of phytochrome A after red-light treatment. High-performance liquid chromatography analyses show that elm1 mutants are unable to convert biliverdin IX alpha to 3Z-phytochromobilin, preventing synthesis of the phytochrome chromophore. Despite the impairment of the phytochrome photoreceptors, elm1 mutants can be grown to maturity in the field. Mature plants retain aspects of the seedling phenotype and flower earlier than wild-type plants under long days. Thus, the elm1 mutant of maize provides the first direct evidence for phytochrome-mediated modulation of flowering time in this agronomically important species. AD - Boyce Thompson Institute, Cornell University, Tower Road, Ithaca, New York 14853, USA. FAU - Sawers, Ruairidh J H AU - Sawers RJ FAU - Linley, Philip J AU - Linley PJ FAU - Farmer, Phyllis R AU - Farmer PR FAU - Hanley, Nicole P AU - Hanley NP FAU - Costich, Denise E AU - Costich DE FAU - Terry, Matthew J AU - Terry MJ FAU - Brutnell, Thomas P AU - Brutnell TP LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Light-Harvesting Protein Complexes) RN - 0 (Photosynthetic Reaction Center Complex Proteins) RN - 0 (phyA phytochrome) RN - 11121-56-5 (Phytochrome) RN - 114-25-0 (Biliverdine) RN - 1406-65-1 (Chlorophyll) RN - 143392-71-6 (phytochromobilin) RN - 36-88-4 (Carotenoids) SB - IM MH - Biliverdine/*analogs & derivatives/metabolism MH - Carotenoids/metabolism MH - Chlorophyll/metabolism MH - Chloroplasts/metabolism MH - Darkness MH - Light MH - Light-Harvesting Protein Complexes MH - Mutation MH - Photosynthesis/physiology/radiation effects MH - Photosynthetic Reaction Center Complex Proteins/genetics/metabolism/radiation effects MH - Phytochrome/*metabolism MH - Plant Stems/genetics/*growth & development/radiation effects MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Signal Transduction/physiology MH - Zea mays/genetics/*growth & development/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.006411 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):155-63. PMID- 12226497 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots. PG - 164-78 AB - Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, beta-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells. AD - Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, Colorado 80523-1173, USA. FAU - Park, Sang-Wook AU - Park SW FAU - Lawrence, Christopher B AU - Lawrence CB FAU - Linden, James C AU - Linden JC FAU - Vivanco, Jorge M AU - Vivanco JM LA - eng SI - GENBANK/AY071928 PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Antifungal Agents) RN - 0 (DNA, Complementary) RN - 0 (Ethylenes) RN - 0 (Plant Proteins) RN - 74-85-1 (ethylene) RN - EC 3.2.1.14 (Chitinase) RN - EC 3.2.1.21 (beta-Glucosidase) RN - EC 3.2.1.58 (Glucan 1,3-beta-Glucosidase) RN - EC 3.4.- (Endopeptidases) SB - IM MH - Amino Acid Sequence MH - Antifungal Agents/pharmacology MH - Base Sequence MH - Cell Surface Extensions/physiology MH - Cell Wall/genetics/metabolism MH - Cells, Cultured MH - Chitinase/metabolism MH - Cloning, Molecular MH - DNA, Complementary/chemistry/genetics MH - Endopeptidases/metabolism MH - Ethylenes/pharmacology MH - Glucan 1,3-beta-Glucosidase MH - Microscopy MH - Molecular Sequence Data MH - Phytolacca americana/genetics/growth & development/*metabolism MH - Plant Proteins/drug effects/*genetics/metabolism MH - Plant Roots/cytology/genetics/*metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Ribosomes/drug effects/genetics/*metabolism MH - Sequence Analysis, DNA MH - Sequence Homology, Amino Acid MH - Sequence Homology, Nucleic Acid MH - beta-Glucosidase/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.000794 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):164-78. PMID- 12226498 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041118 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Engineering herbicide metabolism in tobacco and Arabidopsis with CYP76B1, a cytochrome P450 enzyme from Jerusalem artichoke. PG - 179-89 AB - The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites. We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis. Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification. Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance. Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins. In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea. Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants. Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants. Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites. AD - E.I. DuPont de Nemours and Company, Central Research and Development, DuPont Experimental Station, Wilmington, Delaware 19880-0328, USA. FAU - Didierjean, Luc AU - Didierjean L FAU - Gondet, Laurence AU - Gondet L FAU - Perkins, Roberta AU - Perkins R FAU - Lau, Sze-Mei Cindy AU - Lau SM FAU - Schaller, Hubert AU - Schaller H FAU - O'Keefe, Daniel P AU - O'Keefe DP FAU - Werck-Reichhart, Daniele AU - Werck-Reichhart D LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Carbon Radioisotopes) RN - 0 (DNA, Complementary) RN - 0 (Herbicides) RN - 0 (Phenylurea Compounds) RN - 0 (Recombinant Fusion Proteins) RN - 9035-51-2 (Cytochrome P-450 Enzyme System) RN - EC 1.14.13.- (7-Alkoxycoumarin O-Dealkylase) RN - EC 1.14.13.- (cytochrome P-450 CYP76B1 (Helianthus tuberosus)) RN - EC 1.6.2.4 (NADPH-Ferrihemoprotein Reductase) SB - IM MH - 7-Alkoxycoumarin O-Dealkylase/*genetics/metabolism MH - Adaptation, Physiological/genetics/physiology MH - Amino Acid Sequence MH - Arabidopsis/genetics/*metabolism MH - Carbon Radioisotopes MH - Cytochrome P-450 Enzyme System/*genetics/metabolism MH - DNA, Complementary/genetics MH - Gene Expression Regulation, Plant MH - Genetic Engineering/methods MH - Helianthus/enzymology MH - Herbicides/*metabolism/pharmacology MH - Molecular Sequence Data MH - NADPH-Ferrihemoprotein Reductase/genetics/metabolism MH - *Phenylurea Compounds MH - Plant Leaves/metabolism MH - Plants, Genetically Modified MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Tobacco/genetics/*metabolism MH - Yeasts/genetics/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005801 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):179-89. PMID- 12226499 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - The altered pattern of amylose accumulation in the endosperm of low-amylose barley cultivars is attributable to a single mutant allele of granule-bound starch synthase I with a deletion in the 5'-non-coding region. PG - 190-8 AB - Reasons for the variable amylose content of endosperm starch from waxy cultivars of barley (Hordeum vulgare) were investigated. The mature grains of most such cultivars contain some amylose, although amounts are much lower than in wild-type cultivars. In these low-amylose cultivars, amylose synthesis starts relatively late in grain development. Starch granules in the outer cell layers of the endosperm contain more amylose than those in the center. This distribution corresponds to that of granule-bound starch synthase I (GBSSI), which is more severely reduced in amount in the center of the endosperm than in the outer cell layers, relative to wild-type cultivars. A second GBSSI in the barley plant, GBSSIb, is not detectable in the endosperm and cannot account for amylose synthesis in the low-amylose cultivars. The change in the expression of GBSSI in the endosperm of the low-amylose cultivars appears to be due to a 413-bp deletion of part of the promoter and 5'-untranslated region of the gene. Although these cultivars are of diverse geographical origin, all carry this same deletion, suggesting that the low-amylose cultivars have a common waxy ancestor. Records suggest a probable source in China, first recorded in the 16th century. Two further families of waxy cultivars have no detectable amylose in the endosperm starch. These amylose-free cultivars were selected in the 20th century from chemically mutagenized populations of wild-type barley. In both cases, 1-bp alterations in the GBSSI gene completely eliminate GBSSI activity. AD - John Innes Centre, Norwich Research Park, Colney, Norfolk NR4 7UH, United Kingdom. FAU - Patron, Nicola J AU - Patron NJ FAU - Smith, Alison M AU - Smith AM FAU - Fahy, Brendan F AU - Fahy BF FAU - Hylton, Christopher M AU - Hylton CM FAU - Naldrett, Mike J AU - Naldrett MJ FAU - Rossnagel, Brian G AU - Rossnagel BG FAU - Denyer, Kay AU - Denyer K LA - eng SI - GENBANK/AF486508 SI - GENBANK/AF486509 SI - GENBANK/AF486510 SI - GENBANK/AF486511 SI - GENBANK/AF486512 SI - GENBANK/AF486513 PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 9005-82-7 (Amylose) RN - EC 2.4.1.- (granule-bound starch synthase I) RN - EC 2.4.1.21 (Starch Synthase) SB - IM MH - 5' Flanking Region/*genetics MH - Alleles MH - Amino Acid Sequence MH - Amylose/*metabolism MH - Base Sequence MH - Biological Transport MH - Comparative Study MH - Hordeum/*enzymology/genetics MH - Molecular Sequence Data MH - Mutation MH - Research Support, Non-U.S. Gov't MH - Seeds/*enzymology/genetics MH - Sequence Deletion MH - Sequence Homology, Amino Acid MH - Sequence Homology, Nucleic Acid MH - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MH - Starch Synthase/*genetics/metabolism MH - Triticum/genetics/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005454 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):190-8. PMID- 12226500 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Indole acetic acid distribution coincides with vascular differentiation pattern during Arabidopsis leaf ontogeny. PG - 199-209 AB - We used an anti-indole acetic acid (IAA or auxin) monoclonal antibody-based immunocytochemical procedure to monitor IAA level in Arabidopsis tissues. Using immunocytochemistry and the IAA-driven beta-glucuronidase (GUS) activity of Aux/IAA promoter::GUS constructs to detect IAA distribution, we investigated the role of polar auxin transport in vascular differentiation during leaf development in Arabidopsis. We found that shoot apical cells contain high levels of IAA and that IAA decreases as leaf primordia expand. However, seedlings grown in the presence of IAA transport inhibitors showed very low IAA signal in the shoot apical meristem (SAM) and the youngest pair of leaf primordia. Older leaf primordia accumulate IAA in the leaf tip in the presence or absence of IAA transport inhibition. We propose that the IAA in the SAM and the youngest pair of leaf primordia is transported from outside sources, perhaps the cotyledons, which accumulate more IAA in the presence than in the absence of transport inhibition. The temporal and spatial pattern of IAA localization in the shoot apex indicates a change in IAA source during leaf ontogeny that would influence flow direction and, consequently, the direction of vascular differentiation. The IAA production and transport pattern suggested by our results could explain the venation pattern, and the vascular hypertrophy caused by IAA transport inhibition. An outside IAA source for the SAM supports the notion that IAA transport and procambium differentiation dictate phyllotaxy and organogenesis. AD - Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, California 94720, USA. FAU - Avsian-Kretchmer, Orna AU - Avsian-Kretchmer O FAU - Cheng, Jin-Chen AU - Cheng JC FAU - Chen, Lingjing AU - Chen L FAU - Moctezuma, Edgar AU - Moctezuma E FAU - Sung, Z Renee AU - Sung ZR LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Auxins) RN - 0 (Fluorenes) RN - 0 (Indoleacetic Acids) RN - 0 (Phthalimides) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Triiodobenzoic Acids) RN - 132-66-1 (alpha-naphthylphthalamic acid) RN - 2536-31-4 (chloroflurenol-methyl) RN - 87-51-4 (indoleacetic acid) RN - 88-82-4 (2,3,5-triiodobenzoic acid) RN - EC 3.2.1.31 (Glucuronidase) SB - IM MH - Arabidopsis/cytology/genetics/*metabolism MH - Auxins/metabolism/pharmacology MH - Biological Transport/physiology MH - Cell Differentiation/*physiology MH - Fluorenes/pharmacology MH - Glucuronidase/genetics/metabolism MH - Immunohistochemistry MH - Indoleacetic Acids/*metabolism/pharmacology MH - Meristem/drug effects/growth & development/metabolism MH - Phthalimides/pharmacology MH - Plant Leaves/drug effects/*growth & development/metabolism MH - Plants, Genetically Modified MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Signal Transduction/physiology MH - Triiodobenzoic Acids/pharmacology EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.003228 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):199-209. PMID- 12226501 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Arabidopsis CYP98A3 mediating aromatic 3-hydroxylation. Developmental regulation of the gene, and expression in yeast. PG - 210-20 AB - The general phenylpropanoid pathways generate a wide array of aromatic secondary metabolites that range from monolignols, which are ubiquitous in all plants, to sinapine, which is confined to crucifer seeds. The biosynthesis of these compounds involves hydroxylated and methoxylated cinnamyl acid, aldehyde, or alcohol intermediates. Of the three enzymes originally proposed to hydroxylate the 4-, 3-, and 5-positions of the aromatic ring, cinnamate 4-hydroxylase (C4H), which converts trans-cinnamic acid to p-coumaric acid, is the best characterized and is also the archetypal plant P450 monooxygenase. Ferulic acid 5-hydroxylase (F5H), a P450 that catalyzes 5-hydroxylation, has also been studied, but the presumptive 3-hydroxylase converting p-coumarate to caffeate has been elusive. We have found that Arabidopsis CYP98A3, also a P450, could hydroxylate p-coumaric acid to caffeic acid in vivo when expressed in yeast (Saccharomyces cerevisiae) cells, albeit very slowly. CYP98A3 transcript was found in Arabidopsis stem and silique, resembling both C4H and F5H in this respect. CYP98A3 showed further resemblance to C4H in being highly active in root, but differed from F5H in this regard. In transgenic Arabidopsis, the promoters of CYP98A3 and C4H showed wound inducibility and a comparable developmental regulation throughout the life cycle, except in seeds, where the CYP98A3 promoter construct was inactive while remaining active in silique walls. Within stem and root tissue, the gene product and the promoter activity of CYP98A3 were most abundant in lignifying cells. Collectively, these studies show involvement of CYP98A3 in the general phenylpropanoid metabolism, and suggest a downstream function for CYP98A3 relative to the broader and upstream role of C4H. AD - Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9. FAU - Nair, Ramesh B AU - Nair RB FAU - Xia, Qun AU - Xia Q FAU - Kartha, Cyril J AU - Kartha CJ FAU - Kurylo, Eugen AU - Kurylo E FAU - Hirji, Rozina N AU - Hirji RN FAU - Datla, Raju AU - Datla R FAU - Selvaraj, Gopalan AU - Selvaraj G LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Caffeic Acids) RN - 0 (Coumaric Acids) RN - 0 (Phenylpropionates) RN - 331-39-5 (caffeic acid) RN - 7400-08-0 (4-coumaric acid) RN - 9005-53-2 (Lignin) RN - 9035-51-2 (Cytochrome P-450 Enzyme System) RN - EC 1.- (Mixed Function Oxygenases) RN - EC 1.14.- (cytochrome P-450 CYP98A3 (Arabidopsis thaliana)) RN - EC 1.14.13.11 (trans-cinnamate 4-monooxygenase CYP73) SB - IM MH - Arabidopsis/*enzymology/genetics/growth & development MH - Caffeic Acids/metabolism MH - Comparative Study MH - Coumaric Acids/metabolism MH - Cytochrome P-450 Enzyme System/genetics/*metabolism MH - Gene Expression Regulation, Developmental MH - Gene Expression Regulation, Plant MH - Hydroxylation MH - Immunoblotting MH - Immunohistochemistry MH - Lignin/metabolism MH - Mixed Function Oxygenases/genetics/*metabolism MH - Phenylpropionates/metabolism MH - Plant Roots/metabolism MH - Plant Stems/metabolism MH - Plants, Genetically Modified MH - Saccharomyces cerevisiae/genetics EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.008649 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):210-20. PMID- 12226502 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Regulated expression of Arabidopsis phosphate transporters. PG - 221-33 AB - Phosphorus deficiency is one of the major abiotic stresses affecting plant growth. Plants respond to the persistent deficiency of phosphate (Pi) by coordinating the expression of genes involved in alleviation of the stress. The high-affinity Pi transporters are among the major molecular determinants that are activated during Pi stress. In this study, using three reporter genes (green fluorescent protein, luciferase, and beta-glucuronidase) regulated by two Pi transporter promoters, we have carried out an extensive analysis of transcriptional and spatial regulation of gene expression. Activation of the genes was rapid, repressible, and specific in response to changes in Pi availability. The phytohormones auxin and cytokinin suppressed the expression of the reporter gene driven by the AtPT1 promoter, and that of the native gene, suggesting that hormones may be involved in regulation of some component(s) of Pi starvation response pathway. These studies also provide molecular evidence for a potential role of high-affinity Pi transporters in mobilizing Pi into reproductive organs. The results suggest that members of the Pi transporter family may have similar but nonredundant functions in plants. AD - Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907-1165, USA. FAU - Karthikeyan, Athikkattuvalasu S AU - Karthikeyan AS FAU - Varadarajan, Deepa K AU - Varadarajan DK FAU - Mukatira, Uthappa T AU - Mukatira UT FAU - D'Urzo, Matilde Paino AU - D'Urzo MP FAU - Damsz, Barbara AU - Damsz B FAU - Raghothama, Kashchandra G AU - Raghothama KG LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Auxins) RN - 0 (Cytokinins) RN - 0 (Phosphate Transport Proteins) RN - 0 (Phosphates) RN - 0 (Plant Growth Regulators) RN - 0 (Recombinant Fusion Proteins) RN - EC 1.13.12.- (Luciferases) RN - EC 3.2.1.31 (Glucuronidase) SB - IM MH - Arabidopsis/genetics/growth & development/*metabolism MH - Auxins/pharmacology MH - Biological Transport MH - Cytokinins/pharmacology MH - Gene Expression Regulation, Developmental/drug effects MH - Gene Expression Regulation, Plant/drug effects MH - Glucuronidase/genetics/metabolism MH - Histocytochemistry MH - Luciferases/genetics/metabolism MH - Phosphate Transport Proteins/genetics/*metabolism MH - Phosphates/deficiency/*pharmacology MH - Plant Growth Regulators/pharmacology MH - Plants, Genetically Modified MH - Promoter Regions (Genetics)/genetics MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Trans-Activation (Genetics)/drug effects EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.020007 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):221-33. PMID- 12226503 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Arabidopsis UVR8 regulates ultraviolet-B signal transduction and tolerance and contains sequence similarity to human regulator of chromatin condensation 1. PG - 234-43 AB - To further our understanding of how plants defend against the harmful effects of ultraviolet (UV) light, we characterized an Arabidopsis mutant hypersensitive to UV-B. This mutant, UV resistance locus 8-1 (uvr8-1), contains a single recessive mutation at the bottom of chromosome 5. Fine-scale mapping localized uvr8-1 to a 21-kb locus containing five predicted open reading frames. Sequencing of this entire region revealed that the uvr8-1 allele contains a 15-nucleotide deletion in a gene similar to the human guanine nucleotide exchange factor regulator of chromatin condensation 1. This mutation reduces the UV-B-mediated induction of flavonoids and blocks chalcone synthase mRNA and protein induction. In contrast, uvr8-1 has enhanced induction of PR1 and PR5 proteins in response to UV-B, an indication of increased UV-B injury. These results suggest that UVR8 acts in a UV-B signal transduction pathway leading to induction of flavonoid biosynthesis. AD - The Boyce Thompson Institute for Plant Research and Section of Genetics and Development, Cornell University, Ithaca, New York 14853, USA. Kliebenstein@ucdavis.edu FAU - Kliebenstein, Daniel J AU - Kliebenstein DJ FAU - Lim, Jackie E AU - Lim JE FAU - Landry, Laurie G AU - Landry LG FAU - Last, Robert L AU - Last RL LA - eng SI - GENBANK/AF130441 PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Arabidopsis Proteins) RN - 0 (CHC1 protein, human) RN - 0 (Cell Cycle Proteins) RN - 0 (Flavonoids) RN - 0 (Guanine Nucleotide Exchange Factors) RN - 0 (Nuclear Proteins) RN - 0 (Phenylpropionates) RN - 0 (RNA, Messenger) RN - EC 2.3. (Acyltransferases) RN - EC 2.3.1.74 (flavanone synthetase) SB - IM MH - Acyltransferases/metabolism MH - Adaptation, Physiological/physiology MH - Alleles MH - Arabidopsis/genetics/*physiology/radiation effects MH - Arabidopsis Proteins/*genetics/metabolism/*physiology/radiation effects MH - *Cell Cycle Proteins MH - Chromosome Mapping MH - Comparative Study MH - Flavonoids/biosynthesis MH - Gene Expression Regulation, Plant/radiation effects MH - Genetic Complementation Test MH - Guanine Nucleotide Exchange Factors/*genetics MH - Humans MH - Molecular Sequence Data MH - Mutation MH - *Nuclear Proteins MH - Phenylpropionates/metabolism MH - RNA, Messenger/genetics/metabolism MH - Sequence Analysis, DNA MH - Sequence Deletion MH - Signal Transduction/*physiology MH - Ultraviolet Rays EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005041 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):234-43. PMID- 12226504 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Glycerophosphocholine metabolism in higher plant cells. Evidence of a new glyceryl-phosphodiester phosphodiesterase. PG - 244-55 AB - Glycerophosphocholine (GroPCho) is a diester that accumulates in different physiological processes leading to phospholipid remodeling. However, very little is known about its metabolism in higher plant cells. (31)P-Nuclear magnetic resonance spectroscopy and biochemical analyses performed on carrot (Daucus carota) cells fed with GroPCho revealed the existence of an extracellular GroPCho phosphodiesterase. This enzymatic activity splits GroPCho into sn-glycerol-3-phosphate and free choline. In vivo, sn-glycerol-3-phosphate is further hydrolyzed into glycerol and inorganic phosphate by acid phosphatase. We visualized the incorporation and the compartmentation of choline and observed that the major choline pool was phosphorylated and accumulated in the cytosol, whereas a minor fraction was incorporated in the vacuole as free choline. Isolation of plasma membranes, culture medium, and cell wall proteins enabled us to localize this phosphodiesterase activity on the cell wall. We also report the existence of an intracellular glycerophosphodiesterase. This second activity is localized in the vacuole and hydrolyzes GroPCho in a similar fashion to the cell wall phosphodiesterase. Both extra- and intracellular phosphodiesterases are widespread among different plant species and are often enhanced during phosphate deprivation. Finally, competition experiments on the extracellular phosphodiesterase suggested a specificity for glycerophosphodiesters (apparent K(m) of 50 microM), which distinguishes it from other phosphodiesterases previously described in the literature. AD - Laboratoire de Physiologie Cellulaire Vegetale, Unite Mixte de Recherche 5019, Commissariat a l'Energie Atomique, Centre National de la Recherche Scientifique, Universite Joseph Fourier, Departement de Biologie Moleculaire et Structurale, Grenoble, France. FAU - van der Rest, Benoit AU - van der Rest B FAU - Boisson, Anne-Marie AU - Boisson AM FAU - Gout, Elisabeth AU - Gout E FAU - Bligny, Richard AU - Bligny R FAU - Douce, Roland AU - Douce R LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Carbon Isotopes) RN - 0 (Phosphates) RN - 0 (Phosphorus Isotopes) RN - 312-45-8 (Hemicholinium 3) RN - 563-24-6 (Glycerylphosphorylcholine) RN - 62-49-7 (Choline) RN - EC 3.1.4 (Phosphoric Diester Hydrolases) RN - EC 3.1.4.46 (glycerophosphodiester phosphodiesterase) SB - IM MH - Carbon Isotopes MH - Cell Wall/enzymology MH - Cells, Cultured MH - Choline/metabolism MH - Cytosol/enzymology MH - Glycerylphosphorylcholine/*metabolism MH - Hemicholinium 3/pharmacology MH - Hydrogen-Ion Concentration MH - Hydrolysis MH - Magnetic Resonance Spectroscopy MH - Phosphates/pharmacology MH - Phosphoric Diester Hydrolases/drug effects/isolation & purification/*metabolism MH - Phosphorus Isotopes/metabolism MH - Plants/cytology/*enzymology MH - Protoplasts/enzymology MH - Substrate Specificity MH - Vacuoles/enzymology EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.003392 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):244-55. PMID- 12226505 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Biochemical characterization of the Arabidopsis protein kinase SOS2 that functions in salt tolerance. PG - 256-64 AB - The Arabidopsis Salt Overly Sensitive 2 (SOS2) gene encodes a serine/threonine (Thr) protein kinase that has been shown to be a critical component of the salt stress signaling pathway. SOS2 contains a sucrose-non-fermenting protein kinase 1/AMP-activated protein kinase-like N-terminal catalytic domain with an activation loop and a unique C-terminal regulatory domain with an FISL motif that binds to the calcium sensor Salt Overly Sensitive 3. In this study, we examined some of the biochemical properties of the SOS2 in vitro. To determine its biochemical properties, we expressed and isolated a number of active and inactive SOS2 mutants as glutathione S-transferase fusion proteins in Escherichia coli. Three constitutively active mutants, SOS2T168D, SOS2T168D Delta F, and SOS2T168D Delta 308, were obtained previously, which contain either the Thr-168 to aspartic acid (Asp) mutation in the activation loop or combine the activation loop mutation with removal of the FISL motif or the entire regulatory domain. These active mutants exhibited a preference for Mn(2+) relative to Mg(2+) and could not use GTP as phosphate donor for either substrate phosphorylation or autophosphorylation. The three enzymes had similar peptide substrate specificity and catalytic efficiency. Salt overly sensitive 3 had little effect on the activity of the activation loop mutant SOS2T168D, either in the presence or absence of calcium. The active mutant SOS2T168D Delta 308 could not transphosphorylate an inactive protein (SOS2K40N), which indicates an intramolecular reaction mechanism of SOS2 autophosphorylation. Interestingly, SOS2 could be activated not only by the Thr-168 to Asp mutation but also by a serine-156 or tyrosine-175 to Asp mutation within the activation loop. Our results provide insights into the regulation and biochemical properties of SOS2 and the SOS2 subfamily of protein kinases. AD - Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721, USA. FAU - Gong, Deming AU - Gong D FAU - Guo, Yan AU - Guo Y FAU - Jagendorf, Andre T AU - Jagendorf AT FAU - Zhu, Jian-Kang AU - Zhu JK LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Cations, Divalent) RN - 0 (Recombinant Fusion Proteins) RN - 55520-40-6 (Tyrosine) RN - 56-45-1 (Serine) RN - 56-84-8 (Aspartic Acid) RN - 7439-95-4 (Magnesium) RN - 7439-96-5 (Manganese) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.- (Sos2 protein, kinase) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Adaptation, Physiological/*physiology MH - Amino Acid Sequence MH - Amino Acid Substitution MH - Arabidopsis/*enzymology/genetics MH - Aspartic Acid/metabolism MH - Cations, Divalent/pharmacology MH - Enzyme Activation MH - Escherichia coli/genetics MH - Gene Expression Regulation, Enzymologic MH - Gene Expression Regulation, Plant MH - Glutathione Transferase/genetics/metabolism MH - Hydrogen-Ion Concentration MH - Magnesium/pharmacology MH - Manganese/pharmacology MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - Mutation MH - Phosphorylation MH - Protein-Serine-Threonine Kinases/drug effects/genetics/*metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Sequence Homology, Amino Acid MH - Serine/metabolism MH - Signal Transduction/*physiology MH - Substrate Specificity MH - Tyrosine/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004507 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):256-64. PMID- 12226506 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells. PG - 265-72 AB - Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression. AD - Physiologie Cellulaire et Moleculaire des Plantes, Unite Mixte de Recherche Centre National de la Recherche Scientifique 7632, case 156, Universite Pierre et Marie Curie (Paris VI), 4 place Jussieu, 75252 Paris cedex 05, France. FAU - Hallouin, Matthieu AU - Hallouin M FAU - Ghelis, Thanos AU - Ghelis T FAU - Brault, Mathias AU - Brault M FAU - Bardat, Francoise AU - Bardat F FAU - Cornel, Daniel AU - Cornel D FAU - Miginiac, Emile AU - Miginiac E FAU - Rona, Jean-Pierre AU - Rona JP FAU - Sotta, Bruno AU - Sotta B FAU - Jeannette, Emmanuelle AU - Jeannette E LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Arabidopsis Proteins) RN - 0 (Ion Channels) RN - 0 (rab18 protein, Arabidopsis) RN - 21293-29-8 (Abscisic Acid) RN - EC 3.1.4.3 (Phospholipase C) RN - EC 3.1.4.4 (Phospholipase D) RN - EC 3.6.1.- (rab GTP-Binding Proteins) RN - EC 3.6.1.46 (Heterotrimeric GTP-Binding Proteins) SB - IM MH - Abscisic Acid/*pharmacology MH - Arabidopsis/cytology/enzymology/*genetics MH - Arabidopsis Proteins/*genetics MH - Cell Membrane/drug effects/*physiology MH - Cells, Cultured MH - Enzyme Activation/drug effects MH - Gene Expression Regulation, Enzymologic/drug effects MH - Gene Expression Regulation, Plant/drug effects MH - Heterotrimeric GTP-Binding Proteins/metabolism MH - Ion Channels/drug effects MH - Membrane Potentials/drug effects/physiology MH - Phospholipase C/metabolism MH - Phospholipase D/*metabolism MH - Signal Transduction/drug effects/physiology MH - Substrate Specificity MH - rab GTP-Binding Proteins/*genetics EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004168 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):265-72. PMID- 12226507 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Characterization of a novel lipoxygenase-independent senescence mechanism in Alstroemeria peruviana floral tissue. PG - 273-83 AB - The role of lipoxygenase (lox) in senescence of Alstroemeria peruviana flowers was investigated using a combination of in vitro assays and chemical profiling of the lipid oxidation products generated. Phospholipids and galactolipids were extensively degraded during senescence in both sepals and petals and the ratio of saturated/unsaturated fatty acids increased. Lox protein levels and enzymatic activity declined markedly after flower opening. Stereochemical analysis of lox products showed that 13-lox was the major activity present in both floral tissues and high levels of 13-keto fatty acids were also synthesized. Lipid hydroperoxides accumulated in sepals, but not in petals, and sepals also had a higher chlorophyll to carotenoid ratio that favors photooxidation of lipids. Loss of membrane semipermeability was coincident for both tissue types and was chronologically separated from lox activity that had declined by over 80% at the onset of electrolyte leakage. Thus, loss of membrane function was not related to lox activity or accumulation of lipid hydroperoxides per se and differs in these respects from other ethylene-insensitive floral tissues representing a novel pattern of flower senescence. AD - Department of Plant Genetics and Biotechnology, Horticulture Research International, Wellesbourne, Warwickshire CV35 9EF, United Kingdom. FAU - Leverentz, Michael K AU - Leverentz MK FAU - Wagstaff, Carol AU - Wagstaff C FAU - Rogers, Hilary J AU - Rogers HJ FAU - Stead, Anthony D AU - Stead AD FAU - Chanasut, Usawadee AU - Chanasut U FAU - Silkowski, Helena AU - Silkowski H FAU - Thomas, Brian AU - Thomas B FAU - Weichert, Heiko AU - Weichert H FAU - Feussner, Ivo AU - Feussner I FAU - Griffiths, Gareth AU - Griffiths G LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Anthocyanins) RN - 0 (Antioxidants) RN - 0 (Electrolytes) RN - 0 (Fatty Acids) RN - 0 (Lipids) RN - 0 (Pigments) RN - 0 (Reactive Oxygen Species) RN - 0 (Thiobarbituric Acid Reactive Substances) RN - 1406-65-1 (Chlorophyll) RN - 36-88-4 (Carotenoids) RN - EC 1.13.11.12 (Lipoxygenase) SB - IM MH - Angiosperms/*enzymology/growth & development MH - Anthocyanins/metabolism MH - Antioxidants/metabolism MH - Carotenoids/metabolism MH - Chlorophyll/metabolism MH - Electrolytes/metabolism MH - Fatty Acids/chemistry/metabolism MH - Lipid Peroxidation MH - Lipids/chemistry/metabolism MH - Lipoxygenase/*metabolism MH - Molecular Conformation MH - Pigments/metabolism MH - Plant Stems/*enzymology MH - Reactive Oxygen Species/metabolism MH - Research Support, Non-U.S. Gov't MH - Thiobarbituric Acid Reactive Substances/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.000919 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):273-83. PMID- 12226508 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Mitochondrial-driven bicarbonate transport supports photosynthesis in a marine microalga. PG - 284-91 AB - The CO(2)-concentrating mechanism (CCM) of the marine eustigmatophycean microalga Nannochloropsis gaditana consists of an active HCO(3)(-) transport system and an internal carbonic anhydrase to facilitate accumulation and conversion of HCO(3)(-) to CO(2) for photosynthetic fixation. Aqueous inlet mass spectrometry revealed that a portion of the CO(2) generated within the cells leaked to the medium, resulting in a significant rise in the extracellular CO(2) concentration to a level above its chemical equilibrium that was diagnostic for active HCO(3)(-) transport. The transient rise in extracellular CO(2) occurred in the light and the dark and was resolved from concurrent respiratory CO(2) efflux using H(13)CO(3)(-) stable isotope techniques. H(13)CO(3)(-) pump-(13)CO(2) leak activity of the CCM was unaffected by 10 microM 3(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of chloroplast linear electron transport, although photosynthetic O(2) evolution was reduced by 90%. However, low concentrations of cyanide, azide, and rotenone along with anoxia significantly reduced or abolished (13)CO(2) efflux in the dark and light. These results indicate that H(13)CO(3)(-) transport was supported by mitochondrial energy production in contrast to other algae and cyanobacteria in which it is supported by photosynthetic electron transport. This is the first report of a direct role for mitochondria in the energization and functioning of the CCM in a photosynthetic organism. AD - Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3. FAU - Huertas, I Emma AU - Huertas IE FAU - Colman, Brian AU - Colman B FAU - Espie, George S AU - Espie GS LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Azides) RN - 0 (Bicarbonates) RN - 0 (Carbon Isotopes) RN - 124-38-9 (Carbon Dioxide) RN - 151-50-8 (Potassium Cyanide) RN - 330-54-1 (Diuron) RN - 7782-44-7 (Oxygen) RN - 83-79-4 (Rotenone) RN - EC 4.2.1.1 (Carbonic Anhydrases) SB - IM MH - Algae/drug effects/*physiology/radiation effects MH - Azides/pharmacology MH - Bicarbonates/*metabolism MH - Biological Transport/drug effects MH - Carbon Dioxide/metabolism MH - Carbon Isotopes/metabolism MH - Carbonic Anhydrases/metabolism MH - Cell Respiration/radiation effects MH - Darkness MH - Diuron/pharmacology MH - Electron Transport/drug effects MH - Light MH - Mitochondria/*metabolism MH - Oxygen/metabolism MH - Photosynthesis/*physiology/radiation effects MH - Potassium Cyanide/pharmacology MH - Rotenone/pharmacology EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004598 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):284-91. PMID- 12226509 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Early embryo development in Fucus distichus is auxin sensitive. PG - 292-302 AB - Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [(3)H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development. AD - Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109-7325, USA. FAU - Basu, Swati AU - Basu S FAU - Sun, Haiguo AU - Sun H FAU - Brian, Leigh AU - Brian L FAU - Quatrano, Ralph L AU - Quatrano RL FAU - Muday, Gloria K AU - Muday GK LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Auxins) RN - 0 (Indoleacetic Acids) RN - 0 (Phthalimides) RN - 10028-17-8 (Tritium) RN - 132-66-1 (alpha-naphthylphthalamic acid) RN - 87-51-4 (indoleacetic acid) SB - IM SB - S MH - Algae, Brown/*drug effects/physiology MH - Auxins/*pharmacology MH - Biological Transport/drug effects MH - Cell Division/drug effects MH - Dose-Response Relationship, Drug MH - Indoleacetic Acids/pharmacology MH - Phthalimides/pharmacology MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Spores/cytology/drug effects/growth & development MH - Time Factors MH - Tritium OTO - NASA OT - NASA Discipline Plant Biology OT - Non-NASA Center IR - Muday GK FIR - Muday, G K IRAD- Wake Forest U, Winston-Salem, NC EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004747 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):292-302. PMID- 12226510 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Sterol C-24 methyltransferase type 1 controls the flux of carbon into sterol biosynthesis in tobacco seed. PG - 303-11 AB - The first committed step in the conversion of cycloartenol into Delta(5) C24-alkyl sterols in plants is catalyzed by an S-adenosyl-methionine-dependent sterol-C24-methyltransferase type 1 (SMT1). We report the consequences of overexpressing SMT1 in tobacco (Nicotiana tabacum), under control of either the constitutive carnation etched ring virus promoter or the seed-specific Brassica napus acyl-carrier protein promoter, on sterol biosynthesis in seed tissue. Overexpression of SMT1 with either promoter increased the amount of total sterols in seed tissue by up to 44%. The sterol composition was also perturbed with levels of sitosterol increased by up to 50% and levels of isofucosterol and campesterol increased by up to 80%, whereas levels of cycloartenol and cholesterol were decreased by up to 53% and 34%, respectively. Concomitant with the enhanced SMT1 activity was an increase in endogenous 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, from which one can speculate that reduced levels of cycloartenol feed back to up-regulate 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thereby control the carbon flux into sterol biosynthesis. This potential regulatory role of SMT1 in seed sterol biosynthesis is discussed. AD - Plant Sciences, Colworth House, Unilever Research and Development Laboratory, Sharnbrook, Bedford MK44 1LQ, United Kingdom. niklas.holmberg@alligatorbioscience.com FAU - Holmberg, Niklas AU - Holmberg N FAU - Harker, Mark AU - Harker M FAU - Gibbard, Carl L AU - Gibbard CL FAU - Wallace, Andrew D AU - Wallace AD FAU - Clayton, John C AU - Clayton JC FAU - Rawlins, Sally AU - Rawlins S FAU - Hellyer, Amanda AU - Hellyer A FAU - Safford, Richard AU - Safford R LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Phytosterols) RN - 0 (Sitosterols) RN - 17605-67-3 (fucosterol) RN - 469-38-5 (cycloartenol) RN - 474-62-4 (campesterol) RN - 57-88-5 (Cholesterol) RN - 5779-62-4 (sitosterol) RN - 7440-44-0 (Carbon) RN - 83-48-7 (Stigmasterol) RN - EC 1.1.1.- (Hydroxymethylglutaryl CoA Reductases) RN - EC 2.1.1. (Methyltransferases) RN - EC 2.1.1.- (24-methylene-lophenol-C24-methyltransferase) RN - EC 2.1.1.- (S-adenosyl-L-methionine-cycloartenol methyltransferase) SB - IM MH - Biological Transport MH - Carbon/*metabolism MH - Cholesterol/*analogs & derivatives/metabolism MH - Cloning, Molecular MH - Gene Expression Regulation, Enzymologic MH - Gene Expression Regulation, Plant MH - Hydroxymethylglutaryl CoA Reductases/metabolism MH - Methyltransferases/genetics/*metabolism MH - Phytosterols/*biosynthesis/chemistry/metabolism MH - Plant Leaves/metabolism MH - Plants, Genetically Modified MH - Seeds/metabolism MH - Sitosterols/metabolism MH - Stigmasterol/*analogs & derivatives/metabolism MH - Tobacco/*enzymology/genetics/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004226 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):303-11. PMID- 12226511 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Two new loci, PLEIADE and HYADE, implicate organ-specific regulation of cytokinesis in Arabidopsis. PG - 312-24 AB - In screens for regulators of root morphogenesis in Arabidopsis we isolated six new recessive mutants with irregular cell expansion. Complementation analyses placed the mutations in two loci, PLEIADE (PLE) and HYADE (HYA). Phenotypic analyses revealed multinucleated cells, cell wall stubs, and synchronized cell divisions in incompletely separated cells that are all characteristics of defective cytokinesis. These defects were pronounced in roots and undetectable in aerial organs. In addition, fertility and germination were not affected by the mutations. Thus, the alleles that we have isolated of PLE and HYA suggest that the genes may encode organ-specific components needed primarily during root development. Analysis of microtubule arrays during cell cycle in ple and hya roots indicates that the presence of several synchronized nuclei influences the position of preprophase band, mitotic spindles, and phragmoplasts. The enhanced and synergistic phenotype of PLE/ple.hya/hya seedlings and double mutants point to a role of PLE and HYA in the same process. These mutants provide tools to elucidate the regulation of nuclear cytoskeletal interactions during cell division and cytokinesis. AD - Center of Applied Genetics, University of Agricultural Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria. FAU - Muller, Sabine AU - Muller S FAU - Fuchs, Esther AU - Fuchs E FAU - Ovecka, Miroslav AU - Ovecka M FAU - Wysocka-Diller, Joanna AU - Wysocka-Diller J FAU - Benfey, Philip N AU - Benfey PN FAU - Hauser, Marie-Theres AU - Hauser MT LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Arabidopsis Proteins) SB - IM MH - Arabidopsis/cytology/genetics/*growth & development MH - Arabidopsis Proteins/*genetics/metabolism MH - Cell Division/genetics MH - Cell Nucleus/genetics/metabolism MH - Chromosome Mapping MH - Gene Expression Regulation, Developmental MH - Gene Expression Regulation, Plant MH - Genetic Complementation Test MH - Genotype MH - Microtubules/metabolism MH - Mutation MH - Phenotype MH - Research Support, Non-U.S. Gov't EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004416 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):312-24. PMID- 12226512 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Light-intensity-dependent expression of Lhc gene family encoding light-harvesting chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii. PG - 325-33 AB - Excessive light conditions repressed the levels of mRNAs accumulation of multiple Lhc genes encoding light-harvesting chlorophyll-a/b (LHC) proteins of photosystem (PS)II in the unicellular green alga, Chlamydomonas reinhardtii. The light intensity required for the repression tended to decrease with lowering temperature or CO(2) concentration. The responses of six LhcII genes encoding the major LHC (LHCII) proteins and two genes (Lhcb4 and Lhcb5) encoding the minor LHC proteins of PSII (CP29 and CP26) were similar. The results indicate that the expression of these Lhc genes is coordinately repressed when the energy input through the antenna systems exceeds the requirement for CO(2) assimilation. The Lhc mRNA level repressed under high-light conditions was partially recovered by adding the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting that redox signaling via photosynthetic electron carriers is involved in the gene regulation. However, the mRNA level was still considerably lower under high-light than under low-light conditions even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Repression of the Lhc genes by high light was prominent even in the mutants deficient in the reaction center(s) of PSII or both PSI and PSII. The results indicate that two alternative processes are involved in the repression of Lhc genes under high-light conditions, one of which is independent of the photosynthetic reaction centers and electron transport events. AD - Laboratory for Photobiology (1), Photodynamics Research Center, The Institute of Physical and Chemical Research, Sendai 980-0845, Japan. FAU - Teramoto, Haruhiko AU - Teramoto H FAU - Nakamori, Akira AU - Nakamori A FAU - Minagawa, Jun AU - Minagawa J FAU - Ono, Taka-aki AU - Ono TA LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Algal Proteins) RN - 124-38-9 (Carbon Dioxide) RN - 330-54-1 (Diuron) SB - IM MH - Algal Proteins/genetics/metabolism MH - Animals MH - Carbon Dioxide/pharmacology MH - Chlamydomonas reinhardtii/drug effects/*genetics/radiation effects MH - Diuron/pharmacology MH - Gene Expression Regulation/drug effects/radiation effects MH - Light MH - Multigene Family/genetics MH - Mutation MH - Photosynthesis/drug effects/*genetics/radiation effects MH - Research Support, Non-U.S. Gov't MH - Temperature EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004622 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):325-33. PMID- 12226513 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041201 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase. PG - 334-46 AB - To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [(14)C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells. AD - Institut de Biologie Moleculaire des Plantes, Centre National de la Recherche Scientifique Unite Propre de Recherche 2357, 28 rue Goethe, 67083 Strasbourg, France. FAU - Wentzinger, Laurent F AU - Wentzinger LF FAU - Bach, Thomas J AU - Bach TJ FAU - Hartmann, Marie-Andree AU - Hartmann MA LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Bicyclo Compounds, Heterocyclic) RN - 0 (Carbon Radioisotopes) RN - 0 (Enzyme Inhibitors) RN - 0 (Naphthalenes) RN - 0 (Phytosterols) RN - 0 (Polyisoprenyl Phosphates) RN - 0 (Tricarboxylic Acids) RN - 0 (Triglycerides) RN - 111-02-4 (Squalene) RN - 13058-04-3 (farnesyl pyrophosphate) RN - 142561-96-4 (squalestatin 1) RN - 469-38-5 (cycloartenol) RN - 91161-71-6 (terbinafine) RN - EC 1.1.1.