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PubMed1999

Inactivation and regulation of the aerobic C(4)-dicarboxylate transport (dctA) gene of Escherichia coli.

The gene (dctA) encoding the aerobic C(4)-dicarboxylate transporter (DctA) of Escherichia coli was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the dctA gene: f428 at 79.3 min and the o157a-o424-o328 (or orfQMP) operon at 79.9 min. The f428 gene encodes a homologue of the Sinorhizobium meliloti and Rhizobium leguminosarum H(+)/C(4)-dicarboxylate symporter, DctA, whereas the orfQMP operon encodes homologues of the aerobic periplasmic-binding protein- dependent C(4)-dicarboxylate transport system (DctQ, DctM, and DctP) of Rhodobacter capsulatus. To determine which, if either, of these loci specify the E. coli DctA system, the chromosomal f428 and orfM genes were inactivated by inserting Sp(r) or Ap(r) cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in the f428 mutant and succinate transport was approximately 10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the f428 mutant were complemented by transformation with an f428-containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to the dctA gene and indicate that the orfQMP products play no role in C(4)-dicarboxylate transport. Regulation studies with a dctA-lacZ (f428-lacZ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C(4)-dicarboxylates and citrate. Interestingly, in a dctA mutant, expression of dctA is constitutive with respect to C(4)-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic dctA transcript and confirmed that dctA is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.

Aerobiosis↗
PubMed2003

Involvement of cyclic AMP (cAMP) and cAMP receptor protein in anaerobic respiration of Shewanella oneidensis.

Shewanella oneidensis is a metal reducer that can use several terminal electron acceptors for anaerobic respiration, including fumarate, nitrate, dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nitrite, and insoluble iron and manganese oxides. Two S. oneidensis mutants, SR-558 and SR-559, with Tn5 insertions in crp, were isolated and analyzed. Both mutants were deficient in Fe(III) and Mn(IV) reduction. They were also deficient in anaerobic growth with, and reduction of, nitrate, fumarate, and DMSO. Although nitrite reductase activity was not affected by the crp mutation, the mutants failed to grow with nitrite as a terminal electron acceptor. This growth deficiency may be due to the observed loss of cytochromes c in the mutants. In contrast, TMAO reduction and growth were not affected by loss of cyclic AMP (cAMP) receptor protein (CRP). Fumarate and Fe(III) reductase activities were induced in rich medium by the addition of cAMP to aerobically growing wild-type S. oneidensis. These results indicate that CRP and cAMP play a role in the regulation of anaerobic respiration, in addition to their known roles in catabolite repression and carbon source utilization in other bacteria.

Anaerobiosis↗
PubMed2001

Myocardial metabolism of exogenous FDP is consistent with transport by a dicarboxylate transporter.

The extent to and the mechanism by which fructose-1,6-bisphosphate (FDP) crosses cell membranes are unknown. We hypothesized that its transport is either via band 3 or a dicarboxylate transporter. The question was addressed in isolated Langendorff rat hearts perfused under normoxic conditions. Groups of hearts received the following metabolic substrates (in mM): 5 FDP; 5 FDP + either 5, 10, or 20 fumarate; 10 FDP and either 5, 10, or 20 fumarate; or 5 FDP + 2 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), a band 3 inhibitor. FDP uptake and metabolism were measured as production of [(13)C]lactate from [(13)C]FDP or (14)CO(2) and [(14)C]lactate from uniformly labeled [(14)C]FDP in sample perfusates. During 30 min of perfusion, FDP metabolism was 12.4 +/- 2.6 and 31.2 +/- 3.0 micromol for 5 and 10 mM FDP, respectively. Addition of 20 mM fumarate reduced FDP metabolism over a 30-min perfusion period to 3.1 +/- 0.6 and 6.3 +/- 0.5 micromol for 5 and 10 mM FDP groups, respectively. DNDS did not affect FDP utilization. These data are consistent with transport of FDP by a dicarboxylate transport system.

Animals↗
PubMedDate not supplied

Enhanced biological phosphorus removal from wastewater by biomass with different phosphorus contents, Part II: Anaerobic adenosine triphosphate utilization and acetate uptake rates.

