Inhibitor Of Tricarboxylic Acid Cycle Research Articles

The trehalose-6-phosphate synthase TPS5 negatively regulates ABA signaling in Arabidopsis thaliana.

The TPS5 negatively regulates ABA signaling by mediating ROS level and NR activity during seed germination and stomatal closure in Arabidopsis thaliana. Trehalose metabolism is important in plant growth and development and in abiotic stress response. Eleven TPS genes were identified in Arabidopsis, divided into Class I (TPS1-TPS4) and Class II (TPS5-TPS11). Although Class I has been shown to have TPS activity, the function of most members of Class II remains enigmatic. Here, we characterized the biological function of the trehalose-6-phosphate synthase TPS5 in ABA signaling in Arabidopsis. TPS5 expression was induced by ABA and abiotic stress, and expression in epidermal and guard cells was dramatically increased after ABA treatment. Loss-of-function analysis revealed that tps5 mutants (tps5-1 and tps5-cas9) are more sensitive to ABA during seed germination and ABA-mediated stomatal closure. Furthermore, the H2O2 level increased in the tps5-1 and tps5-cas9 mutants, which was consistent with the changes in the expression of RbohD and RbohF, key genes responsible for H2O2 production. Further, TPS5 knockout reduced the amounts of trehalose and other soluble carbohydrates as well as nitrate reductase (NR) activity. In vitro, trehalose and other soluble carbohydrates promoted NR activity, which was blocked by the tricarboxylic acid cycle inhibitor iodoacetic acid. Thus, this study identified that TPS5 functions as a negative regulator of ABA signaling and is involved in altering the trehalose content and NR activity.

Edwardsiella tarda Tunes Tricarboxylic Acid Cycle to Evade Complement-Mediated Killing.

Evasion of complement-mediated killing is a common phenotype for many different types of pathogens, but the mechanism is still poorly understood. Most of the clinic isolates of Edwardsiella tarda, an important pathogen infecting both of human and fish, are commonly found serum-resistant. To explore the potential mechanisms, we applied gas chromatography-mass spectrometry (GC-MS)-based metabolomics approaches to profile the metabolomes of E. tarda EIB202 in the presence or absence of serum stress. We found that tricarboxylic acid (TCA) cycle was greatly enhanced in the presence of serum. The quantitative real-time PCR (qRT-PCR) and enzyme activity assays validated this result. Furthermore, exogenous succinate that promotes the TCA cycle increased serum resistance, while TCA cycle inhibitors (bromopyruvate and propanedioic acid) that inhibit TCA cycle, attenuated serum resistance. Moreover, the enhanced TCA cycle increased membrane potential, thus decreased the formation of membrane attack complex at cell surface, resulting serum resistance. These evidences suggested a previously unknown membrane potential-dependent mechanism of serum resistance. Therefore, our findings reveal that pathogen mounts a metabolic trick to cope with the serum complement-mediated killing.

Poly-3-hydroxybutyrate production from methanol by Methylosinus trichosporium IMV3011 in the non-sterilized fed-batch fermentation

The methanotrophic bacteria have promising applications in the production of a biopolymer, poly-3-hydroxybutyrate (PHB). The fed-batch fermentation strategies were studied for PHB accumulation by methanol-grown Methylosinus trichosporium IMV3011 under the non-sterilized conditions. The biomass of 2.91 g dry wt/L and PHB accumulation of 47.6% (w/w) were reached in the fed-batch fermentation. In order to increase PHB content, some organic acids were added into the culture as inhibitors of tricarboxylic acid cycle (TCA). It was found that the malic acid should be optimum for PHB accumulation. The biomass and PHB yield would increase to 3.32 g dry wt/L and 58.5% (w/w), respectively by adding 0.2 g/L malic acid. Other key intermediates in the metabolism such as citric acid, succinic acid and acetyl-CoA, also influenced the cell growth and PHB accumulation to a certain extent. The PHB produced in the fermentation process was of steady quality with molecular weights up to 1.7 × 106 Da. Key words: Biosynthesis, poly-3-hydroxybutyrate, methanotrophic bacteria, methanol, fed-batch fermentation.

