Silver nanoparticles-resistance of HeLa cell associated with its unusually high concentration of α-ketoglutarate and glutathione / 生物工程学报

Silver nanoparticles (AgNPs) is known as one of the most valuable metal nanoparticles in antibacterial and anticancer application. AgNPs-resistant bacteria has been documented, but it is unclear whether cancer cells can also escape the anti-cancer effect of AgNPs. In this study, we aimed to investigate this phenomenon and its underlying mechanism. The antibacterial activity and cytotoxicity of AgNPs were measured in the presence of HeLa cell metabolites. The status of AgNPs in the system associated with metabolites were characterized by UV-Vis, Zetasizer Nano ZS, and transmission electron microscopy. Non-targeted metabolomics was used to reveal the metabolites components that bind with AgNPs. HeLa cells were injected intraperitoneally to establish the tumor-bearing mice model, and the stability of AgNPs in mice serum was analyzed. The results manifested that HeLa cell metabolites inhibited the anticancer and antibacterial effects of AgNPs in a dose-dependent manner by causing AgNPs aggregation. Effective metabolites that inhibited the biological activity of AgNPs were stable in 100 ℃, insoluble in chloroform, containing sulfur elements, and had a molecular weight less than 1 kDa in molecular weight. There were 115 compounds bound with AgNPs. In vitro experiments showed that AgNPs aggregation occurred only when the concentration of α-ketoglutarate (AKG) and glutathione (GSH) together reached a certain threshold. Interestingly, the concentration of AKG and GSH in HeLa cellular metabolites was 10 and 6 times higher than that in normal cervical epithelial cells, respectively, which explained why the threshold was reached. Furthermore, the stability of AgNPs in the serum of tumor-bearing mice decreased by 20% (P < 0.05) compared with the healthy mice. In conclusion, our study demonstrates that HeLa cells escaped the anti-cancer effect of AgNPs through the synergistic effect of AKG and GSH, suggesting the need to develop strategies to overcome this limitation.

Rational metabolic engineering of Corynebacterium glutamicum for efficient synthesis of L-glutamate / 生物工程学报

L-glutamic acid is the world's largest bulk amino acid product that is widely used in the food, pharmaceutical and chemical industries. Using Corynebacterium glutamicum G01 as the starting strain, the fermentation by-product alanine content was firstly reduced by knocking out the gene encoding alanine aminotransferase (alaT), a major by-product related to alanine synthesis. Secondly, since the α-ketoglutarate node carbon flow plays an important role in glutamate synthesis, the ribosome-binding site (RBS) sequence optimization was used to reduce the activity of α-ketoglutarate dehydrogenase and enhance the glutamate anabolic flow. The endogenous conversion of α-ketoglutarate to glutamate was also enhanced by screening different glutamate dehydrogenase. Subsequently, the glutamate transporter was rationally desgined to improve the glutamate efflux capacity. Finally, the fermentation conditions of the strain constructed using the above strategy were optimized in 5 L fermenters by a gradient temperature increase combined with a batch replenishment strategy. The glutamic acid production reached (135.33±4.68) g/L, which was 41.2% higher than that of the original strain (96.53±2.32) g/L. The yield was 55.8%, which was 11.6% higher than that of the original strain (44.2%). The combined strategy improved the titer and the yield of glutamic acid, which provides a reference for the metabolic modification of glutamic acid producing strains.

