Science.gov

Sample records for cycle enzymes glutamine

  1. Enzymes in the glutamate-glutamine cycle in the anterior cingulate cortex in postmortem brain of subjects with autism

    PubMed Central

    2013-01-01

    Background Accumulating evidence suggests that dysfunction in the glutamatergic system may underlie the pathophysiology of autism. The anterior cingulate cortex (ACC) has been implicated in autism as well as in glutamatergic neurotransmission. We hypothesized that alterations in the glutamate-glutamine cycle in the ACC might play a role in the pathophysiology of autism. Methods We performed Western blot analyses for the protein expression levels of enzymes in the glutamate-glutamine cycle, including glutamine synthetase, kidney-type glutaminase, liver-type glutaminase, and glutamate dehydrogenases 1 and 2, in the ACC of postmortem brain of individuals with autism (n = 7) and control subjects (n = 13). Results We found that the protein levels of kidney-type glutaminase, but not those of the other enzymes measured, in the ACC were significantly lower in subjects with autism than in controls. Conclusion The results suggest that reduced expression of kidney-type glutaminase may account for putative alterations in glutamatergic neurotransmission in the ACC in autism. PMID:23531457

  2. Glutamine metabolism and cycling in Neurospora crassa.

    PubMed Central

    Mora, J

    1990-01-01

    Evidence for the existence of a glutamine cycle in Neurospora crassa is reviewed. Through this cycle glutamine is converted into glutamate by glutamate synthase and catabolized by the glutamine transaminase-omega-amidase pathway, the products of which (2-oxoglutarate and ammonium) are the substrates for glutamate dehydrogenase-NADPH, which synthesizes glutamate. In the final step ammonium is assimilated into glutamine by the action of a glutamine synthetase (GS), which is formed by two distinct polypeptides, one catalytically very active (GS beta), and the other (GS alpha) less active but endowed with the capacity to modulate the activity of GS alpha. Glutamate synthase uses the amide nitrogen of glutamine to synthesize glutamate; glutamate dehydrogenase uses ammonium, and both are required to maintain the level of glutamate. The energy expended in the synthesis of glutamine drives the cycle. The glutamine cycle is not futile, because it is necessary to drive an effective carbon flow to support growth; in addition, it facilitates the allocation of nitrogen or carbon according to cellular demands. The glutamine cycle which dissipates energy links catabolism and anabolism and, in doing so, buffers variations in the nutrient supply and drives energy generation and carbon flow for optimal cell function. PMID:2145504

  3. Imbalanced expression of glutamate-glutamine cycle enzymes induced by human T-cell lymphotropic virus type 1 Tax protein in cultivated astrocytes.

    PubMed Central

    Akaoka, H; Hardin-Pouzet, H; Bernard, A; Verrier, B; Belin, M F; Giraudon, P

    1996-01-01

    Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent involved in the disease HTLV-1-associated myelopathy, or tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is poorly understood, but it is probable that viral infection has an indirect, deleterious effect on neural function. In this regard, dysfunction in astrocytes may be severely detrimental, as they supply neurons with metabolic precursors, control the extracellular levels of ion and excitatory neurotransmitters, and are electrically coupled with oligodendrocytes. In a model in vitro, we demonstrate that HTLV-1 induces an imbalance in the expression of two astrocyte enzymes, at both the transcriptional and translational levels. In both human astrocyte precursors and rat glial cells, the levels of expression of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were increased and decreased, respectively, after coculture with HTLV-1 T cells. The enhancement of GS expression may result from the action of the protein Tax, which is demonstrated to transactivate the GS gene promoter, while the decreased expression of GDH seems to reflect some compensatory mechanism in response to GS induction. GS and GDH are involved in the conversion of glutamate into glutamine or alpha-ketoglutarate, which then acts as a precursor for glutamatergic and gamma-aminobutyric acid (GABA)-ergic neurons. Metabolism in astrocytes altered by Tax protein may lead to deleterious effects if it modifies the extracellular levels of glutamine, glutamate, and GABA and thus modulates neuronal excitability and osmotic equilibrium in the central nervous system of HTLV-1-infected patients. PMID:8971000

  4. Enzyme-based flow injection analysis system for glutamine and glutamate in mammalian cell culture media.

    PubMed

    Mayer, C; Frauer, A; Schalkhammer, T; Pittner, F

    1999-03-01

    We present the setup of a flow injection analysis system designed for on-line monitoring of glutamate and glutamine. These amino acids represent a major energy source in mammalian cell culture. A cycling assay consisting of glutamate dehydrogenase and aspartate aminotransferase produces NADH proportional to the glutamate concentration in the sample. NADH is then measured spectrophotometrically. Glutamine is determined by conversion to glutamate which is fed into the cycling assay. The conversion of glutamine to glutamate is catalyzed by asparaginase. Asparaginase was used in place of glutaminase due to its relatively high reactivity with glutamine and a pH optimum similar to that of glutamate dehydrogenase. The enzymes were immobilized covalently to activated controlled pore glass beads and integrated into the flow injection analysis system. The application of the immobilized enzymes and the technical setup are presented in this paper.

  5. Enzymes utilizing glutamine as an amide donor.

    PubMed

    Zalkin, H; Smith, J L

    1998-01-01

    Amide nitrogen from glutamine is a major source of nitrogen atoms incorporated biosynthetically into other amino acids, purine and pyrimidine bases, amino-sugars, and coenzymes. A family comprised of at least sixteen amidotransferases are known to catalyze amide nitrogen transfer from glutamine to their acceptor substrates. Recent fine structural advances, largely as a result of X-ray crystallography, now provide structure-based mechanisms that help to explain fundamental aspects of the catalytic and regulatory interactions of several of these aminotransferases. This chapter provides an overview of this recent progress made on the characterization of amidotransferase structure and mechanism. PMID:9559052

  6. GLUTAMINE AND HYPERAMMONEMIC CRISES IN PATIENTS WITH UREA CYCLE DISORDERS

    PubMed Central

    Lee, B.; Diaz, G.A.; Rhead, W.; Lichter-Konecki, U.; Feigenbaum, A.; Berry, S.A.; Le Mons, C.; Bartley, J.; Longo, N.; Nagamani, S.C.; Berquist, W.; Gallagher, R.C.; Harding, C.O.; McCandless, S.E.; Smith, W.; Schulze, A.; Marino, M.; Rowell, R.; Coakley, D.F.; Mokhtarani, M.; Scharschmidt, B.F.

    2016-01-01

    Blood ammonia and glutamine levels are used as biomarkers of control in patients with urea cycle disorders (UCDs). This study was undertaken to evaluate glutamine variability and utility as a predictor of hyperammonemic crises (HACs) in UCD patients. Methods The relationships between glutamine and ammonia levels and the incidence and timing of HACs were evaluated in over 100 adult and pediatric UCD patients who participated in clinical trials of glycerol phenylbutyrate. Results The median (range) intra-subject 24-hour coefficient of variation for glutamine was 15% (8–29%) as compared with 56% (28%–154%) for ammonia, and the correlation coefficient between glutamine and concurrent ammonia levels varied from 0.17 to 0.29. Patients with baseline (fasting) glutamine values >900 µmol/L had higher baseline ammonia levels (mean [SD]: 39.6 [26.2] µmol/L) than patients with baseline glutamine ≤900 µmol/L (26.6 [18.0] µmol/L). Glutamine values >900 µmol/L during the study were associated with an approximately 2-fold higher HAC risk (odds ratio [OR]=1.98; p=0.173). However, glutamine lost predictive significance (OR=1.47; p=0.439) when concomitant ammonia was taken into account, whereas the predictive value of baseline ammonia ≥ 1.0 upper limit of normal (ULN) was highly statistically significant (OR=4.96; p=0.013). There was no significant effect of glutamine >900 µmol/L on time to first HAC crisis (hazard ratio [HR]=1.14; p=0.813), but there was a significant effect of baseline ammonia ≥ 1.0 ULN (HR=4.62; p=0.0011). Conclusions The findings in this UCD population suggest that glutamine is a weaker predictor of HACs than ammonia and that the utility of the predictive value of glutamine will need to take into account concurrent ammonia levels. PMID:26586473

  7. Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored product pests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cereals have storage proteins with high amounts of the amino acids glutamine and proline. Therefore, storage pests need to have digestive enzymes that are efficient in hydrolyzing these types of proteins. Post-glutamine cleaving peptidases (PGP) were isolated from the midgut of the stored product pe...

  8. Glutamine affects glutathione recycling enzymes in a DMBA-induced breast cancer model.

    PubMed

    Kaufmann, Yihong; Todorova, Valentina K; Luo, Shaoke; Klimberg, V Suzanne

    2008-01-01

    Malignancy depletes host glutathione (GSH) levels to increase treatment-related toxicity and increases itself to resist the treatments. Our previous studies have shown that dietary glutamine (GLN) prevented 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors through enhancing gut GSH release and reducing tumor GSH level. In addition, GSH synthesis, metabolism, and recycling are accomplished in gamma-glutamyl cycle. We hypothesized that the GLN prevention might be through a differential regulation of the gamma-glutamyl cycle enzymes. Female Sprague-Dawley rats were randomized into DMBA-tumor bearing, DMBA-treated, and control groups subdivided into GLN and water groups. GLN supplementation was given at 1 g/kg/day by gastric gavage. The activities and messenger RNA levels of gamma-glutamyl transpeptidase (GTP), gamma-glutamylcysteine synthetase (GCS), 5-oxo-L-prolinase (OPase), gamma-glutamyl transferase (GTF), and glutaminase (GLNase) were determined in gut mucosa and breast tumor using specific enzyme assays and semiquantitative reverse transcription polymerase chain reaction. GLN upregulated gut GTP, GCS, OPase, and GLNase in DMBA-tumor bearing, DMBA-treated, and/or control rats; however, it downregulated these enzymes in the tumor. The paradoxical effect of GLN on key GSH recycling enzymes in the gut versus tumor suggests that dietary supplemental GLN could be used in the clinical practice to increase the therapeutic index of cancer treatments by protecting normal tissues from, and sensitizing tumor cells to, chemotherapy and radiation-related injury.

  9. A novel glutamine biosensor based on zinc oxide nanorod and glutaminase enzyme from Hypocria jecorina.

    PubMed

    Albayrak, Dilruba; Karakuş, Emine

    2016-01-01

    A novel biosensor for determination of L-glutamine in pharmaceutical glutamine powder was developed via immobilizing our produced glutaminase enzyme from Hypocria jecorina onto our prepared zinc oxide (ZnO) nanorod and chitosan. ZnO nanorods were prepared as surface-dependent and surface-independent and both were used. The biosensor is specific for L-glutamine and the peculiar analytical properties (linearity range, reproducibility, and accuracy) of it were experimentally determined. The optimum operating conditions of the biosensor such as buffer concentration, buffer pH, and medium temperature effect on the response of biosensor were studied. Km and Vmax values for the our-producing glutaminase enzyme from Hypocria jecorina immobilized on the biosensor were also determined as 0.29 mM and 208.33 mV/min., respectively, from Lineweaver-Burk plot. The biosensor was then used for the determination of glutamine contained in pharmaceutical formulations.

  10. Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport.

    PubMed

    Yang, Chendong; Ko, Bookyung; Hensley, Christopher T; Jiang, Lei; Wasti, Ajla T; Kim, Jiyeon; Sudderth, Jessica; Calvaruso, Maria Antonietta; Lumata, Lloyd; Mitsche, Matthew; Rutter, Jared; Merritt, Matthew E; DeBerardinis, Ralph J

    2014-11-01

    Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

  11. Disruption of glutamate-glutamine-GABA cycle significantly impacts on suicidal behaviour: survey of the literature and own findings on glutamine synthetase.

    PubMed

    Bernstein, Hans-Gert; Tausch, Anne; Wagner, Rebecca; Steiner, Johann; Seeleke, Patrick; Walter, Martin; Dobrowolny, Henrik; Bogerts, Bernhard

    2013-11-01

    The aetiology of suicide is complex and still not completely understood. The present communication, which consists of two parts, aims to shed some light on the role of amino acidergic neurotransmission in suicide. In the first part we provide an overview of the literature showing that with the exception of certain gamma-aminobutyric acid transporters, virtually all components of the glutamate-glutamine- gamma-aminobutyric acid cycle are, in some way or other, abnormal in suicide victims, which indicates a prominent involvement of the glutamatergic and gammaaminobutyric acidergic neurotransmitter systems in suicidal behaviour. In the second part we present own immunohistochemical findings showing that densities of glutamine synthetase expressing glial cells in the mediodorsal thalamus as well as in the dorsolateral prefrontal and orbitofrontal cortex of schizophrenic suicide completers are significantly elevated compared with controls and non-suicide individuals with schizophrenia, thus calling into question the belief that cerebral glutamine synthetase deficit is indicative of suicidal behaviour.

  12. Heat maps for intramolecular communication in an RNP enzyme encoding glutamine.

    PubMed

    Rodríguez-Hernández, Annia; Perona, John J

    2011-03-01

    Allosteric signaling within large ribonucleoproteins modulates both catalytic function and biological specificity, but the spatial extent and quantitative magnitudes of long-distance free-energy couplings have yet to be well characterized. Here, we employ pre-steady-state kinetics to generate a comprehensive mapping of intramolecular communication in the glutaminyl-tRNA synthetase:tRNA(Gln) complex. Alanine substitution at 29 positions across the protein-RNA interface reveals distinct coupling amplitudes for glutamine binding and aminoacyl-tRNA formation on the enzyme, respectively, implying the existence of multiple signaling pathways. Structural models suggest that long-range signal propagation from the tRNA anticodon is dynamically driven, whereas shorter pathways are mediated by induced-fit rearrangements. Seven protein contacts with the distal tRNA vertical arm each weaken glutamine binding affinity across distances up to 40 Å, demonstrating that negative allosteric coupling plays a key role in enforcing the selective RNA-amino acid pairing at the heart of the genetic code. PMID:21397189

  13. Prefrontal changes in the glutamate-glutamine cycle and neuronal/glial glutamate transporters in depression with and without suicide.

    PubMed

    Zhao, J; Verwer, R W H; van Wamelen, D J; Qi, X-R; Gao, S-F; Lucassen, P J; Swaab, D F

    2016-11-01

    There are indications for changes in glutamate metabolism in relation to depression or suicide. The glutamate-glutamine cycle and neuronal/glial glutamate transporters mediate the uptake of the glutamate and glutamine. The expression of various components of the glutamate-glutamine cycle and the neuronal/glial glutamate transporters was determined by qPCR in postmortem prefrontal cortex. The anterior cingulate cortex (ACC) and the dorsolateral prefrontal cortex (DLPFC) were selected from young MDD patients who had committed suicide (MDD-S; n = 17), from MDD patients who died of non-suicide related causes (MDD-NS; n = 7) and from matched control subjects (n = 12). We also compared elderly depressed patients who had not committed suicide (n = 14) with matched control subjects (n = 22). We found that neuronal located components (EAAT3, EAAT4, ASCT1, SNAT1, SNAT2) of the glutamate-glutamine cycle were increased in the ACC while the astroglia located components (EAAT1, EAAT2, GLUL) were decreased in the DLPFC of MDD-S patients. In contrast, most of the components in the cycle were increased in the DLPFC of MDD-NS patients. In conclusion, the glutamate-glutamine cycle - and thus glutamine transmission - is differentially affected in depressed suicide patients and depressed non-suicide patients in an area specific way.

  14. Allosteric regulation of monocyclic interconvertible enzyme cascade systems: use of Escherichia coli glutamine synthetase as an experimental model.

    PubMed

    Rhee, S G; Park, R; Chock, P B; Stadtman, E R

    1978-07-01

    The interconversion of Escherichia coli glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] between its adenylylated and unadenylylated forms has been used to verify the prediction derived from a theoretical analysis of the steady-state functions of a model for a monocyclic interconvertible enzyme cascade system [Stadtman, E. R. & Chock, P. B. (1977) Proc. Natl. Acad. Sci. USA 74, 2761-2770]. Because glutamine and alpha-ketoglutarate are multifunctional effectors and because three active enzyme complexes are involved in both adenylylation and deadenylylation of glutamine synthetase, at least 28 constants are required to describe the glutamine synthetase monocyclic cascade. Of these, 22 constants were determined experimentally and 6 were estimated via computer curve fitting. Despite the complexity, when both adenylylation and deadenylylation reactions are functioning, the number of adenylyl groups bound per mole of enzyme, n, assumes a steady-state level as is predicted by the model. This n value is determined by the mole fraction of P(IIA)-given by ([P(IIA)]/([P(IIA)] + [P(IID)])-and the ratio of glutamine to alpha-ketoglutarate (P(IID) and P(IID) are the unmodified and the uridylylated forms of the P(II) regulatory protein). In the presence of 0.5 mM glutamine and 2 mM alpha-ketoglutarate, the value of n increases as a nearly hyperbolic function in response to increasing mole fractions of P(IIA). When the constant level of alpha-ketoglutarate is gradually increased to 40 muM, the hyperbolic function converts slowly to a parabolic function. When the P(IIA) mole fraction was maintained at 0.6 and alpha-ketoglutarate levels were varied from 1 mM to 4 muM, an 800-fold increase in signal amplification was observed with respect to glutamine activation. In addition, because glutamine activates the adenylylation and inhibits the deadenylylation reaction, a sensitivity index of 2.1 (corresponding to a Hill number of 1.5) was obtained for the

  15. Glutamine Synthetase in Legumes: Recent Advances in Enzyme Structure and Functional Genomics

    PubMed Central

    Betti, Marco; García-Calderón, Margarita; Pérez-Delgado, Carmen M.; Credali, Alfredo; Estivill, Guillermo; Galván, Francisco; Vega, José M.; Márquez, Antonio J.

    2012-01-01

    Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N2 fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1) or plastidic (GS2) isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photorespiration and water stress, in this latter case by means of proline production. Functional genomic analysis using GS2-minus mutant reveals the key role of GS2 in the metabolic control of the plants and, more particularly, in carbon metabolism. PMID:22942686

  16. mTORC2 Responds to Glutamine Catabolite Levels to Modulate the Hexosamine Biosynthesis Enzyme GFAT1.

    PubMed

    Moloughney, Joseph G; Kim, Peter K; Vega-Cotto, Nicole M; Wu, Chang-Chih; Zhang, Sisi; Adlam, Matthew; Lynch, Thomas; Chou, Po-Chien; Rabinowitz, Joshua D; Werlen, Guy; Jacinto, Estela

    2016-09-01

    Highly proliferating cells are particularly dependent on glucose and glutamine for bioenergetics and macromolecule biosynthesis. The signals that respond to nutrient fluctuations to maintain metabolic homeostasis remain poorly understood. Here, we found that mTORC2 is activated by nutrient deprivation due to decreasing glutamine catabolites. We elucidate how mTORC2 modulates a glutamine-requiring biosynthetic pathway, the hexosamine biosynthesis pathway (HBP) via regulation of expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme of the HBP. GFAT1 expression is dependent on sufficient amounts of glutaminolysis catabolites particularly α-ketoglutarate, which are generated in an mTORC2-dependent manner. Additionally, mTORC2 is essential for proper expression and nuclear accumulation of the GFAT1 transcriptional regulator, Xbp1s. Thus, while mTORC1 senses amino acid abundance to promote anabolism, mTORC2 responds to declining glutamine catabolites in order to restore metabolic homeostasis. Our findings uncover the role of mTORC2 in metabolic reprogramming and have implications for understanding insulin resistance and tumorigenesis. PMID:27570073

  17. Glutamine fuels a vicious cycle of autophagy in the tumor stroma and oxidative mitochondrial metabolism in epithelial cancer cells: implications for preventing chemotherapy resistance.

    PubMed

    Ko, Ying-Hui; Lin, Zhao; Flomenberg, Neal; Pestell, Richard G; Howell, Anthony; Sotgia, Federica; Lisanti, Michael P; Martinez-Outschoorn, Ubaldo E

    2011-12-15

    Glutamine metabolism is crucial for cancer cell growth via the generation of intermediate molecules in the tricarboxylic acid (TCA) cycle, antioxidants and ammonia. The goal of the current study was to evaluate the effects of glutamine on metabolism in the breast cancer tumor microenvironment, with a focus on autophagy and cell death in both epithelial and stromal compartments. For this purpose, MCF7 breast cancer cells were cultured alone or co-cultured with non-transformed fibroblasts in media containing high glutamine and low glucose (glutamine +) or under control conditions, with no glutamine and high glucose (glutamine -). Here, we show that MCF7 cells maintained in co-culture with glutamine display increased mitochondrial mass, as compared with control conditions. Importantly, treatment with the autophagy inhibitor chloroquine abolishes the glutamine-induced augmentation of mitochondrial mass. It is known that loss of caveolin-1 (Cav-1) expression in fibroblasts is associated with increased autophagy and an aggressive tumor microenvironment. Here, we show that Cav-1 downregulation which occurs in fibroblasts maintained in co-culture specifically requires glutamine. Interestingly, glutamine increases the expression of autophagy markers in fibroblasts, but decreases expression of autophagy markers in MCF7 cells, indicating that glutamine regulates the autophagy program in a compartment-specific manner. Functionally, glutamine protects MCF7 cells against apoptosis, via the upregulation of the anti-apoptotic and anti-autophagic protein TIGAR. Also, we show that glutamine cooperates with stromal fibroblasts to confer tamoxifen-resistance in MCF7 cancer cells. Finally, we provide evidence that co-culture with fibroblasts (1) promotes glutamine catabolism, and (2) decreases glutamine synthesis in MCF7 cancer cells. Taken together, our findings suggest that autophagic fibroblasts may serve as a key source of energy-rich glutamine to fuel cancer cell mitochondrial

  18. In vivo nuclear magnetic resonance spectroscopy studies of the relationship between the glutamate-glutamine neurotransmitter cycle and functional neuroenergetics.

    PubMed Central

    Rothman, D L; Sibson, N R; Hyder, F; Shen, J; Behar, K L; Shulman, R G

    1999-01-01

    In this article we review recent studies, primarily from our laboratory, using 13C NMR (nuclear magnetic resonance) to non-invasively measure the rate of the glutamate-glutamine neurotransmitter cycle in the cortex of rats and humans. In the glutamate-glutamine cycle, glutamate released from nerve terminals is taken up by surrounding glial cells and returned to the nerve terminals as glutamine. 13C NMR studies have shown that the rate of the glutamate-glutamine cycle is extremely high in both the rat and human cortex, and that it increases with brain activity in an approximately 1:1 molar ratio with oxidative glucose metabolism. The measured ratio, in combination with proposals based on isolated cell studies by P. J. Magistretti and co-workers, has led to the development of a model in which the majority of brain glucose oxidation is mechanistically coupled to the glutamate-glutamine cycle. This model provides the first testable mechanistic relationship between cortical glucose metabolism and a specific neuronal activity. We review here the experimental evidence for this model as well as implications for blood oxygenation level dependent magnetic resonance imaging and positron emission tomography functional imaging studies of brain function. PMID:10466144

  19. Energy metabolism and glutamate-glutamine cycle in the brain: a stoichiometric modeling perspective

    PubMed Central

    2013-01-01

    Background The energetics of cerebral activity critically relies on the functional and metabolic interactions between neurons and astrocytes. Important open questions include the relation between neuronal versus astrocytic energy demand, glucose uptake and intercellular lactate transfer, as well as their dependence on the level of activity. Results We have developed a large-scale, constraint-based network model of the metabolic partnership between astrocytes and glutamatergic neurons that allows for a quantitative appraisal of the extent to which stoichiometry alone drives the energetics of the system. We find that the velocity of the glutamate-glutamine cycle (Vcyc) explains part of the uncoupling between glucose and oxygen utilization at increasing Vcyc levels. Thus, we are able to characterize different activation states in terms of the tissue oxygen-glucose index (OGI). Calculations show that glucose is taken up and metabolized according to cellular energy requirements, and that partitioning of the sugar between different cell types is not significantly affected by Vcyc. Furthermore, both the direction and magnitude of the lactate shuttle between neurons and astrocytes turn out to depend on the relative cell glucose uptake while being roughly independent of Vcyc. Conclusions These findings suggest that, in absence of ad hoc activity-related constraints on neuronal and astrocytic metabolism, the glutamate-glutamine cycle does not control the relative energy demand of neurons and astrocytes, and hence their glucose uptake and lactate exchange. PMID:24112710

  20. Critical Evaluation of the Changes in Glutamine Synthetase Activity in Models of Cerebral Stroke.

    PubMed

    Jeitner, Thomas M; Battaile, Kevin; Cooper, Arthur J L

    2015-12-01

    The following article addresses some seemingly paradoxical observations concerning cerebral glutamine synthetase in ischemia-reperfusion injury. In the brain, this enzyme is predominantly found in astrocytes and catalyzes part of the glutamine-glutamate cycle. Glutamine synthetase is also thought to be especially sensitive to inactivation by the oxygen- and nitrogen-centered radicals generated during strokes. Despite this apparent sensitivity, glutamine synthetase specific activity is elevated in the affected tissues during reperfusion. Given the central role of the glutamine-glutamate cycle in the brain, we sought to resolve these conflicting observations with the view of providing an alternative perspective for therapeutic intervention in stroke.

  1. Glutamine metabolism to glucosamine is necessary for glutamine inhibition of endothelial nitric oxide synthesis.

    PubMed Central

    Wu, G; Haynes, T E; Li, H; Yan, W; Meininger, C J

    2001-01-01

    L-Glutamine is a physiological inhibitor of endothelial NO synthesis. The present study was conducted to test the hypothesis that metabolism of glutamine to glucosamine is necessary for glutamine inhibition of endothelial NO generation. Bovine venular endothelial cells were cultured for 24 h in the presence of 0, 0.1, 0.5 or 2 mM D-glucosamine, or of 0.2 or 2 mM L-glutamine with or without 20 microM 6-diazo-5-oxo-L-norleucine (DON) or with 100 microM azaserine. Both DON and azaserine are inhibitors of L-glutamine:D-fructose-6-phosphate transaminase (isomerizing) (EC 2.6.1.16), the first and rate controlling enzyme in glucosamine synthesis. Glucosamine at 0.1, 0.5 and 2 mM decreased NO production by 34, 45 and 56% respectively compared with controls where glucosamine was lacking. DON (20 microM) and azaserine (100 microM) blocked glucosamine synthesis and prevented the inhibition of NO generation by glutamine. Neither glutamine nor glucosamine had an effect on NO synthase (NOS) activity, arginine transport or cellular tetrahydrobiopterin and Ca(2+) levels. However, both glutamine and glucosamine inhibited pentose cycle activity and decreased cellular NADPH concentrations; these effects of glutamine were abolished by DON or azaserine. Restoration of cellular NADPH levels by the addition of 1 mM citrate also prevented the inhibiting effect of glutamine or glucosamine on NO synthesis. A further increase in cellular NADPH levels by the addition of 5 mM citrate resulted in greater production of NO. Collectively, our results demonstrate that the metabolism of glutamine to glucosamine is necessary for the inhibition of endothelial NO generation by glutamine. Glucosamine reduces the cellular availability of NADPH (an essential cofactor for NOS) by inhibiting pentose cycle activity, and this may be a metabolic basis for the inhibition of endothelial NO synthesis by glucosamine. PMID:11139387

  2. The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme.

    PubMed

    Moreadith, R W; Lehninger, A L

    1984-05-25

    Little evidence has been available on the oxidative pathways of glutamine and glutamate, the major respiratory substrates of cancer cells. Glutamate formed from glutamine by phosphate-dependent glutaminase undergoes quantitative transamination by aerobic tumor mitochondria to yield aspartate. However, when malate is also added there is a pronounced decrease in aspartate production and a large formation of citrate and alanine, in both state 3 and 4 conditions. In contrast, addition of malate to normal rat heart, liver, or kidney mitochondria oxidizing glutamate causes a marked increase in aspartate production. Further analysis showed that extramitochondrial malate is oxidized almost quantitatively to pyruvate + CO2 by NAD(P)+-linked malic enzyme, present in the mitochondria of all tumors tested, but absent in heart, liver, and kidney mitochondria. On the other hand intramitochondrial malate generated from glutamate is oxidized quantitatively to oxalacetate by mitochondrial malate dehydrogenase of tumors. Acetyl-CoA derived from extramitochondrial malate via pyruvate and oxalacetate derived from glutamate via intramitochondrial malate are quantitatively converted into citrate, which is extruded. No evidence was found that malic enzyme of tumor mitochondria converts glutamate-derived malate into pyruvate as postulated in other reports. Possible mechanisms for the integration of mitochondrial malic enzyme and malate dehydrogenase activities in tumors are discussed.

  3. ω-Amidase: an underappreciated, but important enzyme in L-glutamine and L-asparagine metabolism; relevance to sulfur and nitrogen metabolism, tumor biology and hyperammonemic diseases.

    PubMed

    Cooper, Arthur J L; Shurubor, Yevgeniya I; Dorai, Thambi; Pinto, John T; Isakova, Elena P; Deryabina, Yulia I; Denton, Travis T; Krasnikov, Boris F

    2016-01-01

    In mammals, two major routes exist for the metabolic conversion of L-glutamine to α-ketoglutarate. The most widely studied pathway involves the hydrolysis of L-glutamine to L-glutamate catalyzed by glutaminases, followed by the conversion of L-glutamate to α-ketoglutarate by the action of an L-glutamate-linked aminotransferase or via the glutamate dehydrogenase reaction. However, another major pathway exists in mammals for the conversion of L-glutamine to α-ketoglutarate (the glutaminase II pathway) in which L-glutamine is first transaminated to α-ketoglutaramate (KGM) followed by hydrolysis of KGM to α-ketoglutarate and ammonia catalyzed by an amidase known as ω-amidase. In mammals, the glutaminase II pathway is present in both cytosolic and mitochondrial compartments and is most prominent in liver and kidney. Similarly, two routes exist for the conversion of L-asparagine to oxaloacetate. In the most extensively studied pathway, L-asparagine is hydrolyzed to L-aspartate by the action of asparaginase, followed by transamination of L-aspartate to oxaloacetate. However, another pathway also exists for the conversion of L-asparagine to oxaloacetate (the asparaginase II pathway). In this pathway, L-asparagine is first transaminated to α-ketosuccinamate (KSM), followed by hydrolysis of KSM to oxaloacetate by the action of ω-amidase. One advantage of both the glutaminase II and the asparaginase II pathways is that they are irreversible, and thus are important in anaplerosis by shuttling 5-C (α-ketoglutarate) and 4-C (oxaloacetate) units into the TCA cycle. In this review, we briefly mention the importance of the glutaminase II and asparaginase II pathways in microorganisms and plants. However, the major emphasis of the review is related to the importance of these pathways (especially the common enzyme component of both pathways--ω-amidase) in nitrogen and sulfur metabolism in mammals and as a source of anaplerotic carbon moieties in rapidly dividing cells. The

  4. ω-Amidase: an underappreciated, but important enzyme in L-glutamine and L-asparagine metabolism; relevance to sulfur and nitrogen metabolism, tumor biology and hyperammonemic diseases.

    PubMed

    Cooper, Arthur J L; Shurubor, Yevgeniya I; Dorai, Thambi; Pinto, John T; Isakova, Elena P; Deryabina, Yulia I; Denton, Travis T; Krasnikov, Boris F

    2016-01-01

    In mammals, two major routes exist for the metabolic conversion of L-glutamine to α-ketoglutarate. The most widely studied pathway involves the hydrolysis of L-glutamine to L-glutamate catalyzed by glutaminases, followed by the conversion of L-glutamate to α-ketoglutarate by the action of an L-glutamate-linked aminotransferase or via the glutamate dehydrogenase reaction. However, another major pathway exists in mammals for the conversion of L-glutamine to α-ketoglutarate (the glutaminase II pathway) in which L-glutamine is first transaminated to α-ketoglutaramate (KGM) followed by hydrolysis of KGM to α-ketoglutarate and ammonia catalyzed by an amidase known as ω-amidase. In mammals, the glutaminase II pathway is present in both cytosolic and mitochondrial compartments and is most prominent in liver and kidney. Similarly, two routes exist for the conversion of L-asparagine to oxaloacetate. In the most extensively studied pathway, L-asparagine is hydrolyzed to L-aspartate by the action of asparaginase, followed by transamination of L-aspartate to oxaloacetate. However, another pathway also exists for the conversion of L-asparagine to oxaloacetate (the asparaginase II pathway). In this pathway, L-asparagine is first transaminated to α-ketosuccinamate (KSM), followed by hydrolysis of KSM to oxaloacetate by the action of ω-amidase. One advantage of both the glutaminase II and the asparaginase II pathways is that they are irreversible, and thus are important in anaplerosis by shuttling 5-C (α-ketoglutarate) and 4-C (oxaloacetate) units into the TCA cycle. In this review, we briefly mention the importance of the glutaminase II and asparaginase II pathways in microorganisms and plants. However, the major emphasis of the review is related to the importance of these pathways (especially the common enzyme component of both pathways--ω-amidase) in nitrogen and sulfur metabolism in mammals and as a source of anaplerotic carbon moieties in rapidly dividing cells. The

  5. Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii.

    PubMed

    Nitzsche, Richard; Zagoriy, Vyacheslav; Lucius, Richard; Gupta, Nishith

    2016-01-01

    Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells. PMID:26518878

  6. Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii.

    PubMed

    Nitzsche, Richard; Zagoriy, Vyacheslav; Lucius, Richard; Gupta, Nishith

    2016-01-01

    Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.

  7. Activity of the lactate-alanine shuttle is independent of glutamate-glutamine cycle activity in cerebellar neuronal-astrocytic cultures.

    PubMed

    Bak, Lasse K; Sickmann, Helle M; Schousboe, Arne; Waagepetersen, Helle S

    The glutamate-glutamine cycle describes the neuronal release of glutamate into the synaptic cleft, astrocytic uptake, and conversion into glutamine, followed by release for use as a neuronal glutamate precursor. This only explains the fate of the carbon atoms, however, and not that of the ammonia. Recently, a role for alanine has been proposed in transfer of ammonia between glutamatergic neurons and astrocytes, denoted the lactate-alanine shuttle (Waagepetersen et al. [ 2000] J. Neurochem. 75:471-479). The role of alanine in this context has been studied further using cerebellar neuronal cultures and corresponding neuronal-astrocytic cocultures. A superfusion paradigm was used to induce repetitively vesicular glutamate release by N-methyl-D-aspartate (NMDA) in the neurons, allowing the relative activity dependency of the lactate-alanine shuttle to be assessed. [(15)N]Alanine (0.2 mM), [2-(15)N]/[5-(15)N]glutamine (0.25 mM), and [(15)N]ammonia (0.3 mM) were used as precursors and cell extracts were analyzed by mass spectrometry. Labeling from [(15)N]alanine in glutamine, aspartate, and glutamate in cerebellar cocultures was independent of depolarization of the neurons. Employing glutamine with the amino group labeled ([2-(15)N]glutamine) as the precursor, an activity-dependent increase in the labeling of both glutamate and aspartate (but not alanine) was observed in the cerebellar neurons. When the amide group of glutamine was labeled ([5-(15)N]glutamine), no labeling could be detected in the analyzed metabolites. Altogether, the results of this study support the existence of the lactate-alanine shuttle and the associated glutamate-glutamine cycle. No direct coupling of the two shuttles was observed, however, and only the glutamate-glutamine cycle seemed activity dependent.