Biosynthesis of poly-3-hydroxybutyrate with a high molecular weight by methanotroph from methane and methanol

Poly-3-hydroxybutyrate (PHB) can be produced by various species of bacteria. Among the possible carbon sources, both methane and methanol could be a suitable substrate for the production of PHB. Methane is cheap and plentiful not only as natural gas, but also as biogas. Methanol can also maintain methanotrophic activity in some conditions. The methanotrophic strain Methylosinus trichosporium IMV3011 can accumulate PHB with methane and methanol in a brief nonsterile process. Liquid methanol (0.1%) was added to improve the oxidization of methane. The studies were carried out using shake flasks. Cultivation was performed in two stages: a continuous growth phase and a PHB accumulation phase under the conditions short of essential nutrients (ammonium, nitrate, phosphorus, copper, iron (III), magnesium or ethylenediamine tetraacetate (EDTA)) in batch culture. It was found that the most suitable growth time for the cell is 144 h. Then an optimized culture condition for second stage was determined, in which the PHB concentration could be much increased to 0.6 g/L. In order to increase PHB content, citric acid was added as an inhibitor of tricarboxylic acid cycle (TCA). It was found that citric acid is favorable for the PHB accumulation, and the PHB yield was increased to 40% (w/w) from the initial yield of 12% (w/w) after nutrient deficiency cultivation. The PHB produced is of very high quality with molecular weight up to 1.5×10 6Da.

Propionate increases neuronal histone acetylation, but is metabolized oxidatively by glia. Relevance for propionic acidemia

In propionic acidemia, propionate acts as a metabolic toxin in liver cells by accumulating in mitochondria as propionyl-CoA and its derivative, methylcitrate, two tricarboxylic acid cycle inhibitors. Little is known about the cerebral metabolism of propionate, although clinical effects of propionic acidemia are largely neurological. We found that propionate was metabolized oxidatively by glia: [3-(14)C]propionate injected into mouse striatum or cortex, gave a specific activity of glutamine that was 5-6 times that of glutamate, indicating metabolism in cells that express glutamine synthetase, i.e., glia. Further, cultured cerebellar astrocytes metabolized [3-(14)C]propionate; cultured neurons did not. However, both cultured cerebellar neurons and astrocytes took up [3H]propionate, and propionate exposure increased histone acetylation in cultured neurons and astrocytes as well as in hippocampal CA3 pyramidal neurons of wake mice. The inability of neurons to metabolize propionate may be due to lack of mitochondrial propionyl-CoA synthetase activity or transport of propionyl residues into mitochondria, as cultured neurons expressed propionyl-CoA carboxylase, a mitochondrial matrix enzyme, and oxidized isoleucine, which becomes converted into propionyl-CoA intramitochondrially. The glial metabolism of propionate suggests astrocytic vulnerability in propionic acidemia when intramitochondrial propionyl-CoA may accumulate. Propionic acidemia may alter both neuronal and glial gene expression by affecting histone acetylation.

Measurement of the Energy-Generating Capacity of Human Muscle Mitochondria: Diagnostic Procedure and Application to Human Pathology

Diagnosis of mitochondrial disorders usually requires a muscle biopsy to examine mitochondrial function. We describe our diagnostic procedure and results for 29 patients with mitochondrial disorders. Muscle biopsies were from 43 healthy individuals and 29 patients with defects in one of the oxidative phosphorylation (OXPHOS) complexes, the pyruvate dehydrogenase complex (PDHc), or the adenine nucleotide translocator (ANT). Homogenized muscle samples were used to determine the oxidation rates of radiolabeled pyruvate, malate, and succinate in the absence or presence of various acetyl Co-A donors and acceptors, as well as specific inhibitors of tricarboxylic acid cycle or OXPHOS enzymes. We determined the rate of ATP production from oxidation of pyruvate. Each defect in the energy-generating system produced a specific combination of substrate oxidation impairments. PDHc deficiencies decreased substrate oxidation reactions containing pyruvate. Defects in complexes I, III, and IV decreased oxidation of pyruvate plus malate, with normal to mildly diminished oxidation of pyruvate plus carnitine. In complex V defects, pyruvate oxidation improved by addition of carbonyl cyanide 3-chlorophenyl hydrazone, whereas other oxidation rates were decreased. In most patients, ATP production was decreased. The proposed method can be successfully applied to the diagnosis of defects in PDHc, OXPHOS complexes, and ANT.

