Chemotaxis inhibition by Gardnerella vaginalis and succinate producing vaginal anaerobes: composition of vaginal discharge associated with G vaginalis.

The influence of six succinate producing vaginal anaerobes and Gardnerella vaginalis on the chemotactic activity of granulocytes was studied by the under agarose method. G vaginalis, Mobiluncus species, and three Gram negative anaerobes elicited hardly any response, but Peptostreptococcus productus showed clear positive chemotaxis, as did the Escherichia coli strain used as a control. Inhibition of the chemotactic response of white blood cells was found with all strains, but the high succinate producers from the genus Bacteroides showed the most pronounced effect. The inhibition of chemotaxis by succinate producing anaerobes in the pathogenesis of non-specific vaginitis (NSV) is postulated, and B ureolyticus or Mobiluncus spp, rather than G vaginalis, are suggested as possible causes of NSV.

Carbon dioxide abolishes the reverse Pasteur effect in Leishmania major promastigotes.

The products released by Leishmania major promastigotes incubated with [1-13C]glucose as sole exogenous carbon source were identified using nuclear magnetic resonance (NMR). Under aerobic (95% O2/5% CO2) conditions, acetate, succinate, and small amounts of pyruvate, D-lactate, and glycerol were released in addition to CO2. Under anaerobic (95% N2/5% CO2) conditions, the relative amounts of products formed changed and alanine was also released. The changes in the rates of glucose consumption and product formation during the aerobic to anaerobic transition were measured. Under hypoxic conditions (O2 less than 0.2%), glucose consumption was decreased by about 50%. Under completely anaerobic conditions (100% N2), glucose consumption almost ceased (a total reverse Pasteur effect). The inclusion of 5% CO2 in the gas phase restored hypoxic and anaerobic glucose consumption to the aerobic rate, and increased production of succinate, pyruvate, and D-lactate. Thus, CO2 and very low concentrations of O2 have strong regulatory effects on L. major glucose metabolism. A quantitative carbon balance showed that the NMR-identified products accounted for only about 25% of the glucose carbons consumed under aerobic conditions. CO2, measured as the release of 14CO2 from [U-14C]glucose, accounted for an additional 25% of the glucose consumed. About 11% of the glucose carbon was incorporated into trichloroacetic acid-insoluble products, mostly lipid. Large amounts of label from [U-14C]glucose were incorporated into the intracellular pools of alanine, glutamate, glutamine, and aspartate, indicating that CO2 from unlabeled amino acids contributed to the carbon balance. Under anaerobic conditions, all the glucose carbons consumed could be accounted for solely by the NMR-identified products.

Pathways of succinate formation and their contribution to improvement of cardiac function in the hypoxic rat heart.

Hypoxia led to a dramatic acceleration of amino acid breakdown together with succinate synthesis in the rat heart. Our data do not confirm the simultaneous conversion of aspartate and glutamate to succinate, which has been repeatedly assumed in the literature (7, 8, 21, 28-30), but rather suggest that different pathways are involved during developing hypoxia and that glutamate is the sole source for anaerobic succinate production from endogenous sources in the glucose-perfused heart. Perfusion of hypoxic rat hearts with 2-oxoglutarate, malate, and fumarate (5 mM each) increased succinate formation three- to fourfold. The beneficial effects of these substances on left ventricular systolic pressure, end diastolic pressure, and time of recovery may be due to the elevated content of ATP in these hearts compared to hypoxic controls with glucose as the sole substrate. However, the maintenance of a high rate of anaerobic glycolysis in hearts perfused with 2-oxoglutarate, malate, and fumarate and not the small stimulation of succinate synthesis is considered to be the most important mechanism of cardiac protection. A proposed pathway assumes that malate, after dehydration to fumarate, may serve as an alternative electron acceptor for cytosolic NADH during conditions of oxygen deficiency, thereby cancelling glycolytic inhibition.

Rhodoquinone requirement of the Hymenolepis diminuta mitochondrial electron transport system.

