Concentrations Of Fumarate Research Articles

Intracellular Succinylation of 8-Chloroadenosine and Its Effect on Fumarate Levels

8-Chloroadenosine (8-Cl-Ado) is a ribosyl nucleoside analog currently in phase I testing for the treatment of chronic lymphocytic leukemia (CLL). 8-Cl-Ado activity is dependent on adenosine kinase and requires intracellular accumulation of 8-Cl-Ado as mono-, di-, and tri-phosphates. In the current study with four mantle cell lymphoma cell lines, we report a new major metabolic pathway for 8-Cl-Ado intracellular metabolism, the formation of succinyl-8-chloro-adenosine (S-8-Cl-Ado) and its monophosphate (S-8-Cl-AMP). 8-Cl-AMP levels were highly associated with S-8-Cl-AMP levels and reached a steady-state prior to the secondary metabolites, 8-Cl-ATP and S-8-Cl-Ado. Consistent with fumarate as a required substrate for formation of succinyl-8-Cl-adenylate metabolites, the S-8-Cl-adenylate concentrations in multiple cell lines were associated with fumarate loss. The distribution of metabolites was also altered using the energy metabolism modifiers, metformin and oligomycin. The rates of succinyl-8-Cl-adenylate metabolism were enhanced by increasing the intracellular fumarate concentrations after metformin co-treatment. In addition, the S-8-Cl-AMP concentrations were increased after acute inhibition of ATP synthase by oligomycin. We conclude that 8-Cl-Ado metabolism not only affects intracellular purine metabolism; 8-Cl-Ado conversion to succinyl analogs ties its metabolism to the citric acid cycle by reduction of the fumarate pool.

Significant stimulation of o-phthalic acid in biosynthesis of aspergiolide A by a marine fungus Aspergillus glaucus

The effect of o-phthalic acid ( o-PA) on the production of the anti-tumor polyketide compound aspergiolide A by the marine fungus Aspergillus glaucus was investigated. o-PA at 12 mM increased the aspergiolide A production by 77%. A combination of 12 mM acetate with 12 mM o-PA increased the production by 126%, i.e., from 27.1 mg/l to 60.9 mg/l, which was 27% and 75% higher than adding either 12 mM o-PA or 12 mM acetate, respectively. It was also found that A. glaucus could use o-PA as the sole carbon source on Minimal Medium. Ninety percent of o-PA was degraded by the fungus strain, especially when 12 mM acetate was supplemented simultaneously. In culture medium supplemented with o-PA addition, sugar consumption decreased with increasing o-PA concentration, at the same time, the oxalacetate and pyruvate levels increased and the concentration of fumarate decreased. The results indicated that o-PA could stimulate biosynthesis of aspergiolide A effectively by regulating the metabolic pathway.

Fumaric acid diesters deprive cultured primary astrocytes rapidly of glutathione

Fumaric acid esters (FAE) are used for the systemic therapy of psoriasis and are now considered for the treatment of autoimmune-based neurological disorders such as multiple sclerosis. Currently, the cellular metabolism of FAE as well as the mechanisms of their therapeutic action are poorly understood. Since cellular glutathione (GSH) is involved in the detoxification of xenobiotics, we analysed the consequences of an application of FAE on the content of GSH in brain cells using astroglia-rich primary cultures as model system. Micromolar concentrations of dimethyl fumarate (DMF) or diethyl fumarate (DEF) lowered the cellular GSH content in a time- and concentration-dependent manner. Halfmaximal effects after 60min of incubation were observed for 10μM DMF or DEF. In contrast to the diesters, monomethyl fumarate (MMF), monoethyl fumarate (MEF) or fumarate had to be applied in concentrations of 10mM for 60min to significantly lower the cellular GSH content. During 60min exposure, DMF or DEF did not significantly affect the cell viability, increase the cellular content of glutathione disulfide, nor altered the specific activities of glucose-6-phosphate dehydrogenase, glutathione reductase, or lactate dehydrogenase. After removal of DMF or DEF, cultured astrocytes restored their cellular GSH content completely within 4h. These data demonstrate that acute exposure to fumaric acid diesters deprives astrocytes of their GSH, most likely by the reaction of the reactive α,β-unsaturated diesters with GSH.