Oxidative phosphorylation and the tricarboxylic acid cycle are essential for normal development of mouse ovarian follicles

Mouse ovarian follicles are typically grown in upright drops of culture medium. Recently we found that culture of follicles at the medium-gas interface in inverted drops markedly improved follicular development, possibly due to improved access of oxygen to the follicle. In this study, we examined the importance of aerobic energy metabolism for follicle development by culturing mouse follicles (198 6 16.5 initial microm diameter, mean 6 SD) in the presence of phosphorylation and tricarboxylic acid (TCA) cycle inhibitors. All inhibitors were tested in the inverted system using 100 microl medium drops in 96-well plates; certain inhibitors were also tested in upright drops with or without an oil overlay. The oxidative phosphorylation inhibitor rotenone (0.1, 0.5 and 1 micromol/l) totally abolished follicle growth in the inverted system; cyanide (1 mmol/l) totally abolished growth in the upright with oil system but not in the inverted system (possibly due to loss of cyanide gas due to the absence of an oil overlay). The mitochondrial uncoupler 2,4-dinitrophenol (0.5 and 1 mmol/l) also abolished growth in the inverted system. The TCA cycle inhibitor monofluoroacetate (10 mmol/l), significantly inhibited growth in all three culture systems (P < 0.01) but malonate (10 mmol/l) had no effect. Aerobic metabolism and an adequate oxygen supply are essential for normal follicular development.

Staphylococcus epidermidis Polysaccharide Intercellular Adhesin Production Significantly Increases during Tricarboxylic Acid Cycle Stress

Staphylococcal polysaccharide intercellular adhesin (PIA) is important for the development of a mature biofilm. PIA production is increased during growth in a nutrient-replete or iron-limited medium and under conditions of low oxygen availability. Additionally, stress-inducing stimuli such as heat, ethanol, and high concentrations of salt increase the production of PIA. These same environmental conditions are known to repress tricarboxylic acid (TCA) cycle activity, leading us to hypothesize that altering TCA cycle activity would affect PIA production. Culturing Staphylococcus epidermidis with a low concentration of the TCA cycle inhibitor fluorocitrate dramatically increased PIA production without impairing glucose catabolism, the growth rate, or the growth yields. These data lead us to speculate that one mechanism by which staphylococci perceive external environmental change is through alterations in TCA cycle activity leading to changes in the intracellular levels of biosynthetic intermediates, ATP, or the redox status of the cell. These changes in the metabolic status of the bacteria result in the attenuation or augmentation of PIA production.

The Participation of Energy Substrates in the Control of Meiotic Maturation in Murine Oocytes

In this study we have examined the effects of various energy substrate combinations on both the maintenance of meiotic arrest and follicle-stimulating hormone (FSH)-induced germinal vesicle breakdown (GVB) in isolated mouse oocytes treated with 4 mM hypoxanthine (HX). When cumulus cell-enclosed oocytes were cultured in HX minus glucose, the addition of pyruvate dose-dependently sustained oocyte viability and induced GVB (from 25% GVB at 0 mM pyruvate to 98% GVB at 1 mM pyruvate). FSH had a limited stimulatory effect at all pyruvate concentrations. Oxaloacetate, but no other intermediates of the tricarboxylic acid (TCA) cycle, mimicked the action of pyruvate on viability and GVB. Lactate alone induced maturation in the absence of pyruvate but required NAD for this effect when the medium included pyruvate. Of two TCA cycle inhibitors tested, malonate had no effect on pyruvate-induced GVB, and fluoroacetate was inhibitory only at the highest concentration (10 mM). Pyruvate, oxaloacetate, and lactate also stimulated maturation in denuded oocytes. Adding glucose back to the medium produced an inhibitory effect on meiosis in cumulus cell-enclosed oocytes up to a concentration of 23 mM and was associated with increases in ATP. Exposure to the glycolytic inhibitor, iodoacetate, eliminated both the meiotic arrest and ATP production mediated by glucose. High concentrations of glucose (111 mM) proved stimulatory to oocyte maturation. Glucose had no effect on denuded oocytes. FSH-induced maturation was dependent upon the presence of glucose but was not causally related to increases in lactate production. In addition, raising the pyruvate concentration 10-fold did not overcome the meiotic block. Coculture of denuded oocytes with dissociated cumulus cells, but not intact oocyte-cumulus cell complexes, stimulated GVB in HX-containing medium when glucose was added. However, both the cumulus cells and the complexes produced an increase in pyruvate production, indicating that the effect of the dissociated cumulus cells was not due principally to secretion of this carbohydrate into the culture medium. This study has demonstrated the importance of energy substrates in the meiotic response of oocytes in culture. Evidence failed to support a role for increased TCA cycle activity in the stimulatory action of pyruvate. The inhibitory effect of glucose is the result of glycolytic activity that produces increased levels of ATP, but production of ATP and glycolytic intermediates does not appear to be the mechanism whereby glucose mediates a stimulatory action on meiosis, either in response to high glucose levels or FSH treatment. Other avenues of glucose metabolism or interaction may be responsible for this effect.

