Trichomonads, hydrogenosomes and drug resistance

Trichomonas vaginalis and Tritrichomonas foetus are sexually transmitted pathogens of the genito-urinary tract of humans and cattle, respectively. These organisms are amitochondrial anaerobes possessing hydrogenosomes, double membrane-bound organelles involved in catabolic processes extending glycolysis. The oxidative decarboxylation of pyruvate in hydrogenosomes is coupled to ATP synthesis and linked to ferredoxin-mediated electron transport. This pathway is responsible for metabolic activation of 5-nitroimidazole drugs, such as metronidazole, used in chemotherapy of trichomoniasis. Prolonged cultivation of trichomonads under sublethal pressure of metronidazole results in development of drug resistance. In both pathogenic species the resistance develops in a multistep process involving a sequence of stages that differ in drug susceptibility and metabolic activities. Aerobic resistance, similar to that occurring in clinical isolates of T. vaginalis from treatment-refractory patients, appears as the earliest stage. The terminal stage is characterised by stable anaerobic resistance at which the parasites show very high levels of minimal lethal concentration for metronidazole under anaerobic conditions (approximately 1000 microg ml(-1)). The key event in the development of resistance is progressive decrease and eventual loss of the pyruvate:ferredoxin oxidoreductase so that the drug-activating process is averted. In T. vaginalis at least, the development of resistance is also accompanied by decreased expression of ferredoxin. The pyruvate:ferredoxin oxidoreductase deficiency completely precludes metronidazole activation in T. foetus, while T. vaginalis possesses an additional drug-activating system which must be eliminated before the full resistance is acquired. This alternative pathway involves the hydrogenosomal malic enzyme and NAD:ferredoxin oxidoreductase. Metronidazole-resistant trichomonads compensate for the hydrogenosomal deficiency by an increased rate of glycolysis and by changes in their cytosolic pathways. Trichomonas vaginalis enhances lactate fermentation while T. foetus activates pyruvate conversion to ethanol. Drug-resistant T. foetus also increases activity of the cytosolic NADP-dependent malic enzyme, to enhance the pyruvate producing bypass and provide NADPH required by alcohol dehydrogenase. Production of succinate by this species is abolished. Metabolic changes accompanying in-vitro development of metronidazole resistance demonstrate the versatility of trichomonad metabolism and provide an interesting example of how unicellular eukaryotes can adjust their metabolism in response to the pressure of an unfavorable environment.     

Occurrence of transsulfuration in synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus HB8

A cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine gamma-synthesis with O-acetyl-L-homoserine and L-cysteine as substrates but not beta-synthesis with DL-homocysteine and L-serine (or O-acetyl-L-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cystathionine beta-lyase (EC 4.4.1.8) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) were markedly repressed by L-methionine supplemented to the medium. L-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of O-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase. Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of O-acetylserine sulfhydrylase 2.1- to 2.5-fold. A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of O-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine gamma-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine gamma-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of O-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibited O-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis of O-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine gamma-synthase. Although it has been reported that homocysteine is directly synthesized in T. thermophilus HB27 by the catalysis of O-acetylhomoserine sulfhydrylase on the basis of genetic studies (T. Kosuge, D. Gao, and T. Hoshino, J. Biosci. Bioeng. 90:271-279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in T. thermophilus HB8.     

Mutation modifying the serine pathway in methylotrophic bacteria

Methylotrophic bacteria, Gram-positive, with the serine pathway, were shown to have their growth inhibited by 0.5 % glycine. The effects of this amino acid on individual enzyme activities were studied in wild and mutant strains ofMicrococcus varians andBacillus licheniformis. The enzymes studied were glycerate dehydrogenase (EC 1.1.1.29), isocitrate lyase (EC 4.1.3.1), serine hydroxymethyltransferase (EC 2.1.2.1) and glycine—oxaloacetate aminotransferase (EC 2.6.1.35). The last-named enzyme was found to be inhibited, the kinetic constants having been determined for two strain types.     