Effects of fumarate on ruminal ammonia accumulation and fiber digestion in vitro and nutrient utilization in dairy does

The objective of this study was to evaluate effects of fumarate on ruminal ammonia accumulation and fiber digestion in vitro and on feed intake and nutrient utilization in dairy does. Batch cultures of mixed rumen microorganisms were used to study effects of different concentrations of fumarate on fermentation with various N sources (ammonia as ammonium bicarbonate, casein amino acids, casein peptides, gelatin peptides) and feeds (bermudagrass hay, mixed diet of 60% bermudagrass hay plus 40% concentrate) for 6 and 24h, respectively. Substrates were grouped into pairs for separate incubations. Monosodium fumarate was added to incubation tubes to achieve final concentrations of 0, 5, and 10mM fumarate. More ammonia accumulated at the end of incubation with added ammonium bicarbonate. Ammonia concentration was higher for peptide compared with amino acid incubation, and for casein peptide compared with gelatin peptide. Addition of fumarate linearly decreased ammonia for all N sources and for feed substrates. For all substrate types, fumarate treatment increased acetate, propionate, and total volatile fatty acids (VFA), decreased acetate to propionate ratio, and tended to reduce branched-chain VFA. Digestion of feed neutral detergent fiber (NDF) by rumen microorganisms was improved by fumarate along with elevated endoglucanase and xylanase activities. In an animal metabolism experiment, 8 dairy does (4 per treatment) were used in a completely randomized design for 21 d. Does were fed a hay plus concentrate diet without (control) or with fumarate (6 g/head per day) supplementation to determine feed intake, whole-tract nutrient digestibility, and N utilization. Fumarate treatment did not affect weight change or feed intake but increased whole-tract digestion of gross energy, crude protein, and cellulose. Digested N was increased by fumarate supplementation; however, N retention was unaffected. Plasma glucose concentration was elevated with fumarate but urea N concentration remained unchanged. Fumarate addition had significant effects on rumen microbial fermentation by decreasing ammonia and branched-chain VFA, and by increasing acetate and propionate, and NDF digestion. These effects were reflected in the improvement in whole-tract gross energy, crude protein, and cellulose digestion and elevated plasma glucose concentration when dairy does were supplemented with fumarate.

Alterations in Brain Glucose Utilization Accompanying Elevations in Blood Ethanol and Acetate Concentrations in the Rat

Previous studies in humans have shown that alcohol consumption decreased the rate of brain glucose utilization. We investigated whether the major metabolite of ethanol, acetate, could account for this observation by providing an alternate to glucose as an energy substrate for brain and the metabolic consequences of that shift. Rats were infused with solutions of sodium acetate, ethanol, or saline containing (13)C-2-glucose as a tracer elevating the blood ethanol (BEC) and blood acetate (BAcC) concentrations. After an hour, blood was sampled and the brains of animals were removed by freeze blowing. Tissue samples were analyzed for the intermediates of glucose metabolism, Krebs' cycle, acyl-coenzyme A (CoA) compounds, and amino acids. Mean peak BEC and BAcC were approximately 25 and 0.8 mM, respectively, in ethanol-infused animals. Peak blood BAcC increased to 12 mM in acetate-infused animals. Both ethanol and acetate infused animals had a lower uptake of (13)C-glucose into the brain compared to controls and the concentration of brain (13)C-glucose-6-phosphate varied inversely with the BAcC. There were higher concentrations of brain malonyl-CoA and somewhat lower levels of free Mg(2+) in ethanol-treated animals compared to saline controls. In acetate-infused animals the concentrations of brain lactate, alpha-ketoglutarate, and fumarate were higher. Moreover, the free cytosolic [NAD(+)]/[NADH] was lower, the free mitochondrial [NAD(+)]/[NADH] and [CoQ]/[CoQH(2)] were oxidized and the DeltaG' of ATP lowered by acetate infusion from -61.4 kJ to -59.9 kJ/mol. Animals with elevated levels of blood ethanol or acetate had decreased (13)C-glucose uptake into the brain. In acetate-infused animals elevated BAcC were associated with a decrease in (13)C-glucose phosphorylation. The co-ordinate decrease in free cytosolic NAD, oxidation of mitochondrial NAD and Q couples and the decrease in DeltaG' of ATP was similar to administration of uncoupling agents indicating that the metabolism of acetate in brain caused the mitochondrial voltage dependent pore to form.