Metabonomics Study on Urine 1H-NMR in Chronic Superficial Gastritis Patients with Pi-qi Deficiency Syndrome/Pi-Wei Dampness-heat Syndrome / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe metabolomic changes in urine of chronic superficial gastritis (CSG) patients with Pi-qi deficiency syndrome (PQDS) or Pi-Wei dampness-heat syndrome (PWDHS), thereby providing scientific evidence for syndrome typing of them.</p><p><b>METHODS</b>Urine samples were collected from CSG patients with PQDS/PWDHS and healthy volunteers, 10 in each group. Proton nuclear magnetic resonance spectroscopy (1H-NMR) based metabonomic analysis was performed on urine samples. Contents of related biomarkers were analyzed by principal component analysis (PCA), partial least square discriminant analysis (PLS-DA), and urivariate statistical analysis.</p><p><b>RESULTS</b>PLS-DA analysis showed that metabolites among CSG patients with PQDS/PWDHS and healthy volunteers could be mutually distinguished. Seven differentially identified metabolites were screened from urines of CSG patients with PQDS and healthy volunteers included glutamate, methionine, α-oxoglutarate, dimethylglycine, creatinine, taurine, and glucose. Four differentially identified metabolites were screened from urines of CSG patients with PWDHS and healthy volunteers included 2-hydroxybutyric acid, trimethylamine oxide, taurine, and hippuric acid. Eleven differentially identified metabolites were screened from urines of CSG patients with PQDS and PWDHS included fucose, β-hydroxybutyric acid, alanine, glutamate, methionine, succinic acid, citric acid, creatinine, glucose, hippuric acid, and lactic acid.</p><p><b>CONCLUSION</b>The metabolic differences of CSG patients PQDS and PWDHS mainly manifested in glycometabolism, lipid metabolism, and amino acids catabolism, and 1H-NMR based metabonomics may be used in classified study of Chinese medical syndrome typing.</p>

Enzymatic production of α-ketoglutaric acid by L-glutamate oxidase from L-glutamic acid / 生物工程学报

We produced α-ketoglutaric acid (α-KG) from L-glutamic acid, using enzymatic transformation approach with L-glutamate oxidase (LGOX). First, wild strain Streptomyces sp. FMME066 was mutated with NTG, a genetically stable mutant Streptomyces sp. FMME067 was obtained. Under the optimal nutrition conditions with fructose 10 g/L, peptone 7.5 g/L, KH2PO4 1 g/L and CaCl2 0.05 g/L, the maximum LGOX activity reached 0.14 U/mL. The LGOX was stable to pH and temperature, and Mn2+ had a stimulating effect. Finally, after 24 h enzymatic conversion under the optimal conditions, the maximum titer of α-KG reached 38.1 g/L from 47 g/L L-glutamic acid. Enzymatic transformation by LGOX is a potential approach for α-KG production.

Progress in microbial production of alpha-ketoglutarate / 生物工程学报

Alpha-ketogluratate is one of the key intermediates in the TCA cycle, playing an important role in the connection of carbon and nitrogen metabolism. This article aims at stating recent research progress in the production of alpha-ketoglutarate by microbial fermentation. First, a large group of microbes have been screened to accumulate alpha-ketoglutarate including prokaryotes and eukaryotes. Second, physiological characterization of over-accumulation of alpha-ketoglutarate is caused by thiamine defect and nitrogen starvation. Third, the process of fermentation was controlled and optimized by the manipulation of pH, dissolved oxygen and cofactors. Fourth, many metabolic engineering strategies were also presented for alpha-ketoglutarate production focusing on regeneration of cofactor and manipulation of the pathway. Last, we discussed the limitation of current progress and proposed the future research needs for microbial production of alpha-ketoglutarate.

Urine metabonomic study of intervention effects of Morinda officinalis how. on 'kidney-yang deficiency syndrome' / 药学学报

To investigate the intervention effects of Morinda officinalis How. on 'Kidney-yang deficiency syndrome' induced by hydrocortisone in rats, the metabolic profiles of rat urine were characterized using proton nuclear magnetic resonance and principal component analysis (PCA) was applied to study the trajectory of urinary metabolic phenotype of rats with 'Kidney-yang deficiency syndrome' under administration of M. officinalis at different time points. Meanwhile, the intervention effects of M. officinalis on urinary metabolic potential biomarkers associated with 'Kidney-yang deficiency syndrome' were also discussed. The experimental results showed that in accordance to the increased time of administration, an obvious tendency was observed that clustering of the treatment group moved gradually closed to that of the control group. Eight potential biomarkers including citrate, succinate, alpha-ketoglutarate, lactate, betaine, sarcosine, alanine and taurine were definitely up- or down-regulated. In conclusion, the effectiveness of M. oficinalis on 'Kidney-yang deficiency syndrome' is proved using the established metabonomic method and the regulated metabolic pathways involve energy metabolism, transmethylation and transportation of amine. Meanwhile, the administration of M. officinalis can alleviate the kidney impairment induced by 'Kidney-yang deficiency syndrome'.

Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.

Study on influence of glucoside Tripterygium total tablets on metabolism in rats by NMR metabonomic technique / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the toxic effects of Glucoside Tripterygium total on rats with nuclear magnetic resonance (NMR)-based metabonomic method.</p><p><b>METHOD</b>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.</p><p><b>RESULT</b>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.</p><p><b>CONCLUSION</b>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.</p>

Analysis of isocitrate dehydrogenase-1/2 gene mutations in gliomas / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To highlight recent researches which may show promise for histomolecular classification and new treatments for gliomas.</p><p><b>DATA SOURCES</b>All articles cited in this review were mainly searched from PubMed, which were published in English from 1996 to 2010.</p><p><b>STUDY SELECTION</b>Original articles and critical reviews selected were relevant to the isocitrate dehydrogenase-1/2 mutation in gliomas and other tumors.</p><p><b>RESULTS</b>Extraordinary high rates of somatic mutations in isocitrate dehydrogenase-1/2 occur in the majority of World Health Organization grade II and grade III gliomas as well as grade IV secondary glioblastomas. Isocitrate dehydrogenase-1/2 mutations are associated with younger age at diagnosis and a better prognosis in patients with mutated tumors. The functional role of isocitrate dehydrogenase-1/2 mutations in the pathogenesis of gliomas is still unclear.</p><p><b>CONCLUSION</b>Isocitrate dehydrogenase-1/2 mutations define a specific subtype of gliomas and may have great significance in the diagnosis, prognosis, and treatment of patients with these tumors.</p>

Association of rOAT1 with Caveolin-1 in Rat Kidney / 대한신장학회지

PURPOSE: A family of organic anion transporters (OAT) has been identified, and several isoforms have been reported. The regulatory mechanisms of OATs functions, however, still remain to be elucidated. The rat OAT1 contributes to move a number of negatively-charged organic compounds between cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. To address this issue, we investigated the protein-protein interaction between rOAT1 and Cav-1. METHODS: The expressions of rOAT1 and Cav-1 (mRNA and protein) were evaluated using RT-PCR and Western blot analysis. The localization of rOAT1 and Cav-1 was determined in the caveolae-rich membrane fraction isolated by sucrose density gradient ultra-centrifugation. For the direct binding between the rOAT1 and Cav-1 proteins, the immuno-precipitation method and confocal microscopy were employed. To perform functional analysis, a Xenopus oocytes expression system with the antisense oligonucleotides (ODN) technique was used. RESULTS: The expressions of rOAT1 and Cav-1 were detected in the kidney. The caveolae-rich membranous fractions from the kidney contained both rOAT1 and Cav-1 in the same fractions. The immuno-precipitation experiments showed the formation of a complex between the rOAT1 and Cav-1. The confocal microscopy using primary cultured renal proximal epithelial cells also supported the co-localization of rOAT1 and Cav-1 at the plasma membrane. The uptake function of rOAT1, as assessed by using a Xenopus oocytes expression system, was inhibited by the Xenopus Cav-1 antisense ODN. CONCLUSION: rOAT1 co-localizes with caveolin-1 in the caveolae, and caveolin-1 plays an important role in regulating the function of rOAT1.

Global expression profiling of Saccharomyces cerevisiae: metabolic remodeling in post-log phase / 生物工程学报

For the purpose of revealing the mechanism of the reduction of yeasts ethanol production rate after entrance of post-log phase, we used microarray to study expression profiles of the yeast Saccharomyces cerevisiae during the transition from mid-log growth phase to post-log growth. The results demonstrate that the global pattern of gene expression is very stable during the mid-log phase. However, a dramatic metabolic remodeling was found when the yeast entries post-log phase, during which many of amino acid synthesis and metabolism related genes are up-regulated, moreover, ion transport, energy generation and storage related genes are also up regulated during this phase, while a large number of genes involved in transposition and DNA recombination are repressed. Central metabolic pathways also engage in metabolic remodeling, within which the genes involved in succinate and a-ketoglutarate synthesis pathways are up regulated, accordance with those of amino acid synthesis and metabolism. These results demonstrate that the increasing demand for amino acids in post-log phase lead to a metabolic transition into TCA cycle and glyoxylate cycle, which subsequently reduce the ethanol production rate. This suggests a global insight into the process of yeast ethanol fermentation.