The occurrence of rhodoquinone as a mitochondrial membrane component was demonstrated in adult Hymenolepis diminuta. Chromatographic separation of pentane extracts, from lyophilized mitochondrial membranes, coupled with spectral analyses of separated material demonstrated the presence of rhodoquinone. The presence of ubiquinone was not apparent. Rhodoquinone content of membranes was about 1.2 micrograms (mg protein)-1. The rhodoquinone requirement of the H. diminuta electron transport system was demonstrated both in terms of the less active NADH oxidase and the physiologically required, NADH-dependent fumarate reductase employing lyophilized mitochondrial membranes as the source of activities. Pentane extraction of membranes virtually abolished the oxidase and fumarate reductase systems. Supplementation of pentane-treated membranes with H. diminuta rhodoquinone restored oxidase and fumarate reductase activities to levels simulating those of lyophilized membranes. Ubiquinone did not substitute for rhodoquinone. The rhodoquinone-reconstituted membranes displayed rotenone sensitivity. These findings represent the first direct demonstration of the rhodoquinone requirement of helminth electron transport-coupled oxidase and fumarate reductase.

[Mechanisms for improving the energy metabolism and function of the hypoxic myocardium with amino acids]. / Mekhanizmy uluchsheniia énergeticheskogo obmena i funktsii gipoksicheskogo miokarda aminokislotami.

The relationship between metabolism of the main myocardial amino acids, glutamate, aspartate and alanine, and energy state of hypoxic myocardium, was studied. Depression of cardiac contractile function during asphyxia in rats was accompanied by a decrease in glutamate mitochondrial and tissue contents and an increase in the tissue alanine and mitochondrial aspartate. The reduction of mitochondrial glutamate in asphyxia was related to losses of intramitochondrial ATP and state 3 respiration with glutamate and malate. Using NMR technique, exogenous glutamate and oxaloacetate were shown to increase succinate formation coupled with ATP and CTP production in the rat heart mitochondria in absence of aeration. These data suggest that glutamate and products of its transamination decrease the contraction of hypoxic myocardium stimulating anaerobic energy formation in the tricarboxylic acid cycle.

Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells.

It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive. We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized. In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.

[The NMR study of succinate formation from exogenous precursors in anaerobic rat heart mitochondria]. / Izuchenie metodom IaMR obrazovaniia suktsinata iz ékzogennykh predshestvennikov v neaériruemykh mitokhondriiakh serdtsa krysy.

Succinate formation during incubation of isolated rat heart mitochondria with exogenous precursors, malate, alpha-ketoglutarate, oxaloacetate and L-glutamate was studied in the absence of aeration. The formation of succinate, the end product of the tricarboxylic acid cycle, occurs via two pathways: through reduction of oxaloacetate or malate and via oxiation of alpha-ketoglutarate. The highest rate of succinate synthesis was observed when mitochondria were incubated with a mixture of 5 mM L-glutamate and 10 mM oxaloacetate, i.e., when both routes were used simultaneously. The [U-13C]succinate/succinate and aspartate/succinate ratios were equal to 2, when mitochondria were incubated with 5 mM [U-13C]glutamate and 10 mM oxaloacetate. Therefore, the amount of succinate formed from [13C]alpha-ketoglutarate via transamination of [13C]glutamate with oxaloacetate exceeds twice succinate production from oxialoacetate. These data suggest that GTP formation in the succinic thiokinase reaction should exceed twice the ATP yield coupled with NADH-dependent reduction of fumarate.

A 1H NMR study of succinate synthesis from exogenous precursors in oxygen-deprived rat heart mitochondria.

Succinate synthesis from exogenous malate, alpha-ketoglutarate, oxaloacetate and L-glutamate in isolated oxygen-deprived rat heart mitochondria was studied using 1H NMR. The highest rate of succinate synthesis was observed during incubation of mitochondria with a mixture of L-glutamate and oxaloacetate. When mitochondria were incubated with [U-13C] glutamate and oxaloacetate the [U-13C] succinate/succinate and aspartate/succinate ratios were equal to 2. This suggests that the succinate produced from [U-13C] alpha-keto-glutarate formed via transamination of [U-13C] glutamate with oxaloacetate by aspartate aminotransferase exceeds twofold that synthesized via oxaloacetate reduction. It may thus be expected that GTP yield in a reaction catalyzed by the succinic thiokinase will be 2 times higher that of ATP production coupled with NADH-dependent fumarate reduction.