- (Hydroxymethylglutaryl CoA Reductases) RN - EC 1.13. (Oxygenases) RN - EC 1.14.99.7 (Squalene monooxygenase) RN - EC 1.3.- (Squalene Synthetase) RN - EC 2.1.1. (Methyltransferases) RN - EC 2.1.1.- (24-methylene-lophenol-C24-methyltransferase) RN - EC 2.1.1.- (S-adenosyl-L-methionine-cycloartenol methyltransferase) RN - EC 3.1.3 (Phosphoric Monoester Hydrolases) RN - EC 3.1.7.1 (prenyl-pyrophosphatase) SB - IM MH - Bicyclo Compounds, Heterocyclic/pharmacology MH - Carbon Radioisotopes MH - Cell Line MH - Enzyme Inhibitors/pharmacology MH - Gene Expression Regulation, Enzymologic/drug effects MH - Gene Expression Regulation, Plant/drug effects MH - Hydroxymethylglutaryl CoA Reductases/genetics/*metabolism MH - Methyltransferases/metabolism MH - Naphthalenes/pharmacology MH - Oxygenases/antagonists & inhibitors/genetics/*metabolism MH - Phosphoric Monoester Hydrolases/metabolism MH - Phytosterols/biosynthesis MH - Polyisoprenyl Phosphates/biosynthesis MH - Squalene/metabolism MH - Squalene Synthetase/antagonists & inhibitors/genetics/*metabolism MH - Tobacco/cytology/*enzymology/genetics MH - Tricarboxylic Acids/pharmacology MH - Triglycerides/metabolism MH - Up-Regulation/drug effects EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004655 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):334-46. PMID- 12226514 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Probing in vivo metabolism by stable isotope labeling of storage lipids and proteins in developing Brassica napus embryos. PG - 347-61 AB - Developing embryos of Brassica napus accumulate both triacylglycerols and proteins as major storage reserves. To evaluate metabolic fluxes during embryo development, we have established conditions for stable isotope labeling of cultured embryos under steady-state conditions. Sucrose supplied via the endosperm is considered to be the main carbon and energy source for seed metabolism. However, in addition to 220 to 270 mM carbohydrates (sucrose, glucose, and fructose), analysis of endosperm liquid revealed up to 70 mM amino acids as well as 6 to 15 mM malic acid. Therefore, a labeling approach with multiple carbon sources is a precondition to quantitatively reflect fluxes of central carbon metabolism in developing embryos. Mid-cotyledon stage B. napus embryos were dissected from plants and cultured for 15 d on a complex liquid medium containing (13)C-labeled carbohydrates. The (13)C enrichment of fatty acids and amino acids (after hydrolysis of the seed proteins) was determined by gas chromatography/mass spectrometry. Analysis of (13)C isotope isomers of labeled fatty acids and plastid-derived amino acids indicated that direct glycolysis provides at least 90% of precursors of plastid acetyl-coenzyme A (CoA). Unlabeled amino acids, when added to the growth medium, did not reduce incorporation of (13)C label into plastid-formed fatty acids, but substantially diluted (13)C label in seed protein. Approximately 30% of carbon in seed protein was derived from exogenous amino acids and as a consequence, the use of amino acids as a carbon source may have significant influence on the total carbon and energy balance in seed metabolism. (13)C label in the terminal acetate units of C(20) and C(22) fatty acids that derive from cytosolic acetyl-CoA was also significantly diluted by unlabeled amino acids. We conclude that cytosolic acetyl-CoA has a more complex biogenetic origin than plastidic acetyl-CoA. Malic acid in the growth medium did not dilute (13)C label incorporation into fatty acids or proteins and can be ruled out as a source of carbon for the major storage components of B. napus embryos. AD - Michigan State University, Department of Plant Biology, East Lansing, Michigan 48824, USA. FAU - Schwender, Jorg AU - Schwender J FAU - Ohlrogge, John B AU - Ohlrogge JB LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Amino Acids) RN - 0 (Carbohydrates) RN - 0 (Carbon Isotopes) RN - 0 (Culture Media) RN - 0 (Fatty Acids) RN - 0 (Malates) RN - 0 (Plant Proteins) RN - 50-99-7 (Glucose) RN - 57-50-1 (Sucrose) RN - 6915-15-7 (malic acid) RN - 72-89-9 (Acetyl Coenzyme A) RN - 7440-44-0 (Carbon) SB - IM MH - Acetyl Coenzyme A/biosynthesis MH - Amino Acids/metabolism MH - Brassica napus/growth & development/*metabolism MH - Carbohydrates/metabolism MH - Carbon/metabolism MH - Carbon Isotopes/metabolism MH - Chloroplasts/metabolism MH - Culture Media MH - Culture Techniques MH - Fatty Acids/chemistry/*metabolism MH - Glucose/metabolism MH - Glycolysis/physiology MH - Isotope Labeling/*methods MH - Malates/metabolism MH - Mass Fragmentography MH - Plant Proteins/*metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Seeds/growth & development/*metabolism MH - Sucrose/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004275 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):347-61. PMID- 12226515 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Tandemly duplicated Safener-induced glutathione S-transferase genes from Triticum tauschii contribute to genome- and organ-specific expression in hexaploid wheat. PG - 362-73 AB - Glutathione S-transferase (GST) gene expression was examined in several Triticum species, differing in genome constitution and ploidy level, to determine genome contribution to GST expression in cultivated, hexaploid bread wheat (Triticum aestivum). Two tandemly duplicated tau class GST genes (TtGSTU1 and TtGSTU2) were isolated from a single bacterial artificial chromosome clone in a library constructed from the diploid wheat and D genome progenitor to cultivated wheat, Triticum tauschii. The genes are very similar in genomic structure and their encoded proteins are 95% identical. Gene-specific reverse transcriptase-polymerase chain reaction analysis revealed differential transcript accumulation of TtGSTU1 and TtGSTU2 in roots and shoots. Expression of both genes was induced by herbicide safeners, 2,4-dichlorophenoxyacetic acid and abscisic acid, in the shoots of T. tauschii; however, expression of TtGSTU1 was always higher than TtGSTU2. In untreated seedlings, TtGSTU1 was expressed in both shoots and roots, whereas TtGSTU2 expression was only detected in roots. RNA gel-blot analysis of ditelosomic, aneuploid lines that are deficient for 6AS, 6BS, or 6DS chromosome arms of cultivated, hexaploid bread wheat showed differential genome contribution to safener-induced GST expression in shoots compared with roots. The GST genes from the D genome of hexaploid wheat contribute most to safener-induced expression in the shoots, whereas GSTs from the B and D genomes contribute to safener-induced expression in the roots. AD - Department of Crop Sciences, University of Illinois, Urbana, Illinois 61801, USA. FAU - Xu, Fangxiu AU - Xu F FAU - Lagudah, Evans S AU - Lagudah ES FAU - Moose, Stephen P AU - Moose SP FAU - Riechers, Dean E AU - Riechers DE LA - eng SI - GENBANK/AY013753 SI - GENBANK/AY013754 PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Chromosomes, Artificial, Bacterial) RN - 0 (DNA, Plant) RN - 0 (Pesticides) RN - 0 (Plant Growth Regulators) RN - 0 (Plant Proteins) RN - 21293-29-8 (Abscisic Acid) RN - 94-75-7 (2,4-Dichlorophenoxyacetic Acid) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - 2,4-Dichlorophenoxyacetic Acid/pharmacology MH - 5' Flanking Region/genetics MH - Abscisic Acid/pharmacology MH - Amino Acid Sequence MH - Base Sequence MH - Chromosomes, Artificial, Bacterial/genetics MH - DNA, Plant/chemistry/genetics MH - Gene Duplication/drug effects MH - Gene Expression Regulation, Enzymologic/drug effects MH - Gene Expression Regulation, Plant/drug effects MH - Glutathione Transferase/*genetics/metabolism MH - Molecular Sequence Data MH - Pesticides/pharmacology MH - Plant Growth Regulators/pharmacology MH - Plant Proteins/genetics/metabolism MH - Plant Roots/metabolism MH - Plant Shoots/metabolism MH - Polyploidy MH - Research Support, Non-U.S. Gov't MH - Sequence Alignment MH - Sequence Analysis, DNA MH - Sequence Homology, Amino Acid MH - Sequence Homology, Nucleic Acid MH - Species Specificity MH - Substrate Specificity MH - Tandem Repeat Sequences/*genetics MH - Triticum/drug effects/enzymology/*genetics EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004796 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):362-73. PMID- 12226516 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20040219 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Successive glycosyltransfer activity and enzymatic characterization of pectic polygalacturonate 4-alpha-galacturonosyltransferase solubilized from pollen tubes of Petunia axillaris using pyridylaminated oligogalacturonates as substrates. PG - 374-9 AB - Polygalacturonate 4-alpha-galacturonosyltransferase (pectin synthase) was solubilized from pollen tubes of Petunia axillaris and characterized. To accomplish this, an assay method using fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as acceptor substrates was developed. When the pollen tube enzyme was solubilized with 0.5% (v/v) Triton X-100 and was incubated with PA-OGA and UDP-galacturonic acid (UDP-GalUA), successive transfer activity of more than 10 GalUAs from UDP-GalUA to the nonreducing end of PA-OGA was observed by diethylaminoethyl high-performance liquid chromatography. This activity was time- and enzyme concentration-dependent. The optimum enzyme activity was observed at pH 7.0 and 30 degrees C. Among the PA-OGAs investigated, those with a degree of polymerization of more than 10 were preferred as substrates. The crude pollen tube enzyme had an apparent K(m) value of 13 microM for the PA-OGA with a degree of polymerization 11 and 170 microM for UDP-GalUA. The characteristics of the P. axillaris pollen tube enzyme and the usefulness of fluorogenic PA-OGAs for the assay of this enzyme are discussed. AD - Graduate School of Science, Osaka University, 1-1 Machikaneyamacho, Toyonaka, Osaka 560-0043, Japan. FAU - Akita, Kazumasa AU - Akita K FAU - Ishimizu, Takeshi AU - Ishimizu T FAU - Tsukamoto, Tatsuya AU - Tsukamoto T FAU - Ando, Toshio AU - Ando T FAU - Hase, Sumihiro AU - Hase S LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Cations) RN - 0 (Hexuronic Acids) RN - 0 (Oligosaccharides) RN - 0 (Pectins) RN - 0 (Plant Proteins) RN - 0 (oligogalacturonic acid) RN - 14982-50-4 (galacturonic acid) RN - EC 2.4 (Glycosyltransferases) RN - EC 2.4.1.- (alpha-1,4-GalAT protein, plant) SB - IM MH - Cations/pharmacology MH - Glycosyltransferases/drug effects/isolation & purification/*metabolism MH - Hexuronic Acids/*metabolism MH - Hydrogen-Ion Concentration MH - Oligosaccharides/metabolism MH - Pectins/biosynthesis MH - Plant Proteins/drug effects/isolation & purification/*metabolism MH - Pollen/*enzymology/growth & development/metabolism MH - Solanaceae/*enzymology/growth & development/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005587 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):374-9. PMID- 12226517 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Developmentally regulated dual-specificity kinase from peanut that is induced by abiotic stresses. PG - 380-90 AB - Tyrosine (Tyr) phosphorylation represents an important biochemical mechanism to regulate many cellular processes. No Tyr kinase has been cloned so far in plants. Dual-specificity kinases are reported in plants and the function of these kinases remains unknown. A 1.7-kb cDNA that encodes serine/threonine/Tyr (STY) kinase was isolated by screening peanut (Arachis hypogaea) expression library using the anti-phospho-Tyr antibody. The histidine-tagged recombinant kinase histidine-6-STY predominantly autophosphorylated on Tyr and phosphorylated the histone primarily on threonine. Genomic DNA gel-blot analysis revealed that STY kinase is a member of a small multigene family. The transcript of STY kinase is accumulated in the mid-maturation stage of seed development, suggesting a role in the signaling of storage of seed reserves. The STY kinase mRNA expression, as well as kinase activity, markedly increased in response to cold and salt treatments; however, no change in the protein level was observed, suggesting a posttranslational activation mechanism. The activation of the STY kinase is detected after 12 to 48 h of cold and salt treatments, which indicates that the kinase may not participate in the initial response to abiotic stresses, but may play a possible role in the adaptive process to adverse conditions. The transcript levels and kinase activity were unaltered with abscisic acid treatment, suggesting an abscisic acid-independent cold and salt signaling pathway. Here, we report the first identification of a non-MAP kinase cascade dual-specificity kinase involved in abiotic stress and seed development. AD - Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India. FAU - Rudrabhatla, Parvathi AU - Rudrabhatla P FAU - Rajasekharan, Ram AU - Rajasekharan R LA - eng SI - GENBANK/AY027437 PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (DNA, Complementary) RN - 7647-14-5 (Sodium Chloride) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Amino Acid Sequence MH - Arachis hypogaea/enzymology/*genetics/growth & development MH - Cloning, Molecular MH - DNA, Complementary/chemistry/genetics MH - Enzyme Activation/drug effects MH - Gene Expression Regulation, Developmental MH - Gene Expression Regulation, Enzymologic MH - Gene Expression Regulation, Plant MH - Molecular Sequence Data MH - Phylogeny MH - Protein Processing, Post-Translational/drug effects MH - Protein-Serine-Threonine Kinases/*genetics/metabolism MH - Sequence Analysis, DNA MH - Sequence Homology, Amino Acid MH - Sodium Chloride/pharmacology MH - Stress, Mechanical MH - Substrate Specificity MH - Temperature MH - Trans-Activation (Genetics)/drug effects EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005173 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):380-90. PMID- 12226518 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20031114 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - N-acylethanolamines are metabolized by lipoxygenase and amidohydrolase in competing pathways during cottonseed imbibition. PG - 391-401 AB - Saturated and unsaturated N-acylethanolamines (NAEs) occur in desiccated seeds primarily as 16C and 18C species with N-palmitoylethanolamine and N-linoleoylethanolamine (NAE 18:2) being most abundant. Here, we examined the metabolic fate of NAEs in vitro and in vivo in imbibed cotton (Gossypium hirsutum) seeds. When synthetic [1-(14)C]N-palmitoylethanolamine was used as a substrate, free fatty acids (FFA) were produced by extracts of imbibed cottonseeds. When synthetic [1-(14)C]NAE 18:2 was used as a substrate, FFA and an additional lipid product(s) were formed. On the basis of polarity, we presumed that the unidentified lipid was a product of the lipoxygenase (LOX) pathway and that inclusion of the characteristic LOX inhibitors nordihydroguaiaretic acid and eicosatetraynoic acid reduced its formation in vitro and in vivo. The conversion of NAE 18:2 in imbibed cottonseed extracts to 12-oxo-13-hydroxy-N-(9Z)-octadecanoylethanolamine was confirmed by gas chromatography-mass spectrometry, indicating the presence of 13-LOX and 13-allene oxide synthase, which metabolized NAE 18:2. Cell fractionation studies showed that the NAE amidohydrolase, responsible for FFA production, was associated mostly with microsomes, whereas LOX, responsible for NAE 18:2-oxylipin production, was distributed in cytosol-enriched fractions and microsomes. The highest activity toward NAE by amidohydrolase was observed 4 to 8 h after imbibition and by LOX 8 h after imbibition. Our results collectively indicate that two pathways exist for NAE metabolism during seed imbibition: one to hydrolyze NAEs in a manner similar to the inactivation of endocannabinoid mediators in animal systems and the other to form novel NAE-derived oxylipins. The rapid depletion of NAEs by these pathways continues to point to a role for NAE metabolites in seed germination. AD - Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, Texas 76203, USA. FAU - Shrestha, Rhidaya AU - Shrestha R FAU - Noordermeer, Minke A AU - Noordermeer MA FAU - van der Stelt, Marcelis AU - van der Stelt M FAU - Veldink, Gerrit A AU - Veldink GA FAU - Chapman, Kent D AU - Chapman KD LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Carbon Radioisotopes) RN - 0 (Endocannabinoids) RN - 0 (Ethanolamines) RN - 0 (Fatty Acids) RN - 0 (Lipoxygenase Inhibitors) RN - 0 (Palmitic Acids) RN - 1191-85-1 (5,8,11,14-Eicosatetraynoic Acid) RN - 500-38-9 (Nordihydroguaiaretic Acid) RN - 544-31-0 (palmidrol) RN - EC 1.13.11.12 (Lipoxygenase) RN - EC 3.5. (Amidohydrolases) SB - IM MH - 5,8,11,14-Eicosatetraynoic Acid/pharmacology MH - Amidohydrolases/*metabolism MH - Biological Transport/physiology MH - Carbon Radioisotopes MH - Endocannabinoids MH - Ethanolamines/isolation & purification/*metabolism MH - Fatty Acids/metabolism MH - Germination/physiology MH - Gossypium/enzymology/*growth & development MH - Lipoxygenase/drug effects/*metabolism MH - Lipoxygenase Inhibitors/pharmacology MH - Nordihydroguaiaretic Acid/pharmacology MH - Palmitic Acids/metabolism MH - Seeds/enzymology/*growth & development EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.004689 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):391-401. PMID- 12226519 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Loss of nuclear gene expression during the phytochrome A-mediated far-red block of greening response. PG - 402-14 AB - We have examined the expression of the HEMA1 gene, which encodes the key chlorophyll synthesis enzyme glutamyl-tRNA reductase, during the phytochrome A-mediated far-red light (FR) block of greening response in Arabidopsis. Our results demonstrate that the FR block of greening comprises two separate responses: a white light (WL) intensity-independent response that requires 3 d of FR and is associated with a loss of expression of the nuclear genes HEMA1 and Lhcb following the transfer to WL (transcriptionally coupled response) and a WL intensity-dependent response that is induced by 1 d of FR and is transcriptionally uncoupled. Both responses required phytochrome A. The transcriptionally uncoupled response correlated with a deregulation of tetrapyrrole synthesis and potential photooxidative damage and was inhibited by cytokinin. The transcriptionally coupled FR response was additive with the loss of expression following Norflurazon-induced photobleaching and was absent in the presence of sucrose or after lower fluence rate (1 micromol m(-2) s(-1)) FR treatments. Both pathways leading to the loss of nuclear gene expression were inhibited by overexpression of NADPH:protochlorophyllide oxidoreductase, indicating a role for plastid signaling in the FR-mediated pathway. The significance of identifying a distinct phytochrome A-mediated plastid signaling pathway is discussed. AD - School of Biological Sciences, University of Southampton, Southampton SO16 7PX, United Kingdom. FAU - McCormac, Alex C AU - McCormac AC FAU - Terry, Matthew J AU - Terry MJ LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Cytokinins) RN - 0 (Light-Harvesting Protein Complexes) RN - 0 (Nuclear Proteins) RN - 0 (Photosynthetic Reaction Center Complex Proteins) RN - 0 (Pyridazines) RN - 0 (phyA phytochrome) RN - 106-60-5 (Aminolevulinic Acid) RN - 11121-56-5 (Phytochrome) RN - 1406-65-1 (Chlorophyll) RN - 27314-13-2 (norflurazone) RN - 53-59-8 (NADP) RN - 57-50-1 (Sucrose) RN - EC 1. (Oxidoreductases) RN - EC 1.2. (Aldehyde Oxidoreductases) RN - EC 1.2.1.- (glutamyl tRNA reductase) RN - EC 1.3.- (Oxidoreductases Acting on CH-CH Group Donors) RN - EC 1.3.1.33 (protochlorophyllide reductase) SB - IM MH - Aldehyde Oxidoreductases/genetics/metabolism MH - Aminolevulinic Acid/metabolism MH - Arabidopsis/*genetics/metabolism MH - Chlorophyll/*biosynthesis MH - Chloroplasts/drug effects/physiology/radiation effects MH - Cytokinins/pharmacology MH - Gene Expression Regulation, Plant/drug effects/radiation effects MH - Light MH - Light-Harvesting Protein Complexes MH - NADP/metabolism MH - Nuclear Proteins/genetics/metabolism MH - Oxidative Stress MH - Oxidoreductases/genetics/metabolism MH - *Oxidoreductases Acting on CH-CH Group Donors MH - Photosynthetic Reaction Center Complex Proteins/genetics/metabolism MH - Phytochrome/*metabolism MH - Promoter Regions (Genetics)/genetics MH - Pyridazines/pharmacology MH - Research Support, Non-U.S. Gov't MH - Signal Transduction MH - Sucrose/pharmacology MH - Trans-Activation (Genetics) EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.003806 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):402-14. PMID- 12226520 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Short-term boron deprivation inhibits endocytosis of cell wall pectins in meristematic cells of maize and wheat root apices. PG - 415-21 AB - By using immunofluorescence microscopy, we observed rapidly altered distribution patterns of cell wall pectins in meristematic cells of maize (Zea mays) and wheat (Triticum aestivum) root apices. This response was shown for homogalacturonan pectins characterized by a low level (up to 40%) of methylesterification and for rhamnogalacturonan II pectins cross-linked by a borate diol diester. Under boron deprivation, abundance of these pectins rapidly increased in cell walls, whereas their internalization was inhibited, as evidenced by a reduced and even blocked accumulation of these cell wall pectins within brefeldin A-induced compartments. In contrast, root cells of species sensitive to the boron deprivation, like zucchini (Cucurbita pepo) and alfalfa (Medicago sativa), do not internalize cell wall pectins into brefeldin A compartments and do not show accumulation of pectins in their cell walls under boron deprivation. For maize and wheat root apices, we favor an apoplastic target for the primary action of boron deprivation, which signals deeper into the cell via endocytosis-mediated pectin signaling along putative cell wall-plasma membrane-cytoskeleton continuum. AD - Institute of Botany, Rheinische Friedrich-Wilhelms-University of Bonn, Kirschallee 1, D-53115 Bonn, Germany. FAU - Yu, Qin AU - Yu Q FAU - Hlavacka, Andrej AU - Hlavacka A FAU - Matoh, Toru AU - Matoh T FAU - Volkmann, Dieter AU - Volkmann D FAU - Menzel, Diedrik AU - Menzel D FAU - Goldbach, Heiner E AU - Goldbach HE FAU - Baluska, Frantisek AU - Baluska F LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Pectins) RN - 7440-42-8 (Boron) SB - IM SB - S MH - Boron/*deficiency/pharmacology MH - Cell Wall/metabolism MH - Cucurbita/metabolism MH - Cytoskeleton/drug effects MH - Endocytosis/*drug effects MH - Medicago/metabolism MH - Meristem/cytology/*metabolism MH - Microscopy, Fluorescence MH - Pectins/*metabolism MH - Plant Roots/cytology/*metabolism MH - Research Support, Non-U.S. Gov't MH - Time Factors MH - Triticum/metabolism MH - Zea mays/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.006163 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):415-21. PMID- 12226521 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20021211 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - F-actin-dependent endocytosis of cell wall pectins in meristematic root cells. Insights from brefeldin A-induced compartments. PG - 422-31 AB - Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1-->4)-beta-D-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner. AD - Plant Cell Biology, Institute of Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany. baluska@uni-bonn.de FAU - Baluska, Frantisek AU - Baluska F FAU - Hlavacka, Andrej AU - Hlavacka A FAU - Samaj, Jozef AU - Samaj J FAU - Palme, Klaus AU - Palme K FAU - Robinson, David G AU - Robinson DG FAU - Matoh, Toru AU - Matoh T FAU - McCurdy, David W AU - McCurdy DW FAU - Menzel, Diedrik AU - Menzel D FAU - Volkmann, Dieter AU - Volkmann D LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Actins) RN - 0 (Biological Markers) RN - 0 (Epitopes) RN - 0 (Gibberellins) RN - 0 (Pectins) RN - 20350-15-6 (Brefeldin A) RN - EC 3.6.1.- (ADP-Ribosylation Factor 1) SB - IM SB - S MH - ADP-Ribosylation Factor 1/metabolism MH - Actins/*metabolism MH - Biological Markers MH - Biological Transport/drug effects MH - Brefeldin A/*pharmacology MH - Cell Membrane/metabolism MH - Cell Wall/*metabolism MH - Cells, Cultured MH - Cold MH - Endocytosis/*drug effects MH - Endoplasmic Reticulum/metabolism MH - Endosomes/metabolism MH - Epitopes MH - Gibberellins/metabolism MH - Meristem/cytology/*metabolism MH - Microscopy, Fluorescence MH - Pectins/*metabolism MH - Plant Roots/cytology/metabolism MH - Protoplasts/metabolism MH - Zea mays/drug effects/metabolism MH - trans-Golgi Network/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.007526 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):422-31. PMID- 12226522 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Arabinoxylan biosynthesis in wheat. Characterization of arabinosyltransferase activity in Golgi membranes. PG - 432-41 AB - Arabinoxylan arabinosyltransferase (AX-AraT) activity was investigated using microsomes and Golgi vesicles isolated from wheat (Triticum aestivum) seedlings. Incubation of microsomes with UDP-[(14)C]-beta-L-arabinopyranose resulted in incorporation of radioactivity into two different products, although most of the radioactivity was present in xylose (Xyl), indicating a high degree of UDP-arabinose (Ara) epimerization. In isolated Golgi vesicles, the epimerization was negligible, and incubation with UDP-[(14)C]Ara resulted in formation of a product that could be solubilized with proteinase K. In contrast, when Golgi vesicles were incubated with UDP-[(14)C]Ara in the presence of unlabeled UDP-Xyl, the product obtained could be solubilized with xylanase, whereas proteinase K had no effect. Thus, the AX-AraT is dependent on the synthesis of unsubstituted xylan acting as acceptor. Further analysis of the radiolabeled product formed in the presence of unlabeled UDP-Xyl revealed that it had an apparent molecular mass of approximately 500 kD. Furthermore, the total incorporation of [(14)C]Ara was dependent on the time of incubation and the amount of Golgi protein used. AX-AraT activity had a pH optimum at 6, and required the presence of divalent cations, Mn(2+) being the most efficient. In the absence of UDP-Xyl, a single arabinosylated protein with an apparent molecular mass of 40 kD was radiolabeled. The [(14)C]Ara labeling became reversible by adding unlabeled UDP-Xyl to the reaction medium. The possible role of this protein in arabinoxylan biosynthesis is discussed. AD - Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, 1871 Frederiksberg C, Copenhagen, Denmark. FAU - Porchia, Andrea Celia AU - Porchia AC FAU - Sorensen, Susanne Oxenboll AU - Sorensen SO FAU - Scheller, Henrik Vibe AU - Scheller HV LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Carbon Radioisotopes) RN - 0 (Uridine Diphosphate Sugars) RN - 0 (Xylans) RN - 0 (Xylose) RN - 14697-41-7 (uridine diphosphate arabinose) RN - 9040-27-1 (arabinoxylan) RN - EC 2.4.2. (Pentosyltransferases) RN - EC 2.4.2.- (arabinosyltransferase) SB - IM MH - Carbon Radioisotopes MH - Chromatography, Gel MH - Golgi Apparatus/enzymology/*metabolism MH - Intracellular Membranes/drug effects/metabolism MH - Pentosyltransferases/*metabolism MH - Research Support, Non-U.S. Gov't MH - Triticum/drug effects/*enzymology/metabolism MH - Uridine Diphosphate Sugars/pharmacology MH - Xylans/*biosynthesis MH - Xylose/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.003400 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):432-41. PMID- 12226523 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Patterns of expression and normalized levels of the five Arabidopsis phytochromes. PG - 442-56 AB - Using monoclonal antibodies specific for each apoprotein and full-length purified apoprotein standards, the levels of the five Arabidopsis phytochromes and their patterns of expression in seedlings and mature plants and under different light conditions have been characterized. Phytochrome levels are normalized to the DNA content of the various tissue extracts to approximate normalization to the number of cells in the tissue. One phytochrome, phytochrome A, is highly light labile. The other four phytochromes are much more light stable, although among these, phytochromes B and C are reduced 4- to 5-fold in red- or white-light-grown seedlings compared with dark-grown seedlings. The total amount of extractable phytochrome is 23-fold lower in light-grown than dark-grown tissues, and the percent ratios of the five phytochromes, A:B:C:D:E, are measured as 85:10:2:1.5:1.5 in etiolated seedlings and 5:40:15:15:25 in seedlings grown in continuous white light. The four light-stable phytochromes are present at nearly unchanging levels throughout the course of development of mature rosette and reproductive-stage plants and are present in leaves, stems, roots, and flowers. Phytochrome protein expression patterns over the course of seed germination and under diurnal and circadian light cycles are also characterized. Little cycling in response to photoperiod is observed, and this very low amplitude cycling of some phytochrome proteins is out of phase with previously reported cycling of PHY mRNA levels. These studies indicate that, with the exception of phytochrome A, the family of phytochrome photoreceptors in Arabidopsis constitutes a quite stable and very broadly distributed array of sensory molecules. AD - Department of Plant Sciences and Plant Pathology, 119 ABS Building, Montana State University, Bozeman, Montana 59717-3140, USA. sharrock@montana.edu FAU - Sharrock, Robert A AU - Sharrock RA FAU - Clack, Ted AU - Clack T LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Apoproteins) RN - 0 (Arabidopsis Proteins) RN - 0 (DNA, Plant) RN - 0 (Transcription Factors) RN - 0 (phyA phytochrome) RN - 0 (phytochrome C, Arabidopsis) RN - 0 (phytochrome E) RN - 11121-56-5 (Phytochrome) RN - 136250-22-1 (phyB phytochrome) RN - 158379-16-9 (PHYD protein, Arabidopsis) SB - IM MH - Apoproteins/metabolism MH - Arabidopsis/genetics/*metabolism/radiation effects MH - Arabidopsis Proteins/metabolism MH - Circadian Rhythm/physiology MH - DNA, Plant/genetics/metabolism/radiation effects MH - Darkness MH - Gene Expression Regulation, Plant/radiation effects MH - Germination/physiology/radiation effects MH - Immunoblotting MH - Light MH - *Photoreceptors MH - Phytochrome/*metabolism MH - Plant Shoots/growth & development/metabolism/radiation effects MH - Seeds/growth & development/metabolism/radiation effects MH - *Transcription Factors EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005389 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):442-56. PMID- 12226524 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Characterization of a strong dominant phytochrome A mutation unique to phytochrome A signal propagation. PG - 457-65 AB - Here, we report the isolation and characterization of a strong dominant-negative phytochrome A (phyA) mutation (phyA-300D) in Arabidopsis. This mutation carries a single amino acid substitution at residue 631, from valine to methionine (V631M), in the core region within the C-terminal half of PHYA. This PHYA core region contains two protein-interactive motifs, PAS1 and PAS2. Val-631 is located within the PAS1 motif. The phyA-V631M mutant protein is photochemically active and accumulates to a level similar to wild type in dark-grown seedlings. Overexpression of PHYA-V631M in a wild-type background results in a dominant-negative interference with endogenous wild-type phyA, whereas PHYA-V631M in a phyA null mutant background is inactive. To investigate the specificity of this mutation within the phytochrome family, the corresponding amino acid substitution (V664M) was created in the PHYTOCHROME B (PHYB) polypeptide. We found that the phyB-V664M mutant protein is physiologically active in phyB mutant and causes no interfering effect in a wild-type background. Together, our results reveal a unique feature in phyA signal propagation through the C-terminal core region. AD - Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06511-8104, USA. FAU - Fry, Rebecca C AU - Fry RC FAU - Habashi, Jessica AU - Habashi J FAU - Okamoto, Haruko AU - Okamoto H FAU - Deng, Xing Wang AU - Deng XW LA - eng GR - GM47850/GM/NIGMS PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Transcription Factors) RN - 0 (phyA phytochrome) RN - 11121-56-5 (Phytochrome) RN - 136250-22-1 (phyB phytochrome) SB - IM MH - Alleles MH - Amino Acid Sequence MH - Amino Acid Substitution MH - Arabidopsis/*genetics/physiology/radiation effects MH - Darkness MH - Genes, Dominant MH - Hypocotyl/growth & development MH - Light MH - Mutation, Missense MH - *Photoreceptors MH - Phytochrome/*genetics/physiology MH - Plants, Genetically Modified MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Amino Acid MH - Signal Transduction/genetics/radiation effects MH - *Transcription Factors EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005264 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):457-65. PMID- 12226525 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Characterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri. PG - 466-76 AB - A cDNA encoding a protein with 456 amino acids whose sequence shows considerable similarity to plant acyltransferases was identified among 750 Clarkia breweri flower expressed sequence tags. The cDNA was expressed in Escherichia coli, and the protein produced was shown to encode the enzyme benzoyl-coenzyme A (CoA):benzyl alcohol benzoyl transferase (BEBT). BEBT catalyzes the formation of benzylbenzoate, a minor constituent of the C. breweri floral aroma, but it also has activity with a number of other alcohols and acyl CoAs. The BEBT gene is expressed in different parts of the flowers with maximal RNA transcript levels in the stigma, and no expression was observed in the leaves under normal conditions. However, BEBT expression was induced in damaged leaves, reaching a maximum 6 h after damage occurred. We also show here that a closely related tobacco (Nicotiana tabacum) gene previously shown to be induced in leaves after being challenged by phytopathogenic bacteria also has BEBT activity, whereas the most similar protein to BEBT in the Arabidopsis proteome does not use benzoyl CoA as a substrate and instead can use acetyl CoA to catalyze the formation of cis-3-hexen-1-yl acetate, a green-leaf volatile. AD - Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA. FAU - D'Auria, John C AU - D'Auria JC FAU - Chen, Feng AU - Chen F FAU - Pichersky, Eran AU - Pichersky E LA - eng SI - GENBANK/AF500200 GR - 5 T32 GM07544/GM/NIGMS PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Benzoates) RN - 0 (DNA, Complementary) RN - 0 (Esters) RN - 120-51-4 (benzyl benzoate) RN - EC 2.3. (Acyltransferases) SB - IM MH - Acyltransferases/*genetics/isolation & purification/metabolism MH - Amino Acid Sequence MH - Arabidopsis/genetics MH - Benzoates/*metabolism MH - DNA, Complementary/chemistry/genetics MH - Escherichia coli/genetics MH - Esters MH - Gene Expression Regulation, Enzymologic MH - Gene Expression Regulation, Plant MH - Immunoblotting MH - Kinetics MH - Molecular Sequence Data MH - Onagraceae/*enzymology/genetics/metabolism MH - Phylogeny MH - Plant Leaves/metabolism MH - Plant Stems/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Analysis, DNA MH - Sequence Homology, Amino Acid MH - Stress, Mechanical MH - Substrate Specificity MH - Tobacco/genetics MH - Volatilization EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.006460 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):466-76. PMID- 12226526 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Cloning and functional characterization of a beta-pinene synthase from Artemisia annua that shows a circadian pattern of expression. PG - 477-86 AB - Artemisia annua plants produce a broad range of volatile compounds, including monoterpenes, which contribute to the characteristic fragrance of this medicinal species. A cDNA clone, QH6, contained an open reading frame encoding a 582-amino acid protein that showed high sequence identity to plant monoterpene synthases. The prokaryotically expressed QH6 fusion protein converted geranyl diphosphate to (-)-beta-pinene and (-)-alpha-pinene in a 94:6 ratio. QH6 was predominantly expressed in juvenile leaves 2 weeks postsprouting. QH6 transcript levels were transiently reduced following mechanical wounding or fungal elicitor treatment, suggesting that this gene is not directly involved in defense reaction induced by either of these treatments. Under a photoperiod of 12 h/12 h (light/dark), the abundance of QH6 transcripts fluctuated in a diurnal pattern that ebbed around 3 h before daybreak (9th h in the dark phase) and peaked after 9 h in light (9th h in the light phase). The contents of (-)-beta-pinene in juvenile leaves and in emitted volatiles also varied in a diurnal rhythm, correlating strongly with mRNA accumulation. When A. annua was entrained by constant light or constant dark conditions, QH6 transcript accumulation continued to fluctuate with circadian rhythms. Under constant light, advanced cycles of fluctuation of QH6 transcript levels were observed, and under constant dark, the cycle was delayed. However, the original diurnal pattern could be regained when the plants were returned to the normal light/dark (12 h/12 h) photoperiod. This is the first report that monoterpene biosynthesis is transcriptionally regulated in a circadian pattern. AD - National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. FAU - Lu, Shan AU - Lu S FAU - Xu, Ran AU - Xu R FAU - Jia, Jun-Wei AU - Jia JW FAU - Pang, Jihai AU - Pang J FAU - Matsuda, Seiichi P T AU - Matsuda SP FAU - Chen, Xiao-Ya AU - Chen XY LA - eng SI - GENBANK/AF276072 GR - AI41598/AI/NIAID PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Bicyclo Compounds) RN - 0 (DNA, Complementary) RN - 0 (Monoterpenes) RN - 0 (Plant Proteins) RN - 0 (Transcription Factors) RN - 127-91-3 (beta-pinene) RN - 80-56-8 (alpha-pinene) RN - EC 5.5 (Intramolecular Lyases) RN - EC 5.5.