Influence of metabolic and physical factors on production of diacetoxyscirpenol by Fusarium sambucinum Fuckel.

Fusarium sambucinum Fuckel 8099-1 was grown on Czapek-Dox peptone-supplemented medium at 15 degrees C for 14 days, and the cultures were investigated for diacetoxyscirpenol (DAS) production by liquid-liquid extraction and gas chromatography. The addition of 150 mg of sorbic acid, a tricarboxylic acid cycle inhibitor, per liter stimulated both fungal growth and DAS production. Among the beta-hydroxy-beta-methylglutaryl coenzyme A precursors tested, isovaleric acid completely inhibited fungal growth and DAS production, ethyl isovalerate did not support a significant increase in DAS production, and L-leucine partially inhibited DAS production, showing that L-leucine and isovaleric acid catabolisms do not induce trichothecene biosynthesis. Solid particles (cork powder) were necessary for DAS production in stationary cultures but did not influence DAS production in shaken cultures. Shaking strongly stimulated DAS production and fungal growth.

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum

The effects of nitrate and ammonium addition on net and gross photosynthesis, CO(2) efflux and the dissolved inorganic carbon compensation point of nitrogen-limited Selenastrum minutum Naeg. Collins (Chlorophyta) were studied. Cultures pulsed with nitrate or ammonium exhibited a marked decrease in both net and gross photosynthetic carbon fixation. During this period of suppression the specific activity of exogenous dissolved inorganic carbon decreased rapidly in comparison to control cells indicating an increase in the rate of CO(2) efflux in the light. The nitrate and ammmonium induced rates of CO(2) efflux were 31.0 and 33.8 micromoles CO(2) per milligram chlorophyll per hour, respectively, and represented 49 and 48% of the rate of gross photosynthesis. Nitrate addition to cells at dissolved inorganic carbon compensation point caused an increase in compensation point while ammonium had no effect. In the presence of the tricarboxylic acid cycle inhibitor fluoroacetate, the nitrate-induced change in compensation point was greatly reduced suggesting the source of this CO(2) was the tricarboxylic acid cycle. These results are consistent with the mechanism of N-induced photosynthetic suppression outlined by Elrifi and Turpin (1986 Plant Physiol 81: 273-279).

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum

Nitrate-limited chemostat cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) were used to determine the effects of nitrogen addition on photosynthesis, dark respiration, and dark carbon fixation. Addition of NO(3) (-) or NH(4) (+) induced a transient suppression of photosynthetic carbon fixation (70 and 40% respectively). Intracellular ribulose bisphosphate levels decreased during suppression and recovered in parallel with photosynthesis. Photosynthetic oxygen evolution was decreased by N-pulsing under saturating light (650 microeinsteins per square meter per second). Under subsaturating light intensities (<165 microeinsteins per square meter per second) NH(4) (+) addition resulted in O(2) consumption in the light which was alleviated by the presence of the tricarboxylic acid cycle inhibitor fluoroacetate. Addition of NO(3) (-) or NH(4) (+) resulted in a large stimulation of dark respiration (67 and 129%, respectively) and dark carbon fixation (360 and 2080%, respectively). The duration of N-induced perturbations was dependent on the concentration of added N. Inhibition of glutamine 2-oxoglutarate aminotransferase by azaserine alleviated all these effects. It is proposed that suppression of photosynthetic carbon fixation in response to N pulsing was the result of a competition for metabolites between the Calvin cycle and nitrogen assimilation. Carbon skeletons required for nitrogen assimilation would be derived from tricarboxylic acid cycle intermediates. To maintain tricarboxylic acid cycle activity triose phosphates would be exported from the chloroplast. This would decrease the rate of ribulose bisphosphate regeneration and consequently decrease net photosynthetic carbon accumulation. Stoichiometric calculations indicate that the Calvin cycle is one source of triose phosphates for N assimilation; however, during transient N resupply the major demand for triose phosphates must be met by starch or sucrose breakdown. The effects of N-pulsing on O(2) evolution, dark respiration, and dark C-fixation are shown to be consistent with this model.

Studies of the effects of fluoroacetate on ameloblasts in rat incisor.

