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Tricarboxylic Acid Cycle (tricarboxylic + acid_cycle)
Selected AbstractsMolecular responses of Campylobacter jejuni to cadmium stressFEBS JOURNAL, Issue 20 2008Nadeem O. Kaakoush Cadmium ions are a potent carcinogen in animals, and cadmium is a toxic metal of significant environmental importance for humans. Response curves were used to investigate the effects of cadmium chloride on the growth of Camplyobacter jejuni. In vitro, the bacterium showed reduced growth in the presence of 0.1 mm cadmium chloride, and the metal ions were lethal at 1 mm concentration. Two-dimensional gel electrophoresis combined with tandem mass spectrometry analysis enabled identification of 67 proteins differentially expressed in cells grown without and with 0.1 mm cadmium chloride. Cellular processes and pathways regulated under cadmium stress included fatty acid biosynthesis, protein biosynthesis, chemotaxis and mobility, the tricarboxylic acid cycle, protein modification, redox processes and the heat-shock response. Disulfide reductases and their substrates play many roles in cellular processes, including protection against reactive oxygen species and detoxification of xenobiotics, such as cadmium. The effects of cadmium on thioredoxin reductase and disulfide reductases using glutathione as a substrate were studied in bacterial lysates by spectrophotometry and nuclear magnetic resonance spectroscopy, respectively. The presence of 0.1 mm cadmium ions modulated the activities of both enzymes. The interactions of cadmium ions with oxidized glutathione and reduced glutathione were investigated using nuclear magnetic resonance spectroscopy. The data suggested that, unlike other organisms, C. jejuni downregulates thioredoxin reductase and upregulates other disulfide reductases involved in metal detoxification in the presence of cadmium. [source] Transcriptional regulation of nonfermentable carbon utilization in budding yeastFEMS YEAST RESEARCH, Issue 1 2010Bernard Turcotte Abstract Saccharomyces cerevisiae preferentially uses glucose as a carbon source, but following its depletion, it can utilize a wide variety of other carbons including nonfermentable compounds such as ethanol. A shift to a nonfermentable carbon source results in massive reprogramming of gene expression including genes involved in gluconeogenesis, the glyoxylate cycle, and the tricarboxylic acid cycle. This review is aimed at describing the recent progress made toward understanding the mechanism of transcriptional regulation of genes responsible for utilization of nonfermentable carbon sources. A central player for the use of nonfermentable carbons is the Snf1 kinase, which becomes activated under low glucose levels. Snf1 phosphorylates various targets including the transcriptional repressor Mig1, resulting in its inactivation allowing derepression of gene expression. For example, the expression of CAT8, encoding a member of the zinc cluster family of transcriptional regulators, is then no longer repressed by Mig1. Cat8 becomes activated through phosphorylation by Snf1, allowing upregulation of the zinc cluster gene SIP4. These regulators control the expression of various genes including those involved in gluconeogenesis. Recent data show that another zinc cluster protein, Rds2, plays a key role in regulating genes involved in gluconeogenesis and the glyoxylate pathway. Finally, the role of additional regulators such as Adr1, Ert1, Oaf1, and Pip2 is also discussed. [source] Intrahepatic amino acid and glucose metabolism in a D -galactosamine,induced rat liver failure modelHEPATOLOGY, Issue 2 2001Kosuke Arai A better understanding of the hepatic metabolic pathways affected by fulminant hepatic failure (FHF) would help develop nutritional support and other nonsurgical medical therapies for FHF. We used an isolated perfused liver system in combination with a mass-balance model of hepatic intermediary metabolism to generate a comprehensive map of metabolic alterations in the liver in FHF. To induce FHF, rats were fasted for 36 hours, during which they received 2 D -galactosamine injections. The livers were then perfused for 60 minutes via the portal vein with amino acid,supplemented Eagle minimal essential medium containing 3% wt/vol bovine serum albumin and oxygenated with 95% O2/5% CO2. Control rats were fasted for 36 hours with no other treatment before perfusion. FHF rat livers exhibited reduced amino acid uptake, a switch from gluconeogenesis to glycolysis, and a decrease in urea synthesis, but no change in ammonia consumption compared with normal fasted rat livers. Mass-balance analysis showed that hepatic glucose synthesis was inhibited as a result of a reduction in amino acid entry into the tricarboxylic acid cycle by anaplerosis. Furthermore, FHF inhibited intrahepatic aspartate synthesis, which resulted in a 50% reduction in urea cycle flux. Urea synthesis by conversion of