- (pinene cyclase I) SB - IM MH - Amino Acid Sequence MH - Artemisia annua/enzymology/*genetics/growth & development MH - Base Sequence MH - Bicyclo Compounds/metabolism MH - Circadian Rhythm/*physiology MH - Cloning, Molecular MH - DNA, Complementary/chemistry/genetics MH - Gene Expression Regulation, Developmental MH - Gene Expression Regulation, Enzymologic MH - Gene Expression Regulation, Plant MH - Intramolecular Lyases/*genetics/metabolism MH - Light MH - Molecular Sequence Data MH - Monoterpenes/metabolism MH - Phylogeny MH - Plant Leaves/genetics/metabolism MH - Plant Proteins/genetics/metabolism MH - Plant Stems/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Analysis, DNA MH - Transcription Factors/genetics/metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.006544 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):477-86. PMID- 12226527 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Nitric oxide negatively modulates wound signaling in tomato plants. PG - 487-93 AB - Synthesis of proteinase inhibitor I protein in response to wounding in leaves of excised tomato (Lycopersicon esculentum) plants was inhibited by NO donors sodium nitroprusside and S-nitroso-N-acetyl-penicillamine. The inhibition was reversed by supplying the plants with the NO scavenger 2-(4-carboxiphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. NO also blocked the hydrogen peroxide (H(2)O(2)) production and proteinase inhibitor synthesis that was induced by systemin, oligouronides, and jasmonic acid (JA). However, H(2)O(2) generated by glucose oxidase and glucose was not blocked by NO, nor was H(2)O(2)-induced proteinase inhibitor synthesis. Although the expression of proteinase inhibitor genes in response to JA was inhibited by NO, the expression of wound signaling-associated genes was not. The inhibition of wound-inducible H(2)O(2) generation and proteinase inhibitor gene expression by NO was not due to an increase in salicylic acid, which is known to inhibit the octadecanoid pathway. Instead, NO appears to be interacting directly with the signaling pathway downstream from JA synthesis, upstream of H(2)O(2) synthesis. The results suggest that NO may have a role in down-regulating the expression of wound-inducible defense genes during pathogenesis. AD - Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA. FAU - Orozco-Cardenas, Martha L AU - Orozco-Cardenas ML FAU - Ryan, Clarence A AU - Ryan CA LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Benzoates) RN - 0 (Cyclopentanes) RN - 0 (Imidazoles) RN - 0 (Nitric Oxide Donors) RN - 0 (Oligosaccharides) RN - 0 (Peptides) RN - 0 (Plant Proteins) RN - 0 (oligogalacturonic acid) RN - 10102-43-9 (Nitric Oxide) RN - 137181-56-7 (systemin) RN - 145757-47-7 (1,3-dihydroxy-4,4,5,5-tetramethyl-2-(4-carboxyphenyl)tetrahydroimidazole) RN - 15078-28-1 (Nitroprusside) RN - 37228-81-2 (proteinase inhibitor I (plants)) RN - 6894-38-8 (jasmonic acid) RN - 7722-84-1 (Hydrogen Peroxide) RN - 79032-48-7 (S-Nitroso-N-Acetylpenicillamine) SB - IM MH - Benzoates/pharmacology MH - Cyclopentanes/pharmacology MH - Hydrogen Peroxide/antagonists & inhibitors/metabolism MH - Imidazoles/pharmacology MH - Lycopersicon esculentum/drug effects/*physiology MH - Nitric Oxide/physiology MH - Nitric Oxide Donors/*pharmacology MH - Nitroprusside/pharmacology MH - Oligosaccharides/pharmacology MH - Peptides/pharmacology MH - Plant Proteins/biosynthesis MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - S-Nitroso-N-Acetylpenicillamine/pharmacology MH - Signal Transduction/*drug effects MH - Stress, Mechanical EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.008375 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):487-93. PMID- 12226528 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Resistance of cultivated tomato to cell content-feeding herbivores is regulated by the octadecanoid-signaling pathway. PG - 494-503 AB - The octadecanoid signaling pathway has been shown to play an important role in plant defense against various chewing insects and some pathogenic fungi. Here, we examined the interaction of a cell-content feeding arachnid herbivore, the two-spotted spider mite (Tetranychus urticae Koch), with cultivated tomato (Lycopersicon esculentum) and an isogenic mutant line (defenseless-1 [def-1]) that is deficient in the biosynthesis of the octadecanoid pathway-derived signal, jasmonic acid (JA). Spider mite feeding and fecundity on def-1 plants was significantly greater than on wild-type plants. Decreased resistance of def-1 plants was correlated with reduced JA accumulation and expression of defensive proteinase inhibitor (PI) genes, which were induced in mite-damaged wild-type leaves. Treatment of def-1 plants with methyl-JA restored resistance to spider mite feeding and reduced the fecundity of female mites. Plants expressing a 35S::prosystemin transgene that constitutively activates the octadecanoid pathway in a Def-1-dependent manner were highly resistant to attack by spider mites and western flower thrips (Frankliniella occidentalis), another cell-content feeder of economic importance. These findings indicate that activation of the octadecanoid signaling pathway promotes resistance of tomato to a broad spectrum of herbivores. The techniques of amplified fragment length polymorphism (AFLP) and bulk segregant analysis were used to map the Def-1 gene to a region on the long arm of chromosome 3 that is genetically separable from the map position of known JA biosynthetic genes. Tight linkage of Def-1 to a T-DNA insertion harboring the maize (Zea mays) Dissociation transposable element suggests a strategy for directed transposon tagging of the gene. AD - Department of Energy-Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, USA. FAU - Li, Chuanyou AU - Li C FAU - Williams, Mark M AU - Williams MM FAU - Loh, Ying-Tsu AU - Loh YT FAU - Lee, Gyu In AU - Lee GI FAU - Howe, Gregg A AU - Howe GA LA - eng GR - GM57795/GM/NIGMS PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Acetic Acids) RN - 0 (Cyclopentanes) RN - 0 (Plant Growth Regulators) RN - 0 (Stearic Acids) RN - 1211-29-6 (methyl jasmonate) RN - 57-11-4 (stearic acid) RN - 6894-38-8 (jasmonic acid) SB - IM MH - Acetic Acids/pharmacology MH - Animals MH - Arachnida/*growth & development MH - Chromosome Mapping MH - Cyclopentanes/metabolism/pharmacology MH - Female MH - Genotype MH - Immunity, Natural/drug effects MH - Insects/growth & development MH - Lycopersicon esculentum/drug effects/genetics/*parasitology MH - Mutation MH - Plant Diseases/genetics/*parasitology MH - Plant Growth Regulators/pharmacology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction/*physiology MH - Stearic Acids/*metabolism EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005314 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):494-503. PMID- 12226529 OWN - NLM STAT- MEDLINE DA - 20020912 DCOM- 20021209 LR - 20041117 PUBM- Print IS - 0032-0889 VI - 130 IP - 1 DP - 2002 Sep TI - Regulation of transcript levels of the Arabidopsis cytochrome p450 genes involved in brassinosteroid biosynthesis. PG - 504-13 AB - Cytochrome P450 enzymes of the closely related CYP90 and CYP85 families catalyze essential oxidative reactions in the biosynthesis of brassinosteroid (BR) hormones. Arabidopsis CYP90B1/DWF4 and CYP90A1/CPD are responsible for respective C-22 and C-23 hydroxylation of the steroid side chain and CYP85A1 catalyzes C-6 oxidation of 6-deoxo intermediates, whereas the functions of CYP90C1/ROT3, CYP90D1, and CYP85A2 are still unknown. Semiquantitative reverse transcriptase-polymerase chain reaction analyses show that transcript levels of CYP85 and CYP90 genes are down-regulated by brassinolide, the end product of the BR biosynthesis pathway. Feedback control of the CYP90C1, CYP90D1, and CYP85A2 genes by brassinolide suggests that the corresponding enzymes might also participate in BR synthesis. CYP85 and CYP90 mRNAs show strong and transient accumulation during the 1st week of seedling development, as well as characteristic organ-specific distribution. Transcripts of CYP90A1 and CYP85A2 are preferentially represented in shoots and CYP90C1, CYP90D1, and CYP85A1 mRNAs are more abundant in roots, whereas CYP90B1 is ubiquitously expressed. Remarkably, the spatial pattern of CYP90A1 expression is maintained in the BR-insensitive cbb2 mutant, indicating the independence of organ-specific and BR-dependent regulation. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in shoots and roots of Arabidopsis, pea (Pisum sativum), and tomato (Lycopersicon esculentum) reveal similar partitioning patterns of BR intermediates in these species. Inverse correlation between CYP90A1/CPD transcript levels and the amounts of the CYP90A1 substrate 6-deoxocathasterone in shoots and roots suggests that transcriptional regulation plays an important role in controlling BR biosynthesis. AD - Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary. FAU - Bancos, Simona AU - Bancos S FAU - Nomura, Takahito AU - Nomura T FAU - Sato, Tatsuro AU - Sato T FAU - Molnar, Gergely AU - Molnar G FAU - Bishop, Gerard J AU - Bishop GJ FAU - Koncz, Csaba AU - Koncz C FAU - Yokota, Takao AU - Yokota T FAU - Nagy, Ferenc AU - Nagy F FAU - Szekeres, Miklos AU - Szekeres M LA - eng PT - Journal Article PL - United States TA - Plant Physiol JID - 0401224 RN - 0 (Arabidopsis Proteins) RN - 0 (Cholestanols) RN - 0 (RNA, Messenger) RN - 0 (Steroids, Heterocyclic) RN - 72962-43-7 (brassinolide) RN - 9035-51-2 (Cytochrome P-450 Enzyme System) RN - EC 1.14.- (CPD protein, Arabidopsis) RN - EC 1.14.- (Steroid Hydroxylases) SB - IM MH - Arabidopsis/enzymology/*genetics/growth & development MH - *Arabidopsis Proteins MH - Cholestanols/chemistry/*metabolism MH - Cytochrome P-450 Enzyme System/*genetics/metabolism MH - Evolution, Molecular MH - Gene Expression Regulation, Enzymologic MH - Gene Expression Regulation, Plant MH - Germination/genetics MH - Lycopersicon esculentum/genetics/metabolism MH - Peas/genetics/metabolism MH - Phylogeny MH - Plant Roots/genetics/metabolism MH - Plant Shoots/genetics/metabolism MH - RNA, Messenger/genetics/metabolism MH - Seeds/genetics/growth & development/metabolism MH - Steroid Hydroxylases/genetics/metabolism MH - Steroids, Heterocyclic/chemistry/*metabolism MH - Transcription, Genetic EDAT- 2002/09/13 10:00 MHDA- 2002/12/10 04:00 AID - 10.1104/pp.005439 [doi] PST - ppublish SO - Plant Physiol 2002 Sep;130(1):504-13. PMID- 12323077 OWN - NLM STAT- Publisher DA - 20020926 PUBM- Print IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Sep 12 TI - Specificity of DNA triple helix formation analyzed by a FRET assay. PG - 27 AB - BACKGROUND: A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis. RESULTS: We have analyzed triplex formation of two polypurine triplex forming oligodeoxynucleotides (TFOs) using fluorescence resonance energy transfer (FRET). Under our conditions, the TFOs bind to their cognate double strand DNAs with binding constants of 2.6 x 105 and 2.3 x 106 M-1. Our data confirm that the polypurine TFO binds in an antiparallel orientation with respect to the polypurine DNA strand and that triplex formation requires Mg2+ions whereas it is inhibited by K+ions. The rate of formation of triple helices is slow with bimolecular rate constants of 5.6 x 104 and 8.1 x 104 min-1 M-1. Triplex dissociation was not detectable over at least 30 hours. Triplex formation is sequence specific; alteration of a single base pair within the 13 base pairs long TFOs prevents detectable triplex formation. CONCLUSION: We have applied a FRET assay to investigate the specificity of DNA triple helix formation. This assay is homogeneous, continuous and specific, because the appearance of the FRET signal is directly correlated to triplex formation. We show that polypurine TFOs bind highly specifically to polypurine stretches in double stranded DNA. This is a prerequisite for biotechnical applications of triple helices to mediate sequence specific recognition of DNA. AD - Institut fur Biochemie, FB8 Justus-Liebig-Universitat Heinrich-Buff-Ring 58 35392 Giessen Germany. albert.jeltsch@chemie.bio.uni-giessen.de AU - Reither S AU - Jeltsch A LA - ENG PT - JOURNAL ARTICLE TA - BMC Biochem JID - 101084098 EDAT- 2002/09/27 06:00 MHDA- 2002/09/27 06:00 PHST- 2002/08/28 [received] PHST- 2002/09/12 [accepted] PHST- 2002/09/12 [aheadofprint] PST - aheadofprint SO - BMC Biochem 2002 Sep 12;3(1):27. PMID- 12350235 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021218 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Sep 12 TI - Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels. PG - 26 AB - BACKGROUND: Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one alpha and one beta subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. RESULTS: Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and beta1 subunit protein levels. Both sGC activity and beta1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases beta1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in beta1 subunits. CONCLUSIONS: These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing beta1 subunit stability. AD - Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, 28040-Madrid, Spain. rutfb@yahoo.es FAU - Ferrero, Rut AU - Ferrero R FAU - Torres, Magdalena AU - Torres M LA - eng PT - Journal Article DEP - 20020912 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Nitric Oxide Donors) RN - 0 (Nitroso Compounds) RN - 0 (RNA, Messenger) RN - 146724-94-9 (2,2'-(hydroxynitrosohydrazono)bis-ethanamine) RN - 60-92-4 (Cyclic AMP) RN - EC 4.6.1.2 (Guanylate Cyclase) SB - IM MH - Animals MH - Cattle MH - Cells, Cultured MH - Chromaffin Cells/*drug effects/metabolism MH - Cyclic AMP/metabolism/pharmacology MH - Down-Regulation MH - Guanylate Cyclase/genetics/*metabolism MH - Nitric Oxide Donors/*pharmacology MH - Nitroso Compounds/*pharmacology MH - RNA, Messenger/*drug effects/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Reverse Transcriptase Polymerase Chain Reaction MH - Solubility MH - Time Factors EDAT- 2002/09/28 04:00 MHDA- 2002/12/19 04:00 PHST- 2002/06/12 [received] PHST- 2002/09/12 [accepted] PHST- 2002/09/12 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Sep 12;3(1):26. PMID- 12351358 OWN - NLM STAT- MEDLINE DA - 20020927 DCOM- 20021017 LR - 20041117 PUBM- Print IS - 1468-5833 VI - 325 IP - 7366 DP - 2002 Sep 28 TI - Using standardised patients to measure physicians' practice: validation study using audio recordings. PG - 679 AB - OBJECTIVE: To assess the validity of standardised patients to measure the quality of physicians' practice. DESIGN: Validation study of standardised patients' assessments. Physicians saw unannounced standardised patients presenting with common outpatient conditions. The standardised patients covertly tape recorded their visit and completed a checklist of quality criteria immediately afterwards. Their assessments were compared against independent assessments of the recordings by a trained medical records abstractor. SETTING: Four general internal medicine primary care clinics in California. Participants: 144 randomly selected consenting physicians. MAIN OUTCOME MEASURES: Rates of agreement between the patients' assessments and independent assessment. RESULTS: 40 visits, one per standardised patient, were recorded. The overall rate of agreement between the standardised patients' checklists and the independent assessment of the audio transcripts was 91% (kappa=0.81). Disaggregating the data by medical condition, site, level of physicians' training, and domain (stage of the consultation) gave similar rates of agreement. Sensitivity of the standardised patients' assessments was 95%, and specificity was 85%. The area under the receiver operator characteristic curve was 90%. CONCLUSIONS: Standardised patients' assessments seem to be a valid measure of the quality of physicians' care for a variety of common medical conditions in actual outpatient settings. Properly trained standardised patients compare well with independent assessment of recordings of the consultations and may justify their use as a "gold standard" in comparing the quality of care across sites or evaluating data obtained from other sources, such as medical records and clinical vignettes. AD - Veterans Administration, Greater Los Angeles Healthcare System, 11 301 Wilshire Blvd, Los Angeles, CA 90073, USA. FAU - Luck, Jeff AU - Luck J FAU - Peabody, John W AU - Peabody JW LA - eng PT - Journal Article PT - Multicenter Study PT - Validation Studies PL - England TA - BMJ JID - 8900488 SB - AIM SB - IM CIN - BMJ. 2002 Sep 28;325(7366):672. PMID: 12351345 MH - California MH - *Clinical Competence MH - Humans MH - Patient Satisfaction MH - *Patient Simulation MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Sensitivity and Specificity MH - Tape Recording EDAT- 2002/09/28 04:00 MHDA- 2002/10/18 04:00 PST - ppublish SO - BMJ 2002 Sep 28;325(7366):679. PMID- 12351359 OWN - NLM STAT- MEDLINE DA - 20020927 DCOM- 20021017 LR - 20041117 PUBM- Print IS - 1468-5833 VI - 325 IP - 7366 DP - 2002 Sep 28 TI - Spontaneous talking time at start of consultation in outpatient clinic: cohort study. PG - 682-3 AD - Division of Psychosomatic Medicine, Department of Internal Medicine, University Hospital, CH-4031 Basle, Switzerland. wlangewitz@uhbs.ch FAU - Langewitz, Wolf AU - Langewitz W FAU - Denz, Martin AU - Denz M FAU - Keller, Anne AU - Keller A FAU - Kiss, Alexander AU - Kiss A FAU - Ruttimann, Sigmund AU - Ruttimann S FAU - Wossmer, Brigitta AU - Wossmer B LA - eng PT - Journal Article PL - England TA - BMJ JID - 8900488 SB - AIM SB - IM MH - Adolescent MH - Adult MH - Aged MH - Aged, 80 and over MH - Ambulatory Care/*statistics & numerical data MH - Cohort Studies MH - *Communication MH - Female MH - Humans MH - Male MH - Middle Aged MH - Physician-Patient Relations MH - Research Support, Non-U.S. Gov't MH - Switzerland MH - Time Factors EDAT- 2002/09/28 04:00 MHDA- 2002/10/18 04:00 PST - ppublish SO - BMJ 2002 Sep 28;325(7366):682-3. PMID- 12351360 OWN - NLM STAT- MEDLINE DA - 20020927 DCOM- 20021017 LR - 20041117 PUBM- Print IS - 1468-5833 VI - 325 IP - 7366 DP - 2002 Sep 28 TI - Women's attitudes to the sex of medical students in a gynaecology clinic: cross sectional survey. PG - 683-4 AD - Department of General Practice and Primary Care, Guy's, King's, and St Thomas's School of Medicine, London SE11 6SP. norma.o'flynn@kcl.ac.uk FAU - O'Flynn, Norma AU - O'Flynn N FAU - Rymer, Janice AU - Rymer J LA - eng PT - Journal Article PL - England TA - BMJ JID - 8900488 SB - AIM SB - IM CIN - BMJ. 2002 Sep 28;325(7366):683-4. PMID: 12358014 MH - Adolescent MH - Adult MH - Aged MH - *Attitude MH - Cross-Sectional Studies MH - Female MH - Gynecology/*education MH - Humans MH - London MH - Male MH - Middle Aged MH - Research Support, Non-U.S. Gov't MH - *Sex MH - *Students, Medical MH - Women/*psychology MH - *Women's Health Services EDAT- 2002/09/28 04:00 MHDA- 2002/10/18 04:00 PST - ppublish SO - BMJ 2002 Sep 28;325(7366):683-4. PMID- 12351361 OWN - NLM STAT- MEDLINE DA - 20020927 DCOM- 20021017 LR - 20041117 PUBM- Print IS - 1468-5833 VI - 325 IP - 7366 DP - 2002 Sep 28 TI - UK senior doctors' career destinations, job satisfaction, and future intentions: questionnaire survey. PG - 685-6 AD - UK Medical Careers Research Group, Unit of Health-Care Epidemiology, Department of Public Health, University of Oxford, Oxford OX3 7LF. FAU - Davidson, Jean M AU - Davidson JM FAU - Lambert, Trevor W AU - Lambert TW FAU - Goldacre, Michael J AU - Goldacre MJ FAU - Parkhouse, James AU - Parkhouse J LA - eng PT - Journal Article PL - England TA - BMJ JID - 8900488 SB - AIM SB - IM CIN - BMJ. 2002 Sep 28;325(7366):685-6. PMID: 12358015 EIN - BMJ 2002 Nov 9;325(7372):1089 MH - *Attitude of Health Personnel MH - *Career Mobility MH - Female MH - Great Britain MH - Humans MH - *Job Satisfaction MH - Male MH - Medical Staff, Hospital/*psychology MH - Physicians, Family/*psychology MH - Research Support, Non-U.S. Gov't MH - State Medicine EDAT- 2002/09/28 04:00 MHDA- 2002/10/18 04:00 PST - ppublish SO - BMJ 2002 Sep 28;325(7366):685-6. PMID- 12351362 OWN - NLM STAT- MEDLINE DA - 20020927 DCOM- 20021017 LR - 20041117 PUBM- Print IS - 1468-5833 VI - 325 IP - 7366 DP - 2002 Sep 28 TI - Patients' perceptions of entitlement to time in general practice consultations for depression: qualitative study. PG - 687 AB - OBJECTIVE: To investigate patients' perceptions of entitlement to time in general practice consultations for depression. DESIGN: Qualitative study based on interviews with patients with mild to moderate depression. SETTING: Eight general practices in the West Midlands and the regional membership of the Depression Alliance. PARTICIPANTS: 32 general practice patients and 30 respondents from the Depression Alliance. RESULTS: An intense sense of time pressure and a self imposed rationing of time in consultations were key concerns among the interviewees. Anxiety about time affected patients' freedom to talk about their problems. Patients took upon themselves part of the responsibility for managing time in the consultation to relieve the burden they perceived their doctors to be working under. Respondents' accounts often showed a mismatch between their own sense of time entitlement and the doctors' capacity to respond flexibly and constructively in offering extended consultation time when this was necessary. Patients valued time to talk and would often have liked more, but they did not necessarily associate length of consultation with quality. The impression doctors gave in handling time in consultations sent strong messages about legitimising the patients' illness and their decision to consult. CONCLUSIONS: Patients' self imposed restraint in taking up doctors' time has important consequences for the recognition and treatment of depression. Doctors need to have a greater awareness of patients' anxieties about time and should move to allay such anxieties by pre-emptive reassurance and reinforcing patients' sense of entitlement to time. Far from acting as "consumers," patients voluntarily assume responsibility for conserving scarce resources in a health service that they regard as a collective rather than a personal resource. AD - Department of Medicines Management, Keele University, Keele ST5 5BG. k.pollock@keele.ac.uk FAU - Pollock, Kristian AU - Pollock K FAU - Grime, Janet AU - Grime J LA - eng PT - Journal Article PT - Multicenter Study PL - England TA - BMJ JID - 8900488 SB - AIM SB - IM CIN - BMJ. 2002 Sep 28;325(7366):687. PMID: 12358016 MH - Communication MH - Depressive Disorder/*therapy MH - England MH - Family Practice/*organization & administration MH - Humans MH - Patient Satisfaction MH - Physician-Patient Relations MH - Research Support, Non-U.S. Gov't MH - Time Factors EDAT- 2002/09/28 04:00 MHDA- 2002/10/18 04:00 PST - ppublish SO - BMJ 2002 Sep 28;325(7366):687. PMID- 12351363 OWN - NLM STAT- MEDLINE DA - 20020927 DCOM- 20021017 LR - 20041117 PUBM- Print IS - 1468-5833 VI - 325 IP - 7366 DP - 2002 Sep 28 TI - Patient centredness in the MRCGP video examination: analysis of large cohort. Membership of the Royal College of General Practitioners. PG - 691-2 AD - Department of Public Health and Primary Care, Postgraduate School of Medicine, University of Hull, Willerby HU10 6NS. p.d.campion@hull.ac.uk FAU - Campion, Peter AU - Campion P FAU - Foulkes, John AU - Foulkes J FAU - Neighbour, Roger AU - Neighbour R FAU - Tate, Peter AU - Tate P LA - eng PT - Journal Article PL - England TA - BMJ JID - 8900488 SB - AIM SB - IM MH - Cohort Studies MH - Educational Measurement/*methods MH - Family Practice/*education MH - Great Britain MH - Humans MH - *Patient-Centered Care MH - Videotape Recording EDAT- 2002/09/28 04:00 MHDA- 2002/10/18 04:00 PST - ppublish SO - BMJ 2002 Sep 28;325(7366):691-2. PMID- 12351364 OWN - NLM STAT- MEDLINE DA - 20020927 DCOM- 20021017 LR - 20041117 PUBM- Print IS - 1468-5833 VI - 325 IP - 7366 DP - 2002 Sep 28 TI - Competency based medical training: review. PG - 693-6 AD - Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ. wai_chingleung@hotmail.com FAU - Leung, Wai-Ching AU - Leung WC LA - eng PT - Journal Article PL - England TA - BMJ JID - 8900488 SB - AIM SB - IM CIN - BMJ. 2002 Sep 28;325(7366):693-6. PMID: 12358017 MH - *Competency-Based Education MH - Education, Medical, Graduate/*organization & administration MH - Great Britain MH - Humans MH - Teaching/methods EDAT- 2002/09/28 04:00 MHDA- 2002/10/18 04:00 PST - ppublish SO - BMJ 2002 Sep 28;325(7366):693-6. PMID- 12359833 OWN - NLM STAT- MEDLINE DA - 20021002 DCOM- 20021115 LR - 20041117 PUBM- Print IS - 1072-0502 VI - 9 IP - 5 DP - 2002 Sep-Oct TI - Recognition memory for single items and for associations is similarly impaired following damage to the hippocampal region. PG - 238-42 AB - The formation of new associations between items is critical for establishing episodic memories. It has been suggested that the hippocampus is essential for creating such associations but is not involved, or is much less involved, in memory for single items. In Experiment 1, we tested controls and amnesic patients with bilateral lesions thought to be limited primarily to the hippocampal region in both single-item and associative recognition memory tasks. In the single-item task, a conventional recognition memory task was administered in which participants studied either houses or faces and were tested for their ability to recognize the individual items. In the associative task, participants studied paired pictures of houses and faces with instructions that encouraged associating the two stimuli, and were tested for their ability to recognize the specific pairings that were presented at study. Like the controls, the amnesic patients performed more poorly on the associative task. Relative to the controls, the amnesic patients were impaired to a similar extent on the single-item and associative tasks. In Experiment 2, the performance of the amnesic patients was improved by increasing the number of presentations of the study lists (eight presentations instead of one). On both the single-item and associative tests, the performance of the amnesic patients after eight presentations was now identical to the performance of the controls who had been given only one presentation of the study list. Thus, the associative condition was not disproportionally difficult for the amnesic patients. These results are consistent with the idea that the hippocampus is similarly involved in single-item and associative memory. AD - Departments of Psychological and Brain Sciences and of Neuroscience, Johns Hopkins University, Baltimore, Maryland 21218, USA. FAU - Stark, Craig E L AU - Stark CE FAU - Bayley, Peter J AU - Bayley PJ FAU - Squire, Larry R AU - Squire LR LA - eng GR - MH12278/MH/NIMH GR - MH24600/MH/NIMH PT - Journal Article PL - United States TA - Learn Mem JID - 9435678 SB - IM MH - Adult MH - Amnesia/*physiopathology MH - Association Learning/physiology MH - Female MH - Hippocampus/*physiopathology MH - Humans MH - Male MH - Middle Aged MH - Pattern Recognition, Visual/physiology MH - Recognition (Psychology)/*physiology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Temporal Lobe/physiology EDAT- 2002/10/03 04:00 MHDA- 2002/11/26 04:00 AID - 10.1101/lm.51802 [doi] PST - ppublish SO - Learn Mem 2002 Sep-Oct;9(5):238-42. PMID- 12359834 OWN - NLM STAT- MEDLINE DA - 20021002 DCOM- 20021115 LR - 20041117 PUBM- Print IS - 1072-0502 VI - 9 IP - 5 DP - 2002 Sep-Oct TI - Overexpression of hAPPswe impairs rewarded alternation and contextual fear conditioning in a transgenic mouse model of Alzheimer's disease. PG - 243-52 AB - One of the hallmarks of the pathology in Alzheimer's disease is the deposition of amyloid plaques throughout the brain, especially within the hippocampus and amygdala. Transgenic mice that overexpress the Swedish mutation of human amyloid precursor protein (hAPPswe; Tg2576) show age-dependent memory deficits in hippocampus-dependent learning tasks. However, the performance of aged Tg2576 mice in amygdala-dependent learning tasks has not been thoroughly assessed. We trained young (2-4 mo) and old (16-18 mo) Tg2576 and wild-type mice in a T-maze alternation task (hippocampus-dependent) and a Pavlovian fear-conditioning task (amygdala- and hippocampus-dependent). As previously reported, old Tg2576 mice showed impaired acquisition of rewarded alternation; none of these mice reached the criterion of at least five out of six correct responses over three consecutive days. In contrast, old Tg2576 mice showed normal levels of conditional freezing to an auditory conditional stimulus (CS) and acquired a contextual discrimination normally. However, when the salience of the fear-conditioning context was decreased, old (12-14 mo) Tg2576 mice were impaired at acquiring fear to the conditioning context, but not to the tone CS. Histological examination of a subset of the mice verified the existence of amyloid plaques in the cortex, hippocampus, and amygdala of old, but not young, Tg2576 mice. Hence, learning and memory deficits in old Tg2576 mice are limited to hippocampus-dependent tasks, despite widespread amyloid deposition in cortex, hippocampus, and amygdala. AD - Department of Psychology, University of Michigan, Ann Arbor, Michigan 48109, USA. FAU - Corcoran, Kevin A AU - Corcoran KA FAU - Lu, Ye AU - Lu Y FAU - Turner, R Scott AU - Turner RS FAU - Maren, Stephen AU - Maren S LA - eng GR - MH57865/MH/NIMH PT - Journal Article PL - United States TA - Learn Mem JID - 9435678 RN - 0 (Amyloid beta-Protein Precursor) SB - IM MH - Age Factors MH - Alzheimer Disease/genetics/*physiopathology MH - Amyloid beta-Protein Precursor/*genetics MH - Animals MH - Behavior, Animal/physiology MH - Conditioning, Classical/*physiology MH - Discrimination Learning/physiology MH - Disease Models, Animal MH - Electroshock MH - Fear/*physiology MH - Female MH - Gene Expression/physiology MH - Humans MH - Male MH - Maze Learning/physiology MH - Mice MH - Mice, Inbred C57BL MH - Mice, Transgenic MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Reward EDAT- 2002/10/03 04:00 MHDA- 2002/11/26 04:00 AID - 10.1101/lm.51002 [doi] PST - ppublish SO - Learn Mem 2002 Sep-Oct;9(5):243-52. PMID- 12359835 OWN - NLM STAT- MEDLINE DA - 20021002 DCOM- 20021115 LR - 20041117 PUBM- Print IS - 1072-0502 VI - 9 IP - 5 DP - 2002 Sep-Oct TI - Plasticity of the human auditory cortex induced by discrimination learning of non-native, mora-timed contrasts of the Japanese language. PG - 253-67 AB - In this magnetoencephalographic (MEG) study, we examined with high temporal resolution the traces of learning in the speech-dominant left-hemispheric auditory cortex as a function of newly trained mora-timing. In Japanese, the "mora" is a temporal unit that divides words into almost isochronous segments (e.g., na-ka-mu-ra and to-o-kyo-o each comprises four mora). Changes in the brain responses of a group of German and Japanese subjects to differences in the mora structure of Japanese words were compared. German subjects performed a discrimination training in 10 sessions of 1.5 h each day. They learned to discriminate Japanese pairs of words (in a consonant, anni-ani; and a vowel, kiyo-kyo, condition), where the second word was shortened by one mora in eight steps of 15 msec each. A significant increase in learning performance, as reflected by behavioral measures, was observed, accompanied by a significant increase of the amplitude of the Mismatch Negativity Field (MMF). The German subjects' hit rate for detecting durational deviants increased by up to 35%. Reaction times and MMF latencies decreased significantly across training sessions. Japanese subjects showed a more sensitive MMF to smaller differences. Thus, even in young adults, perceptual learning of non-native mora-timing occurs rapidly and deeply. The enhanced behavioral and neurophysiological sensitivity found after training indicates a strong relationship between learning and (plastic) changes in the cortical substrate. AD - Center for Biomagnetism, Institute of Experimental Audiology, Munster, Germany. hans.menning@uni-muenster.de FAU - Menning, Hans AU - Menning H FAU - Imaizumi, Satoshi AU - Imaizumi S FAU - Zwitserlood, Pienie AU - Zwitserlood P FAU - Pantev, Christo AU - Pantev C LA - eng PT - Journal Article PL - United States TA - Learn Mem JID - 9435678 SB - IM MH - Adult MH - Auditory Cortex/*physiology MH - Discrimination Learning/*physiology MH - Evoked Potentials, Auditory/physiology MH - Humans MH - Japan MH - *Language MH - Magnetoencephalography MH - Neuronal Plasticity/*physiology MH - Reaction Time/physiology MH - Research Support, Non-U.S. Gov't MH - Speech Perception/physiology EDAT- 2002/10/03 04:00 MHDA- 2002/11/26 04:00 AID - 10.1101/lm.49402 [doi] PST - ppublish SO - Learn Mem 2002 Sep-Oct;9(5):253-67. PMID- 12359836 OWN - NLM STAT- MEDLINE DA - 20021002 DCOM- 20021115 LR - 20041117 PUBM- Print IS - 1072-0502 VI - 9 IP - 5 DP - 2002 Sep-Oct TI - Rapid adaptation to auditory-visual spatial disparity. PG - 268-78 AB - The so-called ventriloquism aftereffect is a remarkable example of rapid adaptative changes in spatial localization caused by visual stimuli. After exposure to a consistent spatial disparity of auditory and visual stimuli, localization of sound sources is systematically shifted to correct for the deviation of the sound from visual positions during the previous adaptation period. In the present study, this aftereffect was induced by presenting, within 17 min, 1800 repetitive noise or pure-tone bursts in combination with synchronized, and 20 degrees disparate flashing light spots, in total darkness. Post-adaptive sound localization, measured by a method of manual pointing, was significantly shifted 2.4 degrees (noise), 3.1 degrees (1 kHz tones), or 5.8 degrees (4 kHz tones) compared with the pre-adaptation condition. There was no transfer across frequencies; that is, shifts in localization were insignificant when the frequencies used for adaptation and the post-adaptation localization test were different. It is hypothesized that these aftereffects may rely on shifts in neural representations of auditory space with respect to those of visual space, induced by intersensory spatial disparity, and may thus reflect a phenomenon of neural short-term plasticity. AD - Fakultat fur Psychologie, Ruhr-Universitat, D-44780 Bochum, Germany. joerg.lewald@ruhr-uni-bochum.de FAU - Lewald, Jorg AU - Lewald J LA - eng PT - Journal Article PL - United States TA - Learn Mem JID - 9435678 SB - IM MH - Acoustic Stimulation MH - Adaptation, Physiological/*physiology MH - Adult MH - Female MH - Humans MH - Illusions/*physiology MH - Male MH - Photic Stimulation MH - Research Support, Non-U.S. Gov't MH - Sound Localization/*physiology MH - Visual Perception/physiology EDAT- 2002/10/03 04:00 MHDA- 2002/11/26 04:00 AID - 10.1101/lm.51402 [doi] PST - ppublish SO - Learn Mem 2002 Sep-Oct;9(5):268-78. PMID- 12359837 OWN - NLM STAT- MEDLINE DA - 20021002 DCOM- 20021115 LR - 20041117 PUBM- Print IS - 1072-0502 VI - 9 IP - 5 DP - 2002 Sep-Oct TI - Patterns of interference in sequence learning and prism adaptation inconsistent with the consolidation hypothesis. PG - 279-92 AB - The studies reported here used an interference paradigm to determine whether a long-term consolidation process (i.e., one lasting from several hours to days) occurs in the learning of two implicit motor skills, learning of a movement sequence and learning of a visuo-motor mapping. Subjects learned one skill and were tested on that skill 48 h later. Between the learning session and test session, some subjects trained on a second skill. The amount of time between the learning of the two skills varied for different subjects. In both the learning of a movement sequence and the learning of a visuo-motor mapping, we found that remote memories were susceptible to interference, but the passage of time did not afford protection from interference. These results are inconsistent with the long-term consolidation of these motor skills. A possible difference between these tasks and those that do show long-term consolidation is that the present tasks are not dynamic motor skills. AD - Department of Psychology, Pacific Lutheran University, Tacoma, Washington 98447, USA. goedert@plu.edu FAU - Goedert, Kelly M AU - Goedert KM FAU - Willingham, Daniel B AU - Willingham DB LA - eng PT - Journal Article PL - United States TA - Learn Mem JID - 9435678 SB - IM MH - Adaptation, Physiological/*physiology MH - Adult MH - Female MH - Humans MH - Male MH - Mental Recall/physiology MH - Perceptual Distortion/*physiology MH - Psychomotor Performance/*physiology MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Visual Perception/*physiology EDAT- 2002/10/03 04:00 MHDA- 2002/11/26 04:00 AID - 10.1101/lm.50102 [doi] PST - ppublish SO - Learn Mem 2002 Sep-Oct;9(5):279-92. PMID- 12359838 OWN - NLM STAT- MEDLINE DA - 20021002 DCOM- 20021115 LR - 20041117 PUBM- Print IS - 1072-0502 VI - 9 IP - 5 DP - 2002 Sep-Oct TI - Memory consolidation for the discrimination of frequency-modulated tones in mongolian gerbils is sensitive to protein-synthesis inhibitors applied to the auditory cortex. PG - 293-303 AB - Differential conditioning of Mongolian gerbils to linearly frequency-modulated tones (FM) has recently received experimental attention. In the study of the role of cerebral protein synthesis for FM discrimination memory, gerbils received post-training bilateral injections of anisomycin into the auditory cortex under light halothane anesthesia. Compared with saline-treated controls, anisomycin-treated gerbils showed a discrimination decrement during the subsequent three days of training. They markedly improved their performance within training sessions, but started each session at low levels. When repeatedly trained gerbils received post-session injections of anisomycin, discrimination performance during subsequent sessions was similar to the pre-injection performance, indicating that retention, retrieval, reconsolidation, and expression of the established reaction were not affected. However, the improvement of a partially established discrimination reaction was impaired after this treatment. Intracortical injections of emetine confirmed this finding. Neither drug affected FM discrimination learning when given several days before the initial training. Our results suggest that protein-synthesis inhibitors applied to the auditory cortex of gerbils during the post-acquisition phase interfered with learning and memory-related aspects of FM processing. The resulting deficit was evident for a number of post-injection training days. This effect was probably due to impaired consolidation, i.e., processes required for long-term stabilization or retrieval of the memory trace while leaving short-term memory intact. AD - Leibniz Institute for Neurobiology, D-39008 Magdeburg, Germany. FAU - Kraus, Michaela AU - Kraus M FAU - Schicknick, Horst AU - Schicknick H FAU - Wetzel, Wolfram AU - Wetzel W FAU - Ohl, Frank AU - Ohl F FAU - Staak, Sabine AU - Staak S FAU - Tischmeyer, Wolfgang AU - Tischmeyer W LA - eng PT - Journal Article PL - United States TA - Learn Mem JID - 9435678 RN - 0 (Protein Synthesis Inhibitors) RN - 22862-76-6 (Anisomycin) RN - 483-18-1 (Emetine) SB - IM MH - Acoustic Stimulation MH - Animals MH - Anisomycin/*pharmacology MH - Auditory Cortex/drug effects/*physiology MH - Auditory Perception/drug effects/physiology MH - Discrimination Learning/*drug effects/physiology MH - Emetine/pharmacology MH - Gerbillinae MH - Male MH - Memory/*drug effects/physiology MH - Microinjections MH - Protein Synthesis Inhibitors/*pharmacology MH - Research Support, Non-U.S. Gov't EDAT- 2002/10/03 04:00 MHDA- 2002/11/26 04:00 AID - 10.1101/lm.47502 [doi] PST - ppublish SO - Learn Mem 2002 Sep-Oct;9(5):293-303. PMID- 12359839 OWN - NLM STAT- MEDLINE DA - 20021002 DCOM- 20021115 LR - 20041117 PUBM- Print IS - 1072-0502 VI - 9 IP - 5 DP - 2002 Sep-Oct TI - Eyeblink classical conditioning and interpositus nucleus activity are disrupted in adult rats exposed to ethanol as neonates. PG - 304-20 AB - Neonatal exposure to ethanol in rats, during the period of brain development comparable to that of the human third trimester, produces significant, dose-dependent cell loss in the cerebellum and deficits in coordinated motor performance. These rats are also impaired in eyeblink conditioning as weanlings and as adults. The current study examined single-unit neural activity in the interpositus nucleus of the cerebellum in adults following neonatal binge ethanol exposure. Group Ethanol received alcohol doses of 5.25 g/kg/day on postnatal days 4-9. Group Sham Intubated underwent acute intragastric intubation on postnatal days 4-9 but did not receive any infusions. Group Unintubated Control (from separate litters) did not receive any intubations. When rats were 3-7 mo old, pairs of extracellular microelectrodes were implanted in the region of the interpositus nucleus. Beginning 1 wk later, the rats were given either 100 paired or 190 unpaired trials per day for 10 d followed by 4 d of 100 conditioned stimulus (CS)-alone trials per day. As in our previous study, conditioned response acquisition in Group Ethanol rats was impaired. In addition, by session 5 of paired acquisition, Group Sham Intubated and Group Unintubated Control showed significant increases in interpositus nucleus activity, relative to baseline, in the CS-unconditioned stimulus interval. In contrast, Group Ethanol failed to show significant changes in interpositus nucleus activity until later in training. These results indicate that the disruption in eyeblink conditioning after early exposure to ethanol is reflected in alterations in interpositus nucleus activity. AD - Department of Psychology, Indiana University, Bloomington, Indiana 47405-7007, USA. jtgreen@indiana.edu FAU - Green, John T AU - Green JT FAU - Johnson, Timothy B AU - Johnson TB FAU - Goodlett, Charles R AU - Goodlett CR FAU - Steinmetz, Joseph E AU - Steinmetz JE LA - eng GR - AA11945/AA/NIAAA PT - Journal Article PL - United States TA - Learn Mem JID - 9435678 RN - 0 (Central Nervous System Depressants) RN - 64-17-5 (Ethanol) SB - IM MH - Age Factors MH - Animals MH - Animals, Newborn MH - Behavior, Animal/drug effects/physiology MH - Central Nervous System Depressants/blood/*pharmacology MH - Cerebellar Nuclei/drug effects/growth & development/*physiology MH - Conditioning, Classical/*drug effects/physiology MH - Conditioning, Eyelid/*drug effects/physiology MH - Electrophysiology MH - Ethanol/blood/*pharmacology MH - Female MH - Male MH - Pregnancy MH - Rats MH - Rats, Long-Evans MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2002/10/03 04:00 MHDA- 2002/11/26 04:00 AID - 10.1101/lm.47602 [doi] PST - ppublish SO - Learn Mem 2002 Sep-Oct;9(5):304-20. PMID- 12359840 OWN - NLM STAT- MEDLINE DA - 20021002 DCOM- 20021115 LR - 20041117 PUBM- Print IS - 1072-0502 VI - 9 IP - 5 DP - 2002 Sep-Oct TI - Age-related impairment in the 250-millisecond delay eyeblink classical conditioning procedure in C57BL/6 mice. PG - 321-36 AB - In this study we tested 4-, 9-, 12-, and 18-month-old C57BL/6 mice in the 250-msec delay eyeblink classical conditioning procedure to study age-related changes in a form of associative learning. The short life expectancy of mice, complete knowledge about the mouse genome, and the availability of transgenic and knock-out mouse models of age-related impairments make the mouse an excellent species for expanding knowledge on the neurobiologically and behaviorally well-characterized eyeblink classical conditioning paradigm. Based on previous research with delay eyeblink conditioning in rabbits and humans, we predicted that mice would be impaired on this cerebellar-dependent associative learning task in middle-age, at ~9 months. To fully examine age differences in behavior in mice, we used a battery of additional behavioral measures with which to compare young and older mice. These behaviors included the acoustic startle response, prepulse inhibition, rotorod, and the Morris water maze. Mice began to show impairment in cerebellar-dependent tasks such as rotorod and eyeblink conditioning at 9 to 12 months of age. Performance in hippocampally dependent tasks was not impaired in any group, including 18-month-old mice. These results in mice support results in other species, indicating that cerebellar-dependent tasks show age-related deficits earlier in adulthood than do hippocampally dependent tasks. AD - Research and Technology Development, Albert Einstein Healthcare Network, Philadelphia, Pennsylvania 19141, USA. FAU - Vogel, Richard W AU - Vogel RW FAU - Ewers, Michael AU - Ewers M FAU - Ross, Charlene AU - Ross C FAU - Gould, Thomas J AU - Gould TJ FAU - Woodruff-Pak, Diana S AU - Woodruff-Pak DS LA - eng GR - AG19411/AG/NIA PT - Journal Article PL - United States TA - Learn Mem JID - 9435678 SB - IM MH - Acoustic Stimulation MH - Aging/*physiology MH - Animals MH - Conditioning, Classical/*physiology MH - Conditioning, Eyelid/*physiology MH - Female MH - Male MH - Maze Learning/physiology MH - Mice MH - Mice, Inbred C57BL MH - Motor Activity/physiology MH - Musculoskeletal Equilibrium/physiology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Startle Reaction/physiology EDAT- 2002/10/03 04:00 MHDA- 2002/11/26 04:00 AID - 10.1101/lm.50902 [doi] PST - ppublish SO - Learn Mem 2002 Sep-Oct;9(5):321-36. PMID- 12359841 OWN - NLM STAT- MEDLINE DA - 20021002 DCOM- 20021115 LR - 20041117 PUBM- Print IS - 1072-0502 VI - 9 IP - 5 DP - 2002 Sep-Oct TI - Side-specificity of olfactory learning in the honeybee: US input side. PG - 337-48 AB - In honeybees, Apis mellifera L., the proboscis extension reflex (PER) can be conditioned by associating an odor stimulus (CS) with a sucrose reward (US). As the neural structures involved in the detection and integration of CS and US are bilaterally symmetrical in the bee brain, we ask what respective role each brain side plays in the conditioning process. More specifically, the US normally used in conditioning experiments is the compound stimulation of the antennae (which triggers the PER) and of the proboscis (where bees lick the sucrose solution). Anatomically, the brain receives unilateral US input through each antenna, but bilateral input from the proboscis. By controlling each US component, we show that an antenna-US produces unilateral sensitization, whereas a proboscis-US or a compound-US induces bilateral sensitization. Bees can learn a unilateral odor CS with all three USs, but when a proboscis-US is used, new learning is inhibited on the contralateral side, owing to a possible US-preexposure effect. Furthermore, we show that the antenna-US induces both unilateral and bilateral reinforcement processes, whereas the proboscis-US produces only bilateral effects. Based on these data, we propose a functional model of the role of each brain side in processing lateralized CSs and USs in olfactory learning in honeybees. AD - Freie Universitat Berlin, Institut fur Biologie-Neurobiologie, 14195 Berlin, Germany. sandoz@cict.fr FAU - Sandoz, Jean-Christophe AU - Sandoz JC FAU - Hammer, Martin AU - Hammer M FAU - Menzel, Randolf AU - Menzel R LA - eng PT - Journal Article PL - United States TA - Learn Mem JID - 9435678 SB - IM MH - Animals MH - Association Learning/physiology MH - Bees/*physiology MH - Conditioning (Psychology)/*physiology MH - Memory/physiology MH - Odors MH - Research Support, Non-U.S. Gov't MH - Sense Organs/physiology MH - Smell/*physiology MH - Taste/physiology EDAT- 2002/10/03 04:00 MHDA- 2002/11/26 04:00 AID - 10.1101/lm.50502 [doi] PST - ppublish SO - Learn Mem 2002 Sep-Oct;9(5):337-48. PMID- 12359842 OWN - NLM STAT- MEDLINE DA - 20021002 DCOM- 20021115 LR - 20041117 PUBM- Print IS - 1072-0502 VI - 9 IP - 5 DP - 2002 Sep-Oct TI - Memories in drosophila heat-box learning. PG - 349-59 AB - Learning and memory processes of operant conditioning in the heat-box are analyzed. In a search for conditioning parameters leading to high retention scores, intermittent training is shown to give better results than those of continuous training. Immediate retention tests contain two memory components, a spatial preference for one side of the chamber and a "stay-where-you-are-effect." Intermittent training strengthens the latter. In the second part, memory dynamics is investigated. Flies are trained in one chamber and tested in a second one after a brief reminder training. With this direct transfer, memory scores reflect an associative learning process in the first chamber. To investigate memory retention after extended time periods, indirect transfer experiments are performed. The fly is transferred to a different environment between training and test phases. With this procedure, an aftereffect of the training can still be observed 2 h later. Surprisingly, exposure to the chamber without conditioning also leads to a memory effect in the indirect transfer experiment. This exposure effect reveals a dispositional change that facilitates operant learning during the reminder training. The various memory effects are independent of the mushroom bodies. AD - Lehrstuhl fur Genetik und Neurobiologie, Biozentrum, Am Hubland, D97074, Wuerzburg, Germany. FAU - Putz, Gabriele AU - Putz G FAU - Heisenberg, Martin AU - Heisenberg M LA - eng PT - Journal Article PL - United States TA - Learn Mem JID - 9435678 SB - IM MH - Animals MH - Association Learning/physiology MH - Conditioning (Psychology)/*physiology MH - Drosophila melanogaster/*physiology MH - Environment Design MH - Female MH - *Heat MH - Male MH - Memory/*physiology MH - Motor Activity/physiology MH - Mushroom Bodies/physiology MH - Research Support, Non-U.S. Gov't EDAT- 2002/10/03 04:00 MHDA- 2002/11/26 04:00 AID - 10.1101/lm.50402 [doi] PST - ppublish SO - Learn Mem 2002 Sep-Oct;9(5):349-59. PMID- 12361478 OWN - NLM STAT- MEDLINE DA - 20030415 DCOM- 20030423 LR - 20041117 PUBM- Print-Electronic IS - 1472-6963 VI - 2 IP - 1 DP - 2002 Oct 2 TI - Socio-demographic factors and self-reported functional status: the significance of social support. PG - 20 AB - BACKGROUND: The aim of the present work was to investigate the relative importance of socio-demographic and physical health status factors for subjective functioning, as well as to examine the role of social support. METHODS: A cross-sectional health survey was carried out in a Greek municipality. 