The effects of fluoroacetate on the ameloblasts were studied in the rat incisor. Fluoroacetate is an inhibitor of tricarboxylic acid cycle and accumulation of citrate occurred in the animal tissues due to fluoroacetate administration. In the present study, fluoroacetate injection caused severe morphologic changes in the ameloblasts. The most prominent change was observed in the mitochondria. Reduction of the mitochondrial matrix density was the earliest change followed by varying degrees of matrix swelling. Loss of the matrix granules and disintegration of the cristae were also observed. The difference in the mitochondrial activities in regard to the citrate metabolism was found between the matrix formation stage and the maturation stage in the ameloblasts. Extensive dilatation of the rough-surfaced endoplasmic reticulum and grossly enlarged vacuoles were found mainly in the early maturation stage at 12 and 24 hours after the fluoroacetate administration. These abnormally large vacuoles seemed to be caused by the water stored within the endoplasmic reticulum cisternae. Accumulation of plasma citrate and decrease of ionized calcium concentration in the whole blood were observed in the fluoroacetate treated group. These findings suggest that fluoroacetate may cause the lowering of the function of the ameloblasts through the suppression of cell energy production and that both the secretion of the matrix and the calcification of the enamel may be inhibited.

The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves

The chlorophyll-based specific activity of cytochrome oxidase and three exclusively mitochondrial enzymes of the tricarboxylic acid cycle showed little variation between leaves of C(3) and C(4) plants or between mesophyll and bundle sheath cells of Atriplex spongiosa and Sorghum bicolor. However, a large, light-dependent transfer of label from intermediates of the tricarboxylic acid cycle to photosynthetic products was a feature of leaves of C(4) plants. This light-dependent transfer of label was barely detectable in leaves of C(3) plants and in leaves of F(1) and F(3) hybrids of Atriplex rosea (C(4)) and Atriplex patula spp hastata (C(3)). The light-dependent transfer of label to photosynthetic products in leaves of C(4) plants was inhibited by the tricarboxylic acid cycle inhibitors malonate and fluoroacetate. The requirement for continued tricarboxylic acid cycle activity was also indicated in experiments with specifically labeled succinate-(14)C. These experiments, together with the distribution of (14)C in glucose prepared from sucrose-(14)C formed during the metabolism of succinate-2,3-(14)C, confirmed that the photosynthetic metabolism of malate and aspartate derived from the tricarboxylic acid cycle, and not the refixation of respiratory CO(2), was the main path of carbon from the cycle to photosynthesis.

The pathway of glycollate utilization in Chlorella pyrenoidosa.

1. Exogenous glycollate was rapidly metabolized in both the light and the dark by photoautotrophically grown Chlorella pyrenoidosa. 2. The incorporation of (14)C from [1-(14)C]glycollate by these cells was inhibited by the tricarboxylic acid-cycle inhibitors monofluoroacetate, diethylmalonate and arsenite, and also by alpha-hydroxypyrid-2-ylmethanesulphonate and isonicotinylhydrazine. 3. Short-term kinetic experiments showed over 80% of the total (14)C present in the soluble fraction from the cells to be in glycine and serine after 10s. This percentage decreased with time whereas the percentage radioactivity in glycerate increased for up to 30s then remained steady. The percentage of the total radioactivity present in citrate increased over the experimental period. Malate was the only other tricarboxylic acid-cycle intermediate to become labelled. 4. The kinetic and inhibitor experiments supported the following pathway of glycollate incorporation: glycollate --> glyoxylate --> glycine --> serine --> hydroxypyruvate --> glycerate --> 3-phosphoglycerate --> 2-phosphoglycerate --> phosphoenolpyruvate --> pyruvate --> acetyl-CoA. 5. The specific activities of the enzymes catalysing this metabolic sequence in cell-free extracts were great enough to account for the observed rate of glycollate metabolism of 0.25mumol/h per mg dry wt. of cells in the light.

The effect of delayed injections of tricarboxylic acid cycle inhibitors and reticuloendothelial "blocking" agents on Salmonella typhimurium infection in mice.

A series of injections of tricarboxylic acid cycle inhibitors or intermediates given immediately after infection reduces survival time of mice (Berry and Mitchell, 1953a and b), rats (Berry and Derbyshire, 1956), guinea pigs (Berry and Beuzeville), and chickens (Gilnllan, Holtman, and Ross, 1956) infected with certain bacterial pathogens. Without apparent exception, at least one of the substances employed accelerates the course of the disease when different dis-

Modification of pullorum disease in the chick by certain tricarboxylic acid cycle inhibitors and intermediates.

Modification of pullorum disease in the chick by certain tricarboxylic acid cycle inhibitors and intermediates.