exogenous arginine to ornithine was unchanged. Ammonia removal was quantitatively maintained by glutamine synthesis from glutamate and a decrease in the conversion of glutamate to ,-ketoglutarate. Mass-balance analysis of hepatic metabolism will be useful in characterizing changes during FHF, and in elucidating the effects of nutritional supplements and other treatments on hepatic function. [source] DROUGHT STRESS: Role of Carbohydrate Metabolism in Drought-Induced Male Sterility in Rice Anthers,JOURNAL OF AGRONOMY AND CROP SCIENCE, Issue 5 2010G. N. Nguyen Abstract Rice plants exposed to three consecutive days of water stress (,0.5 MPa) show a reduction in male fertility and grain set, which is attributed to increased levels of reactive oxygen species (ROS) and activation of a programmed cell death. This current research was conducted to further investigate the association of sugar metabolism with microspore abortion in rice anthers. Biochemical assays showed that sucrose, glucose and fructose contents were found to be significantly increased in anthers from water stressed plants compared with the control. qRT-PCR analyses and in situ hybridization of metabolic genes (sugar transporters, invertase and phosphotransferase/kinases) demonstrated that the supply of sugars for developing microspores and the initial steps of sugar utilization e.g. glycolysis, were not repressed. However, it appears that the accumulation of sugars in stressed anthers might involve a reduction of mitochondrial activity during the tricarboxylic acid cycle, which could result in excessive production of ROS and a depletion of the ATP pool. These results also suggest that higher levels of sugars at all stages of anther development seemed to be associated with some measure of protection to the anthers against oxidative stress. Induced expression of sugar transporter genes might have maintained the high levels of sugar in the tapetum and the locules, which alleviated oxidant damage caused by excessive ROS generation. Thus, the increased level of sugars might potentially be a natural response in providing protection against oxidant damage by strengthening the antioxidant system in anthers. [source] Electrophysiological study of infant and adult rats under acute intoxication with fluoroacetamideJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2007Sergey V. Kuznetsov Abstract A study was conducted of acute intoxication of infant and adult Wistar rats with fluoroacetamide (FAA), an inhibitor of oxidative metabolism. FAA was administered orally to adult rats at 1/2 LD50 and subcutaneously to infant rats at LD100 or 1/10 LD50. Electrocardiogram (ECG), respiration and motor activity were registered for 7 days. Clinical analysis of ECG and the heart rate variability (HRV) was carried out to assess the state of the vegetative nervous system. In adult rats, FAA caused marked disturbances in the activity of cardiovascular and respiratory systems, including the development of a potentially lethal acute cor pulmonale. Conversely, there were no significant changes of cardiac function and respiration in infant rats; they died because of extreme emaciation accompanied by retardation of development. In adult rats, bursts of associated cardiac and respiratory tachyarrhythmia, as well as regular high amplitude spasmodic sighs having a deca-second rhythm were observed. In both infant and adult rats, FAA caused short-term enhancement of humoral (metabolic) and sympathetic activities, followed by a gradual and stable predominance of parasympathetic influence on HRV. Under conditions of FAA inhibition of the tricarboxylic acid cycle, the observed physiological reactions may be explained by activation of alternative metabolic pathways. This is also supported by a lack of ontogenetically caused inhibition of spontaneous motor activity in infant rats poisoned with FAA, which highlights the significance of the alternative metabolic pathways for implementation of deca-second and minute rhythms and a lack of a rigid dependence of these rhythms upon activity of neuronal networks. Copyright © 2007 John Wiley & Sons, Ltd. [source] Alterations in cerebral metabolism by the neurotoxin kainic acid studied by 13C MRSJOURNAL OF NEUROCHEMISTRY, Issue 2002E. Olstad Kainic acid is a potent agonist at the kainate subclass of ionotropic glutamate receptors, and functional kainate receptors have not only been demonstrated on neurons but also on glial cells in culture. Kainic acid injections are used to induce limbic seizures in rodents. When combined with injections of [1-13C]glucose and [1,2-13C]acetate followed by analyses of forebrain extracts using 13C magnetic resonance spectroscopy (MRS) and HPLC information about glial neuronal interaction can be obtained. Using kainic acid treatment and 24 h later injection of 13C label a significant increase in label derived from [1,2-13C]acetate was observed in glutamine and glutamate. Label derived