1356 adults of the general population were included in the study. Personal interviews were conducted with house-to-house visits. The response rate was 91.2%. Functioning has been measured by five indexes: 'The Social Roles and Mobility' scale (SORM), 'The Self-Care Restrictions' scale (SCR), 'The Serious Limitations' scale (SL), 'The Minor Self-care Limitations' scale (MSCR) and 'The Minor Limitations in Social Roles and Mobility' scale (MSORM). RESULTS: Among the two sets of independent variables, the socio-demographic ones had significant influence on the functional status, except for MSORM. Allowing for these variables, the physical health status indicators had also significant effects on all functioning scales. Living arrangements and marital status had significant effects on four out of five indexes, while arthritis, Parkinson's disease, past stroke and kidney stones had significant effects on the SCR and SL scales. CONCLUSIONS: These results suggest that socio-demographic factors are as important as physical health variables in affecting a person's ability to function normally in their everyday life. Social support appears to play a significant role in explaining differences in subjective functioning: people living alone or only with the spouse, particularly the elderly, seem to be in greater risk for disability problems and should be targeted by preventive programs in the community. AD - Health Planning Division, Department of Social Medicine, Faculty of Medicine, University of Crete, Greece. koukouli@med.uoc.gr FAU - Koukouli, S AU - Koukouli S FAU - Vlachonikolis, I G AU - Vlachonikolis IG FAU - Philalithis, A AU - Philalithis A LA - eng PT - Journal Article DEP - 20021002 PL - England TA - BMC Health Serv Res JID - 101088677 SB - IM MH - Activities of Daily Living/*classification MH - Adolescent MH - Adult MH - Aged MH - Analysis of Variance MH - Chronic Disease/epidemiology MH - Cross-Sectional Studies MH - Disabled Persons MH - Family Characteristics MH - Female MH - Greece/epidemiology MH - Health Services/utilization MH - *Health Status Indicators MH - Humans MH - Male MH - Middle Aged MH - Physical Fitness MH - Regression Analysis MH - Research Support, Non-U.S. Gov't MH - *Self Efficacy MH - *Social Support MH - Socioeconomic Factors EDAT- 2002/10/04 04:00 MHDA- 2003/04/24 05:00 PHST- 2002/04/02 [received] PHST- 2002/10/02 [accepted] PHST- 2002/10/02 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Oct 2;2(1):20. PMID- 12361483 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021218 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Oct 2 TI - The PEST sequence does not contribute to the stability of the cystic fibrosis transmembrane conductance regulator. PG - 29 AB - BACKGROUND: Endoplasmic reticulum retention of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) mutants and their rapid degradation is the major cause of cystic fibrosis (CF). An important goal is to understand the mechanism of how the misfolded proteins are recognized, retained, and targeted for degradation. RESULTS: Using a web-based algorithm, PESTFind, we found a PEST sequence in the regulatory (R) domain of CFTR. The PEST sequence is found in many short-lived eukaryotic proteins and plays a role in their degradation. To determine its role in the stability and degradation of misprocessed CFTR, we introduced a number of site-directed mutations into the PEST sequence in the cDNA of DeltaF508 CFTR, the most prevalent misprocessed mutation found in CF patients. Analysis of these mutants showed that the disruption of the PEST sequence plays a minor role in the degradation of the CFTR mutants. Multiple mutations to the PEST sequence within the R domain of CFTR inhibit maturation of CFTR and prevent the formation of a 100 kDa degradation product. The mutations, however, do not improve the stability of the mutant DeltaF508 CFTR. CONCLUSION: These observations show that disruption of the structure of the R domain of CFTR can inhibit maturation of the protein and that the predicted PEST sequence plays no significant role in the degradation of CFTR. AD - Canadian Institutes for Health Research Group in Membrane Biology, Department of Medicine, University of Toronto, Toronto, Ontario, M5S 1A8, Canada. evayj.chen@utoronto.ca FAU - Chen, Eva Y AU - Chen EY FAU - Clarke, David M AU - Clarke DM LA - eng GR - R01-CA80900/CA/NCI PT - Journal Article DEP - 20021002 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Amino Acids) RN - 0 (Cysteine Proteinase Inhibitors) RN - 0 (Leupeptins) RN - 0 (Multienzyme Complexes) RN - 0 (Plasmids) RN - 126880-72-6 (Cystic Fibrosis Transmembrane Conductance Regulator) RN - 133407-82-6 (benzyloxycarbonylleucyl-leucyl-leucine aldehyde) RN - 147-85-3 (Proline) RN - 56-45-1 (Serine) RN - 56-86-0 (Glutamic Acid) RN - 72-19-5 (Threonine) RN - EC 3.4.22 (Cysteine Endopeptidases) RN - EC 3.4.25.1 (Proteasome Endopeptidase Complex) SB - IM MH - Amino Acid Sequence MH - Amino Acids/*genetics MH - Animals MH - COS Cells MH - Cysteine Endopeptidases/metabolism MH - Cysteine Proteinase Inhibitors/pharmacology MH - Cystic Fibrosis Transmembrane Conductance Regulator/chemistry/genetics/*metabolism MH - Glutamic Acid/genetics MH - Humans MH - Leupeptins/pharmacology MH - Molecular Sequence Data MH - Multienzyme Complexes/antagonists & inhibitors/metabolism MH - Mutation MH - Plasmids/genetics MH - Proline/genetics MH - Proteasome Endopeptidase Complex MH - Protein Folding MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Serine/genetics MH - Threonine/genetics MH - Transfection EDAT- 2002/10/04 04:00 MHDA- 2002/12/19 04:00 PHST- 2002/07/10 [received] PHST- 2002/10/02 [accepted] PHST- 2002/10/02 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Oct 2;3(1):29. PMID- 12364368 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - Acute septic arthritis. PG - 527-44 AB - Acute septic arthritis may develop as a result of hematogenous seeding, direct introduction, or extension from a contiguous focus of infection. The pathogenesis of acute septic arthritis is multifactorial and depends on the interaction of the host immune response and the adherence factors, toxins, and immunoavoidance strategies of the invading pathogen. Neisseria gonorrhoeae and Staphylococcus aureus are used in discussing the host-pathogen interaction in the pathogenesis of acute septic arthritis. While diagnosis rests on isolation of the bacterial species from synovial fluid samples, patient history, clinical presentation, laboratory findings, and imaging studies are also important. Acute nongonococcal septic arthritis is a medical emergency that can lead to significant morbidity and mortality. Therefore, prompt recognition, rapid and aggressive antimicrobial therapy, and surgical treatment are critical to ensuring a good prognosis. Even with prompt diagnosis and treatment, high mortality and morbidity rates still occur. In contrast, gonococcal arthritis is often successfully treated with antimicrobial therapy alone and demonstrates a very low rate of complications and an excellent prognosis for full return of normal joint function. In the case of prosthetic joint infections, the hardware must be eventually removed by a two-stage revision in order to cure the infection. AD - Center for Biofilm Engineering Montana State University, Bozeman, Montana 59717-3980, USA. mshirtliff@erc.montana.edu FAU - Shirtliff, Mark E AU - Shirtliff ME FAU - Mader, Jon T AU - Mader JT LA - eng PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 SB - IM MH - Acute Disease MH - Animals MH - Arthritis, Infectious/diagnosis/*etiology/therapy MH - Bacterial Adhesion MH - Diagnosis, Differential MH - Gonorrhea/diagnosis/etiology/therapy MH - Humans MH - Prognosis MH - Risk Factors RF - 195 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):527-44. PMID- 12364369 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - Atopic dermatitis and fungi. PG - 545-63 AB - Atopic dermatitis (AD) is a chronic, itching, inflammatory skin disease which is associated with asthma and/or hay fever and a familial occurrence of these conditions. Genetic factors are important in the development of AD, but the exact hereditary pathway is still unknown. Dry skin and the weakened barrier function in patients with AD is very important for the patient's reactions to irritants and other external trigger factors including microorganisms. The standard treatments are topical corticosteroids, topical immunomodulating agents, and emollients. If AD cannot be controlled by this type of treatment, systemic immunomodulating agents may be used. UVB, UVA, or psoralen-UVA may also be used for widespread severe lesions. However, some patients do not respond to these standard treatment, and then it is important to consider the role of microorganisms, house dust mites or food. The role of the Malassezia yeasts in AD, especially AD located to the head and neck region, is now documented in several papers. There are also several papers indicating the role of Candida as an aggravating factor in AD. Patients with AD also develop chronic dermatophyte infections more easily, and patients with AD and chronic dermatophyte infections may show improvement in their AD when treated with antifungal drugs. AD - Department of Dermatology, Sahlgrenska University Hospital, Gothenburg, Sweden. jan.faergemann@derm.gu.se FAU - Faergemann, Jan AU - Faergemann J LA - eng PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 RN - 0 (Antibodies, Fungal) RN - 0 (Antigens, Fungal) RN - 0 (Cytokines) RN - 37341-29-0 (Immunoglobulin E) RN - 65277-42-1 (Ketoconazole) SB - IM MH - Animals MH - Antibodies, Fungal/blood MH - Antigens, Fungal/analysis MH - Candida/immunology/*isolation & purification MH - Cytokines/biosynthesis MH - Dermatitis, Atopic/drug therapy/*etiology/microbiology MH - Humans MH - Immunoglobulin E/blood MH - Ketoconazole/therapeutic use MH - Lymphocyte Activation MH - Malassezia/immunology/*isolation & purification RF - 168 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):545-63. PMID- 12364370 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - Evolutionary and historical aspects of the burden of malaria. PG - 564-94 AB - Malaria is among the oldest of diseases. In one form or another, it has infected and affected our ancestors since long before the origin of the human line. During our recent evolution, its influence has probably been greater than that of any other infectious agent. Here we attempt to trace the forms and impacts of malaria from a distant past through historical times to the present. In the last sections, we review the current burdens of malaria across the world and discuss present-day approaches to its management. Only by following, or attempting to follow, malaria throughout its evolution and history can we understand its character and so be better prepared for our future management of this ancient ill. AD - University of Edinburgh, Division of Biological Sciences, ICAPB, Ashworth Laboratories, Edinburgh EH9 3JT, United Kingdom. r.carter@ed.ac.uk FAU - Carter, Richard AU - Carter R FAU - Mendis, Kamini N AU - Mendis KN LA - eng PT - Historical Article PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 RN - 0 (Duffy Blood-Group System) RN - 9008-00-8 (Hemoglobin C) SB - IM EIN - Clin Microbiol Rev. 2003 Jan;16(1):173. MH - Adaptation, Physiological MH - Animals MH - Cost of Illness MH - Duffy Blood-Group System/genetics MH - *Evolution MH - Glucosephosphate Dehydrogenase Deficiency/immunology MH - Hemoglobin C/genetics MH - History, 20th Century MH - History, Ancient MH - Humans MH - *Malaria/epidemiology/etiology/history/transmission MH - Thalassemia/immunology RF - 219 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):564-94. PMID- 12364371 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - History of human parasitology. PG - 595-612 AB - Humans are hosts to nearly 300 species of parasitic worms and over 70 species of protozoa, some derived from our primate ancestors and some acquired from the animals we have domesticated or come in contact with during our relatively short history on Earth. Our knowledge of parasitic infections extends into antiquity, and descriptions of parasites and parasitic infections are found in the earliest writings and have been confirmed by the finding of parasites in archaeological material. The systematic study of parasites began with the rejection of the theory of spontaneous generation and the promulgation of the germ theory. Thereafter, the history of human parasitology proceeded along two lines, the discovery of a parasite and its subsequent association with disease and the recognition of a disease and the subsequent discovery that it was caused by a parasite. This review is concerned with the major helminth and protozoan infections of humans: ascariasis, trichinosis, strongyloidiasis, dracunculiasis, lymphatic filariasis, loasis, onchocerciasis, schistosomiasis, cestodiasis, paragonimiasis, clonorchiasis, opisthorchiasis, amoebiasis, giardiasis, African trypanosomiasis, South American trypanosomiasis, leishmaniasis, malaria, toxoplasmosis, cryptosporidiosis, cyclosporiasis, and microsporidiosis. AD - Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom. frank.cox@lshtm.ac.uk FAU - Cox, F E G AU - Cox FE LA - eng PT - Historical Article PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 SB - IM EIN - Clin Microbiol Rev. 2003 Jan;16(1):174. MH - Animals MH - Civilization MH - Emigration and Immigration MH - Evolution MH - Helminthiasis/*history MH - Helminths/isolation & purification MH - History, 19th Century MH - History, 20th Century MH - History, Ancient MH - Humans MH - Parasitology/history MH - Protozoa/isolation & purification MH - Protozoan Infections/*history MH - Research Support, Non-U.S. Gov't RF - 281 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):595-612. PMID- 12364372 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - What happened to the streptococci: overview of taxonomic and nomenclature changes. PG - 613-30 AB - Since the division of the Streptococcus genus into enterococci, lactococci, and streptococci in 1984, many changes in the nomenclature and taxonomy of the Streptococcus genus have taken place. The application of genetic comparisons has improved the proper classification of the different species. The Lancefield system of serogrouping the streptococci by the expression of beta-hemolysis on blood agar plates is still very useful for the identification of streptococci for patient management. The Lancefield grouping system cannot be used in itself for accurate identification of specific beta-hemolytic species, but it can be a useful part of the identification procedure. Except for identification of the "Streptococcus bovis group" of species and Streptococcus suis, Lancefield grouping is of little value in identification of the non-beta-hemolytic streptococci and related genera. In fact, identification of the non-beta-hemolytic species is problematic for conventional as well as commercially available identification procedures. A combination of conventional tests and specific chromogenic tests suggested by several investigators is presented and discussed. Tables are included that suggest tests and procedures to guide investigators attempting to identify all the species. AD - Streptococcus Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA. rrf2@cdc.gov FAU - Facklam, Richard AU - Facklam R LA - eng PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 SB - IM MH - Streptococcus/*classification MH - *Terminology RF - 151 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):613-30. PMID- 12364373 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - Tularemia. PG - 631-46 AB - Francisella tularensis is the etiological agent of tularemia, a serious and occasionally fatal disease of humans and animals. In humans, ulceroglandular tularemia is the most common form of the disease and is usually a consequence of a bite from an arthropod vector which has previously fed on an infected animal. The pneumonic form of the disease occurs rarely but is the likely form of the disease should this bacterium be used as a bioterrorism agent. The diagnosis of disease is not straightforward. F. tularensis is difficult to culture, and the handling of this bacterium poses a significant risk of infection to laboratory personnel. Enzyme-linked immunosorbent assay- and PCR-based methods have been used to detect bacteria in clinical samples, but these methods have not been adequately evaluated for the diagnosis of pneumonic tularemia. Little is known about the virulence mechanisms of F. tularensis, though there is a large body of evidence indicating that it is an intracellular pathogen, surviving mainly in macrophages. An unlicensed live attenuated vaccine is available, which does appear to offer protection against ulceroglandular and pneumonic tularemia. Although an improved vaccine against tularemia is highly desirable, attempts to devise such a vaccine have been limited by the inability to construct defined allelic replacement mutants and by the lack of information on the mechanisms of virulence of F. tularensis. In the absence of a licensed vaccine, aminoglycoside antibiotics play a key role in the prevention and treatment of tularemia. AD - Defence Science and Technology Laboratory, CBS Porton Down, Salisbury, Wiltshire SP4 0JQ, United Kingdom. FAU - Ellis, Jill AU - Ellis J FAU - Oyston, Petra C F AU - Oyston PC FAU - Green, Michael AU - Green M FAU - Titball, Richard W AU - Titball RW LA - eng PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 RN - 0 (Bacterial Vaccines) SB - IM MH - Animals MH - Bacterial Vaccines/immunology MH - Disease Models, Animal MH - Francisella tularensis/classification/genetics/isolation & purification MH - Gene Expression Regulation, Bacterial MH - Humans MH - *Tularemia/drug therapy/epidemiology/prevention & control MH - Virulence RF - 185 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):631-46. PMID- 12364374 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - Interactions among strategies associated with bacterial infection: pathogenicity, epidemicity, and antibiotic resistance. PG - 647-79 AB - Infections have been the major cause of disease throughout the history of human populations. With the introduction of antibiotics, it was thought that this problem should disappear. However, bacteria have been able to evolve to become antibiotic resistant. Nowadays, a proficient pathogen must be virulent, epidemic, and resistant to antibiotics. Analysis of the interplay among these features of bacterial populations is needed to predict the future of infectious diseases. In this regard, we have reviewed the genetic linkage of antibiotic resistance and bacterial virulence in the same genetic determinants as well as the cross talk between antibiotic resistance and virulence regulatory circuits with the aim of understanding the effect of acquisition of resistance on bacterial virulence. We also discuss the possibility that antibiotic resistance and bacterial virulence might prevail as linked phenotypes in the future. The novel situation brought about by the worldwide use of antibiotics is undoubtedly changing bacterial populations. These changes might alter the properties of not only bacterial pathogens, but also the normal host microbiota. The evolutionary consequences of the release of antibiotics into the environment are largely unknown, but most probably restoration of the microbiota from the preantibiotic era is beyond our current abilities. AD - Departamento de Biotecnologia Microbiana, Centro Nacional de Biotecnologia. Servicio de Microbiologia, Hospital Ramon y Cajal, Madrid, Spain. jlmtnez@cnb.uam.ed FAU - Martinez, Jose L AU - Martinez JL FAU - Baquero, Fernando AU - Baquero F LA - eng PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 RN - 0 (DNA Transposable Elements) RN - 0 (Plasmids) SB - IM MH - Animals MH - Bacteria/classification/genetics/pathogenicity MH - Bacterial Infections/*drug therapy/epidemiology/etiology MH - DNA Transposable Elements MH - *Drug Resistance, Bacterial/genetics MH - Ecology MH - Humans MH - Opportunistic Infections/drug therapy MH - Plasmids MH - Research Support, Non-U.S. Gov't MH - Virulence/genetics RF - 428 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):647-79. PMID- 12364375 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - Diagnosis and management of human cytomegalovirus infection in the mother, fetus, and newborn infant. PG - 680-715 AB - Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection and mental retardation. HCMV infection, while causing asymptomatic infections in most immunocompetent subjects, can be transmitted during pregnancy from the mother with primary (and also recurrent) infection to the fetus. Hence, careful diagnosis of primary infection is required in the pregnant woman based on the most sensitive serologic assays (immunoglobulin M [IgM] and IgG avidity assays) and conventional virologic and molecular procedures for virus detection in blood. Maternal prognostic markers of fetal infection are still under investigation. If primary infection is diagnosed in a timely manner, prenatal diagnosis can be offered, including the search for virus and virus components in fetal blood and amniotic fluid, with fetal prognostic markers of HCMV disease still to be defined. However, the final step for definite diagnosis of congenital HCMV infection is detection of virus in the blood or urine in the first 1 to 2 weeks of life. To date, treatment of congenital infection with antiviral drugs is only palliative both prior to and after birth, whereas the only efficacious preventive measure seems to be the development of a safe and immunogenic vaccine, including recombinant, subunit, DNA, and peptide-based vaccines now under investigation. The following controversial issues are discussed in the light of the most recent advances in the field: the actual perception of the problem; universal serologic screening before pregnancy; the impact of correct counseling on decision making by the couple involved; the role of prenatal diagnosis in ascertaining transmission of virus to the fetus; the impact of preconceptional and periconceptional infections on the prevalence of congenital infection; and the prevalence of congenitally infected babies born to mothers who were immune prior to pregnancy compared to the number born to mothers undergoing primary infection during pregnancy. AD - Servizio di Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy. FAU - Revello, Maria Grazia AU - Revello MG FAU - Gerna, Giuseppe AU - Gerna G LA - eng PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 RN - 0 (Antibodies, Viral) RN - 0 (Cytomegalovirus Vaccines) RN - 0 (DNA, Viral) RN - 0 (Immunoglobulin M) RN - 0 (RNA, Viral) SB - IM MH - Amniotic Fluid/virology MH - Antibodies, Viral/blood MH - Counseling MH - Cytomegalovirus Infections/congenital/*diagnosis/therapy MH - Cytomegalovirus Vaccines/immunology MH - DNA, Viral/blood MH - Disease Transmission, Vertical MH - Diseases in Twins MH - Female MH - Humans MH - Immunoglobulin M/blood MH - Infant, Newborn MH - Pregnancy MH - Pregnancy Complications, Infectious MH - Prenatal Diagnosis MH - RNA, Viral/blood MH - Research Support, Non-U.S. Gov't MH - Serologic Tests RF - 295 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):680-715. PMID- 12364376 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. PG - 716-46 AB - The history, taxonomy, geographic distribution, clinical disease, and therapy of the pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria (RGM) are reviewed. Community-acquired disease and health care-associated disease are highlighted for each species. The latter grouping includes health care-associated outbreaks and pseudo-outbreaks as well as sporadic disease cases. Treatment recommendations for each species and type of disease are also described. Special emphasis is on the Mycobacterium fortuitum group, including M. fortuitum, M. peregrinum, and the unnamed third biovariant complex with its recent taxonomic changes and newly recognized species (including M. septicum, M. mageritense, and proposed species M. houstonense and M. bonickei). The clinical and taxonomic status of M. chelonae, M. abscessus, and M. mucogenicum is also detailed, along with that of the closely related new species, M. immunogenum. Additionally, newly recognized species, M. wolinskyi and M. goodii, as well as M. smegmatis sensu stricto, are included in a discussion of the M. smegmatis group. Laboratory diagnosis of RGM using phenotypic methods such as biochemical testing and high-performance liquid chromatography and molecular methods of diagnosis are also discussed. The latter includes PCR-restriction fragment length polymorphism analysis, hybridization, ribotyping, and sequence analysis. Susceptibility testing and antibiotic susceptibility patterns of the RGM are also annotated, along with the current recommendations from the National Committee for Clinical Laboratory Standards (NCCLS) for mycobacterial susceptibility testing. AD - University of Texas Health Center, Department of Microbiology, Tyler, Texas 75708, USA. barbara.elliott@uthct.edu FAU - Brown-Elliott, Barbara A AU - Brown-Elliott BA FAU - Wallace, Richard J Jr AU - Wallace RJ Jr LA - eng PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 RN - 0 (Anti-Bacterial Agents) SB - IM MH - Anti-Bacterial Agents/therapeutic use MH - Catheterization/adverse effects MH - Community-Acquired Infections/microbiology MH - Humans MH - Microbial Sensitivity Tests/methods MH - Mycobacterium Infections, Atypical/diagnosis/*microbiology/therapy MH - Mycobacterium chelonae/*classification/drug effects/genetics MH - Mycobacterium fortuitum/*classification/drug effects/genetics MH - Mycobacterium smegmatis/*classification/drug effects/genetics MH - Occupational Diseases/microbiology MH - Skin Diseases, Infectious/microbiology MH - Wound Infection/microbiology RF - 226 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):716-46. PMID- 12364377 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - Current consensus guidelines for treatment of neurocysticercosis. PG - 747-56 AB - Taenia solium neurocysticercosis is a common cause of epileptic seizures and other neurological morbidity in most developing countries. It is also an increasingly common diagnosis in industrialized countries because of immigration from areas where it is endemic. Its clinical manifestations are highly variable and depend on the number, stage, and size of the lesions and the host's immune response. In part due to this variability, major discrepancies exist in the treatment of neurocysticercosis. A panel of experts in taeniasis/cysticercosis discussed the evidence on treatment of neurocysticercosis for each clinical presentation, and we present the panel's consensus and areas of disagreement. Overall, four general recommendations were made: (i) individualize therapeutic decisions, including whether to use antiparasitic drugs, based on the number, location, and viability of the parasites within the nervous system; (ii) actively manage growing cysticerci either with antiparasitic drugs or surgical excision; (iii) prioritize the management of intracranial hypertension secondary to neurocysticercosis before considering any other form of therapy; and (iv) manage seizures as done for seizures due to other causes of secondary seizures (remote symptomatic seizures) because they are due to an organic focus that has been present for a long time. AD - Cysticercosis Unit, Instituto Nacional de Ciencias Neurologicas, Universidad Peruana Cayetano Heredia. School of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru. hgarcia@terra.com.pe FAU - Garcia, Hector H AU - Garcia HH FAU - Evans, Carlton A W AU - Evans CA FAU - Nash, Theodore E AU - Nash TE FAU - Takayanagui, Osvaldo M AU - Takayanagui OM FAU - White, A Clinton Jr AU - White AC Jr FAU - Botero, David AU - Botero D FAU - Rajshekhar, Vedantam AU - Rajshekhar V FAU - Tsang, Victor C W AU - Tsang VC FAU - Schantz, Peter M AU - Schantz PM FAU - Allan, James C AU - Allan JC FAU - Flisser, Ana AU - Flisser A FAU - Correa, Dolores AU - Correa D FAU - Sarti, Elsa AU - Sarti E FAU - Friedland, Jon S AU - Friedland JS FAU - Martinez, S Manuel AU - Martinez SM FAU - Gonzalez, Armando E AU - Gonzalez AE FAU - Gilman, Robert H AU - Gilman RH FAU - Del Brutto, Oscar H AU - Del Brutto OH LA - eng PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 RN - 0 (Antiparasitic Agents) SB - IM MH - Animals MH - Antiparasitic Agents/*therapeutic use MH - Humans MH - Neurocysticercosis/diagnosis/*therapy MH - *Practice Guidelines RF - 111 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):747-56. PMID- 12364378 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - Effects of global climate on infectious disease: the cholera model. PG - 757-70 AB - Recently, the role of the environment and climate in disease dynamics has become a subject of increasing interest to microbiologists, clinicians, epidemiologists, and ecologists. Much of the interest has been stimulated by the growing problems of antibiotic resistance among pathogens, emergence and/or reemergence of infectious diseases worldwide, the potential of bioterrorism, and the debate concerning climate change. Cholera, caused by Vibrio cholerae, lends itself to analyses of the role of climate in infectious disease, coupled to population dynamics of pathogenic microorganisms, for several reasons. First, the disease has a historical context linking it to specific seasons and biogeographical zones. In addition, the population dynamics of V. cholerae in the environment are strongly controlled by environmental factors, such as water temperature, salinity, and the presence of copepods, which are, in turn, controlled by larger-scale climate variability. In this review, the association between plankton and V. cholerae that has been documented over the last 20 years is discussed in support of the hypothesis that cholera shares properties of a vector-borne disease. In addition, a model for environmental transmission of cholera to humans in the context of climate variability is presented. The cholera model provides a template for future research on climate-sensitive diseases, allowing definition of critical parameters and offering a means of developing more sophisticated methods for prediction of disease outbreaks. AD - Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202, USA. FAU - Lipp, Erin K AU - Lipp EK FAU - Huq, Anwar AU - Huq A FAU - Colwell, Rita R AU - Colwell RR LA - eng GR - R01NR042701A1/NR/NINR PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - Clin Microbiol Rev JID - 8807282 SB - IM MH - Air Microbiology MH - Animals MH - Cholera/*transmission MH - *Climate MH - Disease Vectors MH - Ecology MH - Humans MH - Phytoplankton/microbiology MH - Research Support, U.S. Gov't, P.H.S. MH - Seasons MH - Water Microbiology MH - Zooplankton/microbiology RF - 165 EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):757-70. PMID- 12364379 OWN - NLM STAT- MEDLINE DA - 20021004 DCOM- 20021120 LR - 20041117 PUBM- Print IS - 0893-8512 VI - 15 IP - 4 DP - 2002 Oct TI - Malaria rapid diagnostic tests: one size may not fit all. PG - 771; discussion 771-2 FAU - Bell, David AU - Bell D LA - eng PT - Comment PT - Journal Article PL - United States TA - Clin Microbiol Rev JID - 8807282 RN - 0 (Proteins) RN - 0 (histidine-rich proteins) SB - IM CON - Clin Microbiol Rev. 2002 Jan;15(1):66-78. PMID: 11781267 MH - Humans MH - Malaria/*diagnosis MH - Proteins/*analysis MH - Sensitivity and Specificity MH - Time Factors EDAT- 2002/10/05 04:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Clin Microbiol Rev 2002 Oct;15(4):771; discussion 771-2. PMID- 12370087 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021218 LR - 20041215 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Oct 8 TI - Inhibition of the MEK1/ERK pathway reduces arachidonic acid release independently of cPLA2 phosphorylation and translocation. PG - 30 AB - BACKGROUND: The 85-kDa cytosolic phospholipase A2 (cPLA2) mediates arachidonic acid (AA) release in MDCK cells. Although calcium and mitogen-activated protein kinases regulate cPLA2, the correlation of cPLA2 translocation and phosphorylation with MAPK activation and AA release is unclear. RESULTS: MEK1 inhibition by U0126 inhibited AA release in response to ATP and ionomycin. This directly correlated with inhibition of ERK activation but not with phosphorylation of cPLA2 on Ser505, which was only partially inhibited by ERK inhibition. Inhibition of AA release by U0126 was still observed when stoichiometric phosphorylation of cPLA2 on Ser505 was maintained by activating p38 with anisomycin. Translocation kinetics of wild-type cPLA2 and cPLA2 containing S505A or S727A mutations to Golgi were similar in response to ATP and ionomycin and were not affected by U0126. CONCLUSIONS: These results suggest that the ability of cPLA2 to hydrolyze membrane phospholipid is reduced by inhibition of the MEK1/ERK pathway and that the reduction in activity is independent of cPLA2 phosphorylation and translocation to membrane. The results also demonstrate that cPLA2 mutated at the phosphorylation sites Ser505 and Ser727 translocated with similar kinetic as wild-type cPLA2. AD - Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA. evansj@njc.org FAU - Evans, John H AU - Evans JH FAU - Fergus, Daniel J AU - Fergus DJ FAU - Leslie, Christina C AU - Leslie CC LA - eng GR - HL10507/HL/NHLBI GR - HL34303/HL/NHLBI GR - HL61378/HL/NHLBI PT - Journal Article DEP - 20021008 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Butadienes) RN - 0 (Enzyme Inhibitors) RN - 0 (Luminescent Proteins) RN - 0 (MAP Kinase Signaling System) RN - 0 (Nitriles) RN - 0 (U 0126) RN - 147336-22-9 (Green Fluorescent Proteins) RN - 506-32-1 (Arachidonic Acid) RN - 56-65-5 (Adenosine Triphosphate) RN - 56092-81-0 (Ionomycin) RN - 7440-70-2 (Calcium) RN - EC 2.7.1.- (MAP Kinase Kinase 1) RN - EC 2.7.1.- (MAP2K1 protein, human) RN - EC 2.7.1.- (Mitogen-Activated Protein Kinase Kinases) RN - EC 2.7.1.37 (Mitogen-Activated Protein Kinases) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 3.1.1.