  8. PGC-1α supports glutamine metabolism in breast cancer

    PubMed Central

    2013-01-01

    Background Glutamine metabolism is a central metabolic pathway in cancer. Recently, reductive carboxylation of glutamine for lipogenesis has been shown to constitute a key anabolic route in cancer cells. However, little is known regarding central regulators of the various glutamine metabolic pathways in cancer cells. Methods The impact of PGC-1α and ERRα on glutamine enzyme expression was assessed in ERBB2+ breast cancer cell lines with quantitative RT-PCR, chromatin immunoprecipitation, and immunoblotting experiments. Glutamine flux was quantified using 13C-labeled glutamine and GC/MS analyses. Functional assays for lipogenesis were performed using 14C-labeled glutamine. The expression of glutamine metabolism genes in breast cancer patients was determined by bioinformatics analyses using The Cancer Genome Atlas. Results We show that the transcriptional coactivator PGC-1α, along with the transcription factor ERRα, is a positive regulator of the expression of glutamine metabolism genes in ERBB2+ breast cancer. Indeed, ERBB2+ breast cancer cells with increased expression of PGC-1α display elevated expression of glutamine metabolism genes. Furthermore, ERBB2+ breast cancer cells with reduced expression of PGC-1α or when treated with C29, a pharmacological inhibitor of ERRα, exhibit diminished expression of glutamine metabolism genes. The biological relevance of the control of glutamine metabolism genes by the PGC-1α/ERRα axis is demonstrated by consequent regulation of glutamine flux through the citric acid cycle. PGC-1α and ERRα regulate both the canonical citric acid cycle (forward) and the reductive carboxylation (reverse) fluxes; the latter can be used to support de novo lipogenesis reactions, most notably in hypoxic conditions. Importantly, murine and human ERBB2+ cells lines display a significant dependence on glutamine availability for their growth. Finally, we show that PGC-1α expression is positively correlated with that of the glutamine pathway

  9. Glutamate-glutamine cycle and exchange in the placenta-fetus unit during late pregnancy.

    PubMed

    Wu, Xin; Xie, Chunyan; Zhang, Yuzhe; Fan, Zhiyong; Yin, Yulong; Blachier, Francois

    2015-01-01

    The present review focuses on the physiological functions of glutamate-glutamine exchange involving placental amino acid transport and umbilical amino acid uptake in mammals (particularly in sows), with special emphasis on the associated regulating mechanisms. Glutamate plus glutamine are among the most abundant and the most utilized amino acids in fetus during late gestation. During pregnancy, amino acids, notably as precursors of macromolecules including proteins and nucleotides are involved in fetal development and growth. Amino acid concentrations in fetus are generally higher than in the mother. Among amino acids, the transport and metabolism of glutamate and glutamine during fetal development exhibit characteristics that clearly emphasize the importance of the interaction between the placenta and the fetal liver. Glutamate is quite remarkable among amino acids, which originate from the placenta, and is cleared from fetal plasma. In addition, the flux of glutamate through the placenta from the fetal plasma is highly correlated with the umbilical glutamate delivery rate. Glutamine plays a central role in fetal carbon and nitrogen metabolism and exhibits one of the highest fetal/maternal plasma ratio among all amino acids in human and other mammals. Glutamate is taken up by placenta from the fetal circulation and then converted to glutamine before being released back into the fetal circulation. Works are required on the glutamate-glutamine metabolism during late pregnancy in physiological and pathophysiological situations since such works may help to improve fetal growth and development both in humans and other mammals. Indeed, glutamine supplementation appears to ameliorate fetal growth retardation in sows and reduces preweaning mortality of piglets.

  10. BLOOD AMMONIA AND GLUTAMINE AS PREDICTORS OF HYPERAMMONEMIC CRISES IN UREA CYCLE DISORDER PATIENTS

    PubMed Central

    Lee, Brendan; Diaz, George A.; Rhead, William; Lichter-Konecki, U.; Feigenbaum, Annette; Berry, Susan A.; Le Mons, C.; Bartley, James A; Longo, Nicola; Nagamani, Sandesh C.; Berquist, William; Gallagher, Renata; Bartholomew, Dennis; Harding, Cary O.; Korson, Mark S.; McCandless, Shawn E.; Smith, Wendy; Cederbaum, Stephen; Wong, Derek; Merritt, J. Lawrence; Schulze, A.; Vockley, Gerard.; Kronn, David; Zori, Roberto; Summar, Marshall; Milikien, D.A.; Marino, M.; Coakley, D.F.; Mokhtarani, M.; Scharschmidt, B.F.

    2014-01-01

    Purpose To examine predictors of ammonia exposure and hyperammonemic crises (HAC) in patients with urea cycle disorders (UCDs). Methods The relationships between fasting ammonia, daily ammonia exposure, and HACs were analyzed in >100 UCD patients. Results Fasting ammonia correlated strongly with daily ammonia exposure (r=0.764, p<0.001). For patients with fasting ammonia levels <0.5 ULN, 0.5 to <1.0 ULN, and ≥1.0 ULN, the probability of a normal average daily ammonia value was 87%, 60%, and 39%, respectively, and 10.3%, 14.1%, and 37.0% of these patients experienced ≥1 HAC over 12 months. Time to first HAC was shorter (p=0.008) and relative risk (4.5×; p=0.011) and rate (~5×, p=0.006) of HACs higher in patients with fasting ammonia ≥1.0 ULN vs. <0.5ULN; relative risk was even greater (20×; p=0.009) in patients ≥6 years. A 10 or 25 μmol/L increase in ammonia exposure increased the relative risk of a HAC by 50% and >200% (p<0.0001), respectively. The relationship between ammonia and HAC risk appeared independent of treatment, age, UCD subtype, dietary protein intake, or blood urea nitrogen. Fasting glutamine correlated weakly with AUC0-24 and was not a significant predictor of HACs. Conclusions Fasting ammonia correlates strongly and positively with daily ammonia exposure and with the risk and rate of HACs, suggesting that UCD patients may benefit from tight ammonia control. PMID:25503497

  11. Astrocyte glutamine synthetase: pivotal in health and disease.

    PubMed

    Rose, Christopher F; Verkhratsky, Alexei; Parpura, Vladimir

    2013-12-01

    The multifunctional properties of astrocytes signify their importance in brain physiology and neurological function. In addition to defining the brain architecture, astrocytes are primary elements of brain ion, pH and neurotransmitter homoeostasis. GS (glutamine synthetase), which catalyses the ATP-dependent condensation of ammonia and glutamate to form glutamine, is an enzyme particularly found in astrocytes. GS plays a pivotal role in glutamate and glutamine homoeostasis, orchestrating astrocyte glutamate uptake/release and the glutamate-glutamine cycle. Furthermore, astrocytes bear the brunt of clearing ammonia in the brain, preventing neurotoxicity. The present review depicts the central function of astrocytes, concentrating on the importance of GS in glutamate/glutamine metabolism and ammonia detoxification in health and disease.

  12. Impairment of glutamine/glutamate-γ-aminobutyric acid cycle in manganese toxicity in the central nervous system.

    PubMed

    Sidoryk-Wegrzynowicz, M

    2014-01-01

    Manganese (Mn) is an essential trace element that is required for maintaining the proper function and regulation of many biochemical and cellular reactions. Despite its essentiality, at excessive levels Mn is toxic to the central nervous system. The overdose accumulation of Mn in specific brain areas, such as the substantia nigra, the globus pallidus and the striatum, triggers neurotoxicity resulting in a neurological brain disorder, referred to as manganism. Manganese toxicity is associated with the disruption of glutamine (Gln)/glutamate (Glu) GABA cycle (GGC). The GGC represents a complex process, since Gln efflux from astrocytes must be met by its influx in neurons. Mn toxicity is associated with the disruption of both of these critical points in the cycle. In cultured astrocytes, pre-treatment with Mn inhibits the initial net uptake of Gln in a concentration-dependent manner. Manganese added directly to astrocytes induces deregulation in the expression of SNAT3, SNAT2, ASCT2 and LAT2 transporters and significantly decreases in Gln uptake mediated by the transporting Systems N and ASC, and a decrease in Gln efflux mediated by Systems N, ASC and L. Further, Mn disrupts Glu transporting systems leading to both a reduction in Glu uptake and elevation in extracellular Glu levels. Interestingly, there appear to be common signaling targets of Mn in GGC cycling in glial cells. Namely, the PKC signaling is affected by Mn in Gln and Glu transporters expression and function. Additionally, Mn was identified to deregulate glutamine synthetase (GS) expression and activity. Those evidences could triggers depletion of Gln synthesis/metabolism in glia cells and consequently diminish astrocytic-derived glutamine, while disruption of Glu removal/transport can mediate dyshomeostasis in neurotransmission of functioning neurons. Overdose and excessive Mn accumulations in astrocytes not only culminate in pathology, but also affect astrocytic protective properties and defect or

  13. Impairment of glutamine/glutamate-γ-aminobutyric acid cycle in manganese toxicity in the central nervous system.

    PubMed

    Sidoryk-Wegrzynowicz, M

    2014-01-01

    Manganese (Mn) is an essential trace element that is required for maintaining the proper function and regulation of many biochemical and cellular reactions. Despite its essentiality, at excessive levels Mn is toxic to the central nervous system. The overdose accumulation of Mn in specific brain areas, such as the substantia nigra, the globus pallidus and the striatum, triggers neurotoxicity resulting in a neurological brain disorder, referred to as manganism. Manganese toxicity is associated with the disruption of glutamine (Gln)/glutamate (Glu) GABA cycle (GGC). The GGC represents a complex process, since Gln efflux from astrocytes must be met by its influx in neurons. Mn toxicity is associated with the disruption of both of these critical points in the cycle. In cultured astrocytes, pre-treatment with Mn inhibits the initial net uptake of Gln in a concentration-dependent manner. Manganese added directly to astrocytes induces deregulation in the expression of SNAT3, SNAT2, ASCT2 and LAT2 transporters and significantly decreases in Gln uptake mediated by the transporting Systems N and ASC, and a decrease in Gln efflux mediated by Systems N, ASC and L. Further, Mn disrupts Glu transporting systems leading to both a reduction in Glu uptake and elevation in extracellular Glu levels. Interestingly, there appear to be common signaling targets of Mn in GGC cycling in glial cells. Namely, the PKC signaling is affected by Mn in Gln and Glu transporters expression and function. Additionally, Mn was identified to deregulate glutamine synthetase (GS) expression and activity. Those evidences could triggers depletion of Gln synthesis/metabolism in glia cells and consequently diminish astrocytic-derived glutamine, while disruption of Glu removal/transport can mediate dyshomeostasis in neurotransmission of functioning neurons. Overdose and excessive Mn accumulations in astrocytes not only culminate in pathology, but also affect astrocytic protective properties and defect or

  14. Glutamine supplementation.

    PubMed

    Wernerman, Jan

    2011-07-18

    Intravenous glutamine supplementation is standard care when parenteral nutrition is given for critical illness. There are data of a reduced mortality when glutamine supplementation is given. In addition, standard commercial products for parenteral nutrition do not contain any glutamine due to glutamine instability in aqueous solutions. For the majority of critical ill patients who are fed enterally, the available evidence is insufficient to recommend glutamine supplementation. Standard formulation of enteral nutrition contains some glutamine: 2-4 g/L. However, this dose is insufficient to normalize glutamine plasma concentration.Plasma concentration of glutamine is low in many patients with critical illness and a low level is an independent risk factor for mortality. A low plasma glutamine concentration is the best indicator of glutamine depletion. Data are emerging about how the endogenous production of glutamine is regulated. We know that skeletal muscle is the major producer of glutamine and that a part of the profound depletion of skeletal muscle seen in critical illness is a reflection of the need to produce glutamine.Glutamine is utilized in rapidly dividing cells in the splanchnic area. Quantitatively most glutamine is oxidized, but the availability of glutamine in surplus is important for the de novo synthesis of nucleotides and necessary for cell division and protein synthesis. More knowledge about the regulation of the endogenous production of glutamine is needed to outline better guidelines for glutamine supplementation in the future.

  15. Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion.

    PubMed

    Zhang, Ji; Fan, Jing; Venneti, Sriram; Cross, Justin R; Takagi, Toshimitsu; Bhinder, Bhavneet; Djaballah, Hakim; Kanai, Masayuki; Cheng, Emily H; Judkins, Alexander R; Pawel, Bruce; Baggs, Julie; Cherry, Sara; Rabinowitz, Joshua D; Thompson, Craig B

    2014-10-23

    Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed, and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.

  16. Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme.

    PubMed

    Seabra, Ana R; Carvalho, Helena G

    2015-01-01

    Glutamine synthetase (GS) catalyzes the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE) and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of GS isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context. PMID:26284094

  17. α-Ketoglutaramate: An overlooked metabolite of glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle

    PubMed Central

    Cooper, Arthur J. L.; Kuhara, Tomiko

    2013-01-01

    Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to α-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ω-amidase. Transamination of glutamine results in formation of the corresponding α-keto acid, namely, α-ketoglutaramate (KGM). KGM is hydrolyzed by ω-amidase to α-ketoglutarate and ammonia. The net glutaminase II reaction is: L-Glutamine + α-keto acid + H2O → α-ketoglutarate + L-amino acid + ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance. PMID:24234505

  18. Patterns of diversity of citric acid cycle enzymes.

    PubMed

    Weitzman, P D

    1987-01-01

    The citric acid cycle performs a dual role in cell metabolism, acting as a source of both 'energy' and biosynthetic starting materials. The widespread occurrence of the cycle throughout Nature is an excellent example of the unity of biochemistry, but closer examination reveals that there is considerable diversity in the citric acid cycle of different organisms with respect to metabolic role, molecular enzymology and mode of regulation. Two enzymes of the cycle--citrate synthase and succinate thiokinase--have been found to exhibit particularly striking patterns of diversity in structure and catalytic and regulatory function. Some of these patterns show a correlation with the taxonomic groupings of the organisms and with their physiological characteristics. Comparative enzyme studies have a contribution to make to an ultimate understanding of the cycle and its cellular operation, and there are substantial benefits to be gained from interactive studies on both prokaryotic and eukaryotic systems.

  19. Microchip CE-LIF method for the hydrolysis of L-glutamine by using L-asparaginase enzyme reactor based on gold nanoparticle.

    PubMed

    Qiao, Juan; Qi, Li; Yan, Huijuan; Li, Yaping; Mu, Xiaoyu

    2013-02-01

    L-Asparaginase (L-Asnase) can suppress the growth of malignant cells by rapid depletion of two essential amino acids, L-glutamine (L-Gln) and L-asparagine (L-Asn). To study the cytotoxic effect and the secondary complications of L-Asnase in the treatment of acute lymphoblastic leukemia, the development of a novel enzyme reactor of L-Asnase for the hydrolysis of L-Gln, employing the enzyme-gold nanoparticle conjugates in capillary, was reported in this work. First, a microchip CE (MCE)-LIF was established for the separation of L-amino acids (L-Gln and L-glutamic acid) and studying the hydrolysis of L-Gln by using L-Asnase enzyme reactor. Then, using L-Gln as target analyte, the enzyme kinetics of L-Asnase in free solution, enzyme-gold nanoparticle conjugates (E-GNP), and the enzyme-gold nanoparticle conjugates immobilized in capillary (E-GNP-C) were investigated in detail with the proposed MCE-LIF method. Moreover, for optimizing the enzymatic reaction efficiency, three important parameters, including the length of capillary, the enzyme concentration reacted with gold nanoparticle and the amount of L-Asnase immobilized on the gold nanoparticle, have been studied. Owing to the high specific activity, the E-GNP-C enzyme reactor exhibited the best performance for the hydrolysis of L-Gln.

  20. Regulation of Glutamine Transport in Escherichia coli.

    PubMed Central

    Willis, R C; Iwata, K K; Furlong, C E

    1975-01-01

    The formation of the high-affinity (Km equal to 0.2 muM) L-glutamine transport system of Escherichia coli strain 7 (Lin) appears to be subject to the same major control as the glutamine synthetase (EC 6.3.1.2) of this gram-negative organism. Culture of cells under nitrogen-limited conditions provides maximum derepression of both the glutamine synthetase and the glutamine transport system. Nutritional conditions providing a rich supply of ammonium salts or available sources of nitrogen, i.e., conditions which repress the formation of glutamine synthetase, provide three- and 20-fold repression, respectively, of the glutamine transport system. Culture of cells with glutamine supplements of 2 mM does not increase the repression of high-affinity glutamine transport system beyond the level observed in the absence of glutamine. A second kinetically distinct low-affinity component of glutamine. A second kinetically distinct low-affinity component of glutamine uptake is observed in cells cultured with a glutamine-depleted nutrient broth. This second component is associated with the appearance of glutaminase A (EC 3.5.1.2) and asparaginase I (EC 3.5.1.1), a periplasmic enzyme. Parallel changes were observed in the levels of the high-affinity glutamine transport system and the glutamine synthetase when cells were cultured with the carbon sources: glucose, glycerol, or succinate. PMID:238938

  1. ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA

    PubMed Central

    Stine, G. J.

    1968-01-01

    Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged. PMID:4384627

  2. Measurement of (15)N enrichment of glutamine and urea cycle amino acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using liquid chromatography-tandem quadrupole mass spectrometry.

    PubMed

    Nakamura, Hidehiro; Karakawa, Sachise; Watanabe, Akiko; Kawamata, Yasuko; Kuwahara, Tomomi; Shimbo, Kazutaka; Sakai, Ryosei

    2015-05-01

    6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent that has been used for sensitive amino acid quantification by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS). In this study, we aimed to evaluate the ability of this method to measure the isotopic enrichment of amino acids and to determine the positional (15)N enrichment of urea cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and M+1 isotopomers of each natural AQC-amino acid was nearly identical to the theoretical distribution. The standard deviation of the (M+1)/M ratio for each amino acid in repeated measurements was approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the measurements of (15)N enrichment was confirmed by measuring a series of (15)N-labeled arginine standards. The positional (15)N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic distribution of unique fragment ions generated at different collision energies. This method was able to identify their positional (15)N enrichment in the plasma of rats fed (15)N-labeled glutamine. These results suggest the utility of LC-MS/MS detection of AQC-amino acids for the measurement of isotopic enrichment in (15)N-labeled amino acids and indicate that this method is useful for the study of nitrogen metabolism in living organisms.

  3. α-Ketoglutaramate: an overlooked metabolite of glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle.

    PubMed

    Cooper, Arthur J L; Kuhara, Tomiko

    2014-12-01

    Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to α-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ω-amidase. Transamination of glutamine results in formation of the corresponding α-keto acid, namely, α-ketoglutaramate (KGM). KGM is hydrolyzed by ω-amidase to α-ketoglutarate and ammonia. The net glutaminase II reaction is: L - Glutamine + α - keto acid + H2O → α - ketoglutarate + L - amino acid + ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance.

  4. Activities of Tricarboxylic Acid Cycle Enzymes, Glyoxylate Cycle Enzymes, and Fructose Diphosphatase in Bakers' Yeast During Adaptation to Acetate Oxidation

    PubMed Central

    Gosling, J. P.; Duggan, P. F.

    1971-01-01

    Bakers' yeast oxidizes acetate at a high rate only after an adaptation period during which the capacity of the glyoxylate cycle is found to increase. There was apparently no necessity for the activity of acetyl-coenzyme A synthetase, the capacity of the tricarboxylic acid cycle, or the concentrations of the cytochromes to increase for this adaptation to occur. Elevation of fructose 1,6 diphosphatase occurred only when acetate oxidation was nearly maximal. Cycloheximide almost completely inhibited adaptation as well as increases in the activities of isocitrate lyase and aconitate hydratase, the only enzymes assayed. p-Fluorophenylalanine was partially effective and chloramphenicol did not inhibit at all. The presence of ammonium, which considerably delayed adaptation of the yeast to acetate oxidation, inhibited the increases in the activities of the glyoxylate cycle enzymes to different degrees, demonstrating noncoordinate control of these enzymes. Under the various conditions, the only enzyme activity increase consistently related to the rising oxygen uptake rate was that of isocitrate lyase which apparently limited the activity of the cycle. PMID:5557595

  5. Reducing the serine availability complements the inhibition of the glutamine metabolism to block leukemia cell growth

    PubMed Central

    Polet, Florence; Corbet, Cyril; Pinto, Adan; Rubio, Laila Illan; Martherus, Ruben; Bol, Vanesa; Drozak, Xavier; Grégoire, Vincent; Riant, Olivier; Feron, Olivier

    2016-01-01

    Leukemia cells are described as a prototype of glucose-consuming cells with a high turnover rate. The role of glutamine in fueling the tricarboxylic acid cycle of leukemia cells was however recently identified confirming its status of major anaplerotic precursor in solid tumors. Here we examined whether glutamine metabolism could represent a therapeutic target in leukemia cells and whether resistance to this strategy could arise. We found that glutamine deprivation inhibited leukemia cell growth but also led to a glucose-independent adaptation maintaining cell survival. A proteomic study revealed that glutamine withdrawal induced the upregulation of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT), two enzymes of the serine pathway. We further documented that both exogenous and endogenous serine were critical for leukemia cell growth and contributed to cell regrowth following glutamine deprivation. Increase in oxidative stress upon inhibition of glutamine metabolism was identified as the trigger of the upregulation of PHGDH. Finally, we showed that PHGDH silencing in vitro and the use of serine-free diet in vivo inhibited leukemia cell growth, an effect further increased when glutamine metabolism was blocked. In conclusion, this study identified serine as a key pro-survival actor that needs to be handled to sensitize leukemia cells to glutamine-targeting modalities. PMID:26625201

  6. Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

    PubMed

    Pinto, John T; Krasnikov, Boris F; Alcutt, Steven; Jones, Melanie E; Dorai, Thambi; Villar, Maria T; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J L

    2014-11-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. PMID:25231977

  7. Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine*

    PubMed Central

    Pinto, John T.; Krasnikov, Boris F.; Alcutt, Steven; Jones, Melanie E.; Dorai, Thambi; Villar, Maria T.; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J. L.

    2014-01-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. PMID:25231977

  8. Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

    PubMed

    Pinto, John T; Krasnikov, Boris F; Alcutt, Steven; Jones, Melanie E; Dorai, Thambi; Villar, Maria T; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J L

    2014-11-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites.

  9. II. Glutamine and glutamate.

    PubMed

    Tapiero, H; Mathé, G; Couvreur, P; Tew, K D

    2002-11-01

    Glutamine and glutamate with proline, histidine, arginine and ornithine, comprise 25% of the dietary amino acid intake and constitute the "glutamate family" of amino acids, which are disposed of through conversion to glutamate. Although glutamine has been classified as a nonessential amino acid, in major trauma, major surgery, sepsis, bone marrow transplantation, intense chemotherapy and radiotherapy, when its consumption exceeds its synthesis, it becomes a conditionally essential amino acid. In mammals the physiological levels of glutamine is 650 micromol/l and it is one of the most important substrate for ammoniagenesis in the gut and in the kidney due to its important role in the regulation of acid-base homeostasis. In cells, glutamine is a key link between carbon metabolism of carbohydrates and proteins and plays an important role in the growth of fibroblasts, lymphocytes and enterocytes. It improves nitrogen balance and preserves the concentration of glutamine in skeletal muscle. Deamidation of glutamine via glutaminase produces glutamate a precursor of gamma-amino butyric acid, a neurotransmission inhibitor. L-Glutamic acid is a ubiquitous amino acid present in many foods either in free form or in peptides and proteins. Animal protein may contain from 11 to 22% and plants protein as much as 40% glutamate by weight. The sodium salt of glutamic acid is added to several foods to enhance flavor. L-Glutamate is the most abundant free amino acid in brain and it is the major excitatory neurotransmitter of the vertebrate central nervous system. Most free L-glutamic acid in brain is derived from local synthesis from L-glutamine and Kreb's cycle intermediates. It clearly plays an important role in neuronal differentiation, migration and survival in the developing brain via facilitated Ca++ transport. Glutamate also plays a critical role in synaptic maintenance and plasticity. It contributes to learning and memory through use-dependent changes in synaptic efficacy and

  10. Abnormality in glutamine-glutamate cycle in the cerebrospinal fluid of cognitively intact elderly individuals with major depressive disorder: a 3-year follow-up study.

    PubMed

    Hashimoto, K; Bruno, D; Nierenberg, J; Marmar, C R; Zetterberg, H; Blennow, K; Pomara, N

    2016-03-01

    Major depressive disorder (MDD), common in the elderly, is a risk factor for dementia. Abnormalities in glutamatergic neurotransmission via the N-methyl-D-aspartate receptor (NMDA-R) have a key role in the pathophysiology of depression. This study examined whether depression was associated with cerebrospinal fluid (CSF) levels of NMDA-R neurotransmission-associated amino acids in cognitively intact elderly individuals with MDD and age- and gender-matched healthy controls. CSF was obtained from 47 volunteers (MDD group, N=28; age- and gender-matched comparison group, N=19) at baseline and 3-year follow-up (MDD group, N=19; comparison group, N=17). CSF levels of glutamine, glutamate, glycine, L-serine and D-serine were measured by high-performance liquid chromatography. CSF levels of amino acids did not differ across MDD and comparison groups. However, the ratio of glutamine to glutamate was significantly higher at baseline in subjects with MDD than in controls. The ratio decreased in individuals with MDD over the 3-year follow-up, and this decrease correlated with a decrease in the severity of depression. No correlations between absolute amino-acid levels and clinical variables were observed, nor were correlations between amino acids and other biomarkers (for example, amyloid-β42, amyloid-β40, and total and phosphorylated tau protein) detected. These results suggest that abnormalities in the glutamine-glutamate cycle in the communication between glia and neurons may have a role in the pathophysiology of depression in the elderly. Furthermore, the glutamine/glutamate ratio in CSF may be a state biomarker for depression.

  11. [Studies on regulation of glutamine synthetase activity from Streptomyces lincolnensis].

    PubMed

    Jin, Z; Jiao, R; Mao, Y

    2001-08-01

    Glutamine synthetase in crude extracts from Streptomyces lincolnensis growing under different nitrogen sources were studied. The results showed that NH4+ in high concentration repressed the biosynthesis of the enzyme. To determine whether Streptomyces lincolnensis has undergone covalent modification, a comparison of the glutamine synthetase isolated from cells grown on different nitrogen sources was made. No significant difference was observed in specific activity, pH optima, divalent cation response, and ultraviolet absorption spectra. Glutamine synthetase activity was not influenced by ammonia shock or snake venom phosphodiesterase treatment. Under these conditions, the activity of glutamine synthetase from K. aerogenes was markedly changed. There was therefore no evidence for enzymatic adenylylation of glutamine synthetase from Streptomyces lincolnensis. Glutamine synthetase was subject to feedback inhibition by end products of glutamine metabolism. Cumulative feedback inhibition of the Mn(2+)-dependent glutamine synthetase activity was demonstrated. These results suggest that glutamine synthetase from Streptomyces lincolnensis is an allosteric enzyme. PMID:12552916

  12. Effects of Helicobacter suis γ- Glutamyl Transpeptidase on Lymphocytes: Modulation by Glutamine and Glutathione Supplementation and Outer Membrane Vesicles as a Putative Delivery Route of the Enzyme

    PubMed Central

    Zhang, Guangzhi; Ducatelle, Richard; Pasmans, Frank; D’Herde, Katharina; Huang, Liping; Smet, Annemieke; Haesebrouck, Freddy; Flahou, Bram

    2013-01-01

    Helicobacter (H.) suis colonizes the stomach of the majority of pigs as well as a minority of humans worldwide. Infection causes chronic inflammation in the stomach of the host, however without an effective clearance of the bacteria. Currently, no information is available about possible mechanisms H. suis utilizes to interfere with the host immune response. This study describes the effect on various lymphocytes of the γ-glutamyl transpeptidase (GGT) from H. suis. Compared to whole cell lysate from wild-type H. suis, lysate from a H. suis ggt mutant strain showed a decrease of the capacity to inhibit Jurkat T cell proliferation. Incubation of Jurkat T cells with recombinantly expressed H. suis GGT resulted in an impaired proliferation, and cell death was shown to be involved. A similar but more pronounced inhibitory effect was also seen on primary murine CD4+ T cells, CD8+ T cells, and CD19+ B cells. Supplementation with known GGT substrates was able to modulate the observed effects. Glutamine restored normal proliferation of the cells, whereas supplementation with reduced glutathione strengthened the H. suis GGT-mediated inhibition of proliferation. H. suis GGT treatment abolished secretion of IL-4 and IL-17 by CD4+ T cells, without affecting secretion of IFN-γ. Finally, H. suis outer membrane vesicles (OMV) were identified as a possible delivery route of H. suis GGT to lymphocytes residing in the deeper mucosal layers. Thus far, this study is the first to report that the effects on lymphocytes of this enzyme, not only important for H. suis metabolism but also for that of other Helicobacter species, depend on the degradation of two specific substrates: glutamine and reduced glutatione. This will provide new insights into the pathogenic mechanisms of H. suis infection in particular and infection with gastric helicobacters in general. PMID:24147103

  13. Extracellular enzyme activity and biogeochemical cycling in restored prairies

    NASA Astrophysics Data System (ADS)

    Lynch, L.; Hernandez, D.; Schade, J. D.

    2011-12-01

    during the spring. Microbial biomass C:N ratios increased from October to March, and decreased through the summer, while production of CBH, LAP and PHOS all showed the opposite pattern, decreasing through March and increasing in the summer. Following snowmelt, enzyme production preceded a recovery in microbial biomass, possibly as a result of increased competition for available resources between plant and microbial communities, or a shift to organic sources of C, N, and P which required a higher investment in enzymes. Due to their rapid growth rates and turnover, microbes are a particularly reactive component of terrestrial ecosystems and significantly influence biogeochemical cycling. Because carbon degradation may be constrained by nutrient availability, understanding how extracellular enzyme production, decomposition rate, and nutrient flux change over time is essential if we are to anticipate ecosystem responses to environmental changes.

  14. Aberrant expression and distribution of enzymes of the urea cycle and other ammonia metabolizing pathways in dogs with congenital portosystemic shunts.

    PubMed

    van Straten, Giora; van Steenbeek, Frank G; Grinwis, Guy C M; Favier, Robert P; Kummeling, Anne; van Gils, Ingrid H; Fieten, Hille; Groot Koerkamp, Marian J A; Holstege, Frank C P; Rothuizen, Jan; Spee, Bart

    2014-01-01

    The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase. PMID:24945279

  15. Mathematical modelling of the citric acid cycle for the analysis of glutamine isotopomers from cerebellar astrocytes incubated with [1(-13)C]glucose.

    PubMed

    Merle, M; Martin, M; Villégier, A; Canioni, P

    1996-08-01

    A mathematical model of the citric acid cycle devoted to the analysis of 13C-NMR data was developed for determining the relative flux of molecules through the anaplerotic versus oxidative pathways and the relative pyruvate carboxylase versus pyruvate dehydrogenase activities. Different variants of the model were considered depending on the reversibility of the conversion of fumarate into malate and oxaloacetate. The model also included the possibility of orientation-conserved transfer of the four-carbon citric acid cycle intermediates, leading to conversion of succinyl-CoA C1 into either malate C1 or C4. It was used to analyse NMR data from glutamine isotopomers produced by cerebellar astrocytes incubated with [1-13C]glucose. Partial cycling (39%) between oxaloacetate and fumarate was evident from the analysis. Application of the model to glutamate isotopomers from granule cells incubated with [1-13C]glucose [Martin, M.. Portais, J.C.. Labouesse. J., Canioni. P, & Merle, M. (1993) Eur. J. Biochem. 217, 617-625] indicated that total cycling of oxaloacetate into fumarate was, in this case, required to get the best fit. The results emphasized some important differences in carbon metabolism between cerebellar astrocytes and granule cells concerning the sources of carbon fuelling the citric acid cycle and the carbon fluxes on different pathways.

  16. Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions

    NASA Astrophysics Data System (ADS)

    Allison, S. D.; Jastrow, J. D.

    2004-12-01

    Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.

  17. Oncogenic Myc Induces Expression of Glutamine Synthetase through Promoter Demethylation.

    PubMed

    Bott, Alex J; Peng, I-Chen; Fan, Yongjun; Faubert, Brandon; Zhao, Lu; Li, Jinyu; Neidler, Sarah; Sun, Yu; Jaber, Nadia; Krokowski, Dawid; Lu, Wenyun; Pan, Ji-An; Powers, Scott; Rabinowitz, Joshua; Hatzoglou, Maria; Murphy, Daniel J; Jones, Russell; Wu, Song; Girnun, Geoffrey; Zong, Wei-Xing

    2015-12-01

    c-Myc is known to promote glutamine usage by upregulating glutaminase (GLS), which converts glutamine to glutamate that is catabolized in the TCA cycle. Here we report that in a number of human and murine cells and cancers, Myc induces elevated expression of glutamate-ammonia ligase (GLUL), also termed glutamine synthetase (GS), which catalyzes the de novo synthesis of glutamine from glutamate and ammonia. This is through upregulation of a Myc transcriptional target thymine DNA glycosylase (TDG), which promotes active demethylation of the GS promoter and its increased expression. Elevated expression of GS promotes cell survival under glutamine limitation, while silencing of GS decreases cell proliferation and xenograft tumor growth. Upon GS overexpression, increased glutamine enhances nucleotide synthesis and amino acid transport. These results demonstrate an unexpected role of Myc in inducing glutamine synthesis and suggest a molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.

  18. Glutamine and cancer.

    PubMed Central

    Souba, W W

    1993-01-01

    OBJECTIVE: This overview on glutamine and cancer discusses the importance of glutamine for tumor growth, summarizes the alterations in interorgan glutamine metabolism that develop in the tumor-bearing host, and reviews the potential benefits of glutamine nutrition in the patient with cancer. SUMMARY BACKGROUND DATA: Glutamine is the most abundant amino acid in the blood and tissues. It is essential for tumor growth and marked changes in organ glutamine metabolism are characteristic of the host with cancer. Because host glutamine depletion has adverse effects, it is important to study the regulation of glutamine metabolism in cancer and to evaluate the impact of glutamine nutrition in the tumor-bearing state. METHODS: Data from a variety of investigations on glutamine metabolism and nutrition related to the host with cancer were compiled and summarized. RESULTS: Numerous studies on glutamine metabolism in cancer indicate that many tumors are avid glutamine consumers in vivo and in vitro. As a consequence of progressive tumor growth, host glutamine depletion develops and becomes a hallmark. This glutamine depletion occurs in part because the tumor behaves as a "glutamine trap" but also because of cytokine-mediated alterations in glutamine metabolism in host tissues. Animal and human studies that have investigated the use of glutamine-supplemented nutrition in the host with cancer suggest that pharmacologic doses of dietary glutamine may be beneficial. CONCLUSIONS: Understanding the control of glutamine metabolism in the tumor-bearing host not only improves the knowledge of metabolic regulation in the patient with cancer but also will lead to improved nutritional support regimens targeted to benefit the host. PMID:8257221

  19. Beyond Vmax and Km: How details of enzyme function influence geochemical cycles

    NASA Astrophysics Data System (ADS)

    Steen, A. D.

    2015-12-01

    Enzymes catalyze the vast majority of chemical reactions relevant to geomicrobiology. Studies of the activities of enzymes in environmental systems often report Vmax (the maximum possible rate of reaction; often proportional to the concentration of enzymes in the system) and sometimes Km (a measure of the affinity between enzymes and their substrates). However, enzyme studies - particularly those related to enzymes involved in organic carbon oxidation - are often limited to only those parameters, and a relatively limited and mixed set of enzymes. Here I will discuss some novel methods to assay and characterize the specific sets of enzymes that may be important to the carbon cycle in aquatic environments. First, kinetic experiments revealed the collective properties of the complex mixtures of extracellular peptidases that occur where microbial communities are diverse. Crystal structures combined with biochemical characterization of specific enzymes can yield more detailed information about key steps in organic carbon transformations. These new techniques have the potential to provide mechanistic grounding to geomicrobiological models.

  20. H1TF2A, the large subunit of a heterodimeric, glutamine-rich CCAAT-binding transcription factor involved in histone H1 cell cycle regulation.

    PubMed Central

    Martinelli, R; Heintz, N

    1994-01-01

    H1TF2 is a CCAAT transcription factor that binds to the histone H1 subtype-specific consensus sequence, which has previously been shown to be necessary for temporal regulation of histone H1 transcription during the cell cycle (F. La Bella, P. Gallinari, J. McKinney, and N. Heintz, Genes Dev. 3:1982-1990, 1989). In this study, we report that H1TF2 is a heteromeric CCAAT-binding protein composed of two polypeptide doublets of 33 and 34 kDa and 43 and 44 kDa that are not antigenically related. The 33- and 34-kDa species were not detected in our previous studies (P. Gallinari, F. La Bella, and N. Heintz, Mol. Cell. Biol. 9:1566-1575, 1989) because of technical problems in detection of these heavily glycosylated subunits. The cloning of H1TF2A, the large subunit of this factor, reveals it to be a glutamine-rich protein with extremely limited similarity to previously cloned CCAAT-binding proteins. A monospecific antiserum produced against bacterially synthesized H1TF2A was used to establish that HeLa cell H1TF2A is phosphorylated in vivo and that, in contrast to the H2b transcription factor Oct1 (S. B. Roberts, N. Segil, and N. Heintz, Science 253:1022-1026, 1991; N. Segil, S. B. Roberts, and N. Heintz, Cold Spring Harbor Symp. Quant. Biol. 56:285-292, 1991), no gross change in H1TF2A phosphorylation is evident during the cell cycle. Further immunoprecipitation studies demonstrated that H1TF2 is heterodimeric in the absence of DNA in vivo and identified several H1TF2-interacting proteins that may play a role in H1TF2 function in vivo. Images PMID:7969168

  1. Effect of glutamine supplementation on neutrophil function in male judoists.

    PubMed

    Sasaki, Eiji; Umeda, Takashi; Takahashi, Ippei; Arata, Kojima; Yamamoto, Yousuke; Tanabe, Masaru; Oyamada, Kazuyuki; Hashizume, Erika; Nakaji, Shigeyuki

    2013-01-01

    Glutamine is an important amino acid for immune function. Though high intensity and prolonged exercise decreases plasma glutamine concentration and causes immune suppression, the relationship between neutrophil functions and glutamine has not yet been found. The purpose of this study was to investigate the impacts of glutamine supplementation on neutrophil function. Twenty-six male university judoists were recruited. Subjects were classified into glutamine and control groups. The glutamine group ingested 3000 mg of glutamine per day and the control group ingested placebo for 2 weeks. Examinations were performed at the start of preunified loading exercise (pre-ULE), then 1 and 2 weeks after ULE (post-ULE). Reactive oxygen species (ROS) production, phagocytic activity, serum opsonic activity and serum myogenic enzymes were measured. Differences between the levels obtained in pre-ULE and post-ULE for the two groups were compared. In the glutamine group, ROS production activity increased 1 week after ULE, whereas it was not observed in the control group (P < 0.001). Though myogenic enzymes increased significantly after ULE (P < 0.001), the glutamine group remained unchanged by supplementation during ULE. Glutamine supplementation has prevented excessive muscle damage and suppression of neutrophil function, especially in ROS production activity, even during an intensive training period. PMID:23348981

  2. Effect of glutamine supplementation on neutrophil function in male judoists.

    PubMed

    Sasaki, Eiji; Umeda, Takashi; Takahashi, Ippei; Arata, Kojima; Yamamoto, Yousuke; Tanabe, Masaru; Oyamada, Kazuyuki; Hashizume, Erika; Nakaji, Shigeyuki

    2013-01-01

    Glutamine is an important amino acid for immune function. Though high intensity and prolonged exercise decreases plasma glutamine concentration and causes immune suppression, the relationship between neutrophil functions and glutamine has not yet been found. The purpose of this study was to investigate the impacts of glutamine supplementation on neutrophil function. Twenty-six male university judoists were recruited. Subjects were classified into glutamine and control groups. The glutamine group ingested 3000 mg of glutamine per day and the control group ingested placebo for 2 weeks. Examinations were performed at the start of preunified loading exercise (pre-ULE), then 1 and 2 weeks after ULE (post-ULE). Reactive oxygen species (ROS) production, phagocytic activity, serum opsonic activity and serum myogenic enzymes were measured. Differences between the levels obtained in pre-ULE and post-ULE for the two groups were compared. In the glutamine group, ROS production activity increased 1 week after ULE, whereas it was not observed in the control group (P < 0.001). Though myogenic enzymes increased significantly after ULE (P < 0.001), the glutamine group remained unchanged by supplementation during ULE. Glutamine supplementation has prevented excessive muscle damage and suppression of neutrophil function, especially in ROS production activity, even during an intensive training period.