from [1-13C]glucose was unchanged in most metabolites, however, a decrease was observed in [2-13C]GABA. It should be noted that only astrocytes are able to utilize acetate as a substrate, whereas acetyl CoA derived from glucose is metabolized predominantly in the neuronal tricarboxylic acid cycle. These results indicate that turnover of metabolites was increased predominantly in astrocytes whereas glutamatergic neurons were not affected. However, GABAergic neurons showed decreased GABA labelling, possibly due to reduced GABA release 24 h after kainic acid injection. Taken together with results obtained 2 weeks after kainic acid injection, it can be suggested that increased astrocytic activity one day after epileptic seizures results, subsequently, in an increased amino acid turnover in neurons. Cell culture work was also performed, results will be presented at the meeting. [source] Development and validation of a ultra performance LC-ESI/MS method for analysis of metabolic phenotypes of healthy men in day and night urine samplesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 16-17 2008Xijun Wang Abstract Ultra-performance LC coupled to quadrupole TOF/MS (UPLC-QTOF/MS) in positive and negative ESI was developed and validated to analyze metabolite profiles for urine from healthy men during the day and at night. Data analysis using principal components analysis (PCA) revealed differences between metabolic phenotypes of urine in healthy men during the day and at night. Positive ions with mass-to-charge ratio (m/z) 310.24 (5.35 min), 286.24 (4.74 min) and 310.24 (5.63 min) were elevated in the urine from healthy men at night compared to that during the day. Negative ions elevated in day urine samples of healthy men included m/z 167.02 (0.66 min), 263.12 (2.55 min) and 191.03 (0.73 min), whilst ions m/z 212.01 (4.77 min) were at a lower concentration in urine of healthy men during the day compared to that at night. The ions m/z 212.01 (4.77 min), 191.03 (0.73 min) and 310.24 (5.35 min) preliminarily correspond to indoxyl sulfate, citric acid and N -acetylneuraminic acid, providing further support for an involvement of phenotypic difference in urine of healthy men in day and night samples, which may be associated with notably different activities of gut microbiota, velocity of tricarboxylic acid cycle and activity of sialic acid biosynthesis in healthy men as regulated by circadian rhythm of the mammalian bioclock. [source] A path from predation to mutualismMOLECULAR MICROBIOLOGY, Issue 6 2010Antoine Danchin Summary Luminescent bacteria and nematodes associate in a strategy where the bacteria act as virulent pathogens of insects, used as their food supply, while the nematodes graze on them. Upon reaching high density, the bacteria produce light and metabolites that turn the nematodes into hosts permitting them to be carried over to further nematode preys. In this issue of Molecular Microbiology, Lango and Clarke show that the corresponding shift in lifestyle is triggered by a metabolic switch closely linked to the tricarboxylic acid cycle, but apparently not by the well-known acetate switch that monitors entry of bacteria into the stationary phase of growth. [source] Role of glial metabolism in diabetic encephalopathy as detected by high resolution 13C NMRNMR IN BIOMEDICINE, Issue 6-7 2003María A. García-Espinosa Abstract The roles of glial energetics and of the glutamine cycle in diabetic encephalopathy have been investigated ex vivo by 13C NMR in extracts of adult rat brain. Streptozotocin-induced diabetic or euglycemic animals received intravenous infusions of (1- 13C) glucose in the absence and presence of trifluoroacetic acid or methionine sulfoximine, two selective inhibitors of the glial tricarboxylic acid cycle or of glutamine synthase, respectively. (1- 13C) glucose infusions resulted in smaller 13C incorporation in all carbons of cerebral glutamate, glutamine and GABA in the diabetic animals. Co-infusion of trifluoroacetic acid with (1- 13C) glucose further reduced the 13C enrichments in cerebral glutamate and glutamine, the decrease being larger in the diabetic animals than in the corresponding euglycemic controls. Methionine sulfoximine decreased to undetectable levels the fractional 13C enrichment in the carbons of cerebral glutamine in both groups and had no significant effect on 13C incorporation in glutamate and GABA, suggesting that glutamine is not the main precursor of glutamate and GABA. Additional animals were infused with (1,2- 13C2) acetate, a major substrate of glial metabolism. In this case, (1,2- 13C2) acetate infusions resulted in increased 13C incorporation in all carbons of glutamate, glutamine and GABA in the diabetic animals. Together, these results reveal that diabetic encephalopathy has an important effect in astroglial metabolism, decreasing glucose transport and metabolism and increasing the relative contribution of glial oxidative metabolism