The anaerobic heart: succinate formation and mechanical performance of cat papillary muscle.

Quiescent or paced, isometrically contracting right ventricular papillary muscles of cat heart were incubated under oxygenated or hypoxic (Po2 less than 10 Torr) conditions in order to investigate lactate and succinate formation. Both compounds were produced and released into the incubation medium during oxygen deficiency. Mechanical performance stimulated synthesis of both compounds under hypoxic as well as oxygenated conditions. Pacing led to the accumulation of large amounts of glutamine both under oxygenated and hypoxic conditions. Hypoxia resulted in a marked depletion of high-energy phosphates and concomitantly mechanical performance was impaired, i.e. developed tension fell rapidly and relaxation rate decreased. Supplying hypoxic, contracting muscles with aspartate (2 mM) resulted in maintenance of muscular function to some extent and led to augmented release of succinate and lactate. The data indicate that anaerobic succinate formation is correlated to the energy requiring processes of the myocardium. Maintenance of myocardial function by the supply of amino acids may be related to their conversion into succinate and to the stimulation of glycolysis.

The acid end-products of glucose metabolism of oral and other haemophili.

The acids produced in broth culture by various species of oral haemophili and by stock strains of capsulated and other haemophili were identified and measured by gas-liquid chromatography. Succinic acid was the major acid end-product of all strains, with acetic acid also being regularly produced but in smaller amounts. A stock strain, Haemophilus parainfluenzae NCTC 4101, produced less succinic acid than other strains of haemophili. Strain NCTC 4101 possessed all the enzymes of the tricarboxylic acid cycle, as previously reported, but in the other haemophili examined only succinic dehydrogenase, fumarase and malate dehydrogenase could be detected. No other enzymes of the tricarboxylic acid cycle were detected and isocitrate lyase, malate synthase and pyruvate carboxylase were also absent. Phosphoenolpyruvate-carboxylase was present in all strains. A partial tricarboxylic acid cycle and marked malate dehydrogenase activity appear to be characteristic of haemophili. The pathway to succinate in haemophili appears to be via carboxylation of phosphoenolpyruvate to oxalacetate and thence via malate and fumarate. The results of tracer studies on a single oral strain of H. parainfluenzae using various labelled substrates were in keeping with this proposed metabolic pathway.

Glucose metabolism of Treponema bryantii, an anaerobic rumen spirochete.

The pathway of glucose metabolism by Treponema bryantii, an obligately anaerobic spirochete isolated from bovine rumen contents, was studied. Washed cell suspensions of the spirochete consumed glucose and CO2 and produced equimolar amounts of acetate, formate, and succinate. Carbon dioxide was essential for glucose metabolism. Determination of radioactivity in products formed from 14C-labelled glucose and NaH14CO3 and assays of enzyme activities in cell-free extracts were used to determine the pathway of glucose metabolism. Treponema bryantii catabolized glucose to pyruvate via the Embden--Meyerhof--Parnas pathway. The spirochete used a coliform pyruvate-formate lyase to degrade pyruvate and produce formate and acetate. Succinate was formed by a pathway which involved the condensation of CO2 with pyruvate (or phospho(enol)pyruvate) formed from the breakdown of glucose.

Glycosomal and mitochondrial malate dehydrogenases in epimastigotes of Trypanosoma cruzi.

