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B J Berger

Publications and source records attributed to B J Berger.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of the ornithine aminotransferase from Plasmodium falciparum.

The ornithine aminotransferase from Plasmodium falciparum 3D7 was cloned, functionally expressed, and characterized. The gene exists as a single copy in the malarial genome and is located on chromosomes 6/7/8. The deduced amino acid sequence was found to be 85% identical to a similar sequence discovered in Plasmodium yoelii, 82% identical to a partial sequence from Plasmodium vivax, and 42-53% identical to ornithine aminotransferases from other eukaryotes. The enzyme had a very narrow substrate specificity, and could only catalyze the transamination of alpha-ketoglutarate with ornithine or N-acetylornithine, and of glutamate-5-semialdehyde with glutamate and alanine. The aminooxy analogue of ornithine, canaline, was found to inhibit the ornithine aminotransferase uncompetatively with a Ki of 492+/-98 nM. As the enzyme effectively catalyzed both ornithine catabolism and formation, its potential role in ornithine biosynthesis from glutamine, via glutamate, glutamate-5-phosphate, and glutamate-5-semialdehyde, was examined. Over the course of a 3.5 h incubation, P. falciparum converted 34% of exogenous, radiolabeled glutamine to glutamate and 0.68% to ornithine. This low level of conversion suggests that the parasite may have alternative mechanisms for obtaining ornithine for polyamine biosynthesis.

Amino Acid Sequence↗
PubMed2001

Methionine regeneration and aspartate aminotransferase in parasitic protozoa.

Aspartate aminotransferases have been cloned and expressed from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549-561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a K(i) of 27 microm.

Amino Acid Sequence↗
PubMed2000

Antimalarial activities of aminooxy compounds.

Twenty-three aminooxy compounds have been examined for their ability to inhibit the growth of the malaria parasite Plasmodium falciparum in vitro. Eight of these compounds were found to have 50% inhibitory concentrations less than 10 microM, with the best drugs being canaline (the aminooxy analogue of ornithine) and CGP51905A at 297 +/- 23.6 nM and 242 +/- 18.8 nM, respectively. Canaline was also assayed in combination with the ornithine decarboxylase inhibitor difluoromethylornithine, and the two drugs were found to be synergistic in antimalarial activity.

Aminobutyrates↗
PubMed1999

Tyrosine aminotransferase catalyzes the final step of methionine recycling in Klebsiella pneumoniae.

An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of alpha-ketomethiobutyrate to methionine, has been purified from Klebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, and Rhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with alpha-ketomethiobutyrate and of glutamate with alpha-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with alpha-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.

Amino Acid Sequence↗
PubMed1998

Methionine formation from alpha-ketomethiobutyrate in the trypanosomatid Crithidia fasciculata.

Methionine consumed during the synthesis of polyamines can be recycled in most organisms by a unique pathway wherein the final step is the transaminative conversion of alpha-ketomethiobutyrate to methionine (KMAT activity). In the trypanosomatid Crithidia fasciculata, three separate aminotransferases (KMAT-A, -B, -T) were found to catalyse this activity. All three aminotransferases were found to utilise aromatic amino acids as the amino donor for the KMAT reaction, but KMAT-A functioned optimally with histidine and KMAT-B with arginine as amino donors. KMAT-T was found to operate best with aromatic amino acids and glutamate as amino donors, and was also found to catalyse aspartate aminotransferase and tyrosine aminotransferase activities. Amino acid sequencing of internal peptides from KMAT-T yielded a sequence with very high identity to vertebrate, cytosolic aspartate aminotransferase. As pig heart cytosolic aspartate and alanine aminotransferases were found to be unable to catalyse KMAT activity, the crithidial enzyme appears to be an aspartate aminotransferase with unusual catalytic properties. Inhibition studies on C. fasciculata homogenates showed that carboxymethoxylamine, canaline, and nitrophenylalanine were effective inhibitors of total KMAT activity (63-100% inhibition at 1 mM in the presence of 1 mM alpha-ketomethiobutyrate and 30 mM total amino acid as substrates) and the individual, isolated enzymes. At 1 mg ml-1, canaline was found to inhibit cell growth in vitro by 62%, and carboxymethoxylamine caused cell death within 24 h.