Data from laboratory-scale sequencing batch reactors operated in an anaerobic-aerobic cycle showed that a low influent phosphorus/chemical oxygen demand (COD) ratio feed favored a glycogen-accumulating metabolism (GAM)-dominated culture and that a high influent phosphorus/COD ratio feed favored a polyphosphate-accumulating metabolism (PAM)-dominated culture. The PAM-dominated culture anaerobically took up acetate approximately 7 times faster than the GAM-dominated culture. Adenosine triphosphate (ATP) balances were performed assuming eight different metabolic scenarios that included the Entner-Doudoroff or the Embden-Myerhof glycolytic pathway, acetyl-coenzyme A (CoA) synthase or the acetate kinase-phospho-transacetylase (AK-PTA) system for acetyl-CoA synthesis, and ATP synthesis or no ATP synthesis during fumarate reduction. The ATP available for transport of acetate into the cell (2) was calculated using these balances. The assumed quantity of ATP produced during fumarate reduction had a relatively small effect on alpha, particularly when PAM was dominant. When GAM was dominant, little or no ATP was available for acetate transport depending on the assumed scenario, and the Embden-Myerhof pathway was more feasible. The value of alpha increased with increasing PAM dominance for all eight metabolic pathways. The maximum calculated alpha value of 0.5 mol ATP/C-mol acetate uptake occurred at maximum PAM dominance and when the Embden-Myerhof pathway was active, when ATP was produced during fumarate reduction, and when the AK-PTA system was active. This value of alpha was higher than previously calculated values with the same metabolic assumptions. An acetate uptake mechanism was suggested that included acetyl-CoA synthetase and direct regeneration of the proton motive force by a proton-translocating pyrophosphatase. Polyphosphate-accumulating metabolism may have a competitive advantage over GAM through a higher anaerobic acetate uptake rate made possible by a greater use of energy for acetate uptake, by use of a different acetate uptake mechanism, or both.

Acetate-CoA Ligase↗
PubMed1992

Effect of feeding organic acids on selected intestinal content measurements at varying times postweaning in pigs.

Pigs weaned at 21 d of age (n = 72) were fed a 20% CP corn-soybean meal-based diet (control) with 1.5% fumaric or 1.5% citric acid added to observe the effect of these acids on the pH, chloride ion concentration (Cl-), VFA profile, and microflora population in the stomach, jejunum, cecum, and lower colon contents at -2, 0, 3, 7, 14, and 21 d postweaning. Feeding organic acids had no appreciable effect (P greater than .10) on stomach jejunum, cecum, or lower colon pH, Cl-, VFA profile, or microflora populations, except for increasing the concentration of fumaric acid in the stomachs of pigs fed fumaric acid. The pH of the gastrointestinal tract generally decreased from -2 to 21 d postweaning with no corresponding change in Cl- over time. No age effects on total anaerobic culture counts were observed except in the stomach, where counts decreased from -2 to 3 d postweaning. Clostridia counts generally decreased after weaning in all intestinal sections. Lactobacillus counts were usually lower at d 0 and 3 and greatest at d 7 postweaning in the stomach, jejunum, and lower colon, but no age effect was observed for concentration of cecum lactobacilli. Escherichia coli counts generally increased after weaning to 3 and 7 d postweaning. Intestinal content measurements were affected by postweaning age but were not affected by feeding organic acids.

Age Factors↗
PubMed1982

Effects of some metabolic intermediates on AMP-deaminase activity in denervation atrophy.

Effects in vitro of glutamate, aspartate, fumarate and malate on substrate activation kinetics of AMP-deaminase in denervated and contralateral gastrocnemius muscles of the frog, Rana hexadactyla were studied. Glutamate increase the enzyme activity of both muscles by increasing maximal velocity (V) and decreasing Michaelis-Menten constant (Km), whereas, aspartate did not show any significant effect. Fumarate and malate exerted mixed type of inhibition by decreasing V and increasing Km of enzymes in both muscle preparations. The inhibitor constant (Ki) values suggest that the denervated muscle enzyme was more sensitive to fumarate and malate as compared to the contralateral muscle enzyme.