to the support of glutamatergic and GABAergic neurotransmissions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Comparison of protein expression in human deltoideus and vastus lateralis muscles using two-dimensional gel electrophoresisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2005Daniele Capitanio Abstract We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to study the expression of contractile and regulatory proteins in human vastus lateralis and deltoideus muscles, in order to understand protein turnover and isoform switching in muscles with the same fiber-type composition but different functional properties. We demonstrate a two- to six-fold overexpression of enzymes associated with glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, and substrate transport in vastus lateralis compared to deltoideus. Expression levels of contractile protein isoforms correlated to the proportion of slow-twitch fibers in deltoideus compared to vastus lateralis are consistent with the different contractile properties of the two muscles. Two proteins involved in free radical homeostasis were differentially expressed, suggesting a direct relationship between radical scavenging and the muscle function. The application of 2-DE and MS to studies of muscle physiology thus offers a more comprehensive assessment of the molecular determinants of muscle function than traditional approaches. [source] The Corynebacterium glutamicum aconitase repressor: scratching around for crystalsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Javier García-Nafría Imperfections on the surfaces of crystallization containers are known to influence crystal formation and are thought to do so by helping to overcome the nucleation barrier. The intentional creation of imperfections has been widely applied to induce crystallization of small molecules, but has not been reported for protein crystallization. Here, the crystallization and preliminary X-ray analysis of the TetR-type aconitase repressor are reported. This regulator was the first transcription factor to be identified in the regulation of the tricarboxylic acid cycle in Corynebacterium glutamicum, an organism that is of special industrial interest and is an emerging model organism for Corynebacterineae. Successful crystallization involved introducing manual scratches on the surface of standard commercial plates, which led to a substantial improvement in crystal nucleation and quality. [source] Kinetic characterization of vero cell metabolism in a serum-free batch culture processBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Emma Petiot Abstract A global kinetic study of the central metabolism of Vero cells cultivated in a serum-free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum-free medium which is a valuable help for the implementation of this cell line in serum-free industrial production processes. Biotechnol. Bioeng. 2010;107: 143,153. © 2010 Wiley Periodicals, Inc. [source] Importance of NADPH supply for improved L -valine formation in Corynebacterium glutamicumBIOTECHNOLOGY PROGRESS, Issue 2 2010Tobias Bartek Abstract Cofactor recycling is known to be crucial for amino acid synthesis. Hence, cofactor supply was now analyzed for L -valine to identify new targets for an improvement of production. The central carbon metabolism was analyzed by stoichiometric modeling to estimate the influence of cofactors and to quantify the theoretical yield of L -valine on glucose. Three different optimal routes for L -valine biosynthesis were identified by elementary mode (EM) analysis. The modes differed mainly in the manner of NADPH regeneration, substantiating that the cofactor supply may be crucial for efficient L -valine production. Although the isocitrate dehydrogenase as an NADPH source within the tricarboxylic acid cycle only enables an L -valine yield of YVal/Glc = 0.5 mol L -valine/mol glucose (mol Val/mol Glc), the pentose phosphate pathway seems to be the most promising NADPH source. Based on the theoretical calculation of EMs, the gene encoding phosphoglucoisomerase (PGI) was deleted to achieve this EM with a theoretical yield YVal/Glc = 0.86 mol Val/mol Glc during the production phase. The intracellular NADPH concentration was significantly increased in the PGI-deficient mutant. L -Valine yield increased from 0.49 ± 0.13 to 0.67 ± 0.03 mol Val/mol Glc, and, concomitantly, the formation of by-products such as pyruvate was reduced. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Metabolic profiling as a tool for understanding defense response of Taxus Cuspidata cells to shear stressBIOTECHNOLOGY PROGRESS, Issue 5 2009Pei-Pei Han Abstract To obtain a better understanding of responsive mechanism of plant cells in response to hydrodynamic mechanical stress, a metabolic profiling approach was used to profile metabolite changes of Taxus cuspidata cells under laminar shear stress. A total of 65 intracellular metabolites were identified and quantified, using gas chromatography coupled to time-of-flight mass spectrometry. Potential biomarkers were found by the principal component analysis as well as partial least squares combined with variable influence in the projection. Trehalose, sorbitol, ascorbate, sucrose, and gluconic acid were mainly responsible for the discrimination between shear stress induced cells and control cells. Further analysis by mapping measured metabolite concentrations onto the metabolic network revealed that shear stress imposed restrictions on primary metabolic pathways by inhibiting tricarboxylic acid cycle, glycolysis, and N metabolism. To adapt to the shear condition, cells responded by starting defensive programs. These defensive programs included coinduction of glycolysis and sucrose metabolism, accumulation of compatible solutes, and antioxidative strategy. A strategy of defense mechanisms at the level of metabolites for T. cuspidata cells when challenged with the shear stress was proposed. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Insights into the Central Metabolism of Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4(Tn -5) Insect Cells by Radiolabeling StudiesBIOTECHNOLOGY PROGRESS, Issue 1 2005Chouki Benslimane The insect cell baculovirus expression vector system (BEVS) is one of the most commonly used expression systems for recombinant protein production. This system is also widely used for the production of recombinant virus and virus-like particles. Although several published reports exist on recombinant protein expression using insect cells, information dealing with their metabolism in vitro is relatively scarce. In this work we have analyzed the metabolism of glucose and glutamine, the main carbon and/or energy compounds, of the two most commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and the Trichoplusia niBTI-Tn-5B1 - 4 (Tn-5). Radiolabeled substrates have been used to determine the flux of glucose carbon entering the tricarboxylic acid cycle (TCA) and the pentose phosphate (PP) pathway by direct measurement of 14CO2 produced. The percentage of total glucose metabolized to CO2 via the TCA cycle was higher in the case of the Sf-9 (2.7%) compared to Tn-5 (0.6%) cells, while the percentage of glucose that is metabolized via the PP pathway was comparable at 14% and 16% for the two cell lines, respectively. For both cell lines, the remaining 83% of glucose is metabolized through other pathways generating, for example, lactate, alanine, etc. The percentage of glutamine oxidized in the TCA cycle was approximately 5-fold higher in the case of the Tn-5 (26.1%) as compared to the Sf-9 cells (4.6%). Furthermore, the changes in the metabolic fluxes of glucose and glutamine in Tn-5-PYC cells, which have been engineered to express a cytosolic pyruvate carboxylase, have been studied and compared to the unmodified cells Tn-5. As a result of this metabolic engineering, significant increase in the percentage of glucose oxidized in the TCA cycle (3.2%) as well as in the flux through the PP pathway (34%) of the Tn-5-PYC were observed. [source] Robustness Analysis of the Escherichiacoli Metabolic NetworkBIOTECHNOLOGY PROGRESS, Issue 6 2000Jeremy S. Edwards Genomic, biochemical, and strain-specific data can be assembled to define an in silico representation of the metabolic network for a select group of single cellular organisms. Flux-balance analysis and phenotypic phase planes derived therefrom have been developed and applied to analyze the metabolic capabilities and characteristics of Escherichia coli K-12. These analyses have shown the existence of seven essential reactions in the central metabolic pathways (glycolysis, pentose phosphate pathway, tricarboxylic acid cycle) for the growth in glucose minimal media. The corresponding seven gene products can be grouped into three categories: (1) pentose phosphate pathway genes, (2) three-carbon glycolytic genes, and (3) tricarboxylic acid cycle genes. Here we develop a procedure that calculates the sensitivity of optimal cellular growth to altered flux levels of these essential gene products. The results indicate that the E. coli metabolic network is robust with respect to the flux levels of these enzymes. The metabolic flux in the transketolase and the tricarboxylic acid cycle reactions can be reduced to 15% and 19%, respectively, of the optimal value without significantly influencing the optimal growth flux. The metabolic network also exhibited robustness with respect to the ribose-5-phosphate isomerase, and the ribose-5-phosephate isomerase flux was reduced to 28% of the optimal value without significantly effecting the optimal growth flux. The metabolic network exhibited limited robustness to the three-carbon glycolytic fluxes both increased and decreased. The development presented another dimension to the use of FBA to study the capabilities of metabolic networks. [source] | ||||||||||||||||