Effect of regulatory mutations of sulphur metabolism on the levels of cysteine- and homocysteine-synthesizing enzymes in Neurospora crassa

1. Regulation of four enzymes involved in cysteine and homocysteine synthesis, i.e. cysteine synthase (EC 4.2.99.8), homocysteine synthase (EC 4.1.99.10), cystathionine beta-synthase (EC 2.1.22) and gamma-cystathionase (EC 4.4.1.1) was studied in the wild type and sulphur regulatory mutants of Neurospora crassa. 2. Homocysteine synthase and cystathionine beta-synthase were found to be regulatory enzymes but only the former is under control of the cys-3 - scon system regulating several enzymes of sulphur metabolism, including gamma-cystathionase. 3. The results obtained with the mutants strongly suggest that homocysteine synthase plays a physiological role as an enzyme of the alternative pathway of methionine synthesis. Cysteine synthase activity was similar in all strains examined irrespective of growth conditions. 4. The sconc strain with derepressed enzymes of sulphur metabolism showed an increased pool of sulphur amino acids, except for methionine. Particularly characteristic for this pool is a high content of hypotaurine, a product of cysteine catabolism.     

Aspartate: 2-oxoglutarate aminotransferase from trichomonas vaginalis. Identity of aspartate aminotransferase and aromatic amino acid aminotransferase.

Aspartate: 2-oxoglutarate aminotransferase from the anaerobic protozoon Trichomonas vaginalis was purified to homogeneity and characterized. It is a dimeric protein of overall Mr approx. 100000. Only a single isoenzyme was found in T. vaginalis. The overall molecular and catalytic properties have features in common with both the vertebrate cytoplasmic and mitochondrial isoenzymes. The purified aspartate aminotransferase from T. vaginalis showed very high rates of activity with aromatic amino acids as donors and 2-oxoglutarate as acceptor. This broad-spectrum activity was restricted to aromatic amino acids and aromatic 2-oxo acids, and no significant activity was seen with other common amino acids, other than with the substrates and products of the aspartate: 2-oxoglutarate aminotransferase reaction. Co-purification and co-inhibition, by the irreversible inhibitor gostatin, of the aromatic amino acid aminotransferase and aspartate aminotransferase activities, in conjunction with competitive substrate experiments, strongly suggest that a single enzyme is responsible for both activities. Such high rates of aromatic amino acid aminotransferase activity have not been reported before in eukaryotic aspartate aminotransferase.     

Effects of homocysteine on the binding of extracellular-superoxide dismutase to the endothelial cell surface

Homocysteine is known to be a risk factor for several vascular diseases. Previously, we found a significant association between plasma homocysteine and plasma extracellular-superoxide dismutase (EC-SOD) levels. The binding of EC-SOD to human and bovine aortic endothelial cell cultures showed significant decreases after incubation with 10 microM homocysteine, whereas the expression of EC-SOD in fibroblast cell cultures was inhibited with a high concentration (1 mM) of homocysteine. Furthermore, binding of EC-SOD to heparin immobilized on plates was decreased with homocysteine. These observations suggested that homocysteine decreases the binding of EC-SOD to vascular endothelial cell surfaces by degradation of endothelial heparan sulfate proteoglycan, which results in a loss of the ability to protect endothelial cell surfaces from oxidative stress.     

Purification, biochemical properties and active sites of N-acetyl-beta-D-hexosaminidases from human seminal plasma.