  3. Structural and functional insights into enzymes of the vitamin K cycle.

    PubMed

    Tie, J-K; Stafford, D W

    2016-02-01

    Vitamin K-dependent proteins require carboxylation of certain glutamates for their biological functions. The enzymes involved in the vitamin K-dependent carboxylation include: gamma-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR) and an as-yet-unidentified vitamin K reductase (VKR). Due to the hydrophobicity of vitamin K, these enzymes are likely to be integral membrane proteins that reside in the endoplasmic reticulum. Therefore, structure-function studies on these enzymes have been challenging, and some of the results are notably controversial. Patients with naturally occurring mutations in these enzymes, who mainly exhibit bleeding disorders or are resistant to oral anticoagulant treatment, provide valuable information for the functional study of the vitamin K cycle enzymes. In this review, we discuss: (i) the discovery of the enzymatic activities and gene identifications of the vitamin K cycle enzymes; (ii) the identification of their functionally important regions and their active site residues; (iii) the membrane topology studies of GGCX and VKOR; and (iv) the controversial issues regarding the structure and function studies of these enzymes, particularly, the membrane topology, the role of the conserved cysteines and the mechanism of active site regeneration of VKOR. We also discuss the possibility that a paralogous protein of VKOR, VKOR-like 1 (VKORL1), is involved in the vitamin K cycle, and the importance of and possible approaches for identifying the unknown VKR. Overall, we describe the accomplishments and the remaining questions in regard to the structure and function studies of the enzymes in the vitamin K cycle.

  4. Krebs cycle metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes.

    PubMed

    Wu, Fei; Pelster, Lindsey N; Minteer, Shelley D

    2015-01-25

    Dynamics of metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.

  5. {open_quotes}The effects of diabetes on the activity of the enzyme glutamine: fructose-6-phosphate amindotransferase{close_quotes}

    SciTech Connect

    Nelson, S.P.

    1994-12-31

    Hexsoamine synthetic pathway (HexNSP) controls the supply of essential substrates for glycoprotein synthesis. In vitro studies suggest that increased flux of glucose via the hexsoamine synthetic pathway may play a role in glucose induced insulin resistance of glucose transport. Glutamine: fructose-6-phosphate amindotransferase (GFAT) controls flux into the hexsoamine synthetic pathway; the major products are UDPN-acetylhexosamines (UDP.HexNac=UDP.GlcNAc= UDP.GalNac). I examined whether diabetes ({approximately} 7 days post intravenous streptozotocin, and genetically linked) affects the activity of glutamine: fructose-6-phosphate in rat and mouse skeletal muscle in vivo. Nucleotide linked HexNAc were analyzed by high pressure liquid chromatography(HPLC) in deproteinized hind limb muscle extracts.

  6. Effects of inhibiting glutamine synthetase and blocking glutamate uptake on b-wave generation in the isolated rat retina

    PubMed Central

    WINKLER, BARRY S.; KAPOUSTA-BRUNEAU, NATALIA; ARNOLD, MATTHEW J.; GREEN, DANIEL G.

    2006-01-01

    The purpose of the present experiments was to evaluate the contribution of the glutamate-glutamine cycle in retinal glial (Müller) cells to photoreceptor cell synaptic transmission. Dark-adapted isolated rat retinas were superfused with oxygenated bicarbonate-buffered media. Recordings were made of the b-wave of the electroretinogram as a measure of light-induced photoreceptor to ON-bipolar neuron transmission. L-methionine sulfoximine (1–10 mM) was added to superfusion media to inhibit glutamine synthetase, a Müller cell specific enzyme, by more than 99% within 5–10 min, thereby disrupting the conversion of glutamate to glutamine in the Müller cells. Threo-hydroxyaspartic acid and D-aspartate were used to block glutamate transporters. The amplitude of the b-wave was well maintained for 1–2 h provided 0.25 mM glutamate or 0.25 mM glutamine was included in the media. Without exogenous glutamate or glutamine the amplitude of the b-wave declined by about 70% within 1 h. Inhibition of glutamate transporters led to a rapid (2–5 min) reversible loss of the b-wave in the presence and absence of the amino acids. In contrast, inhibition of glutamine synthetase did not alter significantly either the amplitude of the b-wave in the presence of glutamate or glutamine or the rate of decline of the b-wave found in the absence of these amino acids. Excellent recovery of the b-wave was found when 0.25 mM glutamate was resupplied to L-methionine sulfoximine–treated retinas. The results suggest that in the isolated rat retina uptake of released glutamate into photoreceptors plays a more important role in transmitter recycling than does uptake of glutamate into Müller cells and its subsequent conversion to glutamine. PMID:10367968

  7. Waiting cycle times and generalized Haldane equality in the steady-state cycle kinetics of single enzymes.

    PubMed

    Ge, Hao

    2008-01-10

    Enzyme kinetics are cyclic. A more realistic reversible three-step mechanism of the Michaelis-Menten kinetics is investigated in detail, and three kinds of waiting cycle times T, T+, T- are defined. It is shown that the mean waiting cycle times T, T+, and T- are the reciprocal of the steady-state cycle flux Jss, the forward steady-state cycle flux Jss+ and the backward steady-state cycle flux Jss, respectively. We also show that the distribution of T+ conditioned on T+cycle time of T+ conditioned on T+cycle fluxes and waiting cycle times. Furthermore, we extend the same results to the n-step cycle, and finally, experimental and theoretically based evidence are also included. PMID:18069809

  8. Measuring in vivo elasticities of Calvin cycle enzymes: network structure and patterns of modulations.

    PubMed

    Kreim, Michael; Giersch, Christoph

    2007-01-01

    To measure the kinetics of enzymes, the proteins are usually assayed in vitro after isolation from their parent organisms. We make an attempt to show how one might determine enzyme elasticities in an intact system by a multiple modulation approach. Certain target enzymes are modulated in their activities and the changes in metabolite concentrations and flux rates upon the modulations are used to calculate the enzyme elasticities. Central to this approach is that the modulations must be independent of each other, and an algorithm is developed for finding all independent modulations that allow determining the elasticities of a given enzyme. This approach is applied to a mass-action model of the Calvin cycle. The goal is to determine the elasticities of as many enzymes as possible by modulating the activities of as few of them as possible. It is shown that the elasticities of 20 (out of 22) Calvin cycle enzymes can be determined by modulating just five reactions. Moreover, visualization of independence of modulations may be used to decompose the Calvin cycle into several sections that are independent of each other regarding flow of matter and information.

  9. Phloem-specific expression of Yang cycle genes and identification of novel Yang cycle enzymes in Plantago and Arabidopsis.

    PubMed

    Pommerrenig, Benjamin; Feussner, Kirstin; Zierer, Wolfgang; Rabinovych, Valentyna; Klebl, Franz; Feussner, Ivo; Sauer, Norbert

    2011-05-01

    The 5-methylthioadenosine (MTA) or Yang cycle is a set of reactions that recycle MTA to Met. In plants, MTA is a byproduct of polyamine, ethylene, and nicotianamine biosynthesis. Vascular transcriptome analyses revealed phloem-specific expression of the Yang cycle gene 5-METHYLTHIORIBOSE KINASE1 (MTK1) in Plantago major and Arabidopsis thaliana. As Arabidopsis has only a single MTK gene, we hypothesized that the expression of other Yang cycle genes might also be vascular specific. Reporter gene studies and quantitative analyses of mRNA levels for all Yang cycle genes confirmed this hypothesis for Arabidopsis and Plantago. This includes the Yang cycle genes 5-METHYLTHIORIBOSE-1-PHOSPHATE ISOMERASE1 and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1. We show that these two enzymes are sufficient for the conversion of methylthioribose-1-phosphate to 1,2-dihydroxy-3-keto-5-methylthiopentene. In bacteria, fungi, and animals, the same conversion is catalyzed in three to four separate enzymatic steps. Furthermore, comparative analyses of vascular and nonvascular metabolites identified Met, S-adenosyl Met, and MTA preferentially or almost exclusively in the vascular tissue. Our data represent a comprehensive characterization of the Yang cycle in higher plants and demonstrate that the Yang cycle works primarily in the vasculature. Finally, expression analyses of polyamine biosynthetic genes suggest that the Yang cycle in leaves recycles MTA derived primarily from polyamine biosynthesis.

  10. Phloem-specific expression of Yang cycle genes and identification of novel Yang cycle enzymes in Plantago and Arabidopsis.

    PubMed

    Pommerrenig, Benjamin; Feussner, Kirstin; Zierer, Wolfgang; Rabinovych, Valentyna; Klebl, Franz; Feussner, Ivo; Sauer, Norbert

    2011-05-01

    The 5-methylthioadenosine (MTA) or Yang cycle is a set of reactions that recycle MTA to Met. In plants, MTA is a byproduct of polyamine, ethylene, and nicotianamine biosynthesis. Vascular transcriptome analyses revealed phloem-specific expression of the Yang cycle gene 5-METHYLTHIORIBOSE KINASE1 (MTK1) in Plantago major and Arabidopsis thaliana. As Arabidopsis has only a single MTK gene, we hypothesized that the expression of other Yang cycle genes might also be vascular specific. Reporter gene studies and quantitative analyses of mRNA levels for all Yang cycle genes confirmed this hypothesis for Arabidopsis and Plantago. This includes the Yang cycle genes 5-METHYLTHIORIBOSE-1-PHOSPHATE ISOMERASE1 and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1. We show that these two enzymes are sufficient for the conversion of methylthioribose-1-phosphate to 1,2-dihydroxy-3-keto-5-methylthiopentene. In bacteria, fungi, and animals, the same conversion is catalyzed in three to four separate enzymatic steps. Furthermore, comparative analyses of vascular and nonvascular metabolites identified Met, S-adenosyl Met, and MTA preferentially or almost exclusively in the vascular tissue. Our data represent a comprehensive characterization of the Yang cycle in higher plants and demonstrate that the Yang cycle works primarily in the vasculature. Finally, expression analyses of polyamine biosynthetic genes suggest that the Yang cycle in leaves recycles MTA derived primarily from polyamine biosynthesis. PMID:21540433

  11. Sodium butyrate reverses the inhibition of Krebs cycle enzymes induced by amphetamine in the rat brain.

    PubMed

    Valvassori, Samira S; Calixto, Karen V; Budni, Josiane; Resende, Wilson R; Varela, Roger B; de Freitas, Karolina V; Gonçalves, Cinara L; Streck, Emilio L; Quevedo, João

    2013-12-01

    There is increasing interest in the possibility that mitochondrial impairment may play an important role in bipolar disorder (BD). The Krebs cycle is the central point of oxidative metabolism, providing carbon for biosynthesis and reducing agents for generation of ATP. Recently, studies have suggested that histone deacetylase (HDAC) inhibitors may have antimanic effects. The present study aims to investigate the effects of sodium butyrate (SB), a HDAC inhibitor, on Krebs cycle enzymes activity in the brain of rats subjected to an animal model of mania induced by D-amphetamine (D-AMPH). Wistar rats were first given D-AMPH or saline (Sal) for 14 days, and then, between days 8 and 14, rats were treated with SB or Sal. The citrate synthase (CS), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) were evaluated in the prefrontal cortex, hippocampus, and striatum of rats. The D-AMPH administration inhibited Krebs cycle enzymes activity in all analyzed brain structures and SB reversed D-AMPH-induced dysfunction analyzed in all brain regions. These findings suggest that Krebs cycle enzymes' inhibition can be an important link for the mitochondrial dysfunction seen in BD and SB exerts protective effects against the D-AMPH-induced Krebs cycle enzymes' dysfunction.

  12. Allosteric interactions and bifunctionality make the response of glutamine synthetase cascade system of Escherichia coli robust and ultrasensitive.

    PubMed

    Mutalik, Vivek K; Shah, Parag; Venkatesh, K V

    2003-07-18

    Glutamine synthetase (GS) regulation in Escherichia coli by reversible covalent modification cycles is a prototype of signal transduction by enzyme cascades. Such enzyme cascades are known to exhibit ultrasensitive response to primary stimuli and act as signal integration systems. Here, we have quantified GS bicyclic cascade based on steady state analysis by evaluating Hill coefficient. We demonstrate that adenylylation of GS with glutamine as input is insensitive to total enzyme concentrations of GS, uridylyltransferase/uridylyl-removing enzyme, regulatory protein PII, and adenylyltransferase/adenylyl-removing enzyme. This robust response of GS adenylylation is also observed for change in system parameters. From numerical analyses, we show that the robust ultrasensitive response of bicyclic cascade is because of allosteric interactions of glutamine and 2-ketoglutarate, bifunctionality of converter enzymes, and closed loop bicyclic cascade structure. By system level quantification of the GS bicyclic cascade, we conclude that such a robust response may help the cell in adapting to different carbon and nitrogen status. PMID:12676964

  13. Glutamine deprivation initiates reversible assembly of mammalian rods and rings.

    PubMed

    Calise, S John; Carcamo, Wendy C; Krueger, Claire; Yin, Joyce D; Purich, Daniel L; Chan, Edward K L

    2014-08-01

    Rods and rings (RR) are protein assemblies composed of cytidine triphosphate synthetase type 1 (CTPS1) and inosine monophosphate dehydrogenase type 2 (IMPDH2), key enzymes in CTP and GTP biosynthesis. Small-molecule inhibitors of CTPS1 or IMPDH2 induce RR assembly in various cancer cell lines within 15 min to hours. Since glutamine is an essential amide nitrogen donor in these nucleotide biosynthetic pathways, glutamine deprivation was examined to determine whether it leads to RR formation. HeLa cells cultured in normal conditions did not show RR, but after culturing in media lacking glutamine, short rods (<2 μm) assembled after 24 h, and longer rods (>5 μm) formed after 48 h. Upon supplementation with glutamine or guanosine, these RR underwent almost complete disassembly within 15 min. Inhibition of glutamine synthetase with methionine sulfoximine also increased RR assembly in cells deprived of glutamine. Taken together, our data support the hypothesis that CTP/GTP biosynthetic enzymes polymerize to form RR in response to a decreased intracellular level of glutamine. We speculate that rod and ring formation is an adaptive metabolic response linked to disruption of glutamine homeostasis.

  14. Effects of aluminum on activity of krebs cycle enzymes and glutamate dehydrogenase in rat brain homogenate.

    PubMed

    Zatta, P; Lain, E; Cagnolini, C

    2000-05-01

    Aluminum is a neurotoxic agent for animals and humans that has been implicated as an etiological factor in several neurodegenerative diseases and as a destabilizer of cell membranes. Due to its high reactivity, Al3+ is able to interfere with several biological functions, including enzymatic activities in key metabolic pathways. In this paper we report that, among the enzymes that constitute the Krebs cycle, only two are activated by aluminum: alpha-ketoglutarate dehydrogenase and succinate dehydrogenase. In contrast, aconitase, shows decreased activity in the presence of the metal ion. Al3+ also inhibits glutamate dehydrogenase, an allosteric enzyme that is closely linked to the Krebs cycle. A possible correlation between aluminum, the Krebs cycle and aging processes is discussed.

  15. Glutamine as an immunonutrient.

    PubMed

    Kim, Hyeyoung

    2011-11-01

    Dietary supplementation with nutrients enhancing immune function is beneficial in patients with surgical and critical illness. Malnutrition and immune dysfunction are common features in hospitalized patients. Specific nutrients with immunological and pharmacological effects, when consumed in amounts above the daily requirement, are referred to as immune-enhancing nutrients or immunonutrients. Supplementation of immunonutrients is important especially for patients with immunodeficiency, virus or overwhelming infections accompanied by a state of malnutrition. Representative immunonutrients are arginine, omega-3 fatty acids, glutamine, nucleotides, beta-carotene, and/or branched-chain amino acids. Glutamine is the most abundant amino acid and performs multiple roles in human body. However, glutamine is depleted from muscle stores during severe metabolic stress including sepsis and major surgery. Therefore it is considered conditionally essential under these conditions. This review discusses the physiological role of glutamine, mode and dose for glutamine administration, as well as improvement of certain disease state after glutamine supplementation. Even though immunonutrition has not been widely assimilated by clinicians other than nutritionists, immunonutrients including glutamine may exert beneficial influence on diverse patient populations.

  16. MARINE SULFUR CYCLE. Identification of the algal dimethyl sulfide-releasing enzyme: A missing link in the marine sulfur cycle.

    PubMed

    Alcolombri, Uria; Ben-Dor, Shifra; Feldmesser, Ester; Levin, Yishai; Tawfik, Dan S; Vardi, Assaf

    2015-06-26

    Algal blooms produce large amounts of dimethyl sulfide (DMS), a volatile with a diverse signaling role in marine food webs that is emitted to the atmosphere, where it can affect cloud formation. The algal enzymes responsible for forming DMS from dimethylsulfoniopropionate (DMSP) remain unidentified despite their critical role in the global sulfur cycle. We identified and characterized Alma1, a DMSP lyase from the bloom-forming algae Emiliania huxleyi. Alma1 is a tetrameric, redox-sensitive enzyme of the aspartate racemase superfamily. Recombinant Alma1 exhibits biochemical features identical to the DMSP lyase in E. huxleyi, and DMS released by various E. huxleyi isolates correlates with their Alma1 levels. Sequence homology searches suggest that Alma1 represents a gene family present in major, globally distributed phytoplankton taxa and in other marine organisms. PMID:26113722

  17. MARINE SULFUR CYCLE. Identification of the algal dimethyl sulfide-releasing enzyme: A missing link in the marine sulfur cycle.

    PubMed

    Alcolombri, Uria; Ben-Dor, Shifra; Feldmesser, Ester; Levin, Yishai; Tawfik, Dan S; Vardi, Assaf

    2015-06-26

    Algal blooms produce large amounts of dimethyl sulfide (DMS), a volatile with a diverse signaling role in marine food webs that is emitted to the atmosphere, where it can affect cloud formation. The algal enzymes responsible for forming DMS from dimethylsulfoniopropionate (DMSP) remain unidentified despite their critical role in the global sulfur cycle. We identified and characterized Alma1, a DMSP lyase from the bloom-forming algae Emiliania huxleyi. Alma1 is a tetrameric, redox-sensitive enzyme of the aspartate racemase superfamily. Recombinant Alma1 exhibits biochemical features identical to the DMSP lyase in E. huxleyi, and DMS released by various E. huxleyi isolates correlates with their Alma1 levels. Sequence homology searches suggest that Alma1 represents a gene family present in major, globally distributed phytoplankton taxa and in other marine organisms.

  18. Krebs cycle enzymes from livers of old mice are differentially regulated by caloric restriction.

    PubMed

    Hagopian, Kevork; Ramsey, Jon J; Weindruch, Richard

    2004-08-01

    Krebs cycle enzyme activities and levels of five metabolites were determined from livers of old mice (30 months) maintained either on control or on long-term caloric restriction (CR) diets (28 months). In CR mice, the cycle was divided into two major blocks, the first containing citrate synthase, aconitase and NAD-dependent isocitrate dehydrogenase which showed decreased activities, while the second block, containing the remaining enzymes, displayed increased activity (except for fumarase, which was unchanged). CR also resulted in decreased levels of citrate, glutamate and alpha-ketoglutarate, increased levels of malate, and unchanged levels of aspartate. The alpha-ketoglutarate/glutamate and malate/alpha-ketoglutarate ratios were higher in CR, in parallel with previously reported increases with CR in pyruvate carboxylase activity and glucagon levels, respectively. The results indicate that long-term CR induces a differential regulation of Krebs cycle in old mice and this regulation may be the result of changes in gene expression levels, as well as a complex interplay between enzymes, hormones and other effectors. Truncation of Krebs cycle by CR may be an important adaptation to utilize available substrates for the gluconeogenesis necessary to sustain glycolytic tissues, such as brain.

  19. Sarcocystis fusiformis: some Krebs cycle enzymes in various fractions of sarcocysts of buffalo (Bubalus bubalis).

    PubMed

    Gupta, R S; Kushwah, H S; Kushwah, A

    1995-01-01

    A biochemical investigation was carried out on the relative presence of some enzymes of the Krebs cycle and of the associated energy metabolism in various fractions (namely, cyst wall, cyst fluid and zoites) of sarcocysts of Sarcocystis fusiformis from the oesophageal muscles of naturally infected Indian water buffalo (Bubalus bubalis). Except for malate dehydrogenase, the activities of aconitase, isocitrate dehydrogenase, succinate dehydrogenase and fumarase were beyond detectable limits, pointing to a non-functional Krebs cycle in the cysts of this parasite. The activities of adenosine triphosphatase and cytochromes were lowest in cyst fluid and were maximally depicted by cyst wall and zoites.

  20. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  1. Reduced density of glutamine synthetase immunoreactive astrocytes in different cortical areas in major depression but not in bipolar I disorder.

    PubMed

    Bernstein, Hans-Gert; Meyer-Lotz, Gabriela; Dobrowolny, Henrik; Bannier, Jana; Steiner, Johann; Walter, Martin; Bogerts, Bernhard

    2015-01-01

    There is increasing evidence for disturbances within the glutamate system in patients with affective disorders, which involve disruptions of the glutamate-glutamine-cycle. The mainly astroglia-located enzyme glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a central role in glutamate and glutamine homoeostasis. However, GS is also expressed in numerous oligodendrocytes (OLs), another class of glial cells implicated in mood disorder pathology. To learn more about the role of glia-associated GS in mental illnesses, we decided to find out if numerical densities of glial cells immunostained for the enzyme protein differ between subjects with major depressive disorder, bipolar disorder (BD), and psychically healthy control cases. Counting of GS expressing astrocytes (ACs) and OLs in eight cortical and two subcortical brain regions of subjects with mood disorder (N = 14), BD (N = 15), and controls (N = 16) revealed that in major depression the densities of ACs were significantly reduced in some cortical but not subcortical gray matter areas, whereas no changes were found for OLs. In BD no alterations of GS-immunoreactive glia were found. From our findings we conclude that (1) GS expressing ACs are prominently involved in glutamate-related disturbances in major depression, but not in BD and (2) GS expressing OLs, though being present in significant numbers in prefrontal cortical areas, play a minor (if any) role in mood disorder pathology. The latter assumption is supported by findings of others showing that - at least in the mouse brain cortex - GS immunoreactive oligodendroglial cells are unable to contribute to the glutamate-glutamine-cycle due to the complete lack of amino acid transporters (Takasaki et al., 2010).

  2. Reduced density of glutamine synthetase immunoreactive astrocytes in different cortical areas in major depression but not in bipolar I disorder

    PubMed Central

    Bernstein, Hans-Gert; Meyer-Lotz, Gabriela; Dobrowolny, Henrik; Bannier, Jana; Steiner, Johann; Walter, Martin; Bogerts, Bernhard

    2015-01-01

    There is increasing evidence for disturbances within the glutamate system in patients with affective disorders, which involve disruptions of the glutamate–glutamine-cycle. The mainly astroglia-located enzyme glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a central role in glutamate and glutamine homoeostasis. However, GS is also expressed in numerous oligodendrocytes (OLs), another class of glial cells implicated in mood disorder pathology. To learn more about the role of glia-associated GS in mental illnesses, we decided to find out if numerical densities of glial cells immunostained for the enzyme protein differ between subjects with major depressive disorder, bipolar disorder (BD), and psychically healthy control cases. Counting of GS expressing astrocytes (ACs) and OLs in eight cortical and two subcortical brain regions of subjects with mood disorder (N = 14), BD (N = 15), and controls (N = 16) revealed that in major depression the densities of ACs were significantly reduced in some cortical but not subcortical gray matter areas, whereas no changes were found for OLs. In BD no alterations of GS-immunoreactive glia were found. From our findings we conclude that (1) GS expressing ACs are prominently involved in glutamate-related disturbances in major depression, but not in BD and (2) GS expressing OLs, though being present in significant numbers in prefrontal cortical areas, play a minor (if any) role in mood disorder pathology. The latter assumption is supported by findings of others showing that – at least in the mouse brain cortex – GS immunoreactive oligodendroglial cells are unable to contribute to the glutamate–glutamine-cycle due to the complete lack of amino acid transporters (Takasaki et al., 2010). PMID:26321908

  3. Glutamine Modulates Macrophage Lipotoxicity

    PubMed Central

    He, Li; Weber, Kassandra J.; Schilling, Joel D.

    2016-01-01

    Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs), activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli. PMID:27077881

  4. CP12-mediated protection of Calvin-Benson cycle enzymes from oxidative stress.

    PubMed

    Marri, Lucia; Thieulin-Pardo, Gabriel; Lebrun, Régine; Puppo, Rémy; Zaffagnini, Mirko; Trost, Paolo; Gontero, Brigitte; Sparla, Francesca

    2014-02-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two energy-consuming enzymes of the Calvin-Benson cycle, whose regulation is crucial for the global balance of the photosynthetic process under different environmental conditions. In oxygen phototrophs, GAPDH and PRK regulation involves the redox-sensitive protein CP12. In the dark, oxidized chloroplast thioredoxins trigger the formation of a GAPDH/CP12/PRK complex in which both enzyme activities are down-regulated. In this report, we show that free GAPDH (A4-isoform) and PRK are also inhibited by oxidants like H2O2, GSSG and GSNO. Both in the land plant Arabidopsis thaliana and in the green microalga Chlamydomonas reinhardtii, both enzymes can be glutathionylated as shown by biotinylated-GSSG assay and MALDI-ToF mass spectrometry. CP12 is not glutathionylated but homodisulfides are formed upon oxidant treatments. In Arabidopsis but not in Chlamydomonas, the interaction between oxidized CP12 and GAPDH provides full protection from oxidative damage. In both organisms, preformed GAPDH/CP12/PRK complexes are protected from GSSG or GSNO oxidation, and in Arabidopsis also from H2O2 treatment. Overall, the results suggest that the role of CP12 in oxygen phototrophs needs to be extended beyond light/dark regulation, and include protection of enzymes belonging to Calvin-Benson cycle from oxidative stress.

  5. CP12-mediated protection of Calvin-Benson cycle enzymes from oxidative stress.

    PubMed

    Marri, Lucia; Thieulin-Pardo, Gabriel; Lebrun, Régine; Puppo, Rémy; Zaffagnini, Mirko; Trost, Paolo; Gontero, Brigitte; Sparla, Francesca

    2014-02-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two energy-consuming enzymes of the Calvin-Benson cycle, whose regulation is crucial for the global balance of the photosynthetic process under different environmental conditions. In oxygen phototrophs, GAPDH and PRK regulation involves the redox-sensitive protein CP12. In the dark, oxidized chloroplast thioredoxins trigger the formation of a GAPDH/CP12/PRK complex in which both enzyme activities are down-regulated. In this report, we show that free GAPDH (A4-isoform) and PRK are also inhibited by oxidants like H2O2, GSSG and GSNO. Both in the land plant Arabidopsis thaliana and in the green microalga Chlamydomonas reinhardtii, both enzymes can be glutathionylated as shown by biotinylated-GSSG assay and MALDI-ToF mass spectrometry. CP12 is not glutathionylated but homodisulfides are formed upon oxidant treatments. In Arabidopsis but not in Chlamydomonas, the interaction between oxidized CP12 and GAPDH provides full protection from oxidative damage. In both organisms, preformed GAPDH/CP12/PRK complexes are protected from GSSG or GSNO oxidation, and in Arabidopsis also from H2O2 treatment. Overall, the results suggest that the role of CP12 in oxygen phototrophs needs to be extended beyond light/dark regulation, and include protection of enzymes belonging to Calvin-Benson cycle from oxidative stress. PMID:24211189

  6. Glutamine as an immunoenhancing nutrient.

    PubMed

    Saito, H; Furukawa, S; Matsuda, T

    1999-01-01

    New strategies for immunonutritional support include administration of special nutrients such as glutamine. Glutamine is important in several key metabolic processes of immune cells and enterocytes. Exogenous glutamine augments the functions of lymphocytes and macrophages. Neutrophils also reportedly utilize glutamine at a significant rate. Our recent studies demonstrated that glutamine enhances neutrophil function. This article focuses on the effects of glutamine on neutrophil function in surgical stress. Enteral glutamine administration enhanced peritoneal and hepatic bacterial clearance in our rat peritonitis model. Furthermore, IV glutamine supplementation improved the outcome of animals with severe surgical stress. Our in vitro study revealed that supplemental glutamine augmented the bacterial killing function of neutrophils from postoperative patients. Glutamine increased phagocytosis of the neutrophils. In addition, glutamine dose-dependently increased production of reactive oxygen intermediates (ROI) by neutrophils. Thus, our studies suggest that glutamine supplementation may improve bactericidal function of neutrophils by increasing both phagocytosis and ROI production. In conclusion, glutamine plays an important role in neutrophil function. Glutamine may be useful for the prevention, and treatment, of severe infection in critical illness and trauma.

  7. Role of glutamine in cerebral nitrogen metabolism and ammonia neurotoxicity.

    PubMed

    Cooper, A J

    2001-01-01

    Ammonia enters the brain by diffusion from the blood or cerebrospinal fluid, or is formed in situ from the metabolism of endogenous nitrogen-containing substances. Despite its central importance in nitrogen homeostasis, excess ammonia is toxic to the central nervous system and its concentration in the brain must be kept low. This is accomplished by the high activity of glutamine synthetase, which is localized in astrocytes and which permits efficient detoxification of incoming or endogenously generated ammonia. The location also permits the operation of an intercellular glutamine cycle. In this cycle, glutamate released from nerve terminals is taken up by astrocytes where it is converted to glutamine. Glutamine is released to the extracellular fluid to be taken up into the nerve cells, where it is converted back to glutamate by the action of glutaminase. Most extrahepatic organs lack a complete urea cycle, and for many organs, including the brain, glutamine represents a temporary storage form of waste nitrogen. As such, glutamine was long thought to be harmless to the brain. However, recent evidence suggests that excess glutamine is neurotoxic. Hyperammonemic syndromes (e.g., liver disease, inborn errors of the urea cycle, Reye's disease) consistently cause astrocyte pathology. Evidence has been presented that hyperammonemia results in increased formation of glutamine directly in astrocytes, thereby generating an osmotic stress to these cells. This osmotic stress results in impaired astrocyte function, which in turn leads to neuronal dysfunction. In this review a brief overview is presented of the role of glutamine in normal brain metabolism and in the pathogenesis of hyperammonemic syndromes.

  8. Fat-to-glucose interconversion by hydrodynamic transfer of two glyoxylate cycle enzyme genes

    PubMed Central

    Cordero, P; Campion, J; Milagro, FI; Marzo, F; Martinez, JA

    2008-01-01

    The glyoxylate cycle, which is well characterized in higher plants and some microorganisms but not in vertebrates, is able to bypass the citric acid cycle to achieve fat-to-carbohydrate interconversion. In this context, the hydrodynamic transfer of two glyoxylate cycle enzymes, such as isocytrate lyase (ICL) and malate synthase (MS), could accomplish the shift of using fat for the synthesis of glucose. Therefore, 20 mice weighing 23.37 ± 0.96 g were hydrodinamically gene transferred by administering into the tail vein a bolus with ICL and MS. After 36 hours, body weight, plasma glucose, respiratory quotient and energy expenditure were measured. The respiratory quotient was increased by gene transfer, which suggests that a higher carbohydrate/lipid ratio is oxidized in such animals. This application could help, if adequate protocols are designed, to induce fat utilization for glucose synthesis, which might be eventually useful to reduce body fat depots in situations of obesity and diabetes. PMID:19077206

  9. Troglitazone suppresses glutamine metabolism through a PPAR-independent mechanism

    PubMed Central

    Reynolds, Miriam R.; Clem, Brian F.

    2016-01-01

    Enhanced glutamine metabolism is required for tumor cell growth and survival, which suggests that agents targeting glutaminolysis may have utility within anti-cancer therapies. Troglitazone, a PPARγ agonist, exhibits significant anti-tumor activity and can alter glutamine metabolism in multiple cell types. Therefore, we examined whether troglitazone would disrupt glutamine metabolism in tumor cells and whether its action was reliant on PPARγ activity. We found that troglitazone treatment suppressed glutamine uptake and the expression of the glutamine transporter, ASCT2, and glutaminase. In addition, troglitazone reduced 13C-glutamine incorporation into the TCA cycle, decreased [ATP], and resulted in an increase in reactive oxygen species (ROS). Further, troglitazone treatment decreased tumor cell growth, which was partially rescued with the addition of the TCA-intermediate, alpha-ketoglutarate, or the anti-oxidant N-acetylcysteine. Importantly, troglitazone’s effects on glutamine uptake or viable cell number were found to be PPARγ-independent. In contrast, troglitazone caused a decrease in c-Myc levels, while the proteasomal inhibitor, MG132, rescued c-Myc, ASCT2 and GLS1 expression, as well as glutamine uptake and cell number. Lastly, combinatorial treatment of troglitazone and metformin resulted in a synergistic decrease in cell number. Therefore, characterizing new anti-tumor properties of previously approved FDA therapies supports the potential for repurposing of these agents. PMID:25872876

  10. Reciprocal enzyme regulation as a source of bistability in covalent modification cycles.

    PubMed

    Straube, Ronny; Conradi, Carsten

    2013-08-01

    Covalent modification cycles (CMCs) are the building blocks of many regulatory networks in biological systems. Under proper kinetic conditions such mono-cyclic enzyme systems can show a higher sensitivity to effectors than enzymes subject to direct allosteric regulation. Using methods from reaction network theory it has been argued that CMCs can potentially exhibit multiple steady states if the converter enzymes are regulated in a reciprocal manner, but the underlying mechanism as well as the kinetic requirements for the emergence of such a behavior remained unclear. Here, we reinvestigate CMCs with reciprocal regulation of the converter enzymes for two common regulatory mechanisms: allosteric regulation and covalent modification. To analyze the steady state behavior of the corresponding mass-action equations, we derive reduced models by means of a quasi-steady state approximation (QSSA). We also derive reduced models using the total QSSA which often better reproduces the transient dynamics of enzyme-catalyzed reaction systems. Through a steady state analysis of the reduced models we show that the occurrence of bistability can be associated with the presence of a double negative feedback loop. We also derive constraints for the model parameters which might help to evaluate the potential significance of the mechanisms described here for the generation of bistability in natural systems. In particular, our results support the view of a possible bistable response in the metabolic PFK1/F1,6BPase cycle as observed experimentally in rat liver extracts, and it suggests an alternative view on the origin of bistability in the Cdk1-Wee1-Cdc25 system.

  11. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    SciTech Connect

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  12. Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1).

    PubMed

    O'Neill, Ellis C; Stevenson, Clare E M; Tantanarat, Krit; Latousakis, Dimitrios; Donaldson, Matthew I; Rejzek, Martin; Nepogodiev, Sergey A; Limpaseni, Tipaporn; Field, Robert A; Lawson, David M

    2015-12-11

    The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The "gate" is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.