AMP Deaminase↗
PubMed2001

The effect of change in pH on the solubility of iron bis-glycinate chelate and other iron compounds.

The effect of a pH change from 2 to 6 was tested on the solubility of ferrous sulfate, ferrous fumarate, iron bis-glycine chelate (Ferrochel) and sodium-iron ethylenediaminetetraacetic acid (NaFeEDTA). It was found that at pH 2 ferrous sulfate, Ferrochel and NaFeEDTA were completely soluble and only 75% of iron from ferrous fumarate was soluble. When pH was raised to 6, iron from amino acid chelate and NaFeEDTA remained completely soluble while solubility from ferrous sulfate and ferrous fumarate decreased 64 and 74%, respectively compared to the amount of iron initially soluble at pH 2. These results suggest that iron solubility from iron bis-glycine chelate and NaFeEDTA is not affected by pH changes within the ranges tested, probably because iron remained associated to the respective compounds.

Edetic Acid↗
PubMed1991

A comparative study of the effects of phebrol on the respiratory chains of rat liver, Biomphalaria glabrata and Oncomelania nosophora.

1. Phebrol (sodium 2,5-dichloro-4-bromophenol), a synthetic molluscicide against Oncomelania nosophora, showed a dual effect on rat liver submitochondria, acting as an uncoupler at low concentrations (approximately 10 microM) and an inhibitor of succinate-cytochrome c reductase at high concentrations. 2. Phebrol also inhibited the enzymes responsible for succinate-fumarate conversion, i.e. the succinate-cytochrome c reductase, fumarate reductase and NADH-cytochrome c reductase of the mitochondrial fraction from Biomphalaria glabrata. 3. Kinetic inhibition studies showed succinate-cytochrome c reductase of B. glabrata and O. nosophora to be more sensitive than that of rat liver toward phebrol. 4. Phebrol accumulated in whole tissues of B. glabrata and O. nosophora and had significant effects on the production of succinate, fumarate and malate by these snails. 5. On the basis of these results, the possible sites of inhibition by phebrol of snail respiratory chains are proposed.

Animals↗
PubMed1991

Cooperativity in the dopamine beta-monooxygenase reaction. Evidence for ascorbate regulation of enzyme activity.

The steady-state kinetic behavior of dopamine beta-monooxygenase (D beta M) has been examined over a 1000-fold range of ascorbate concentrations. Kinetic plots exhibit extreme curvature indicative of apparent negative cooperativity in the interaction of D beta M with ascorbate, with a calculated Hill coefficient of 0.15-0.30. The observed cooperativity is found to be independent of enzyme concentration and tyramine and oxygen concentrations, as well as the pH employed for the assay. Similar kinetic data have been obtained with both soluble and purified membrane-derived forms of enzyme. An investigation of the effect of the anion activator fumarate upon the observed kinetic patterns has demonstrated a conversion to a less cooperative kinetic pattern at low pH and high concentrations of fumarate. This phenomenon is attributed to an inhibitory binding of the structurally similar monoanionic species of fumarate to the ascorbate reductant site. A simple model has been used to assess the change in apparent Vmax and Km parameters with increased ascorbate concentrations. At all pH values examined, there is a dramatic decrease in the affinity of D beta M for ascorbate from a Km of approximately 0.05-0.10 mM (ascorbate concentration less than 1 mM) to Km greater than 10 mM at limiting ascorbate; at the same time there is a 3- to 4-fold increase in the limiting Vmax value. Several models have been considered to explain the observed activation of D beta M by high levels of ascorbic acid.

Adrenal Medulla↗
PubMed1987

[Changes in organic acids in plasma and cerebrospinal fluid in cerebral infarct].