Two isoenzymes of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) (Hex A and Hex B) from human seminal plasma were purified to homogeneity with specific activities of 26 and 60 units/mg of protein respectively. N-Acetyl-beta-D-glucosaminidase activity was inseparable from N-acetyl-beta-D-galactosaminidase activity in both Hex A and Hex B by various conventional chromatographic procedures. Although Km values of N-acetyl-beta-glucosaminidase activity of Hex A and Hex B were similar (1.33 mM), those of N-acetyl-beta-galactosaminidase activity were 0.14 mM for Hex A and 0.40 mM for Hex B. However, pH optima and temperature optima were identical for N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities of both isoenzymes; Hex A was far more heat-sensitive than Hex B. Thiol-reactive compounds such as silver salts, mercuric salts, p-chloromercuribenzoate and thimerosal strongly inhibited the N-acetyl-beta-glucosaminidase activities of both isoenzymes. GSH protected the enzyme activities from inactivation caused by these reagents, confirming the presence of thiol groups at the active centres. Inhibitions of N-acetyl-beta-glucosaminidase activities of both isoenzymes by metal salts and organic anions were comparable; acetate and arsenite were effective inhibitors for both isoenzymes. In contrast, inhibitions of N-acetyl-beta-glucosaminidase activities of the two isoenzymes by iodoacetic acid, iodoacetamide and ethylmaleimide were not comparable; Hex B was more susceptible to inhibition by these agents at 20 mM concentration. The N-acetyl-beta-glucosaminidase activities of both isoenzymes are strongly inhibited, in decreasing order, by N-acetyl-galactosamine, mannosamine, disaccharic acid lactone, N-acetylglucosamine and gluconolactone. The Ki values of the N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities for N-acetylhexosamines and results from mixed-substrate kinetics indicated that the activities for the two substrates are located at different sites in Hex A and at the same site in Hex B. The Mr values of Hex A and Hex B were determined to be 195,000 and 210,000 respectively by gel filtration through Sephadex G-200. SDS/polyacrylamide-gel electrophoresis revealed that Hex A and Hex B are each composed of four subunits corresponding to Mr about 50,000 each. No further polypeptide chain was obtained after reduction and alkylation of Hex A and Hex B with 10 mM-dithiothreitol and 10 mM-iodoacetamide.     

Characteristics of hepatic serine-pyruvate aminotransferase in different mammalian species.

1. Serine-pyruvate aminotransferase was purified from mouse, rat, dog and cat liver. Each enzyme preparation was homogeneous as judged by polyacrylamide-disc-gel electrophoresis in the presence of sodium dodecyl sulphate. However, isoelectric focusing resulted in the detection of two or more active forms from enzyme preparations from dog, cat and mouse. A single active form was obtained with the rat enzyme. All four enzyme preparations had similar pH optima and molecular weights. 2. Both mouse and rat preparations catalysed transamination between a number of L-amino acids (serine, leucine, asparagine, methionine, glutamine, ornithine, histidine, phenylalanine or tyrosine) and pyruvate. Effective amino acceptors were pyruvate, phenylpyruvate and glyoxylate with serine as amino donor. The reverse transamination activity, with hydroxypyruvate and alanine as subtrates, was lower than with serine and pyruvate for both species. Serine-pyruvate aminotransferase activities were inhibited by isonicotinic acid hydrazide. 3. In contrast, both dog and cat enzyme preparations were highly specific for serine as amino donor with pyruvate, and utilized pyruvate and glyoxylate as effective amino acceptors. A little activity was detected with phenylpyruvate. The reverse activity was higher than with serine and pyruvate for both species. Serine-pyruvate amino-transferase activities were not inhibited by isonicotinic acid hydrazide.     

Homocysteine and other structurally-diverse amino thiols can alter pancreatic beta cell function without evoking cellular damage

Homocysteine and related amino thiols, homocysteic acid, cysteic acid, homocysteine sulphinic acid and cysteine sulphinic acid have been labelled as neurotoxins. Homocysteine thiolactone, a metabolic derivative of homocysteine, is cytotoxic to endothelial cells and other cell lineages. Since pancreatic beta cells share many phenotypic similarities with neuronal cells, the present study uses clonal pancreatic BRIN-BD11 cells to investigate possible detrimental effects of these amino thiols on insulin secretion and pancreatic beta cell function. Insulin secretion was concentration-dependently inhibited at both basal (1.1 mM) and stimulatory (16.7 mM) glucose by homocysteine, homocysteine thiolactone and homocysteine sulphinic acid. Cysteic acid concentration-dependently inhibited insulin secretion at 16.7 mM glucose. Cell viability was not compromised by any of the amino thiols. Insulin secretory responses to alanine were inhibited by homocysteine, homocysteine thiolactone, homocysteic acid and cysteic acid. Insulin secretion in the presence of elevated Ca(2+) and forskolin were lowered by all amino thiols, except homocysteic acid. The secretory responsiveness to PMA, GLP-1 and KCl were only impaired in the presence of homocysteine and homocysteine thiolactone. These findings indicate that homocysteine, homocysteine thiolactone and, to a lesser extent, other amino thiols cause dysfunctional insulin secretion from pancreatic beta cells.     