Alanine↗
PubMed1997

Studies on the mechanism of antimalarial action of a novel arylene bis(methylketone).

2-Amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium chloride (CNI-H0294) is a novel arylene bis(methylketone) compound that displays antimalarial activity against chloroquine- and pyrimethamine-resistant Plasmodium falciparum clones. The compound has been found to be concentrated into infected erythrocytes, with 80-179 microM accumulated when parasites were cultured in the presence of 1.0 microM CNI-H0294. Uninfected erythrocytes, in contrast, only accumulated 2.5-3.4 microM CNI-H0294 under identical conditions. Using postmitochondrial supernatants from a number of parasite clones, the compound was found to inhibit dihydrofolate reductase (EC 1.5.1.3) activity with an IC50 of 243-483 microM. Thus, while CNI-H0294 is not a powerful inhibitor of plasmodial dihydrofolate reductase, the accumulation of the compound into infected erythrocytes, when correlated to the external ED50 concentration against parasite growth in vitro, reaches concentrations sufficient to inhibit the malarial enzyme.

Animals↗
PubMed1997

Studies on the pharmacological properties of novel arylene bis(methylketone) compounds using solid-phase extraction and high-performance liquid chromatography.

A method utilising solid-phase extraction followed by high-performance liquid chromatography has been developed to quantify novel arylene bis(methylketone) chemotherapeutics present in biological samples. The samples are extracted over cyanopropylsilane solid-phase extraction cartridges using 10 mM heptanesulfonate-10 mM tetramethylammonium chloride-4.2 mM H3PO4-95% CH3CN as the eluent. Analytical chromatography utilises a diisopropyl-C8 reversed-phase column and a 7.5-45% CH3CN gradient in 10 mM heptanesulfonate-10 mM tetramethylammonium chloride-4.2 mM H3PO4-H2O. Detection was by ultraviolet spectrophotometry at 300 or 240 nm. The linear response of the assay was found to extend from at least 100 microg/ml down to 97.66 ng/ml for a 100 microl injection. The assay system was utilised to determine the plasma kinetics of the compounds in mice, where all the drugs were found to display rapid absorption and elimination following intraperitoneal dosing. In vitro and in vivo studies of metabolism demonstrated that each of the compounds produced several metabolites, and that this conversion could be extensive in vivo.

Aniline Compounds↗
PubMed1997

Synergism of cimetidine with anti-malarial agents.

The histamine type-2 receptor antagonist and cytochrome P450 inhibitor cimetidine was examined for its antimalarial properties in the presence or absence of chloroquine or pyrimethamine. When used alone, cimetidine displayed little activity against a number of Plasmodium falciparum clones in vitro, with an IC50 of 300-700 microM. The compound was found to be highly synergistic in combination with chloroquine and also displayed a degree of synergism when used in combination with pyrimethamine. These synergistic effects were independent of the chloroquine- or pyrimethamine-resistance status of the clones. The cytochrome P450 inhibitor proadifen displayed weak synergism or antagonism with chloroquine, depending on the clone tested, and clear antagonism with pyrimethamine. The results with proadifen indicate that cimetidine was exerting its synergistic activity via a mechanism distinct from inhibition of cytochrome P450. Additional experiments have demonstrated that cimetidine interferes with neither plasmodial sterol metabolism nor heme polymerization.

Animals↗
PubMed1996

Aromatic amino acid transamination and methionine recycling in trypanosomatids.

Although trypanosomatids are known to rapidly transaminate exogenous aromatic amino acids in vitro and in vivo, the physiological significance of this reaction is not understood. In postmitochondrial supernatants prepared from Trypanosoma brucei brucei and Crithidia fasciculata, we have found that aromatic amino acids were the preferred amino donors for the transamination of alpha-ketomethiobutyrate to methionine. Intact C. fasciculata grown in the presence of [15N]tyrosine were found to contain detectable [15N]methionine, demonstrating that this reaction occurs in situ in viable cells. This process is the final step in the recycling of methionine from methylthioadenosine, a product of decarboxylated S-adenosylmethionine from the polyamine synthetic pathway. Mammalian liver, in contrast, preferentially used glutamine for this reaction and utilized a narrower range of amino donors than seen with the trypanosomatids. Studies with methylthioadenosine showed that this compound was readily converted to methionine, demonstrating a fully functional methionine-recycling pathway in trypanosomatids.