Seventeen patients with recent cerebral infarction were included in the study. The general condition of the patients was evaluated within 3 days after the incidence (rating 1) and 7-9 days after the incidence (rating 2) using a clinical rating scale for internal, psychiatric and neurological findings. The statistical evaluation showed a clinical improvement of the score of 32%. The extent of the brain lesions was determined by cranial computed tomography. Routine analysis of the cerebrospinal fluid included determination of protein, cell count, cell type, glucose, lactic acid, and hemoglobin. In addition, spinal fluid and serum concentration of pyruvic acid, citric acid, fumaric acid, alpha-ketoglutaric acid and alpha-keto-beta-methylvaleric acid (3-MKV) were determined at the two rating times. CSF and serum values of fumaric acid, 3-MKV and pyruvic acid correlated at rating 1, while at rating 2 a correlation existed only for fumaric acid. A trend analysis infarction - postinfarction period showed the concentration of all metabolites to decrease significantly. This is interpreted as a reconstitution of the initially disturbed blood brain barrier function.

Aged↗
PubMed1997

Long-term prevention of atrial fibrillation after coronary artery surgery.

UNLABELLED: We researched the necessity of quinidine fumarate or acebutolol prophylaxis in patients in whom atrial fibrillation occurred in the postdischarge period and returned to sinus rhythm after coronary artery surgery. DESIGN: Prospective review. PATIENTS: Since 1992, 60 patients were chosen in whom atrial fibrillation occurred in early postoperative period. There were no significant differences between them and they were separated into 3 groups. In group I (20 patients) we did not give any drug, in group II (20 patients) quinidine fumarate was given and in group III (20 patients) acebutolol was given and patients were controlled for 90 days. RESULT: Atrial fibrillation occurred in one patient in group I, (5%), two in group II (10%) and two in group III (10%), (p < 0.05). Different from the other groups, atrial fibrillation was asymptomatic with low ventricular response in group III. CONCLUSIONS: There were no significant differences among three groups statistically, so we suggested that long-term prevention of atrial fibrillation with quinidine fumarate or acebutolol was not necessary after coronary artery surgery.

Acebutolol↗
PubMed1989

Effect of iron overload on spontaneous and xenobiotic-induced lipid peroxidation in vivo.

To study the effect of iron-overload on hepatic lipid peroxidation, two rat models of haemochromatosis were employed: in the first model resembling secondary haemochromatosis, repeated i.p. injections with Fe-dextran led to an accumulation of Fe in Kupffer cells, while in the second model resembling hereditary haemochromatosis, iron was located mainly in periportal hepatocytes after feeding on a diet containing 3.5% Fe-fumarate for 3 weeks. In both models, total hepatic iron content was elevated four- to fivefold over controls. In vivo lipid peroxidation (ethane exhalation) was enhanced only in the second model, indicating that the hepatocytes are the main targets of Fe-induced lipid peroxidation. Low hepatotoxicity was observed in the second model. Additional treatment of the rats with hepatotoxic agents led to different results: with ethanol and bromobenzene, lipid peroxidation was only evident in both models of iron-overload, while paracetamol-induced lipid peroxidation was seen only in Fe-fumarate-fed rats. CCl4-induced lipid peroxidation was strongly enhanced in both models of haemochromatosis. Hepatotoxicity was enhanced by iron overload only in the case of CCl4-treated, Fe-fumarate-fed rats. The activities of phase I and phase II enzymes of xenobiotic metabolism were not markedly altered in livers of iron-overloaded rats. This implies that neither the bioactivation nor the detoxification of the agents studied were affected in experimental haemochromatosis.

Alanine Transaminase↗
PubMed1986

Drug-excipient interactions resulting from powder mixing. IV: Role of lubricants and their effect on in vitro dissolution.

Two lubricants, magnesium stearate and sodium stearyl fumarate, were compared under identical mixing conditions to study their roles in drug-excipient interactions. After prolonged mixing, sodium stearyl fumarate did not interact with the drug or excipients; as a result, the disintegration time and drug dissolution rate from hand-filled, uncompacted capsules were not adversely affected. In contrast, magnesium stearate did exhibit drug-excipient interactions which resulted in lamination and subsequent adhesion of the lubricant to the drug-crospovidone agglomerates. These interactions adversely affected the disintegration time and drug dissolution rate from hand-filled, uncompacted capsules. Although the initial specific surface area of magnesium stearate was higher than that of sodium stearyl fumarate, flaking of magnesium stearate due to particulate-particulate interactions caused a large increase in the surface area. The adhesion of the magnesium stearate flakes to the drug-crospovidone agglomerates resulted in a decrease in the drug dissolution rate.