The degradation of glucose by Trypanosoma cruzi leads to the excretion of succinate. Malate dehydrogenase (MDH) participates in this process by reducing to malate the oxaloacetate synthesized by the glycosomal enzyme, phosphoenolpyruvate carboxykinase. The best coupling for these two sequential reactions would be attained if both enzymes were placed in the same subcellular compartment. The intracellular distribution of the MDH activity in epimastigotes of T. cruzi was studied by two methods. Selective disruption of cellular membranes with increasing concentrations of digitonin, indicated that trypanosomal MDH is particulate. Isopycnic centrifugation in a sucrose gradient of a large granule fraction, obtained by grinding the cells with silicon carbide, showed the presence of two MDH activities: one banding together with the glycosomal marker phosphoenolpyruvate carboxykinase, the other with the mitochondrial marker citrate synthase. Isoelectrofocusing of cell-free extracts led to the separation of two enzyme forms, with pI values of about 3.5 (MDHa) and 9.4 (MDHb). These forms had similar molecular weights (approx. 60 000) and apparent Km values, but showed a small but consistent difference in their pH optima (9.23 for MDHa and 9.05 for MDHb), and in their activation by inorganic phosphate (apparent Ka values of 33 mM and 87 mM, for MDHa and MDHb, respectively). Determination of the pH optima of the enzyme forms separated by isopycnic centrifugation suggests that the glycosomal enzyme form is MDHa, and the mitochondrial one is MDHb.

Regulation of biosynthesis of guanidinosuccinic acid in isolated rat hepatocytes and in vivo.

To clarify the metabolic pathway of guanidinosuccinic acid (GSA), we investigated the relationship between GSA synthesis and the urea cycle. In isolated rat hepatocytes, GSA formation increased as urea concentration was raised; the effect of urea was not modified by the addition of ammonium chloride. Other urea cycle intermediates, including arginine and cyanate, a degradation product of urea, failed to stimulate GSA synthesis. Ornithine and arginine, which stimulate urea synthesis, strongly inhibited urea-stimulated GSA synthesis in the presence of 10 mM ammonium chloride, but the inhibitory effect of ornithine was not observed when ammonium chloride was not present. Citrulline (5 mM) strongly inhibited urea-stimulated GSA synthesis with or without ammonium chloride. D,L-norvaline, which inhibits urea cycle enzymes, strongly inhibited GSA synthesis. Following urea injection, hepatic GSA levels also increased in vivo, but there was little change in hepatic arginine. However, the addition of ornithine or D,L-norvaline inhibited the production of hepatic GSA, although arginine was increased substantially. These results indicate that GSA synthesis occurs in rat hepatocytes and is stimulated by urea. The data also suggest that the urea cycle enzymes catalyze some of the biochemical reactions in the GSA synthetic pathway.

Two succinic semialdehyde dehydrogenases are induced when Escherichia coli K-12 Is grown on gamma-aminobutyrate.

When Escherichia coli K-12 was grown on gamma-aminobutyrate, a second succinic semialdehyde dehydrogenase, dependent upon oxidized nicotinamide adenine dinucleotide or oxidized nicotinamide adenine dinucleotide phosphate and distinct from that induced by gamma-aminobutyrate, was gratuitously induced by succinic semialdehyde.

Influence of heme and vitamin B12 on growth and fermentations of Bacteroides species.

We examined the effects of heme on the growth and fermentations of Bacteroides species. Bacteroides fragilis ATCC 25285 required heme for growth and produced malate and lactate as major products of glucose fermentation when the concentration of heme was 1 ng/ml. With 1 microgram of heme per ml, malate was not formed, lactate production decreased, and succinate and acetate were the major fermentation products. B. eggerthii ATCC 27754 grew without heme, with the production of mainly malate and lactate from glucose. Its fermentation with 1 microgram of heme per ml was similar to that of B. fragilis grown with the same concentration of heme. B. splanchicus VPI 6842 grew without heme, with the production of mainly malate, acetate, and H2 from glucose. With 1 microgram of heme per ml, malate disappeared, H2 decreased significantly, and succinate, acetate, and butyrate were the major products. The addition of vitamin B12 to media containing 1 microgram of heme per ml caused all species to produce propionate at the expense of succinate and, with B. splanchnicus, also at the expense of butyrate. Thus, the concentration of heme and the presence of vitamin B12 significantly influenced the course of glucose fermentation by these bacteria.