Amino Acids↗
PubMed1996

High-performance liquid chromatographic method for guanylhydrazone compounds.

A high-performance liquid chromatographic method has been developed for a series of aromatic guanylhydrazones that have demonstrated therapeutic potential as anti-inflammatory agents. The compounds were separated using octadecyl or diisopropyloctyl reversed-phase columns, with an acetonitrile gradient in water containing heptane sulfonate, tetramethylammonium chloride, and phosphoric acid. The method was used to reliably quantify levels of analyte as low as 785 ng/ml, and the detector response was linear to at least 50 micrograms/ml using a 100 microliters injection volume. The assay system was used to determine the basic pharmacokinetics of a lead compound, CNI-1493, from serum concentrations following a single intravenous injection in rats.

Animals↗
PubMed1996

Antimalarial activity of novel arylene bis(methylketone) compounds.

Because of the spread of drug-resistant Plasmodium species, there is an urgent need for novel effective antimalarial agents. A series of arylene bis(methylketone) compounds were screened in vitro against a number of Plasmodium falciparum clones and in vivo against Plasmodium berghei. 2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium chloride (Cytokine Network Inc. [CNI]-H0294) was the most effective of the compounds in vitro, with an IC50 of 1.5-4.0 microM against parasite clones with a wide range of sensitivities to chloroquine and pyrimethamine. Other compounds in the series had in vitro IC50 values of 20-25 microM. In a 4-day test for suppression of P. berghei parasitemia in vivo, 50 mg/kg/day CNI-H0294 significantly decreased parasitemia by >90%. The compound was found to have low toxicity in mice, with an LD50 of 590 +/- 66 mg/kg intraperitoneally, and rapid plasma kinetics. These results show that CNI-H0294 has considerable antimalarial activity and merits further study.

Animals↗
PubMed1995

Uptake of diamidine drugs by the P2 nucleoside transporter in melarsen-sensitive and -resistant Trypanosoma brucei brucei.

The African trypanosome, Trypanosoma brucei brucei, possesses at least two nucleoside transporter systems designated P1 and P2, the latter being implicated in the selective uptake of melaminophenyl arsenical drugs. Since arsenical-resistant trypanosomes show cross-resistance in vivo to aromatic diamidines, we have investigated whether these drugs are also substrates for the P2 nucleoside transporter. In melarsen-sensitive T. b. brucei, the diamidines, including the commonly used trypanocides, pentamidine and berenil, were found to abrogate lysis induced by the P2 transport of melarsen oxide in vitro. Measurement of [ring-3H]pentamidine transport in melarsen-sensitive T. b. brucei, demonstrated that uptake is carrier-mediated, with a Km of 0.84 microM and a Vmax of 9.35 pmol s-1 (10(8) cells)-1. Pentamidine transport appears to be P2-mediated in these cells, as pentamidine strongly inhibited uptake of [2',5',8-3H]adenosine by the P2 transporter, with a Ki of 0.56 microM. Furthermore, [ring-3H]pentamidine transport was blocked by a number of P2 transporter substrates and inhibitors, as well as by other diamidine drugs. Analysis of the uptake of pentamidine and other diamidines in melarsen-resistant trypanosomes in vitro and in vivo, which also show differential levels of resistance to these compounds in vivo, indicated that P2 transport was altered in these cells and that accumulation of these drugs was markedly reduced.

Adenosine↗
PubMed1995

High-performance liquid chromatographic analysis of biological and chemical heme polymerization.

Free hematin can be converted to a stable polymer both chemically, by heating hematin in acid suspensions, or biologically, in the food vacuoles of malaria. A high-performance liquid chromatographic assay has been developed which can separate and quantitate both free hematin and the polymer (beta-hematin), based on the differential solubility of the two compounds. Ion-pair reverse-phase chromatography, utilizing tetramethylammonium chloride and heptane sulfonate as the ion-pair agents in the presence of 40% acetonitrile, was performed on a polymeric-resin-based column with a phenyl bonded phase. Initiating the runs at pH 2.5 led to elution only of the free hematin, and a subsequent shift to pH 12.0 converted the beta-hematin back to hematin which then eluted separately. The method was found to have a linear range of detection from 78 pmol to 20 nmol injected hematin and intra- and interday variations of 9.71 and 12.46%, respectively. The assay was used to study several basic aspects of heme polymerization in vitro, including effects of hematin and beta-hematin concentration on the rate of polymerization.

