| Microculture screening assay for primary in vitro evaluation of drugs against Pneumocystis carinii. | |
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MedLine Citation:
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PMID: 1759815 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Pneumocystis carinii inoculated into 96-well filtration plate assemblies was shown to synthesize radiolabeled folates de novo from [para-3H]aminobenzoic acid ([3H]pABA). At the end of each incubation with [3H]pABA, a vacuum manifold was used to remove the medium and wash P. carinii. The membrane at the base of each well was dried and punched out, and the level of 3H retained was determined by direct scintillation counting. High-pressure liquid chromatography analysis of duplicate filters confirmed that direct counting of 3H retained on membranes (after correction for unmetabolized [3H]pABA) was an accurate reflection of total [3H]pABA incorporation by P. carinii. Greater than 95% of the 3H recovered was shown to be present as polyglutamated species. After digestion with rat plasma folic acid gamma-glutamyl hydrolase, para-aminobenzoylglutamate, N10-formyltetrahydrofolate, and tetrahydrofolate were identified as the major 3H-labeled components. para-Aminobenzoylglutamate was presumed to have arisen from folylpolyglutamates synthesized by P. carinii and was therefore included in the calculation of total [3H]pABA incorporation. P. carinii incorporation of [3H]pABA under optimal conditions was used as a selective measure of in vitro viability against which the inhibitory effects of some antipneumocystis agents (pentamidine, sulfamethoxazole, 566C80, and piritrexim) were quantitated. The concentrations of pentamidine, sulfamethoxazole, 566C80, and piritrexim required for 50% inhibition in this assay were 7.3, 0.1, 1.4, and approximately 100 microM, respectively. The results suggest that this 96-well [3H]pABA incorporation assay has considerable potential for objective in vitro drug screening against P. carinii. |
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Authors:
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J C Comley; R J Mullin; L A Wolfe; M H Hanlon; R Ferone |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Antimicrobial agents and chemotherapy Volume: 35 ISSN: 0066-4804 ISO Abbreviation: Antimicrob. Agents Chemother. Publication Date: 1991 Oct |
Date Detail:
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Created Date: 1992-01-31 Completed Date: 1992-01-31 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 0315061 Medline TA: Antimicrob Agents Chemother Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 1965-74 Citation Subset: IM |
Affiliation:
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Department of Molecular Sciences, Wellcome Research Laboratories, Beckenham, Kent, United Kingdom. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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4-Aminobenzoic Acid
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metabolism Anti-Infective Agents / pharmacology* Chromatography, High Pressure Liquid Microbial Sensitivity Tests Pneumocystis / drug effects*, metabolism Temperature |
| Chemical | |
Reg. No./Substance:
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0/Anti-Infective Agents; 150-13-0/4-Aminobenzoic Acid |
| Comments/Corrections | |
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