Downregulation of Gnas, Got2 and Snord32a following tenofovir exposure of primary osteoclasts

Clinical observations have implicated the antiretroviral drug tenofovir with bone density loss during the management of HIV infection. The goal of this study was to investigate the in vitro effects of tenofovir exposure of primary osteoclasts in order to gain insights into the potential mechanisms for the drug-induced bone density loss. We hypothesized that tenofovir may alter the expression of key genes involved in osteoclast function. To test this, primary osteoclasts were exposed to physiologically relevant concentrations of the prodrug tenofovir disoproxil fumarate (TDF), then intensive microarray analysis was done to compare tenofovir-treated versus untreated cells. Specific downregulation of Gnas, Got2 and Snord32a were observed in the TDF-treated cells. The functions of these genes help to explain the basis for tenofovir-associated bone density loss. Our studies represent the first analysis of the effects of tenofovir on osteoclast gene expression and help to explain the basis of tenofovir-associated bone density loss in HIV-infected individuals.

Dipropionylcysteine Ethyl Ester Compensates for Loss of Citric Acid Cycle Intermediates During Post Ischemia Reperfusion in the Pig Heart

During reperfusion, following myocardial ischemia, uncompensated loss of citric acid cycle (CAC) intermediates may impair CAC flux and energy transduction. Propionate has an anaplerotic effect when converted to the CAC intermediate succinyl-CoA, and may improve contractile recovery during reperfusion. Antioxidant therapy with N-acetylcysteine decreases reperfusion injury. To synergize the antioxidant effects of cysteine with the anaplerotic effects of propionate, we synthesized a novel bi-functional compound, N,S-dipropionyl cysteine ethyl ester (DPNCE) and tested its anaplerotic and anti-oxidative capacity in anesthetized pigs. Ischemia was induced by a 70% reduction in left anterior descending coronary artery flow for one hour, followed by 1 h of reperfusion. After 30 min of ischemia and throughout reperfusion animals were treated with saline or intravenous DPNCE (1.5 mg x kg(-1) x min(-1), n = 8/group). Arterial concentrations and myocardial propionate, cysteine, free fatty acids, glucose and lactate uptakes, cardiac mechanical functions, myocardial content of CAC intermediates and oxidative stress were assessed. Ischemia resulted in reduction in myocardial tissue concentration of CAC intermediates. DPNCE treatment elevated arterial propionate and cysteine concentrations and myocardial propionate uptake, and increased myocardial concentrations of citrate, succinate, fumarate, and malate compared to saline treated animals. DPNCE treatment did not affect blood pressure or myocardial contractile function, but increased arterial free fatty acid concentration and myocardial fatty acid uptake. Arterial cysteine concentration was elevated by DPNCE, but there was negligible myocardial cysteine uptake, and no change in markers of oxidative stress. DPNCE elevated arterial cysteine and propionate, and increased myocardial concentration of CAC intermediates, but did not affect mechanical function or oxidative stress.