Further studies on the surface charge of various strains of Trichomonas vaginalis and Tritrichomonas foetus

The surface charge of three strains of Trichomonas vaginalis and five strains of Tritrichomonas foetus was determined by direct measurement of the mean cellular electrophoretic mobility (EPM) of cells suspended in solutions of different ionic strength and pH. No differences were observed in the mean EPM among the two species, although significant differences among the strains exist. Strains that are more pathogenic to mouse, as measured using the subcutaneous assay, had a surface more negative. Treatment of the parasites with trypsin or neuraminidase reduced significantly their mean EPM and increased their isoelectric point. Tritrichomonas foetus was more sensitive to the enzyme treatment than T. vaginalis. Enzyme-treated cells recovered their normal EPM if, after enzyme treatment, they were incubated in fresh culture medium. The recovery process of trypsin-treated cells was inhibited 10-20% by addition of inhibitors of either protein synthesis (puromycin) or N-glycosylation of proteins (tunicamycin) to the incubation medium, suggesting that a cytoplasmic pool of sialoglycoproteins may exist. The recovering of the EPM of T. foetus and T. vaginalis previously treated with neuraminidase was inhibited by puromycin or tunicamycin about 40-50% and 17-30%, respectively. These observations suggest that sialoglycolipids exist on the surface of both parasite species, and that they contribute more to the surface charge of T. vaginalis than to that of T. foetus.     

Homocysteine inhibits angiogenesis in vitro and in vivo

Homocysteine has been reported to inhibit endothelial cell proliferation, which is closely related to angiogenesis. However, the relationship between homocysteine and angiogenesis is unknown. To clarify whether homocysteine would inhibit angiogenesis in vitro and in vivo, we examined the effect of homocysteine on tube formation by bovine aortic endothelial cells (BAECs) and by human microvessel endothelial cell-1 (HMEC-1) in vitro, and on angiogenesis in vivo using the chorioallantoic membrane (CAM) assay, as well as on BAEC proliferation and migration. Homocysteine, but not cysteine, inhibited BAEC proliferation, migration, and tube formation in a dose-dependent manner at concentrations from 0 to 10 mM. Homocysteine also inhibited tube formation by HMEC-1s. In these assay, 50% inhibition was induced by about 1 mM homocysteine. In the in vivo CAM assay, 0, 10, 100, 500, and 1000 microgram homocysteine induced an avascular zone by 0, 0, 16.7, 53.3 and 76.5%, respectively, also showing a dose-dependent effect. It was suggested that homocysteine inhibited angiogenesis by preventing proliferation and migration of endothelial cells.     

Homocysteine Thiolactone and Human Cholinesterases

1. The cholinergic system is important in cognition and behavior as well as in the function of the cerebral vasculature. 2. Hyperhomocysteinemia is a risk factor for development of both dementia and cerebrovascular disease. 3. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are serine hydrolase enzymes that catalyze the hydrolysis of the neurotransmitter acetylcholine, a key process in the regulation of the cholinergic system. 4. It has been hypothesized that the deleterious effects of elevated homocysteine may, in part, be due to its actions on cholinesterases. 5. To further test this hypothesis, homocysteine and a number of its metabolites and analogues were examined for effects on the activity of human cholinesterases. 6. Homocysteine itself did not have any measurable effect on the activity of these enzymes. 7. Homocysteine thiolactone, the cyclic metabolite of homocysteine, slowly and irreversibly inhibited the activity of human AChE. 8. Conversely, this metabolite and some of its analogues significantly enhanced the activity of human BuChE. 9. Structure–activity studies indicated that the unprotonated amino group of homocysteine thiolactone and related compounds represents the essential feature for activation of BuChE, whereas the thioester linkage appears to be responsible for the slow AChE inactivation. 10. It is concluded that hyperhomocysteinemia may exert its adverse effects, in part, through the metabolite of homocysteine, homocysteine thiolactone, which is capable of altering the activity of human cholinesterases, the most pronounced effect being BuChE activation.     