Chemistry, Pharmaceutical↗
PubMed1994

Peroxynitrite inactivates thiol-containing enzymes of Trypanosoma cruzi energetic metabolism and inhibits cell respiration.

Activated macrophages release peroxynitrite anion (ONOO-), which has been recently shown to be highly cytotoxic against Trypanosoma cruzi epimastigotes. In this work, we report that two critical enzymes for the energetic metabolism of the parasite, succinate dehydrogenase and fumarate reductase, are inactivated by biologically relevant concentrations of peroxynitrite. Enzyme inactivation was accompanied by a significant inhibition of succinate-dependent respiration in intact cells as well as in the membrane-rich fraction. Peroxynitrite also inhibited NADH-dependent oxygen consumption which depends almost exclusively on fumarate reductase activity in T. cruzi epimastigotes. Direct reactions of peroxynitrite anion with critical sulfhydryl residues of the two enzymes were responsible for most of the observed inactivation as indicated by the protection afforded by peroxynitrite scavengers and the reactivation of the enzymes by dithiothreitol. We propose that peroxynitrite-mediated inactivation of succinate dehydrogenase and fumarate reductase may be a key mechanism of macrophage-mediated cytotoxicity to T. cruzi, through inhibition of the energetic metabolism of the parasite.

Adenosine Triphosphatases↗
PubMed1996

Stabilization of the tetrameric structure of human and bovine hemoglobins by pseudocrosslinking with muconic acid.

In previous studies mono-3,5-dibromosalicyl-fumarate was used to introduce an intramolecular crosslink (pseudo-crosslink) in the beta cleft between hemoglobin beta subunits. Sedimentation velocity analysis indicated that the product had a mean molecular weight indicating a tetramer with low dissociability. The product had a P50 higher than that of native hemoglobin and a plasma retention time in the rat of about 3 h, i.e., four times longer than untreated hemoglobin. However, the product contained a fraction which was rapidly eliminated in the urine and which had a short plasma half-time of about 20 min, indicating the presence of a dissociable fraction. We have attempted to further enhance the tetrameric stability of hemoglobin and prevent urine elimination by positioning a longer chain carboxylic acid than fumaric acid into the beta cleft. We reason that a longer molecule would allow for greater stabilizing interactions across the beta cleft. In the present study human and bovine hemoglobins were reacted with mono-3-5-dibromosalicyl muconate. Muconic acid is two carbons longer than fumaric acid. The products were acylated at the beta 82 (human) and beta 81 (bovine) lysines of the beta-cleft and had a low degree of dissociability. For reasons not presently understood, urine excretion was high and plasma half-time was not increased above that of untreated hemoglobin. In conclusion, it appears that only covalently crosslinked hemoglobins which are completely nondissociable tetramers escape filtration; tetramers with any degree of dissociability into dimers are filterable.

Amino Acid Sequence↗
PubMed1998

Metabolic effects of citrate in liver during cold hypoxia studied by 1H NMR spectroscopy.

We propose the use of 1H nuclear magnetic resonance (NMR) spectroscopy to investigate metabolite fluxes in the mammalian liver during cold hypoxia. Rat livers were flushed with one of four different preservation solutions and stored on ice in the same solution. The preservation solutions were: Marshall's hypertonic citrate (HC); carnosine modified HC (HC-C); modified University of Wisconsin (mod UW); and Bretschneider's histidine--typtophan--ketoglutarate (HTK). Liver biopsies were taken before and at 1, 2.5. 4, 24, and 48 h after storage, and freeze-clamped. The liver was extracted with perchloric acid and analyzed by 1H NMR spectroscopy. Components of the individual preservation solutions, such as citrate, histidine, mannitol, and raffinose, were detected in the extracts. Lactate was increased over the first 4 h in all stored livers, but only continued to increase in those stored in HC-C and HTK, reaching significantly high levels of 15 and 14 mumol/g, respectively, by 48 h storage (P < 0.05 and P < 0.01, respectively). Levels of succinate and fumarate in all livers were generally unchanged in the first 0-4 h of storage. However, after 4 h of storage, succinate levels rose in the HC and HC-C livers, while remaining unchanged in mod UW and HTK livers. The presence of citrate in the preservation solutions appeared to enhance the late hepatic synthesis of succinate. Fumarate levels were significantly decreased by 48 h of cold storage, indicating continued fumarate consumption at low temperatures. Despite cold hypoxic conditions, some carbon-substrate cycling appears to continue in mammalian liver via pathways other than glycolysis, and citrate from the preservation solution appears to influence this.