Metabolic viability assessment of cystic echinococcosis using high‐field 1H MRS of cyst contents

Cystic echinococcosis is a worldwide disease caused by larval stages of the parasite Echinococcus granulosus (canine tapeworm). In clinical practice, staging of cyst development by ultrasonography (US) has allowed treatment options to be tailored to individual patient needs. However, the empirical correlation between cyst morphology and parasite viability is not always dependable and has, until now, required confirmation by invasive assessment of cyst content by light microscopy (LM), for example. Alternatively, high-field 1H MRS may be used to examine cyst fluid ex vivo and prepare detailed quantitative metabolite profiles, enabling a multivariate metabolomics approach to cyst staging. One-dimensional and two-dimensional 1H and 1H/13C MRS at 600 MHz (14.1 T) was used to analyze 50 cyst aspirates of various US and LM classes. MR parameters and concentrations relative to internal valine were determined for 44 metabolites and four substance classes. The high concentrations of succinate, fumarate, malate, acetate, alanine, and lactate found in earlier studies of viable cysts were confirmed, and additional metabolites such as myo-inositol, sorbitol, 1,5-anhydro-D-glucitol, betaine, and 2-hydroxyisovalerate were identified. Data analysis and cyst classification were performed using univariate (succinate), bivariate (succinate vs fumarate), and multivariate partial least squares discriminant analysis (PSL-DA) methods (with up to 48 metabolite variables). Metabolic classification of 23 viable and 18 nonviable cysts on the basis of succinate alone agreed with LM results. However, for seven samples, LM and MRS gave opposing results. Reclassification of these samples and two unclassified samples by PLS-DA prediction techniques led to a set of 50 samples that could be completely separated into viable and nonviable MRS classes with no overlap, using as few as nine variables: succinate, formate, malate, 2-hydroxyisovalerate, acetate, total protein content, 1,5-anhydro-D-glucitol, alanine, and betaine. Thus, future noninvasive in vivo applications of MRS would appear promising.

Composition of organic solutes and respiration in soils derived from alkaline and non-alkaline parent materials

Parent material greatly influences pedogenesis and soil nutrient availability and consequently we hypothesized that it would significantly affect the amount of organic solutes in soil, many of which have been implicated in rhizosphere processes linked to plant nutrient uptake. Consequently, we investigated the influence of two contrasting parent materials in which calcite was present or absent (alkaline and non-alkaline soils) on the concentrations of dissolved organic carbon (DOC), low-molecular weight organic acids (LMWOA) and glucose in soil solution. Both soils were under Norway spruce. The dynamics of LMWOAs in soil were also investigated using 14C-labelled citrate and oxalate. Some of the mineral horizons of the alkaline soils showed significantly higher concentrations of DOC, phenolics, and fumarate in soil solution and also a higher basal respiration. No major differences were seen in organic solute status in the organic horizons of the two soil types. LMWOAs were present at low concentrations in soil solution (< 1 to 25 µM). Their mineralization rate significantly decreased with soil depth, however, overall neither their concentration or half-life in soil was markedly affected by parent material. The alkaline soils had significantly higher CO 2-to-soil organic C (SOC) ratios, and consequently SOC in the alkaline soils did not seem more chemically stable against mineralization. Considering possible DOC and CO 2 efflux rates it was suggested that the equal or larger SOC stocks in alkaline mineral soils were most likely linked to a higher net primary productivity. In conclusion, our study found that parent material exerted only a small effect on the concentration and dynamics of organic solutes in soil solution. This suggests that in comparison to other factors (e.g. vegetation cover, climate etc) parent material may not be a major regulator of the organic solute pool in soil.

Succination of Protein Thiols during Adipocyte Maturation

Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased > or =5-fold during adipogenesis in medium containing 30 mm glucose, producing a > or =10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one- and two-dimensional electrophoresis and approximately 60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.

A meta-analysis of fumarate effects on methane production in ruminal batch cultures

