Concentrations Of 2-oxoglutarate Research Articles

Revealing metabolic alterations in brucellosis patients by targeted metabolomics

Brucellosis, a zoonotic disease caused by brucella infection, presents metabolic profile changes in patients that have not been extensively explored. This study utilized an ultra-high performance liquid chromatography tandem mass spectrometry based targeted metabolomic approach to comprehensively investigated metabolic changes in Brucella patients. Serum samples of brucellosis 50 patients and 50 well-matched healthy controls were analyzed for 228 metabolites, revealing significant alterations in 83 metabolites in brucellosis patients. Notably, disruptions were observed in key metabolite pathways, such as amino acid metabolism, urea cycle, tricarboxylic acid cycle (TCA), and fatty acid metabolism. Patients diagnosed with Brucellosis exhibited distinct differences in the levels of aspartate, glutamate, β-alanine, and asparagine when compared to controls. Within the urea cycle, a significant downregulation of arginine was observed, whereas ornithine levels were considerably upregulated. In the TCA cycle, concentrations of 2-oxoglutarate, succinate, and malate were significantly elevated, while citrate levels demonstrated a notable decrease. Due to the interruption of the TCA cycle, glycolysis was accelerated to compensate for the resultant energy deficit in Brucella patients. Concurrently, there was a significant increase in the levels of short and medium-chain fatty acids, while long-chain fatty acids showed a marked decrease. The study systematically revealed significant metabolic alterations in Brucellosis patients and further explored the potential correlation between these changes and clinic symptoms, including fatigue, muscle soreness and prolonged fever. The results enhanced our understanding of Brucellosis, offering valuable insights potentially beneficial in formulating more effective treatment strategies and improving prognostic approaches.

A mechanistic study of the influence of nitrogen and energy availability on the NH4+ sensitivity of nitrogen assimilation in Synechococcus.

In most algae, NO3- assimilation is tightly controlled and is often inhibited by the presence of NH4+. In the marine, non-colonial, non-diazotrophic cyanobacterium Synechococcus UTEX 2380, NO3- assimilation is sensitive to NH4+ only when N does not limit growth. We sequenced the genome of Synechococcus UTEX 2380, studied the genetic organization of the nitrate assimilation related (NAR) genes, and investigated expression and kinetics of the main NAR enzymes, under N or light limitation. We found that Synechococcus UTEX 2380 is a β-cyanobacterium with a full complement of N uptake and assimilation genes and NAR regulatory elements. The nitrate reductase of our strain showed biphasic kinetics, previously observed only in freshwater or soil diazotrophic Synechococcus strains. Nitrite reductase and glutamine synthetase showed little response to our growth treatments, and their activity was usually much higher than that of nitrate reductase. NH4+ insensitivity of NAR genes may be associated with the stimulation of the binding of the regulator NtcA to NAR gene promoters by the high 2-oxoglutarate concentrations produced under N limitation. NH4+ sensitivity in energy-limited cells fits with the fact that, under these conditions, the use of NH4+ rather than NO3- decreases N-assimilation cost, whereas it would exacerbate N shortage under N limitation.

Increasing the Ascomycin Yield by Relieving the Inhibition of Acetyl/Propionyl-CoA Carboxylase by the Signal Transduction Protein GlnB.

Ascomycin (FK520) is a multifunctional antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus. In this study, we demonstrated that the inactivation of GlnB, a signal transduction protein belonging to the PII family, can increase the production of ascomycin by strengthening the supply of the precursors malonyl-CoA and methylmalonyl-CoA, which are produced by acetyl-CoA carboxylase and propionyl-CoA carboxylase, respectively. Bioinformatics analysis showed that Streptomyces hygroscopicus var. ascomyceticus contains two PII family signal transduction proteins, GlnB and GlnK. Protein co-precipitation experiments demonstrated that GlnB protein could bind to the α subunit of acetyl-CoA carboxylase, and this binding could be disassociated by a sufficient concentration of 2-oxoglutarate. Coupled enzyme activity assays further revealed that the interaction between GlnB protein and the α subunit inhibited both the activity of acetyl-CoA carboxylase and propionyl-CoA carboxylase, and this inhibition could be relieved by 2-oxoglutarate in a concentration-dependent manner. Because GlnK protein can act redundantly to maintain metabolic homeostasis under the control of the global nitrogen regulator GlnR, the deletion of GlnB protein enhanced the supply of malonyl-CoA and methylmalonyl-CoA by restoring the activity of acetyl-CoA carboxylase and propionyl-CoA carboxylase, thereby improving the production of ascomycin to 390 ± 10 mg/L. On this basis, the co-overexpression of the β and ε subunits of propionyl-CoA carboxylase further increased the ascomycin yield to 550 ± 20 mg/L, which was 1.9-fold higher than that of the parent strain FS35 (287 ± 9 mg/L). Taken together, this study provides a novel strategy to increase the production of ascomycin, providing a reference for improving the yield of other antibiotics.