- (Phospholipases A) SB - IM MH - Adenosine Triphosphate/pharmacology MH - Animals MH - Arachidonic Acid/*secretion MH - Binding Sites/genetics MH - Biological Transport/drug effects MH - Butadienes/*pharmacology MH - Calcium/metabolism MH - Cell Line MH - Cytosol/enzymology MH - Enzyme Inhibitors/*pharmacology MH - Green Fluorescent Proteins MH - Humans MH - Ionomycin/pharmacology MH - Luminescent Proteins/genetics/metabolism MH - MAP Kinase Kinase 1 MH - MAP Kinase Signaling System/drug effects MH - Microscopy, Fluorescence MH - Mitogen-Activated Protein Kinase Kinases/*antagonists & inhibitors/metabolism MH - Mitogen-Activated Protein Kinases/*antagonists & inhibitors/metabolism MH - Mutation MH - Nitriles/*pharmacology MH - Phospholipases A/genetics/*metabolism MH - Phosphorylation/drug effects MH - Protein-Serine-Threonine Kinases/*antagonists & inhibitors/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2002/10/09 04:00 MHDA- 2002/12/19 04:00 PHST- 2002/06/07 [received] PHST- 2002/10/08 [accepted] PHST- 2002/10/08 [aheadofprint] PST - ppublish SO - BMC Biochem 2002 Oct 8;3(1):30. Epub 2002 Oct 8. PMID- 12370088 OWN - NLM STAT- Publisher DA - 20021008 PUBM- Print IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Sep 20 TI - MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function. PG - 28 AB - BACKGROUND: Three macromolecular assemblages, the lid complex of the proteasome, the COP9-Signalosome (CSN) and the eIF3 complex, all consist of multiple proteins harboring MPN and PCI domains. Up to now, no specific function for any of these proteins has been defined, nor has the importance of these motifs been elucidated. In particular Rpn11, a lid subunit, serves as the paradigm for MPN-containing proteins as it is highly conserved and important for proteasome function. RESULTS: We have identified a sequence motif, termed the MPN+ motif, which is highly conserved in a subset of MPN domain proteins such as Rpn11 and Csn5/Jab1, but is not present outside of this subfamily. The MPN+ motif consists of five polar residues that resemble the active site residues of hydrolytic enzyme classes, particularly that of metalloproteases. By using site-directed mutagenesis, we show that the MPN+ residues are important for the function of Rpn11, while a highly conserved Cys residue outside of the MPN+ motif is not essential. Single amino acid substitutions in MPN+ residues all show similar phenotypes, including slow growth, sensitivity to temperature and amino acid analogs, and general proteasome-dependent proteolysis defects. CONCLUSIONS: The MPN+ motif is abundant in certain MPN-domain proteins, including newly identified proteins of eukaryotes, bacteria and archaea thought to act outside of the traditional large PCI/MPN complexes. The putative catalytic nature of the MPN+ motif makes it a good candidate for a pivotal enzymatic function, possibly a proteasome-associated deubiquitinating activity and a CSN-associated Nedd8/Rub1-removing activity. AD - Dept, of Biology and Institute for Catalysis Science and Technology (ICST) Technion - Israel Institute of Technology, Israel. glickman@tx.technion.ac.il AU - Maytal-Kivity V AU - Reis N AU - Hofmann K AU - Glickman MH LA - ENG PT - JOURNAL ARTICLE TA - BMC Biochem JID - 101084098 EDAT- 2002/10/09 04:00 MHDA- 2002/10/09 04:00 PHST- 2002/07/24 [received] PHST- 2002/09/20 [accepted] PHST- 2002/09/20 [aheadofprint] PST - aheadofprint SO - BMC Biochem 2002 Sep 20;3(1):28. PMID- 12398769 OWN - NLM STAT- MEDLINE DA - 20021025 DCOM- 20021203 LR - 20041117 PUBM- Print-Electronic IS - 1364-8535 VI - 6 IP - 5 DP - 2002 Oct TI - Hypothermia and neurologic outcome in patients following cardiac arrest: should we be hot to cool off our patients? PG - 377-80 AB - Hypothermia as a protectant of neurologic function in the treatment of cardiac arrest patients, although not a new concept, is now supported by two recent randomized, prospective clinical trials. The basic science research in support of the effects of hypothermia at the cellular and animal levels is extensive. The process of cooling for cerebral protection holds potential promise for human resuscitation efforts in multiple realms. It appears that, at least, those patients who suffer a witnessed cardiac arrest with ventricular fibrillation and early restoration of spontaneous circulation, such as those who were included in the European and Australian trials (discussed here), should be considered for hypothermic therapy. AD - Neuroscience Critical Care, and Clinical Instructor of Neurology, University of Virginia School of Medicine, Charlottesville, Virginia, USA. tls2u@virginia.edu FAU - Smith, Teresa L AU - Smith TL FAU - Bleck, Thomas P AU - Bleck TP LA - eng PT - Journal Article PT - Review PT - Review, Tutorial DEP - 20020816 PL - England TA - Crit Care JID - 9801902 SB - IM MH - Animals MH - Australia MH - Europe MH - Heart Arrest/*therapy MH - Humans MH - Hypothermia, Induced/adverse effects/*methods MH - Randomized Controlled Trials MH - Treatment Outcome RF - 22 EDAT- 2002/10/26 04:00 MHDA- 2002/12/04 04:00 PHST- 2002/08/16 [aheadofprint] PST - ppublish SO - Crit Care 2002 Oct;6(5):377-80. Epub 2002 Aug 16. PMID- 12398770 OWN - NLM STAT- MEDLINE DA - 20021025 DCOM- 20021203 LR - 20041117 PUBM- Print-Electronic IS - 1364-8535 VI - 6 IP - 5 DP - 2002 Oct TI - The International Sepsis Forum's controversies in sepsis: corticosteroids should be used to treat septic shock. PG - 381-3 AB - The use of corticosteroids in septic shock remains controversial. It has been demonstrated that high doses of steroids (30 mg/kg methylprednisolone) for short periods of time are not beneficial. More recent studies using smaller doses (200-300 mg/day hydrocortisone) for longer periods of time have shown beneficial effects. These positive effects have included reversal of shock, trends toward decreased organ system dysfunction and decreased mortality. Based on the high proportion of patients who have relative adrenal insufficiency, the benefits of low doses of steroids and the minimal risks, steroids should be used to treat septic shock. AD - Department of Anesthesiology and Critical Care Medicine, Hadassah Hebrew University Medical Center, The Hebrew University of Jerusalem, Israel. FAU - Goodman, Sergey AU - Goodman S FAU - Sprung, Charles L AU - Sprung CL CN - International Sepsis Forum. LA - eng PT - Journal Article PT - Review PT - Review, Tutorial DEP - 20020717 PL - England TA - Crit Care JID - 9801902 RN - 0 (Adrenal Cortex Hormones) RN - 9002-60-2 (Corticotropin) SB - IM MH - Adrenal Cortex Hormones/*therapeutic use MH - Corticotropin/administration & dosage/*therapeutic use MH - Humans MH - Randomized Controlled Trials MH - Shock, Septic/*drug therapy/mortality MH - Treatment Outcome RF - 10 EDAT- 2002/10/26 04:00 MHDA- 2002/12/04 04:00 PHST- 2002/07/17 [aheadofprint] PST - ppublish SO - Crit Care 2002 Oct;6(5):381-3. Epub 2002 Jul 17. PMID- 12398771 OWN - NLM STAT- MEDLINE DA - 20021025 DCOM- 20021203 LR - 20041117 PUBM- Print-Electronic IS - 1364-8535 VI - 6 IP - 5 DP - 2002 Oct TI - The International Sepsis Forum's controversies in sepsis: corticosteroids should not be routinely used to treat septic shock. PG - 384-6 AB - Corticosteroid treatment of severe sepsis has been one of the most controversial clinical issues in critical care. In fact, few agents can claim to have been evaluated in scores of studies spanning 3-4 decades. Yet, convincing proof that corticosteroids are useful pharmacologic agents in the treatment of this major clinical problem remains elusive. Recently, interest has resurfaced but this time the focus is on a steroid replacement approach for what has now been termed "relative adrenal insufficiency" rather than relying on the pharmacologic effects of steroids. This route holds promise, but proof remains lacking. AD - Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennessee, USA. Gordon.Bernard@Vanderbilt.edu FAU - Bernard, Gordon AU - Bernard G CN - International Sepsis Forum. LA - eng PT - Journal Article PT - Review PT - Review, Tutorial DEP - 20020717 PL - England TA - Crit Care JID - 9801902 RN - 0 (Adrenal Cortex Hormones) RN - 50-23-7 (Hydrocortisone) SB - IM MH - Adrenal Cortex Hormones/adverse effects/*therapeutic use MH - Humans MH - Hydrocortisone/blood MH - Randomized Controlled Trials MH - Shock, Septic/blood/*drug therapy/mortality RF - 19 EDAT- 2002/10/26 04:00 MHDA- 2002/12/04 04:00 PHST- 2002/07/17 [aheadofprint] PST - ppublish SO - Crit Care 2002 Oct;6(5):384-6. Epub 2002 Jul 17. PMID- 12398772 OWN - NLM STAT- MEDLINE DA - 20021025 DCOM- 20021203 LR - 20041117 PUBM- Print-Electronic IS - 1364-8535 VI - 6 IP - 5 DP - 2002 Oct TI - Paediatric intensive care: out of commission. PG - 387-8 AB - Problems with commissioning paediatric intensive care stem both from difficulties in recruitment and retention of nurses, and from incoherent or nonexistent national audit. Pyramidal career structures and patterns of remuneration that concentrate on administrative responsibility over clinical skills underlie the former, whereas poor audit conceals variations in both service quality and demand. Epidemiologically superior data are required if we are to solve commissioning problems. We need to know what happened to every child from a defined population receiving intensive care and whether a lack of resources means that some children are denied intensive care. AD - Birmingham Childrens Hospital, Steelhouse Lane, Birmingham, UK. Gale.Pearson@Bhamchildrens.wmids.nhs.uk FAU - Pearson, Gale A AU - Pearson GA LA - eng PT - Journal Article DEP - 20020709 PL - England TA - Crit Care JID - 9801902 SB - IM MH - Child MH - Critical Care/*trends MH - Great Britain MH - Humans MH - Intensive Care Units, Pediatric/manpower/*trends MH - Mortality MH - *Quality of Health Care EDAT- 2002/10/26 04:00 MHDA- 2002/12/04 04:00 PHST- 2002/07/09 [aheadofprint] PST - ppublish SO - Crit Care 2002 Oct;6(5):387-8. Epub 2002 Jul 9. PMID- 12398773 OWN - NLM STAT- MEDLINE DA - 20021025 DCOM- 20021203 LR - 20041117 PUBM- Print-Electronic IS - 1364-8535 VI - 6 IP - 5 DP - 2002 Oct TI - Is insulin an endogenous cardioprotector? PG - 389-93 AB - Stress hyperglycemia and diabetes mellitus with myocardial infarction are associated with increased risk for in-hospital mortality, congestive heart failure, or cardiogenic shock. Hyperglycemia triggers free radical generation and suppresses endothelial nitric oxide generation, and thus initiates and perpetuates inflammation. Conversely, insulin suppresses production of tumor necrosis factor-alpha and free radicals, enhances endothelial nitric oxide generation, and improves myocardial function. It is proposed that the balance between insulin and plasma glucose levels is critical to recovery and/or complications that occur following acute myocardial infarction and in the critically ill. Adequate attention should be given to maintaining euglycemia (plasma glucose FAU - Soto, Carlos M AU - Soto CM FAU - Kleinman, Kenneth P AU - Kleinman KP FAU - Simon, Steven R AU - Simon SR LA - eng PT - Journal Article DEP - 20021210 PL - England TA - BMC Health Serv Res JID - 101088677 RN - 0 (Pharmaceutical Preparations) SB - IM MH - Ambulatory Care Information Systems/*standards MH - Documentation/*standards MH - Female MH - Guideline Adherence/statistics & numerical data MH - Health Maintenance Organizations/*organization & administration/standards MH - Health Services Research MH - Humans MH - Immunization MH - Internal Medicine/organization & administration MH - Male MH - Mass Screening/standards MH - Massachusetts MH - Medical Records Systems, Computerized/*standards MH - Pediatrics/organization & administration MH - Pharmaceutical Preparations/adverse effects MH - Primary Health Care/*organization & administration MH - Quality Control MH - Research Support, Non-U.S. Gov't MH - Self Administration MH - Smoking Cessation EDAT- 2002/12/11 04:00 MHDA- 2003/04/23 05:00 PHST- 2002/09/09 [received] PHST- 2002/12/10 [accepted] PHST- 2002/12/10 [aheadofprint] PST - epublish SO - BMC Health Serv Res 2002 Dec 10;2(1):22. PMID- 12509223 OWN - NLM STAT- MEDLINE DA - 20030206 DCOM- 20030307 LR - 20050111 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Dec 31 TI - Src kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells. PG - 32 AB - BACKGROUND: The cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor) - dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1) and Flt-1 (Fms-like tyrosine kinase-1). However, to date, it has not been determined which VEGF receptor (VEGFR) is involved in binding to and activating Src kinase following VEGF stimulation of the receptors. RESULTS: In this report, we demonstrate that Src preferentially associates with KDR/Flk-1 rather than Flt-1 in human umbilical vein endothelial cells (HUVECs), and that VEGF stimulation resulted in an increase of Src activity associated with activated KDR/Flk-1. These findings were determined through immunoprecipitation-kinase experiments and coimmunoprecipitation studies, and were further confirmed by GST-pull-down assays and Far Western studies. However, Fyn and Yes, unlike Src, were found to associate preferentially with Flt-1. CONCLUSIONS: Thus, Src preferentially associates with KDR/Flk-1, rather than with Flt-1, upon VEGF stimulation in endothelial cells. Our findings further highlight the potential significance of upregulated KDR/Flk-1-associated Src activity in the process of angiogenesis, and help to elucidate more clearly the specific roles and mechanisms involving Src family tyrosine kinase in VEGF-stimulated signal transduction events. AD - Department of Biochemistry and Molecular Biology, University of Calgary Health Sciences Center, 3330 Hospital Dr, N,W, Calgary, AB, Canada T2N 4N1. mthchou@ucalgary.ca FAU - Chou, Mary T AU - Chou MT FAU - Wang, Jing AU - Wang J FAU - Fujita, Donald J AU - Fujita DJ LA - eng PT - Journal Article DEP - 20021231 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Endothelial Growth Factors) RN - 0 (Intercellular Signaling Peptides and Proteins) RN - 0 (Lymphokines) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Vascular Endothelial Growth Factor A) RN - 0 (Vascular Endothelial Growth Factors) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.112 (Vascular Endothelial Growth Factor Receptor-1) RN - EC 2.7.1.112 (Vascular Endothelial Growth Factor Receptor-2) RN - EC 2.7.1.112 (protein-tyrosine kinase c-src) RN - EC 2.7.1.112 (proto-oncogene protein c-fyn) RN - EC 2.7.1.112 (proto-oncogene protein c-yes) RN - EC 2.7.1.112 (src-Family Kinases) SB - IM MH - Cells, Cultured MH - Cytoplasm/enzymology MH - Endothelial Growth Factors/isolation & purification/*metabolism MH - Endothelium, Vascular/chemistry/cytology/*enzymology/*metabolism MH - Enzyme Activation/physiology MH - Glutathione Transferase/biosynthesis/genetics MH - Humans MH - Intercellular Signaling Peptides and Proteins/isolation & purification/*metabolism MH - Lymphokines/isolation & purification/*metabolism MH - Peptide Mapping MH - Precipitin Tests MH - Protein-Tyrosine Kinase/biosynthesis/genetics/*metabolism/physiology MH - Proto-Oncogene Proteins/isolation & purification MH - Recombinant Fusion Proteins/biosynthesis/genetics MH - Research Support, Non-U.S. Gov't MH - Time Factors MH - Umbilical Veins MH - Vascular Endothelial Growth Factor A MH - Vascular Endothelial Growth Factor Receptor-1/immunology/isolation & purification/*metabolism/physiology MH - Vascular Endothelial Growth Factor Receptor-2/isolation & purification/*metabolism/physiology MH - Vascular Endothelial Growth Factors MH - src Homology Domains/physiology MH - *src-Family Kinases EDAT- 2003/01/02 04:00 MHDA- 2003/03/08 04:00 PHST- 2002/11/15 [received] PHST- 2002/12/31 [accepted] PHST- 2002/12/31 [aheadofprint] PST - ppublish SO - BMC Biochem 2002 Dec 31;3(1):32. Epub 2002 Dec 31. PMID- 12915527 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Reovirus receptors and pathogenesis. PG - 9109-15 AD - Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. FAU - Forrest, J Craig AU - Forrest JC FAU - Dermody, Terence S AU - Dermody TS LA - eng GR - AI38296/AI/NIAID GR - AI50080/AI/NIAID PT - Journal Article PT - Review PT - Review, Tutorial PL - United States TA - J Virol JID - 0113724 RN - 0 (Capsid Proteins) RN - 0 (Carbohydrates) RN - 0 (Cell Adhesion Molecules) RN - 0 (Receptors, Virus) RN - 0 (junctional adhesion molecule) RN - 0 (viral cell attachment protein sigma 1) RN - 131-48-6 (N-Acetylneuraminic Acid) SB - IM MH - Animals MH - Apoptosis MH - Capsid Proteins/chemistry/physiology MH - Carbohydrates/metabolism MH - Cell Adhesion Molecules/physiology MH - Humans MH - Mice MH - Models, Biological MH - Models, Molecular MH - N-Acetylneuraminic Acid/metabolism MH - Receptors, Virus/*physiology MH - Reoviridae/*pathogenicity/*physiology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. RF - 74 EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9109-15. PMID- 12915528 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Chimeric influenza A viruses with a functional influenza B virus neuraminidase or hemagglutinin. PG - 9116-23 AB - Reassortment of influenza A and B viruses has never been observed in vivo or in vitro. Using reverse genetics techniques, we generated recombinant influenza A/WSN/33 (WSN) viruses carrying the neuraminidase (NA) of influenza B virus. Chimeric viruses expressing the full-length influenza B/Yamagata/16/88 virus NA grew to titers similar to that of wild-type influenza WSN virus. Recombinant viruses in which the cytoplasmic tail or the cytoplasmic tail and the transmembrane domain of the type B NA were replaced with those of the type A NA were impaired in tissue culture. This finding correlates with reduced NA content in virions. We also generated a recombinant influenza A virus expressing a chimeric hemagglutinin (HA) protein in which the ectodomain is derived from type B/Yamagata/16/88 virus HA, whereas both the cytoplasmic and the transmembrane domains are derived from type A/WSN virus HA. This A/B chimeric HA virus did not grow efficiently in MDCK cells. However, after serial passage we obtained a virus population that grew to titers as high as wild-type influenza A virus in MDCK cells. One amino acid change in position 545 (H545Y) was found to be responsible for the enhanced growth characteristics of the passaged virus. Taken together, we show here that the absence of reassortment between influenza viruses belonging to different A and B types is not due to spike glycoprotein incompatibility at the level of the full-length NA or of the HA ectodomain. AD - Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, USA. FAU - Flandorfer, Astrid AU - Flandorfer A FAU - Garcia-Sastre, Adolfo AU - Garcia-Sastre A FAU - Basler, Christopher F AU - Basler CF FAU - Palese, Peter AU - Palese P LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Hemagglutinin Glycoproteins, Influenza Virus) RN - 0 (Recombinant Proteins) RN - EC 3.2.1.18 (Neuraminidase) SB - IM MH - Amino Acid Substitution MH - Animals MH - Cell Line MH - Dogs MH - Genes, Viral MH - Hemagglutinin Glycoproteins, Influenza Virus/chemistry/*genetics MH - Humans MH - Influenza A virus/*genetics/growth & development/physiology MH - Influenza B virus/*genetics/growth & development/physiology MH - Neuraminidase/chemistry/*genetics MH - Protein Structure, Tertiary MH - Recombinant Proteins/chemistry/genetics MH - *Recombination, Genetic MH - Research Support, U.S. Gov't, P.H.S. EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9116-23. PMID- 12915529 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Targeting of the turnip yellow mosaic virus 66K replication protein to the chloroplast envelope is mediated by the 140K protein. PG - 9124-35 AB - Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two replication proteins, 140K and 66K, both being required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase, and the 66K protein encompasses the RNA-dependent RNA polymerase domain. During viral infection, the 66K protein localizes to virus-induced chloroplastic membrane vesicles, which are closely associated with TYMV RNA replication. To investigate the determinants of its subcellular localization, the 66K protein was expressed in plant protoplasts from separate plasmids. Green fluorescent protein (GFP) fusion and immunofluorescence experiments demonstrated that the 66K protein displayed a cytoplasmic distribution when expressed individually but that it was relocated to the chloroplast periphery under conditions in which viral replication occurred. The 66K protein produced from an expression vector was functional in viral replication since it could transcomplement a defective replication template. Targeting of the 66K protein to the chloroplast envelope in the course of the viral infection appeared to be solely dependent on the expression of the 140K protein. Analysis of the subcellular localization of the 140K protein fused to GFP demonstrated that it is targeted to the chloroplast envelope in the absence of other viral factors and that it induces the clumping of the chloroplasts, one of the typical cytological effects of TYMV infection. These results suggests that the 140K protein is a key organizer of the assembly of the TYMV replication complexes and a major determinant for their chloroplastic localization and retention. AD - Laboratoire de Virologie Moleculaire, Institut Jacques Monod, UMR 7592, CNRS-Universites Paris 6-Paris 7, 75251 Paris Cedex 05, France. FAU - Prod'homme, Delphine AU - Prod'homme D FAU - Jakubiec, Anna AU - Jakubiec A FAU - Tournier, Vincent AU - Tournier V FAU - Drugeon, Gabriele AU - Drugeon G FAU - Jupin, Isabelle AU - Jupin I LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (Plasmids) RN - 0 (RNA, Viral) RN - 0 (Recombinant Proteins) RN - 0 (Viral Proteins) SB - IM MH - Arabidopsis/virology MH - Base Sequence MH - Brassica napus/virology MH - Chloroplasts/*virology MH - DNA, Viral/genetics MH - Intracellular Membranes/virology MH - Molecular Weight MH - Open Reading Frames MH - Plasmids/genetics MH - RNA, Viral/biosynthesis/genetics MH - Recombinant Proteins/chemistry/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Transfection MH - Tymovirus/genetics/*pathogenicity/*physiology MH - Viral Proteins/chemistry/genetics/*physiology MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9124-35. PMID- 12915530 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - A wild-type porcine encephalomyocarditis virus containing a short poly(C) tract is pathogenic to mice, pigs, and cynomolgus macaques. PG - 9136-46 AB - Previous studies using wild-type Encephalomyocarditis virus (EMCV) and Mengo virus, which have long poly(C) tracts (61 to 146 C's) at the 5' nontranslated region of the genome, and variants of these viruses genetically engineered to truncate or substitute the poly(C) tracts have produced conflicting data on the role of the poly(C) tract in the virulence of these viruses. Analysis of the nucleotide sequence of an EMCV strain isolated from an aborted swine fetus (EMCV 30/87) revealed that the virus had a poly(C) tract that was 7- to 10-fold shorter than the poly(C) tracts of other EMCV strains and 4-fold shorter than that of Mengo virus. Subsequently, we investigated the virulence and pathogenesis of this naturally occurring short-poly(C)-tract-containing virus in rodents, pigs, and nonhuman primates. Infection of C57BL/6 mice, pigs, and cynomolgus macaques resulted in similar EMCV 30/87 pathogenesis, with the heart and brain as the primary sites of infections in all three animals, but with different disease phenotypes. Sixteen percent of EMCV 30/87-infected pigs developed acute fatal cardiac failure, whereas the rest of the pigs were overtly asymptomatic for as long as 90 days postinfection (p.i.), despite extensive myocardial and central nervous system (CNS) pathological changes. In contrast, mice infected with >/==" BORDER="0">4 PFU of EMCV 30/87 developed acute encephalitis that resulted in the death of all animals (n = 25) between days 2 and 7 p.i. EMCV 30/87-infected macaques remained overtly asymptomatic for 45 days, despite extensive myocardial and CNS pathological changes and viral persistence in more than 50% of the animals. The short poly(C) tract in EMCV 30/87 (CUC(5)UC(8)) was comparable to that of strain 2887A/91 (C(10)UCUC(3)UC(10)), another recent porcine isolate. AD - Department of Veterinary Pathobiology, University of Minnesota, St. Paul, Minnesota 55108, USA. FAU - LaRue, Rebecca AU - LaRue R FAU - Myers, Suzanne AU - Myers S FAU - Brewer, Laurie AU - Brewer L FAU - Shaw, Daniel P AU - Shaw DP FAU - Brown, Corrie AU - Brown C FAU - Seal, Bruce S AU - Seal BS FAU - Njenga, M Kariuki AU - Njenga MK LA - eng GR - HL04369/HL/NHLBI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (RNA, Viral) RN - 30811-80-4 (Poly C) SB - IM MH - Animals MH - Base Sequence MH - Brain/pathology/virology MH - Cardiovirus Infections/*etiology/pathology/virology MH - Comparative Study MH - Encephalomyelitis, Enzootic Porcine/*etiology/pathology/virology MH - Encephalomyocarditis virus/classification/*genetics/*pathogenicity MH - Heart/virology MH - Humans MH - Macaca fascicularis MH - Mengovirus/genetics/pathogenicity MH - Mice MH - Mice, Inbred C57BL MH - Myocardium/pathology MH - Phylogeny MH - Poly C/genetics MH - RNA, Viral/genetics MH - Research Support, U.S. Gov't, P.H.S. MH - Species Specificity MH - Sus scrofa MH - Virulence/genetics EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9136-46. PMID- 12915531 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Competition between the Sendai virus N mRNA start site and the genome 3'-end promoter for viral RNA polymerase. PG - 9147-55 AB - The genomic and antigenomic 3'-end replication promoters of Sendai virus are bipartite in nature and symmetrical, composed of le or tr sequences; a gene start or gene end site, respectively; and a simple hexameric repeat. The relative strengths of these 3'-end promoters determines the ratios of genomes and antigenomes formed during infection and whether model mini-genomes can be rescued from DNA by nondefective helper viruses. Using these tests of promoter strength, we have confirmed that tr is stronger than le in this respect. We have also found that the presence of a gene start site within either 3'-end promoter strongly reduces 3'-end promoter strength. The negative effects of the gene start site on the 3'-end promoter suggest that these closely spaced RNA start sites compete with each other for a common pool of viral RNA polymerase. The manner in which this competition could occur for polymerase off the template (in trans) and polymerase on the template (in cis) adds insight into how the viral RNA polymerase switches between its dual functions as transcriptase and replicase. AD - Department of Genetics and Microbiology, University of Geneva School of Medicine, CH1211 Geneva, Switzerland. FAU - Le Mercier, Philippe AU - Le Mercier P FAU - Garcin, Dominique AU - Garcin D FAU - Garcia, Eduardo AU - Garcia E FAU - Kolakofsky, Daniel AU - Kolakofsky D LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (NP protein) RN - 0 (Nucleoproteins) RN - 0 (RNA, Messenger) RN - 0 (RNA, Viral) RN - 0 (Viral Core Proteins) RN - EC 2.7.7.6 (DNA-Directed RNA Polymerases) SB - IM MH - Animals MH - Binding Sites/genetics MH - Binding, Competitive MH - Cell Line MH - DNA-Directed RNA Polymerases/genetics/*metabolism MH - Defective Viruses/genetics/physiology MH - Genome, Viral MH - Helper Viruses/genetics/physiology MH - Humans MH - Models, Molecular MH - Mutagenesis, Site-Directed MH - Nucleoproteins/chemistry/*genetics MH - Promoter Regions (Genetics) MH - RNA, Messenger/*genetics/metabolism MH - RNA, Viral/*genetics MH - Recombination, Genetic MH - Research Support, Non-U.S. Gov't MH - Sendai virus/*genetics/*physiology MH - Viral Core Proteins/chemistry/*genetics MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9147-55. PMID- 12915532 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Respiratory syncytial virus infection sensitizes cells to apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand. PG - 9156-72 AB - Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease worldwide, especially in the pediatric population. For viruses in general, apoptotic death of infected cells is a mechanism for reducing virus replication. Apoptosis can also be an important factor in augmenting antigen presentation and the host immune response. We examined apoptosis in response to RSV infection of primary small airway cells, primary tracheal-bronchial cells, and A549 and HEp-2 cell lines. The primary cells and the A549 cell line gave generally similar responses, indicating their appropriateness as models in contrast to HEp-2 cells. With the use of RNase protection assays with probes representing 33 common apoptosis factors, we found strong transcriptional activation of both pro- and antiapoptotic factors in response to RSV infection, which were further studied at the protein level and by functional assays. In particular, RSV infection strongly up-regulated the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors death receptor 4 (DR4) and DR5. Furthermore, RSV-infected cells became highly sensitive to apoptosis induced by exogenous TRAIL. These findings suggest that RSV-infected cells in vivo are susceptible to killing through the TRAIL pathway by immune cells such as natural killer and CD4(+) cells that bear membrane-bound TRAIL. RSV infection also induced several proapoptotic factors of the Bcl-2 family and caspases 3, 6, 7, 8, 9, and 10, representing both the death receptor- and mitochondrion-dependent apoptotic pathways. RSV also mediated the strong induction of antiapoptotic factors of the Bcl-2 family, especially Mcl-1, which might account for the delayed induction of apoptosis in RSV-infected cells in the absence of exogenous induction of the TRAIL pathway. AD - Respiratory Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. FAU - Kotelkin, Alexander AU - Kotelkin A FAU - Prikhod'ko, Elena A AU - Prikhod'ko EA FAU - Cohen, Jeffrey I AU - Cohen JI FAU - Collins, Peter L AU - Collins PL FAU - Bukreyev, Alexander AU - Bukreyev A LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (MCL1 protein) RN - 0 (Membrane Glycoproteins) RN - 0 (Neoplasm Proteins) RN - 0 (Proto-Oncogene Proteins c-bcl-2) RN - 0 (RNA, Messenger) RN - 0 (Receptors, Tumor Necrosis Factor) RN - 0 (TNF-related apoptosis-inducing ligand) RN - 0 (Tumor Necrosis Factor-alpha) RN - 0 (death receptor-4) RN - 0 (death receptor-5) RN - EC 3.4.22.- (Caspases) SB - IM MH - Apoptosis/*physiology MH - Caspases/genetics/metabolism MH - Cell Line MH - Cells, Cultured MH - Enzyme Activation MH - Epithelial Cells/pathology/virology MH - Humans MH - Kinetics MH - Lung/pathology/virology MH - Membrane Glycoproteins/genetics/*physiology MH - Neoplasm Proteins/genetics MH - Proto-Oncogene Proteins c-bcl-2/genetics MH - RNA, Messenger/genetics/metabolism MH - Receptors, Tumor Necrosis Factor/genetics MH - Respiratory Syncytial Virus Infections/etiology/genetics/*pathology/virology MH - Respiratory Syncytial Virus, Human/*pathogenicity/physiology MH - Tumor Necrosis Factor-alpha/genetics/*physiology MH - Up-Regulation MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9156-72. PMID- 12915533 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Tsg101 control of human immunodeficiency virus type 1 Gag trafficking and release. PG - 9173-82 AB - The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55(gag), contains a proline-rich motif (PTAP) called the "late domain" in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of gag-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding. AD - Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794-5222, USA. FAU - Goff, A AU - Goff A FAU - Ehrlich, L S AU - Ehrlich LS FAU - Cohen, S N AU - Cohen SN FAU - Carter, C A AU - Carter CA LA - eng GR - GM 48294/GM/NIGMS PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA-Binding Proteins) RN - 0 (Gene Products, gag) RN - 0 (Peptide Fragments) RN - 0 (Recombinant Proteins) RN - 0 (Transcription Factors) RN - 0 (Tsg101 protein) RN - 0 (Ubiquitin) SB - IM MH - Animals MH - Binding Sites/genetics MH - COS Cells MH - DNA-Binding Proteins/chemistry/genetics/*physiology MH - Gene Products, gag/genetics/*physiology MH - HIV-1/genetics/*physiology MH - Humans MH - Mutagenesis, Site-Directed MH - Peptide Fragments/chemistry/genetics/metabolism MH - Protein Transport MH - Recombinant Proteins/chemistry/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Transcription Factors/chemistry/genetics/*physiology MH - Transfection MH - Ubiquitin/metabolism MH - Virus Assembly EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9173-82. PMID- 12915534 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Adenovirus type 11 uses CD46 as a cellular receptor. PG - 9183-91 AB - The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type 11 (Ad11; of species B) binds to Chinese hamster ovary (CHO) cells transfected with CD46 (membrane cofactor protein)-cDNA at least 10 times more strongly than to CHO cells transfected with cDNAs encoding CAR or CD55 (decay accelerating factor). Nonpermissive CHO cells were rendered permissive to Ad11 infection upon transfection with CD46-cDNA. Soluble Ad11 fiber knob but not Ad7 or Ad5 knob inhibited binding of Ad11 virions to CD46-transfected cells, and anti-CD46 antibodies inhibited both binding of and infection by Ad11. From these results we conclude that CD46 is a cellular receptor for Ad11. AD - Department of Virology, Umea University, SE-90185 Umea, Sweden. FAU - Segerman, Anna AU - Segerman A FAU - Atkinson, John P AU - Atkinson JP FAU - Marttila, Marko AU - Marttila M FAU - Dennerquist, Veronica AU - Dennerquist V FAU - Wadell, Goran AU - Wadell G FAU - Arnberg, Niklas AU - Arnberg N LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antibodies, Monoclonal) RN - 0 (Antigens, CD) RN - 0 (Antigens, CD55) RN - 0 (CAR receptor) RN - 0 (CD46 antigen) RN - 0 (DNA, Recombinant) RN - 0 (Membrane Glycoproteins) RN - 0 (Receptors, Virus) RN - 0 (Recombinant Proteins) RN - 0 (adenovirus receptor) RN - 7439-96-5 (Manganese) RN - 7440-70-2 (Calcium) RN - EC 3.4.21.4 (Trypsin) SB - IM MH - Adenoviruses, Human/classification/pathogenicity/*physiology MH - Animals MH - Antibodies, Monoclonal MH - Antigens, CD/genetics/*physiology MH - Antigens, CD55/genetics/physiology MH - Base Sequence MH - CHO Cells MH - Calcium/metabolism MH - Cell Line MH - DNA, Recombinant/genetics MH - Hamsters MH - Humans MH - Manganese/metabolism MH - Membrane Glycoproteins/genetics/*physiology MH - Receptors, Virus/genetics/*physiology MH - Recombinant Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Transfection MH - Trypsin/pharmacology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9183-91. PMID- 12915535 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Association of the herpes simplex virus type 1 Us11 gene product with the cellular kinesin light-chain-related protein PAT1 results in the redistribution of both polypeptides. PG - 9192-203 AB - The herpes simplex virus type 1 (HSV-1) Us11 gene encodes a multifunctional double-stranded RNA (dsRNA)-binding protein that is expressed late in infection and packaged into the tegument layer of the virus particle. As a tegument component, Us11 associates with nascent capsids after its synthesis late in the infectious cycle and is delivered into newly infected cells at times prior to the expression of viral genes. Us11 is also an abundant late protein that regulates translation through its association with host components and contains overlapping nucleolar retention and nuclear export signals, allowing its accumulation in both nucleoli and the cytosol. Thus, at various times during the viral life cycle and in different intracellular compartments, Us11 has the potential to execute discrete tasks. The analysis of these functions, however, is complicated by the fact that Us11 is not essential for viral replication in cultured cells. To discover new host targets for the Us11 protein, we searched for cellular proteins that interact with Us11 and have identified PAT1 as a Us11-binding protein according to multiple, independent experimental criteria. PAT1 binds microtubules, participates in amyloid precursor protein trafficking, and has homology to the kinesin light chain (KLC) in its carboxyl terminus. The carboxyl-terminal dsRNA-binding domain of Us11, which also contains the nucleolar retention and nuclear export signals, binds PAT1, whereas 149 residues derived from the KLC homology region of PAT1 are important for binding to Us11. Both PAT1 and Us11 colocalize within a perinuclear area in transiently transfected and HSV-1-infected cells. The 149 amino acids derived from the KLC homology region are required for colocalization of the two polypeptides. Furthermore, although PAT1 normally accumulates in the nuclear compartment, Us11 expression results in the exclusion of PAT1 from the nucleus and its accumulation in the perinuclear space. Similarly, Us11 does not accumulate in the nucleoli of infected cells that overexpress PAT1. These results establish that Us11 and PAT1 can associate, resulting in an altered subcellular distribution of both polypeptides. The association between PAT1, a cellular trafficking protein with homology to KLC, and Us11, along with a recent report demonstrating an interaction between Us11 and the ubiquitous kinesin heavy chain (R. J. Diefenbach et al., J. Virol. 76:3282-3291, 2002), suggests that these associations may be important for the intracellular movement of viral components. AD - Department of Microbiology and NYU Cancer Institute, New York University School of Medicine, New York, New York 10016, USA. FAU - Benboudjema, Louisa AU - Benboudjema L FAU - Mulvey, Matthew AU - Mulvey M FAU - Gao, Yuehua AU - Gao Y FAU - Pimplikar, Sanjay W AU - Pimplikar SW FAU - Mohr, Ian AU - Mohr I LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Amyloid beta-Protein Precursor) RN - 0 (Carrier Proteins) RN - 0 (Microtubule-Associated Proteins) RN - 0 (RNA-Binding Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (US11 protein, virus) RN - 0 (Viral Proteins) RN - 0 (kinesin light-chain proteins) SB - IM MH - Amyloid beta-Protein Precursor/metabolism MH - Animals MH - Binding Sites MH - COS Cells MH - Carrier Proteins/chemistry/*metabolism MH - Cell Line MH - Dogs MH - Genes, Viral MH - Herpesvirus 1, Human/*genetics/*metabolism MH - Humans MH - Microtubule-Associated Proteins/metabolism MH - Protein Structure, Tertiary MH - RNA-Binding Proteins/chemistry/*genetics/*metabolism MH - Recombinant Fusion Proteins/chemistry/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Subcellular Fractions/metabolism/virology MH - Viral Proteins/chemistry/*genetics/*metabolism EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9192-203. PMID- 12915536 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells. PG - 9204-10 AB - The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations. Although the human liver is the primary site for HCV replication, it is not known whether extrahepatic tissues are also infected by the virus and whether nonprimate cells are permissive for RNA replication. Because HCV exists as a quasispecies, it is conceivable that a viral population may include variants that can replicate in different cell types and in other species. We have tested this hypothesis and found that subgenomic HCV RNAs can replicate in mouse hepatoma and nonhepatic human epithelial cells. Replicons isolated from these cell lines carry new mutations that could be involved in the control of tropism of the virus. Our results demonstrated that translation and RNA-directed RNA replication of HCV do not depend on hepatocyte or primate-specific factors. Moreover, our results could open the path for the development of animal models for HCV infection. AD - Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. FAU - Zhu, Qing AU - Zhu Q FAU - Guo, Ju-Tao AU - Guo JT FAU - Seeger, Christoph AU - Seeger C LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (RNA, Viral) SB - IM CIN - Hepatology. 2004 Mar;39(3):835-8. PMID: 14999704 MH - Animals MH - Cell Line MH - Epithelial Cells/virology MH - *Genome, Viral MH - Hela Cells MH - Hepacivirus/*genetics/*physiology MH - Hepatitis C/virology MH - Humans MH - Liver Neoplasms, Experimental/virology MH - Mice MH - Mutation MH - RNA, Viral/biosynthesis/genetics MH - Replicon/genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Transcription, Genetic MH - Tumor Cells, Cultured MH - Virus Replication/*genetics EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9204-10. PMID- 12915537 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Production of human papillomavirus type 16 virus-like particles in transgenic plants. PG - 9211-20 AB - Cervical cancer is linked to infection with human papillomaviruses (HPV) and is the third most common cancer among women worldwide. There is a strong demand for the development of an HPV preventive vaccine. Transgenic plants expressing the HPV major capsid protein L1 could be a system to produce virus-like particles for prophylactic vaccination or could even be used as edible vaccines to induce an L1-specific prophylactic immune response. Here, we describe the generation of transgenic tobacco and potato plants carrying the HPV type 16 major structural gene L1 under the control of the cauliflower mosaic virus 35S promoter. All attempts to express either the original, unmodified L1 gene or an L1 gene with a codon usage optimized for expression in plants failed. Surprisingly, small amounts of the protein were detected using an L1 gene optimized for expression in human cells. However, Northern blot analysis revealed that most of the L1 transcripts were degraded. Introduction of the translational enhancer Omega derived from the tobacco mosaic virus strongly increased transcript stability and resulted in accumulation of L1 protein to approximately 0.5 to 0.2% of total soluble protein in transgenic tobacco and potato plants, respectively. The plant-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles. Furthermore, we did not find any indications of protein modification of the L1 protein produced in plants. Plant-derived L1 was as immunogenic as L1 expressed in baculovirus-infected insect cells. Feeding of tubers from transgenic potatoes to mice induced an anti-L1 antibody response in 3 out of 24 mice, although this response was only transient in two of the mice. Our data, however, indicate that an anti-L1 response was primed in about half of the 24 animals. AD - Institut fur Pflanzengenetik und Kulturpflanzenforschung, 06466 Gatersleben, Germany. FAU - Biemelt, Sophia AU - Biemelt S FAU - Sonnewald, Uwe AU - Sonnewald U FAU - Galmbacher, Petra AU - Galmbacher P FAU - Willmitzer, Lothar AU - Willmitzer L FAU - Muller, Martin AU - Muller M LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Capsid Proteins) RN - 0 (Oncogene Proteins, Viral) RN - 0 (Vaccines, Edible) RN - 0 (Vaccines, Synthetic) RN - 0 (Viral Vaccines) RN - 0 (oncogene viral capsid protein, L1 human papillomavirus type 16) SB - IM MH - Animals MH - *Capsid Proteins MH - Enhancer Elements (Genetics) MH - Female MH - Gene Expression MH - Genes, Viral MH - Humans MH - Mice MH - Mice, Inbred BALB C MH - Oncogene Proteins, Viral/biosynthesis/genetics/immunology MH - Papillomavirus Infections/immunology/prevention & control MH - Papillomavirus, Human/*genetics/immunology/pathogenicity/physiology MH - Plants, Genetically Modified MH - Potatoes/genetics MH - Research Support, Non-U.S. Gov't MH - Tobacco/genetics MH - Tobacco Mosaic Virus/genetics MH - Tumor Virus Infections/immunology/prevention & control MH - Vaccines, Edible/genetics MH - Vaccines, Synthetic/genetics/isolation & purification MH - Viral Vaccines/genetics/isolation & purification EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9211-20. PMID- 12915538 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Mutations in the N termini of herpes simplex virus type 1 and 2 gDs alter functional interactions with the entry/fusion receptors HVEM, nectin-2, and 3-O-sulfated heparan sulfate but not with nectin-1. PG - 9221-31 AB - Multiple cell surface molecules (herpesvirus entry mediator [HVEM], nectin-1, nectin-2, and 3-O-sulfated heparan sulfate) can serve as entry receptors for herpes simplex virus type 1 (HSV-1) or HSV-2 and also as receptors for virus-induced cell fusion. Viral glycoprotein D (gD) is the ligand for these receptors. A previous study showed that HVEM makes contact with HSV-1 gD at regions within amino acids 7 to 15 and 24 to 32 at the N terminus of gD. In the present study, amino acid substitutions and deletions were introduced into the N termini of HSV-1 and HSV-2 gDs to determine the effects on interactions with all of the known human and mouse entry/fusion receptors, including mouse HVEM, for which data on HSV entry or cell fusion were not previously reported. A cell fusion assay was used to assess functional activity of the gD mutants with each entry/fusion receptor. Soluble gD:Fc hybrids carrying each mutation were tested for the ability to bind to cells expressing the entry/fusion receptors. We found that deletions overlapping either or both of the HVEM contact regions, in either HSV-1 or HSV-2 gD, severely reduced cell fusion and binding activity with all of the human and mouse receptors except nectin-1. Amino acid substitutions described previously for HSV-1 (L25P, Q27P, and Q27R) were individually introduced into HSV-2 gD and, for both serotypes, were found to be without effect on cell fusion and the binding activity for nectin-1. Each of these three substitutions in HSV-1 gD enhanced fusion with cells expressing human nectin-2 (ordinarily low for wild-type HSV-1 gD), but the same substitutions in HSV-2 gD were without effect on the already high level of cell fusion observed with the wild-type protein. The Q27P or Q27R substitution in either HSV-1 and HSV-2 gD, but not the L25P substitution, significantly reduced cell fusion and binding activity for both human and mouse HVEM. Each of the three substitutions in HSV-1 gD, as well as the deletions mentioned above, reduced fusion with cells bearing 3-O-sulfated heparan sulfate. Thus, the N terminus of HSV-1 or HSV-2 gD is not necessary for functional interactions with nectin-1 but is necessary for all of the other receptors tested here. The sequence of the N terminus determines whether nectin-2 or 3-O-sulfated heparan sulfate, as well as HVEM, can serve as entry/fusion receptors. AD - Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. FAU - Yoon, Miri AU - Yoon M FAU - Zago, Anna AU - Zago A FAU - Shukla, Deepak AU - Shukla D FAU - Spear, Patricia G AU - Spear PG LA - eng GR - R01 CA21776/CA/NCI GR - R37 AI36293/AI/NIAID GR - U19 AI31494/AI/NIAID PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Cell Adhesion Molecules) RN - 0 (DNA, Recombinant) RN - 0 (Receptors, Tumor Necrosis Factor) RN - 0 (Receptors, Virus) RN - 0 (Recombinant Proteins) RN - 0 (Viral Envelope Proteins) RN - 0 (glycoprotein D, Human herpesvirus 1) RN - 0 (glycoprotein D-herpes simplex virus type 2) RN - 0 (herpesvirus entry mediator) RN - 0 (nectin) RN - 9050-30-0 (Heparitin Sulfate) SB - IM MH - Amino Acid Sequence MH - Amino Acid Substitution MH - Animals MH - Base Sequence MH - Binding Sites/genetics MH - CHO Cells MH - Cell Adhesion Molecules/physiology MH - DNA, Recombinant/genetics MH - Hamsters MH - Heparitin Sulfate/physiology MH - Herpesvirus 1, Human/*genetics/*physiology MH - Herpesvirus 2, Human/*genetics/*physiology MH - Humans MH - Membrane Fusion/physiology MH - Mice MH - Molecular Sequence Data MH - Mutation MH - Receptors, Tumor Necrosis Factor/physiology MH - Receptors, Virus/physiology MH - Recombinant Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Viral Envelope Proteins/*genetics/*physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9221-31. PMID- 12915539 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Effects of modification of the transcription initiation site context on citrus tristeza virus subgenomic RNA synthesis. PG - 9232-43 AB - Citrus tristeza virus (CTV), a member of the Closteroviridae, has a positive-sense RNA genome of about 20 kb organized into 12 open reading frames (ORFs). The last 10 ORFs are expressed through 3'-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of 10 sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5' termini of intermediate- (CP and p13) and low- (p18) produced sgRNAs that, like the two highly abundant sgRNAs (p20 and p23) previously mapped, all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5' or 3' of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism. AD - Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, Florida 33850, USA. FAU - Ayllon, Maria A AU - Ayllon MA FAU - Gowda, Siddarame AU - Gowda S FAU - Satyanarayana, Tatineni AU - Satyanarayana T FAU - Karasev, Alexander V AU - Karasev AV FAU - Adkins, Scott AU - Adkins S FAU - Mawassi, Munir AU - Mawassi M FAU - Guerri, Jose AU - Guerri J FAU - Moreno, Pedro AU - Moreno P FAU - Dawson, William O AU - Dawson WO LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (RNA, Viral) SB - IM MH - Base Sequence MH - Citrus/virology MH - Closteroviridae/*genetics/*physiology MH - DNA, Viral/genetics MH - Genome, Viral MH - Mutagenesis, Site-Directed MH - RNA, Viral/*biosynthesis/*genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Transcription, Genetic MH - Virus Replication/genetics EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9232-43. PMID- 12915540 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Characterization of the RNA-binding domains in the replicase proteins of tomato bushy stunt virus. PG - 9244-58 AB - Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine- and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus. AD - Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546, USA. FAU - Rajendran, K S AU - Rajendran KS FAU - Nagy, Peter D AU - Nagy PD LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (RNA, Viral) RN - 0 (Recombinant Proteins) RN - EC 2.7.7.48 (RNA Replicase) SB - IM MH - Amino Acid Sequence MH - Base Sequence MH - Binding Sites/genetics MH - DNA, Viral/genetics MH - Kinetics MH - Molecular Sequence Data MH - Nucleic Acid Conformation MH - Peptide Mapping MH - Protein Structure, Tertiary MH - RNA Replicase/*chemistry/genetics/*metabolism MH - RNA, Viral/chemistry/*genetics/*metabolism MH - Recombinant Proteins/chemistry/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Tombusvirus/*genetics/*metabolism EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9244-58. PMID- 12915541 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20040818 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Identification of a mutation in editing of defective Newcastle disease virus recombinants that modulates P-gene mRNA editing and restores virus replication and pathogenicity in chicken embryos. PG - 9259-65 AB - Editing of P-gene mRNA of Newcastle disease virus (NDV) enables the formation of two additional proteins (V and W) by inserting one or two nontemplated G residues at a conserved editing site (5'-AAAAAGGG). The V protein of NDV plays an important role in virus replication and is also a virulence factor presumably due to its ability to counteract the antiviral effects of interferon. A recombinant virus possessing a nucleotide substitution within the A-stretch (5'-AAgAAGGG) produced 20-fold-less V protein and, in consequence, was impaired in replication capacity and completely attenuated in pathogenicity for chicken embryos. However, in a total of seven serial passages, restoration of replication and pathogenic capacity in 9- to 11-day-old chicken embryos was noticed. Determining the sequence around the editing site of the virus at passage 7 revealed a C-to-U mutation at the second nucleotide immediately upstream of the 5'-A(5) stretch (5'-GuUAAgAAGGG). The V mRNA increased from an undetectable level at passage 5 to ca. 1 and 5% at passages 6 and 7, respectively. In addition, similar defects in another mutant possessing a different substitution mutation (5'-AAAcAGGG) were restored in an identical manner within a total of seven serial passages. Introduction of the above C-to-U mutation into the parent virus (5'-GuUAAAAAGGG) altered the frequency of P, V, and W mRNAs from 68, 28, and 4% to 15, 44, and 41%, respectively, demonstrating that the U at this position is a key determinant in modulating P-gene mRNA editing. The results indicate that this second-site mutation is required to compensate for the drop in edited mRNAs and consequently to restore the replication capacity, as well as the pathogenic potential, of editing-defective NDV recombinants. AD - Department of Virology, Intervet International B.V., 5830 AA Boxmeer, The Netherlands. teshome.mebatsion@intervet.com FAU - Mebatsion, Teshome AU - Mebatsion T FAU - de Vaan, Leonie T C AU - de Vaan LT FAU - de Haas, Niels AU - de Haas N FAU - Romer-Oberdorfer, Angela AU - Romer-Oberdorfer A FAU - Braber, Marian AU - Braber M LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (P phosphoprotein, Newcastle disease virus) RN - 0 (Phosphoproteins) RN - 0 (RNA, Messenger) RN - 0 (RNA, Viral) RN - 0 (Viral Proteins) SB - IM EIN - J Virol. 