Lysine- and lysine-plus-threonine-inhibitable aspartokinases in Bacillus brevis.

Two aspartokinase (ATP:L-aspartate 4-phosphotrasferase, EC 2.7.2.4) enzyme activities have been identified and partially purified from Bacillus brevis. Aspartokinase I is subject to both inhibition and repression by lysine, and has a molecular weight in the region of 110 000. Aspartokinase II is a lysine-stabilised enzyme, inhibited multivalently by lysine plus theonine and has a molecular weight in the region of 95 000. This attern of aspartokinase activity has not been described previously and is unusual in that one end product (lysine) regulates two isoenzymes catalysing the first reaction of a branced biosynthetic pathway. In the absence of lysine, aspartokinase II changes to a more unstable non-inhibitable enzyme. Both enzymes are stabilised by sulphydryl reducing agents and have similar affinities for ATP, aspartate and lysine. However, there is no evidence for a view that they are products of a common gene. Problem concerned with the regulation of aspartokinase activities in Bacillus species are discussed.     

Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance

Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica , Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.     

Metronidazole inhibition of hydrogen production in vivo in drug-sensitive and resistant strains of Trichomonas vaginalis

H2 production by the human protozoan parasite Trichomonas vaginalis was monitored continuously under a mobile gas phase using a membrane-inlet mass spectrometer. Simultaneous and continuous measurement of dissolved H2, O2 and CO2 indicated that H2 evolution was inhibited at levels of O2 (less than 0.25 microM) undetectable by the technique, whereas CO2 production was stimulated. Respiration was not stimulated by admitting H2 to the gas phase. Metronidazole inhibited both H2 and CO2 production. Values of K1 for inhibition of H2 formation in strain ATCC 30001 (metronidazole sensitive) of 0.16 mM and in strain 85 (metronidazole resistant) of 1.0 mM were obtained. These data suggest that metronidazole not only competes with protons as electron acceptor but that the drug itself or a product of reduction actively inhibits some hydrogenosomal enzyme or electron carrier involved in H2 production. Under these conditions metronidazole inhibition leads to irreversible loss of cell motility.     

Folate deprivation reduces homocysteine remethylation in a human intestinal epithelial cell culture model: role of serine in one-carbon donation

Little is known about homocysteine metabolism in intestine. To address this question, we investigated homocysteine metabolism under conditions of folate adequacy and folate deprivation in the Caco-2 cell line, a model of human intestinal mucosal cells. Caco-2 cells were cultured in media enriched with [3-(13)C]serine and [U-(13)C(5)]methionine tracers, and enrichments of intracellular free amino acid pools of these amino acids as well as homocysteine, cystathionine, and cysteine were measured by using gas chromatography/mass spectrometry. Homocysteine transsulfuration plus folate-dependent and total remethylation were quantified from these amino acid enrichments. Homocysteine remethylation accounted for 19% of the intracellular free methionine pool in cells cultured with supplemental folate, and nearly all one-carbon units used for remethylation originated from the three carbon of serine via folate-dependent remethylation. Labeling of cystathionine and cysteine indicated the presence of a complete transsulfuration pathway in Caco-2 cells, and this pathway produced 13% of the intracellular free cysteine pool. Appearance of labeled homocysteine and cystathionine in culture medium suggests export of these metabolites from intestinal cells. Remethylation was reduced by one-third in folate-restricted cell cultures (P < 0.001), and only approximately 50% of the one-carbon units used for remethylation originated from the three carbon of serine under these conditions. In conclusion, the three carbon of serine is the primary source of one-carbon units used for homocysteine remethylation in folate-supplemented Caco-2 cell cultures. Remethylation is reduced as a result of folate restriction in this mucosal cell model, and one-carbon sources other than the three carbon of serine contribute to remethylation under this condition.     