Expeller-Pressed Canola (Brassica napus) Meal Modulates the Structure and Function of the Cecal Microbiota, and Alters the Metabolome of the Pancreas, Liver, and Breast Muscle of Broiler Chickens.

Simple SummaryThe impact of a broiler chicken diet that is supplemented with canola meal (CM) on the cecal microbiota, growth, and metabolome of broiler chickens was examined. CM did not affect feed consumption, weight gain, nor the richness, evenness, or diversity of the cecal bacterial community. However, CM affected the structure of the bacterial microbiota, the concentrations of short-chain fatty acids (SCFAs) were elevated, and the abundance of bacterial taxa known to ferment dietary fiber were more abundant in the ceca of birds fed the CM diet. Nuclear magnetic resonance spectroscopy metabolomics was applied, and a number of metabolites that were associated with SCFA metabolism were differentially regulated in cecal digesta. The supplementation of diet with CM also affected the metabolic profiles of the pancreas, liver, and breast muscle (e.g., metabolites that are associated with energy production, protection against oxidative stress, and pathways of amino acid and glycerophospholipid metabolism). In summary, broiler chickens that were fed a diet supplemented with CM showed equivalent feed consumption and growth, but CM affected the composition and function of the cecal microbiota, and the metabolome of the pancreas, liver, and breast muscle of birds indicating the possibility of an increased disease risk. The study also demonstrated the utility of using metabolomics to ascertain impacts of diet on the microbiota and host tissues, and identified biomarkers to facilitate subsequent evaluation in production settings.The inoculation of one-day-old broiler chicks with the cecal contents from a mature broiler breeder resulted in a highly diverse and uniform cecal bacterial community. CM did not affect feed consumption, weight gain, nor the richness, evenness, or diversity of the cecal bacterial community. However, the structure of the bacterial community was altered in birds fed the CM diet. Although the CM diet was formulated to contain equivalent metabolizable energy to the control diet, it contained more dietary fiber. The abundance of bacterial families, including those that are known to contain species able to metabolize fiber was altered (e.g., bacteria within the families, Methanobacteriaceae, Atopobiaceae, Prevotellaceae, Clostridiales Family XIII, Peptostreptococcaceae, and Succinivibrionaceae), and concentrations of SCFAs were higher in the ceca of birds fed the CM diet. Moreover, concentrations of isoleucine, isobutyrate, glutamate, and 2-oxoglutarate were higher, whereas concentrations of phenyllactic acid, indole, glucose, 3-phenylpropionate, and 2-oxobutyrate were lower in the digesta of chickens that were fed CM. The metabolic profiles of pancreas, liver, and breast muscle tissues of birds fed the CM diet differed from control birds. Metabolites that were associated with energy production, protection against oxidative stress, and pathways of amino acid and glycerophospholipid metabolism had altered concentrations in these tissues. Some of the observed changes in metabolite levels may indicate an increased disease risk in birds fed the CM diet (e.g., pancreatitis), and others suggested that birds mounted metabolic response to offset the adverse impacts of CM (e.g., oxidative stress in the liver).

Redox interference in nitrogen status via oxidative stress is mediated by 2-oxoglutarate in cyanobacteria.