  13. Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1)*

    PubMed Central

    O'Neill, Ellis C.; Stevenson, Clare E. M.; Tantanarat, Krit; Latousakis, Dimitrios; Donaldson, Matthew I.; Rejzek, Martin; Nepogodiev, Sergey A.; Limpaseni, Tipaporn; Field, Robert A.; Lawson, David M.

    2015-01-01

    The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The “gate” is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms. PMID:26504082

  14. Viral affects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection

    PubMed Central

    Yu, Yongjun; Clippinger, Amy J.; Alwine, James C.

    2011-01-01

    Human cytomegalovirus (HCMV) infection causes dramatic alterations of intermediary metabolism, similar to those found in tumor cells. In infected cells, glucose carbon is not completely broken down by the tricarboxylic acid (TCA) cycle for energy; instead it is used biosynthetically. This process requires increased glucose uptake, increased glycolysis and the diversion of glucose carbon, in the form of citrate, from the TCA cycle for use in HCMV-induced fatty acid biosynthesis. The diversion of citrate from the TCA cycle (cataplerosis) requires induction of enzymes to promote glutaminolysis, the conversion of glutamine to -ketoglutarate in order to maintain the TCA cycle (anaplerosis) and ATP production. Such changes could result in heretofore uncharacterized pathogenesis, potentially implicating HCMV as a subtle co-factor in many maladies, including oncogenesis. Recognition of the effects of HCMV, and other viruses, on host cell metabolism will provide new understanding of viral pathogenesis and novel avenues for antiviral therapy. PMID:21570293

  15. Evolutionary History of the Enzymes Involved in the Calvin-Benson Cycle in Euglenids.

    PubMed

    Markunas, Chelsea M; Triemer, Richard E

    2016-05-01

    Euglenids are an ancient lineage that may have existed as early as 2 billion years ago. A mere 65 years ago, Melvin Calvin and Andrew A. Benson performed experiments on Euglena gracilis and elucidated the series of reactions by which carbon was fixed and reduced during photosynthesis. However, the evolutionary history of this pathway (Calvin-Benson cycle) in euglenids was more complex than Calvin and Benson could have imagined. The chloroplast present today in euglenophytes arose from a secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga. A long period of evolutionary time existed before this secondary endosymbiotic event took place, which allowed for other endosymbiotic events or gene transfers to occur prior to the establishment of the green chloroplast. This research revealed the evolutionary history of the major enzymes of the Calvin-Benson cycle throughout the euglenid lineage and showed that the majority of genes for Calvin-Benson cycle enzymes shared an ancestry with red algae and/or chromophytes suggesting they may have been transferred to the nucleus prior to the acquisition of the green chloroplast.

  16. Targeting Glutamine Metabolism in Breast Cancer with Aminooxyacetate

    PubMed Central

    Korangath, Preethi; Teo, Wei Wen; Sadik, Helen; Han, Liangfeng; Mori, Noriko; Huijts, Charlotte M.; Wildes, Flonne; Bharti, Santosh; Zhang, Zhe; Santa-Maria, Cesar A.; Tsai, Hualing; Dang, Chi V.; Stearns, Vered; Bhujwalla, Zaver M.; Sukumar, Saraswati

    2015-01-01

    Purpose Glutamine addiction in c-MYC–overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However, the mechanism of ensuing cell death remains unresolved. Experimental Design A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by magnetic resonance spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell-cycle and bromodeoxyuridine (BrdUrd) uptake analysis, and activation of the endoplasmic reticulum (ER) stress–mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model. Results We established a direct correlation between c-MYC overexpression, suppression of glutaminolysis, and AOA sensitivity in most breast cancer cells. MRS, cell-cycle analysis, and BrdUrd uptake measurements indicated depletion of aspartic acid and alanine leading to cell-cycle arrest at S-phase by AOA. Activation of components of the ER stress–mediated pathway, initiated through GRP78, led to apoptotic cell death. AOA inhibited growth of SUM159, SUM149, and MCF-7 xenografts and c-myc–overexpressing transgenic mouse mammary tumors. In MDA-MB-231, AOA was effective only in combination with chemotherapy. Conclusions AOA mediates its cytotoxic effects largely through the stress response pathway. The preclinical data of AOA’s effectiveness provide a strong rationale for further clinical development, particularly for c-MYC–overexpressing breast cancers. PMID:25813021

  17. Immunomodulation. Part IV: Glutamine.

    PubMed

    Bell, Susan Givens

    2006-01-01

    Glutamine, a nonessential amino acid that appears to be conditionally essential during periods of physiologic stress, plays important physiologic roles in the immune system. However, neither enteral nor parenteral glutamine supplementation makes a difference in the rate of systemic infection or of NEC in very low birth weight infants. Thus, the search for agents to enhance the neonate's immune system and to serve as safe and effective adjuvants to antibiotics continues. Part V, the final article in this immunomodulation series, will explore the use of probiotics to support the neonatal immune system.

  18. Photosynthesis in Rhodospirillum rubrum. III. Metabolic Control of Reductive Pentose Phosphate and Tricarboxylic Acid Cycle Enzymes 1

    PubMed Central

    Anderson, Louise; Fuller, R. C.

    1967-01-01

    Enzymes of the reductive pentose phosphate cycle including ribulose-diphosphate carboxylase, ribulose-5-phosphate kinase, ribose-5-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and alkaline fructose-1,6-diphos-phatase were shown to be present in autotrophically grown Rhodospirillum rubrum. Enzyme levels were measured in this organism grown photo- and dark heterotrophically as well. Several, but not all, of these enzymes appeared to be under metabolic control, mediated by exogenous carbon and nitrogen compounds. Light had no effect on the presence or levels of any of these enzymes in this photosynthetic bacterium. The enzymes of the tricarboxylic acid cycle and enolase were shown to be present in R. rubrum cultured aerobically, autotrophically, or photoheterotrophically, both in cultures evolving hydrogen and under conditions where hydrogen evolution is not observed. Light had no clearly demonstrable effect on the presence or levels of any of these enzymes. PMID:6042359

  19. MYC-induced reprogramming of glutamine catabolism supports optimal virus replication

    PubMed Central

    Thai, Minh; Thaker, Shivani K.; Feng, Jun; Du, Yushen; Hu, Hailiang; Ting Wu, Ting; Graeber, Thomas G.; Braas, Daniel; Christofk, Heather R.

    2015-01-01

    Viruses rewire host cell glucose and glutamine metabolism to meet the bioenergetic and biosynthetic demands of viral propagation. However, the mechanism by which viruses reprogram glutamine metabolism and the metabolic fate of glutamine during adenovirus infection have remained elusive. Here, we show MYC activation is necessary for adenovirus-induced upregulation of host cell glutamine utilization and increased expression of glutamine transporters and glutamine catabolism enzymes. Adenovirus-induced MYC activation promotes increased glutamine uptake, increased use of glutamine in reductive carboxylation and increased use of glutamine in generating hexosamine pathway intermediates and specific amino acids. We identify glutaminase (GLS) as a critical enzyme for optimal adenovirus replication and demonstrate that GLS inhibition decreases replication of adenovirus, herpes simplex virus 1 and influenza A in cultured primary cells. Our findings show that adenovirus-induced reprogramming of glutamine metabolism through MYC activation promotes optimal progeny virion generation, and suggest that GLS inhibitors may be useful therapeutically to reduce replication of diverse viruses. PMID:26561297

  20. Expression of glutamine synthetase in balloon cells: a basis of their antiepileptic role?

    PubMed

    Buccoliero, Anna Maria; Barba, Carmen; Giordano, Flavio; Baroni, Gianna; Genitori, Lorenzo; Guerrini, Renzo; Taddei, Gian Luigi

    2015-01-01

    Glutamine synthetase is an enzyme involved in the clearance of glutamate, the most potent excitatory neurotransmitter. We studied the immunohistochemical expression of glutamine synthetase in neocortical samples from 5 children who underwent surgery for pharmacoresistant epilepsy and a histological diagnosis of focal cortical dysplasia IIb. In all cases, balloon cells, but not dysmorphic neurons, were immunopositive for glutamine synthetase. This finding suggests that balloon cells can be involved in the neutralization of glutamate and play a protective anti-seizure role.

  1. Evaluation of Krebs cycle enzymes in the brain of rats after chronic administration of antidepressants.

    PubMed

    Scaini, Giselli; Santos, Patricia M; Benedet, Joana; Rochi, Natália; Gomes, Lara M; Borges, Lislaine S; Rezin, Gislaine T; Pezente, Daiana P; Quevedo, João; Streck, Emilio L

    2010-05-31

    Several works report brain impairment of metabolism as a mechanism underlying depression. Citrate synthase and succinate dehydrogenase are enzymes localized within cells in the mitochondrial matrix and are important steps of Krebs cycle. In addition, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase and succinate dehydrogenase activities from rat brain after chronic administration of paroxetine, nortriptiline and venlafaxine. Adult male Wistar rats received daily injections of paroxetine (10mg/kg), nortriptiline (15mg/kg), venlafaxine (10mg/kg) or saline in 1.0mL/kg volume for 15 days. Twelve hours after the last administration, the rats were killed by decapitation, the hippocampus, striatum and prefrontal cortex were immediately removed, and activities of citrate synthase and succinate dehydrogenase were measured. We verified that chronic administration of paroxetine increased citrate synthase activity in the prefrontal cortex, hippocampus, striatum and cerebral cortex of adult rats; cerebellum was not affected. Chronic administration of nortriptiline and venlafaxine did not affect the enzyme activity in these brain areas. Succinate dehydrogenase activity was increased by chronic administration of paroxetine and nortriptiline in the prefrontal cortex, hippocampus, striatum and cerebral cortex of adult rats; cerebellum was not affected either. Chronic administration of venlafaxine increased succinate dehydrogenase activity in prefrontal cortex, but did not affect the enzyme activity in cerebellum, hippocampus, striatum and cerebral cortex. Considering that metabolism impairment is probably involved in the pathophysiology of depressive disorders, an increase in these enzymes by antidepressants may be an important mechanism of action of these drugs.

  2. Regulation of the activity of the Bacillus licheniformis A5 glutamine synthetase.

    PubMed

    Donohue, T J; Bernlohr, R W

    1981-10-01

    The regulation of glutamine synthetase activity by positive and negative effectors of enzyme activity singularly and in combinations was studied by using a homogeneous enzyme preparation from Bacillus licheniformis A5. Phosphorylribosyl pyrophosphate at concentrations greater than 2mM stimulated glutamine synthetase activity by approximately 70%. The concentration of phosphorylribosyl pyrophosphate required for half-maximal stimulation of enzyme activity was 0.4 mM. Results obtained from studies of fractional inhibition of glutamine synthetase activity were consistent with the presence of one allosteric site for glutamine binding (apparent I0.5, 2.2mM) per active enzyme unit at a glutamate concentration of 50 mM. At a glutamate concentration of 30 mM or less, the data were consistent with the enzyme containing two binding sites for glutamine (one of which was an allosteric site with an apparent I0.5 of 0.4 mM). Bases on an analysis of the response of glutamine synthetase activity to positive and negative effectors in vitro and to the intracellular concentration of these effectors in vivo, the primary modulators of glutamine synthetase activity in B. licheniformis A5 appear to be glutamine and alanine (apparent I0.5, 5.2mM). PMID:6169702

  3. Glutamine alimentation in catabolic state.

    PubMed

    Boelens, P G; Nijveldt, R J; Houdijk, A P; Meijer, S; van Leeuwen, P A

    2001-09-01

    Glutamine should be reclassified as a conditionally essential amino acid in the catabolic state because the body's glutamine expenditures exceed synthesis and low glutamine levels in plasma are associated with poor clinical outcome. After severe stress, several amino acids are mobilized from muscle tissue to supply energy and substrate to the host. Glutamine is one of the most important amino acids that provide this function. Glutamine acts as the preferred respiratory fuel for lymphocytes, hepatocytes and intestinal mucosal cells and is metabolized in the gut to citrulline, ammonium and other amino acids. Low concentrations of glutamine in plasma reflect reduced stores in muscle and this reduced availability of glutamine in the catabolic state seems to correlate with increased morbidity and mortality. Adding glutamine to the nutrition of clinical patients, enterally or parenterally, may reduce morbidity. Several excellent clinical trials have been performed to prove efficacy and feasibility of the use of glutamine supplementation in parenteral and enteral nutrition. The increased intake of glutamine has resulted in lower septic morbidity in certain critically ill patient populations. This review will focus on the efficacy and the importance of glutamine supplementation in diverse catabolic states.

  4. Comparative Biochemical and Immunological Studies of Bacterial Glutamine Synthetases

    PubMed Central

    Tronick, Steven R.; Ciardi, Joseph E.; Stadtman, E. R.

    1973-01-01

    Antisera prepared against adenylylated and unadenylylated Escherichia coli glutamine synthetase cross-reacted with the glutamine synthetases from a number of gram-negative bacteria and one gram-variable species as demonstrated by immunodiffusion and inhibition of enzyme activity. In contrast, the antisera did not cross-react with the glutamine synthetases from gram-positive bacteria (with one exception) nor with the synthetases of higher organisms. Modification of the various glutamine synthetases by covalent attachment of adenosine 5′-monophosphate (or other nucleotides) was tested for by determining whether or not snake venom phosphodiesterase altered catalytic activity in a manner similar to its effect on adenylylated E. coli glutamine synthetase. Only the activity of the glutamine synthetases from gram-negative bacteria grown with specific levels of nitrogen sources could be altered by snake venom phosphodiesterase. In addition, a relative order of antigenic homology between cross-reacting enzymes was suggested based on the patterns of spur formation in the immunodiffusion assay. Images PMID:4125585

  5. Exchange of glutamine-217 to glutamate of Clostridium limosum exoenzyme C3 turns the asparagine-specific ADP-ribosyltransferase into an arginine-modifying enzyme.

    PubMed

    Vogelsgesang, Martin; Aktories, Klaus

    2006-01-24

    C3-like ADP-ribosyltransferaseses are produced by Clostridium species, Bacillus cereus, and various Staphylococcus aureus strains. The exoenzymes modify the low-molecular-mass GTPases RhoA, B, and C. In structural studies of C3-like exoenzymes, an ARTT-motif (ADP-ribosylating turn-turn motif) was identified that appears to be involved in substrate specificity and recognition (Han, S., Arvai, A. S., Clancy, S. B., Tainer, J. A. (2001) J. Mol. Biol. 305, 95-107). Exchange of Gln217, which is a key residue of the ARTT-motif, to Glu in C3 from Clostridium limosum results in inhibition of ADP-ribosyltransferase activity toward RhoA. The mutant protein is still capable of NAD-binding and possesses NAD+ glycohydrolase activity. Whereas recombinant wild-type C3 modifies Rho proteins specifically at an asparagine residue (Asn41), Gln217Glu-C3 is capable of ADP-ribosylation of poly-arginine but not poly-asparagine. Soybean trypsin inhibitor, a model substrate for many arginine-specific ADP-ribosyltransferases, is modified by the Gln217Glu-C3 transferase. Also in C3 ADP-ribosyltransferases from Clostridium botulinum and B. cereus, the exchange of the equivalent Gln residue to Glu blocked asparagine modification of RhoA but elicited arginine-specific ADP-ribosylation. Moreover, the Gln217Glu-C3lim transferase was able to ADP-ribosylate recombinant wild-type C3lim at Arg86, resulting in decrease in ADP-ribosyltransferase activity of the wild-type enzyme. The data indicate that the exchange of one amino acid residue in the ARTT-motif turns the asparagine-modifying ADP-ribosyltransferases of the C3 family into arginine-ADP-ribosylating transferases.

  6. Clinical use of glutamine supplementation.

    PubMed

    Wernerman, Jan

    2008-10-01

    Endogenous production of glutamine may become insufficient during critical illness. The shortage of glutamine is reflected as a decrease in plasma concentration, which is a prognostic factor for poor outcome in sepsis. Because glutamine is a precursor for nucleotide synthesis, rapidly dividing cells are most likely to suffer from a shortage. Therefore, exogenous glutamine supplementation is necessary. In particular, when i.v. nutrition is given, extra glutamine supplementation becomes critical, because most present formulations for i.v. use do not contain any glutamine for technical reasons. The major part of endogenously produced glutamine comes from skeletal muscle. For patients staying a long time in the intensive care unit (ICU), the muscle mass decreases rapidly, which leaves a tissue of diminishing size to maintain the export of glutamine. The metabolic and nutritional adaptation in long-staying ICU patients is poorly studied and is one of the fields that needs more scientific evidence for clinical recommendations. To date, there is evidence to support the clinical use of glutamine supplementation in critically ill patients, in hematology patients, and in oncology patients. Strong evidence is presently available for i.v. glutamine supplementation to critically ill patients on parenteral nutrition. This must be regarded as the standard of care. For patients on enteral nutrition, more evidence is needed. To guide administration of glutamine, there are good arguments to use measurement of plasma glutamine concentration for guidance. This will give an indication for treatment as well as proper dosing. Most patients will have a normalized plasma glutamine concentration by adding 20-25 g/24 h. Furthermore, there are no reported adverse or negative effects attributable to glutamine supplementation.

  7. Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion

    PubMed Central

    Shanware, Naval P.; Bray, Kevin; Eng, Christina H.; Wang, Fang; Follettie, Maximillian; Myers, Jeremy; Fantin, Valeria R.; Abraham, Robert T.

    2014-01-01

    The non-essential amino acid, glutamine, exerts pleiotropic effects on cell metabolism, signalling and stress resistance. Here we demonstrate that short-term glutamine restriction triggers an endoplasmic reticulum (ER) stress response that leads to production of the pro-inflammatory chemokine, interleukin-8 (IL-8). Glutamine deprivation-induced ER stress triggers colocalization of autophagosomes, lysosomes and the Golgi into a subcellular structure whose integrity is essential for IL-8 secretion. The stimulatory effect of glutamine restriction on IL-8 production is attributable to depletion of tricarboxylic acid cycle intermediates. The protein kinase, mTOR, is also colocalized with the lysosomal membrane clusters induced by glutamine deprivation, and inhibition of mTORC1 activity abolishes both endomembrane reorganization and IL-8 secretion. Activated mTORC1 elicits IL8 gene expression via the activation of an IRE1-JNK signalling cascade. Treatment of cells with a glutaminase inhibitor phenocopies glutamine restriction, suggesting that these results will be relevant to the clinical development of glutamine metabolism inhibitors as anticancer agents. PMID:25254627

  8. Structure-function relationships in the Na,K-ATPase. cap alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    SciTech Connect

    Price, E.M.; Lingrel, J.B.

    1988-11-01

    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the ..cap alpha..1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat ..cap alpha..1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase ..cap alpha.. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep ..cap alpha..1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep ..cap alpha..1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep ..cap alpha..1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat ..cap alpha..1 cDNA, the rat/sheep chimera, or the mutant sheep ..cap alpha..1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, /sup 86/Rb/sup +/ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase ..cap alpha.. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep ..cap alpha..1 subunit (glutamine and asparagine) are somehow involved in ouabain binding.

  9. Bidirectional substrate fluxes through the System N (SNAT5) glutamine transporter may determine net glutamine flux in rat liver

    PubMed Central

    Baird, F E; Beattie, K J; Hyde, A R; Ganapathy, V; Rennie, M J; Taylor, P M

    2004-01-01

    System N (SNAT3 and SNAT5) amino acid transporters are key mediators of glutamine transport across the plasma membrane of mammalian cell types, including hepatocytes and astrocytes. We demonstrate that SNAT5 shows simultaneous bidirectional glutamine fluxes when overexpressed in Xenopus oocytes. Influx and efflux are both apparently Na+ dependent but, since they are not directly coupled, the carrier is capable of mediating net amino acid movement across the cell membrane. The apparent Km values for glutamine influx and efflux are similar (∼1 mm) and the transporter behaviour is consistent with a kinetic model in which re-orientation of the carrier from outside- to inside-facing conformations (either empty or substrate loaded) is the limiting step in the transport cycle. In perfused rat liver, the observed relationship between influent (portal) glutamine concentration and net hepatic glutamine flux may be described by a simple kinetic model, assuming the balance between influx and efflux through System N determines net flux, where under physiological conditions efflux is generally saturated owing to high intracellular glutamine concentration. SNAT5 shows a more periportal mRNA distribution than SNAT3 in rat liver, indicating that SNAT5 may have particular importance for modulation of net hepatic glutamine flux. PMID:15218073

  10. Comparative analysis on the key enzymes of the glycerol cycle metabolic pathway in Dunaliella salina under osmotic stresses.

    PubMed

    Chen, Hui; Lu, Yan; Jiang, Jian-Guo

    2012-01-01

    The glycerol metabolic pathway is a special cycle way; glycerol-3-phosphate dehydrogenase (G3pdh), glycerol-3-phosphate phosphatase (G3pp), dihydroxyacetone reductase (Dhar), and dihydroxyacetone kinase (Dhak) are the key enzymes around the pathway. Glycerol is an important osmolyte for Dunaliella salina to resist osmotic stress. In this study, comparative activities of the four enzymes in D. salina and their activity changes under various salt stresses were investigated, from which glycerol metabolic flow direction in the glycerol metabolic pathway was estimated. Results showed that the salinity changes had different effects on the enzymes activities. NaCl could stimulate the activities of all the four enzymes in various degrees when D. salina was grown under continuous salt stress. When treated by hyperosmotic or hypoosmotic shock, only the activity of G3pdh in D. salina was significantly stimulated. It was speculated that, under osmotic stresses, the emergency response of the cycle pathway in D. salina was driven by G3pdh via its response to the osmotic stress. Subsequently, with the changes of salinity, other three enzymes started to respond to osmotic stress. Dhar played a role of balancing the cycle metabolic pathway by its forward and backward reactions. Through synergy, the four enzymes worked together for the effective flow of the cycle metabolic pathways to maintain the glycerol requirements of cells in order to adapt to osmotic stress environments.

  11. [Imbalance of system of glutamin - glutamic acid in the placenta and amniotic fluid at placental insufficiency].

    PubMed

    Pogorelova, T N; Gunko, V O; Linde, V A

    2014-01-01

    Metabolism of glutamine and glutamic acid has been investigated in the placenta and amniotic fluid under conditions of placental insufficiency. The development of placental insufficiency is characterized by the increased content of glutamic acid and a decrease of glutamine in both placenta and amniotic fluid. These changes changes were accompanied by changes in the activity of enzymes involved in the metabolism of these amino acids. There was a decrease in glutamate dehydrogenase activity and an increase in glutaminase activity with the simultaneous decrease of glutamine synthetase activity. The compensatory decrease in the activity of glutamine keto acid aminotransferase did not prevent a decrease in the glutamine level. The impairments in the system glutamic acid-glutamine were more pronounced during the development of premature labor.

  12. Glutamine Involvement in Nitrogen Control of Gibberellic Acid Production in Gibberella fujikuroi

    PubMed Central

    Muñoz, Gastón A.; Agosin, Eduardo

    1993-01-01

    When the fungus Gibberella fujikuroi ATCC 12616 was grown in fermentor cultures, both intracellular kaurene biosynthetic activities and extracellular GA3 accumulation reached high levels when exogenous nitrogen was depleted in the culture. Similar patterns were exhibited by several nonrelated enzymatic activities, such as formamidase and urease, suggesting that all are subject to nitrogen regulation. The behavior of the enzymes involved in nitrogen assimilation (glutamine synthetase, glutamate dehydrogenase, and glutamate synthase) during fungal growth in different nitrogen sources suggests that glutamine is the final product of nitrogen assimilation in G. fujikuroi. When ammonium or glutamine was added to hormone-producing cultures, extracellular GA3 did not accumulate. However, when the conversion of ammonium into glutamine was inhibited by L-methionine-DL-sulfoximine, only glutamine maintained this effect. These results suggest that glutamine may well be the metabolite effector in nitrogen repression of GA3 synthesis, as well as in other nonrelated enzymatic activities in G. fujikuroi. PMID:16349128

  13. Supplementation with L-Glutamine and L-Alanyl-L-Glutamine Changes Biochemical Parameters and Jejunum Morphophysiology in Type 1 Diabetic Wistar Rats

    PubMed Central

    da Rosa, Carlos Vinicius D.; Azevedo, Silvia C. S. F.; Bazotte, Roberto B.; Peralta, Rosane M.; Buttow, Nilza C.; Pedrosa, Maria Montserrat D.; de Godoi, Vilma A. F.; Natali, Maria Raquel M.

    2015-01-01

    We evaluated the effects of the supplementation with L-glutamine and glutamine dipeptide (GDP) on biochemical and morphophysiological parameters in streptozotocin-diabetic rats. For this purpose, thirty animals were distributed into six groups treated orally (gavage) during thirty days: non diabetic rats (Control) + saline, diabetic + saline; Control + L-glutamine (248 mg/kg), Diabetic + L-glutamine (248 mg/kg), Control + GDP (400 mg/kg), Diabetic + GDP (400 mg/kg). Diabetes was induced by an intravenous injection of streptozotocin (60 mg/kg) and confirmed by fasting glucose ≥ 200 mg/dL. Physiological parameters, i.e., body mass, food intake, blood glucose, water intake, urine and faeces were evaluated during supplementation. After the period of supplementation, the animals were euthanized. The blood was collected for biochemical assays (fructosamine, transaminases, lipid profile, total protein, urea, ammonia). Moreover, the jejunum was excised and stored for morphophysiological assays (intestinal enzyme activity, intestinal wall morphology, crypt proliferative index, number of serotoninergic cells from the mucosa, and vipergic neurons from the submucosal tunica). The physiological parameters, protein metabolism and intestinal enzyme activity did not change with the supplementation with L-glutamine or GDP. In diabetic animals, transaminases and fructosamine improved with L-glutamine and GDP supplementations, while the lipid profile improved with L-glutamine. Furthermore, both forms of supplementation promoted changes in jejunal tunicas and wall morphometry of control and diabetic groups, but only L-glutamine promoted maintenance of serotoninergic cells and vipergic neurons populations. On the other hand, control animals showed changes that may indicate negative effects of L-glutamine. Thus, the supplementation with L-glutamine was more efficient for maintaining intestinal morphophysiology and the supplementation with GDP was more efficient to the organism as a

  14. Interrelationships between glutamine and citrulline metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This article analyzes the contribution of glutamine to the synthesis of citrulline and reviews the evidence that glutamine supplementation increases citrulline production. Glutamine supplementation has been proposed in the treatment of critically ill patients; however, a recent large multicenter ran...

  15. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets.

    PubMed

    Pinto-Fernandez, Adan; Kessler, Benedikt M

    2016-01-01

    Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors. PMID:27516771

  16. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase

    PubMed Central

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70–4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes. PMID:27537184

  17. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets

    PubMed Central

    Pinto-Fernandez, Adan; Kessler, Benedikt M.

    2016-01-01

    Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors. PMID:27516771

  18. Coevolution and Life Cycle Specialization of Plant Cell Wall Degrading Enzymes in a Hemibiotrophic Pathogen

    PubMed Central

    Brunner, Patrick C.; Torriani, Stefano F.F.; Croll, Daniel; Stukenbrock, Eva H.; McDonald, Bruce A.

    2013-01-01

    Zymoseptoria tritici is an important fungal pathogen on wheat that originated in the Fertile Crescent. Its closely related sister species Z. pseudotritici and Z. ardabiliae infect wild grasses in the same region. This recently emerged host–pathogen system provides a rare opportunity to investigate the evolutionary processes shaping the genome of an emerging pathogen. Here, we investigate genetic signatures in plant cell wall degrading enzymes (PCWDEs) that are likely affected by or driving coevolution in plant-pathogen systems. We hypothesize four main evolutionary scenarios and combine comparative genomics, transcriptomics, and selection analyses to assign the majority of PCWDEs in Z. tritici to one of these scenarios. We found widespread differential transcription among different members of the same gene family, challenging the idea of functional redundancy and suggesting instead that specialized enzymatic activity occurs during different stages of the pathogen life cycle. We also find that natural selection has significantly affected at least 19 of the 48 identified PCWDEs. The majority of genes showed signatures of purifying selection, typical for the scenario of conserved substrate optimization. However, six genes showed diversifying selection that could be attributed to either host adaptation or host evasion. This study provides a powerful framework to better understand the roles played by different members of multigene families and to determine which genes are the most appropriate targets for wet laboratory experimentation, for example, to elucidate enzymatic function during relevant phases of a pathogen’s life cycle. PMID:23515261

  19. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase.

    PubMed

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan; Trnka, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.

  20. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase.

    PubMed

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan; Trnka, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes. PMID:27537184

  1. The glutamine-alpha-ketoglutarate (AKG) metabolism and its nutritional implications.

    PubMed

    Xiao, Dingfu; Zeng, Liming; Yao, Kang; Kong, Xiangfeng; Wu, Guoyao; Yin, Yulong

    2016-09-01

    L-Glutamine is a nutritionally semi-essential amino acid for proper growth in most cells and tissues, and plays an important role in the determination and guarding of the normal metabolic processes of the cells. With the help of transport systems, extracellular L-glutamine crosses the plasma membrane and is converted into alpha-ketoglutarate (AKG) through two pathways, namely, the glutaminase (GLS) I and II pathway. Reversely, AKG can be converted into glutamine by glutamate dehydrogenase (GDH) and glutamine synthetase (GS), or be converted into CO2 via the tricarboxylic acid (TCA) cycle and provide energy for the cells. Different steps of glutamine metabolism (the glutamine-AKG axis) are regulated by several factors, rendering the glutamine-AKG axis a potential target to counteract cancer. Moreover, intracellular glutamine plays an important role in cellular homeostasis not only as a precursor for protein synthesis, but also for its nutritional roles in cell growth, lipid metabolism, insulin secretion, and so on. The main objective of this review is to highlight the metabolic pathways of glutamine to AKG, with special emphasis on nutritional and therapeutic use of glutamine-AKG axis to improve the health and well-being of animals and humans. PMID:27161106

  2. Glutamine and leucine nitrogen kinetics and their relation to urea nitrogen in newborn infants.

    PubMed

    Parimi, Prabhu S; Devapatla, Srisatish; Gruca, Lourdes; O'Brien, Alicia M; Hanson, Richard W; Kalhan, Satish C

    2002-03-01

    Glutamine kinetics and its relation to transamination of leucine and urea synthesis were quantified in 16 appropriate-for-gestational-age infants, four small-for-gestational-age infants, and seven infants of diabetic mothers. Kinetics were measured between 4 and 5 h after the last feed (fasting) and in response to formula feeding using [5-(15)N]glutamine, [1-(13)C,(15)N]leucine, [(2)H(5)]phenylalanine, and [(15)N(2)]urea tracers. Leucine nitrogen and glutamine kinetics during fasting were significantly higher than those reported in adults. De novo synthesis accounted for approximately 85% of glutamine turnover. In response to formula feeding, a significant increase (P = 0.04) in leucine nitrogen turnover was observed, whereas a significant decrease (P = 0.002) in glutamine and urea rate of appearance was seen. The rate of appearance of leucine nitrogen was positively correlated (r(2) = 0.59, P = 0.001) with glutamine turnover. Glutamine flux was negatively correlated (r(2) = 0.39, P = 0.02) with the rate of urea synthesis. These data suggest that, in the human newborn, glutamine turnover is related to a high anaplerotic flux into the tricarboxylic acid cycle as a consequence of a high rate of protein turnover. The negative relationship between glutamine turnover and the irreversible oxidation of protein (urea synthesis) suggests an important role of glutamine as a nitrogen source for other synthetic processes and accretion of body proteins.

  3. Cell cycle effect on the activity of deoxynucleoside analogue metabolising enzymes

    SciTech Connect

    Fyrberg, Anna; Albertioni, Freidoun; Lotfi, Kourosh . E-mail: koulo@imv.liu.se

    2007-06-15

    Deoxynucleoside analogues (dNAs) are cytotoxic towards both replicating and indolent malignancies. The impact of fluctuations in the metabolism of dNAs in relation to cell cycle could have strong implications regarding the activity of dNAs. Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) are important enzymes for phosphorylation/activation of dNAs. These drugs can be dephosphorylated/deactivated by 5'-nucleotidases (5'-NTs) and elevated activities of 5'-NTs and decreased dCK and/or dGK activities represent resistance mechanisms towards dNAs. The activities of dCK, dGK, and three 5'-NTs were investigated in four human leukemic cell lines in relationship to cell cycle progression and cytotoxicity of dNAs. Synchronization of cell cultures to arrest in G0/G1 by serum-deprivation was performed followed by serum-supplementation for cell cycle progression. The activities of dCK and dGK increased up to 3-fold in CEM, HL60, and MOLT-4 cells as they started to proliferate, while the activity of cytosolic nucleotidase I was reduced in proliferating cells. CEM, HL60, and MOLT-4 cells were also more sensitive to cladribine, cytarabine, 9-{beta}-D-arabinofuranosylguanine and clofarabine than K562 cells which demonstrated lower levels and less alteration of these enzymes and were least susceptible to the cytotoxic effects of most dNAs. The results suggest that, in the cell lines studied, the proliferation process is associated with a general shift in the direction of activation of dNAs by inducing activities of dCK/dGK and reducing the activity of cN-I which is favourable for the cytotoxic effects of cladribine, cytarabine and, 9-{beta}-D-arabinofuranosylguanine. These results emphasize the importance of cellular proliferation and dNA metabolism by both phosphorylation and dephosphorylation for susceptibility to dNAs. It underscores the need to understand the mechanisms of action and resistance to dNAs in order to increase efficacy of dNAs treatment by new rational.

  4. IKKβ promotes metabolic adaptation to glutamine deprivation via phosphorylation and inhibition of PFKFB3.

    PubMed

    Reid, Michael A; Lowman, Xazmin H; Pan, Min; Tran, Thai Q; Warmoes, Marc O; Ishak Gabra, Mari B; Yang, Ying; Locasale, Jason W; Kong, Mei

    2016-08-15

    Glutamine is an essential nutrient for cancer cell survival and proliferation. Enhanced utilization of glutamine often depletes its local supply, yet how cancer cells adapt to low glutamine conditions is largely unknown. Here, we report that IκB kinase β (IKKβ) is activated upon glutamine deprivation and is required for cell survival independently of NF-κB transcription. We demonstrate that IKKβ directly interacts with and phosphorylates 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase isoform 3 (PFKFB3), a major driver of aerobic glycolysis, at Ser269 upon glutamine deprivation to inhibit its activity, thereby down-regulating aerobic glycolysis when glutamine levels are low. Thus, due to lack of inhibition of PFKFB3, IKKβ-deficient cells exhibit elevated aerobic glycolysis and lactate production, leading to less glucose carbons contributing to tricarboxylic acid (TCA) cycle intermediates and the pentose phosphate pathway, which results in increased glutamine dependence for both TCA cycle intermediates and reactive oxygen species suppression. Therefore, coinhibition of IKKβ and glutamine metabolism results in dramatic synergistic killing of cancer cells both in vitro and in vivo. In all, our results uncover a previously unidentified role of IKKβ in regulating glycolysis, sensing low-glutamine-induced metabolic stress, and promoting cellular adaptation to nutrient availability. PMID:27585591

  5. Novel subunit-subunit interactions in the structure of glutamine synthetase.

    PubMed

    Almassy, R J; Janson, C A; Hamlin, R; Xuong, N H; Eisenberg, D

    We present an atomic model for glutamine synthetase, an enzyme of central importance in bacterial nitrogen metabolism, from X-ray crystallography. The 12 identical subunits are arranged as the carbon atoms in two face-to-face benzene rings, with unusual subunit contacts. Our model, which places the active sites at the subunit interfaces, suggests a mechanism for the main functional role of glutamine synthetase: how the enzyme regulates the rate of synthesis of glutamine in response to covalent modification and feedback inhibition. PMID:2876389

  6. Glutamate, GABA, and glutamine are synchronously upregulated in the mouse lateral septum during the postpartum period

    PubMed Central

    Zhao, Changjiu; Gammie, Stephen C.

    2014-01-01

    Dramatic structural and functional remodeling occurs in the postpartum brain for the establishment of maternal care, which is essential for the growth and development of young offspring. Glutamate and GABA signaling are critically important in modulating multiple behavioral performances. Large scale signaling changes occur in the postpartum brain, but it is still not clear to what extent the neurotransmitters glutamate and GABA change and whether the ratio of glutamate/GABA remains balanced. In this study, we examined the glutamate/GABA-glutamine cycle in the lateral septum (LS) of postpartum female mice. In postpartum females (relative to virgins), tissue levels of glutamate and GABA were elevated in LS and increased mRNA was found for the respective enzymes producing glutamate and GABA, glutaminase (Gls) and glutamate decarboxylase 1 and 2 (Gad1 and Gad2). The common precursor, glutamine, was elevated as was the enzyme that produces it, glutamate-ammonia ligase (Glul). Additionally, glutamate, GABA, and glutamine were positively correlated and the glutamate/GABA ratio was almost identical in the postpartum and virgin females. Collectively, these findings indicate that glutamate and GABA signaling are increased and that the ratio of glutamate/GABA is well balanced in the maternal LS. The postpartum brain may provide a useful model system for understanding how glutamate and GABA are linked despite large signaling changes. Given that some mental health disorders, including depression and schizophrenia display dysregulated glutamate/GABA ratio, and there is increased vulnerability to mental disorders in mothers, it is possible that these postpartum disorders emerge when glutamate and GABA changes are not properly coordinated. PMID:25451092

  7. Glutamate, GABA, and glutamine are synchronously upregulated in the mouse lateral septum during the postpartum period.