2003 Oct;77(20):11299 MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Chick Embryo MH - Genes, Viral MH - Mutation MH - Newcastle disease virus/*genetics/pathogenicity/physiology MH - Phosphoproteins/*genetics MH - RNA Editing/*genetics MH - RNA, Messenger/genetics/metabolism MH - RNA, Viral/genetics/metabolism MH - Recombination, Genetic MH - Viral Proteins/*genetics MH - Virulence/genetics MH - Virus Replication/genetics EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9259-65. PMID- 12915542 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Local character of readthrough activation in adenovirus type 5 early region 1 transcription control. PG - 9266-77 AB - Wild-type early activity of the adenovirus 5 E1b gene promoter requires readthrough transcription originating from the adjacent upstream E1a gene. This unusual mode of viral transcription activation was identified by genetic manipulation of the mouse beta(maj)-globin gene transcription termination sequence (GGT) inserted into the E1a gene. To facilitate further study of the mechanism of readthrough activation, the activities of GGT and a composite termination sequence CT were tested in recombinant adenoviruses containing luciferase reporters driven by the E1b promoter. There was a strict correlation between readthrough and substantial downstream gene expression, indicating that interference with downstream transcription was not a unique property of GGT. Blockage of readthrough transcription of E1a had no apparent effect on early expression of the major late promoter, the next active promoter downstream of E1b. A test for epistatic interaction between termination sequence insertions and E1a enhancer mutations suggested that readthrough activation and E1a enhancer activation of the E1b promoter are mechanistically distinct. In addition, substitution of the human cytomegalovirus major immediate-early promoter for the E1b promoter suppressed the requirement for readthrough. These results suggest that readthrough activation is a "local" effect of a direct interaction between the invading transcription elongation complex and the E1b promoter. DNase I hypersensitivity footprinting provided evidence that this interaction altered an extensive E1b promoter DNA-protein complex that was assembled in the absence of readthrough transcription. AD - Department of Microbiology and Immunology and Inter-College Graduate Degree Program in Genetics, College of Medicine, The Pennsylvania State University, Hershey, Pennsylvania 17033, USA. FAU - Shen, Li AU - Shen L FAU - Spector, David J AU - Spector DJ LA - eng GR - GM058214/GM/NIGMS PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Adenovirus E1A Proteins) RN - 0 (Adenovirus E1B Proteins) RN - 0 (DNA, Recombinant) RN - 0 (DNA, Viral) SB - IM MH - Adenovirus E1A Proteins/genetics MH - Adenovirus E1B Proteins/*genetics MH - Adenoviruses, Human/*genetics MH - Base Sequence MH - Cell Line MH - DNA, Recombinant/genetics MH - DNA, Viral/genetics MH - Enhancer Elements (Genetics) MH - Hela Cells MH - Humans MH - Promoter Regions (Genetics) MH - Research Support, U.S. Gov't, P.H.S. MH - *Trans-Activation (Genetics) EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9266-77. PMID- 12915543 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic. PG - 9278-86 AB - Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development. AD - Center for Biodefense and Emerging Infectious Diseases, Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USA. FAU - Paessler, Slobodan AU - Paessler S FAU - Fayzulin, Rafik Z AU - Fayzulin RZ FAU - Anishchenko, Michael AU - Anishchenko M FAU - Greene, Ivorlyne P AU - Greene IP FAU - Weaver, Scott C AU - Weaver SC FAU - Frolov, Ilya AU - Frolov I LA - eng GR - AI10984/AI/NIAID GR - AI48807/AI/NIAID GR - AI50537/AI/NIAID GR - AIO7536/AI/NIAID PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (RNA, Viral) RN - 0 (RNA, recombinant) RN - 0 (Vaccines, Attenuated) RN - 0 (Vaccines, Synthetic) RN - 0 (Viral Vaccines) RN - 63231-63-0 (RNA) SB - IM MH - Animals MH - Base Sequence MH - Cell Line MH - Cercopithecus aethiops MH - Encephalitis Virus, Venezuelan Equine/*genetics/*immunology/pathogenicity/physiology MH - Encephalomyelitis, Venezuelan Equine/immunology/prevention & control MH - Female MH - Hamsters MH - Male MH - Mice MH - Molecular Sequence Data MH - RNA/genetics MH - RNA, Viral/genetics MH - Recombination, Genetic MH - Research Support, U.S. Gov't, P.H.S. MH - Sindbis Virus/*genetics/*immunology/pathogenicity/physiology MH - Vaccines, Attenuated/genetics MH - Vaccines, Synthetic/genetics MH - Vero Cells MH - Viral Vaccines/genetics MH - Virulence MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9278-86. PMID- 12915544 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Human cytomegalovirus US2 causes similar effects on both major histocompatibility complex class I and II proteins in epithelial and glial cells. PG - 9287-94 AB - The human cytomegalovirus (HCMV) glycoprotein US2 specifically binds to major histocompatibility complex (MHC) class I heavy chain (HC) and class II proteins DRalpha and DMalpha, triggering their degradation by proteasomes. Effects of US2 on class II proteins were originally characterized in HCMV- or adenovirus vector-infected U373 astroglioma cells. Here, we have extended characterization of US2-mediated degradation of class II DRalpha to two other cell lines, including biologically relevant epithelial cells. Comparison of the effects of US2 in cells expressing both class I and II proteins demonstrated only a slight preference for class I HC. Moreover, US2 caused degradation of DRalpha and DMalpha when these proteins were expressed by transfection without DRbeta, invariant chain (Ii), or DMbeta. Therefore, US2 binds to alpha chains of DR and DM and triggers endoplasmic reticulum degradation without formation of class II DR alphabeta/Ii or DM alphabeta complexes. Similar levels of degradation of class II alpha were observed in cells expressing vastly different amounts of class II, suggesting that cellular factors, other than class II, were limiting. We concluded that US2 has broad effects in a variety of cells that express both class I and II proteins and is relevant to HCMV infection in vivo. AD - Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon 97239-3098, USA. FAU - Hegde, Nagendra R AU - Hegde NR FAU - Johnson, David C AU - Johnson DC LA - eng GR - CA73996/CA/NCI GR - EY11245/EY/NEI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Genetic Vectors) RN - 0 (HCMV US2 protein) RN - 0 (HLA-D Antigens) RN - 0 (HLA-DMB) RN - 0 (HLA-DR Antigens) RN - 0 (Histocompatibility Antigens Class I) RN - 0 (Histocompatibility Antigens Class II) RN - 0 (Membrane Glycoproteins) RN - 0 (Recombinant Proteins) RN - 0 (Viral Proteins) SB - IM MH - Adenoviridae/genetics MH - Cell Line MH - Cytomegalovirus/genetics/immunology/pathogenicity/*physiology MH - Epithelial Cells/immunology/virology MH - Genetic Vectors MH - HLA-D Antigens/metabolism MH - HLA-DR Antigens/metabolism MH - Hela Cells MH - Histocompatibility Antigens Class I/*metabolism MH - Histocompatibility Antigens Class II/*metabolism MH - Humans MH - Kinetics MH - Membrane Glycoproteins/genetics/*physiology MH - Neuroglia/immunology/virology MH - Protein Binding MH - Recombinant Proteins/genetics/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Transfection MH - Viral Proteins/genetics/*physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9287-94. PMID- 12915545 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Heterologous human immunodeficiency virus type 1 lentiviral vectors packaging a simian immunodeficiency virus-derived genome display a specific postentry transduction defect in dendritic cells. PG - 9295-304 AB - Heterologous lentiviral vectors (LVs) represent a way to address safety concerns in the field of gene therapy by decreasing the possibility of genetic recombination between vector and packaging constructs and the generation of replication-competent viruses. Using described LVs based on human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus MAC251 (SIV(MAC251)), we asked whether heterologous virion particles in which trans-acting factors belonged to HIV-1 and cis elements belonged to SIV(MAC251) (HIV-siv) would behave as parental homologous vectors in all cell types. To our surprise, we found that although the heterologous HIV-siv vector was as infectious as its homologous counterpart in most human cells, it was defective in the transduction of dendritic cells (DCs) and, to a lesser extent, macrophages. In DCs, the main postentry defect was observed in the formation of two-long-terminal-repeat circles, despite the fact that full-length proviral DNA was being synthesized and was associated with the nucleus. Taken together, our data suggest that heterologous HIV-siv vectors display a cell-dependent infectivity defect, most probably at a post-nuclear entry migration step. As homologous HIV and SIV vectors do transduce DCs, we believe that these results underscore the importance of a conserved interaction between cis elements and trans-acting viral factors that is lost or suboptimal in heterologous vectors and essential only in the transduction of certain cell types. For gene therapy purposes, these findings indicate that the cellular tropism of LVs can be modulated not only through the use of distinct envelope proteins or tissue-specific promoters but also through the specific combinatorial use of packaging and transfer vector constructs. AD - INSERM U412, Ecole Normale Superieure de Lyon. Etablissement Francais du Sang, Lyon, France. FAU - Goujon, Caroline AU - Goujon C FAU - Jarrosson-Wuilleme, Loraine AU - Jarrosson-Wuilleme L FAU - Bernaud, Jeanine AU - Bernaud J FAU - Rigal, Dominique AU - Rigal D FAU - Darlix, Jean-Luc AU - Darlix JL FAU - Cimarelli, Andrea AU - Cimarelli A LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (Genetic Vectors) SB - IM MH - Animals MH - Base Sequence MH - Cell Nucleus/virology MH - DNA, Viral/genetics MH - Dendritic Cells/virology MH - *Genetic Vectors MH - Genome, Viral MH - HIV-1/*genetics MH - HL-60 Cells MH - Hela Cells MH - Humans MH - Macrophages/virology MH - Proviruses/genetics MH - Recombination, Genetic MH - Research Support, Non-U.S. Gov't MH - SIV/*genetics MH - Transduction, Genetic MH - U937 Cells EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9295-304. PMID- 12915546 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Rotavirus infection stimulates the Cl- reabsorption process across the intestinal brush-border membrane of young rabbits. PG - 9305-11 AB - Rotavirus is a major cause of infantile gastroenteritis worldwide. However, the mechanisms underlying fluid and electrolyte secretion associated with diarrhea remain largely unknown. We investigated the hypothesis that loss of Cl(-) into the luminal contents during rotavirus infection may be caused by a dysfunction in the chloride absorptive capacity across the intestinal brush-border membrane (BBM). The luminal Cl(-) concentrations in the entire small intestine of young rabbits infected with lapine rotavirus decreased at 1 and 2 days postinfection (dpi), indicating net Cl(-) absorption. At 7 dpi, luminal Cl(-) concentrations were slightly increased, indicating a moderate net Cl(-) secretion. By using a rapid filtration technique, (36)Cl uptake across BBM was quantified by modulating the alkali-metal ion, electrical, chloride, and/or proton gradients. Rotavirus infection caused an identical, 127% +/- 24% increase in all Cl(-) uptake activities (Cl(-)/H(+) symport, Cl(-) conductance, and Cl(-)/anion exchange) observed across the intestinal BBM. The rotavirus activating effects on the symporter started at 1 dpi and persisted up to 7 dpi. Kinetic analyses revealed that rotavirus selectively affected the capacity parameter characterizing the symporter. We report the novel observation that rotavirus infection stimulated the Cl(-) reabsorption process across the intestinal BBM. We propose that the massive Cl(-) reabsorption in villi could partly overwhelm chloride secretion in crypt cells, which possibly increases during rotavirus diarrhea, the resulting imbalance leading to a moderate net chloride secretion. AD - Institut National de la Sante et de la Recherche Medicale, Unite 510, Faculte de Pharmacie, Universite de Paris XI, 92296 Chatenay-Malabry, France. FAU - Lorrot, Mathie AU - Lorrot M FAU - Martin, Sandra AU - Martin S FAU - Vasseur, Monique AU - Vasseur M LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antiporters) RN - 0 (Chlorides) RN - 0 (hydrogen-chloride symporter) SB - IM MH - Animals MH - Antiporters/metabolism MH - Chlorides/*metabolism MH - Gastroenteritis/*metabolism MH - Humans MH - Hydrogen-Ion Concentration MH - Infant MH - Intestines/*metabolism MH - Ion Transport MH - Kinetics MH - Microvilli/metabolism MH - Rabbits MH - Research Support, Non-U.S. Gov't MH - Rotavirus Infections/*metabolism EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9305-11. PMID- 12915547 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Novel recombinant parapoxvirus vectors induce protective humoral and cellular immunity against lethal herpesvirus challenge infection in mice. PG - 9312-23 AB - Orf virus (ORFV; Parapoxvirus ovis) was used to develop a novel vector system for the generation of effective and safe live vaccines. Based on the attenuated ORFV strain D1701-V, recombinants were produced that express the glycoproteins gC (D1701-VrVgC) or gD (D1701-VrVgD) of the alphaherpesvirus of swine, pseudorabies virus (PRV). Expression of gC and gD was also demonstrated on the surface of recombinant virus-infected murine cells that do not produce infectious ORFV. Single or combined immunization with the ORFV recombinants protected different mouse strains of a host species nonpermissive for ORFV against a fulminant, lethal PRV challenge infection equal to immunization with PRV live vaccine. Most notably, even a single immunization with D1701-VrVgC was protective, whereas two applications of D1701-VrVgD were required for immune protection. The higher protective capacity of D1701-VrVgC correlated with the induction of a strong specific humoral immune response. This suggestion was supported by transfer experiments using sera from recombinant-immunized mice, which resulted in partial gC but not gD antibody-mediated protection of the naive recipients. Remarkably, immunization of different immune-deficient mice demonstrated that the application of the PRV gC-expressing recombinant controlled the challenge infection in the absence of either CD4(+) or CD8(+) T cells, B cells, or an intact perforin pathway. In contrast, D1701-VrVgD-immunized mice lacking CD4(+) T cells exhibited reduced protection, whereas animals lacking CD8(+) T cells, B cells, or perforin resisted the challenge infection. The present study demonstrates the potential of these new vector vaccines to efficiently prime both protective humoral and cell-mediated immune mechanisms in a host species nonpermissive for the vector virus. AD - Federal Research Centre for Virus Diseases of Animals, Institute of Immunology, D-72076 Tuebingen, Germany. FAU - Fischer, Timo AU - Fischer T FAU - Planz, Oliver AU - Planz O FAU - Stitz, Lothar AU - Stitz L FAU - Rziha, Hanns-Joachim AU - Rziha HJ LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antibodies, Viral) RN - 0 (Genetic Vectors) RN - 0 (Vaccines, Synthetic) RN - 0 (Viral Envelope Proteins) RN - 0 (Viral Vaccines) RN - 0 (glycoprotein D, pseudorabies virus) RN - 0 (pseudorabies virus glycoproteins) SB - IM MH - Animals MH - Antibodies, Viral/biosynthesis MH - *Genetic Vectors MH - Genome, Viral MH - Herpesvirus 1, Suid/*genetics/*immunology MH - Immunity, Cellular MH - Immunization, Passive MH - Immunologic Deficiency Syndromes/genetics/immunology MH - Mice MH - Mice, Inbred BALB C MH - Mice, Inbred C57BL MH - Mice, Knockout MH - Parapoxvirus/*genetics MH - Pseudorabies/immunology/prevention & control MH - Recombination, Genetic MH - Research Support, Non-U.S. Gov't MH - Vaccines, Synthetic/genetics/pharmacology MH - Viral Envelope Proteins/genetics/immunology MH - Viral Vaccines/genetics/pharmacology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9312-23. PMID- 12915548 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Transforming growth factor beta1 receptor II is downregulated by E1A in adenovirus-infected cells. PG - 9324-36 AB - Transforming growth factor beta1 (TGF-beta1) signaling is compromised in many tumors, thereby allowing the tumor to escape the growth-inhibitory and proapoptotic activities of the cytokine. Human adenoviruses interfere with a number of cellular pathways involved in cell cycle regulation and apoptosis, initially placing the cell in a "tumor-like" state by forcing quiescent cells into the cell cycle and also inhibiting apoptosis. We report that adenovirus-infected cells resemble tumor cells in that TGF-beta1 signaling is inhibited. The levels of TGF-beta1 receptor II (TbetaRII) in adenovirus-infected cells were decreased, and this decrease was mapped, by using virus mutants, to the E1A gene and to amino acids 2 to 36 and the C-terminal binding protein binding site in the E1A protein. The decrease in the TbetaRII protein was accompanied by a decrease in TbetaRII mRNA. The decrease in TbetaRII protein levels in adenovirus-infected cells was greater than the decrease in TbetaRII mRNA, suggesting that downregulation of the TbetaRII protein may occur through more than one mechanism. Surprisingly in this context, the half-lives of the TbetaRII protein in infected and uninfected cells were similar. TGF-beta1 signaling was compromised in cells infected with wild-type adenovirus, as measured with 3TP-lux, a TGF-beta-sensitive reporter plasmid expressing luciferase. Adenovirus mutants deficient in TbetaRII downregulation did not inhibit TGF-beta1 signaling. TGF-beta1 pretreatment reduced the relative abundance of adenovirus structural proteins in infected cells, an effect that was potentiated when cells were infected with mutants incapable of modulating the TGF-beta signaling pathway. These results raise the possibility that inhibition of TGF-beta signaling by E1A is a means by which adenovirus counters the antiviral defenses of the host. AD - Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA. vera@tarakanov.com FAU - Tarakanova, Vera L AU - Tarakanova VL FAU - Wold, William S M AU - Wold WS LA - eng GR - CA58538/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Adenovirus E1A Proteins) RN - 0 (RNA, Messenger) RN - 0 (Receptors, Transforming Growth Factor beta) RN - 0 (Transforming Growth Factor beta) RN - 0 (transforming growth factor beta1) RN - 0 (transforming growth factor-beta type II receptor) SB - IM MH - Adenovirus E1A Proteins/genetics/*physiology MH - Adenoviruses, Human/genetics/immunology/*physiology MH - Cell Line MH - Down-Regulation MH - Humans MH - Mutation MH - RNA, Messenger/genetics/metabolism MH - Receptors, Transforming Growth Factor beta/*genetics/*metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction MH - Transforming Growth Factor beta/metabolism MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9324-36. PMID- 12915549 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Herpes simplex virus type 2 virion host shutoff protein regulates alpha/beta interferon but not adaptive immune responses during primary infection in vivo. PG - 9337-45 AB - The herpes simplex virus (HSV) virion host shutoff (vhs) protein, the product of the UL41 (vhs) gene, is an important determinant of HSV virulence. vhs has been implicated in HSV interference with host antiviral immune responses, down-regulating expression of major histocompatibility complex molecules to help HSV evade host adaptive immunity. The severe attenuation of vhs-deficient viruses in vivo could reflect their inability to escape immune detection. To test this hypothesis, BALB/c or congenic SCID mice were infected intravaginally (i.vag.) with the HSV type 2 (HSV-2) vhs null mutant 333d41 or the vhs rescue virus 333d41(R). vhs-deficient virus remained severely attenuated in SCID mice compared with rescue virus, indicating that vhs regulation of adaptive immune responses does not influence HSV pathogenesis during acute infection. Innate antiviral effectors remain intact in SCID mice; prominent among these is alpha/beta interferon (IFN-alpha/beta). The attenuation of HSV-2 vhs mutants could reflect their failure to suppress IFN-alpha/beta-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) mice were infected i.vag. with wild-type virus, vhs null mutants 333-vhsB or 333d41, or the vhs rescue virus 333d41(R). Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type or vhs rescue viruses, they were restored to nearly wild-type levels of replication in IFN-alpha/betaR(-/-) mice over the first 2 days postinfection. Only wild-type and vhs rescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-alpha/betaR(-/-) mice restored the virulence of vhs-deficient viruses. vhs-deficient viruses replicated as vigorously as wild-type and rescue viruses in the nervous systems of IFN-alpha/betaR(-/-) mice. Restoration was specific for the vhs mutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-alpha/betaR(-/-) mice. Furthermore, the defect in the IFN-alpha/beta response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-alpha/beta-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-alpha/beta response in vivo, because an HSV-1 vhs null mutant did not recover replication and virulence after i.vag. inoculation into IFN-alpha/betaR(-/-) mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-alpha/beta-mediated antiviral response. AD - Department of Molecular Microbiology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA. FAU - Murphy, Jenny A AU - Murphy JA FAU - Duerst, Rebecca J AU - Duerst RJ FAU - Smith, Tracy J AU - Smith TJ FAU - Morrison, Lynda A AU - Morrison LA LA - eng GR - CA75052/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Interferon-alpha) RN - 0 (Receptors, Interferon) RN - 0 (Viral Proteins) RN - 0 (herpes simplex virus type 2 protein UL41) RN - 156986-95-7 (interferon alpha-beta receptor) RN - 77238-31-4 (Interferon-beta) SB - IM MH - Animals MH - Female MH - Herpes Genitalis/etiology/*immunology/virology MH - Herpesvirus 2, Human/genetics/*immunology/pathogenicity/physiology MH - Interferon-alpha/*biosynthesis MH - Interferon-beta/*biosynthesis MH - Mice MH - Mice, Congenic MH - Mice, Inbred BALB C MH - Mice, Knockout MH - Mice, SCID MH - Mutation MH - Receptors, Interferon/deficiency/genetics/physiology MH - Research Support, U.S. Gov't, P.H.S. MH - Time Factors MH - Viral Proteins/genetics/immunology/*physiology MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9337-45. PMID- 12915550 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Activation of mitogen-activated protein kinase and NF-kappaB pathways by a Kaposi's sarcoma-associated herpesvirus K15 membrane protein. PG - 9346-58 AB - The K15 gene of Kaposi's sarcoma-associated herpesvirus (also known as human herpesvirus 8) consists of eight alternatively spliced exons and has been predicted to encode membrane proteins with a variable number of transmembrane regions and a common C-terminal cytoplasmic domain with putative binding sites for SH2 and SH3 domains, as well as for tumor necrosis factor receptor-associated factors. These features are reminiscent of the latent membrane proteins LMP-1 and LMP2A of Epstein-Barr virus and, more distantly, of the STP, Tip, and Tio proteins of the related gamma(2)-herpesviruses herpesvirus saimiri and herpesvirus ateles. These viral membrane proteins can activate a number of intracellular signaling pathways. We have therefore examined the abilities of different K15-encoded proteins to initiate intracellular signaling. We found that a 45-kDa K15 protein derived from all eight K15 exons and containing 12 predicted transmembrane domains in addition to the cytoplasmic domain activated the Ras/mitogen-activated protein kinase (MAPK) and NF-kappaB pathways, as well as (more weakly) the c-Jun N-terminal kinase/SAPK pathway. Activation of the MAPK and NF-kappaB pathways required phosphorylation of tyrosine residue 481 within a putative SH2-binding site (YEEVL). This motif was phosphorylated by the tyrosine kinases Src, Lck, Yes, Hck, and Fyn. The region containing the YEEVL motif interacted with tumor necrosis factor receptor-associated factor 2 (TRAF-2), and a dominant negative TRAF-2 mutant inhibited the K15-mediated activation of the Ras/MAPK pathway, suggesting the involvement of TRAF-2 in the initiation of these signaling routes. In contrast, several smaller K15 protein isoforms activated these pathways only weakly. All of the K15 isoforms tested were, however, localized in lipid rafts, suggesting that incorporation into lipid rafts is not sufficient to initiate signaling. Additional regions of K15, located presumably in exons 2 to 5, may therefore contribute to the activation of these pathways. These findings illustrate that the 45-kDa K15 protein engages pathways similar to LMP1, LMP2A, STP, Tip, and Tio but combines functional features that are separated between LMP1 and LMP2A or STP and Tip. AD - Institut fur Virologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany. FAU - Brinkmann, Melanie M AU - Brinkmann MM FAU - Glenn, Mark AU - Glenn M FAU - Rainbow, Lucille AU - Rainbow L FAU - Kieser, Arnd AU - Kieser A FAU - Henke-Gendo, Cornelia AU - Henke-Gendo C FAU - Schulz, Thomas F AU - Schulz TF LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (K15 protein, Human herpesvirus 8) RN - 0 (MAP Kinase Signaling System) RN - 0 (NF-kappa B) RN - 0 (Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (TNF Receptor-Associated Factor 2) RN - 0 (Transcription Factor AP-1) RN - 0 (Viral Proteins) RN - EC 2.7.1.112 (src-Family Kinases) RN - EC 2.7.1.37 (Mitogen-Activated Protein Kinase 1) RN - EC 2.7.1.37 (Mitogen-Activated Protein Kinase 8) RN - EC 2.7.1.37 (Mitogen-Activated Protein Kinases) SB - IM MH - Amino Acid Motifs MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Cell Line MH - Genes, Viral MH - Herpesvirus 8, Human/genetics/pathogenicity/*physiology MH - Humans MH - MAP Kinase Signaling System MH - Membrane Microdomains/virology MH - Mitogen-Activated Protein Kinase 1/biosynthesis/genetics MH - Mitogen-Activated Protein Kinase 8 MH - Mitogen-Activated Protein Kinases/biosynthesis/*metabolism MH - Mutation MH - NF-kappa B/*metabolism MH - Phosphorylation MH - Protein Binding MH - Proteins/genetics/metabolism MH - Recombinant Fusion Proteins/chemistry/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - TNF Receptor-Associated Factor 2 MH - Transcription Factor AP-1/metabolism MH - Viral Proteins/chemistry/genetics/*physiology MH - src-Family Kinases/metabolism EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9346-58. PMID- 12915551 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041222 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Interferon regulatory factor 7 regulates expression of Epstein-Barr virus latent membrane protein 1: a regulatory circuit. PG - 9359-68 AB - We have shown previously that interferon regulatory factor 7 (IRF7), a multifunctional protein intimately involved in latent Epstein-Barr virus (EBV) infection, is induced as well as activated by EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein. Since the LMP1 promoter (LMP1p) contains an interferon-stimulated response element (ISRE), we hypothesized that IRF7 might be able to regulate LMP1 expression and thus participate in a regulatory circuit between these two genes. In this study, IRF7 was shown first to activate LMP1p in transient transfection assays. Compared with EBV nuclear antigen 2 (EBNA2), the most potent viral transactivator of LMP1p, IRF7 has a lesser effect (approximately 10% that of EBNA2) on induction of LMP1p. Study with IRF7 deletion mutants showed that IRF7 functional domains have similar effects on both the beta interferon (IFN-beta) and LMP1 promoters in BJAB and 293 cells, and study with IRF7 phosphomimetic mutants showed that IRF7 phosphorylation may be involved in the activation of these two promoters. Further, the ISRE in LMP1p responds to IRF7 induction and IRF7 binds to this element. In the EBV-positive cell line P3HR1, which lacks the complete EBNA2 and EBV-encoded leader protein genes and hence expresses low-level LMP1, IRF7 alone can notably increase the endogenous LMP1 mRNA and protein levels. These results indicate that LMP1 is regulated by this host cell gene in addition to the viral factor, EBNA2, and may help to explain how LMP1 is expressed in type II latency in the absence of EBNA2. Moreover, IRF7 can regulate a viral gene in addition to a host cellular gene such as the IFN-beta gene. Together with the previous data that LMP1 can induce IRF7 expression and facilitate IRF7 phosphorylation and nuclear translocation, these results suggest a positive regulatory circuit between IRF7 and LMP1. AD - Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. FAU - Ning, Shunbin AU - Ning S FAU - Hahn, Angela M AU - Hahn AM FAU - Huye, Leslie E AU - Huye LE FAU - Pagano, Joseph S AU - Pagano JS LA - eng GR - AI 42372-01/AI/NIAID GR - CA 19014/CA/NCI GR - T32-CA09156-27/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (DNA-Binding Proteins) RN - 0 (EBNA2 protein) RN - 0 (EBV-associated membrane antigen, Epstein-Barr virus) RN - 0 (Epstein-Barr Virus Nuclear Antigens) RN - 0 (Recombinant Proteins) RN - 0 (Viral Matrix Proteins) RN - 0 (interferon regulatory factor-7) SB - IM MH - Active Transport, Cell Nucleus MH - Amino Acid Sequence MH - Base Sequence MH - Cell Line MH - DNA, Viral/genetics MH - DNA-Binding Proteins/chemistry/genetics/*physiology MH - Epstein-Barr Virus Nuclear Antigens/genetics/physiology MH - Gene Expression Regulation, Viral MH - Herpesvirus 4, Human/*genetics/*physiology MH - Humans MH - Models, Biological MH - Mutagenesis, Site-Directed MH - Phosphorylation MH - Promoter Regions (Genetics) MH - Protein Binding MH - Protein Structure, Tertiary MH - Recombinant Proteins/chemistry/genetics/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Transfection MH - Up-Regulation MH - Viral Matrix Proteins/*genetics/*physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9359-68. PMID- 12915552 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Passive immunization with neutralizing antibodies interrupts the mouse mammary tumor virus life cycle. PG - 9369-77 AB - Mouse mammary tumor virus (MMTV) infects the host via mucosal surfaces and exploits the host immune system for systemic spread and chronic infection. We have tested a neutralizing rat monoclonal antibody specific for the retroviral envelope glycoprotein gp52 for its efficiency in preventing acute and chronic mucosal and systemic infection. The antibody completely inhibits the superantigen response and chronic viral infection following systemic or nasal infection. Surprisingly however, the antibody only partially inhibits the early infection of antigen-presenting cells in the draining lymph node. Despite this initially inefficient protection from infection, superantigen-specific B- and T-cell responses and systemic viral spread are abolished, leading to complete clearance of the retroviral infection and hence interruption of the viral life cycle. In conclusion, systemic neutralizing monoclonal antibodies can provide an efficient protection against chronic retroviral amplification and persistence. AD - Swiss Institute for Experimental Cancer Research. Institute of Biochemistry, University of Lausanne, Switzerland. FAU - Mpandi, M AU - Mpandi M FAU - Otten, L A AU - Otten LA FAU - Lavanchy, C AU - Lavanchy C FAU - Acha-Orbea, H AU - Acha-Orbea H FAU - Finke, D AU - Finke D LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antibodies, Monoclonal) RN - 0 (Antibodies, Viral) RN - 0 (Antigens, Viral, Tumor) RN - 0 (DNA, Viral) RN - 0 (glycoprotein 52 antigen, Mouse mammary tumor virus) SB - IM MH - Animals MH - Antibodies, Monoclonal/administration & dosage MH - Antibodies, Viral/administration & dosage MH - Antigens, Viral, Tumor/genetics/immunology MH - B-Lymphocytes/immunology/pathology MH - Base Sequence MH - Cell Differentiation MH - DNA, Viral/genetics MH - Female MH - Immunity, Mucosal MH - Immunization, Passive MH - Mammary Neoplasms, Experimental/immunology/prevention & control MH - Mammary Tumor Virus, Mouse/genetics/*growth & development/*immunology MH - Mice MH - Mice, Inbred BALB C MH - Neutralization Tests MH - Rats MH - Rats, Inbred Lew MH - Research Support, Non-U.S. Gov't MH - Retroviridae Infections/immunology/prevention & control MH - Tumor Virus Infections/immunology/prevention & control EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9369-77. PMID- 12915553 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Silent point mutation in an avian retrovirus RNA processing element promotes c-myb-associated short-latency lymphomas. PG - 9378-87 AB - The avian leukosis virus DeltaLR-9 causes a high frequency of B-cell lymphomas within weeks after injection into 10-day-old chicken embryos. These lymphomas result from proviral integrations into the oncogene c-myb. In contrast, LR-9, which lacks the 42-nucleotide gag gene deletion of DeltaLR-9, does not cause a high frequency of c-myb-associated short-latency lymphomas. Although viral replication rates and spliced env mRNA levels were found to be similar for both viruses, DeltaLR-9 exhibited an increase in readthrough transcription compared to LR-9. The DeltaLR-9 deletion is located in the region of the gag gene corresponding to the matrix (MA) protein as well as in the negative regulator of splicing (NRS) element. To test whether disruption of the NRS or of the MA protein was responsible for inducing short-latency lymphomas, we generated viruses with NRS point mutations that maintained the wild-type Gag amino acid sequence. One of the mutant viruses induced an even higher incidence than DeltaLR-9 of short-latency lymphomas with viral integrations into c-myb. Thus, we propose that disruption of the NRS sequence promotes readthrough transcription and splicing to the downstream myb gene, causing overexpression of a slightly truncated Myb protein, which induces short-latency tumors. AD - Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA. FAU - Polony, Tatjana S AU - Polony TS FAU - Bowers, Sandra J AU - Bowers SJ FAU - Neiman, Paul E AU - Neiman PE FAU - Beemon, Karen L AU - Beemon KL LA - eng GR - R01CA20068/CA/NCI GR - R01CA48746/CA/NCI GR - T32GM07231/GM/NIGMS PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (Oncogene Proteins v-myb) RN - 0 (RNA, Messenger) RN - 0 (RNA, Viral) RN - 0 (Viral Matrix Proteins) SB - IM MH - Animals MH - Avian Leukosis/*etiology/genetics/virology MH - Base Sequence MH - Chick Embryo MH - DNA, Viral/genetics MH - Genes, env MH - *Genes, myb MH - Leukosis Virus, Avian/*genetics/*pathogenicity/physiology MH - Lymphoma, B-Cell/*etiology/genetics/virology MH - Oncogene Proteins v-myb/genetics/physiology MH - Point Mutation MH - RNA Processing, Post-Transcriptional/genetics MH - RNA Splicing/genetics MH - RNA, Messenger/genetics/metabolism MH - RNA, Viral/genetics/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Deletion MH - Viral Matrix Proteins/genetics/physiology MH - Virus Integration/genetics MH - Virus Replication/genetics EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9378-87. PMID- 12915554 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Link between genome packaging and rate of budding for Rous sarcoma virus. PG - 9388-98 AB - The subcellular location at which genomic RNA is packaged by Gag proteins during retrovirus assembly remains unknown. Since the membrane-binding (M) domain is most critical for targeting Gag to the plasma membrane, changes to this determinant might alter the path taken through the cell and reduce the efficiency of genome packaging. In this report, a Rous sarcoma virus (RSV) mutant having two acidic-to-basic substitutions in the M domain is described. This mutant, designated Super M, produced particles much faster than the wild type, but the mutant virions were noninfectious and contained only 1/10 the amount of genomic RNA found in wild-type particles. To identify the cause(s) of these defects, we considered data that suggest that RSV Gag traffics through the nucleus to package the viral genome. Although inhibition of the CRM-1 pathway of nuclear export caused the accumulation of wild-type Gag in the nucleus, nuclear accumulation did not occur with Super M. The importance of the nucleocapsid (NC) domain in membrane targeting was also determined, and, importantly, deletion of the NC sequence prevented plasma membrane localization by wild-type Gag but not by Super M Gag. Based on these results, we reasoned that the enhanced membrane-targeting