Reactive oxygen species (ROS) are generated naturally in photosynthetic organisms by respiration and photosynthesis. Therefore, detoxification of these compounds, avoiding oxidative stress, is essential for proper cell function. In cyanobacteria, some observations point to a crosstalk between ROS homeostasis, in particular hydrogen peroxide, and nitrogen metabolism by a mechanism independent of known redox regulators. Using glutamine synthetase (GS), a finely regulated enzyme essential for nitrogen assimilation, as a tool, we were able to monitor nitrogen metabolism in relation to oxidative stress. We show that hydrogen peroxide clearly alters the expression of different genes related to nitrogen metabolism, both in the wild-type strain of the cyanobacterium Synechocystis sp. PCC 6803 and in a mutant strain lacking the catalase-peroxidase encoded by the katG gene and therefore highly sensitive to oxidative stress. As cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate (2-OG) concentrations, the hydrogen peroxide effect was analysed under different nitrogen conditions in the wild-type, the ∆katG strain and in a strain able to transport 2-OG. The results obtained demonstrate that hydrogen peroxide interferes with signalling of cellular carbon:nitrogen status by decreasing the intracellular concentrations of 2-OG and hence altering the function of the 2-OG-sensing global nitrogen regulator NtcA.

2-Oxoglutarate cooperativity and biphasic ammonium saturation of Aspergillus niger NADP-glutamate dehydrogenase are structurally coupled

NADP-glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoidal 2-oxoglutarate saturation. Despite sharing 88% amino acid identity, the homologous enzyme from Aspergillus terreus (AtGDH) shows hyperbolic 2-oxoglutarate saturation. In order to address the structural origins of this phenomenon, six AnGDH-AtGDH chimeras were constructed and characterized. The C-terminal sequence (residues 315–460, named the D-segment) was implicated in the AnGDH cooperativity. The D-segment residues largely contribute to the monomer-monomer interface of each trimer in the native hexamer and are far removed from the enzyme active site. The D-segment appears to be a part of the allosteric network responsible for 2-oxoglutarate homotropic interactions in AnGDH. AnGDH and its C415S mutant, but not AtGDH, also showed atypical, biphasic ammonium saturation, particularly at sub-saturating 2-oxoglutarate concentrations. We found that the sigmoidal 2-oxoglutarate saturation and the biphasic ammonium response are tightly coupled; the analysis of AnGDH-AtGDH chimeras ascribes the two features to the AnGDH D-segment. The two non-Michaelis-Menten substrate saturations of AnGDH were influenced by ionic strength. Increase in ionic strength reduced the nH of 2-oxoglutarate saturation as well as abolished the biphasic response, suggesting that polar/ionic interactions determine the allosteric, inter-subunit communications. The biochemical analysis in the context of available structural data implicates the D-segment of AnGDH in the allosteric feature of this enzyme. The coupling of sigmoidal 2-oxoglutarate saturation and the biphasic ammonium response could possibly confer growth advantage to A. niger experiencing carbon and/or nitrogen limitation.

Administration of chitosan-tripolyphosphate-DNA nanoparticles to knockdown glutamate dehydrogenase expression impairs transdeamination and gluconeogenesis in the liver

Glutamate dehydrogenase (GDH) plays a major role in amino acid catabolism. To increase the current knowledge of GDH function, we analysed the effect of GDH silencing on liver intermediary metabolism from gilthead sea bream (Sparus aurata). Sequencing of GDH cDNA from S. aurata revealed high homology with its vertebrate orthologues and allowed us to design short hairpin RNAs (shRNAs) to knockdown GDH expression. Following validation of shRNA-dependent downregulation of S. aurata GDH in vitro, chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a selected shRNA (pCpG-sh2GDH) were produced to address the effect of GDH silencing on S. aurata liver metabolism. Seventy-two hours following intraperitoneal administration of chitosan-TPP-pCpG-sh2GDH, GDH mRNA levels and immunodetectable protein decreased in the liver, leading to reduced GDH activity in both oxidative and reductive reactions to about 53–55 % of control values. GDH silencing decreased glutamate, glutamine and aspartate aminotransferase activity, while increased 2-oxoglutarate content, 2-oxoglutarate dehydrogenase activity and 6-phosphofructo-1-kinase/fructose-1,6-bisphosphatase activity ratio. Our findings show for the first time that GDH silencing reduces transdeamination and gluconeogenesis in the liver, hindering the use of amino acids as gluconeogenic substrates and enabling protein sparing and metabolisation of dietary carbohydrates, which would reduce environmental impact and production costs of aquaculture.