Animals↗
PubMed1995

p-(4,6-Diamino-s-triazin-2-yl)aminophenylarsonic acid dihydrate.

The structure of the title compound C9 H11 AsN6O3-2H2O, contains a planar organic moiety bonded to pentavalent arsenic. The lattice is stabilized by a combination of aromatic stacking interactions and by a hydrogen-bonding network involving water molecules of crystallization.

Arsenicals↗
PubMed1995

Characterisation of pentamidine-resistant Trypanosoma brucei brucei.

Following selection in vitro by exposure to increasing concentrations of the aromatic diamidine pentamidine, a Trypanosoma brucei brucei clone has been characterised in vivo and in vitro. The resistant clone, designated T.b. brucei S427/118/PR32.6, was found to be less virulent than the parental clone T.b. bruci S427/118, with an intraperitoneal injection of 2.5 x 10(6) resistant organisms required to produce a course of disease equivalent to 1 x 10(4) sensitive trypanosomes. This lowered virulence is not associated with an increased susceptibility to the host's immune system, and is not due to the in vitro culturing process. The pentamidine-resistant clone was found to be 26- and 4.5-fold resistant to pentamidine in vitro and in vivo, respectively. Although not cross-resistant in vivo to any other aromatic diamidines (stilbamidine, berenil and propamidine), a 2.4-fold increase in resistance to the melaminophenylarsine melarsoprol was observed. While pentamidine completely inhibited uptake of 1 microM [3H]adenosine in the presence of 1 mM inosine, suggesting that pentamidine is transported by the inosine-insensitive P2 transporter, the pentamidine-resistant clone appeared to have a fully functional P2-adenosine transport system. Both resistant and parental cloned lines accumulated approx. 6 nmol pentamidine (10(8) cells)-1 over the course of 3 h, representing an internal concentration of 0.7-1.0 mM. Thus, unlike previously characterised drug-resistant trypanosomes, T.b. brucei PR32.6 is not deficient in drug accumulation, suggesting that other resistance mechanisms are likely to be involved.

Adenosine↗
PubMed1995

Bacterial vaginosis in lesbians: a sexually transmitted disease.

Sexual transmission of bacterial vaginosis (BV), a common syndrome in sexually active women, has not been previously established. Because no male counterpart for BV has been found, a population of lesbians is an ideal one in which to test the hypothesis that BV is sexually transmitted. We studied 103 homosexual women (lesbians) who sought gynecologic care at a community clinic and in a private gynecology practice in New York City. Participants were asked to refer their sexual partners for evaluation. In this cross-sectional prevalence study, all participants were evaluated for the presence of BV, and pairs of monogamous sexual partners were analyzed for concordance of their vaginal secretions. Twenty-nine (28.7%) of the 101 participants from whom satisfactory vaginal wash samples were available had BV. There were 21 pairs of monogamous partners. Of 11 index women who had BV, eight (72.7%) had partners who also had BV. Of 10 index women who did not have BV, only one (10%) had a partner with BV. The likelihood of a partner's having BV was 19.7 times greater if the index case had BV (P < .008; 95% CI, 2.1-588.0). We conclude that with respect to BV, lesbians in monogamous relationships usually have concordant vaginal secretions. This concordance probably reflects the sexual transmission of BV between lesbians.

Female↗
PubMed1995

Chloroquine resistance is not associated with drug metabolism in Plasmodium falciparum.

Chloroquine-sensitive and -resistant clones of Plasmodium falciparum were incubated in vitro with [3H]chloroquine for 16 hr, and the resulting culture supernatants and cell pellets were analyzed by high-performance liquid chromatography for evidence of chloroquine metabolism. After separation by normal- or reverse-phase chromatography, there was no evidence of drug metabolism by the chloroquine-resistant P. falciparum. However, a single, unidentified, radiolabeled metabolite, which did not coelute with desethylchloroquine, was produced by 1 of the chloroquine-sensitive clones. Thus, chloroquine resistance does not appear to be due to drug metabolism by the resistant parasites.

Animals↗