properties of Super M inhibit genome packaging. Consistent with this interpretation, substitutions that reestablished the wild-type number of basic and acidic residues in the Super M Gag M domain reduced the budding efficiency and restored genome packaging and infectivity. Therefore, these data suggest that Gag targeting and genome packaging are normally linked to ensure that RSV particles contain viral RNA. AD - Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17036, USA. FAU - Callahan, Eric M AU - Callahan EM FAU - Wills, John W AU - Wills JW LA - eng GR - CA47482/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (Gene Products, gag) RN - 0 (RNA, Viral) SB - IM MH - Animals MH - Base Sequence MH - Cell Line MH - DNA, Viral/genetics MH - Gene Products, gag/chemistry/genetics/physiology MH - Genome, Viral MH - Microscopy, Electron MH - Mutagenesis, Site-Directed MH - Phenotype MH - RNA, Viral/genetics MH - Research Support, U.S. Gov't, P.H.S. MH - Sarcoma Viruses, Avian/*genetics/*physiology/ultrastructure MH - Suppression, Genetic MH - Virus Assembly EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9388-98. PMID- 12915555 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 transcriptional activator Rta is an oligomeric DNA-binding protein that interacts with tandem arrays of phased A/T-trinucleotide motifs. PG - 9399-411 AB - Kaposi's sarcoma associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) encodes an immediate early transcriptional activator, Rta, which mediates viral reactivation from latency and lytic viral replication. Here we report the purification and characterizations of HHV-8 Rta and its interaction with Rta-responsive DNA elements. The Rta response element (RtaRE) in the promoter of the KSHV/HHV-8 K8 open reading frame was mapped to a 47-bp sequence (RtaRE1) and a 60-bp sequence (RtaRE2) upstream of the TATA motif. A comparison of the K8 RtaREs with other viral RtaREs revealed a pattern of multiple A/T triplets spaced with a periodicity of 10 or 20 bp. Substitutions of the in-phase A/T trinucleotides of the RtaRE1 with G/C bases greatly diminished Rta responsiveness and Rta binding. By contrast, base substitutions in an out-of-phase A/T-trinucleotide sequence had no effect. Importantly, multimers of (A/T)(3)N(7) and N(5)(A/T)(5)N(6)(A/T)(4) motifs supported a strong Rta response in a copy number-dependent manner. No specific sequence motifs in the spacer regions could be discerned. Potent Rta response, however, was obtained with phased A/T trinucleotides with 7-bp spacers of arbitrary sequences with high G/C content. Lengthening of the phased A/T motifs or lowering of the G/C content of the spacers resulted in a reduction in Rta response. Finally, Escherichia coli-derived Rta is an oligomer of 440 kDa in molecular size and binds RtaRE as an oligomer. These results support a model of Rta transactivation wherein the subunits of the Rta oligomer make multiple contacts with a tandem array of phased A/T triplets in the configuration of (A/T)(3)(G/C)(7) repeats. AD - Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA. FAU - Liao, Wei AU - Liao W FAU - Tang, Yong AU - Tang Y FAU - Kuo, Yu-Liang AU - Kuo YL FAU - Liu, Bao-Ying AU - Liu BY FAU - Xu, Chi-Jie AU - Xu CJ FAU - Giam, Chou-Zen AU - Giam CZ LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (Immediate-Early Proteins) RN - 0 (Rta protein, Human herpesvirus 8) RN - 0 (Trans-Activators) RN - 0 (Viral Proteins) SB - IM MH - Base Sequence MH - Binding Sites/genetics MH - Cell Line MH - DNA, Viral/genetics/metabolism MH - Genes, Viral MH - Herpesvirus 8, Human/genetics/*physiology MH - Humans MH - Immediate-Early Proteins/chemistry/genetics/*physiology MH - Molecular Sequence Data MH - Open Reading Frames MH - Promoter Regions (Genetics) MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Tandem Repeat Sequences MH - Trans-Activators/chemistry/genetics/*physiology MH - Viral Proteins/chemistry/genetics/*physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9399-411. PMID- 12915556 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Edmonston measles virus prevents increased cell surface expression of peptide-loaded major histocompatibility complex class II proteins in human peripheral monocytes. PG - 9412-21 AB - Gamma interferon (IFN-gamma) induces expression of the gene products of the major histocompatibility complex (MHC), whereas IFN-alpha/beta can interfere with or suppress class II protein expression. In separate studies, measles virus (MV) was reported to induce IFN-alpha/beta and to up-regulate MHC class II proteins. In an attempt to resolve this paradox, we examined the surface expression of MHC class I and class II proteins in MV-infected peripheral monocytes in the presence and absence of IFN-alpha/beta. Infection of purified monocytes with Edmonston B MV resulted in an apparent increase in cell surface expression of HLA-A, -B, and -C class I proteins, but it had no effect on the expression of HLA-DR class II proteins. MV-infected purified monocytes expressed IFN-alpha/beta, but no measurable IFN-gamma expression was detected in supernatant fluids. Class II protein expression could be enhanced by coculture of purified monocytes with uninfected peripheral blood mononuclear cell (PBMC) supernatant. MV infection of PBMCs also did not affect expression of class II proteins, but the expression of HLA-A, -B, and -C class I proteins was increased two- to threefold in most donor cells. A direct role for IFN-alpha/beta suppression of MHC class II protein expression was not evident in monocytes since MV suppressed class II protein expression in the absence of IFN-alpha/beta. Taken together, these data suggest that MV interferes with the expression of peptide-loaded class II complexes, an effect that may potentially alter CD4(+)-T-cell proliferation and the cell-mediated immune responses that they help to regulate. AD - Respiratory and Enteric Viruses Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA. mby7@cdc.gov FAU - Yilla, Mamadi AU - Yilla M FAU - Hickman, Carole AU - Hickman C FAU - McGrew, Marcia AU - McGrew M FAU - Meade, Elizabeth AU - Meade E FAU - Bellini, William J AU - Bellini WJ LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antigens, CD14) RN - 0 (Histocompatibility Antigens Class I) RN - 0 (Histocompatibility Antigens Class II) RN - 0 (Interferon Type I, Recombinant) RN - 0 (Interferon-gamma, Recombinant) SB - IM MH - Adult MH - Antigens, CD14/metabolism MH - Cell Line MH - Cells, Cultured MH - Histocompatibility Antigens Class I/metabolism MH - Histocompatibility Antigens Class II/*metabolism MH - Humans MH - In Vitro MH - Interferon Type I, Recombinant/pharmacology MH - Interferon-gamma, Recombinant/pharmacology MH - Measles virus/*immunology/*pathogenicity MH - Monocytes/drug effects/*immunology/*virology MH - Research Support, Non-U.S. Gov't EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9412-21. PMID- 12915557 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Variability at human immunodeficiency virus type 1 subtype C protease cleavage sites: an indication of viral fitness? PG - 9422-30 AB - Naturally occurring polymorphisms in the protease of human immunodeficiency virus type 1 (HIV-1) subtype C would be expected to lead to adaptive (compensatory) changes in protease cleavage sites. To test this hypothesis, we examined the prevalences and patterns of cleavage site polymorphisms in the Gag, Gag-Pol, and Nef cleavage sites of C compared to those in non-C subtypes. Codon-based maximum-likelihood methods were used to assess the natural selection and evolutionary history of individual cleavage sites. Seven cleavage sites (p17/p24, p24/p2, NC/p1, NC/TFP, PR/RT, RT/p66, and p66/IN) were well conserved over time and in all HIV-1 subtypes. One site (p1/p6(gag)) exhibited moderate variation, and four sites (p2/NC, TFP/p6(pol), p6(pol)/PR, and Nef) were highly variable, both within and between subtypes. Three of the variable sites are known to be major determinants of polyprotein processing and virion production. P2/NC controls the rate and order of cleavage, p6(gag) is an important phosphoprotein required for virion release, and TFP/p6(pol), a novel cleavage site in the transframe domain, influences the specificity of Gag-Pol processing and the activation of protease. Overall, 58.3% of the 12 HIV-1 cleavage sites were significantly more diverse in C than in B viruses. When analyzed as a single concatenated fragment of 360 bp, 96.0% of group M cleavage site sequences fell into subtype-specific phylogenetic clusters, suggesting that they coevolved with the virus. Natural variation at C cleavage sites may play an important role, not only in regulation of the viral cycle but also in disease progression and response to therapy. AD - HIV-1 Molecular Virology and Bioinformatics Unit, Africa Centre for Health and Population Studies, and the Nelson R. Mandela School of Medicine, University of Natal, Durban, South Africa. FAU - de Oliveira, Tulio AU - de Oliveira T FAU - Engelbrecht, Susan AU - Engelbrecht S FAU - Janse van Rensburg, Estrelita AU - Janse van Rensburg E FAU - Gordon, Michelle AU - Gordon M FAU - Bishop, Karen AU - Bishop K FAU - zur Megede, Jan AU - zur Megede J FAU - Barnett, Susan W AU - Barnett SW FAU - Cassol, Sharon AU - Cassol S LA - eng GR - N01-AI-05396/AI/NIAID PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Fusion Proteins, gag-pol) RN - 0 (Gene Products, gag) RN - EC 3.4.23.- (HIV Protease) SB - IM MH - Amino Acid Sequence MH - Binding Sites/genetics MH - Comparative Study MH - Evolution, Molecular MH - Fusion Proteins, gag-pol/genetics/physiology MH - Gene Products, gag/genetics/physiology MH - Genes, gag MH - Genes, pol MH - HIV Infections/virology MH - HIV Protease/*physiology MH - HIV-1/classification/*genetics/pathogenicity/*physiology MH - Humans MH - In Vitro MH - Phylogeny MH - Polymorphism, Genetic MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Variation (Genetics) EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9422-30. PMID- 12915558 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Involvement of the matrix and nucleocapsid domains of the bovine leukemia virus Gag polyprotein precursor in viral RNA packaging. PG - 9431-8 AB - The RNA packaging process for retroviruses involves a recognition event of the genome-length viral RNA by the viral Gag polyprotein precursor (PrGag), an important step in particle morphogenesis. The mechanism underlying this genome recognition event for most retroviruses is thought to involve an interaction between the nucleocapsid (NC) domain of PrGag and stable RNA secondary structures that form the RNA packaging signal. Presently, there is limited information regarding PrGag-RNA interactions involved in RNA packaging for the deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2, respectively). To address this, alanine-scanning mutagenesis of BLV PrGag was done with a virus-like particle (VLP) system. As predicted, mutagenesis of conserved basic residues as well as residues of the zinc finger domains in the BLV NC domain of PrGag revealed residues that led to a reduction in viral RNA packaging. Interestingly, when conserved basic residues in the BLV MA domain of PrGag were mutated to alanine or glycine, but not when mutated to another basic residue, reductions in viral RNA packaging were also observed. The ability of PrGag to be targeted to the cell membrane was not affected by these mutations in MA, indicating that PrGag membrane targeting was not associated with the reduction in RNA packaging. These observations indicate that these basic residues in the MA domain of PrGag influence RNA packaging, without influencing Gag membrane localization. It was further observed that (i) a MA/NC double mutant had a more severe RNA packaging defect than either mutant alone, and (ii) RNA packaging was not found to be associated with transient localization of Gag in the nucleus. In summary, this report provides the first direct evidence for the involvement of both the BLV MA and NC domains of PrGag in viral RNA packaging. AD - Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio 43210, USA. FAU - Wang, Huating AU - Wang H FAU - Norris, Kendra M AU - Norris KM FAU - Mansky, Louis M AU - Mansky LM LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (Gene Products, gag) RN - 0 (Protein Precursors) RN - 0 (RNA, Viral) RN - 0 (Recombinant Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - COS Cells MH - Cattle MH - DNA, Viral/genetics MH - Gene Products, gag/*chemistry/genetics/*physiology MH - Humans MH - Leukemia Virus, Bovine/genetics/growth & development/*physiology MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - Protein Precursors/*chemistry/genetics/*physiology MH - Protein Structure, Tertiary MH - RNA, Viral/genetics/*physiology MH - Recombinant Proteins/chemistry/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Transfection EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9431-8. PMID- 12915559 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells. PG - 9439-50 AB - The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression. AD - Division of Research Immunology/BMT, Children's Hospital Los Angeles, Los Angeles, California 90027, USA. FAU - Haas, Dennis L AU - Haas DL FAU - Lutzko, Carolyn AU - Lutzko C FAU - Logan, Aaron C AU - Logan AC FAU - Cho, Gerald J AU - Cho GJ FAU - Skelton, Dianne AU - Skelton D FAU - Jin Yu, Xiao AU - Jin Yu X FAU - Pepper, Karen A AU - Pepper KA FAU - Kohn, Donald B AU - Kohn DB LA - eng GR - 1P01 CA59318/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (Genetic Vectors) RN - 0 (Peptide Elongation Factor 1) RN - 0 (Ubiquitin C) SB - IM MH - Animals MH - Base Sequence MH - Binding Sites/genetics MH - Bone Marrow Transplantation MH - Cell Line MH - DNA, Viral/*genetics/metabolism MH - Gene Expression Regulation, Viral MH - Genetic Vectors MH - Genome, Viral MH - Hematopoietic Stem Cells/*virology MH - Humans MH - In Vitro MH - Lentivirus/genetics MH - Mice MH - Moloney murine leukemia virus/*genetics/pathogenicity/*physiology MH - Peptide Elongation Factor 1/genetics MH - Promoter Regions (Genetics) MH - Research Support, U.S. Gov't, P.H.S. MH - Simian virus 40/genetics MH - Ubiquitin C/genetics EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9439-50. PMID- 12915560 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Comparative study of regulation of RTA-responsive genes in Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8. PG - 9451-62 AB - Replication and transcription activator (RTA) (also referred to as ORF50), an immediate-early gene product of Kaposi's sarcoma-associated herpesvirus (KSHV)/(human herpesvirus 8), plays a critical role in balancing the viral life cycle between latency and lytic replication. RTA has been shown to act as a strong transcription activator for several downstream genes of KSHV. Direct binding of RTA to DNA is thought to be one of the important mechanisms for transactivation of target genes, while indirect mechanisms are also implicated in RTA transactivation of certain selected genes. This study demonstrated direct binding of the DNA-binding domain of RTA (Rdbd) to a Kaposin (Kpsn) promoter sequence, which is highly homologous to the RTA-responsive element (RRE) of the PAN promoter. We undertook a comparative study of the RREs of PAN RNA, ORF57, vIL-6, and Kpsn to understand how RTA regulates gene expression during lytic replication. Comparing RNA abundance and transcription initiation rates of these RTA target genes in virus-infected cells suggested that the transcription initiation rate of the promoters is a major determinant of viral gene expression, rather than stability of the transcripts. RTA-mediated transactivation of reporters containing each RRE showed that their promoter strengths in a transient-transfection system were comparable to their transcription rates during reactivation. Moreover, our electrophoretic mobility shift assays of each RRE demonstrated that the highly purified Rdbd protein directly bound to the RREs. Based on these results, we conclude that direct binding of RTA to these target sequences contributes to their gene expression to various extents during the lytic life cycle of KSHV. AD - Department of Molecular and Medical Pharmacology, UCLA AIDS Institute, Jonsson Comprehensive Cancer Center, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA. FAU - Song, Moon Jung AU - Song MJ FAU - Deng, Hongyu AU - Deng H FAU - Sun, Ren AU - Sun R LA - eng GR - CA83525/CA/NCI GR - CA91791/CA/NCI GR - DE14153/DE/NIDCR PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (Immediate-Early Proteins) RN - 0 (RNA, Viral) RN - 0 (Rta protein, Human herpesvirus 8) RN - 0 (Trans-Activators) RN - 0 (Viral Proteins) RN - 0 (kaposin B protein, Human herpesvirus 8) SB - IM MH - Base Sequence MH - Cell Line MH - Comparative Study MH - DNA, Viral/genetics/metabolism MH - Gene Expression Regulation, Viral MH - Genes, Reporter MH - Genes, Viral MH - Herpesvirus 8, Human/*genetics/pathogenicity/*physiology MH - Humans MH - Immediate-Early Proteins/*genetics/*physiology MH - Promoter Regions (Genetics) MH - RNA, Viral/genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Trans-Activation (Genetics) MH - Trans-Activators/*genetics/*physiology MH - Viral Proteins/*genetics/*physiology MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9451-62. PMID- 12915561 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity. PG - 9463-73 AB - The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4(+) T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD(49-82), gD(146-179), gD(228-257), and gD(332-358)), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4(+) Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2(d) mice with the four newly identified CD4(+) Th1 peptide epitopes but not with four CD4(+) Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes. AD - Department of Ophthalmology, University of California-Irvine, College of Medicine, Orange, California 92868, USA. Lbenmoha@uci.edu FAU - BenMohamed, Lbachir AU - BenMohamed L FAU - Bertrand, Georges AU - Bertrand G FAU - McNamara, Cory D AU - McNamara CD FAU - Gras-Masse, Helene AU - Gras-Masse H FAU - Hammer, Juergen AU - Hammer J FAU - Wechsler, Steven L AU - Wechsler SL FAU - Nesburn, Anthony B AU - Nesburn AB LA - eng GR - EY09392/EY/NEI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antigens, Viral) RN - 0 (Herpesvirus Vaccines) RN - 0 (Immunodominant Epitopes) RN - 0 (Interleukin-2) RN - 0 (Viral Envelope Proteins) RN - 0 (glycoprotein D, Human herpesvirus 1) RN - 82115-62-6 (Interferon Type II) SB - IM MH - Algorithms MH - Amino Acid Sequence MH - Animals MH - Antigens, Viral/chemistry/*genetics MH - Herpes Simplex/immunology/prevention & control MH - Herpesvirus 1, Human/*genetics/*immunology MH - Herpesvirus Vaccines/genetics/pharmacology MH - Immunization MH - Immunodominant Epitopes/chemistry/*genetics MH - Interferon Type II/biosynthesis MH - Interleukin-2/biosynthesis MH - Lymphocyte Depletion MH - Mice MH - Mice, Inbred BALB C MH - Mice, Inbred C3H MH - Mice, Inbred C57BL MH - Molecular Sequence Data MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Th1 Cells/*immunology MH - Viral Envelope Proteins/*genetics/*immunology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9463-73. PMID- 12915562 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - The Mason-Pfizer monkey virus PPPY and PSAP motifs both contribute to virus release. PG - 9474-85 AB - Late (L) domains are required for the efficient release of several groups of enveloped viruses. Three amino acid motifs have been shown to provide L-domain function, namely, PPXY, PT/SAP, or YPDL. The retrovirus Mason-Pfizer monkey virus (MPMV) carries closely spaced PPPY and PSAP motifs. Mutation of the PPPY motif results in a complete loss of virus release. Here, we show that the PSAP motif acts as an additional L domain and promotes the efficient release of MPMV but requires an intact PPPY motif to perform its function. Examination of HeLaP4 cells expressing PSAP mutant virus by electron microscopy revealed mostly late budding structures and chains of viruses accumulating at the cell surface with little free virus. In the case of the PPPY mutant virus, budding appeared to be mostly arrested at an earlier stage before induction of membrane curvature. The cellular protein TSG101, which interacts with the human immunodeficiency virus type 1 (HIV-1) PTAP L domain, was packaged into MPMV in a PSAP-dependent manner. Since TSG101 is crucial for HIV-1 release, this result suggests that the Gag-TSG101 interaction is responsible for the virus release function of the MPMV PSAP motif. Nedd4, which has been shown to interact with viral PPPY motifs, was also detected in MPMV particles, albeit at much lower levels. Consistent with a role of VPS4A in the budding of both PPPY and PTAP motif-containing viruses, the overexpression of ATPase-defective GFP-VPS4A fusion proteins blocked both wild-type and PSAP mutant virus release. AD - Abteilung Virologie, Universitatsklinikum Heidelberg, D-69120 Heidelberg, Germany. FAU - Gottwein, Eva AU - Gottwein E FAU - Bodem, Jochen AU - Bodem J FAU - Muller, Barbara AU - Muller B FAU - Schmechel, Ariane AU - Schmechel A FAU - Zentgraf, Hanswalter AU - Zentgraf H FAU - Krausslich, Hans-Georg AU - Krausslich HG LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Calcium-Binding Proteins) RN - 0 (DNA-Binding Proteins) RN - 0 (Gene Products, gag) RN - 0 (Transcription Factors) RN - 0 (Tsg101 protein) RN - EC 6. (Ligases) RN - EC 6.3.2.19 (Nedd4 ubiquitin protein ligases) RN - EC 6.3.2.19 (Ubiquitin-Protein Ligases) SB - IM MH - Amino Acid Motifs MH - Amino Acid Sequence MH - Animals MH - Calcium-Binding Proteins/metabolism MH - Cell Line MH - DNA-Binding Proteins/metabolism MH - Gene Products, gag/chemistry/genetics/physiology MH - HIV-1/genetics/physiology MH - Hela Cells MH - Humans MH - Kinetics MH - Ligases/metabolism MH - Mason-Pfizer monkey virus/*genetics/*physiology/ultrastructure MH - Microscopy, Electron MH - Molecular Sequence Data MH - Protein Processing, Post-Translational MH - Research Support, Non-U.S. Gov't MH - Sequence Deletion MH - Transcription Factors/metabolism MH - Transfection MH - *Ubiquitin-Protein Ligases MH - Virus Assembly EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9474-85. PMID- 12915563 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Monkey rotavirus binding to alpha2beta1 integrin requires the alpha2 I domain and is facilitated by the homologous beta1 subunit. PG - 9486-501 AB - Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen. AD - Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Victoria 3010, Australia. FAU - Londrigan, Sarah L AU - Londrigan SL FAU - Graham, Kate L AU - Graham KL FAU - Takada, Yoshikazu AU - Takada Y FAU - Halasz, Peter AU - Halasz P FAU - Coulson, Barbara S AU - Coulson BS LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antibodies, Monoclonal) RN - 0 (Integrin alpha2beta1) RN - 0 (Protein Subunits) RN - 0 (Recombinant Proteins) SB - IM MH - Animals MH - Antibodies, Monoclonal MH - Binding Sites MH - CHO Cells MH - Caco-2 Cells MH - Cell Line MH - Hamsters MH - Humans MH - Integrin alpha2beta1/chemistry/genetics/*metabolism MH - Precipitin Tests MH - Protein Structure, Tertiary MH - Protein Subunits MH - Recombinant Proteins/chemistry/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Rotavirus/immunology/pathogenicity/*physiology MH - Transfection EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9486-501. PMID- 12915564 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Complementation of a deletion in the rubella virus p150 nonstructural protein by the viral capsid protein. PG - 9502-10 AB - Rubella virus (RUB) replicons with an in-frame deletion of 507 nucleotides between two NotI sites in the P150 nonstructural protein (DeltaNotI) do not replicate (as detected by expression of a reporter gene encoded by the replicon) but can be amplified by wild-type helper virus (Tzeng et al., Virology 289:63-73, 2001). Surprisingly, virus with DeltaNotI was viable, and it was hypothesized that this was due to complementation of the NotI deletion by one of the virion structural protein genes. Introduction of the capsid (C) protein gene into DeltaNotI-containing replicons as an in-frame fusion with a reporter gene or cotransfection with both DeltaNotI replicons and RUB replicon or plasmid constructs containing the C gene resulted in replication of the DeltaNotI replicon, confirming the hypothesis that the C gene was the structural protein gene responsible for complementation and demonstrating that complementation could occur either in cis or in trans. Approximately the 5' one-third of the C gene was necessary for complementation. Mutations that prevented translation of the C protein while minimally disturbing the C gene sequence abrogated complementation, while synonymous codon mutations that changed the C gene sequence without affecting the amino acid sequence at the 5' end of the C gene had no effect on complementation, indicating that the C protein, not the C gene RNA, was the moiety responsible for complementation. Complementation occurred at a basic step in the virus replication cycle, because DeltaNotI replicons failed to accumulate detectable virus-specific RNA. AD - Department of Biology, Georgia State University, Atlanta, Georgia 30302-4010, USA. FAU - Tzeng, Wen-Pin AU - Tzeng WP FAU - Frey, Teryl K AU - Frey TK LA - eng GR - AI21389/AI/NIAID PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Capsid Proteins) RN - 0 (RNA, Viral) RN - 0 (Viral Nonstructural Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Capsid Proteins/*genetics/*physiology MH - Cercopithecus aethiops MH - Genes, Reporter MH - Genes, Viral MH - Genetic Complementation Test MH - In Vitro MH - Molecular Sequence Data MH - Mutation MH - RNA, Viral/genetics MH - Replicon/genetics MH - Research Support, U.S. Gov't, P.H.S. MH - Rubella virus/*genetics/*physiology MH - Sequence Deletion MH - Sequence Homology, Amino Acid MH - Vero Cells MH - Viral Nonstructural Proteins/*genetics/*physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9502-10. PMID- 12915565 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus. PG - 9511-21 AB - The susceptibility of the tree shrew Tupaia belangeri to human hepatitis B virus (HBV) has been demonstrated both in vivo and in vitro. In this study, we show that purified HBV infects primary T. belangeri hepatocyte cultures in a very specific manner, as detected by HBV covalently closed circular DNA, mRNA, HBV e antigen, and HBsAg production. A monoclonal antibody (MAb), MA18/7, directed against the pre-S1 domain of the large HBs protein, which has been shown to neutralize infectivity of HBV for primary human hepatocytes, also blocked infection of primary Tupaia hepatocytes. MAbs against the pre-S2 domain of HBs inhibited infection only partially, whereas an S MAb and polyvalent anti-HBs antibodies neutralized infection completely. Thus, both pre-S1 and S antigens are necessary for infection in the tupaia. Using subviral particles, >70% of primary Tupaia hepatocytes are capable of specific binding of pre-S1-rich HBsAg, showing localization in distinct membrane areas. The data show that the early steps of HBV infection in Tupaia hepatocyte cultures are comparable to those in the human system. AD - Institute of Medical Virology. Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, 35392 Giessen, Germany. dieter.glebe@viro.med.unigiessen.de FAU - Glebe, Dieter AU - Glebe D FAU - Aliakbari, Mehriar AU - Aliakbari M FAU - Krass, Peter AU - Krass P FAU - Knoop, Eva V AU - Knoop EV FAU - Valerius, Klaus P AU - Valerius KP FAU - Gerlich, Wolfram H AU - Gerlich WH LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (Hepatitis B Antibodies) RN - 0 (Hepatitis B Surface Antigens) RN - 0 (Protein Precursors) RN - 0 (presurface protein 1, hepatitis B surface antigen) SB - IM MH - Animals MH - Base Sequence MH - Cells, Cultured MH - DNA, Viral/genetics MH - Hepatitis B Antibodies MH - Hepatitis B Surface Antigens/genetics/*physiology MH - Hepatitis B virus/genetics/immunology/*pathogenicity/physiology MH - Hepatocytes/*virology MH - Humans MH - Kinetics MH - Neutralization Tests MH - Protein Precursors/genetics/*physiology MH - Research Support, Non-U.S. Gov't MH - Tupaia MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9511-21. PMID- 12915566 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Newcastle disease virus V protein is a determinant of host range restriction. PG - 9522-32 AB - It has been demonstrated that the V protein of Newcastle disease virus (NDV) functions as an alpha/beta interferon (IFN-alpha/beta) antagonist (M. S. Park, M. L. Shaw, J. Munoz-Jordan, J. F. Cros, T. Nakaya, N. Bouvier, P. Palese, A. Garcia-Sastre, and C. F. Basler, J. Virol. 77:1501-1511, 2003). We now show that the NDV V protein plays an important role in host range restriction. In order to study V functions in vivo, recombinant NDV (rNDV) mutants, defective in the expression of the V protein, were generated. These rNDV mutants grow poorly in both embryonated chicken eggs and chicken embryo fibroblasts (CEFs) compared to the wild-type (wt) rNDV. However, insertion of the NS1 gene of influenza virus A/PR8/34 into the NDV V(-) genome [rNDV V(-)/NS1] restores impaired growth to wt levels in embryonated chicken eggs and CEFs. These data indicate that for viruses infecting avian cells, the NDV V protein and the influenza NS1 protein are functionally interchangeable, even though there are no sequence similarities between the two proteins. Interestingly, in human cells, the titer of wt rNDV is 10 times lower than that of rNDV V(-)/NS1. Correspondingly, the level of IFN secreted by human cells infected with wt rNDV is much higher than that secreted by cells infected with the NS1-expressing rNDV. This suggests that the IFN antagonist activity of the NDV V protein is species specific. Finally, the NDV V protein plays an important role in preventing apoptosis in a species-specific manner. The rNDV defective in V induces apoptotic cell death more rapidly in CEFs than does wt rNDV. Taken together, these data suggest that the host range of NDV is limited by the ability of its V protein to efficiently prevent innate host defenses, such as the IFN response and apoptosis. AD - Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, USA. FAU - Park, Man-Seong AU - Park MS FAU - Garcia-Sastre, Adolfo AU - Garcia-Sastre A FAU - Cros, Jerome F AU - Cros JF FAU - Basler, Christopher F AU - Basler CF FAU - Palese, Peter AU - Palese P LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (36 kDa V protein, Newcastle disease virus) RN - 0 (INS1 protein, influenza virus) RN - 0 (Interferon-alpha) RN - 0 (RNA, Messenger) RN - 0 (RNA, Viral) RN - 0 (Viral Nonstructural Proteins) RN - 0 (Viral Proteins) RN - 77238-31-4 (Interferon-beta) SB - IM MH - Amino Acid Sequence MH - Animals MH - Apoptosis MH - Base Sequence MH - Cell Line MH - Cells, Cultured MH - Cercopithecus aethiops MH - Chick Embryo MH - Humans MH - Interferon-alpha/antagonists & inhibitors/biosynthesis MH - Interferon-beta/antagonists & inhibitors/biosynthesis MH - Mutagenesis, Site-Directed MH - Newcastle disease virus/genetics/immunology/pathogenicity/*physiology MH - RNA Editing/genetics MH - RNA, Messenger/genetics MH - RNA, Viral/genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Deletion MH - Species Specificity MH - Vero Cells MH - Viral Nonstructural Proteins/genetics/physiology MH - Viral Proteins/genetics/immunology/*physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9522-32. PMID- 12915567 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Latent herpes simplex virus infection of sensory neurons alters neuronal gene expression. PG - 9533-41 AB - The persistence of herpes simplex virus (HSV) and the diseases that it causes in the human population can be attributed to the maintenance of a latent infection within neurons in sensory ganglia. Little is known about the effects of latent infection on the host neuron. We have addressed the question of whether latent HSV infection affects neuronal gene expression by using microarray transcript profiling of host gene expression in ganglia from latently infected versus mock-infected mouse trigeminal ganglia. (33)P-labeled cDNA probes from pooled ganglia harvested at 30 days postinfection or post-mock infection were hybridized to nylon arrays printed with 2,556 mouse genes. Signal intensities were acquired by phosphorimager. Mean intensities (n = 4 replicates in each of three independent experiments) of signals from mock-infected versus latently infected ganglia were compared by using a variant of Student's t test. We identified significant changes in the expression of mouse neuronal genes, including several with roles in gene expression, such as the Clk2 gene, and neurotransmission, such as genes encoding potassium voltage-gated channels and a muscarinic acetylcholine receptor. We confirmed the neuronal localization of some of these transcripts by using in situ hybridization. To validate the microarray results, we performed real-time reverse transcriptase PCR analyses for a selection of the genes. These studies demonstrate that latent HSV infection can alter neuronal gene expression and might provide a new mechanism for how persistent viral infection can cause chronic disease. AD - Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA. FAU - Kramer, Martha F AU - Kramer MF FAU - Cook, W James AU - Cook WJ FAU - Roth, Frederick P AU - Roth FP FAU - Zhu, Jia AU - Zhu J FAU - Holman, Holly AU - Holman H FAU - Knipe, David M AU - Knipe DM FAU - Coen, Donald M AU - Coen DM LA - eng GR - P01 NS 35138/NS/NINDS PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (RNA, Messenger) SB - IM MH - Animals MH - Gene Expression MH - Gene Expression Profiling MH - Genes, MHC Class II MH - Genes, Viral MH - Herpes Simplex/etiology/genetics/physiopathology MH - Humans MH - Male MH - Mice MH - Mice, Inbred ICR MH - Neurons, Afferent/*physiology/*virology MH - Oligonucleotide Array Sequence Analysis MH - RNA, Messenger/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Reverse Transcriptase Polymerase Chain Reaction MH - Simplexvirus/genetics/*pathogenicity EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9533-41. PMID- 12915568 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry. PG - 9542-52 AB - Herpes simplex virus (HSV) entry requires the interaction of glycoprotein D (gD) with a cellular receptor such as herpesvirus entry mediator (HVEM or HveA) or nectin-1 (HveC). However, the fusion mechanism is still not understood. Since cholesterol-enriched cell membrane lipid rafts are involved in the entry of other enveloped viruses such as human immunodeficiency virus and Ebola virus, we tested whether HSV entry proceeds similarly. Vero cells and cells expressing either HVEM or nectin-1 were treated with cholesterol-sequestering drugs such as methyl-beta-cyclodextrin or nystatin and then exposed to virus. In all cases, virus entry was inhibited in a dose-dependent manner, and the inhibitory effect was fully reversible by replenishment of cholesterol. To examine the association of HVEM and nectin-1 with lipid rafts, we analyzed whether they partitioned into nonionic detergent-insoluble glycolipid-enriched membranes (DIG). There was no constitutive association of either receptor with DIG. Binding of soluble gD or virus to cells did not result in association of nectin-1 with the raft-containing fractions. However, during infection, a fraction of gB but not gC, gD, or gH associated with DIG. Similarly, when cells were incubated with truncated soluble glycoproteins, soluble gB but not gC was found associated with DIG. Together, these data favor a model in which HSV uses gB to rapidly mobilize lipid rafts that may serve as a platform for entry and cell signaling. It also suggests that gB may interact with a cellular molecule associated with lipid rafts. AD - Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. fbender@biochem.dental.upen.edu FAU - Bender, Florent C AU - Bender FC FAU - Whitbeck, J Charles AU - Whitbeck JC FAU - Ponce de Leon, Manuel AU - Ponce de Leon M FAU - Lou, Huan AU - Lou H FAU - Eisenberg, Roselyn J AU - Eisenberg RJ FAU - Cohen, Gary H AU - Cohen GH LA - eng GR - AI-18289/AI/NIAID GR - NS-30606/NS/NINDS GR - NS-36731/NS/NINDS PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Cell Adhesion Molecules) RN - 0 (Receptors, Tumor Necrosis Factor) RN - 0 (Receptors, Virus) RN - 0 (Recombinant Proteins) RN - 0 (Viral Envelope Proteins) RN - 0 (glycoprotein D, Human herpesvirus 1) RN - 0 (herpesvirus entry mediator) RN - 0 (nectin) RN - 57-88-5 (Cholesterol) SB - IM MH - Animals MH - Cell Adhesion Molecules/genetics/physiology MH - Cercopithecus aethiops MH - Cholesterol/metabolism MH - Herpesvirus 1, Human/genetics/*pathogenicity/*physiology MH - Humans MH - Membrane Microdomains/metabolism/*virology MH - Models, Biological MH - Receptors, Tumor Necrosis Factor/genetics/physiology MH - Receptors, Virus/genetics/physiology MH - Recombinant Proteins/genetics/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Solubility MH - Vero Cells MH - Viral Envelope Proteins/genetics/*physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9542-52. PMID- 12915569 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods. PG - 9553-66 AB - A major impediment to the use of adenovirus as a gene therapy vector and for vaccine applications is the host immune response to adenovirus hexon-the major protein component of the icosahedral capsid. A solution may lie in novel vectors with modified or chimeric hexons designed to evade the immune response. To facilitate this approach, we have distinguished the portion of hexon that all serotypes have in common from the hypervariable regions that are responsible for capsid diversity and type-specific immunogenicity. The common hexon core-conserved because it forms the viral capsid-sets boundaries to the regions where modifications can be made to produce nonnative hexons. The core has been defined from the large and diverse set of known hexon sequences by an accurate alignment based on the newly refined crystal structures of human adenovirus types 2 (Ad2) and Ad5 hexon. Comparison of the two hexon models, which are the most accurate so far, reveals that over 90% of the residues in each have three-dimensional positions that closely match. Structures for more distant hexons were predicted by building molecular models of human Ad4, chimpanzee adenovirus (AdC68), and fowl adenovirus 1 (FAV1 or CELO). The five structures were then used to guide the alignment of the 40 full-length (>900 residues) hexon sequences in public databases. Distance- and parsimony-based phylogenetic trees are consistent and reveal evolutionary relationships between adenovirus types that parallel those of their animal hosts. The combination of crystallography, molecular modeling, and phylogenetic analysis defines a conserved molecular core that can serve as the armature for the directed design of novel hexons. AD - The Wistar Institute, Philadelphia, Pennsylvania 19104, USA. FAU - Rux, John J AU - Rux JJ FAU - Kuser, Paula R AU - Kuser PR FAU - Burnett, Roger M AU - Burnett RM LA - eng GR - AI-17270/AI/NIAID GR - CA 09171/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Capsid Proteins) RN - 0 (Genetic Vectors) RN - 0 (Protein Subunits) RN - 0 (hexon capsid protein, Adenovirus) SB - IM MH - Adenoviruses, Human/*chemistry/genetics/immunology MH - Amino Acid Sequence MH - Animals MH - Capsid Proteins/*chemistry/genetics/immunology MH - Crystallography, X-Ray MH - Gene Therapy MH - Genetic Vectors MH - Humans MH - Models, Molecular MH - Molecular Sequence Data MH - Phylogeny MH - Protein Structure, Quaternary MH - Protein Subunits MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Amino Acid EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9553-66. PMID- 12915570 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events. PG - 9567-77 AB - Toroviruses (family Coronaviridae, order Nidovirales) are enveloped, positive-stranded RNA viruses that have been implicated in enteric disease in cattle and possibly in humans. Despite their potential veterinary and clinical relevance, little is known about torovirus epidemiology and molecular genetics. Here, we present the first study into the diversity among toroviruses currently present in European swine and cattle herds. Comparative sequence analysis was performed focusing on the genes for the structural proteins S, M, HE, and N, with fecal specimens serving as sources of viral RNA. Sequence data published for animal and human torovirus variants were included. Four genotypes, displaying 30 to 40% divergence, were readily distinguished, exemplified by bovine torovirus (BToV) Breda, porcine torovirus (PToV) Markelo, equine torovirus Berne, and the putative human torovirus. The ungulate toroviruses apparently display host species preference. In phylogenetic analyses, all PToV variants clustered, while the recent European BToVs mostly resembled the New World BToV variant Breda, identified 19 years ago. However, we found ample evidence for recurring intertypic recombination. All newly characterized BToV variants seem to have arisen from a genetic exchange, during which the 3' end of the HE gene, the N gene, and the 3' nontranslated region of a Breda virus-like parent had been swapped for those of PToV. Moreover, some PToV and BToV variants carried chimeric HE genes, which apparently resulted from recombination events involving hitherto unknown toroviruses. From these observations, the existence of two additional torovirus genotypes can be inferred. Toroviruses may be even more promiscuous than their closest relatives, the coronaviruses and arteriviruses. AD - Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. FAU - Smits, S L AU - Smits SL FAU - Lavazza, A AU - Lavazza A FAU - Matiz, K AU - Matiz K FAU - Horzinek, M C AU - Horzinek MC FAU - Koopmans, M P AU - Koopmans MP FAU - de Groot, R J AU - de Groot RJ LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) SB - IM MH - Animals MH - Base Sequence MH - Cattle MH - Cattle Diseases/epidemiology/virology MH - DNA, Viral/genetics MH - Epidemiology, Molecular MH - Europe/epidemiology MH - Evolution, Molecular MH - Humans MH - Microscopy, Electron MH - Models, Genetic MH - Molecular Sequence Data MH - Phylogeny MH - Recombination, Genetic MH - Sequence Homology, Nucleic Acid MH - Sus scrofa MH - Swine Diseases/epidemiology/virology MH - Torovirus/*classification/*genetics/isolation & purification MH - Torovirus Infections/epidemiology/veterinary/virology MH - Variation (Genetics) EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9567-77. PMID- 12915571 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Epidemiology, genetic diversity, and evolution of endemic feline immunodeficiency virus in a population of wild cougars. PG - 9578-89 AB - Within the large body of research on retroviruses, the distribution and evolution of endemic retroviruses in natural host populations have so far received little attention. In