Effect of cadmium on chlorophyll biosynthesis and enzymes of nitrogen assimilation in greening maize leaf segments: role of 2-oxoglutarate.

Supply of cadmium chloride (0.5 mM) inhibited chlorophyll formation in greening maize leaf segments, while lower concentration of Cd (0.01 mM) slightly enhanced it. Inclusion of 2-oxoglutarate (2-OG, 0.1-10 mM) in the incubation mixture increased chlorophyll content in the absence as well as presence of Cd. Substantial inhibition of chlorophyll formation by Cd was observed at longer treatment both in the absence and presence of 2-OG. When the tissue was pre-incubated with 2-OG or Cd, the inhibition (%) of chlorophyll formation by Cd was lowered in the presence of 2-OG. Treatment with Cd inhibited ALAD activity and ALA formation and the inhibition (%) of ALA formation by Cd was strongly reduced in the presence of 2-OG. Glutamate dehydrogenase (GDH) activity was increased by the supply of Cd both in the absence as well as presence of 2-OG. In the presence of 2-OG, Cd supply significantly increased glutamate synthase (GOGAT) activity and reduced inhibition (%) of glutamine synthetase (GS) activity. The results suggested the involvement of the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway of ammonia assimilation to provide the precursor, glutamate, for ALA synthesis under Cd toxicity and 2-OG supplementation.

Effect of pre-treatment of alpha-ketoglutarate on cyanide-induced toxicity and alterations in various physiological variables in rodents / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the effects of pre-treatment of alpha-ketoglutarate (alpha-KG) on cyanide-induced lethality and changes in various physiological parameters in rodents.</p><p><b>METHODS</b>The LD50 of potassium cyanide (KCN) given orally (po), intraperitoneally (ip), subcutaneously (sc) or intravenously (iv) was determined in male mice, in the presence or absence alpha-KG given po, ip or iv. alpha-KG was administered 10, 20 or 40 min prior to KCN at 0.50, 1.0 or 2.0 g/kg by po or ip route, and at 0.10, 0.20 or 0.40 g/kg by iv route. Protection index (PI) was calculated as the ratio of LD50 of KCN in the presence of alpha-KG (protected animals) and LD50 of KCN in the absence of alpha-KG (unprotected animals). In a separate experiment, several physiological variables viz. mean arterial pressure (MAP), heart rate (HR), respiratory rate (RR), neuromuscular transmission (NMT) and rectal temperature (RT) were measured in anesthetized female rats pre-treated (-10 min) with po (2.0 g/kg) or iv (0.125 g/kg) alpha-KG and then administered sub-lethal (0.75 LD50) or lethal (2.0, 4.0 or 8.0 LD50) doses of KCN (po).</p><p><b>RESULTS</b>PI of 4.52, 6.40 and 7.60 at -10 min, 3.20, 5.40 and 6.40 at -20 min, and 1.40, 3.20 and 5.40 at -40 min of po administration with a-KG was observed for 0.50, 1.0 and 2.0 g/kg doses, respectively, against KCN given by po route. When KCN was given ip, a PI of 3.38, 4.79 and 5.70 was observed for 0.50, 1.0 and 2.0 g/kg alpha-KG given ip (-10 min), respectively. A lower PI of 3.37, 2.83 and 2.38 was observed when KCN given sc was challenged by 2.0 g/kg alpha-KG given ip at -10, -20 or -40 min, respectively. Similarly, a PI of 3.37, 2.83 and 2.0 was noted when KCN given sc was antagonized by 2.0 g/kg alpha-KG given po at -10, -20 or -40 min, respectively. No appreciable protection was observed when lower doses of alpha-KG (ip or po) challenged KCN given by sc route. Pre-treatment of iv or po administration of alpha-KG did not afford any protection against KCN given po or iv route. Oral treatment of 0.75 LD50 KCN caused significant decrease in MAP and HR after 15 min, RR after 30 min and NMT after 60 min. There was no effect on RT. No reduction in MAP, HR, RR and RT was observed when rats received 2.0 or 4.0 LD50 KCN after pre-treatment of alpha-KG (po; 2.0 g/kg). However, no protection was observed on NMT. Protective efficacy of alpha-KG was not observed on MAP, HR, RR, and NMT decreased by 8.0 LD50 KCN. Decrease in MAP and NMT caused by 2.0 LD50 KCN (po) was resolved by iv administration of alpha-KG.</p><p><b>CONCLUSIONS</b>Cyanide antagonism by alpha-KG is best exhibited when both alpha-KG and KCN are given by po route. The protective effect of a-KG on cyanide-induced changes in several physiological parameters also indicates a promising role of alpha-KG as an alternative cyanide antidote.</p>

