Folate metabolism and chemotherapy in Pneumocystis carinii and Plasmodium falciparum.
Item
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Title
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Folate metabolism and chemotherapy in Pneumocystis carinii and Plasmodium falciparum.
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Identifier
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AAI9315489
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identifier
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9315489
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Creator
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Merali, Salim.
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Contributor
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Adviser: Steven Mechnick
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Date
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1993
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biology, Microbiology | Health Sciences, Pharmacology
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Abstract
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In the studies reported here, we examined three steps of the folate biosynthetic pathway as targets for chemotherapy. These were the synthesis of para-aminobenzoic acid (PABA), synthesis of dihydropteroate, and synthesis of folates.;Using a new reversed-phase HPLC assay procedure, dihydropteroate synthetase (DHPS), activity was detected from P. carinii (14 units/mg). Concentrations required for 50% inhibition (IC{dollar}\sb{lcub}50{rcub}{dollar}) and the Michaelis-Menton inhibitory constants (K{dollar}\sb{lcub}\rm i{rcub}){dollar} for several sulfa drugs were determined. Dapsone (IC{dollar}\sb{lcub}50{rcub}{dollar} = 215 {dollar}\mu{dollar}M, K{dollar}\sb{lcub}\rm i{rcub}{dollar} = 9 {dollar}\mu{dollar}M) was the most potent inhibitor, whereas sulfadiazine was the weakest. Sulfaquinoxaline, sulfamethoxazole, and sulfadoxine all displayed intermediate inhibition, with IC{dollar}\sb{lcub}50{rcub}{dollar} of 400-600 {dollar}\mu{dollar}M and K{dollar}\sb{lcub}\rm i{rcub}{dollar} of 60-150 {dollar}\mu{dollar}M.;A culture system using mink lung cells (MV1-LU) as supporting cells maintained P. carinii growth for up to 5 days. Because quantitation of P. carinii by staining and counting is difficult and inaccurate, we developed a new reliable in vitro assay for quantitation of parasite growth. This assay measured the incorporation of ({dollar}\sp3{dollar}H) -PABA into folate. The folate synthesized by the microbe was bound by folate binding protein and unincoporated PABA was removed by binding to charcoal-coated dextran. Using this assay, it was determined that the microbe accumulates 1.8 {dollar}\pm{dollar} 0.6 fmol of radioactive folate/mg protein. Several sulfa drugs were tested for their ability to inhibit folate synthesis; it was again determined that dapsone was the most potent inhibitor.;The sensitivity of Pneumocystis carinii to artemisinin (qinghaosu), a potent antimalarial agent, was determined in a short-term primary culture.;Deferoxamine, an iron chelator, was previously shown to have an anti-pneumocystis activity but it has a short half-life in vivo.;The folate assay described above was utilized to investigate the importance of exogenous PABA. It was shown that the parasite-infected red cells accumulate 5.4 {dollar}\pm{dollar} 0.8 fmol radioactive folates per mg of protein from ({dollar}\sp3{dollar}H) PABA.;Using HPLC, it was determined that Plasmodium falciparum-infected red cells incubated in PABA- and folate-free media, contained 95.4 {dollar}\pm{dollar} 17 pmol of PABA per mg protein (n = 3) while the uninfected red cells had {dollar}<{dollar}1 pmol of PABA per mg of protein.;Four P. falciparum mutants auxotrophic for PABA were analyzed for their intracellular PABA content. Although, all four strains were comparable to the parent clone in regards to PABA accumulation, only two strains were able to synthesize PABA de novo. (Abstract shortened by UMI.).
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