The objective of this study was to understand the effects of fumarate addition on methane (CH4) and VFA production in the rumen through a meta-analysis of its effects on ruminal batch cultures. Because the reduction of fumarate to succinate can draw electrons away from ruminal methanogenesis, fumarate has been studied as a potential feed additive to decrease CH4 production in ruminants. The average decrease in CH4 in batch cultures was of 0.037 micromol/micromol of added fumarate, which is considerably lower than 0.25 micromol/micromol, the decrease predicted from the stoichiometry of the reactions involved. One reason that fumarate was not effective at decreasing CH4 in batch cultures was that only an average of 48% of added fumarate appeared to be converted to propionate. Secondly, the incorporation of reducing equivalents in the conversion of fumarate to propionate was almost entirely offset by their release from an average of 20% of added fumarate that appeared to be converted to acetate. Thermodynamic calculations indicated that the conversion of added fumarate to both propionate and acetate was feasible. Fumarate appears to be more effective in decreasing CH4 production and increasing propionate in continuous culture than in batch culture. This suggests that microbial adaptation to fumarate metabolism can be important. Variation in populations of fumarate-reducers, methanogens, and protozoa could all be involved. Fumarate supplementation for an extended period may result in the amplification of otherwise small populations of fumarate-reducers. Addition of some of these organisms may be helpful to improve fumarate conversion to propionate. Strategies based on enhancing the rumen's capacity to convert fumarate to propionate by maintaining a low fumarate concentration have been effective. Thermodynamic considerations should be taken into account when designing strategies for CH4 abatement through the addition of external electron acceptors.

Redirecting metabolic fluxes through cofactor engineering: Role of CoA-esters pool during l(−)-carnitine production by Escherichia coli

Cofactor engineering, defined as the purposeful modification of the pool of intracellular cofactors, has been demonstrated to be a very suitable strategy for the improvement of l(−)-carnitine production in Escherichia coli strains. The overexpression of CaiB (CoA-transferase) and CaiC (CoA-ligase), both enzymes involved in coenzyme A transfer and substrate activation during the bioprocess, led to an increase in l(−)-carnitine production. Under optimal concentrations of inducer and fumarate (used as electron acceptors) yields reached 10- and 50-fold, respectively, that obtained for the wild type strain. However, low levels of coenzyme A limited the activity of these two enzymes since the addition of pantothenate increased production. Growth on substrates whose assimilation yields acetyl-CoA (such as acetate or pyruvate) further inhibited l(−)-carnitine production. Interestingly, control steps in the metabolism of acetyl-CoA of E. coli were detected. The glyoxylate shunt and anaplerotic pathways limit the bioprocess since strains carrying deletions of isocitrate lyase and isocitrate dehydrogenase phosphatase/kinase yielded 20–25% more l(−)-carnitine than the control. On the other hand, the deletion of phosphotransacetylase strongly inhibited the bioprocess, suggesting that an adequate flux of acetyl-CoA and the connection of the phosphoenolpyruvate–glyoxylate cycle together with the acetate metabolism are crucial for the biotransformation.

Causes of metabolic acidosis in canine hemorrhagic shock: role of unmeasured ions

IntroductionMetabolic acidosis during hemorrhagic shock is common and conventionally considered to be due to hyperlactatemia. There is increasing awareness, however, that other nonlactate, unmeasured anions contribute to this type of acidosis.MethodsEleven anesthetized dogs were hemorrhaged to a mean arterial pressure of 45 mm Hg and were kept at this level until a metabolic oxygen debt of 120 mLO2/kg body weight had evolved. Blood pH, partial pressure of carbon dioxide, and concentrations of sodium, potassium, magnesium, calcium, chloride, lactate, albumin, and phosphate were measured at baseline, in shock, and during 3 hours post-therapy. Strong ion difference and the amount of weak plasma acid were calculated. To detect the presence of unmeasured anions, anion gap and strong ion gap were determined. Capillary electrophoresis was used to identify potential contributors to unmeasured anions.ResultsDuring induction of shock, pH decreased significantly from 7.41 to 7.19. The transient increase in lactate concentration from 1.5 to 5.5 mEq/L during shock was not sufficient to explain the transient increases in anion gap (+11.0 mEq/L) and strong ion gap (+7.1 mEq/L), suggesting that substantial amounts of unmeasured anions must have been generated. Capillary electrophoresis revealed increases in serum concentration of acetate (2.2 mEq/L), citrate (2.2 mEq/L), α-ketoglutarate (35.3 μEq/L), fumarate (6.2 μEq/L), sulfate (0.1 mEq/L), and urate (55.9 μEq/L) after shock induction.ConclusionLarge amounts of unmeasured anions were generated after hemorrhage in this highly standardized model of hemorrhagic shock. Capillary electrophoresis suggested that the hitherto unmeasured anions citrate and acetate, but not sulfate, contributed significantly to the changes in strong ion gap associated with induction of shock.