Metabonomics profile of urine from rats administrated with different treatment period of isoniazid / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the effect of different treatment period, of isoniazid (INH) on the metabonomic profile of rat urine and its relationship with traditional toxicity evaluation of blood biochemical indicators and histopathology and to explore the feasibility of metabonomics in the application of drug toxicity.</p><p><b>METHODS</b>Sixty male Wistar rats were orally administrated with 0, 50, 100, 200, and 400 mg x kg(-1) INH for 3, 7, and 14 days, respectively. Rat urine was then collected and its 1H nuclear magnetic resonance (NMR) spectra were acquired. All animals underwent traditional toxicity evaluation.</p><p><b>RESULTS</b>Hepatotoxicity was revealed by traditional toxicity evaluation in rats treated with higher dosage and longer treatment of INH. Time-response relationship existed during the treatment. Time-dependent metabonomics changes conformed with the results of traditional toxicity evaluation. The urine metabonomics showed a trajectory bias from those of the controls or pre-administration, and such bias exaggerated along with the prolongation of treatment, indicating a severer toxic injury. Along with the increase of the concentrations of urinary taurine and glucose and the decrease of the concentrations of urinary citrate and 2-oxoglutarate, the 1H NMR spectra of urine in rats treated with INH also changed.</p><p><b>CONCLUSIONS</b>The metabonomics technique can distinguish the onset and development of toxicity, which helps track and identify biomarkers. The hepatic toxicity induced by INH is related to the injury of mitochondrial function, reduction of energy metabolism in tricarboxylic acid cycle, and perturbations in the metabolism of glucose and lipid. The effect of INH on the rat urine metabonomic profile is related with INH toxicology. Therefore, metabonomics can be recognized as an ideal technique to explore and evaluate the drug toxicities.</p>

Biochemical Characteristics of The Na-alpha-Ketoglutarate Cotransport System in Proximal Convoluted and Straight Tubules of the Rabbit Kidney / 대한신장학회잡지

PURPOSE: alpha-Ketoglutarate (alphaKG), a Krebs cycle intermediate, is extensively used in the kidney as a fuel substrate and as a counter anion for organic acid secretion. It is known to be taken up by the proximal tubule cells via the brush-border as well as basolateral membranes. We explored biochemical characteristics of the brush-border and basolateral alphaKG transport systems in pars convoluta and pars recta of the proximal tubule, respectively. METHODS: Brush-border and basolateral membrane vesicles (BBMV and BLMV) were isolated from rabbit renal outer cortex and outer medulla by Percoll gradient centrifugation. Vesicular uptake of alphaKG was determined by rapid Millipore filtration method using alpha-14[C]KG as a substrate. RESULTS: Both BBMV and BLMV showed a Na-gradient dependent uphill transport of alphaKG. The systems in both membranes were similarly inhibited by Li and activated by Na (Hill coefficient of 1.4). Kinetic analyses indicated that the Na-alphaKG cotransporters in the BBMV had a lower substrate affinity as compared with those in the BLMV. The transport systems in BLMVs showed a similar Km but different Vmax between the outer cortex (Km: 34 uM, Vmax: 3.3 nmol/mg protein/10s) and outer medulla (Km: 37, Vmax: 1.8). On the other hand, the systems in BBMVs were different in both Km and Vmax between the outer cortex (Km: 194, Vmax: 3.3) and outer medulla (Km: 89, Vmax: 1.7). CONCLUSION: The findings suggest that both axial and apical to basolateral heterogeneity of the Na-alphaKG cotransport system in proximal tubules may be due to a physiological adaptation to efficiently utilize alphaKG in the kidney.