Differential NtcA Responsiveness to 2-Oxoglutarate Underlies the Diversity of C/N Balance Regulation in Prochlorococcus.

Previous studies showed differences in the regulatory response to C/N balance in Prochlorococcus with respect to other cyanobacteria, but no information was available about its causes, or the ecological advantages conferred to thrive in oligotrophic environments. We addressed the changes in key enzymes (glutamine synthetase, isocitrate dehydrogenase) and the ntcA gene (the global nitrogen regulator) involved in C/N metabolism and its regulation, in three model Prochlorococcus strains: MED4, SS120, and MIT9313. We observed a remarkable level of diversity in their response to azaserine, a glutamate synthase inhibitor which increases the concentration of the key metabolite 2-oxoglutarate, used to sense the C/N balance by cyanobacteria. Besides, we studied the binding between the global nitrogen regulator (NtcA) and the promoter of the glnA gene in the same Prochlorococcus strains, and its dependence on the 2-oxoglutarate concentration, by using isothermal titration calorimetry, surface plasmon resonance, and electrophoretic mobility shift. Our results show a reduction in the responsiveness of NtcA to 2-oxoglutarate in Prochlorococcus, especially in the MED4 and SS120 strains. This suggests a trend to streamline the regulation of C/N metabolism in late-branching Prochlorococcus strains (MED4 and SS120), in adaptation to the rather stable conditions found in the oligotrophic ocean gyres where this microorganism is most abundant.

Metabolomics and Gene Expression Analysis Reveal Down-regulation of the Citric Acid (TCA) Cycle in Non-diabetic CKD Patients

Chronic kidney disease (CKD) is a public health problem with very high prevalence and mortality. Yet, there is a paucity of effective treatment options, partly due to insufficient knowledge of underlying pathophysiology. We combined metabolomics (GCMS) with kidney gene expression studies to identify metabolic pathways that are altered in adults with non-diabetic stage 3–4 CKD versus healthy adults. Urinary excretion rate of 27 metabolites and plasma concentration of 33 metabolites differed significantly in CKD patients versus controls (estimate range−68% to +113%). Pathway analysis revealed that the citric acid cycle was the most significantly affected, with urinary excretion of citrate, cis-aconitate, isocitrate, 2-oxoglutarate and succinate reduced by 40–68%. Reduction of the citric acid cycle metabolites in urine was replicated in an independent cohort. Expression of genes regulating aconitate, isocitrate, 2-oxoglutarate and succinate were significantly reduced in kidney biopsies. We observed increased urine citrate excretion (+74%, p=0.00009) and plasma 2-oxoglutarate concentrations (+12%, p=0.002) in CKD patients during treatment with a vitamin-D receptor agonist in a randomized trial. In conclusion, urinary excretion of citric acid cycle metabolites and renal expression of genes regulating these metabolites were reduced in non-diabetic CKD. This supports the emerging view of CKD as a state of mitochondrial dysfunction.

Metabolic profiling by gas chromatography-mass spectrometry of energy metabolism in high-fat diet-fed obese mice.

A novel, selective and sensitive single-ion monitoring (SIM) gas chromatography-mass spectrometry (GCMS) method was developed and validated for the determination of energy metabolites related to glycolysis, the tricarboxylic acid (TCA) cycle, glutaminolysis, and fatty acid β-oxidation. This assay used N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) containing 1% tert-butyldimethylchlorosilane (TBDMCS) as derivatizing reagent and was highly reproducible, sensitive, specific and robust. The assay was used to analyze liver tissue and serum from C57BL/6N obese mice fed a high-fat diet (HFD) and C57BL/6N mice fed normal chow for 8 weeks. HFD-fed mice serum displayed statistically significantly reduced concentrations of pyruvate, citrate, succinate, fumarate, and 2-oxoglutarate, with an elevated concentration of pantothenic acid. In liver tissue, HFD-fed mice exhibited depressed levels of glycolysis end-products pyruvate and lactate, glutamate, and the TCA cycle intermediates citrate, succinate, fumarate, malate, and oxaloacetate. Pantothenate levels were 3-fold elevated accompanied by a modest increased gene expression of Scl5a6 that encodes the pantothenate transporter SLC5A6. Since both glucose and fatty acids inhibit coenzyme A synthesis from pantothenate, it was concluded that these data were consistent with downregulated fatty acid β-oxidation, glutaminolysis, glycolysis, and TCA cycle activity, due to impaired anaplerosis. The novel SIM GCMS assay provided new insights into metabolic effects of HFD in mice.