Effects of tributyltin on the energy metabolism of pen shell ( Atrina pectinata japonica)

We examined the effects of tributyltin (TBT) on aerobic and anaerobic energy metabolism of pen shell ( Atrina pectinata japonica). We exposed pen shells to TBT at nominal concentrations of 0 (control) and 1.0 μg/l for 72 h under aerobic condition. At the end of the exposure, half of the pen shells in each treatment were wrapped in plastic wrap to simulate exposure to hypoxia and held at 25 °C for another 12 h. The concentrations of the products of energy metabolism, namely lactate, pyruvate, fumarate and succinate, in adductor muscle were measured. The exposure to TBT under aerobic condition significantly elevated lactate, pyruvate and fumarate concentrations ( p < 0.001). After subsequent exposure to anaerobic condition, the mean concentration of succinate in the TBT treatment group was 64% of that in the control group, but there were no significant differences. Our results suggest that the energy metabolism of pen shell is disrupted by exposure to TBT.

Dipropionylcysteine ethyl ester compensates for the leakage of citric acid cycle intermediates in the pig heart with ischemia/reperfusion.

During post-ischemic reperfusion uncompensated cataplerosis disrupts the proper operation of citric acid cycle and impairs energy production. Conventional anti-oxidative therapy utilizing N-acetylcysteine has limited success in organ reperfusion injury. To synergize an anti-oxidative capacity of cysteine with anaplerotic propionate, we synthesized a novel bi-functional compound, namely N,S-dipropionyl cysteine ethyl ester (DPrNCE). We tested DPrNCE on an anesthetized, open-chest low-flow-ischemia pig model, which was induced by 70%-reduction in LAD coronary artery flow for 1 hour, followed by 1 hour of reperfusion. Animals were either untreated (n=8), or treated with intravenous DPrNCE (1.5 mg/kg−1min−1, n=8) after 30 min. of flow reduction and continued throughout reperfusion. The treated animals had elevated propionate arterial levels and increased myocardial concentrations of succinate, fumarate, and malate, compared to untreated control. No difference was observed between the 2 groups with regards to blood pressure, heart rate, regional wall motion, and total glutathione concentration. In conclusion, an intravenous infusion of DPrNCE safely increases myocardial tissue contents of citric acid cycle intermediates and arterial concentration of propionate without any effect on cardiac mechanical function. Supported by NIH Roadmap Grant R33DK070291, and AHA Grant # 0465221.

Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme)

Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the K(m) values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate K(m) values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The K(act) values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.

Transcriptional Expression of the tceA Gene in a Dehalococcoides- Containing Microbial Enrichment

Dynamic changes in the transcriptional expression of the tceA gene, which encodes a trichloroethene reductive dehalogenase, were characterized in a Dehalococcoides-containing microbial enrichment culture. Expression was quantified by real-time PCR as the number of tceA transcripts per tceA gene. Expression of tceA increased 40-fold after chlorinated ethene-starved cells were exposed to trichloroethene (TCE), cis-dichloroethene (DCE), or 1,1-DCE but did not increase after exposure to tetrachloroethene or vinyl chloride. Surprisingly, tceA expression also increased 30-fold after cellular exposure to the nonmetabolic substrate trans-DCE, indicating that expression of tceA is induced by both growth-supporting and non-growth-supporting chlorinated ethenes. Additional experiments revealed that the level of tceA expression was independent of the concentration of chlorinated ethenes (sum concentrations of TCE and DCEs of 2.2 to 333 microM), the concentration of the electron donor hydrogen (concentrations of 12 nM to 17 microM), and the presence of alternate bacterial electron acceptors (5 mM concentrations of fumarate, sulfate, sulfite, thiosulfate, nitrate, or nitrite) but was highly dependent on incubation temperature.