Flow Cytoenzymology of the Early Differentiation of Mouse Embryonal Carcinoma Cells

Dual parameter flow cytoenzymology was used to detect biochemical differentiation of embryonal carcinoma cells, the undifferentiated, multipotent stem cells of teratocarcinomas. With the use of fluorogenic substrates, two enzyme systems, alkaline phosphatase (EC 3.1.3.1.) and carboxyl esterase (EC 3.1.1.), were studied. Embryonal carcinoma cells passaged in vitro for several years retained high alkaline phosphatase activities similar to those of embryonal carcinoma cells in embryoid bodies grown in vivo. Similar to the embryonal carcinoma cells in vivo, the in vitro embryonal carcinoma cells were capable of giving rise to progeny with greatly decreased levels of alkaline phosphatase. The embryonal carcinoma cell alkaline phosphatase was inhibited by l-p-bromotetramisole, suggesting a relationship between this enzyme and somatic, nonintestinal alkaline phosphatase isoenzymes. Determinations of esterase activities in viable teratocarcinoma cells showed that prior to any evidence of morphologic differentiation, the embryonal carcinoma cells are quite heterogeneous with regard to esterase activities.     

Mechanistic, inhibitory and stereochemical studies on cytoplasmic and mitochondrial serine transhydorxymethylases.

By using cytoplasmic and mitochondrial serine transhydroxymethylase isoenzymes from rabbit liver, it was shown that both enzymes exhibited similar ratios of serine transhydroxymethylase/threonine aldolase activities. Both enzymes catalysed the removal of the pro-S hydrogen atom of glycine, which was greatly enhanced by the presence of tetrahydrofolate. The cytoplasmic as well as the mitochondrial enzyme catalysed the synthesis of serine from glycine and [3H2]formaldehyde in the absence of tetrahydrofolate. The results are consistent with our previous suggestion that a role of tetrahydrofolate in the serine transhydroxymethylase reaction is to transport formaldehyde in and out of the active site (Jordan & Akhtar, 1970). The isoenzymes, however, showed remarkable differences in their inactivation by inhibitors. The serine transhydroxymethylase as well as the threonine aldolase activities of the cytoplasmic enzyme were inactivated in a similar fashion by chloroacetaldehyde, iodoacetamide, bromopyruvate and glycidaldehyde (2,3-epoxypropionaldehyde). These inhibitors had no effect on the two activities of the mitochondrial enzyme. The rate of inactivation of the cytoplasmic enzyme by glycidaldehyde was enhanced by the presence of glycine but decreased by the presence of serine. The implications of these results to the mechanism of catalysis and the nature of the active site of the enzymes are discussed.     

Plasma total homocysteine level and methylenetetrahydrofolate reductase 677C>T genetic polymorphism in Japanese patients with rheumatoid arthritis

Hyperhomocysteinemia is a known risk factor of cardiovascular disease. Homocysteine has been also linked to inflammation in rheumatoid arthritis (RA). In this study, we investigated the relationship between plasma homocysteine levels and single nucleotide polymorphism (SNP) of the gene coding for methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the biosynthesis of homocysteine, and the correlation between the plasma homocysteine levels and generally used inflammatory markers (C-reactive protein, erythrocyte sedimentation rate and matrix metalloproteinase-3) in 96 Japanese patients with RA. Plasma homocysteine levels in patients with the MTHFR 677TT genotype were significantly higher than in those with the 677CC genotype (p < 0.05). In addition, plasma homocysteine levels were increased along with the elevation of general inflammatory markers. Therefore, we conclude that homocysteine might affect the inflammatory status of patients, and the MTHFR 677C>T SNP could be a predictive factor of hyperhomocysteinemia in patients with RA.