Loss of Mitochondrial Malate Dehydrogenase Activity Alters Seed Metabolism Impairing Seed Maturation and Post-Germination Growth in Arabidopsis.

Mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37) has multiple roles; the most commonly described is its catalysis of the interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. The roles of mMDH in Arabidopsis (Arabidopsis thaliana) seed development and germination were investigated in mMDH1 and mMDH2 double knockout plants. A significant proportion of mmdh1mmdh2 seeds were nonviable and developed only to torpedo-shaped embryos, indicative of arrested seed embryo growth during embryogenesis. The viable mmdh1mmdh2 seeds had an impaired maturation process that led to slow germination rates as well as retarded post-germination growth, shorter root length, and decreased root biomass. During seed development, mmdh1mmdh2 showed a paler green phenotype than the wild type and exhibited deficiencies in reserve accumulation and reduced final seed biomass. The respiration rate of mmdh1mmdh2 seeds was significantly elevated throughout their maturation, consistent with the previously reported higher respiration rate in mmdh1mmdh2 leaves. Mutant seeds showed a consistently higher content of free amino acids (branched-chain amino acids, alanine, serine, glycine, proline, and threonine), differences in sugar and sugar phosphate levels, and lower content of 2-oxoglutarate. Seed-aging assays showed that quiescent mmdh1mmdh2 seeds lost viability more than 3 times faster than wild-type seeds. Together, these data show the important role of mMDH in the earliest phases of the life cycle of Arabidopsis.

Physiological Regulation of Isocitrate Dehydrogenase and the Role of 2-Oxoglutarate in Prochlorococcus sp. Strain PCC 9511

The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.

Comparative Proteomics Reveals That a Saxitoxin-Producing and a Nontoxic Strain ofAnabaena circinalisAre Two Different Ecotypes

In Australia, saxitoxin production is restricted to the cyanobacterial species Anabaena circinalis and is strain-dependent. We aimed to characterize a saxitoxin-producing and nontoxic strain of A. circinalis at the proteomic level using iTRAQ. Seven proteins putatively involved in saxitoxin biosynthesis were identified within our iTRAQ experiment for the first time. The proteomic profile of the toxic A. circinalis was significantly different from the nontoxic strain, indicating that each is likely to inhabit a unique ecological niche. Under control growth conditions, the saxitoxin-producing A. circinalis displayed a higher abundance of photosynthetic, carbon fixation and nitrogen metabolic proteins. Differential abundance of these proteins suggests a higher intracellular C:N ratio and a higher concentration of intracellular 2-oxoglutarate in our toxic strain compared with the nontoxic strain. This may be a novel site for posttranslational regulation because saxitoxin biosynthesis putatively requires a 2-oxoglutarate-dependent dioxygenase. The nontoxic A. circinalis was more abundant in proteins, indicating cellular stress. Overall, our study has provided the first insight into fundamental differences between a toxic and nontoxic strain of A. circinalis, indicating that they are distinct ecotypes.

Competitive HIF Prolyl Hydroxylase Inhibitors Show Protection against Oxidative Stress by a Mechanism Partially Dependent on Glycolysis

The hypoxia inducible factor 1 (HIF-1) is a central transcription factor involved in the cellular and molecular adaptation to hypoxia and low glucose supply. The level of HIF-1 is to a large degree regulated by the HIF prolyl hydroxylase enzymes (HPHs) belonging to the Fe(II) and 2-oxoglutarate-dependent dioxygenase superfamily. In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12). Although the competitive HPH-inhibitor compounds were found to be pharmacologically more potent than the non-competitive compounds at inhibiting HPH2 and HPH1, this was not translated into the cellular effects of the compounds, where the non-competitive inhibitors were actually more potent than the competitive in stabilizing and translocatingHIF1αto the nucleus (quantified with Cellomics ArrayScan technology). This could be explained by the high cellular concentrations of the cofactor 2-oxoglutarate (2-OG) as the competitive inhibitors act by binding to the 2-OG site of the HPH enzymes. Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress. In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.