High-performance liquid chromatography-based methods of enzymatic analysis: electron transport chain activity in mitochondria from human skeletal muscle

This study addresses an application of pyridine nucleotide enzymatic analyses to evaluate the activity of the mitochondrial electron transport chain (reduced nicotinamide adenine dinucleotide (NADH) oxidase) and Complexes I and II in samples of human muscle as small as ∼10 mg wet weight. Key aspects in this adaptation are the use of high-performance liquid chromatography with fluorescence detection of NADH and use of alamethicin, a channel-forming antibiotic that enables an unrestricted access of substrates into the mitochondrial matrix. The procedure includes disintegration of tissue by Polytron homogenizer, extraction of myosin from myofibrillar fragments by KCl/pyrophosphate to facilitate release of mitochondria, and preparation of fractions of subsarcolemmal and intermyofibrillar mitochondria. Oxidation of NADH or succinate is assayed in the presence of 40 μg/ml alamethicin and the reaction is terminated by H 2SO 4, which also destroys the remaining NADH. Nicotinamide adenine dinucleotide (NAD) or fumarate concentrations are measured using alcohol dehydrogenase or fumarase plus malic dehydrogenase reactions, respectively. Generation of NADH, assessed in auxiliary reactions in the presence of hydrazine, is strictly proportional to NAD or fumarate content across a concentration range of 1–20 μM. NADH is quantitatively analyzed with a detection limit of 3–5 pmol by HPLC using a reverse-phase Hypersil ODS column connected to a fluorescence detector.

Influence of different concentrations of disodium fumarate on methane production and fermentation of concentrate feeds by rumen micro-organisms in vitro.

Batch cultures of mixed rumen micro-organisms were used to study the effects of different concentrations of disodium fumarate on the fermentation of five concentrate feeds (maize, barley, wheat, sorghum and cassava meal). Rumen contents were collected from four Merino sheep fed lucerne hay ad libitum and supplemented with 300 g concentrate/d. Disodium fumarate was added to the incubation bottles to achieve final concentrations of 0, 4, 7 and 10 mM-fumarate. In 17 h incubations, the final pH and total volatile fatty acid production increased (P<0.001) linearly for all substrates as fumarate concentration increased from 0 to 10 mm. Propionate and acetate production increased (P<0.05), while the value of the acetate:propionate ratio decreased (P<0.05) linearly with increasing doses of fumarate. In contrast, l-lactate and NH3-N concentrations in the cultures were not affected (P>0.05) by the addition of fumarate. For all substrates, fumarate treatment decreased (P<0.05) CH4 production, the mean values of the decrease being 2.3, 3.8 and 4.8 % for concentrations of 4, 7 and 10 mM-fumarate respectively. Addition of fumarate did not affect (P>0.05) the total gas production. If the results of the present experiment are confirmed in vivo, fumarate could be used as a feed additive for ruminant animals fed high proportions of cereal grains, because it increased pH, acetate and propionate production and it decreased CH4 production.

Transmembrane structure and function ofdctPQM encoding C4-dicarboxylate transport proteins from nitrogen-fixingP. stutzeri A1501

C4-dicarboxylate transport proteins of diazotrophPseudomonas stutzen were encoded bydctPQM genes. Nucleotide sequence analysis indicated thatdctP, dctQ, anddctM grouped together. Its nucleotide and amino acid sequence shared high homology with that ofdctP gene encoding periplasmic C4-dicarboxylate-binding protein anddctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixingAotobacter vinelandii. Structural analysis showed that DctP ofP. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively. The fragment containing the completedctPQM genes was cloned into the Tn5 transposon region of suicide mobilization plasmid pSZ21. The resultant plasmid was named pSZY6. By triparental mating, Tn5 transposon carrying thedctPQM genes inserted into the genome of the wild type strain A1501, randomly. The recombinant strain A-142 which harboured an extra copy ofdctPQM genes was constructed and identified by PCR amplification ofnpt II gene. When A-142 was grown in minimal medium with different concentrations (20, 10 and 5 mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A1501. This result was established that an extra copy ofdctPQM genes could increase the activity of nitrogen fixation ofP. stutzeri strain A1501.