Effect of alpha-ketoglutarate on cyanide-induced biochemical alterations in rat brain and liver / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the biochemical changes in rat brain and liver following acute exposure to a lethal dose of cyanide, and its response to treatment of alpha-ketoglutarate (alpha-KG) in the absence or presence of sodium thiosulfate (STS).</p><p><b>METHODS</b>Female rats were administered 2.0 LD50 potassium cyanide (KCN; oral) in the absence or presence of pre-treatment (-10 min), simultaneous treatment (0 min) or post-treatment (+2-3 min) of alpha-KG (2.0 g/kg, oral) and/or STS (1.0 g/kg, intraperitoneal, -15 min, 0 min or + 2-3 min). At the time of onset of signs and symptoms of KCN toxicity (2-4 min) and at the time of death (5-15 min), various parameters particularly akin to oxidative stress viz. cytochrome oxidase (CYTOX), superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH) and oxidized glutathione (GSSG) in brain, and CYTOX, sorbitol dehydrogenase (SDH), alkaline phosphatase (ALP), GSH and GSSG in liver homogenate were measured.</p><p><b>RESULTS</b>At both time intervals brain CYTOX, SOD, GPx, and GSH significantly reduced (percent inhibition compared to control) to 24%, 56%, 77%, and 65%, and 44%, 46%, 78%, and 57%, respectively. At the corresponding time points liver CYTOX and GSH reduced to 74% and 63%, and 44% and 68%, respectively. The levels of GSSG in the brain and liver, and hepatic ALP and SDH were unchanged. Pre-treatment and simultaneous treatment of a-KG alone or with STS conferred significant protection on above variables. Post-treatment was effective in restoring the changes in liver but failed to normalize the changes in the brain.</p><p><b>CONCLUSIONS</b>Oral treatment with alpha-KG alone or in combination with STS has protective effects on cyanide-induced biochemical alterations in rat brain and liver.</p>

Inhibition of chlorophyll biosynthesis by mercury in excised etiolated maize leaf segments during greening: effect of 2-oxoglutarate.

Mercury (0.01-1.0 mM) inhibited chlorophyll formation in greening maize leaf segments. However, supplementing incubation medium with 2-oxoglutarate, maintained substantially higher level of chlorophyll in absence of metal after an initial period of 8 hr. On preincubation of leaf segments with HgCl2, per cent inhibition of chlorophyll synthesis by metal was same in the presence and absence of 2-oxoglutarate. Supply of 2-oxoglutarate (0.1-10.0 mM) exerted concentration dependent effect on chlorophyll formation in absence or presence of metal. Increase in delta-amino levulinic acid dehydratase as well as NADH-glutamate synthase activity and decrease in NADH-glutamate dehydrogenase activity by 2-oxoglutarate in the presence of Hg suggested that glutamate for delta-amino levulinic acid synthesis could be made available from NH4+ assimilation via., glutamine synthetase/glutamate synthase pathway during mercury toxicity.