Integrated Histopathological and Urinary Metabonomic Investigation of the Pathogenesis of Microcystin-LR Toxicosis

Patterns of change of endogenous metabolites may closely reflect systemic and organ-specific toxic changes. The authors examined the metabolic effects of the cyanobacterial (blue-green algal) toxin microcystin-LR by (1)H-nuclear magnetic resonance (NMR) analysis of urinary endogenous metabolites. Rats were treated with a single sublethal dose, either 20 or 80 µg/kg intraperitoneally, and sacrificed at 2 or 7 days post dosing. Changes in the high-dose, 2-day sacrifice group included centrilobular hepatic necrosis and congestion, accompanied in some animals by regeneration and neovascularization. By 7 days, animals had recovered, the necrotizing process had ended, and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. There was considerable interanimal variation in the histologic process and severity, which correlated with the changes in patterns of endogenous metabolites in the urine, thus providing additional validation of the biomarker and biochemical changes. Similarity of the shape of the metabolic trajectories suggests that the mechanisms of toxic effects and recovery are similar among the individual animals, albeit that the magnitude and timing are different for the individual animals. Initial decreases in urinary citrate, 2-oxoglutarate, succinate, and hippurate concentrations were accompanied by a temporary increase in betaine and taurine, then creatine from 24 to 48 hours. Further changes were an increase in guanidinoacetate, dimethylglycine, urocanic acid, and bile acids. As a tool, urine can be repeatedly and noninvasively sampled and metabonomics utilized to study the onset and recovery after toxicity, thus identifying time points of maximal effect. This can help to employ histopathological examination in a guided and effective fashion.

Metabonomic Responses in Rat Urine Following Subacute Exposure to Propoxur

Metabolic profiling of urine from pesticide-treated rats was investigated by the nuclear magnetic resonance (NMR)-based metabonomic strategy. Twenty-four-hour urine samples of rats were collected after administration with propoxur at doses of 0.85, 1.70, and 8.51 mg/kg, respectively, for 28 consecutive days. Liver tissue was fixed and the histopathological alterations were examined. The results showed that propoxur at high dose induced liver histopathological injury. Metabonomic analysis demonstrated that the levels of creatine and taurine markedly increased together with slight elevation of hippurate, glucose, and amino acids in low- and medium-dose groups. However, concentrations of urinary lactate, acetate, acetone, succinate, citrate, and 2-oxoglutarate increased in high-dose group. All these results suggested that propoxur could inhibit liver function through altering the energy and lipid metabolism. These data also supported the contention that the NMR-based metabonomic approach represents a promising new technology for the development of pesticide toxicity screening and mechanism exploration.

Antisense suppression of the small chloroplast protein CP12 in tobacco alters carbon partitioning and severely restricts growth.

The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PRK and GAPDH are inactivated when present in this complex, a process shown in vitro to be dependent upon oxidized CP12. The importance of CP12 in vivo in higher plants, however, has not been investigated. Here, antisense suppression of CP12 in tobacco (Nicotiana tabacum) was observed to impact on NAD-induced PRK and GAPDH complex formation but had little effect on enzyme activity. Additionally, only minor changes in photosynthetic carbon fixation were observed. Despite this, antisense plants displayed changes in growth rates and morphology, including dwarfism and reduced apical dominance. The hypothesis that CP12 is essential to separate oxidative pentose phosphate pathway activity from Calvin-Benson cycle activity, as proposed in cyanobacteria, was tested. No evidence was found to support this role in tobacco. Evidence was seen, however, for a restriction to malate valve capacity, with decreases in NADP-malate dehydrogenase activity (but not protein levels) and pyridine nucleotide content. Antisense repression of CP12 also led to significant changes in carbon partitioning, with increased carbon allocation to the cell wall and the organic acids malate and fumarate and decreased allocation to starch and soluble carbohydrates. Severe decreases were also seen in 2-oxoglutarate content, a key indicator of cellular carbon sufficiency. The data presented here indicate that in tobacco, CP12 has a role in redox-mediated regulation of carbon partitioning from the chloroplast and provides strong in vivo evidence that CP12 is required for normal growth and development in plants.