    PubMed

    Zhao, Changjiu; Gammie, Stephen C

    2014-12-01

    Dramatic structural and functional remodeling occurs in the postpartum brain for the establishment of maternal care, which is essential for the growth and development of young offspring. Glutamate and GABA signaling are critically important in modulating multiple behavioral performances. Large scale signaling changes occur in the postpartum brain, but it is still not clear to what extent the neurotransmitters glutamate and GABA change and whether the ratio of glutamate/GABA remains balanced. In this study, we examined the glutamate/GABA-glutamine cycle in the lateral septum (LS) of postpartum female mice. In postpartum females (relative to virgins), tissue levels of glutamate and GABA were elevated in LS and increased mRNA was found for the respective enzymes producing glutamate and GABA, glutaminase (Gls) and glutamate decarboxylase 1 and 2 (Gad1 and Gad2). The common precursor, glutamine, was elevated as was the enzyme that produces it, glutamate-ammonia ligase (Glul). Additionally, glutamate, GABA, and glutamine were positively correlated and the glutamate/GABA ratio was almost identical in the postpartum and virgin females. Collectively, these findings indicate that glutamate and GABA signaling are increased and that the ratio of glutamate/GABA is well balanced in the maternal LS. The postpartum brain may provide a useful model system for understanding how glutamate and GABA are linked despite large signaling changes. Given that some mental health disorders, including depression and schizophrenia display dysregulated glutamate/GABA ratio, and there is increased vulnerability to mental disorders in mothers, it is possible that these postpartum disorders emerge when glutamate and GABA changes are not properly coordinated.

  8. Phloem-Specific Expression of Yang Cycle Genes and Identification of Novel Yang Cycle Enzymes in Plantago and Arabidopsis[C][W

    PubMed Central

    Pommerrenig, Benjamin; Feussner, Kirstin; Zierer, Wolfgang; Rabinovych, Valentyna; Klebl, Franz; Feussner, Ivo; Sauer, Norbert

    2011-01-01

    The 5-methylthioadenosine (MTA) or Yang cycle is a set of reactions that recycle MTA to Met. In plants, MTA is a byproduct of polyamine, ethylene, and nicotianamine biosynthesis. Vascular transcriptome analyses revealed phloem-specific expression of the Yang cycle gene 5-METHYLTHIORIBOSE KINASE1 (MTK1) in Plantago major and Arabidopsis thaliana. As Arabidopsis has only a single MTK gene, we hypothesized that the expression of other Yang cycle genes might also be vascular specific. Reporter gene studies and quantitative analyses of mRNA levels for all Yang cycle genes confirmed this hypothesis for Arabidopsis and Plantago. This includes the Yang cycle genes 5-METHYLTHIORIBOSE-1-PHOSPHATE ISOMERASE1 and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1. We show that these two enzymes are sufficient for the conversion of methylthioribose-1-phosphate to 1,2-dihydroxy-3-keto-5-methylthiopentene. In bacteria, fungi, and animals, the same conversion is catalyzed in three to four separate enzymatic steps. Furthermore, comparative analyses of vascular and nonvascular metabolites identified Met, S-adenosyl Met, and MTA preferentially or almost exclusively in the vascular tissue. Our data represent a comprehensive characterization of the Yang cycle in higher plants and demonstrate that the Yang cycle works primarily in the vasculature. Finally, expression analyses of polyamine biosynthetic genes suggest that the Yang cycle in leaves recycles MTA derived primarily from polyamine biosynthesis. PMID:21540433

  9. Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements.

    PubMed Central

    Gray, N K; Pantopoulos, K; Dandekar, T; Ackrell, B A; Hentze, M W

    1996-01-01

    The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8643505

  10. Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson-Calvin cycle enzyme enslaved to its cysteine residues.

    PubMed

    Thieulin-Pardo, Gabriel; Remy, Thérèse; Lignon, Sabrina; Lebrun, Régine; Gontero, Brigitte

    2015-04-01

    Phosphoribulokinase (PRK) in the green alga Chlamydomonas reinhardtii is a finely regulated and well-studied enzyme of the Benson-Calvin cycle. PRK can form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small chloroplast protein CP12. This study aimed to determine the molecular determinants on PRK involved in the complex and the mechanism of action of a recently described novel regulation of PRK that involves glutathionylation. A combination of mass spectrometry, mutagenesis and activity analyses showed that Cys16, besides its role as the binding site of ATP, was also the site for S-glutathionylation. Previous kinetic analysis of the C55S mutant showed that in the oxidized inactive form of PRK, this residue formed a disulfide bridge with the Cys16 residue. This is the only bridge reported for PRK in the literature. Our data show for the first time that a disulfide bridge between Cys243 and Cys249 on PRK is required to form the PRK-GAPDH-CP12 complex. These results uncover a new mechanism for the PRK-GAPDH-CP12 formation involving a thiol disulfide exchange reaction with CP12 and identify Cys16 of PRK as a target of glutathionylation acting against oxidative stress. Although Cys16 is the key residue involved in binding ATP and acting as a defense against oxidative damage, the formation of the algal ternary complex requires the formation of another disulfide bridge on PRK involving Cys243 and Cys249.

  11. Comprehensive expression analysis of prostanoid enzymes and receptors in the human endometrium across the menstrual cycle.

    PubMed

    Catalano, Rob D; Wilson, Martin R; Boddy, Sheila C; Jabbour, Henry N

    2011-03-01

    Prostanoids are well-described primary mediators of inflammatory processes and are essential for the normal physiological function of the female reproductive system. The aim of this study was to determine the temporal expression of the prostanoid biosynthetic enzymes (PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, AKR1C3, CBR1, HPGDS, PTGDS, PTGIS, TBXAS1 and HPGD) and the prostanoid receptors (PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGDR, GPR44, PTGIR and TBXA2R) in the human endometrium throughout the menstrual cycle. The analysis identified PTGFR to have a distinct expression profile compared with other components of the prostanoid system, as expression is maximal during the proliferative phase. Immunohistochemical analysis for PTGER1 suggests a dual function for this receptor depending on its temporal (proliferative versus secretory) and spatial (nuclear versus cell membrane) expression. The expression profiles of the PGF(2α) synthases identified AKR1B1 and CBR1 as the likely regulators of PGF(2α) production during the menstrual phase. Immunohistochemical analysis for AKR1B1, CBR1 and AKR1C3 suggest expression to be in the glandular epithelium and vasculature. This study represents the first comprehensive analysis of the components of prostanoid biosynthetic and signalling pathway in the human endometrium. The expression profiles described have the potential to identify specific prostanoid components that may be dysregulated in inflammatory-associated disorders of the endometrium. PMID:21112968

  12. Glutamine Assimilation and Feedback Regulation of L-acetyl-N-glutamate Kinase Activity in Chlorella variabilis NC64A Results in Changes in Arginine Pools.

    PubMed

    Minaeva, Ekaterina; Forchhammer, Karl; Ermilova, Elena

    2015-11-01

    Glutamine is a metabolite of central importance in nitrogen metabolism of microorganisms and plants. The Chlorella PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine/arginine biosynthesis pathway, N-acetyl-L-glutamate kinase (NAGK) that leads to arginine formation. We provide evidence that glutamine promotes effective growth of C. variabilis strain NC64A. The present study shows that externally supplied glutamine directly influences the internal pool of arginine in NC64A. Glutamine synthetase (GS) catalyzes the ATP-dependent conversion of glutamate and ammonium to glutamine. The results of this study demonstrate that glutamine acts as a negative effector of GS activity. These data emphasize the importance of glutamine-dependent coupling of metabolism and signaling as components of an efficient pathway allowing the maintenance of metabolic homeostasis and sustaining growth of Chlorella.

  13. Glutamine Assimilation and Feedback Regulation of L-acetyl-N-glutamate Kinase Activity in Chlorella variabilis NC64A Results in Changes in Arginine Pools.

    PubMed

    Minaeva, Ekaterina; Forchhammer, Karl; Ermilova, Elena

    2015-11-01

    Glutamine is a metabolite of central importance in nitrogen metabolism of microorganisms and plants. The Chlorella PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine/arginine biosynthesis pathway, N-acetyl-L-glutamate kinase (NAGK) that leads to arginine formation. We provide evidence that glutamine promotes effective growth of C. variabilis strain NC64A. The present study shows that externally supplied glutamine directly influences the internal pool of arginine in NC64A. Glutamine synthetase (GS) catalyzes the ATP-dependent conversion of glutamate and ammonium to glutamine. The results of this study demonstrate that glutamine acts as a negative effector of GS activity. These data emphasize the importance of glutamine-dependent coupling of metabolism and signaling as components of an efficient pathway allowing the maintenance of metabolic homeostasis and sustaining growth of Chlorella. PMID:26356535

  14. Persistent reduction of hippocampal glutamine synthetase expression after status epilepticus in immature rats.

    PubMed

    van der Hel, W Saskia; Hessel, Ellen V S; Bos, Ineke W M; Mulder, Sandra D; Verlinde, Suzanne A M W; van Eijsden, Pieter; de Graan, Pierre N E

    2014-12-01

    Mesiotemporal sclerosis (MTS), the most frequent form of drug-resistant temporal lobe epilepsy, often develops after an initial precipitating injury affecting the immature brain. To analyse early processes in epileptogenesis we used the juvenile pilocarpine model to study status epilepticus (SE)-induced changes in expression of key components in the glutamate-glutamine cycle, known to be affected in MTS patients. SE was induced by Li(+) /pilocarpine injection in 21-day-old rats. At 2-19 weeks after SE hippocampal protein expression was analysed by immunohistochemistry and neuron damage by FluoroJade staining. Spontaneous seizures occurred in at least 44% of animals 15-18 weeks after SE. As expected in this model, we did not observe loss of principal hippocampal neurons. Neuron damage was most pronounced in the hilus, where we also detected progressive loss of parvalbumin-positive GABAergic interneurons. Hilar neuron loss (or end-folium sclerosis), a common feature in patients with MTS, was accompanied by a progressively decreased glutamine synthetase (GS)-immunoreactivity from 2 (-15%) to 19 weeks (-33.5%) after SE. Immunoreactivity for excitatory amino-acid transporters, vesicular glutamate transporter 1 and glial fibrillary acidic protein was unaffected. Our data show that SE elicited in 21-day-old rats induces a progressive reduction in hilar GS expression without affecting other key components of the glutamate-glutamine cycle. Reduced expression of glial enzyme GS was first detected 2 weeks after SE, and thus clearly before spontaneous recurrent seizures occurred. These results support the hypothesis that reduced GS expression is an early event in the development of hippocampal sclerosis in MTS patients and emphasize the importance of astrocytes in early epileptogenesis.

  15. Right on TARGET: glutamine metabolism in cancer

    PubMed Central

    Ratnikov, Boris; Jeon, Young Joo; Smith, Jeffrey W.; Ronai, Ze'ev A.

    2015-01-01

    Recent studies highlight the importance of glutamine metabolism in metabolic reprogramming, which underlies cancer cell addiction to glutamine. Examples for the dependence on glutamine metabolism are seen across different tumor types as during different phases of cancer development, progression and response to therapy. In this perspective, we assess the possibility of targeting glutamine metabolism as a therapeutic modality for cancer. PMID:26425657

  16. Exogenous glutamine: the clinical evidence.

    PubMed

    Bongers, Thomas; Griffiths, Richard D; McArdle, Anne

    2007-09-01

    We know that critically ill patients suffering from undernutrition with a limited nutritional reserve have a poorer outcome. Furthermore, having a low body mass index has been shown to be an independent predictor of excess mortality in multiple organ failure. Therefore, nutritional support has gained increasing interest in critical illness with the hope of preventing or attenuating the effects of malnutrition. A negative nitrogen balance is the characteristic metabolic feature in critical illness, with the major protein loss derived from skeletal muscle. In particular, glutamine concentrations are rapidly reduced in plasma and muscle. Over the last 20 yrs or so, increasing evidence is emerging to support the use of glutamine supplementation in critical illness. Clinical trials have found a mortality and morbidity advantage with glutamine supplementation. The advantage appears to be greater the more glutamine is given and greater again when given parenterally. Various modes of action have been postulated. Glutamine seems to have an effect on the immune system, antioxidant status, glucose metabolism, and heat shock protein response. However, the benefit of exogenous glutamine on morbidity and mortality is not universally accepted. This review critically appraises the current clinical evidence regarding glutamine supplementation in critical illness.

  17. Polymorphisms in Genes of Tricarboxylic Acid Cycle Key Enzymes Are Associated with Early Recurrence of Hepatocellular Carcinoma.

    PubMed

    Wan, Shaogui; Wu, Yousheng; Zhou, Xingchun; Chen, Yibing; An, Jiaze; Yu, Xiaohe; Zhang, Huiqing; Yang, Hushan; Xing, Jinliang

    2015-01-01

    Alterations of activity and expression in tricarboxylic acid (TCA) cycle key enzymes have been indicated in several malignancies, including hepatocellular carcinoma (HCC). They play an important role in the progression of cancer. However, the impact of single nucleotide polymorphisms (SNPs) in genes encoding these key enzymes on the recurrence of HCC has not been investigated. In this study, we genotyped 17 SNPs in genes encoding TCA cycle key enzymes and analyzed their association with recurrence-free survival (RFS) in a cohort of 492 Chinese HCC patients by Cox proportional hazard model and survival tree analysis. We identified 7 SNPs in SDHC, SDHD, FH, and IDH2 genes to be significantly associated with the RFS of HCC patients. Moreover, all these SNPs were associated with the early recurrence (within 2 years after surgery) risk of diseases. Cumulative effect analysis showed that these SNPs exhibited a dose-dependent effect on the overall and early recurrence. Further stratified analysis suggested that number of risk genotypes modified the protective effect on HCC recurrence conferred by transcatheter arterial chemoembolization treatment. Finally, the survival tree analysis revealed that SNP rs10789859 in SDHD gene was the primary factor contributing to HCC recurrence in our population. To the best of our knowledge, we for the first time observed the association between SNPs in genes encoding TCA cycle key enzymes and HCC recurrence risk. Further observational and functional studies are needed to validate our findings and generalize its clinical usage. PMID:25894340

  18. Chloroplastic thioredoxin m functions as a major regulator of Calvin cycle enzymes during photosynthesis in vivo.

    PubMed

    Okegawa, Yuki; Motohashi, Ken

    2015-12-01

    Thioredoxins (Trxs) regulate the activity of various chloroplastic proteins in a light-dependent manner. Five types of Trxs function in different physiological processes in the chloroplast of Arabidopsis thaliana. Previous in vitro experiments have suggested that the f-type Trx (Trx f) is the main redox regulator of chloroplast enzymes, including Calvin cycle enzymes. To investigate the in vivo contribution of each Trx isoform to the redox regulatory system, we first quantified the protein concentration of each Trx isoform in the chloroplast stroma. The m-type Trx (Trx m), which consists of four isoforms, was the most abundant type. Next, we analyzed several Arabidopsis Trx-m-deficient mutants to elucidate the physiological role of Trx m in vivo. Deficiency of Trx m impaired plant growth and decreased the CO2 assimilation rate. We also determined the redox state of Trx target enzymes to examine their photo-reduction, which is essential for enzyme activation. In the Trx-m-deficient mutants, the reduction level of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase was lower than that in the wild type. Inconsistently with the historical view, our in vivo study suggested that Trx m plays a more important role than Trx f in the activation of Calvin cycle enzymes.

  19. Changes in the activities of key enzymes of glycolysis during the cell cycle in yeast: a rectification.

    PubMed

    de Koning, W; Groeneveld, K; Oehlen, L J; Berden, J A; van Dam, K

    1991-04-01

    Activities of glycolytic enzymes were determined in elutriation fractionated cultures of Saccharomyces cerevisiae grown on different carbon sources. Almost pure fractions of single cells at the G1 state of cell division were obtained for some of the growth conditions tested, whereas other stages were enriched in particular fractions. Specific activities of glucose-6-phosphate dehydrogenase and alcohol dehydrogenase were found to be constant during the cell cycle, as reported by van Doorn et al. (1988a), Journal of Bacteriology 170, 4808-4815, and (1988b), Journal of General Microbiology 134, 785-790. In contrast to the earlier reports, the activities of hexokinase, phosphofructokinase, pyruvate kinase and trehalase were also constant in different states of the cell cycle. For hexokinase and phosphofructokinase it was shown that the apparent specific activity in a cell-free extract strongly diminished when extracts contained less that 0.5-1 mg protein ml-1. In the experiments of van Doorn et al. (1988a) the protein content of the outer fractions was up to 20 times lower than that of the central fractions, suggesting an alternative explanation for the observed changes in enzyme activities during the cell cycle. Therefore, we want to rectify the observations presented by van Doorn et al. (1988a), and conclude that the activities of the glycolytic enzymes do not vary greatly during the cell cycle of S. cervisiae. PMID:1856683

  20. Vitamin A deficiency increases protein catabolism and induces urea cycle enzymes in rats.

    PubMed

    Esteban-Pretel, Guillermo; Marín, M Pilar; Cabezuelo, Francisco; Moreno, Verónica; Renau-Piqueras, Jaime; Timoneda, Joaquín; Barber, Teresa

    2010-04-01

    Chronic vitamin A deficiency induces a substantial delay in the rates of weight and height gain in both humans and experimental animals. This effect has been associated with an impaired nutrient metabolism and loss of body protein. Therefore, we analyzed the effect of vitamin A deficiency on endogenous proteolysis and nitrogen metabolism and its reversibility with all-trans retinoic acid (RA). Male weanling rats, housed in pairs, were pair-fed a vitamin A-deficient (VAD) or control diet until they were 60 d old. A group of deficient rats were further treated with daily intraperitoneal injections of all-trans RA for 10 d. Final body and tissue (i.e. liver and heart) weights were significantly lower and tissue:body weight ratios were similar in VAD rats and in controls. Conversely, the epididymal white fat:body weight ratio and the plasma concentrations of alanine aminotransferase and adiponectin were significantly higher in VAD rats, which also had hepatic macrovesicular lipid accumulations. Plasma and gastrocnemius muscle 3-methylhistidine, urine nitrogen, and plasma and urine urea concentrations were all significantly higher in the VAD group. The expression of the genes encoding urea cycle enzymes and their activities increased in VAD livers. These changes were partially reverted by all-trans RA. We propose that fuel partitioning in vitamin A deficiency may shift from fatty acids to protein catabolism as an energy source. Our results emphasize the importance of vitamin A on the energy balance control system and they provide an explanation for the role of vitamin A in protein turnover, development, and growth.

  1. [Lactate dehydrogenase and Krebs cycle enzyme activity in rat liver during the growth of transplanted and spontaneous tumors].

    PubMed

    Morozkina, T S

    1978-03-01

    Certain distinctions in the mouse and rat liver responses to transplanted and spontaneous tumours have been discovered at the initial periods of their growth. The most pronounced changes (the mosaic distribution of enzymatic activity in the lobe) are observed in the case of spontaneous tumours. Activities the Krebs cycle enzymes, especially of NAD-dependent enzymes are seen inhibited in the tumour-bearing liver at the terminal periods of growth of both spontaneous and transplanted tumours; lactate dehydrogenase activity increases (with the exception of mitochondrial lactate dehydrogenase in the rat liver with transplanted sarcomas). PMID:684845

  2. Protection from radiation injury by elemental diet: does added glutamine change the effect?

    PubMed

    McArdle, A H

    1994-01-01

    The feeding of a protein hydrolysate based 'elemental' diet supplemented with added glutamine did not provide superior protection to the small intestine of dogs subjected to therapeutic pelvic irradiation. Comparison of diets with and without the added glutamine showed significant protection of the intestine from radiation injury. Both histological examination and electron microscopy showed lack of tissue injury with both diets. The activity of the free radical generating enzymes, scavengers, and antioxidants were similar in the intestinal mucosa of dogs fed either diet. After radiation, however, the activity of xanthine oxidase, superoxide dismutase, and glutathione peroxidase were significantly (p < 0.002) higher in the intestine of dogs fed elemental diet without the added glutamine. If the activities of these enzymes are important in the protection of the intestine from radiation injury, then the addition of extra glutamine may provide no benefit.

  3. Kinetics and spatial distribution of enzymes of carbon, nitrogen and phosphorus cycles in earthworm biopores

    NASA Astrophysics Data System (ADS)

    Hoang Thi Thu, Duyen; Razavi, Bahar S.

    2016-04-01

    Earthworms boost microbial activities and consequently form hotspots in soil. The distribution of enzyme activities inside the earthworm biopores is completely unknown. For the first time, we analyzed enzyme kinetics and visualized enzyme distribution inside and outside biopores by in situ soil zymography. Kinetic parameters (Vmax and Km) of 6 enzymes β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) were determined in biopores formed by Lumbricus terrestris L.. The spatial distributions of GLU, NAG and APT become visible via zymograms in comparison between earthworm-inhabited and earthworm-free soil. Zymography showed heterogeneous distribution of hotspots in the rhizosphere and biopores. The hotspot areas were 2.4 to 14 times larger in the biopores than in soil without earthworms. The significantly higher Vmax values for GLU, CBH, XYL, NAG and APT in biopores confirmed the stimulation of enzyme activities by earthworms. For CBH, XYL and NAG, the 2- to 3-fold higher Km values in biopores indicated different enzyme systems with lower substrate affinity compared to control soil. The positive effects of earthworms on Vmax were cancelled by the Km increase for CBH, XYL and NAG at a substrate concentration below 20 μmol g-1 soil. The change of enzyme systems reflected a shift in dominant microbial populations toward species with lower affinity to holo-celluloses and to N-acetylglucosamine, and with higher affinity to proteins as compared to the biopores-free soil. We conclude that earthworm biopores are microbial hotspots with much higher and dense distribution of enzyme activities compared to bulk soil. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77. Blagodatskaya, E., Kuzyakov, Y., 2013. Review paper: Active microorganisms in soil

  4. ALDH Enzyme Expression Is Independent of the Spermatogenic Cycle, and Their Inhibition Causes Misregulation of Murine Spermatogenic Processes.

    PubMed

    Kent, Travis; Arnold, Samuel L; Fasnacht, Rachael; Rowsey, Ross; Mitchell, Debra; Hogarth, Cathryn A; Isoherranen, Nina; Griswold, Michael D

    2016-01-01

    Perturbations in the vitamin A metabolism pathway could be a significant cause of male infertility, as well as a target toward the development of a male contraceptive, necessitating the need for a better understanding of how testicular retinoic acid (RA) concentrations are regulated. Quantitative analyses have recently demonstrated that RA is present in a pulsatile manner along testis tubules. However, it is unclear if the aldehyde dehydrogenase (ALDH) enzymes, which are responsible for RA synthesis, contribute to the regulation of these RA concentration gradients. Previous studies have alluded to fluctuations in ALDH enzymes across the spermatogenic cycle, but these inferences have been based primarily on qualitative transcript localization experiments. Here, we show via various quantitative methods that the three well-known ALDH enzymes (ALDH1A1, ALDH1A2, and ALDH1A3), and an ALDH enzyme previously unreported in the murine testis (ALDH8A1), are not expressed in a stage-specific manner in the adult testis, but do fluctuate throughout juvenile development in perfect agreement with the first appearance of each advancing germ cell type. We also show, via treatments with a known ALDH inhibitor, that lowered testicular RA levels result in an increase in blood-testis barrier permeability, meiotic recombination, and meiotic defects. Taken together, these data further our understanding of the complex regulatory actions of RA on various spermatogenic events and, in contrast with previous studies, also suggest that the ALDH enzymes are not responsible for regulating the recently measured RA pulse.

  5. Identification of the glutamine synthetase adenylyltransferase of Azospirillum brasilense.

    PubMed

    Van Dommelen, Anne; Spaepen, Stijn; Vanderleyden, Jozef

    2009-04-01

    Glutamine synthetase, a key enzyme in nitrogen metabolism of both prokaryotes and eukaryotes, is strictly regulated. One means of regulation is the modulation of activity through adenylylation catalyzed by adenylyltransferases. Using PCR primers based on conserved sequences in glutamine synthetase adenylyltransferases, we amplified part of the glnE gene of Azospirillum brasilense Sp7. The complete glnE sequence of A. brasilense Sp245 was retrieved from the draft genome sequence of this organism (http://genomics.ornl.gov/research/azo/). Adenylyltransferase is a bifunctional enzyme consisting of an N-terminal domain responsible for deadenylylation activity and a C-terminal domain responsible for adenylylation activity. Both domains are partially homologous to each other. Residues important for catalytic activity were present in the deduced amino acid sequence of the A. brasilense Sp245 glnE sequence. A glnE mutant was constructed in A. brasilense Sp7 by inserting a kanamycin resistance cassette between the two active domains of the enzyme. The resulting mutant was unable to adenylylate the glutamine synthetase enzyme and was impaired in growth when shifted from nitrogen-poor to nitrogen-rich medium.

  6. Tryptophan boost caused by senescence occurred independently of cytoplasmic glutamine synthetase.

    PubMed

    Park, Sangkyu; Lee, Kyungjin; Kang, Kiyoon; Kim, Young Soon; Lee, Sungbeom; Kweon, Soon-Jong; Back, Kyoungwhan

    2010-01-01

    We examined to determine whether senescence-induced tryptophan levels are positively associated with levels of glutamine synthetase (GS1), the initial enzyme in tryptophan biosynthesis. We generated transgenic rice plants in which GS1 was suppressed by RNA interference technology. The transgenic line showed a dramatic decrease in GS1 protein and glutamine content, but the levels of tryptophan and mRNA of the key tryptophan biosynthetic genes upon senescence were comparable to those of the wild type.

  7. Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

    PubMed

    Tardito, Saverio; Oudin, Anaïs; Ahmed, Shafiq U; Fack, Fred; Keunen, Olivier; Zheng, Liang; Miletic, Hrvoje; Sakariassen, Per Øystein; Weinstock, Adam; Wagner, Allon; Lindsay, Susan L; Hock, Andreas K; Barnett, Susan C; Ruppin, Eytan; Mørkve, Svein Harald; Lund-Johansen, Morten; Chalmers, Anthony J; Bjerkvig, Rolf; Niclou, Simone P; Gottlieb, Eyal

    2015-12-01

    L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

  8. Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

    PubMed

    Tardito, Saverio; Oudin, Anaïs; Ahmed, Shafiq U; Fack, Fred; Keunen, Olivier; Zheng, Liang; Miletic, Hrvoje; Sakariassen, Per Øystein; Weinstock, Adam; Wagner, Allon; Lindsay, Susan L; Hock, Andreas K; Barnett, Susan C; Ruppin, Eytan; Mørkve, Svein Harald; Lund-Johansen, Morten; Chalmers, Anthony J; Bjerkvig, Rolf; Niclou, Simone P; Gottlieb, Eyal

    2015-12-01

    L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes. PMID:26595383

  9. Glutamine Synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma

    PubMed Central

    Tardito, Saverio; Oudin, Anaïs; Ahmed, Shafiq U.; Fack, Fred; Keunen, Olivier; Zheng, Liang; Miletic, Hrvoje; Sakariassen, Per Øystein; Weinstock, Adam; Wagner, Allon; Lindsay, Susan L.; Hock, Andreas K.; Barnett, Susan C.; Ruppin, Eytan; Mørkve, Svein Harald; Lund-Johansen, Morten; Chalmers, Anthony J.; Bjerkvig, Rolf; Niclou, Simone P.; Gottlieb, Eyal

    2015-01-01

    L-Glutamine (Gln) functions physiologically to balance tissue requirements of carbon and nitrogen. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle and, that inhibiting glutaminolysis does not affect proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by Glutamine Synthetase (GS) (cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, 13C-glucose tracing showed that GS produces Gln from TCA cycle-derived carbons. Finally, while it is contributed only marginally by the circulation, the Gln required for the growth of GBM tumours is either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes. PMID:26595383

  10. Glutamine synthetase gene evolution: A good molecular clock

    SciTech Connect

    Pesole, G.; Lanvave, C.; Saccone, C. ); Bozzetti, M.P. ); Preparata, G. )

    1991-01-15

    Glutamine synthetase gene evolution in various animals, plants, and bacteria was evaluated by a general stationary Markov model. The evolutionary process proved to be unexpectedly regular even for a time span as long as that between the divergence of prokaryotes from eukaryotes. This enabled us to draw phylogenetic trees for species whose phylogeny cannot be easily reconstructed from the fossil record. The calculation of the times of divergence of the various organelle-specific enzymes led us to hypothesize that the pea and bean chloroplast genes for these enzymes originated from the duplication of nuclear genes as a result of the different metabolic needs of the various species. The data indicate that the duplication of plastid glutamine synthetase genes occurred long after the endosymbiotic events that produced the organelles themselves.

  11. Glutamine synthetase gene evolution: a good molecular clock.

    PubMed Central

    Pesole, G; Bozzetti, M P; Lanave, C; Preparata, G; Saccone, C

    1991-01-01

    Glutamine synthetase (EC 6.3.1.2) gene evolution in various animals, plants, and bacteria was evaluated by a general stationary Markov model. The evolutionary process proved to be unexpectedly regular even for a time span as long as that between the divergence of prokaryotes from eukaryotes. This enabled us to draw phylogenetic trees for species whose phylogeny cannot be easily reconstructed from the fossil record. Our calculation of the times of divergence of the various organelle-specific enzymes led us to hypothesize that the pea and bean chloroplast genes for these enzymes originated from the duplication of nuclear genes as a result of the different metabolic needs of the various species. Our data indicate that the duplication of plastid glutamine synthetase genes occurred long after the endosymbiotic events that produced the organelles themselves. PMID:1671172

  12. Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase.

    PubMed

    Knight, T J; Langston-Unkefer, P J

    1988-08-15

    The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked

  13. Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer.

    PubMed

    Schnarrenberger, Claus; Martin, William

    2002-02-01

    The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha

  14. Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer.

    PubMed

    Schnarrenberger, Claus; Martin, William

    2002-02-01

    The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha

  15. Nitric oxide inhibits specific enzymes in the Krebs cycle and the respiratory chain of rat hepatocyte mitochondria

    SciTech Connect

    Stadler, J.; Billiar, T.R.; Curran, R.D.; Kim, R.; Simmons, R.L. )

    1990-02-26

    Nitric oxide (NO) is a highly-reactive molecule produced from L-arginine as recently described. In macrophages and tumor cells, NO inhibits specific mitochondrial enzymes presumably by attacking their intrinsic 4Fe-4S centers. The susceptible enzymes include aconitase of the Krebs cycle and oxidoreductase (complex II) of the electron transport chain. The authors have recently demonstrated that hepatocytes (HC) produce NO in large amounts in response to endotoxin and inflammatory cytokines. To determine whether HC suffer a similar enzyme inhibition, the authors exposed rat HC to increasing concentrations of NO solutions for 5 minutes. The activity of aconitase, complex 1, complex 2, and complex 4 (cytochrome oxidase) was determined by measuring O{sub 2} consumption after addition of enzyme-specific substrates. An NO concentration-dependent inhibition of aconitase, complex 1, and complex 2 was measured. After exposure to 0.6 mM solution, the activity of aconitase was blocked to non-measurable values while complex 1 was reduced to 11 + 8%, and complex 2 to 36 + 2% of the activity of control HC. Complex 4 of the respiratory chain remained intact at 100 + 8%. These data indicate that HC, like other cell types, are susceptible to inhibition of important steps of energy production by NO. As NO is produced in response to septic stimuli, this mechanism may play a role in the metabolic dysfunction of HC in sepsis.

  16. Impact of repeated dry-wet cycles on soil greenhouse gas emissions, extracellular enzyme activity and nutrient cycling in a temperate forest

    NASA Astrophysics Data System (ADS)

    Leitner, Sonja; Zimmermann, Michael; Bockholt, Jan; Schartner, Markus; Brugner, Paul; Holtermann, Christian; Zechmeister-Boltenstern, Sophie

    2014-05-01

    Climate change research predicts that both frequency and intensity of weather extremes such as long drought periods and heavy rainfall events will increase in mid Europe over the next decades. Soil moisture is one of the major factors controlling microbial soil processes, and it has been widely agreed that feedback effects between altered precipitation and changed soil fluxes of the greenhouse gases CO2, CH4 and N2O could intensify climate change. In a field experiment in an Austrian beech forest, we established a precipitation manipulation experiment, which will be conducted for 3 years. We use roofs to exclude rainfall from reaching the forest soil and simulate drought periods, and a sprinkler system to simulate heavy rainfall events. We applied repeated dry-wet cycles in two intensities: one treatment received 6 cycles of 1 month drought followed by 75mm irrigation within 2 hours, and a parallel treatment received 3 cycles of 2 months drought followed by 150mm irrigation within 3 hours. We took soil samples 1 day before, 1 day after and 1 week after rewetting events and analyzed them for soil nutrients and extracellular enzyme activities. Soil fluxes of CO2, N2O and CH4 were constantly monitored with an automated flux chamber system, and environmental parameters were recorded via dataloggers. In addition, we determined fluxes and nutrient concentrations of bulk precipitation, throughfall, stemflow, litter percolate and soil water. Next we plan to analyze soil microbial community composition via PLFAs to investigate microbial stress resistance and resilience, and we will use ultrasonication to measure soil aggregate stability and protection of soil organic matter in stressed and control plots. The results of the first year show that experimental rainfall manipulation has influenced soil extracellular enzymes. Potential phenoloxidase activity was significantly reduced in stressed treatments compared to control plots. All measured hydrolytic enzymes (cellulase

  17. Inhibitors of the Glyoxylate Cycle Enzyme ICL1 in Candida albicans for Potential Use as Antifungal Agents

    PubMed Central

    Cheah, Hong-Leong; Lim, Vuanghao; Sandai, Doblin

    2014-01-01

    Candida albicans is an opportunistic pathogen that causes candidiasis in humans. In recent years, metabolic pathways in C. albicans have been explored as potential antifungal targets to treat candidiasis. The glyoxylate cycle, which enables C. albicans to survive in nutrient-limited host niches and its. Key enzymes (e.g., isocitrate lyase (ICL1), are particularly attractive antifungal targets for C. albicans. In this study, we used a new screening approach that better reflects the physiological environment that C. albicans cells experience during infection to identify potential inhibitors of ICL. Three compounds (caffeic acid (CAFF), rosmarinic acid (ROS), and apigenin (API)) were found to have antifungal activity against C. albicans when tested under glucose-depleted conditions. We further confirmed the inhibitory potential of these compounds against ICL using the ICL enzyme assay. Lastly, we assessed the bioavailability and toxicity of these compounds using Lipinski's rule-of-five and ADMET analysis. PMID:24781056

  18. Identification of the algal dimethyl sulfide-releasing enzyme: A missing link in the marine sulfur cycle

    NASA Astrophysics Data System (ADS)

    Alcolombri, Uria; Ben-Dor, Shifra; Feldmesser, Ester; Levin, Yishai; Tawfik, Dan S.; Vardi, Assaf

    2015-06-01

    Algal blooms produce large amounts of dimethyl sulfide (DMS), a volatile with a diverse signaling role in marine food webs that is emitted to the atmosphere, where it can affect cloud formation. The algal enzymes responsible for forming DMS from dimethylsulfoniopropionate (DMSP) remain unidentified despite their critical role in the global sulfur cycle. We identified and characterized Alma1, a DMSP lyase from the bloom-forming algae Emiliania huxleyi. Alma1 is a tetrameric, redox-sensitive enzyme of the aspartate racemase superfamily. Recombinant Alma1 exhibits biochemical features identical to the DMSP lyase in E. huxleyi, and DMS released by various E. huxleyi isolates correlates with their Alma1 levels. Sequence homology searches suggest that Alma1 represents a gene family present in major, globally distributed phytoplankton taxa and in other marine organisms.

  19. Distribution of Two C Cycle Enzymes in Soil Aggregates of a Prairie Chronosequence

    SciTech Connect

    Fansler, Sarah J.; Smith, Jeffery L.; Bolton, Harvey; Bailey, Vanessa L.