CaCO3 stimulates alpha-ketoglutarate accumulation during pyruvate fermentation by Torulopsis glabrata / 生物工程学报

A large amount of alpha-ketoglutarate (alpha-KG) (6.8 g/L) was accumulated in flask culture when CaCO3 was used as a buffering agent in the production of pyruvate by multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019. In a 5 L jar-fermentor, less alpha-KG (1.3 g/L) was produced when NaOH was used to adjust the pH, while more alpha-KG (11.5 g/L) detected when CaCO3 was used as the buffer. In the latter case, the molar carbon ratio of pyruvate to alpha-KG (C(PYR)/ CalphaKG) was similar to that obtained in flask culture, suggesting the accumulation of alpha-ketoglutarate was related to the addition of CaCO3. Furthermore, it was found that: (1) delaying the addition time of CaCO3 decreased the a-ketoglutarate formation but increased C(PYR)/ C(alphaKG); and (2) under vitamin limitation conditions increasing the concentration of CaCO3 led to an increased a-KG accumulation at the expenses of pyruvate. To study which ions in CaCO3 was responsible for the accumulation of alpha-KG, the effects of different pH buffers on the a-KG accumulation were studied. The level of alpha-KG was found to correlate with the levels of both Ca2+ and CO3(2-), with Ca2+ played a dominant role and CO3(2-) played a minor role. To find out which pathway was responsible for the accumulation of alpha-KG, the effects of biotin and thiamine on alpha-KG accumulation was investigated. The increase in biotin concentration led to an increase in alpha-KG accumulation and a decrease in C(PYR)/ C(alpha-KG), while the levels of alpha-KG and C(PYR)/C(alphaKG) were not affected by thiamine concentration. The activity of pyruvate carboxylase was increased as much as 40% when the medium was supplemented with Ca2+ . On the other hand, the activity of the pyruvate dehydrogenase complex was unaffected by the presence of Ca2+. To conclude, the higher level of a-KG was caused by higher activity of pyruvate carboxylase stimulated by Ca2+, with CO3(2-) served as the substrate of the reaction.

Protective influences of alpha-ketoglutarate on lipid peroxidation and antioxidant status in ammonium acetate treated rats.

The effects of alpha-ketoglutarate on ammonium acetate induced hyperammonemia were studied biochemically in experimental rats. The levels of circulatory, non-protein nitrogen, serum transaminases and thiobarbituric acid reactive substances were significantly increased in ammonium acetate treated rats. These levels were significantly decreased in alpha-ketoglutarate and ammonium acetate treated rats. Similar patterns of alterations were observed in the levels of free fatty acids, triglycerides, phopholipids and cholesterol inbetween various groups. Further non-enzymatic (vitamins C and E) and enzymatic (superoxide dismutase and catalase) antioxidants were significantly decreased in ammonium acetate treated rats; and were significantly increased in alpha-ketoglutarate and ammonium acetate treated rats. The biochemical alterations during alpha-ketoglutarate treatment could be due to (i) the detoxification of excess ammonia, (ii) by participating in the non-enzymatic oxidative decarboxylation in the hydrogen peroxide decomposition process and (iii) by enhancing the proper metabolism of fats which could suppress oxygen radicals generation and thus prevent the lipid peroxidative damages in rats.

Response of sugar interconversion, starch and protein accumulation to supplied metabolites during pod filling in chickpea.

Detached chickpea inflorescences bearing pods at 20 days after flowering (DAF) were cultured for 5 days in complete liquid medium supplemented separately with asparate, myo-inositol, alpha-ketoglutarate and phytic acid. Effect of these metabolites on sugar interconvestion and starch and protein accumulation in developing pods was studied. Substituting asparate (62.5 mM) for glutamine in culture medium decreased relative proportion of sucrose in all pod tissues but increased the level of sugars, starch and protein in pod wall and cotyledons. In cotyledons, whereas myo-inositol (75 mM) reduced the accumulation of starch without affecting protein level, alpha-ketoglutarate (44 mM) increased both starch and protein accumulation. Both myo-inositol and alpha-ketoglutarate increased relative proportion of sucrose in cotyledons. Phytic acid (1 mM) decreased in cotyledons 14C incorporation from glucose into EtOH extract (principally constituted by sugars), amino acids and proteins but increased the same into starch. In cotyledons, phytic acid also increased 14C incorporation from glutamate into amino acids but this increase was negatively correlated with protein synthesis. Phytic acid decreased the relative distribution of 14C from glucose and glutamate into sucrose from pod wall but enhanced the same into EtOH extract from embryo. Based on the results, it is suggested that mode of metabolic response to exogenously supplied metabolites widely differs in pod tissues of chickpea.