Overcoming Fluctuation and Leakage Problems in the Quantification of Intracellular 2-Oxoglutarate Levels in Escherichia coli

2-Oxoglutarate is located at the junction between central carbon and nitrogen metabolism, serving as an intermediate for both. In nitrogen metabolism, 2-oxoglutarate acts as both a carbon skeletal carrier and an effector molecule. There have been only sporadic reports of its internal concentrations. Here we describe a sensitive and accurate method for determination of the 2-oxoglutarate pool concentration in Escherichia coli. The detection was based on fluorescence derivatization followed by reversed-phase high-pressure liquid chromatography separation. Two alternative cell sampling strategies, both of which were based on a fast filtration protocol, were sequentially developed to overcome both its fast metabolism and contamination from 2-oxoglutarate that leaks into the medium. We observed rapid changes in the 2-oxoglutarate pool concentration upon sudden depletion of nutrients: decreasing upon carbon depletion and increasing upon nitrogen depletion. The latter was studied in mutants lacking either of the two enzymes using 2-oxoglutarate as the carbon substrate for glutamate biosynthesis. The results suggest that flux restriction on either reaction greatly influences the internal 2-oxoglutarate level. Additional study indicates that KgtP, a 2-oxoglutarate proton symporter, functions to recover the leakage loss of 2-oxoglutarate. This recovery mechanism benefits the measurement of cellular 2-oxoglutarate level in practice by limiting contamination from 2-oxoglutarate leakage.

Study on influence of glucoside Tripterygium total tablets on metabolism in rats by NMR metabonomic technique

To investigate the toxic effects of Glucoside Tripterygium total on rats with nuclear magnetic resonance (NMR)-based metabonomic method. The influence of intragastric administration of Glucoside Tripterygium total suspension at two different doses on endogenetic metabolites in normal rat urine was determined with bio-NMR method then analyzed by pattern recognition technique and partial least-squares discriminant analysis (PLS-DA). Histopathological analysis was carried out. Escalations of concentrations of urinary taurine, TMAO and glucose as well as reductions of concentrations of urinary citrate and 2-oxoglutarate were found by analysis of the 1H-NMR spectra, which was coincident with the result of histopathological analysis. The result of pathological examination indicated that pathologic change was not observed in nephridial tissue, but there were obvious changes in hepatic tissue. The urinary metabomic spectra were closely associated with the hepatic toxicity, which manifested the mitochondrial dysfunctions, the abnormal energy metabolism in TCA cycle as well as the abnormal glucose metabolism.

Endogenous 2-oxoglutarate levels impact potencies of competitive HIF prolyl hydroxylase inhibitors

The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by oxygen-dependent hydroxylation that is catalyzed by three HIF prolyl 4-hydroxylases (HPHs). Use of HPH inhibition as a mean for HIF-upregulation has recently gained interest as a potential treatment paradigm against neurodegenerative diseases like ischemia and Parkinson's disease. In the present investigation we report the development of a new and robust assay to measure HPH activity. The assay is based on capture of hydroxylated peptide product by the von Hippel–Lindau protein which is directly measured in a scintillation proximity assay. In addition we describe the determination of HPH subtype potencies of HPH inhibitors which either directly or indirectly inhibit the HPH enzyme. The potencies of the HPH inhibitors displayed almost identical IC50 values toward the HPH1 and HPH2 subtype while the potency against the HPH3 subtype was increased for several of the compounds. For the most potent compound, a hydroxyl thiazole derivative, the potency against HPH2 and HPH3 was 7nM and 0.49nM, respectively corresponding to a 14-fold difference. These results suggest that HPH subtype-selective compounds may be developed. In addition we determined the 2-oxoglutarate concentration in brain tissue and neuronal cell lines as 2-oxoglutarate is an important co-factor used by the HPH enzyme during the hydroxylation reaction. The high intracellular 2-oxoglutarate concentration provides an explanation for the diminished cellular HIF activating potency of a competitive HPH inhibitor compared to its orders of magnitude higher HPH inhibiting potency. The present reported data suggest that in the development of specific Hif prolyl hydroxylase inhibitors the high 2-oxoglutarate tissue level should be taken into account as this might affect the cellular potency. Thus to specifically inhibit the intracellular HPH enzymatic reaction a competitive inhibitor with a low Ki should be developed.