    2005-11-01

    Recently attention has focused on the potential of using soil as a sink for atmospheric CO2. The objective of this study was to use soil enzymes and classical methods of soil aggregate fractionation to explore the relationship between microbial community function and soil structure of a tallgrass prairie chronosequence. The soils within the chronosequence were: (1) remnant native prairie, (2) agricultural soil, and (3, 4) tallgrass prairies restored from agriculture in 1979 and 1993. β-glucosidase (E.C. 3.2.1.21) and N-acetyl-β-glucosaminidase (NAGase, EC 3.2.1.30) assays were conducted on four different aggregate size fractions (>2 mm, 1 -2 mm, 250µm-1 mm, and 2 - 250 µm) from each soil. Specific activities for both enzymes (µg PNP g-1 soil h-1) were greatest in the microaggregate (2 µm -250 µm) fractions across the chronosequence; however, this size fraction makes up only a small proportion of the whole soil. Therefore, it is the larger macroaggregate-derived enzyme activities that have the greatest impact on the activity of the whole soil. Analyzing both enzymes and the physical structure, a reversion from an agricultural soil through the restored to more like the prairie soil, was not detected. It appears that the function of these microbial community systems in the native tallgrass prairie and agricultural soils of the chronosequence are in equilibria while the lands restored to tallgrass prairie are in an ongoing state of recovery.

  20. [Progress and application prospects of glutamine synthase in plants].

    PubMed

    Feng, Wanjun; Xing, Guofang; Niu, Xulong; Dou, Chen; Han, Yuanhuai

    2015-09-01

    Nitrogen is one of the most important nutrient elements for plants and a major limiting factor in plant growth and crop productivity. Glutamine synthase (GS) is a key enzyme involved in the nitrogen assimilation and recycling in plants. So far, members of the glutamine synthase gene family have been characterized in many plants such as Arabidopsis, rice, wheat, and maize. Reports show that GS are involved in the growth and development of plants, in particular its role in seed production. However, the outcome has generally been inconsistent, which are probably derived from the transcriptional and post-translational regulation of GS genes. In this review, we outlined studies on GS gene classification, QTL mapping, the relationship between GS genes and plant growth with nitrogen and the distribution characters, the biological functions of GS genes, as well as expression control at different regulation levels. In addition, we summarized the application prospects of glutamine synthetase genes in enhancing plant growth and yield by improving the nitrogen use efficiency. The prospects were presented on the improvement of nitrogen utility efficiency in crops and plant nitrogen status diagnosis on the basis of glutamine synthase gene regulation. PMID:26955708

  1. [Progress and application prospects of glutamine synthase in plants].

    PubMed

    Feng, Wanjun; Xing, Guofang; Niu, Xulong; Dou, Chen; Han, Yuanhuai

    2015-09-01

    Nitrogen is one of the most important nutrient elements for plants and a major limiting factor in plant growth and crop productivity. Glutamine synthase (GS) is a key enzyme involved in the nitrogen assimilation and recycling in plants. So far, members of the glutamine synthase gene family have been characterized in many plants such as Arabidopsis, rice, wheat, and maize. Reports show that GS are involved in the growth and development of plants, in particular its role in seed production. However, the outcome has generally been inconsistent, which are probably derived from the transcriptional and post-translational regulation of GS genes. In this review, we outlined studies on GS gene classification, QTL mapping, the relationship between GS genes and plant growth with nitrogen and the distribution characters, the biological functions of GS genes, as well as expression control at different regulation levels. In addition, we summarized the application prospects of glutamine synthetase genes in enhancing plant growth and yield by improving the nitrogen use efficiency. The prospects were presented on the improvement of nitrogen utility efficiency in crops and plant nitrogen status diagnosis on the basis of glutamine synthase gene regulation.

  2. Glutamine supplementation in a child with inherited GS deficiency improves the clinical status and partially corrects the peripheral and central amino acid imbalance.

    PubMed

    Häberle, Johannes; Shahbeck, Noora; Ibrahim, Khalid; Schmitt, Bernhard; Scheer, Ianina; O'Gorman, Ruth; Chaudhry, Farrukh A; Ben-Omran, Tawfeg

    2012-07-25

    Glutamine synthetase (GS) is ubiquitously expressed in mammalian organisms and is a key enzyme in nitrogen metabolism. It is the only known enzyme capable of synthesising glutamine, an amino acid with many critical roles in the human organism. A defect in GLUL, encoding for GS, leads to congenital systemic glutamine deficiency and has been described in three patients with epileptic encephalopathy. There is no established treatment for this condition.Here, we describe a therapeutic trial consisting of enteral and parenteral glutamine supplementation in a four year old patient with GS deficiency. The patient received increasing doses of glutamine up to 1020 mg/kg/day. The effect of this glutamine supplementation was monitored clinically, biochemically, and by studies of the electroencephalogram (EEG) as well as by brain magnetic resonance imaging and spectroscopy.Treatment was well tolerated and clinical monitoring showed improved alertness. Concentrations of plasma glutamine normalized while levels in cerebrospinal fluid increased but remained below the lower reference range. The EEG showed clear improvement and spectroscopy revealed increasing concentrations of glutamine and glutamate in brain tissue. Concomitantly, there was no worsening of pre-existing chronic hyperammonemia.In conclusion, supplementation of glutamine is a safe therapeutic option for inherited GS deficiency since it corrects the peripheral biochemical phenotype and partially also improves the central biochemical phenotype. There was some clinical improvement but the patient had a long standing severe encephalopathy. Earlier supplementation with glutamine might have prevented some of the neuronal damage.

  3. Distribution of immunoreactive glutamine synthetase in the adult human and mouse brain. Qualitative and quantitative observations with special emphasis on extra-astroglial protein localization.

    PubMed

    Bernstein, Hans-Gert; Bannier, Jana; Meyer-Lotz, Gabriela; Steiner, Johann; Keilhoff, Gerburg; Dobrowolny, Henrik; Walter, Martin; Bogerts, Bernhard

    2014-11-01

    Glutamine synthetase catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a pivotal role in glutamate and glutamine homoeostasis. Despite a plethora of studies on this enzyme, knowledge about the regional and cellular distribution of this enzyme in human brain is still fragmentary. Therefore, we mapped fourteen post-mortem brains of psychically healthy individuals for the distribution of the glutamine synthetase immunoreactive protein. It was found that glutamine synthetase immunoreactivity is expressed in multiple gray and white matter astrocytes, but also in oligodendrocytes, ependymal cells and certain neurons. Since a possible extra-astrocytic expression of glutamine synthetase is highly controversial, we paid special attention to its appearance in oligodendrocytes and neurons. By double immunolabeling of mouse brain slices and cultured mouse brain cells for glutamine synthetase and cell-type-specific markers we provide evidence that besides astrocytes subpopulations of oligodendrocytes, microglial cells and neurons express glutamine synthetase. Moreover, we show that glutamine synthetase-immunopositive neurons are not randomly distributed throughout human and mouse brain, but represent a subpopulation of nitrergic (i.e. neuronal nitric oxide synthase expressing) neurons. Possible functional implications of an extra-astrocytic localization of glutamine synthetase are discussed.

  4. The glutamine synthetase gene family in Populus

    PubMed Central

    2011-01-01

    Background Glutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is a key enzyme in ammonium assimilation and metabolism of higher plants. The current work was undertaken to develop a more comprehensive understanding of molecular and biochemical features of GS gene family in poplar, and to characterize the developmental regulation of GS expression in various tissues and at various times during the poplar perennial growth. Results The GS gene family consists of 8 different genes exhibiting all structural and regulatory elements consistent with their roles as functional genes. Our results indicate that the family members are organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1) and 1 which codes for the choroplastic GS isoform (GS2). Our analysis shows that Populus trichocarpa is the first plant species in which it was observed the complete GS family duplicated. Detailed expression analyses have revealed specific spatial and seasonal patterns of GS expression in poplar. These data provide insights into the metabolic function of GS isoforms in poplar and pave the way for future functional studies. Conclusions Our data suggest that GS duplicates could have been retained in order to increase the amount of enzyme in a particular cell type. This possibility could contribute to the homeostasis of nitrogen metabolism in functions associated to changes in glutamine-derived metabolic products. The presence of duplicated GS genes in poplar could also contribute to diversification of the enzymatic properties for a particular GS isoform through the assembly of GS polypeptides into homo oligomeric and/or hetero oligomeric holoenzymes in specific cell types. PMID:21867507

  5. Reactive oxygen species production and antioxidant enzyme expression after Epstein-Barr virus lytic cycle induction in Raji cell line.

    PubMed

    Gargouri, Bochra; Nasr, Rihab; ben Mansour, Riadh; Lassoued, Saloua; Mseddi, Malek; Attia, Hammadi; El Feki, Abd el Fatteh; Van Pelt, Jos

    2011-12-01

    In a previous study, we have described oxidative stress during Epstein-Barr virus lytic cycle induction. Oxidative stress was evidenced by the observed high MDA levels and the decreased activities of antioxidant enzymes. We hypothesised that the lower activities of the antioxidant enzymes decrease were the result of either the excessive production of reactive oxygen radical species (ROS) or a negative regulation of the antioxidant enzyme gene expressions. In an attempt to clarify this situation, EBV lytic cycle was induced in Raji cell line by a non-stressing dose of 12-0-tetradecanoylphorbol-13-acetate. BZLF-1, superoxide dismutase, and catalase gene expressions were then analysed using semi-quantitative RT-PCR, simultaneously at a kinetic of 6, 12, 24, 36, and 48 h. ROS production was evaluated by chemiluminescence. A study was conducted to establish whether ROS production, BZLF-1, and the expression of antioxidant genes were inter-correlated. Induction of the lytic cycle resulted in increased expressions of the genes of superoxide dismutase and catalase, which began at 24 h (p < 0.05) and reached a peak at 48 h (p < 0.05). Significant increases of the ROS levels were observed in TPA-treated Raji cell line at 12 h, as compared with untreated cells, reaching a peak at 48 h after EBV lytic cycle induction. ROS production correlates positively with BZLF-1, SOD, and CAT gene expressions (p < 0.05; r = 0.913, r = 0.978, and r = 0.955, respectively). A positive correlation was also observed between BZLF-1 and antioxidant gene expressions (p < 0.05; r = 0.961 and r = 0.987, respectively). In conclusion, the observed increases of the SOD and CAT gene expressions eliminate the hypothesis of a repression of the respective genes during the induction of the lytic cycle. On the other hand, the observed direct correlation between the BZLF-1 gene expression and the ROS production is indicative of a role of this gene in oxidative stress.

  6. Phosphotransferase-dependent accumulation of (p)ppGpp in response to glutamine deprivation in Caulobacter crescentus

    PubMed Central

    Ronneau, Séverin; Petit, Kenny; De Bolle, Xavier; Hallez, Régis

    2016-01-01

    The alarmone (p)ppGpp is commonly used by bacteria to quickly respond to nutrient starvation. Although (p)ppGpp synthetases such as SpoT have been extensively studied, little is known about the molecular mechanisms stimulating alarmone synthesis upon starvation. Here, we describe an essential role of the nitrogen-related phosphotransferase system (PTSNtr) in controlling (p)ppGpp accumulation in Caulobacter crescentus. We show that cells sense nitrogen starvation by way of detecting glutamine deprivation using the first enzyme (EINtr) of PTSNtr. Decreasing intracellular glutamine concentration triggers phosphorylation of EINtr and its downstream components HPr and EIIANtr. Once phosphorylated, both HPr∼P and EIIANtr∼P stimulate (p)ppGpp accumulation by modulating SpoT activities. This burst of second messenger primarily impacts the non-replicative phase of the cell cycle by extending the G1 phase. This work highlights a new role for bacterial PTS systems in stimulating (p)ppGpp accumulation in response to metabolic cues and in controlling cell cycle progression and cell growth. PMID:27109061

  7. Phosphotransferase-dependent accumulation of (p)ppGpp in response to glutamine deprivation in Caulobacter crescentus.

    PubMed

    Ronneau, Séverin; Petit, Kenny; De Bolle, Xavier; Hallez, Régis

    2016-04-25

    The alarmone (p)ppGpp is commonly used by bacteria to quickly respond to nutrient starvation. Although (p)ppGpp synthetases such as SpoT have been extensively studied, little is known about the molecular mechanisms stimulating alarmone synthesis upon starvation. Here, we describe an essential role of the nitrogen-related phosphotransferase system (PTS(Ntr)) in controlling (p)ppGpp accumulation in Caulobacter crescentus. We show that cells sense nitrogen starvation by way of detecting glutamine deprivation using the first enzyme (EI(Ntr)) of PTS(Ntr). Decreasing intracellular glutamine concentration triggers phosphorylation of EI(Ntr) and its downstream components HPr and EIIA(Ntr). Once phosphorylated, both HPr∼P and EIIA(Ntr)∼P stimulate (p)ppGpp accumulation by modulating SpoT activities. This burst of second messenger primarily impacts the non-replicative phase of the cell cycle by extending the G1 phase. This work highlights a new role for bacterial PTS systems in stimulating (p)ppGpp accumulation in response to metabolic cues and in controlling cell cycle progression and cell growth.

  8. Comparison of metabolic flux distributions for MDCK cell growth in glutamine- and pyruvate-containing media.

    PubMed

    Sidorenko, Yury; Wahl, Aljoscha; Dauner, Michael; Genzel, Yvonne; Reichl, Udo

    2008-01-01

    In mammalian cell cultures, ammonia that is released into the medium as a result of glutamine metabolism and lactate that is excreted due to incomplete glucose oxidation are both known to essentially inhibit the growth of cells. For some cell lines, for example, hybridoma cells, excreted ammonia also has an effect on product formation. Although glutamine has been generally considered as the major energy source for mammalian cells, it was recently found that various adherent cell lines (MDCK, CHO-K1, and BHK21) can grow as well in glutamine-free medium, provided glutamine is substituted with pyruvate. In such a medium the level of both ammonia and lactate released was significantly reduced. In this study, metabolic flux analysis (MFA) was applied to Madin Darby Canine Kidney (MDCK) cells cultivated in glutamine-containing and glutamine-free medium. The results of the MFA allowed further investigation of the influence of glutamine substitution with pyruvate on the metabolism of MDCK cells during different growth stages of adherent cells, e.g., early exponential and late contact-inhibited phase. Pyruvate seemed to directly enter the TCA cycle, whereas most of the glucose consumed was excreted as lactate. Although the exact mechanisms are not clear so far, this resulted in a reduction of the glucose uptake necessary for cellular metabolism in glutamine-free medium. Furthermore, consumption of ATP by futile cycles seemed to be significantly reduced when substituting glutamine with pyruvate. These findings imply that glutamine-free medium favors a more efficient use of nutrients by cells. However, a number of metabolic fluxes were similar in the two cultivations considered, e.g., most of the amino acid uptake and degradation rates or fluxes through the branch of the TCA cycle converting alpha-ketoglutarate to malate, which is responsible for the mitochondrial ATP synthesis. Besides, the specific rate of cell growth was approximately the same in both cultivations. Thus

  9. The effects of acidosis and alkalosis on the metabolism of glutamine and glutamate in renal cortex slices

    PubMed Central

    Kamm, Donald E.; Strope, Gerald L.

    1972-01-01

    Studies of the metabolism of glutamine and glutamate by renal cortex slices from acidotic, alkalotic, and control rats were performed. 88-95% of the glutamine and 104-115% of the glutamate taken up from the medium could be accounted for by the products found. Acidosis increased glutamine uptake and conversion to ammonia, CO2, glucose, lactate, pyruvate, lipid, and protein. The increase in glutamine conversion to ammonia after acidosis could be completely accounted for by the associated increase in its conversion to glucose, glutamate, lactate, and pyruvate. When glutamate metabolism was examined, acidosis did not affect substrate uptake but did increase its conversion to ammonia, glucose, lactate, CO2, and lipid. The increase in 14CO2 from U-14C-glutamine and U-14C-glutamate found with cortex slices from acidotic animals could be explained by the CO2 production calculated to be associated with the enhanced conversion of these substrates to other products during acidosis. 14CO2 production from 1.2-14C-acetate was found to be significantly increased in alkalosis rather than acidosis. These studies suggest that in the rat, the rate at which glutamine is completely oxidized in the Krebs cycle is not a factor regulating renal ammonia production. A comparison of the effects of acidbase status on glutamine and glutamate metabolism suggests that either glutamine transport or glutamine transaminase activity are significantly increased by acidosis. Images PMID:5057130

  10. Plasma glutamine levels and falciparum malaria.

    PubMed

    Cowan, G; Planche, T; Agbenyega, T; Bedu-Addo, G; Owusu-Ofori, A; Adebe-Appiah, J; Agranoff, D; Woodrow, C; Castell, L; Elford, B; Krishna, S

    1999-01-01

    Glutamine deficiency is associated with increased rates of sepsis and mortality, which can be prevented by glutamine supplementation. Changes in glutamine concentration were examined in Ghanaian children with acute falciparum malaria and control cases. The mean (SD) plasma glutamine concentration was lower in patients with acute malaria (401 (82) mumol/L, n = 50) than in control patients (623 (67) mumol/L, n = 7; P < 0.001). Plasma glutamine concentrations all rose in convalescence. The mean (SD) increase in plasma glutamine was 202 (123) mumol/L (n = 18; P < 0.001) compared with acute infection. We conclude that acute falciparum malaria is associated with large decreases in plasma glutamine and these falls may increase susceptibility to sepsis and dyserythropoeisis.

  11. Isolation and compositional analysis of a CP12-associated complex of calvin cycle enzymes from Nicotiana tabacum.

    PubMed

    Carmo-Silva, A Elizabete; Marri, Lucia; Sparla, Francesca; Salvucci, Michael E

    2011-06-01

    Two Calvin Cycle enzymes, NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a multiprotein complex with CP12, a small intrinsically-unstructured protein. Under oxidizing conditions, association with CP12 confers redox-sensitivity to the otherwise redox-insensitive A isoform of GAPDH (GapA) and provides an additional level of down-regulation to the redox-regulated PRK. To determine if CP12-mediated regulation is specific for GAPDH and PRK in vivo, a high molecular weight complex containing CP12 was isolated from tobacco chloroplasts and leaves and its protein composition was characterized. Gel electrophoresis and immunoblot analyses after separation of stromal proteins by size fractionation verified that the GAPDH (both isoforms) and PRK co-migrated with CP12 in dark- but not light-adapted chloroplasts. Nano-liquid-chromatography-mass-spectrometry of the isolated complex identified only CP12, GAPDH and PRK. Since nearly all of the CP12 from darkened chloroplasts migrates with GADPH and PRK as a high molecular mass species, these data indicate that the tight association of tobacco CP12 with GAPDH and PRK is specific and involves no other Calvin Cycle enzymes.

  12. Effect of dexamethasone on fetal hepatic glutamine-glutamate exchange.

    PubMed

    Timmerman, M; Teng, C; Wilkening, R B; Fennessey, P; Battaglia, F C; Meschia, G

    2000-05-01

    Intravenous infusion of dexamethasone (Dex) in the fetal lamb causes a two- to threefold increase in plasma glutamine and other glucogenic amino acids and a decrease of plasma glutamate to approximately one-third of normal. To explore the underlying mechanisms, hepatic amino acid uptake and conversion of L-[1-(13)C]glutamine to L-[1-(13)C]glutamate and (13)CO(2) were measured in six sheep fetuses before and in the last 2 h of a 26-h Dex infusion. Dex decreased hepatic glutamine and alanine uptakes (P < 0.01) and hepatic glutamate output (P < 0.001). Hepatic outputs of the glutamate (R(Glu,Gln)) and CO(2) formed from plasma glutamine decreased to 21 (P < 0.001) and 53% (P = 0.009) of control, respectively. R(Glu,Gln), expressed as a fraction of both outputs, decreased (P < 0.001) from 0.36 +/- 0.02 to 0.18 +/- 0.04. Hepatic glucose output remained virtually zero throughout the experiment. We conclude that Dex decreases fetal hepatic glutamate output by increasing the routing of glutamate carbon into the citric acid cycle and by decreasing the hepatic uptake of glucogenic amino acids. PMID:10780940

  13. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

    PubMed Central

    Metallo, Christian M.; Gameiro, Paulo A.; Bell, Eric L.; Mattaini, Katherine R.; Yang, Juanjuan; Hiller, Karsten; Jewell, Christopher M.; Johnson, Zachary R.; Irvine, Darrell J.; Guarente, Leonard; Kelleher, Joanne K.; Vander Heiden, Matthew G.; Iliopoulos, Othon; Stephanopoulos, Gregory

    2013-01-01

    Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and adenosine triphosphate citrate lyase (ACL) in the cytosol1-3. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle (TCA) and fatty acid synthesis4. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis5, the regulation and utilization of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells employ reductive metabolism of alpha-ketoglutarate (αKG) to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase 1 (IDH1) dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived αKG for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau (VHL) tumor suppressor protein preferentially utilize reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon utilization in order to produce AcCoA and support lipid synthesis in mammalian cells. PMID:22101433

  14. Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

    SciTech Connect

    Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa; Chang, Andrew; Gerratana, Barbara

    2012-08-31

    Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligand complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

  15. Effect of oral glutamine supplementation on human neutrophil lipopolysaccharide-stimulated degranulation following prolonged exercise.

    PubMed

    Walsh, N P; Blannin, A K; Bishop, N C; Robson, P J; Gleeson, M

    2000-03-01

    Recent studies have shown that neutrophils can utilize glutamine and that glutamine supplementation can improve neutrophil function in postoperative and burn patients. The present study investigated the influence of oral glutamine supplementation on stimulated neutrophil degranulation and oxidative burst activity following prolonged exercise. Subjects, 7 well-trained men, reported to the laboratory following an overnight fast and cycled for 2 hrs at 60% VO2max on two occasions a week apart. They were randomly assigned to either a glutamine or placebo treatment. For both trials, subjects consumed a sugar-free lemon drink at 15-min intervals until 90 minutes, then a lemon flavored glutamine drink (GLN) or sugar-free lemon drink (PLA) was consumed at 15-min intervals for the remaining exercise and the 2-hr recovery period. Venous blood samples were taken pre-, during, and postexercise. Glutamine supplementation had no effect on the magnitude of postexercise leukocytosis, the plasma elastase concentration following exercise (which increased in both trials), or the plasma elastase release in response to bacterial stimulation (which fell in both trials). Neutrophil function assessed by oxidative burst activity of isolated cells did not change following exercise in either trial. These findings therefore suggest that the fall in plasma glutamine concentration does not account for the decrease in neutrophil function (degranulation response) following prolonged exercise.

  16. The krebs cycle enzyme α-ketoglutarate decarboxylase is an essential glycosomal protein in bloodstream African trypanosomes.

    PubMed

    Sykes, Steven; Szempruch, Anthony; Hajduk, Stephen

    2015-03-01

    α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei.

  17. Glutamine supplementation in the newborn infant.

    PubMed

    Parimi, Prabhu S; Kalhan, Satish C

    2007-02-01

    Glutamine is a non-essential amino acid that can be synthesized de novo from glutamate. This synthesis can be increased by intravenous infusion of carbon precursors (alpha-ketoglutarate or amino acids) in adults and in infants. The metabolism of glutamine is highly compartmentalized between the splanchnic tissues and the periphery, so that orally administered glutamine is completely metabolized in the splanchnic compartment. Data from studies in adults and children show that plasma levels of glutamine decline during acute stress and illness. Because of its importance in several physiological functions (the demonstrated benefits of supplemental glutamine in adult burns and trauma patients and the inhibitory effect on proteolysis in the skeletal muscle in vitro), it has been suggested that during 'acute stress' the demands of glutamine outweigh its de novo synthesis, resulting in a fall in plasma glutamine levels. As a consequence, glutamine has been considered a 'conditionally essential' amino acid. Because of its instability in solution, glutamine is not routinely added to the parenteral amino acid mixtures. A number of clinical trials of parenteral and enteral supplementation of glutamine have been performed. The outcome measures examined have varied between acute effects and long-term complex clinical events such as mortality and risk of infections. Although acute studies in LBW babies have shown some beneficial effects such as changes in protein metabolism and activation of immune system, these have not been translated into prolonged advantages such as reduction in mortality or in nosocomial infection. The reasons for these differences are discussed.

  18. Resistance to vanadium in Pseudomonas fluorescens ATCC 17400 caused by mutations in TCA cycle enzymes.

    PubMed

    Denayer, Sarah; Matthijs, Sandra; Cornelis, Pierre

    2006-11-01

    Vanadium inhibits the growth of Pseudomonas fluorescens ATCC 17400 in the low-iron casamino acids medium and even more when iron is added to the medium. Analysis of transposon mutants allowed the isolation of two mutants with increased resistance to vanadium. One mutant had an insertion in the idh gene coding for the tricarboxylic acid enzyme isocitrate dehydrogenase. The second mutant had the transposon inserted into acnD, one out of three genes coding for a 2-methyl-isocitrate dehydratase (aconitase). In this mutant, there was a higher level of acnB aconitase transcripts while the levels of acnA transcripts were unchanged. A nonpolar idh mutant was obtained, which showed the same level of resistance against vanadium as the original transposon mutant. PMID:17020548

  19. Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities.

    PubMed

    Nordeste, Ricardo F; Trainer, Maria A; Charles, Trevor C

    2010-01-01

    Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

  20. Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities.

    PubMed

    Nordeste, Ricardo F; Trainer, Maria A; Charles, Trevor C

    2010-01-01

    Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries. PMID:20830568

  1. Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle.

    PubMed

    Maalcke, Wouter J; Reimann, Joachim; de Vries, Simon; Butt, Julea N; Dietl, Andreas; Kip, Nardy; Mersdorf, Ulrike; Barends, Thomas R M; Jetten, Mike S M; Keltjens, Jan T; Kartal, Boran

    2016-08-12

    Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N2, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N2H4). Previously, we identified N2-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one of 10 paralogs related to octaheme hydroxylamine (NH2OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAO-related hydroxylamine-oxidizing enzyme kustc1061 from K. stuttgartiensis Interestingly, the HDH trimers formed octamers in solution, each octamer harboring an amazing 192 c-type heme moieties. Whereas HAO and kustc1061 are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well defined HAO and kustc1061. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (P460) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N2-producing enzyme, next to nitrous oxide (N2O) reductase from denitrifying microorganisms. PMID:27317665

  2. Endogenous glutamine production in critically ill patients: the effect of exogenous glutamine supplementation

    PubMed Central

    2014-01-01

    Introduction Glutamine rate of appearance (Ra) may be used as an estimate of endogenous glutamine production. Recently a technique employing a bolus injection of isotopically labeled glutamine was introduced, with the potential to allow for multiple assessments of the glutamine Ra over time in critically ill patients, who may not be as metabolically stable as healthy individuals. Here the technique was used to evaluate the endogenous glutamine production in critically ill patients in the fed state with and without exogenous glutamine supplementation intravenously. Methods Mechanically ventilated patients (n = 11) in the intensive care unit (ICU) were studied on two consecutive days during continuous parenteral feeding. To allow the patients to be used as their own controls, they were randomized for the reference measurement during basal feeding without supplementation, before or after the supplementation period. Glutamine Ra was determined by a bolus injection of 13C-glutamine followed by a period of frequent sampling to establish the decay-curve for the glutamine tracer. Exogenous glutamine supplementation was given by intravenous infusion of a glutamine containing dipeptide, L-alanyl-L-glutamine, 0.28 g/kg during 20 hours. Results A 14% increase of endogenous glutamine Ra was seen at the end of the intravenous supplementation period as compared to the basal measurements (P = 0.009). Conclusions The bolus injection technique to measure glutamine Ra to estimate the endogenous production of glutamine in critically ill patients was demonstrated to be useful for repetitive measurements. The hypothesized attenuation of endogenous glutamine production during L-alanyl-L-glutamine infusion given as a part of full nutrition was not seen. PMID:24731231

  3. Enhanced mitochondrial glutamine anaplerosis suppresses pancreatic cancer growth through autophagy inhibition

    PubMed Central

    Jeong, Seung Min; Hwang, Sunsook; Park, Kyungsoo; Yang, Seungyeon; Seong, Rho Hyun

    2016-01-01

    Cancer cells use precursors derived from tricarboxylic acid (TCA) cycle to support their unlimited growth. However, continuous export of TCA cycle intermediates results in the defect of mitochondrial integrity. Mitochondria glutamine metabolism plays an essential role for the maintenance of mitochondrial functions and its biosynthetic roles by refilling the mitochondrial carbon pool. Here we report that human pancreatic ductal adenocarcinoma (PDAC) cells have a distinct dependence on mitochondrial glutamine metabolism. Whereas glutamine flux into mitochondria contributes to proliferation of most cancer cells, enhanced glutamine anaplerosis results in a pronounced suppression of PDAC growth. A cell membrane permeable α-ketoglutarate analog or overexpression of glutamate dehydrogenase lead to decreased proliferation and increased apoptotic cell death in PDAC cells but not other cancer cells. We found that enhanced glutamine anaplerosis inhibits autophagy, required for tumorigenic growth of PDAC, by activating mammalian TORC1. Together, our results reveal that glutamine anaplerosis is a crucial regulator of growth and survival of PDAC cells, which may provide novel therapeutic approaches to treat these cancers. PMID:27477484

  4. Enhanced mitochondrial glutamine anaplerosis suppresses pancreatic cancer growth through autophagy inhibition.

    PubMed

    Jeong, Seung Min; Hwang, Sunsook; Park, Kyungsoo; Yang, Seungyeon; Seong, Rho Hyun

    2016-01-01

    Cancer cells use precursors derived from tricarboxylic acid (TCA) cycle to support their unlimited growth. However, continuous export of TCA cycle intermediates results in the defect of mitochondrial integrity. Mitochondria glutamine metabolism plays an essential role for the maintenance of mitochondrial functions and its biosynthetic roles by refilling the mitochondrial carbon pool. Here we report that human pancreatic ductal adenocarcinoma (PDAC) cells have a distinct dependence on mitochondrial glutamine metabolism. Whereas glutamine flux into mitochondria contributes to proliferation of most cancer cells, enhanced glutamine anaplerosis results in a pronounced suppression of PDAC growth. A cell membrane permeable α-ketoglutarate analog or overexpression of glutamate dehydrogenase lead to decreased proliferation and increased apoptotic cell death in PDAC cells but not other cancer cells. We found that enhanced glutamine anaplerosis inhibits autophagy, required for tumorigenic growth of PDAC, by activating mammalian TORC1. Together, our results reveal that glutamine anaplerosis is a crucial regulator of growth and survival of PDAC cells, which may provide novel therapeutic approaches to treat these cancers. PMID:27477484

  5. Effect of glucose deprivation on rat glutamine synthetase in cultured astrocytes.

    PubMed Central

    Rosier, F; Lambert, D; Mertens-Strijthagen, M

    1996-01-01

    Glutamine synthetase was purified from the cerebral cortex of adult rats and characterized. Polyclonal rabbit antibodies were raised against the enzyme, purified and their specific anti-(glutamine synthetase) activity determined. A primary astroglial culture was prepared from newborn Sprague-Dawley rats. Astrocytes at different ages of development were incubated in the presence and absence of glucose. In glucose-deprived conditions the specific activity of glutamine synthetase decreased. This decrease was more pronounced in 8-day-old than in 21-day-old cultures. Kinetic analysis demonstrated that the reduction in activity was mainly related to a decrease in Vmax. By immunoprecipitation, it was shown that the number of enzyme molecules in astrocytes was decreased in glucose-deprived conditions. On addition of glucose, a total recovery of glutamine synthetase was obtained after 36 h in 8-day-old culture. Rates of degradation and synthesis were investigated. When compared with an incubation in the presence of glucose, glucose deprivation increased enzyme turnover, as estimated from the first-order disappearance of radioactivity from glutamine synthetase. Synthesis rate was estimated from the incorporation of [35S]methionine during a 2 h incubation period and was decreased in glucose-deprived conditions. Trichloroacetate-precipitable proteins changed only slightly in the experimental conditions, and total protein did not vary significantly during the experimental period. A mathematical model is presented which attempts to integrate degradation and synthesis in our experimental model. PMID:8615836

  6. Molecular basis of an adult form of Sandhoff disease: substitution of glutamine for arginine at position 505 of the beta-chain of beta-hexosaminidase results in a labile enzyme.

    PubMed

    Bolhuis, P A; Ponne, N J; Bikker, H; Baas, F; Vianney de Jong, J M

    1993-09-01

    Sandhoff disease is a lysosomal storage disorder characterized by accumulation of GM2 ganglioside due to mutations in the beta-chain of beta-hexosaminidase. Hexosaminidase activity is negligible in infantile Sandhoff disease whereas residual activity is present in juvenile and adult forms. Here we report the molecular basis of the first described adult form of Sandhoff disease. Southern analysis of chromosomal DNA indicated the absence of chromosomal deletions in the gene encoding the beta-chain. Northern analysis of RNA from cultured fibroblasts demonstrated that at least one of the beta-chain alleles was transcribed into normal-length mRNA. Sequence analysis of the entire cDNA prepared from poly-adenylated RNA showed that only one point mutation was present, consisting of a G-->A transition at nucleotide position 1514. This mutation changes the electric charge at amino acid position 505 by substitution of glutamine for arginine in a highly conserved part of the beta-chain, present even in the slime mold Dictyostelium discoideum. The nucleotide transition generated a new restriction site for DdeI, which was present in only one of the alleles of the patient. Reverse transcription of mRNA followed by restriction with DdeI resulted in complete digestion at the mutation site, demonstrating that the second allele was of an mRNA-negative type. Transfection of COS cells with a cDNA construct containing the mutation but otherwise the normal sequence resulted in the expression of a labile form of beta-hexosaminidase. These results show that the patient's is a genetic compound, and that the lability of beta-hexosaminidase found in this form of Sandhoff disease is based on a single nucleotide transition. PMID:8357844

  7. Monitoring 6 weeks of progressive endurance training with plasma glutamine.

    PubMed

    Kargotich, S; Keast, D; Goodman, C; Bhagat, C I; Joske, D J L; Dawson, B; Morton, A R

    2007-03-01

    The distinction between positive and negative training adaptation is an important prerequisite in the identification of any marker for monitoring training in athletes. To investigate the glutamine responses to progressive endurance training, twenty healthy males were randomly assigned to a training group or a non-exercising control group. The training group performed a progressive (3 to 6 x 90 minute sessions per week at 70 % V.O (2max)) six-week endurance training programme on a cycle ergometer, while the control group did not participate in any exercise during this period. Performance assessments (V.O (2max) and time to exhaustion) and resting blood samples (for haemoglobin concentration, haematocrit, cortisol, ferritin, creatine kinase, glutamine, uric acid and urea analysis) were obtained prior to the commencement of training (Pre) and at the end of week 2, week 4 and week 6. The training group showed significant improvements in time to exhaustion (p < 0.01), and V.O (2max) (p < 0.05) at all time points (except week 2 for V.O (2max)), while the control group performance measures did not change. In the training group, haemoglobin concentration and haematocrit were significantly lower (p < 0.01) than pretraining values at week 2 and 4, as percentage changes in plasma volume indicated a significant (p < 0.01) haemodilution (+ 6 - 9 %) was present at week 2, 4 and 6. No changes were seen in the control group. In the training group, plasma glutamine (week 2, 4 and 6), creatine kinase (week 2 and 4), uric acid (week 2 and 4) and urea (week 2 and 4) all increased significantly from pretraining levels. No changes in cortisol or ferritin were found in the training group and no changes in any blood variables were present in the control group. Plasma glutamine was the only blood variable to remain significantly above pretraining (966 +/- 32 micromol . 1 (-1)) levels at week 6 (1176 +/- 24 micromol . 1 (-1); p < 0.05) The elevation seen here in glutamine levels, after 6

  8. In vivo and in vitro liver cancer metabolism observed with hyperpolarized [5-13C]glutamine

    NASA Astrophysics Data System (ADS)

    Cabella, C.; Karlsson, M.; Canapè, C.; Catanzaro, G.; Colombo Serra, S.; Miragoli, L.; Poggi, L.; Uggeri, F.; Venturi, L.; Jensen, P. R.; Lerche, M. H.; Tedoldi, F.

    2013-07-01

    Glutamine metabolism is, with its many links to oncogene expression, considered a crucial step in cancer metabolism and it is thereby a key target for alteration in cancer development. In particular, strong correlations have been reported between oncogene expression and expression and activity of the enzyme glutaminase. This mitochondrial enzyme, which is responsible for the deamidation of glutamine to form glutamate, is overexpressed in many tumour tissues. In animal models, glutaminase expression is correlated with tumour growth rate and it is readily possible to limit tumour growth by suppression of glutaminase activity. In principle, hyperpolarized 13C MR spectroscopy can provide insight to glutamine metabolism and should hence be a valuable tool to study changes in glutaminase activity as tumours progress. However, no such successful in vivo studies have been reported, even though several good biological models have been tested. This may, at least partly, be due to problems in preparing glutamine for hyperpolarization. This paper reports a new and improved preparation of hyperpolarized [5-13C]glutamine, which provides a highly sensitive 13C MR marker. With this preparation of hyperpolarized [5-13C]glutamine, glutaminase activity in vivo in a rat liver tumour was investigated. Moreover, this marker was also used to measure response to drug treatment in vitro in cancer cells. These examples of [5-13C]glutamine used in tumour models warrant the new preparation to allow metabolic studies with this conditionally essential amino acid.