this study, the epidemiology, genetic diversity, and molecular evolution of feline immunodeficiency virus specific to cougars (FIVpco) was examined using blood samples collected over several years from a free-ranging cougar population in the western United States. The virus prevalence was 58% in this population (n = 52) and increased significantly with host age. Based on phylogenetic analysis of fragments of envelope (env) and polymerase (pol) genes, two genetically distinct lineages of FIVpco were found to cooccur in the population but not in the same individuals. Within each of the virus lineages, geographically nearby isolates formed monophyletic clusters of closely related viruses. Sequence diversity for env within a host rarely exceeded 1%, and the evolution of this gene was dominated by purifying selection. For both pol and env, our data indicate mean rates of molecular evolution of 1 to 3% per 10 years. These results support the premise that FIVpco is well adapted to its cougar host and provide a basis for comparing lentivirus evolution in endemic and epidemic infections in natural hosts. AD - Wildlife Biology Program, University of Montana, Missoula, Montana 59812, USA. FAU - Biek, Roman AU - Biek R FAU - Rodrigo, Allen G AU - Rodrigo AG FAU - Holley, David AU - Holley D FAU - Drummond, Alexei AU - Drummond A FAU - Anderson, Charles R Jr AU - Anderson CR Jr FAU - Ross, Howard A AU - Ross HA FAU - Poss, Mary AU - Poss M LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antibodies, Viral) RN - 0 (DNA, Viral) SB - IM MH - Animals MH - Antibodies, Viral/blood MH - Base Sequence MH - Carnivora/*virology MH - DNA, Viral/genetics MH - Epidemiology, Molecular MH - Evolution, Molecular MH - Female MH - Genes, env MH - Genes, pol MH - Immunodeficiency Virus, Feline/*genetics/immunology/isolation & purification MH - Lentivirus Infections/epidemiology/immunology/veterinary/virology MH - Male MH - Molecular Sequence Data MH - Phylogeny MH - Research Support, Non-U.S. Gov't MH - United States/epidemiology MH - Variation (Genetics) EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9578-89. PMID- 12915572 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - CCAAT/enhancer-binding protein-alpha is induced during the early stages of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic cycle reactivation and together with the KSHV replication and transcription activator (RTA) cooperatively stimulates the viral RTA, MTA, and PAN promoters. PG - 9590-612 AB - During the immediate-early (IE) phase of reactivation from latency, the Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator protein (RTA) (or ORF50) is thought to be the most critical trigger that upregulates expression of many downstream viral lytic cycle genes, including the delayed-early (DE) gene encoding the replication-associated protein (RAP) (or K8). RAP physically interacts with and stabilizes the cellular transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha), leading to upregulated expression of the cellular C/EBPalpha and p21(CIP-1) proteins followed by G(0)/G(1) cell cycle arrest. Furthermore, RTA also interacts with C/EBPalpha, and both RAP and RTA cooperate with C/EBPalpha to activate the RAP promoter through binding to a strong proximal C/EBP binding site that also serves as an RTA-responsive element (RRE). Here we show that C/EBPalpha also activates the IE RTA promoter in transient-cotransfection reporter gene assays and that addition of either RTA or RAP enhances the effect. Electrophoretic mobility shift assay and deletion analysis revealed three C/EBP binding sites that mediate cooperative transactivation of the RTA promoter by C/EBPalpha and RTA. Furthermore, chromatin immunoprecipitation assay results showed that the endogenous C/EBPalpha, RTA, and RAP proteins all associate with RTA promoter sequences in tetradecanoyl phorbol acetate-induced primary effusion lymphoma (PEL) cells. Induction of endogenous KSHV RTA mRNA in PEL cells by exogenously introduced C/EBPalpha was confirmed by reverse transcription-PCR analysis and by double-label indirect immunofluorescence assays. Reciprocally, expression of exogenous RTA also led to an increase of endogenous C/EBPalpha expression that could be detected by Western immunoblot assays even in KSHV-negative DG75 cells. Cotransfected RTA also increased positive C/EBPalpha autoregulation of the C/EBPalpha promoter in transient-cotransfection reporter gene assays. Finally, C/EBPalpha proved to strongly activate the promoters of two other KSHV DE genes encoding PAN (polyadenylated nuclear) RNA and MTA (ORF57), which was again mediated by C/EBP binding sites that also contribute to RTA activation. Overall, these results support a model in which the cellular transcription factor C/EBPalpha and RTA:C/EBPalpha interactions play important roles both upstream and downstream of the two major KSHV regulatory proteins RTA and RAP during the early stages of lytic cycle reactivation. AD - Molecular Virology Laboratories, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231-1000, USA. FAU - Wang, Shizhen Emily AU - Wang SE FAU - Wu, Frederick Y AU - Wu FY FAU - Yu, Yanxing AU - Yu Y FAU - Hayward, Gary S AU - Hayward GS LA - eng GR - R01 CA73585/CA/NCI GR - R01 CA81400/CA/NCI GR - T32 CA09243/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (CCAAT-Enhancer-Binding Protein-alpha) RN - 0 (Carrier Proteins) RN - 0 (DNA, Viral) RN - 0 (Immediate-Early Proteins) RN - 0 (K8 protein, Human herpesvirus 8) RN - 0 (RNA, Messenger) RN - 0 (RNA, Viral) RN - 0 (Rta protein, Human herpesvirus 8) RN - 0 (Trans-Activators) RN - 0 (Viral Proteins) SB - IM MH - Base Sequence MH - Binding Sites/genetics MH - CCAAT-Enhancer-Binding Protein-alpha/*biosynthesis/genetics MH - Carrier Proteins/genetics/physiology MH - Cell Line MH - DNA, Viral/genetics MH - Gene Expression Regulation, Viral MH - Genes, Reporter MH - Genes, Viral MH - Hela Cells MH - Herpesvirus 8, Human/genetics/*pathogenicity/physiology MH - Humans MH - Immediate-Early Proteins/*genetics/*physiology MH - Molecular Sequence Data MH - Promoter Regions (Genetics) MH - RNA, Messenger/genetics/metabolism MH - RNA, Viral/genetics/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Trans-Activation (Genetics) MH - Trans-Activators/*genetics/*physiology MH - Transfection MH - Viral Proteins/*genetics/*physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9590-612. PMID- 12915573 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Point mutations in exon I of the herpes simplex virus putative terminase subunit, UL15, indicate that the most conserved residues are essential for cleavage and packaging. PG - 9613-21 AB - The herpes simplex virus UL15 and UL28 genes are believed to encode two subunits of the terminase involved in cleavage and packaging of viral genomes. Analysis of the UL15 protein sequence and its herpesvirus homologues revealed the presence of 20 conserved regions. Twelve of the twenty regions conserved among herpesviruses are also conserved in terminases from DNA bacteriophage. Point mutations in UL15 were designed in four conserved regions: L120N (CR1), Q205E (CR2), Q251E (CR3), G263A (CR3), and Y285S (CR4). Transfection experiments indicated that each mutant gene could produce stable UL15 protein at wild-type levels; however, only one mutant (Q251E) was able to complement the UL15-null virus. Each mutation was introduced into the viral genome by marker transfer, and all mutants except Q251E were unable to form plaques on Vero cells. Furthermore, failure to form plaques on Vero cells correlated with a defect in cleavage and packaging. Immunofluorescence experiments indicated that in cells infected with all mutant viruses the UL15 protein could be detected and was found to localize to replication compartments. Although wild-type and mutant Q251E were able to produce A, B, and C capsids, the rest of the mutants were only able to produce B capsids, a finding consistent with their defects in cleavage and packaging. In addition, all mutant UL15 proteins retained their ability to interact with B capsids. Therefore, amino acid residues 120, 205, 263, and 285 are essential for the cleavage and packaging process rather than for association with capsids or localization to replication compartments. AD - Department of Microbiology, University of Connecticut Heath Center, Farmington, Connecticut 06030, USA. FAU - Przech, Angela J AU - Przech AJ FAU - Yu, Dong AU - Yu D FAU - Weller, Sandra K AU - Weller SK LA - eng GR - AI37549/AI/NIAID PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Capsid Proteins) RN - 0 (DNA, Viral) RN - 0 (UL15 protein, herpes simplex virus 1) RN - 0 (Viral Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Capsid Proteins/metabolism MH - Cell Line MH - Cercopithecus aethiops MH - Conserved Sequence MH - DNA, Viral/genetics/metabolism MH - Exons MH - Genes, Viral MH - Herpesvirus 1, Human/*enzymology/*genetics/growth & development/physiology MH - Molecular Sequence Data MH - Point Mutation MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Amino Acid MH - Vero Cells MH - Viral Proteins/chemistry/*genetics/*metabolism MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9613-21. PMID- 12915574 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Antiviral activity of limitin against encephalomyocarditis virus, herpes simplex virus, and mouse hepatitis virus: diverse requirements by limitin and alpha interferon for interferon regulatory factor 1. PG - 9622-31 AB - Limitin has sequence homology with alpha interferon (IFN-alpha) and IFN-beta and utilizes the IFN-alpha/beta receptor. However, it has no influence on the proliferation of normal myeloid and erythroid progenitors. In this study, we show that limitin has antiviral activity in vitro as well as in vivo. Limitin inhibited not only cytopathic effects in encephalomyocarditis virus- or herpes simplex virus (HSV) type 1-infected L929 cells, but also plaque formation in mouse hepatitis virus (MHV) type 2-infected DBT cells. In addition, administration of limitin to mice suppressed MHV-induced hepatitis and HSV-induced death. The antiviral activity may be mediated in part by 2',5'-oligoadenylate synthetase, RNA-dependent protein kinase, and Mx protein, which inhibit viral replication or degrade viral components, because limitin induced their mRNA expression and enzyme activity. While limitin has antiviral activity as strong as that of IFN-alpha in vitro (the concentration that provided 50% inhibition of cytopathic effect is approximately 30 pg/ml), IFN regulatory factor 1 (IRF-1) dependencies for induction of an antiviral state were different for limitin and IFN-alpha. In IRF-1-deficient fibroblasts, a higher concentration of limitin than of IFN-alpha was required for the induction of antiviral activity and the transcription of proteins from IFN-stimulated response element. The unique signals and the fewer properties of myelosuppression suggest that a human homolog of limitin may be used as a new antiviral drug. AD - Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan. FAU - Kawamoto, Shin-Ichiro AU - Kawamoto S FAU - Oritani, Kenji AU - Oritani K FAU - Asada, Hideo AU - Asada H FAU - Takahashi, Isao AU - Takahashi I FAU - Ishikawa, Jun AU - Ishikawa J FAU - Yoshida, Hitoshi AU - Yoshida H FAU - Yamada, Masahide AU - Yamada M FAU - Ishida, Naoko AU - Ishida N FAU - Ujiie, Hidetoshi AU - Ujiie H FAU - Masaie, Hiroaki AU - Masaie H FAU - Tomiyama, Yoshiaki AU - Tomiyama Y FAU - Matsuzawa, Yuji AU - Matsuzawa Y LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antiviral Agents) RN - 0 (Cytokines) RN - 0 (DNA-Binding Proteins) RN - 0 (Interferon Type I, Recombinant) RN - 0 (Phosphoproteins) RN - 0 (Recombinant Proteins) RN - 0 (interferon regulatory factor-1) RN - 0 (limitin) SB - IM MH - Animals MH - Antiviral Agents/*pharmacology MH - Cell Line MH - Cytokines/*pharmacology MH - Cytopathogenic Effect, Viral/drug effects MH - DNA-Binding Proteins/*physiology MH - Encephalomyocarditis virus/*drug effects/pathogenicity/physiology MH - Gene Expression Regulation/drug effects MH - Herpesvirus 1, Human/*drug effects/pathogenicity/physiology MH - Interferon Type I, Recombinant/*pharmacology MH - Mice MH - Murine hepatitis virus/*drug effects/pathogenicity/physiology MH - Phosphoproteins/*physiology MH - Recombinant Proteins/pharmacology MH - Research Support, Non-U.S. Gov't MH - Virus Replication/drug effects EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9622-31. PMID- 12915575 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Maedi-visna virus and caprine arthritis encephalitis virus genomes encode a Vpr-like but no Tat protein. PG - 9632-8 AB - A small open reading frame (ORF) in maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) was initially named "tat" by analogy with a similarly placed ORF in the primate lentiviruses. The encoded "Tat" protein was ascribed the function of up regulation of the viral transcription from the long terminal repeat (LTR) promoter, but we have recently reported that MVV and CAEV Tat proteins lack trans-activation function activity under physiological conditions (S. Villet, C. Faure, B. Bouzar, G. Verdien, Y. Chebloune, and C. Legras, Virology 307:317-327, 2003). In the present work, we show that MVV Tat localizes to the nucleus of transfected cells, probably through the action of a nuclear localization signal in its C-terminal portion. We also show that, unlike the human immunodeficiency virus (HIV) Tat protein, MVV Tat was not secreted into the medium by transfected human or caprine cells in the absence of cell lysis but that, like the primate accessory protein Vpr, MVV and CAEV Tat proteins were incorporated into viral particles. In addition, analysis of the primary protein structures showed that small-ruminant lentivirus (SRLV) Tat proteins are more similar to the HIV type 1 (HIV-1) Vpr protein than to HIV-1 Tat. We also demonstrate a functional similarity between the SRLV Tat proteins and the HIV-1 Vpr product in the induction of a specific G(2) arrest of the cell cycle in MVV Tat-transfected cells, which increases the G(2)/G(1) ratio 2.8-fold. Together, these data strongly suggest that the tat ORF in the SRLV genomes does not code for a regulatory transactivator of the LTR but, rather, for a Vpr-like accessory protein. AD - UMR 754 INRA/ENVL/UCBL, Retrovirus et pathologie comparee, "Virologie Cellulaire, Moleculaire, et Maladies Emergentes," Universite Claude Bernard Lyon-1, Lyon, France. FAU - Villet, Stephanie AU - Villet S FAU - Bouzar, Baya Amel AU - Bouzar BA FAU - Morin, Thierry AU - Morin T FAU - Verdier, Gerard AU - Verdier G FAU - Legras, Catherine AU - Legras C FAU - Chebloune, Yahia AU - Chebloune Y LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (Gene Products, tat) RN - 0 (Gene Products, vpr) SB - IM MH - Animals MH - Arthritis-Encephalitis Virus, Caprine/*genetics/pathogenicity/physiology MH - Base Sequence MH - Cells, Cultured MH - Comparative Study MH - DNA, Viral/genetics MH - G2 Phase MH - Gene Products, tat/genetics/physiology MH - Gene Products, vpr/*genetics/physiology MH - Genes, tat MH - Genes, vpr MH - *Genome, Viral MH - Goats MH - Hela Cells MH - Humans MH - Open Reading Frames MH - Research Support, Non-U.S. Gov't MH - Subcellular Fractions/virology MH - Transfection MH - Visna-maedi virus/*genetics/pathogenicity/physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9632-8. PMID- 12915576 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Existence of transdominant and potentiating mutants of UL9, the herpes simplex virus type 1 origin-binding protein, suggests that levels of UL9 protein may be regulated during infection. PG - 9639-51 AB - UL9 is a multifunctional protein required for herpes simplex virus type 1 (HSV-1) replication in vivo. UL9 is a member of the superfamily II helicases and exhibits helicase and origin-binding activities. We have previously shown that mutations in the conserved helicase motifs of UL9 can have either a transdominant or potentiating effect on the plaque-forming ability of infectious DNA from wild-type virus (A. J. Malik and S. K. Weller, J. Virol. 70:7859-7866, 1996). In this paper, the mechanisms of transdominance and potentiation are explored. We show that the motif V mutant protein containing a G to A substitution at residue 354 is unstable when expressed by transfection and is either processed to a 38-kDa N-terminal fragment or degraded completely. The overexpression of the MV mutant protein is able to influence the steady-state protein levels of wild-type UL9 and to override the inhibitory effects of wild-type UL9. Potentiation correlates with the ability of the UL9 variants containing the G354A mutation to be processed or degraded to the 38-kDa form. We propose that the MV mutant protein is able to interact with full-length UL9 and that this interaction results in a decrease in the steady-state levels of UL9, which in turn leads to enhanced viral infection. Furthermore, we demonstrate that inhibition of HSV-1 infection can be obtained by overexpression of full-length UL9, the C-terminal third of the protein containing the origin-binding domain, or the N-terminal two-thirds of UL9 containing the conserved helicase motifs and the putative dimerization domain. Our results suggest that transdominance can be mediated by overexpression, origin-binding activity, and dimerization, whereas potentiation is most likely caused by the ability of the UL9 MV mutant to influence the steady-state levels of wild-type UL9. Taken together, the results presented in this paper suggest that the regulation of steady-state levels of UL9 may play an important role in controlling viral infection. AD - Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA. FAU - Marintcheva, Boriana AU - Marintcheva B FAU - Weller, Sandra K AU - Weller SK LA - eng GR - AI21747/AI/NIAID PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) RN - 0 (DNA-Binding Proteins) RN - 0 (Peptide Fragments) RN - 0 (Plasmids) RN - 0 (Recombinant Proteins) RN - 0 (Viral Proteins) RN - 115004-77-8 (UL9 protein, Human herpesvirus 1) RN - EC 5.99.- (DNA Helicases) SB - IM MH - Animals MH - Base Sequence MH - Cell Line MH - Cercopithecus aethiops MH - DNA Helicases/chemistry/*genetics/*physiology MH - DNA, Viral/genetics MH - DNA-Binding Proteins/chemistry/*genetics/*physiology MH - Genes, Viral MH - Herpesvirus 1, Human/*genetics/pathogenicity/*physiology MH - Models, Biological MH - Mutation MH - Peptide Fragments/chemistry/genetics/metabolism MH - Plasmids/genetics MH - Recombinant Proteins/chemistry/genetics/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Spodoptera MH - Transfection MH - Vero Cells MH - Viral Proteins/chemistry/*genetics/*physiology MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9639-51. PMID- 12915577 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20050204 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Cell cycle regulation by Kaposi's sarcoma-associated herpesvirus K-bZIP: direct interaction with cyclin-CDK2 and induction of G1 growth arrest. PG - 9652-61 AB - In order to cope with hostile host environments, many viruses have developed strategies to perturb the cellular machinery to suit their replication needs. Some herpesvirus genes protect cells from undergoing apoptosis to prolong the lives of infected cells, while others, such as Epstein-Barr virus Zta, slow down the G(1)/S transition phase to allow ample opportunity for transcription and translation of viral genes before the onset of cellular genomic replication. In this study, we investigated whether Kaposi's sarcoma-associated herpesvirus (KSHV) K-bZIP, a homologue of the Epstein-Barr virus transcription factor BZLF1 (Zta), plays a role in cell cycle regulation. Here we show that K-bZIP physically associates with cyclin-CDK2 and downmodulates its kinase activity. The association can be detected in the natural environment of KSHV-infected cells without artificial overexpression of either component. With purified protein, it can be shown that the interaction between K-bZIP and cyclin-CDK2 is direct and that K-bZIP alone is sufficient to inhibit CDK2 activity. The interacting domain of K-bZIP has been mapped to the basic region. The result of these associations is a prolonged G(1) phase, accompanied by the induction of p21 and p27 in a naturally infected B-cell line. Thus, in addition to the previously described transcription and genome replication functions, a new role of K-bZIP in KSHV replication is identified in this report. AD - Department of Biological Chemistry, School of Medicine, University of California, Davis, UC Davis Cancer Center, Sacramento, California 95817, USA. FAU - Izumiya, Yoshihiro AU - Izumiya Y FAU - Lin, Su-Fang AU - Lin SF FAU - Ellison, Thomas J AU - Ellison TJ FAU - Levy, Alon M AU - Levy AM FAU - Mayeur, Greg L AU - Mayeur GL FAU - Izumiya, Chie AU - Izumiya C FAU - Kung, Hsing-Jien AU - Kung HJ LA - eng GR - CA46613/CA/NCI GR - CA91574/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Carrier Proteins) RN - 0 (K8 protein, Human herpesvirus 8) RN - 0 (Recombinant Proteins) RN - 0 (Viral Proteins) RN - EC 2.7.1.- (cyclin-dependent kinase 2) RN - EC 2.7.1.37 (CDC2-CDC28 Kinases) RN - EC 2.7.1.37 (Cyclin-Dependent Kinases) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - B-Lymphocytes/cytology/metabolism/virology MH - Binding Sites MH - *CDC2-CDC28 Kinases MH - Carrier Proteins/chemistry/genetics/*physiology MH - Cell Cycle/*physiology MH - Cell Line MH - Cyclin-Dependent Kinases/*physiology MH - G1 Phase/physiology MH - Gene Expression MH - Genes, Viral MH - Hela Cells MH - Herpesvirus 8, Human/genetics/pathogenicity/*physiology MH - Humans MH - Protein-Serine-Threonine Kinases/*physiology MH - Recombinant Proteins/chemistry/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Deletion MH - Transfection MH - Viral Proteins/chemistry/genetics/*physiology MH - Virus Replication/physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9652-61. PMID- 12915578 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Chromosomal distribution of endogenous Jaagsiekte sheep retrovirus proviral sequences in the sheep genome. PG - 9662-8 AB - A family of endogenous retroviruses (enJSRV) closely related to Jaagsiekte sheep retrovirus (JSRV) is ubiquitous in domestic and wild sheep and goats. Southern blot hybridization studies indicate that there is little active replication or movement of the enJSRV proviruses in these species. Two approaches were used to investigate the distribution of proviral loci in the sheep genome. Fluorescence in situ hybridization (FISH) to metaphase chromosome spreads using viral DNA probes was used to detect loci on chromosomes. Hybridization signals were reproducibly detected on seven sheep chromosomes and eight goat chromosomes in seven cell lines. In addition, a panel of 30 sheep-hamster hybrid cell lines, each of which carries one or more sheep chromosomes and which collectively contain the whole sheep genome, was examined for enJSRV sequences. DNA from each of the lines was used as a template for PCR with JSRV gag-specific primers. A PCR product was amplified from 27 of the hybrid lines, indicating that JSRV gag sequences are found on at least 15 of the 28 sheep chromosomes, including those identified by FISH. Thus, enJSRV proviruses are essentially randomly distributed among the chromosomes of sheep and goats. FISH and/or Southern blot hybridization on DNA from several of the sheep-hamster hybrid cell lines suggests that loci containing multiple copies of enJSRV are present on chromosomes 6 and 9. The origin and functional significance of these arrays is not known. AD - Department of Microbiology, Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, Colorado 80523, USA. jcarlson@colostate.edu FAU - Carlson, Jonathan AU - Carlson J FAU - Lyon, Monique AU - Lyon M FAU - Bishop, Jeanette AU - Bishop J FAU - Vaiman, Anne AU - Vaiman A FAU - Cribiu, Edmond AU - Cribiu E FAU - Mornex, Jean-Francois AU - Mornex JF FAU - Brown, Susan AU - Brown S FAU - Knudson, Dennis AU - Knudson D FAU - DeMartini, James AU - DeMartini J FAU - Leroux, Caroline AU - Leroux C LA - eng GR - 1R01 CA 59116/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (DNA, Viral) SB - IM MH - Animals MH - Base Sequence MH - Cell Line MH - Chromosomes/genetics/virology MH - DNA, Viral/genetics/isolation & purification MH - Genes, gag MH - Genome MH - Goats/genetics/virology MH - Hamsters MH - Hybrid Cells MH - In Situ Hybridization, Fluorescence MH - Ovine pulmonary adenocarcinoma virus/*genetics/*isolation & purification MH - Physical Chromosome Mapping MH - Polymerase Chain Reaction MH - Proviruses/genetics/isolation & purification MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sheep/*genetics/*virology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9662-8. PMID- 12915579 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Surface downregulation of major histocompatibility complex class I, PE-CAM, and ICAM-1 following de novo infection of endothelial cells with Kaposi's sarcoma-associated herpesvirus. PG - 9669-84 AB - Under selective pressure from host cytotoxic T lymphocytes, many viruses have evolved to downregulate major histocompatibility complex (MHC) class I and/or T-cell costimulatory molecules from the surface of infected cells. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two proteins, MIR-1 and MIR-2, that serve this function during lytic replication. In vivo, however, KSHV exists in a predominantly latent state, with less than 5% of infected cells expressing discernible lytic gene products. Thus, mechanisms of immune evasion that depend on genes expressed only during lytic replication are unlikely to be active in most KSHV-infected cells. As a result, we searched for evidence of similar defensive strategies extant during latency, employing culture systems that strongly favor latent KSHV infection. We measured cell surface levels of immunomodulatory proteins on both primary dermal microvascular endothelial cells (pDMVEC) infected through coculture with induced primary effusion lymphoma cells and telomerase-immortalized DMVEC infected directly with cell-free virus. Employing a panel of antibodies against several endothelial cell surface proteins, we show that de novo infection with KSHV leads to the downregulation of MHC class I, CD31 (PE-CAM), and CD54 (ICAM-I) but not CD58 (LFA-3) or CD95 (Fas). Furthermore, flow cytometry with a fluorescently labeled monoclonal antibody to the latency-associated nuclear antigen (LANA) revealed that downregulation occurred predominantly on KSHV-infected (LANA-positive) cells. Although the vast majority of infected cells displayed this downregulation, less than 1% expressed either immediate-early or late lytic proteins detectable by immunofluorescence. Together, these results suggest that downregulation of immunomodulatory proteins on the surface of target cells may represent a constitutive mode of immune evasion employed by KSHV following de novo infection. AD - Myles H. Thaler Center for AIDS and Human Retrovirus Research, Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA. FAU - Tomescu, Costin AU - Tomescu C FAU - Law, Wai K AU - Law WK FAU - Kedes, Dean H AU - Kedes DH LA - eng GR - CA88768-01/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antigens, CD31) RN - 0 (Antigens, CD58) RN - 0 (Histocompatibility Antigens Class I) RN - 126547-89-5 (Intercellular Adhesion Molecule-1) RN - EC 2.7.7.- (Telomerase) RN - EC 2.7.7.- (telomerase reverse transcriptase) SB - IM MH - Antigens, CD31/*metabolism MH - Antigens, CD58/metabolism MH - Cell Line, Transformed MH - Cell Membrane/immunology MH - Cells, Cultured MH - Coculture Techniques MH - Down-Regulation MH - Endothelium, Vascular/*immunology/*virology MH - Fluorescent Antibody Technique MH - Herpesvirus 8, Human/*immunology/*pathogenicity MH - Histocompatibility Antigens Class I/*metabolism MH - Humans MH - Intercellular Adhesion Molecule-1/*metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Telomerase/genetics EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9669-84. PMID- 12915580 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - A bromodomain-containing protein from tomato specifically binds potato spindle tuber viroid RNA in vitro and in vivo. PG - 9685-94 AB - For the identification of RNA-binding proteins that specifically interact with potato spindle tuber viroid (PSTVd), we subjected a tomato cDNA expression library prepared from viroid-infected leaves to an RNA ligand screening procedure. We repeatedly identified cDNA clones that expressed a protein of 602 amino acids. The protein contains a bromodomain and was termed viroid RNA-binding protein 1 (VIRP1). The specificity of interaction of VIRP1 with viroid RNA was studied by different methodologies, which included Northwestern blotting, plaque lift, and electrophoretic mobility shift assays. VIRP1 interacted strongly and specifically with monomeric and oligomeric PSTVd positive-strand RNA transcripts. Other RNAs, for example, U1 RNA, did not bind to VIRP1. Further, we could immunoprecipitate complexes from infected tomato leaves that contained VIRP1 and viroid RNA in vivo. Analysis of the protein sequence revealed that VIRP1 is a member of a newly identified family of transcriptional regulators associated with chromatin remodeling. VIRP1 is the first member of this family of proteins, for which a specific RNA-binding activity is shown. A possible role of VIRP1 in viroid replication and in RNA mediated chromatin remodeling is discussed. AD - Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Crete, Greece. FAU - Martinez de Alba, Angel Emilio AU - Martinez de Alba AE FAU - Sagesser, Rudolf AU - Sagesser R FAU - Tabler, Martin AU - Tabler M FAU - Tsagris, Mina AU - Tsagris M LA - eng SI - GENBANK/AJ249592 SI - GENBANK/AJ249593 SI - GENBANK/AJ249594 SI - GENBANK/AJ249595 PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Plant Proteins) RN - 0 (RNA, Messenger) RN - 0 (RNA, Viral) RN - 0 (RNA-Binding Proteins) SB - IM MH - Amino Acid Sequence MH - Base Sequence MH - Gene Expression MH - Genes, Plant MH - Lycopersicon esculentum/genetics/*metabolism/*virology MH - Molecular Sequence Data MH - Plant Proteins/chemistry/genetics/*metabolism MH - Protein Processing, Post-Translational MH - Protein Structure, Tertiary MH - RNA, Messenger/genetics/metabolism MH - RNA, Viral/genetics/*metabolism MH - RNA-Binding Proteins/chemistry/genetics/*metabolism MH - Research Support, Non-U.S. Gov't MH - Viroids/*genetics/growth & development/pathogenicity/*physiology MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9685-94. PMID- 12915581 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Differences in the N termini of herpes simplex virus type 1 and 2 gDs that influence functional interactions with the human entry receptor Nectin-2 and an entry receptor expressed in Chinese hamster ovary cells. PG - 9695-9 AB - Amino acid differences at seven positions in the N termini of the glycoproteins D (gDs) specified by herpes simplex virus type 1 (HSV-1) and HSV-2 are largely responsible for the significantly higher cell fusion activity of HSV-2 gD with Chinese hamster ovary cells expressing human nectin-2 or only an endogenous hamster receptor. AD - Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. FAU - Zago, Anna AU - Zago A FAU - Spear, Patricia G AU - Spear PG LA - eng GR - R01 CA21776/CA/NCI GR - R37 AI36293/AI/NIAID GR - U19 AI31494/AI/NIAID PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Cell Adhesion Molecules) RN - 0 (Plasmids) RN - 0 (Receptors, Virus) RN - 0 (Viral Envelope Proteins) RN - 0 (glycoprotein D, Human herpesvirus 1) RN - 0 (glycoprotein D-herpes simplex virus type 2) RN - 0 (nectin) SB - IM MH - Amino Acid Sequence MH - Animals MH - CHO Cells MH - Cell Adhesion Molecules/genetics/*physiology MH - Hamsters MH - Humans MH - Membrane Fusion MH - Models, Molecular MH - Molecular Sequence Data MH - Plasmids/genetics MH - Protein Structure, Tertiary MH - Receptors, Virus/genetics/physiology MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Amino Acid MH - Viral Envelope Proteins/*chemistry/genetics/*physiology EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9695-9. PMID- 12915582 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Murid herpesvirus 4 strain 68 M2 protein is a B-cell-associated antigen important for latency but not lymphocytosis. PG - 9700-9 AB - This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vbeta4 T-cell-receptor-positive CD8(+) T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells. AD - Department of Medical Microbiology and Genitourinary Medicine, Centre for Comparative Infectious Diseases, University of Liverpool, Liverpool, United Kingdom. FAU - Macrae, Alastair I AU - Macrae AI FAU - Usherwood, Edward J AU - Usherwood EJ FAU - Husain, S Mazher AU - Husain SM FAU - Flano, Emilio AU - Flano E FAU - Kim, In-Jeong AU - Kim IJ FAU - Woodland, David L AU - Woodland DL FAU - Nash, Anthony A AU - Nash AA FAU - Blackman, Marcia A AU - Blackman MA FAU - Sample, Jeffery T AU - Sample JT FAU - Stewart, James P AU - Stewart JP LA - eng GR - AI42927/AI/NIAID GR - CA90208/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Antigens, Viral) RN - 0 (DNA, Viral) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Matrix Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Antigens, Viral/*genetics MH - B-Lymphocytes/*immunology/*virology MH - Base Sequence MH - Cell Line MH - DNA, Viral/genetics MH - Genes, Viral MH - Hamsters MH - Herpesviridae Infections/immunology/virology MH - In Vitro MH - Lung/immunology/virology MH - Lymphocytosis/etiology MH - Mice MH - Mice, Inbred BALB C MH - Mice, Inbred C57BL MH - Mutation MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Rhadinovirus/genetics/*immunology/*pathogenicity/physiology MH - Spleen/immunology/virology MH - T-Lymphocyte Subsets/immunology/virology MH - Transfection MH - Tumor Virus Infections/immunology/virology MH - Viral Matrix Proteins/*genetics/*immunology MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9700-9. PMID- 12915583 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Protective efficacy of an AIDS vaccine, a single DNA priming followed by a single booster with a recombinant replication-defective Sendai virus vector, in a macaque AIDS model. PG - 9710-5 AB - We previously demonstrated the excellent protective efficacy of DNA priming followed by Gag-expressing Sendai virus (SeV) boosting (DNA prime/SeV-Gag boost vaccine) against a pathogenic simian-human immunodeficiency virus (SHIV89.6PD) infection in macaques. Here we show that we established a practical, safer AIDS vaccine protocol, a single DNA priming followed by a single booster with a recently developed replication-defective F deletion SeV-expressing Gag, and show its protective efficacy against SHIV89.6PD infections. AD - Department of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan. FAU - Takeda, Akiko AU - Takeda A FAU - Igarashi, Hiroko AU - Igarashi H FAU - Nakamura, Hiromi AU - Nakamura H FAU - Kano, Munehide AU - Kano M FAU - Iida, Akihiro AU - Iida A FAU - Hirata, Takahiro AU - Hirata T FAU - Hasegawa, Mamoru AU - Hasegawa M FAU - Nagai, Yoshiyuki AU - Nagai Y FAU - Matano, Tetsuro AU - Matano T LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (AIDS Vaccines) RN - 0 (DNA, Viral) RN - 0 (Genetic Vectors) RN - 0 (SAIDS Vaccines) RN - 0 (Vaccines, DNA) SB - IM MH - AIDS Vaccines/*administration & dosage/genetics MH - Acquired Immunodeficiency Syndrome/immunology/prevention & control MH - Animals MH - CD4-Positive T-Lymphocytes/immunology MH - CD8-Positive T-Lymphocytes/immunology MH - DNA, Viral/*administration & dosage/genetics MH - Defective Viruses/genetics MH - Disease Models, Animal MH - Genes, gag MH - Genetic Vectors MH - Humans MH - Immunization, Secondary MH - Macaca fascicularis MH - Macaca mulatta MH - Research Support, Non-U.S. Gov't MH - SAIDS Vaccines/administration & dosage/genetics MH - Sendai virus/genetics MH - Simian Acquired Immunodeficiency Syndrome/immunology/prevention & control MH - Vaccines, DNA/*administration & dosage/genetics EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9710-5. PMID- 12915584 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - High circulating frequencies of tumor necrosis factor alpha- and interleukin-2-secreting human T-lymphotropic virus type 1 (HTLV-1)-specific CD4+ T cells in patients with HTLV-1-associated neurological disease. PG - 9716-22 AB - Significantly higher frequencies of tumor necrosis factor alpha- and interleukin-2-secreting human T-lymphotropic virus type 1 (HTLV-1)-specific CD4(+) T cells were present in the peripheral blood mononuclear cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients than in those of asymptomatic carriers with similar provirus loads. The data suggest that HTLV-1-specific CD4(+) T cells play a role in the pathogenesis of HAM/TSP. AD - Department of Immunology, Imperial College London, London W2 1PG, United Kingdom. FAU - Goon, Peter K C AU - Goon PK FAU - Igakura, Tadahiko AU - Igakura T FAU - Hanon, Emmanuel AU - Hanon E FAU - Mosley, Angelina J AU - Mosley AJ FAU - Asquith, Becca AU - Asquith B FAU - Gould, Keith G AU - Gould KG FAU - Taylor, Graham P AU - Taylor GP FAU - Weber, Jonathan N AU - Weber JN FAU - Bangham, Charles R M AU - Bangham CR LA - eng PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Enterotoxins) RN - 0 (Interleukin-2) RN - 0 (Lymphotoxin) RN - 0 (Tumor Necrosis Factor-alpha) RN - 16561-29-8 (Tetradecanoylphorbol Acetate) RN - 39424-53-8 (enterotoxin B, staphylococcal) RN - 82115-62-6 (Interferon Type II) SB - IM MH - CD4 Lymphocyte Count MH - CD4-Positive T-Lymphocytes/drug effects/*immunology/*virology MH - Carrier State/immunology MH - Enterotoxins/pharmacology MH - Human T-lymphotropic virus 1/*immunology/isolation & purification MH - Humans MH - In Vitro MH - Interferon Type II/biosynthesis MH - Interleukin-2/*biosynthesis MH - Lymphotoxin/biosynthesis MH - Paraparesis, Tropical Spastic/etiology/*immunology MH - Proviruses/immunology/isolation & purification MH - Research Support, Non-U.S. Gov't MH - Tetradecanoylphorbol Acetate/pharmacology MH - Tumor Necrosis Factor-alpha/*biosynthesis EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9716-22. PMID- 12915585 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Early- and intermediate-stage variants of simian immunodeficiency virus replicate efficiently in cells lacking CCR5. PG - 9723-7 AB - Primate lentiviruses are thought to use the chemokine receptor CCR5 as the major coreceptor for entry into cells. Here we show that some variants of simian immunodeficiency virus (SIV) replicate efficiently in peripheral blood mononuclear cells (PBMCs) lacking a functional CCR5. There were differences in the replication patterns of sequential variants that evolved during SIVMne infection; the late-stage pathogenic variants were unable to replicate in PBMCs lacking CCR5, whereas the early- and intermediate-stage viruses replicated as well in PBMCs lacking CCR5 as they did in cells with wild-type CCR5. The coreceptor specificities of these sequential variants were compared using indicator cell lines expressing known SIV coreceptors. Among the known SIV coreceptors, there were none that were functional for the early and intermediate variants but not the late-stage variants, suggesting that the coreceptor used for replication in PBMCs may be a coreceptor that has not yet been described. Because some variants replicate with high efficiency in peripheral blood cells using this as yet uncharacterized cellular receptor, this coreceptor may be important for viral entry of some target cell populations in the host. AD - Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. FAU - Forte, Serene AU - Forte S FAU - Harmon, Mary-Elizabeth AU - Harmon ME FAU - Pineda, Mario J AU - Pineda MJ FAU - Overbaugh, Julie AU - Overbaugh J LA - eng GR - R01 AI34251/AI/NIAID GR - T32 CA09229/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Gag protein p27, Simian immunodeficiency virus) RN - 0 (Gene Products, gag) RN - 0 (Receptors, CCR5) SB - IM MH - Animals MH - Cell Line MH - Gene Products, gag/biosynthesis MH - HIV-1/genetics/immunology/physiology MH - Humans MH - Leukocytes, Mononuclear/immunology/virology MH - Receptors, CCR5/*deficiency/genetics/physiology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - SIV/genetics/immunology/*physiology MH - Sequence Deletion MH - Transfection MH - Variation (Genetics) MH - Virus Replication EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9723-7. PMID- 12915586 OWN - NLM STAT- MEDLINE DA - 20030813 DCOM- 20030924 LR - 20041117 PUBM- Print IS - 0022-538X VI - 77 IP - 17 DP - 2003 Sep TI - Susceptibility of human hepatitis delta virus RNAs to small interfering RNA action. PG - 9728-31 AB - In animal cells, small interfering RNAs (siRNA), when exogenously provided, have been reported to be capable of inhibiting replication of several different viruses. In preliminary studies, siRNA species were designed and tested for their ability to act on the protein expressed in Huh7 cells transfected with DNA-directed mRNA constructs containing hepatitis delta virus (HDV) target sequences. The aim was to achieve siRNA specific for each of the three RNAs of HDV replication: (i) the 1,679-nucleotide circular RNA genome, (ii) its exact complement, the antigenome, and (iii) the less abundant polyadenylated mRNA for the small delta protein. Many of the 16 siRNA tested gave >80% inhibition in this assay. Next, these three classes of siRNA were tested for their ability to act during HDV genome replication. It was found that only siRNA targeted against HDV mRNA sequences could interfere with HDV genome replication. In contrast, siRNA targeted against genomic and antigenomic RNA sequences had no detectable effect on the accumulation of these RNAs. Reconstruction experiments with nonreplicating HDV RNA sequences support the interpretation that neither the potential for intramolecular rod-like RNA folding nor the presence of the delta protein conferred resistance to siRNA. In terms of replicating HDV RNAs, it is considered more likely that the genomic and antigenomic RNAs are resistant because their location within the nucleus makes them inaccessible to siRNA-mediated degradation. AD - Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111-2497, USA. FAU - Chang, Jinhong AU - Chang J FAU - Taylor, John M AU - Taylor JM LA - eng GR - AI-26522/AI/NIAID GR - CA-06927/CA/NCI PT - Journal Article PL - United States TA - J Virol JID - 0113724 RN - 0 (Hepatitis Delta Virus) RN - 0 (RNA, Messenger) RN - 0 (RNA, Small Interfering) RN - 0 (RNA, Viral) SB - IM MH - Animals MH - Base Sequence MH - Cell Line MH - Genome, Viral MH - Hepatitis Delta Virus/drug effects/*genetics/physiology MH - Humans MH - RNA Interference MH - RNA, Messenger/genetics MH - RNA, Small Interfering/*genetics/*pharmacology MH - RNA, Viral/genetics MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Transfection MH - Virus Replication/drug effects/genetics EDAT- 2003/08/14 05:00 MHDA- 2003/09/25 05:00 PST - ppublish SO - J Virol 2003 Sep;77(17):9728-31.