Adenosine↗
PubMed1983

Biosynthesis of amino acids from sucrose and Krebs cycle metabolites by Rhizobium lupini bacteroids.

The possibility of amino acids biosynthesis from sucrose, metabolites of Krebs cycle or glyoxylate and ammonium by intact bacteroids has been studied. The suspension of intact Rhizobium lupini bacteroids in phosphate buffer solution pH 7.8 was shown to catalyse the biosynthesis from sucrose and ammonium of some amino acids, such as alanine, aspartic and glutamic acids, glycine and serine. The yield of alanine and aspartic acid was 2.5-3 times higher than that of other amino acids, which were formed in almost equal quantities. Intact bacteroids were also found to catalyse the biosynthesis of aspartic and glutamic acids, alanine and glycine from ammonium and Krebs cycle metabolites such as fumaric acid (FA), oxaloacetic acid (OAA), pyruvic acid (PA), alpha-ketoglutaric acid (alpha-KGA), malic acid (MA), as well as from glyoxylic acid (GOA). The biosynthesis of aspartic acid from fumaric acid was dominant. Besides that, the suspension of intact bacteroids catalysed transamination of aspartic and glutamic acids, the transamination of aspartic acid being especially intense with alpha-KGA and GOA. Aspartic acid was synthesized most efficiently through the amination of fumaric acid, while glutamic acid was better synthesized through the transamination of aspartic acid with alpha-KGA than through reductive amination of alpha-KGA. The experimental data proved that intact bacteroids possess Krebs cycle enzymes and primary ammonia assimilation enzymes. This enzyme complex permits bacteroids to detoxify ammonia, which they produce using sucrose and metabolites of Krebs cycle as the sources of carbon. The data obtained are of great interest as they prove the importance of bacteroids in the synthesis of amino acids from ammonium which is formed in the course of N2-fixation, and sucrose available from leaves.

Amino Acids↗
PubMed1976

Energy metabolism of some representatives of the Haemophilus group.

The purpose of this investigation was to characterize the carbohydrate catabolism and the constellation of the respiratory chain components of Haemophilus influenzae RAMC 18 Bensted, H. parainfluenzae 1 Fleming, H. parainfluenzae 429 Pittman and H. aegyptius 180a Pittman. These strains represent several physiological types with respect to respiratory quinones and glucose catabolism. On addition of glucose or lactate to the complex growth medium a remarkable increase in cell mass was observed. Depending on the growth rate, carbohydrate degradation varied with the strains examined so that at the end of the exponential growth phase only small amounts of the supplements could be demonstrated. All strains were found to possess functional enzymes of Embden-Meyerhof-Parnas-, Entner-Doudoroff-pathways, hexosemonophosphate shunt, tricarboxylic acid cycle and gluconeogenesis with an extremely high activity of malate dehydrogenase. The concentration of cytochromes varied according to culture conditions. The cytochromes a1, d, o and b + c were found to occur under aerobic conditions. In cells grown anaerobically in the presence of fumarate cytochromes a1 and d could not be demonstrated. Under aerobic conditions preparations of H. parainfluenzae 1 Fleming exhibited an alpha-maximum at 558 nm, whereas under anaerobic culture conditions with fumarate as terminal electron acceptor an alpha-maximum at 552 nm occurred, suggesting different roles of b and c type cytochromes in aerobic and anaerobic electron transport to fumarate, respectively.

Aerobiosis↗