  9. Acute Carnosine Administration Increases Respiratory Chain Complexes and Citric Acid Cycle Enzyme Activities in Cerebral Cortex of Young Rats.

    PubMed

    Macedo, Levy W; Cararo, José H; Maravai, Soliany G; Gonçalves, Cinara L; Oliveira, Giovanna M T; Kist, Luiza W; Guerra Martinez, Camila; Kurtenbach, Eleonora; Bogo, Maurício R; Hipkiss, Alan R; Streck, Emilio L; Schuck, Patrícia F; Ferreira, Gustavo C

    2016-10-01

    Carnosine (β-alanyl-L-histidine) is an imidazole dipeptide synthesized in excitable tissues of many animals, whose biochemical properties include carbonyl scavenger, anti-oxidant, bivalent metal ion chelator, proton buffer, and immunomodulating agent, although its precise physiological role(s) in skeletal muscle and brain tissues in vivo remain unclear. The aim of the present study was to investigate the in vivo effects of acute carnosine administration on various aspects of brain bioenergetics of young Wistar rats. The activity of mitochondrial enzymes in cerebral cortex was assessed using a spectrophotometer, and it was found that there was an increase in the activities of complexes I-III and II-III and succinate dehydrogenase in carnosine-treated rats, as compared to vehicle-treated animals. However, quantitative real-time RT-PCR (RT-qPCR) data on mRNA levels of mitochondrial biogenesis-related proteins (nuclear respiratory factor 1 (Nrf1), peroxisome proliferator-activated receptor-γ coactivator 1-α (Ppargc1α), and mitochondrial transcription factor A (Tfam)) were not altered significantly and therefore suggest that short-term carnosine administration does not affect mitochondrial biogenesis. It was in agreement with the finding that immunocontent of respiratory chain complexes was not altered in animals receiving carnosine. These observations indicate that acute carnosine administration increases the respiratory chain and citric acid cycle enzyme activities in cerebral cortex of young rats, substantiating, at least in part, a neuroprotector effect assigned to carnosine against oxidative-driven disorders. PMID:26476839

  10. Acute Carnosine Administration Increases Respiratory Chain Complexes and Citric Acid Cycle Enzyme Activities in Cerebral Cortex of Young Rats.

    PubMed

    Macedo, Levy W; Cararo, José H; Maravai, Soliany G; Gonçalves, Cinara L; Oliveira, Giovanna M T; Kist, Luiza W; Guerra Martinez, Camila; Kurtenbach, Eleonora; Bogo, Maurício R; Hipkiss, Alan R; Streck, Emilio L; Schuck, Patrícia F; Ferreira, Gustavo C

    2016-10-01

    Carnosine (β-alanyl-L-histidine) is an imidazole dipeptide synthesized in excitable tissues of many animals, whose biochemical properties include carbonyl scavenger, anti-oxidant, bivalent metal ion chelator, proton buffer, and immunomodulating agent, although its precise physiological role(s) in skeletal muscle and brain tissues in vivo remain unclear. The aim of the present study was to investigate the in vivo effects of acute carnosine administration on various aspects of brain bioenergetics of young Wistar rats. The activity of mitochondrial enzymes in cerebral cortex was assessed using a spectrophotometer, and it was found that there was an increase in the activities of complexes I-III and II-III and succinate dehydrogenase in carnosine-treated rats, as compared to vehicle-treated animals. However, quantitative real-time RT-PCR (RT-qPCR) data on mRNA levels of mitochondrial biogenesis-related proteins (nuclear respiratory factor 1 (Nrf1), peroxisome proliferator-activated receptor-γ coactivator 1-α (Ppargc1α), and mitochondrial transcription factor A (Tfam)) were not altered significantly and therefore suggest that short-term carnosine administration does not affect mitochondrial biogenesis. It was in agreement with the finding that immunocontent of respiratory chain complexes was not altered in animals receiving carnosine. These observations indicate that acute carnosine administration increases the respiratory chain and citric acid cycle enzyme activities in cerebral cortex of young rats, substantiating, at least in part, a neuroprotector effect assigned to carnosine against oxidative-driven disorders.

  11. Differentiated effect of ageing on the enzymes of Krebs' cycle, electron transfer complexes and glutamate metabolism of non-synaptic and intra-synaptic mitochondria from cerebral cortex.

    PubMed

    Villa, R F; Gorini, A; Hoyer, S

    2006-11-01

    The effect of ageing on the activity of enzymes linked to Krebs' cycle, electron transfer chain and glutamate metabolism was studied in three different types of mitochondria of cerebral cortex of 1-year old and 2-year old male Wistar rats. We assessed the maximum rate (V(max)) of the mitochondrial enzyme activities in non-synaptic perikaryal mitochondria, and in two populations of intra-synaptic mitochondria. The results indicated that: (i) in normal, steady-state cerebral cortex the values of the catalytic activities of the enzymes markedly differed in the various populations of mitochondria; (ii) in intra-synaptic mitochondria, ageing affected the catalytic properties of the enzymes linked to Krebs' cycle, electron transfer chain and glutamate metabolism; (iii) these changes were more evident in intra-synaptic "heavy" than "light" mitochondria. These results indicate a different age-related vulnerability of subpopulations of mitochondria in vivo located into synapses than non-synaptic ones.

  12. Coupled effects of light and nitrogen source on the urea cycle and nitrogen metabolism over a diel cycle in the marine diatom Thalassiosira pseudonana.

    PubMed

    Bender, Sara J; Parker, Micaela S; Armbrust, E Virginia

    2012-03-01

    Diatoms are photoautotrophic organisms capable of growing on a variety of inorganic and organic nitrogen sources. Discovery of a complete urea cycle in diatoms was surprising, as this pathway commonly functions in heterotrophic organisms to rid cells of waste nitrogen. To determine how the urea cycle is integrated into cellular nitrogen metabolism and energy management, the centric diatom Thalassiosira pseudonana was maintained in semi-continuous batch cultures on nitrate, ammonium, or urea as the sole nitrogen source, under a 16: 8 light: dark cycle and at light intensities that were low, saturating, or high for growth. Steady-state transcript levels were determined for genes encoding enzymes linked to the urea cycle, urea hydrolysis, glutamine synthesis, pyrimidine synthesis, photorespiration, and energy storage. Transcript abundances were significantly affected by nitrogen source, light intensity and a diel cycle. The impact of N source on differential transcript accumulation was most apparent under the highest light intensity. Models of cellular metabolism under high light were developed based on changes in transcript abundance and predicted enzyme localizations. We hypothesize that the urea cycle is integrated into nitrogen metabolism through its connection to glutamine and in the eventual production of urea. These findings have important implications for nitrogen flow in the cell over diel cycles at surface ocean irradiances.

  13. Time course of the uridylylation and adenylylation states in the glutamine synthetase bicyclic cascade.

    PubMed Central

    Varón-Castellanos, R; Havsteen, B H; García-Moreno, M; Valero-Ruiz, E; Molina-Alarcón, M; García-Cánovas, F

    1993-01-01

    A kinetic analysis of the glutamine synthetase bicyclic cascade is presented. It includes the dependence on time from the onset of the reaction of both the uridylylation of Shapiro's regulatory protein and the adenylylation of the glutamine synthetase. The transient phase equations obtained allow an estimation of the time elapsed until the states of uridylylation and adenylylation reach their steady-states, and therefore an evaluation of the effective sensitivity of the system. The contribution of the uridylylation cycle to the adenylylation cycle has been studied, and an equation relating the state of adenylylation at any time to the state of uridylylation at the same instant has been derived. PMID:8104399

  14. Neuropsychiatric manifestations in late-onset urea cycle disorder patients.

    PubMed

    Serrano, Mercedes; Martins, Cecilia; Pérez-Dueñas, Belén; Gómez-López, Lilian; Murgui, Empar; Fons, Carmen; García-Cazorla, Angels; Artuch, Rafael; Jara, Fernando; Arranz, José A; Häberle, Johannes; Briones, Paz; Campistol, Jaume; Pineda, Mercedes; Vilaseca, Maria A

    2010-03-01

    Inherited urea cycle disorders represent one of the most common groups of inborn errors of metabolism. Late-onset urea cycle disorders caused by partial enzyme deficiencies may present with unexpected clinical phenotypes. We report 9 patients followed up in our hospital presenting late-onset urea cycle disorders who initially manifested neuropsychiatric/neurodevelopmental symptoms (the most prevalent neuropsychiatric/neurodevelopmental diagnoses were mental retardation, attention-deficit hyperactivity disorder [ADHD], language disorder, and delirium). Generally, these clinical pictures did not benefit from pharmacological treatment. Conversely, dietary treatment improved the symptoms. Regarding biochemical data, 2 patients showed normal ammonium but high glutamine levels. This study highlights the fact that neuropsychiatric/neurodevelopmental findings are common among the initial symptomatology of late-onset urea cycle disorders. The authors recommend that unexplained or nonresponsive neuropsychiatric/neurodevelopmental symptoms appearing during childhood or adolescence be followed by a study of ammonia and amino acid plasmatic levels to rule out a urea cycle disorder.

  15. Turnover of bacterial glutamine synthetase: oxidative inactivation precedes proteolysis.

    PubMed

    Levine, R L; Oliver, C N; Fulks, R M; Stadtman, E R

    1981-04-01

    We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover.

  16. Turnover of bacterial glutamine synthetase: oxidative inactivation precedes proteolysis.

    PubMed Central

    Levine, R L; Oliver, C N; Fulks, R M; Stadtman, E R

    1981-01-01

    We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover. Images PMID:6113590

  17. Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511.

    PubMed

    Gómez-Baena, Guadalupe; Domínguez-Martín, María Agustina; Donaldson, Robert P; García-Fernández, José Manuel; Diez, Jesús

    2015-01-01

    Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed. PMID:26270653

  18. Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511.

    PubMed

    Gómez-Baena, Guadalupe; Domínguez-Martín, María Agustina; Donaldson, Robert P; García-Fernández, José Manuel; Diez, Jesús

    2015-01-01

    Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed.

  19. Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511

    PubMed Central

    Gómez-Baena, Guadalupe; Domínguez-Martín, María Agustina; Donaldson, Robert P.; García-Fernández, José Manuel; Diez, Jesús

    2015-01-01

    Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed. PMID:26270653

  20. Depletion of glutamine enhances sodium butyrate-induced erythroid differentiation of K562 cells.

    PubMed

    Canh Hiep, Nguyen; Kinohira, Seiko; Furuyama, Kazumichi; Taketani, Shigeru

    2012-12-01

    Human erytholeukemia K562 cells are induced to differentiate along the erythroid lineage by a variety of chemical compounds, including hemin, sodium butyrate and 1-β-d-arabinofuranosylcytosine. We have investigated the induction of erythroid differentiation of K562 cells by glutamine depletion. When K562 cells were cultured in glutamine-minus medium, the induction of hemoglobin synthesis, accompanied by those of heme-biosynthetic enzymes and erythroid transcriptional factors, was observed. This induction was dependent on the temporally marked decrease of intracellular level of glutathione, followed by the marked activation of p38MAPK and SAPK/JNK, but not ERK. Under glutamine-deficient conditions, the treatment of K562 cells with sodium butyrate resulted in the marked enhancement of the induction of heme biosynthesis. Glutamine depletion also accelerated the expressions of erythroid-related factors including α-globin and heme-biosynthetic enzymes, GATA-1 and NF-E2, in sodium butyrate-induced K562 cells. The transcriptional activity of β-globin gene promoter-reporter was markedly enhanced by these treatments, indicating that glutamine deficiency in combination with sodium butyrate treatment gives high efficiency of chemical-induced differentiation in the hematopoiesis process.

  1. Oncogenic PIK3CA mutations reprogram glutamine metabolism in colorectal cancer

    PubMed Central

    Hao, Yujun; Samuels, Yardena; Li, Qingling; Krokowski, Dawid; Guan, Bo-Jhih; Wang, Chao; Jin, Zhicheng; Dong, Bohan; Cao, Bo; Feng, Xiujing; Xiang, Min; Xu, Claire; Fink, Stephen; Meropol, Neal J.; Xu, Yan; Conlon, Ronald A.; Markowitz, Sanford; Kinzler, Kenneth W.; Velculescu, Victor E.; Brunengraber, Henri; Willis, Joseph E.; LaFramboise, Thomas; Hatzoglou, Maria; Zhang, Guo-Fang; Vogelstein, Bert; Wang, Zhenghe

    2016-01-01

    Cancer cells often require glutamine for growth, thereby distinguishing them from most normal cells. Here we show that PIK3CA mutations reprogram glutamine metabolism by upregulating glutamate pyruvate transaminase 2 (GPT2) in colorectal cancer (CRC) cells, making them more dependent on glutamine. Compared with isogenic wild-type (WT) cells, PIK3CA mutant CRCs convert substantially more glutamine to α-ketoglutarate to replenish the tricarboxylic acid cycle and generate ATP. Mutant p110α upregulates GPT2 gene expression through an AKT-independent, PDK1–RSK2–ATF4 signalling axis. Moreover, aminooxyacetate, which inhibits the enzymatic activity of aminotransferases including GPT2, suppresses xenograft tumour growth of CRCs with PIK3CA mutations, but not with WT PIK3CA. Together, these data establish oncogenic PIK3CA mutations as a cause of glutamine dependency in CRCs and suggest that targeting glutamine metabolism may be an effective approach to treat CRC patients harbouring PIK3CA mutations. PMID:27321283

  2. Diel variation in ammonia excretion, glutamine levels, and hydration status in two species of terrestrial isopods.

    PubMed

    Wright, Jonathan C; Peña-Peralta, Mariasol

    2005-01-01

    Terrestrial isopods (suborder Oniscidea) excrete most nitrogen diurnally as volatile ammonia, and ammonia-loaded animals accumulate nonessential amino acids, which may constitute the major nocturnal nitrogen pool. This study explored the relationship between ammonia excretion, glutamine storage/mobilization, and water balance, in two sympatric species Ligidium lapetum (section Diplocheta), a hygric species; and Armadillidium vulgare (Section Crinocheta), a xeric species capable of water-vapor absorption (WVA). Ammonia excretion (12-h), tissue glutamine levels, and water contents were measured following field collection of animals at dusk and dawn. In both species, diurnal ammonia excretion exceeded nocturnal excretion four- to fivefold while glutamine levels increased four- to sevenfold during the night. Most glutamine was accumulated in the somatic tissues ("body wall"). While data support the role of glutamine in nocturnal nitrogen storage, potential nitrogen mobilization from glutamine breakdown (162 micromol g(-1) in A. vulgare) exceeds measured ammonia excretion (2.5 micromol g(-1)) over 60-fold. This may serve to generate the high hemolymph ammonia concentrations (and high P(NH3)) seen during volatilization. The energetic cost of ammonia volatilization is discussed in the light of these findings. Mean water contents were similar at dusk and dawn in both species, indicating that diel cycles of water depletion and replenishment were not occurring. PMID:15578188

  3. Diel variation in ammonia excretion, glutamine levels, and hydration status in two species of terrestrial isopods.

    PubMed

    Wright, Jonathan C; Peña-Peralta, Mariasol

    2005-01-01

    Terrestrial isopods (suborder Oniscidea) excrete most nitrogen diurnally as volatile ammonia, and ammonia-loaded animals accumulate nonessential amino acids, which may constitute the major nocturnal nitrogen pool. This study explored the relationship between ammonia excretion, glutamine storage/mobilization, and water balance, in two sympatric species Ligidium lapetum (section Diplocheta), a hygric species; and Armadillidium vulgare (Section Crinocheta), a xeric species capable of water-vapor absorption (WVA). Ammonia excretion (12-h), tissue glutamine levels, and water contents were measured following field collection of animals at dusk and dawn. In both species, diurnal ammonia excretion exceeded nocturnal excretion four- to fivefold while glutamine levels increased four- to sevenfold during the night. Most glutamine was accumulated in the somatic tissues ("body wall"). While data support the role of glutamine in nocturnal nitrogen storage, potential nitrogen mobilization from glutamine breakdown (162 micromol g(-1) in A. vulgare) exceeds measured ammonia excretion (2.5 micromol g(-1)) over 60-fold. This may serve to generate the high hemolymph ammonia concentrations (and high P(NH3)) seen during volatilization. The energetic cost of ammonia volatilization is discussed in the light of these findings. Mean water contents were similar at dusk and dawn in both species, indicating that diel cycles of water depletion and replenishment were not occurring.

  4. Methylphenidate treatment leads to abnormalities on krebs cycle enzymes in the brain of young and adult rats.

    PubMed

    Réus, Gislaine Z; Scaini, Giselli; Furlanetto, Camila B; Morais, Meline O S; Jeremias, Isabela C; Mello-Santos, Lis Mairá; Freitas, Karolina V; Quevedo, João; Streck, Emilio L

    2013-08-01

    Studies have shown a relationship between energy metabolism and methylphenidate (MPH); however, there are no studies evaluating the effects of MPH in Krebs cycle. So, we investigated if MPH treatment could alter the activity of citrate synthase (CS), malate dehydrogenase (MD), and isocitrate dehydrogenase (ID) in the brain of young and adult Wistar rats. Our results showed that MPH (2 and 10 mg/kg) reduced CS in the striatum and prefrontal cortex (PF), with MPH at all doses in the cerebellum and hippocampus after chronic treatment in young rats. In adult rats the CS was reduced in the cerebellum after acute treatment with MPH at all doses, and after chronic treatment in the PF and cerebellum with MPH (10 mg/kg), and in the hippocampus with MPH (2 and 10 mg/kg). The ID decreased in the hippocampus and striatum with MPH (2 and 10 mg/kg), and in the cortex (10 mg/kg) after acute treatment in young rats. In adult rats acute treatment with MPH (2 and 10 mg/kg) reduced ID in the cerebellum, and with MPH (10 mg/kg) in the cortex; chronic treatment with MPH (10 mg/kg) decreased ID in the PF; with MPH (2 and 10 mg/kg) in the cerebellum, and with MPH at all doses in the hippocampus. The MD did not alter. In conclusion, our results suggest that MPH can alter enzymes of Krebs cycle in brain areas involved with circuits related with attention deficit hyperactivity disorder; however, such effects depend on age of animal and treatment regime.

  5. Glutamine-fueled mitochondrial metabolism is decoupled from glycolysis in melanoma

    PubMed Central

    Filipp, Fabian V.; Ratnikov, Boris; De Ingeniis, Jessica; Smith, Jeffrey W.; Osterman, Andrei L.; Scott, David A.

    2013-01-01

    Summary In this perspective, we revise the historic notion that cancer is a disease of mitochondria. We summarize recent findings on the function and rewiring of central carbon metabolism in melanoma. Metabolic profiling studies using stable isotope tracers show that glycolysis is decoupled from the tricarboxylic acid (TCA) cycle. This decoupling is not ‘dysfunction’ but rather an alternate wiring required by tumor cells to remain metabolically versatile. In large part, this requirement is met by glutamine feeding the TCA cycle as an alternative source of carbon. Glutamine is also used in non-conventional ways, like traveling in reverse through the TCA flux to feed fatty acid biosynthesis. The biosynthetic networks linked with non-essential amino acids alanine, serine, arginine, and proline are also significantly impacted by the use of glutamine as an alternate carbon source. PMID:22846158

  6. The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state.

    PubMed

    Jiang, P; Peliska, J A; Ninfa, A J

    1998-09-15

    The regulation of Escherichia coli glutamine synthetase (GS) by reversible adenylylation has provided one of the classical paradigms for signal transduction by cyclic cascades. Yet, many mechanistic features of this regulation remain to be elucidated. We examined the regulation of GS adenylylation state in a reconstituted system containing GS, adenylyltransferase (ATase), the PII signal transduction protein that controls ATase, and the uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which has a role in regulating PII. In this reconstituted bicyclic cascade system, the adenylylation state of GS was regulated reciprocally by the small molecule effectors 2-ketoglutarate and glutamine at physiological effector concentrations. By examination of the individual regulatory monocycles and comparison to the bicyclic system and existing data, we could deduce that the only sensors of 2-ketoglutarate were PII and PII-UMP. At physiological conditions, we observed that the main role of 2-ketoglutarate in bringing about the deadenylylation of GS was to inhibit GS adenylylation, and this was due to the allosteric regulation of PII activity. Glutamine acted as an allosteric regulator of both ATase and UTase/UR. We also compared the regulation of GS adenylylation state to the regulation of phosphorylation state of the transcription factor NRI (NtrC) in a reconstituted bicyclic system containing NRI, the bifunctional kinase/phosphatase NRII (NtrB), PII, and the UTase/UR. This comparison indicated that, at a fixed 2-ketoglutarate concentration, the regulation of GS adenylylation state by glutamine was sharper and occurred at a higher concentration than did the regulation of NRI phosphorylation. The possible biological implications of this regulatory arrangement are discussed. PMID:9737857

  7. Amino acids, glutamine, and protein metabolism in very low birth weight infants.

    PubMed

    Parimi, Prabhu S; Kadrofske, Mark M; Gruca, Lourdes L; Hanson, Richard W; Kalhan, Satish C

    2005-12-01

    Glutamine has been proposed to be conditionally essential for premature infants, and the currently used parenteral nutrient mixtures do not contain glutamine. De novo glutamine synthesis (DGln) is linked to inflow of carbon into and out of the tricarboxylic acid (TCA) cycle. We hypothesized that a higher supply of parenteral amino acids by increasing the influx of amino acid carbon into the TCA cycle will enhance the rate of DGln. Very low birth weight infants were randomized to receive parenteral amino acids either 1.5 g/kg/d for 20 h followed by 3.0 g/kg/d for 5 h (AA1.5) or 3.0 g/kg/d for 20 h followed by 1.5 g/kg/d for 5 h (AA3.0). A third group of babies received amino acids 1.5 g/kg/d for 20 h followed by 3.0 g/kg/d for 20 h (AA-Ext). Glutamine and protein/nitrogen kinetics were examined using [5-(15)N]glutamine, [2H5]phenylalanine, [1-(13)C,15N]leucine, and [15N2]urea tracers. An acute increase in parenteral amino acid infusion for 5 h (AA1.5) resulted in decrease in rate of appearance (Ra) of phenylalanine and urea, but had no effect on glutamine Ra. Infusion of amino acids at 3.0 g/kg/d for 20 h resulted in increase in DGln, leucine transamination, and urea synthesis, but had no effect on Ra phenylalanine (AA-Ext). These data show an acute increase in parenteral amino acid-suppressed proteolysis, however, such an effect was not seen when amino acids were infused for 20 h and resulted in an increase in glutamine synthesis.

  8. 2H2O incorporation into hepatic acetyl-CoA and de novo lipogenesis as measured by Krebs cycle-mediated 2H-enrichment of glutamate and glutamine.

    PubMed

    Silva, Ana Maria; Martins, Fatima; Jones, John G; Carvalho, Rui

    2011-12-01

    Deuterated water is widely used for measuring de novo lipogenesis on the basis of quantifying lipid (2)H-enrichment relative to that of body water. However, incorporation of (2)H-enrichment from body water into newly synthesized lipid molecules is incomplete therefore the true lipid precursor enrichment differs from that of body water. We describe a novel measurement of de novo lipogenesis that is based on a true precursor-product analysis of hepatic acetyl-CoA and triglyceride methyl enrichments from deuterated water. After deuterated water administration to seven in situ and seven perfused livers, acetyl-CoA methyl enrichment was inferred from (2)H nuclear magnetic resonance analysis of hepatic glutamate/glutamine (Glx) enrichment and triglyceride methyl enrichment was directly determined by (2)H nuclear magnetic resonance of triglycerides. Acetyl-CoA (2) H-enrichment was 71% ± 1% that of body water for in situ livers and 53% ± 2% of perfusate water for perfused livers. From the ratio of triglyceride-methyl/acetyl-CoA enrichments, fractional de novo lipogenesis rates of 0.97% ± 0.09%/2 hr and 7.92% ± 1.47%/48 hr were obtained for perfused and in situ liver triglycerides, respectively. Our method reveals that acetyl-CoA enrichment is significantly less than body water both for in situ and perfused livers. Furthermore, the difference between acetyl-CoA and body water enrichments is sensitive to the experimental setting.

  9. The effect of portacaval anastomosis on the expression of glutamine synthetase and ornithine aminotransferase in perivenous hepatocytes.

    PubMed

    da Silva, Robin; Levillain, Oliver; Brosnan, John T; Araneda, Silvia; Brosnan, Margaret E

    2013-05-01

    There is functional zonation of metabolism across the liver acinus, with glutamine synthetase restricted to a narrow band of cells around the terminal hepatic venules. Portacaval anastomosis, where there is a major rerouting of portal blood flow from the portal vein directly to the vena cava bypassing the liver, has been reported to result in a marked decrease in the activity of glutamine synthetase. It is not known whether this represents a loss of perivenous hepatocytes or whether there is a specific loss of glutamine synthetase. To answer this question, we have determined the activity of glutamine synthetase and another enzyme from the perivenous compartment, ornithine aminotransferase, as well as the immunochemical localization of both glutamine synthetase and ornithine aminotransferase in rats with a portacaval shunt. The portacaval shunt caused a marked decrease in glutamine synthetase activity and an increase in ornithine aminotransferase activity. Immunohistochemical analysis showed that the glutamine synthetase and ornithine aminotransferase proteins maintained their location in the perivenous cells. These results indicate that there is no generalized loss of perivenous hepatocytes, but rather, there is a significant alteration in the expression of these proteins and hence metabolism in this cell population. PMID:23656379

  10. The effect of portacaval anastomosis on the expression of glutamine synthetase and ornithine aminotransferase in perivenous hepatocytes.

    PubMed

    da Silva, Robin; Levillain, Oliver; Brosnan, John T; Araneda, Silvia; Brosnan, Margaret E

    2013-05-01

    There is functional zonation of metabolism across the liver acinus, with glutamine synthetase restricted to a narrow band of cells around the terminal hepatic venules. Portacaval anastomosis, where there is a major rerouting of portal blood flow from the portal vein directly to the vena cava bypassing the liver, has been reported to result in a marked decrease in the activity of glutamine synthetase. It is not known whether this represents a loss of perivenous hepatocytes or whether there is a specific loss of glutamine synthetase. To answer this question, we have determined the activity of glutamine synthetase and another enzyme from the perivenous compartment, ornithine aminotransferase, as well as the immunochemical localization of both glutamine synthetase and ornithine aminotransferase in rats with a portacaval shunt. The portacaval shunt caused a marked decrease in glutamine synthetase activity and an increase in ornithine aminotransferase activity. Immunohistochemical analysis showed that the glutamine synthetase and ornithine aminotransferase proteins maintained their location in the perivenous cells. These results indicate that there is no generalized loss of perivenous hepatocytes, but rather, there is a significant alteration in the expression of these proteins and hence metabolism in this cell population.

  11. Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity.

    PubMed

    Willemoës, Martin; Mølgaard, Anne; Johansson, Eva; Martinussen, Jan

    2005-02-01

    GTP is an allosteric activator of CTP synthase and acts to increase the k(cat) for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain of CTP synthase. In the GATase domain of the recently published structures of the Escherichia coli and Thermus thermophilus CTP synthases a loop region immediately proceeding amino acid residues forming the oxy-anion hole and named lid L11 is shown for the latter enzyme to be flexible and change position depending on the presence or absence of glutamine in the glutamine binding site. Displacement or rearrangement of this loop may provide a means for the suggested role of allosteric activation by GTP to optimize the oxy-anion hole for glutamine hydrolysis. Arg359, Gly360 and Glu362 of the Lactococcus lactis enzyme are highly conserved residues in lid L11 and we have analyzed their possible role in GTP activation. Characterization of the mutant enzymes R359M, R359P, G360A and G360P indicated that both Arg359 and Gly360 are involved in the allosteric response to GTP binding whereas the E362Q enzyme behaved like wild-type enzyme. Apart from the G360A enzyme, the results from kinetic analysis of the enzymes altered at position 359 and 360 showed a 10- to 50-fold decrease in GTP activation of glutamine dependent CTP synthesis and concomitant four- to 10-fold increases in K(A) for GTP. The R359M, R359P and G360P also showed no GTP activation of the uncoupled glutaminase reaction whereas the G360A enzyme was about twofold more active than wild-type enzyme. The elevated K(A) for GTP and reduced GTP activation of CTP synthesis of the mutant enzymes are in agreement with a predicted interaction of bound GTP with lid L11 and indicate that the GTP activation of glutamine dependent CTP synthesis may be explained by structural rearrangements around the oxy-anion hole of the GATase

  12. No effect of glutamine supplementation and hyperoxia on oxidative metabolism and performance during high-intensity exercise.

    PubMed

    Marwood, Simon; Bowtell, Jo

    2008-08-01

    Glutamine enhances the exercise-induced expansion of the tricarboxylic acid intermediate pool. The aim of the present study was to determine whether oral glutamine, alone or in combination with hyperoxia, influenced oxidative metabolism and cycle time-trial performance. Eight participants consumed either placebo or 0.125 g kg body mass(-1) of glutamine in 5 ml kg body mass(-1) placebo 1 h before exercise in normoxic (control and glutamine respectively) or hyperoxic (FiO(2) = 50%; hyperoxia and hyperoxia + glutamine respectively) conditions. Participants then cycled for 6 min at 70% maximal oxygen uptake (VO(2max)) immediately before completing a brief high-intensity time-trial (approximately 4 min) during which a pre-determined volume of work was completed as fast as possible. The increment in pulmonary oxygen uptake during the performance test (DeltaVO(2max), P = 0.02) and exercise performance (control: 243 s, s(x) = 7; glutamine: 242 s, s(x) = 3; hyperoxia: 231 s, s(x) = 3; hyperoxia + glutamine: 228 s, s(x) = 5; P < 0.01) were significantly improved in hyperoxic conditions. There was some evidence that glutamine ingestion increased DeltaVO(2max) in normoxia, but not hyperoxia (interaction drink/FiO(2), P = 0.04), but there was no main effect or impact on performance. Overall, the data show no effect of glutamine ingestion either alone or in combination with hyperoxia, and thus no limiting effect of the tricarboxylic acid intermediate pool size, on oxidative metabolism and performance during maximal exercise. PMID:18608833

  13. No effect of glutamine supplementation and hyperoxia on oxidative metabolism and performance during high-intensity exercise.

    PubMed

    Marwood, Simon; Bowtell, Jo

    2008-08-01

    Glutamine enhances the exercise-induced expansion of the tricarboxylic acid intermediate pool. The aim of the present study was to determine whether oral glutamine, alone or in combination with hyperoxia, influenced oxidative metabolism and cycle time-trial performance. Eight participants consumed either placebo or 0.125 g kg body mass(-1) of glutamine in 5 ml kg body mass(-1) placebo 1 h before exercise in normoxic (control and glutamine respectively) or hyperoxic (FiO(2) = 50%; hyperoxia and hyperoxia + glutamine respectively) conditions. Participants then cycled for 6 min at 70% maximal oxygen uptake (VO(2max)) immediately before completing a brief high-intensity time-trial (approximately 4 min) during which a pre-determined volume of work was completed as fast as possible. The increment in pulmonary oxygen uptake during the performance test (DeltaVO(2max), P = 0.02) and exercise performance (control: 243 s, s(x) = 7; glutamine: 242 s, s(x) = 3; hyperoxia: 231 s, s(x) = 3; hyperoxia + glutamine: 228 s, s(x) = 5; P < 0.01) were significantly improved in hyperoxic conditions. There was some evidence that glutamine ingestion increased DeltaVO(2max) in normoxia, but not hyperoxia (interaction drink/FiO(2), P = 0.04), but there was no main effect or impact on performance. Overall, the data show no effect of glutamine ingestion either alone or in combination with hyperoxia, and thus no limiting effect of the tricarboxylic acid intermediate pool size, on oxidative metabolism and performance during maximal exercise.

  14. Cerebral glutamine metabolism under hyperammonemia determined in vivo by localized 1H and 15N NMR spectroscopy

    PubMed Central

    Cudalbu, Cristina; Lanz, Bernard; Duarte, João MN; Morgenthaler, Florence D; Pilloud, Yves; Mlynárik, Vladimir; Gruetter, Rolf

    2012-01-01

    Brain glutamine synthetase (GS) is an integral part of the glutamate–glutamine cycle and occurs in the glial compartment. In vivo Magnetic Resonance Spectroscopy (MRS) allows noninvasive measurements of the concentrations and synthesis rates of metabolites. 15N MRS is an alternative approach to 13C MRS. Incorporation of labeled 15N from ammonia in cerebral glutamine allows to measure several metabolic reactions related to nitrogen metabolism, including the glutamate–glutamine cycle. To measure 15N incorporation into the position 5N of glutamine and position 2N of glutamate and glutamine, we developed a novel 15N pulse sequence to simultaneously detect, for the first time, [5-15N]Gln and [2-15N]Gln+Glu in vivo in the rat brain. In addition, we also measured for the first time in the same experiment localized 1H spectra for a direct measurement of the net glutamine accumulation. Mathematical modeling of 1H and 15N MRS data allowed to reduce the number of assumptions and provided reliable determination of GS (0.30±0.050 μmol/g per minute), apparent neurotransmission (0.26±0.030 μmol/g per minute), glutamate dehydrogenase (0.029±0.002 μmol/g per minute), and net glutamine accumulation (0.033±0.001 μmol/g per minute). These results showed an increase of GS and net glutamine accumulation under hyperammonemia, supporting the concept of their implication in cerebral ammonia detoxification. PMID:22167234

  15. Dependence on glutamine uptake and glutamine addiction characterize myeloma cells: a new attractive target.

    PubMed

    Bolzoni, Marina; Chiu, Martina; Accardi, Fabrizio; Vescovini, Rosanna; Airoldi, Irma; Storti, Paola; Todoerti, Katia; Agnelli, Luca; Missale, Gabriele; Andreoli, Roberta; Bianchi, Massimiliano G; Allegri, Manfredi; Barilli, Amelia; Nicolini, Francesco; Cavalli, Albertina; Costa, Federica; Marchica, Valentina; Toscani, Denise; Mancini, Cristina; Martella, Eugenia; Dall'Asta, Valeria; Donofrio, Gaetano; Aversa, Franco; Bussolati, Ovidio; Giuliani, Nicola

    2016-08-01

    The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138(+) cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138(+) cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138(+) cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM. PMID:27268090

  16. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    PubMed Central

    Torreira, Eva; Seabra, Ana Rita; Marriott, Hazel; Zhou, Min; Llorca, Óscar; Robinson, Carol V.; Carvalho, Helena G.; Fernández-Tornero, Carlos; Pereira, Pedro José Barbosa

    2014-01-01

    The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants. PMID:24699643

  17. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture.

    PubMed

    Torreira, Eva; Seabra, Ana Rita; Marriott, Hazel; Zhou, Min; Llorca, Óscar; Robinson, Carol V; Carvalho, Helena G; Fernández-Tornero, Carlos; Pereira, Pedro José Barbosa

    2014-04-01

    The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants. PMID:24699643

  18. Plasma Glutamine Concentrations in Liver Failure

    PubMed Central

    Helling, Gunnel; Wahlin, Staffan; Smedberg, Marie; Pettersson, Linn; Tjäder, Inga; Norberg, Åke; Rooyackers, Olav; Wernerman, Jan

    2016-01-01

    Background Higher than normal plasma glutamine concentration at admission to an intensive care unit is associated with an unfavorable outcome. Very high plasma glutamine levels are sometimes seen in both acute and chronic liver failure. We aimed to systematically explore the relation between different types of liver failure and plasma glutamine concentrations. Methods Four different groups of patients were studies; chronic liver failure (n = 40), acute on chronic liver failure (n = 20), acute fulminant liver failure (n = 20), and post-hepatectomy liver failure (n = 20). Child-Pugh and Model for End-stage Liver Disease (MELD) scores were assessed as indices of liver function. All groups except the chronic liver failure group were followed longitudinally during hospitalisation. Outcomes were recorded up to 48 months after study inclusion. Results All groups had individuals with very high plasma glutamine concentrations. In the total group of patients (n = 100), severity of liver failure correlated significantly with plasma glutamine concentration, but the correlation was not strong. Conclusion Liver failure, regardless of severity and course of illness, may be associated with a high plasma glutamine concentration. Further studies are needed to understand whether high glutamine levels should be regarded as a biomarker or as a contributor to symptomatology in liver failure. PMID:26938452

  19. Cross-Species Analysis of Protein Dynamics Associated with Hydride and Proton Transfer in the Catalytic Cycle of the Light-Driven Enzyme Protochlorophyllide Oxidoreductase.

    PubMed

    Hoeven, Robin; Hardman, Samantha J O; Heyes, Derren J; Scrutton, Nigel S

    2016-02-16

    Experimental interrogation of the relationship between protein dynamics and enzyme catalysis is challenging. Light-activated protochlorophyllide oxidoreductase (POR) is an excellent model for investigating this relationship because photoinitiation of the reaction cycle enables coordinated turnover in a "dark-assembled" ternary enzyme-substrate complex. The catalytic cycle involves sequential hydride and proton transfers (from NADPH and an active site tyrosine residue, respectively) to the substrate protochlorophyllide. Studies with a limited cross-species subset of POR enzymes (n = 4) have suggested that protein dynamics associated with hydride and proton transfer are distinct [Heyes, D. J., Levy, C., Sakuma, M., Robertson, D. L., and Scrutton, N. S. (2011) J. Biol. Chem. 286, 11849-11854]. Here, we use steady-state assays and single-turnover laser flash spectroscopy to analyze hydride and proton transfer dynamics in an extended series of POR enzymes taken from many species, including cyanobacteria, algae, embryophytes, and angiosperms. Hydride/proton transfer in all eukaryotic PORs is faster compared to prokaryotic PORs, suggesting active site architecture has been optimized in eukaryotic PORs following endosymbiosis. Visible pump-probe spectroscopy was also used to demonstrate a common photoexcitation mechanism for representative POR enzymes from different branches of the phylogenetic tree. Dynamics associated with hydride transfer are localized to the active site of all POR enzymes and are conserved. However, dynamics associated with proton transfer are variable. Protein dynamics associated with proton transfer are also coupled to solvent dynamics in cyanobacterial PORs, and these networks are likely required to optimize (shorten) the donor-acceptor distance for proton transfer. These extended networks are absent in algal and plant PORs. Our analysis suggests that extended networks of dynamics are disfavored, possibly through natural selection. Implications for

  20. Cross-Species Analysis of Protein Dynamics Associated with Hydride and Proton Transfer in the Catalytic Cycle of the Light-Driven Enzyme Protochlorophyllide Oxidoreductase.

    PubMed

    Hoeven, Robin; Hardman, Samantha J O; Heyes, Derren J; Scrutton, Nigel S

    2016-02-16

    Experimental interrogation of the relationship between protein dynamics and enzyme catalysis is challenging. Light-activated protochlorophyllide oxidoreductase (POR) is an excellent model for investigating this relationship because photoinitiation of the reaction cycle enables coordinated turnover in a "dark-assembled" ternary enzyme-substrate complex. The catalytic cycle involves sequential hydride and proton transfers (from NADPH and an active site tyrosine residue, respectively) to the substrate protochlorophyllide. Studies with a limited cross-species subset of POR enzymes (n = 4) have suggested that protein dynamics associated with hydride and proton transfer are distinct [Heyes, D. J., Levy, C., Sakuma, M., Robertson, D. L., and Scrutton, N. S. (2011) J. Biol. Chem. 286, 11849-11854]. Here, we use steady-state assays and single-turnover laser flash spectroscopy to analyze hydride and proton transfer dynamics in an extended series of POR enzymes taken from many species, including cyanobacteria, algae, embryophytes, and angiosperms. Hydride/proton transfer in all eukaryotic PORs is faster compared to prokaryotic PORs, suggesting active site architecture has been optimized in eukaryotic PORs following endosymbiosis. Visible pump-probe spectroscopy was also used to demonstrate a common photoexcitation mechanism for representative POR enzymes from different branches of the phylogenetic tree. Dynamics associated with hydride transfer are localized to the active site of all POR enzymes and are conserved. However, dynamics associated with proton transfer are variable. Protein dynamics associated with proton transfer are also coupled to solvent dynamics in cyanobacterial PORs, and these networks are likely required to optimize (shorten) the donor-acceptor distance for proton transfer. These extended networks are absent in algal and plant PORs. Our analysis suggests that extended networks of dynamics are disfavored, possibly through natural selection. Implications for

  1. [Inhibition of glutamine synthetase activity by biologically active derivatives of glutamic acid].

    PubMed

    Firsova, N A; Selivanova, K M; Alekseeva, L V; Evstigneeva, Z G

    1986-05-01

    The inhibition of activity of glutamine synthetase from Chlorella and porcine brain by 4-hydroxy-D-4-fluoro-D,L- and 4-amino-D,L-glutamic acids diastereoisomers was studied. Each compound was shown to exert the same inhibiting effect on glutamine synthetase from both sources. In case of threo-4-hydroxy-D-glutamic acid the inhibition of the Chlorella enzyme was of a competitive and of a completely mixed type. The enzyme inhibition by 4-fluoro-D, L-glutamic acids seemed to be of a completely non-competitive type. The Ki values for all inhibition reactions were determined. A comparison of biochemical parameters and biological activity revealed that the most effective inhibitors of the enzyme exert a most potent antitumour and antiviral action.

  2. Therapeutic strategies impacting cancer cell glutamine metabolism

    PubMed Central

    Lukey, Michael J; Wilson, Kristin F; Cerione, Richard A

    2014-01-01

    The metabolic adaptations that support oncogenic growth can also render cancer cells dependent on certain nutrients. Along with the Warburg effect, increased utilization of glutamine is one of the metabolic hallmarks of the transformed state. Glutamine catabolism is positively regulated by multiple oncogenic signals, including those transmitted by the Rho family of GTPases and by c-Myc. The recent identification of mechanistically distinct inhibitors of glutaminase, which can selectively block cellular transformation, has revived interest in the possibility of targeting glutamine metabolism in cancer therapy. Here, we outline the regulation and roles of glutamine metabolism within cancer cells and discuss possible strategies for, and the consequences of, impacting these processes therapeutically. PMID:24047273

  3. Glutamine facilitates chemotherapy while reducing toxicity.

    PubMed

    Klimberg, V S; Nwokedi, E; Hutchins, L F; Pappas, A A; Lang, N P; Broadwater, J R; Read, R C; Westbrook, K C

    1992-01-01

    Dose intensification of chemotherapy is thought to increase survival. With recent advances in hemopoietic cell modulators such as granulocyte colony stimulating factor, the limiting toxicity of intensifying chemotherapeutic regimens has become the severity of the associated enterocolitis. In animal models, glutamine protects the host from methotrexate-induced enterocolitis. This study evaluates the effects of a glutamine-supplemented diet on the tumoricidal effectiveness of methotrexate. Sarcoma-bearing Fisher 344 rats (n = 30) were pair-fed an isocaloric elemental diet containing 1% glutamine or an isonitrogenous amount of glycine beginning on day 25 of the study. Rats from each group received two intraperitoneal injections of methotrexate (5 mg/kg) or saline on days 26 and 33 of the study. On day 40, rats were killed, tumor volume and weight were recorded, and tumor glutaminase activity and tumor morphometrics were measured. Blood was taken for arterial glutamine content, complete blood count, and blood culture. The gut was processed for glutaminase activity and synthesis phase of the deoxyribonucleic acid. In rats receiving methotrexate, the tumor volume loss was nearly doubled when glutamine was added to the diet. Significant differences in tumor glutaminase activity and morphometrics were not detected. The toxicity to the host was ameliorated. Significantly increased synthesis phase of deoxyribonucleic acid of the whole jejunum, decreased bacteremia, "sepsis," and mortality were demonstrated. Glutamine supplementation enhances the tumoricidal effectiveness of methotrexate while reducing its morbidity and mortality in this sarcoma rat model.

  4. TM-15GLUTAMINE BASED PET IMAGING FACILITATES ENHANCED METABOLIC DETECTION OF GLIOMAS IN VIVO

    PubMed Central

    Venneti, Sriram; Dunphy, Mark; Zhang, Hanwen; Pitter, Kenneth; Campos, Carl; Carlin, Sean; Lyashchenko, Serge; Plöessl, Carl; Rohle, Daniel; Omuro, Antonio; Cross, Justin; Brennan, Cameron; Weber, Wolfgang; Holland, Eric; Mellinghoff, Ingo; Kung, Hank; Lewis, Jason; Thompson, Craig

    2014-01-01

    Glutamine is the most abundant plasma amino acid and many cancers show altered glutamine metabolism. We evaluated glutamine uptake and metabolism in gliomas using PET imaging and biochemical approaches. We demonstrate that glutamine is a key TCA cycle anaplerotic substrate and is metabolized to generate 2-HG in IDH1-mutant gliomas. PET imaging with18F-labeled glutamine (18F-FGln) showed high uptake in gliomas in vivo but low background uptake in the surrounding brain in RCAS-PDGF/PTEN null and IDH1-mutant glioma animal models, facilitating clear tumor delineation in contrast to that seen with 18F-FDG. We did not observe 18F-FGln uptake in animals with neuroinflammation or animals with a disrupted BBB. Further, 18F-FGln uptake was specifically reduced on chemo/radiation therapy. Finally, 18F-FGln showed high avidity in human glioma with low uptake in the surrounding brain. These data suggest that 18F-FGln is specifically taken up by gliomas, can be used to assess the metabolic state of gliomas in vivo and may serve as a valuable tool in the clinical management of gliomas.

  5. Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle.

    PubMed

    Grabarczyk, Daniel B; Chappell, Paul E; Johnson, Steven; Stelzl, Lukas S; Lea, Susan M; Berks, Ben C

    2015-12-29

    The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a "suicide enzyme" strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein-protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway.

  6. Glutamine prevents oxidative stress in a model of mesenteric ischemia and reperfusion

    PubMed Central

    Zabot, Gilmara Pandolfo; Carvalhal, Gustavo Franco; Marroni, Norma Possa; Hartmann, Renata Minuzzo; da Silva, Vinícius Duval; Fillmann, Henrique Sarubbi

    2014-01-01

    AIM: To evaluate preventative effects of glutamine in an animal model of gut ischemia/reperfusion (I/R). METHODS: Male Wistar rats were housed in a controlled environment and allowed access to food and water ad libitum. Twenty male Wistar rats were divided into four experimental groups: (1) control group (control) - rats underwent exploratory laparotomy; (2) control + glutamine group (control-GLU) - rats were subjected to laparotomy and treated intraperitoneally with glutamine 24 and 48 h prior to surgery; (3) I/R group - rats were subjected to occlusion of the superior mesenteric artery for 30 min followed by 15 min of reperfusion; and (4) ischemia/reperfusion + glutamine group (G + I/R) - rats were treated intraperitoneally with glutamine 24 and 48 h before I/R. Local and systemic injuries were determined by evaluating intestinal and lung segments for oxidative stress using lipid peroxidation and the activity of superoxide dismutase (SOD), interleukin-6 (IL-6) and nuclear factor kappa beta (NF-κB) after mesenteric I/R. RESULTS: Lipid peroxidation of the membrane was increased in the animals subjected to I/R (P < 0.05). However, the group that received glutamine 24 and 48 h before the I/R procedure showed levels of lipid peroxidation similar to the control groups (P < 0.05). The activity of the antioxidant enzyme SOD was decreased in the gut of animals subjected to I/R when compared with the control group of animals not subjected to I/R (P < 0.05). However, the group that received glutamine 24 and 48 h before I/R showed similar SOD activity to both control groups not subjected to I/R (P < 0.05). The mean area of NF-κB staining for each of the control groups was similar. The I/R group showed the largest area of staining for NF-κB. The G + I/R group had the second highest amount of staining, but the mean value was much lower than that of the I/R group (P < 0.05). For IL-6, control and control-GLU groups showed similar areas of staining. The I/R group contained

  7. Plant nutritional status modulates glutamine synthetase levels in ripe tomatoes (Solanum lycopersicum cv. Micro-Tom).

    PubMed

    Scarpeci, Telma E; Marro, Martin L; Bortolotti, Santiago; Boggio, Silvana B; Valle, Estela M

    2007-02-01

    Tomato (Solanum lycopersicum) fruit ripening implies that chloroplastic proteins are degraded and new proteins are synthesized. Supplementary nutrition is frequently required when tomato plants begin to fruit and continues until the end of the plant's life cycle. Ammonium assimilation is crucial in these fruit maturation and ripening processes. Glutamine synthetase (GS; EC 6.3.1.2), the main ammonium-fixing enzyme in plants, could not be detected in red fruits of several tomato varieties when growing under standard nutrition. In this paper, we analyze the influence of the nutritional status on the ammonium assimilation capacity of ripe tomato (cv. Micro-Tom) fruit. For this purpose, GS expression and protein profiles were followed in mature green and red fruits harvested from plants grown under standard or supplemented nutrition. Under standard nutrient regime (weekly supplied with 0.5 x Hoagland solution) GS activity was found in chloroplasts (GS2) of mature green fruits, but it was not detected either in the chromoplasts or in the cytosol of red fruits. When plants were shifted to a supplemented nutritional regime (daily supplied with 0.5 x Hoagland solution), GS was found in red fruits. Also, cytosolic transcripts (gs1) preferentially accumulated in red fruits under high nutrition. These results indicate that mature green Micro-Tom fruits assimilate ammonia through GS2 under standard nutrition, while ripe red fruits accumulate GS1 under high nutrition, probably in order to assimilate the extra N-compounds made available through supplemented nutrition.

  8. Glutamine supplementation maintains intramuscular glutamine concentrations and normalizes lymphocyte function in infected early weaned pigs.

    PubMed

    Yoo, S S; Field, C J; McBurney, M I

    1997-11-01

    Numerous studies in humans and rats have shown that glutamine supplementation during stressful conditions has favorable outcomes. However, the requirements for glutamine during weaning are unknown. Thus, the effects of glutamine supplementation in healthy and infected weaned pigs were investigated. At 21 d of age, pigs were weaned to an elemental diet supplemented with glutamine (+Gln) or an isonitrogenous diet containing nonessential amino acids (-Gln). At 26 d of age, pigs were intraperitoneally injected with Escherichia coli (+Ecoli) or buffered saline (-Ecoli) and killed at 28 d of age. Infection decreased (P < 0.05) plasma and intramuscular glutamine concentrations, but infected pigs that received +Gln diets had higher intramuscular glutamine levels than those that received -Gln diets. Infected pigs had elevated (P < 0.05) total leukocyte counts, and blood lymphocyte responses ([3H]-thymidine incorporation) to a mixture of phorbol myristate acetate and ionomycin were reduced. White blood cell counts were greater (P < 0.05) in +Gln than -Gln pigs. The peak responses to concanavalin A (Con A) by lymphocytes of +Ecoli+Gln pigs were greater (P < 0.05) than those of +Ecoli-Gln pigs and not different than those of noninfected pigs. Hence, glutamine supplementation maintained muscular glutamine concentrations and normalized lymphocyte function in infected pigs. PMID:9349855

  9. Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle

    PubMed Central

    Grabarczyk, Daniel B.; Chappell, Paul E.; Johnson, Steven; Stelzl, Lukas S.; Berks, Ben C.

    2015-01-01

    The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a “suicide enzyme” strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein–protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway. PMID:26655737

  10. Expression, purification, and characterization of recombinant human glutamine synthetase.

    PubMed Central

    Listrom, C D; Morizono, H; Rajagopal, B S; McCann, M T; Tuchman, M; Allewell, N M

    1997-01-01

    A bacterial expression system has been engineered for human glutamine synthetase (EC 6.3.1.2) that produces approximately 60 mg of enzyme (20% of the bacterial soluble protein) and yields approx. 8 mg of purified enzyme per litre of culture. The recombinant enzyme was purified 5-fold to apparent homogeneity and characterized. It has a subunit molecular mass of approx. 45000 Da. The Vmax value obtained using a radioactive assay with ammonia and l-[G-3H]glutamic acid as substrates was 15.9 micromol/min per mg, 40% higher than that obtained in the colorimetric assay (9.9 micromol/min per mg) with hydroxylamine replacing ammonia as a substrate. Km values for glutamate were 3.0 mM and 3.5 mM, and for ATP they were 2.0 mM and 2. 9 mM for the radioactive and spectrophotometric assays respectively. The Km for ammonia in the radioactive assay was 0.15 mM. The midpoint of thermal inactivation was 49.7 degrees C. Hydroxylamine, Mg(II) and Mg(II)-ATP stabilized the enzyme against thermal inactivation, whereas ATP promoted inactivation. The pure enzyme is stable for several months in storage and provides a source for additional studies, including X-ray crystallography. PMID:9359847

  11. Glutamine synthetase desensitizes differentiated adipocytes to proinflammatory stimuli by raising intracellular glutamine levels.

    PubMed

    Palmieri, Erika Mariana; Spera, Iolanda; Menga, Alessio; Infantino, Vittoria; Iacobazzi, Vito; Castegna, Alessandra

    2014-12-20

    The role of glutamine synthetase (GS) during adipocyte differentiation is unclear. Here, we assess the impact of GS on the adipocytic response to a proinflammatory challenge at different differentiation stages. GS expression at the late stages of differentiation desensitized mature adipocytes to bacterial lipopolysaccharide (LPS) by increasing intracellular glutamine levels. Furthermore, LPS-activated mature adipocytes were unable to produce inflammatory mediators; LPS sensitivity was rescued following GS inhibition and the associated drop in intracellular glutamine levels. The ability of adipocytes to differentially respond to LPS during differentiation negatively correlates to GS expression and intracellular glutamine levels. Hence, modulation of intracellular glutamine levels by GS expression represents an endogenous mechanism through which mature adipocytes control the inflammatory response.

  12. Oxidative modification of glutamine synthetase. I. Inactivation is due to loss of one histidine residue.

    PubMed

    Levine, R L

    1983-10-10

    Intracellular proteolytic degradation of glutamine synthetase occurs in two distinct steps in Escherichia coli (Levine, R. L., Oliver, C. N., Fulks, R. M., and Stadtman, E. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2120-2124). In the first step, a mixed function oxidation modifies the glutamine synthetase. The modified enzyme, which is catalytically inactive, becomes susceptible to proteolytic attack. In the second step, a protease specific for the modified enzyme catalyzes the actual proteolytic degradation. The oxidatively modified glutamine synthetase was studied to determine the chemical differences between it and the native enzyme. Only a single alteration was found; one of sixteen histidine residues/subunit was altered by the oxidative modification. The modification introduced a carbonyl group into the protein, permitting isolation of a stable dinitrophenylhydrazone. No other differences were detected between the native and modified proteins. Specifically, the cysteine, methionine, phenylalanine, tyrosine, and tryptophan contents were not altered. A number of other prokaryotic and eukaryotic enzymes are also susceptible to oxidative modification. This covalent modification may be important in intracellular proteolysis, in mammalian host defense systems, in prevention of autolysis, in aging processes, and in oxygen toxicity.

  13. The effect of glial glutamine synthetase inhibition on recognition and temporal memories in the rat.

    PubMed

    Kant, Deepika; Tripathi, Shweta; Qureshi, Munazah F; Tripathi, Shweta; Pandey, Swati; Singh, Gunjan; Kumar, Tankesh; Mir, Fayaz A; Jha, Sushil K

    2014-02-01

    The glutamate neurotransmitter is intrinsically involved in learning and memory. Glial glutamine synthetase enzyme synthesizes glutamine, which helps maintain the optimal neuronal glutamate level. However, the role of glutamine synthetase in learning and memory remains unclear. Using associative trace learning task, we investigated the effects of methionine sulfoximine (MSO) (glutamine synthetase inhibitor) on recognition and temporal memories. MSO and vehicle were injected (i.p.) three hours before training in separate groups of male Wistar rats (n=11). Animals were trained to obtain fruit juice after following a set of sequential events. Initially, house-light was presented for 15s followed by 5s trace interval. Thereafter, juice was given for 20s followed by 20s inter-presentation interval. A total of 75 presentations were made over five sessions during the training and testing periods. The average number of head entries to obtain juice per session and during individual phases at different time intervals was accounted as an outcome measure of recognition and temporal memories. The total head entries in MSO and vehicle treated animals were comparable on training and testing days. However, it was 174.90% (p=0.08), 270.61% (p<0.05), 143.20% (p<0.05) more on training day and 270.33% (p<0.05), 157.94% (p<0.05), 170.42% (p<0.05) more on testing day, during the house-light, trace-interval and inter-presentation interval phases in MSO animals. Glutamine synthetase inhibition did not induce recognition memory deficit, while temporal memory was altered, suggesting that glutamine synthetase modulates some aspects of mnemonic processes.

  14. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    SciTech Connect

    Torreira, Eva; Seabra, Ana Rita; Marriott, Hazel; Zhou, Min; Llorca, Óscar; Robinson, Carol V.; Carvalho, Helena G.; Fernández-Tornero, Carlos; Pereira, Pedro José Barbosa

    2014-04-01

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

  15. Glutamine supplementation in bone marrow transplantation.

    PubMed

    Ziegler, Thomas R

    2002-01-01

    An increasing number of clinical investigations have focused on supplementation of specialized enteral and parenteral nutrition with the amino acid glutamine. This interest derives from strong evidence in animal models and emerging clinical data on the efficacy of glutamine administration following chemotherapy, trauma, sepsis and other catabolic conditions. Glutamine has protein-anabolic effects in stressed patients and, among many key metabolic functions, is used as a major fuel/substrate by cells of the gastrointestinal epithelium and the immune system. These effects may be particularly advantageous in patients undergoing bone marrow transplantation (BMT), who exhibit post-transplant body protein wasting, gut mucosal injury and immunodeficiency. Studies to date indicate that enteral and parenteral glutamine supplementation is well tolerated and potentially efficacious after high-dose chemotherapy or BMT for cancer treatment. Although not all studies demonstrate benefits, sufficient positive data have been published to suggest that this nutrient should be considered as adjunctive metabolic support of some individuals undergoing marrow transplant. However, BMT is a rapidly evolving clinical procedure with regard to the conditioning and supportive protocols utilized. Thus, additional randomized, double-blind, controlled clinical trials are indicated to define the efficacy of glutamine with current BMT regimens.

  16. Neuromuscular Dysfunction in Experimental Sepsis and Glutamine

    PubMed Central

    Çankayalı, İlkin; Boyacılar, Özden; Demirağ, Kubilay; Uyar, Mehmet; Moral, Ali Reşat

    2016-01-01

    Background: Electrophysiological studies show that critical illness polyneuromyopathy appears in the early stage of sepsis before the manifestation of clinical findings. The metabolic response observed during sepsis causes glutamine to become a relative essential amino acid. Aims: We aimed to assess the changes in neuromuscular transmission in the early stage of sepsis after glutamine supplementation. Study Design: Animal experimentation. Methods: Twenty male Sprague-Dawley rats were randomized into two groups. Rats in both groups were given normal feeding for one week. In the study group, 1 g/kg/day glutamine was added to normal feeding by feeding tube for one week. Cecal ligation and perforation (CLP) surgery was performed at the end of one week. Before and 24 hours after CLP, compound muscle action potentials were recorded from the gastrocnemius muscle. Results: Latency measurements before and 24 hours after CLP were 0.68±0.05 ms and 0.80±0.09 ms in the control group and 0.69±0.07 ms and 0.73±0.07 ms in the study group (p<0.05). Conclusion: Since enteral glutamine prevented compound muscle action potentials (CMAP) latency prolongation in the early phase of sepsis, it was concluded that enteral glutamine replacement might be promising in the prevention of neuromuscular dysfunction in sepsis; however, further studies are required. PMID:27308070

  17. Glutamine: An Obligatory Parenteral Nutrition Substrate in Critical Care Therapy

    PubMed Central

    Stehle, Peter; Kuhn, Katharina S.

    2015-01-01

    Critical illness is characterized by glutamine depletion owing to increased metabolic demand. Glutamine is essential to maintain intestinal integrity and function, sustain immunologic response, and maintain antioxidative balance. Insufficient endogenous availability of glutamine may impair outcome in critically ill patients. Consequently, glutamine has been considered to be a conditionally essential amino acid and a necessary component to complete any parenteral nutrition regimen. Recently, this scientifically sound recommendation has been questioned, primarily based on controversial findings from a large multicentre study published in 2013 that evoked considerable uncertainty among clinicians. The present review was conceived to clarify the most important questions surrounding glutamine supplementation in critical care. This was achieved by addressing the role of glutamine in the pathophysiology of critical illness, summarizing recent clinical studies in patients receiving parenteral nutrition with intravenous glutamine, and describing practical concepts for providing parenteral glutamine in critical care. PMID:26495301

  18. Glutamine supplementation in the critically ill: friend or foe?

    PubMed

    Oudemans-van Straaten, Heleen M; van Zanten, Arthur R H

    2014-05-19

    In the previous issue of Critical Care, Mori and colleagues demonstrate that glutamine supplementation in mechanically ventilated patients as part of parenteral nutrition increases plasma glutamine concentration and glutamine utilization, but does not mitigate protein degradation and even increases de novo glutamine production. Studies suggest that protein degradation is regulated by the degree of inflammation. Immune cells utilize large amounts of glutamine and derive their glutamine requirements from muscle protein degradation. We hypothesize that the effects of glutamine supplementation depend on the degree of inflammation. Infusing large amounts of exogenous glutamine into patients with inflammatory conditions like sepsis and multiple organ failure may not only enhance immune competence, but may potentially augment the inflammatory response and thereby negatively influence outcome.

  19. Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin.

    PubMed

    Pascual, María Belén; Jing, Zhong Ping; Kirby, Edward G; Cánovas, Francisco M; Gallardo, Fernando

    2008-01-01

    Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar (Populus tremula x P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Cantón, F.R., García-Gutiérrez, A., Cánovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19-26; Fu, J., Sampalo, R., Gallardo, F., Cánovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411-418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Cánovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137-145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729-736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31-39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha(-1), growth

  20. The relationship between leaf rolling and ascorbate-glutathione cycle enzymes in apoplastic and symplastic areas of Ctenanthe setosa subjected to drought stress.

    PubMed

    Saruhan, Neslihan; Terzi, Rabiye; Saglam, Aykut; Kadioglu, Asim

    2009-01-01

    The ascorbate-glutathione (ASC-GSH) cycle has an important role in defensive processes against oxidative damage generated by drought stress. In this study, the changes that take place in apoplastic and symplastic ASC-GSH cycle enzymes of the leaf and petiole were investigated under drought stress causing leaf rolling in Ctenanthe setosa (Rose.) Eichler (Marantaceae). Apoplastic and symplastic extractions of leaf and petiole were performed at different visual leaf rolling scores from 1 to 4 (1 is unrolled, 4 is tightly rolled and the others are intermediate forms). Glutathione reductase (GR), a key enzyme in the GSH regeneration cycle, and ascorbate (ASC) were present in apoplastic spaces of the leaf and petiole, whereas dehydroascorbate reductase (DHAR), which uses glutathione as reductant, monodehydroascorbate reductase (MDHAR), which uses NAD(P)H as reductant, and glutathione were absent. GR, DHAR and MDHAR activities increased in the symplastic and apoplastic areas of the leaf. Apoplastic and symplastic ASC and dehydroascorbate (DHA), the oxidized form of ascorbate, rose at all scores except score 4 of symplastic ASC in the leaf. On the other hand, while reduced glutathione (GSH) content was enhanced, oxidized glutathione (GSSG) content decreased in the leaf during rolling. As for the petiole, GR activity increased in the apoplastic area but decreased in the symplastic area. DHAR and MDHAR activities increased throughout all scores, but decreased to the score 1 level at score 4. The ASC content of the apoplast increased during leaf rolling. Conversely, symplastic ASC content increased at score 2, however decreased at the later scores. While the apoplastic DHA content declined, symplastic DHA rose at score 2, but later was down to the level of score 1. While GSH content enhanced during leaf rolling, GSSG content did not change except at score 2. As well, there were good correlations between leaf rolling and ASC-GSH cycle enzyme activities in the leaf (GR and DHAR

  1. The relationship between leaf rolling and ascorbate-glutathione cycle enzymes in apoplastic and symplastic areas of Ctenanthe setosa subjected to drought stress.

    PubMed

    Saruhan, Neslihan; Terzi, Rabiye; Saglam, Aykut; Kadioglu, Asim

    2009-01-01

    The ascorbate-glutathione (ASC-GSH) cycle has an important role in defensive processes against oxidative damage generated by drought stress. In this study, the changes that take place in apoplastic and symplastic ASC-GSH cycle enzymes of the leaf and petiole were investigated under drought stress causing leaf rolling in Ctenanthe setosa (Rose.) Eichler (Marantaceae). Apoplastic and symplastic extractions of leaf and petiole were performed at different visual leaf rolling scores from 1 to 4 (1 is unrolled, 4 is tightly rolled and the others are intermediate forms). Glutathione reductase (GR), a key enzyme in the GSH regeneration cycle, and ascorbate (ASC) were present in apoplastic spaces of the leaf and petiole, whereas dehydroascorbate reductase (DHAR), which uses glutathione as reductant, monodehydroascorbate reductase (MDHAR), which uses NAD(P)H as reductant, and glutathione were absent. GR, DHAR and MDHAR activities increased in the symplastic and apoplastic areas of the leaf. Apoplastic and symplastic ASC and dehydroascorbate (DHA), the oxidized form of ascorbate, rose at all scores except score 4 of symplastic ASC in the leaf. On the other hand, while reduced glutathione (GSH) content was enhanced, oxidized glutathione (GSSG) content decreased in the leaf during rolling. As for the petiole, GR activity increased in the apoplastic area but decreased in the symplastic area. DHAR and MDHAR activities increased throughout all scores, but decreased to the score 1 level at score 4. The ASC content of the apoplast increased during leaf rolling. Conversely, symplastic ASC content increased at score 2, however decreased at the later scores. While the apoplastic DHA content declined, symplastic DHA rose at score 2, but later was down to the level of score 1. While GSH content enhanced during leaf rolling, GSSG content did not change except at score 2. As well, there were good correlations between leaf rolling and ASC-GSH cycle enzyme activities in the leaf (GR and DHAR

  2. The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis.

    PubMed

    Hauf, Ksenia; Kayumov, Airat; Gloge, Felix; Forchhammer, Karl

    2016-02-12

    TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated L-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA. PMID:26635369

  3. The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis.

    PubMed

    Hauf, Ksenia; Kayumov, Airat; Gloge, Felix; Forchhammer, Karl

    2016-02-12

    TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated L-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA.

  4. Contribution of active-site glutamine to rate enhancement in ubiquitin carboxy terminal hydrolases

    PubMed Central

    Boudreaux, David; Chaney, Joseph; Maiti, Tushar K.; Das, Chittaranjan

    2012-01-01

    Ubiquitin carboxy terminal hydrolases (UCHs) are cysteine proteases featuring a classical cysteine-histidine-aspartate catalytic triad, also a highly conserved glutamine thought to be a part of the oxyanion hole. However, the contribution of this side chain to the catalysis by UCH enzymes is not known. Herein, we demonstrate that the glutamine side chain contributes to rate enhancement in UCHL1, UCHL3 and UCHL5. Mutation of the glutamine to alanine in these enzymes impairs the catalytic efficiency mainly due to a 16 to 30-fold reduction in kcat, which is consistent with a loss of approximately 2 kcal/mol in transition-state stabilization. However, the contribution to transition-state stabilization observed here is rather modest for the side chain’s role in oxyanion stabilization. Interestingly, we discovered that the carbonyl oxygen of this side chain is engaged in a C—H•••O hydrogen-bonding contact with the CεH group of the catalytic histidine. Upon further analysis, we found that this interaction is a common active-site structural feature in most cysteine proteases, including papain, belonging to families with the QCH(N/D) type of active-site configuration. It is possible that removal of the glutamine side chain might have abolished the C—H•••O interaction, which typically accounts for 2 kcal/mol of stabilization, leading to the effect on catalysis observed here. Additional studies performed on UCHL3 by mutating the glutamine to glutamate (strong C—H•••O acceptor but oxyanion destabilizer) and to lysine (strong oxyanion stabilizer but lacking C—H•••O hydrogen-bonding property) suggest that the C—H•••O hydrogen bond could contribute to catalysis. PMID:22284438

  5. Malonyl-Coenzyme A Reductase from Chloroflexus aurantiacus, a Key Enzyme of the 3-Hydroxypropionate Cycle for Autotrophic CO2 Fixation

    PubMed Central

    Hügler, Michael; Menendez, Castor; Schägger, Hermann; Fuchs, Georg

    2002-01-01

    The 3-hydroxypropionate cycle is a new autotrophic CO2 fixation pathway in Chloroflexus aurantiacus and some archaebacteria. The initial step is acetyl-coenzyme A (CoA) carboxylation to malonyl-CoA by acetyl-CoA carboxylase, followed by NADPH-dependent reduction of malonyl-CoA to 3-hydroxypropionate. This reduction step was studied in Chloroflexus aurantiacus. A new enzyme was purified, malonyl-CoA reductase, which catalyzed the two-step reduction malonyl-CoA + NADPH + H+ → malonate semialdehyde + NADP+ + CoA and malonate semialdehyde + NADPH + H+ → 3-hydroxypropionate + NADP+. The bifunctional enzyme (aldehyde dehydrogenase and alcohol dehydrogenase) had a native molecular mass of 300 kDa and consisted of a single large subunit of 145 kDa, suggesting an α2 composition. The N-terminal amino acid sequence was determined, and the incomplete gene was identified in the genome database. Obviously, the enzyme consists of an N-terminal short-chain alcohol dehydrogenase domain and a C-terminal aldehyde dehydrogenase domain. No indication of the presence of a prosthetic group was obtained; Mg2+ and Fe2+ stimulated and EDTA inhibited activity. The enzyme was highly specific for its substrates, with apparent Km values of 30 μM malonyl-CoA and 25 μM NADPH and a turnover number of 25 s−1 subunit−1. The specific activity in autotrophically grown cells was 0.08 μmol of malonyl-CoA reduced min−1 (mg of protein)−1, compared to 0.03 μmol min−1 (mg of protein)−1 in heterotrophically grown cells, indicating downregulation under heterotrophic conditions. Malonyl-CoA reductase is not required in any other known pathway and therefore can be taken as a characteristic enzyme of the 3-hydroxypropionate cycle. Furthermore, the enzyme may be useful for production of 3-hydroxypropionate and for a coupled spectrophotometric assay for activity screening of acetyl-CoA carboxylase, a target enzyme of potent herbicides. PMID:11948153

  6. Glutamine: the nonessential amino acid for performance enhancement.

    PubMed

    Phillips, George C

    2007-07-01

    Glutamine is a popular dietary supplement consumed for purported ergogenic benefits of increased strength, quicker recovery, decreased frequency of respiratory infections, and prevention of overtraining. From a biochemical standpoint, glutamine does play a physiologic role in each of these areas, but it remains only one of a host of factors involved. This review examines the effects of glutamine on exercise and demonstrates a lack of evidence for definitive positive ergogenic benefits as a result of glutamine supplementation. PMID:17618004

  7. A Tracer Bolus Method for Investigating Glutamine Kinetics in Humans

    PubMed Central

    Mori, Maiko; Smedberg, Marie; Klaude, Maria; Tjäder, Inga; Norberg, Åke; Rooyackers, Olav; Wernerman, Jan

    2014-01-01

    Glutamine transport between tissues is important for the outcome of critically ill patients. Investigation of glutamine kinetics is, therefore, necessary to understand glutamine metabolism in these patients in order to improve future intervention studies. Endogenous glutamine production can be measured by continuous infusion of a glutamine tracer, which necessitates a minimum measurement time period. In order to reduce this problem, we used and validated a tracer bolus injection method. Furthermore, this method was used to measure the glutamine production in healthy volunteers in the post-absorptive state, with extra alanine and with glutamine supplementation and parenteral nutrition. Healthy volunteers received a bolus injection of [1-13C] glutamine, and blood was collected from the radial artery to measure tracer enrichment over 90 minutes. Endogenous rate of appearance (endoRa) of glutamine was calculated from the enrichment decay curve and corrected for the extra glutamine supplementation. The glutamine endoRa of healthy volunteers was 6.1±0.9 µmol/kg/min in the post-absorptive state, 6.9±1.0 µmol/kg/min with extra alanyl-glutamine (p = 0.29 versus control), 6.1±0.4 µmol/kg/min with extra alanine only (p = 0.32 versus control), and 7.5±0.9 µmol/kg/min with extra alanyl-glutamine and parenteral nutrition (p = 0.049 versus control). In conclusion, a tracer bolus injection method to measure glutamine endoRa showed good reproducibility and small variation at baseline as well as during parenteral nutrition. Additionally, we showed that parenteral nutrition including alanyl-glutamine increased glutamine endoRa in healthy volunteers, which was not attributable to the alanine part of the dipeptide. PMID:24810895

  8. Intestinal and hepatic metabolism of glutamine and citrulline in humans.

    PubMed

    van de Poll, Marcel C G; Ligthart-Melis, Gerdien C; Boelens, Petra G; Deutz, Nicolaas E P; van Leeuwen, Paul A M; Dejong, Cornelis H C

    2007-06-01

    Glutamine plays an important role in nitrogen homeostasis and intestinal substrate supply. It has been suggested that glutamine is a precursor for arginine through an intestinal-renal pathway involving inter-organ transport of citrulline. The importance of intestinal glutamine metabolism for endogenous arginine synthesis in humans, however, has remained unaddressed. The aim of this study was to investigate the intestinal conversion of glutamine to citrulline and the effect of the liver on splanchnic citrulline metabolism in humans. Eight patients undergoing upper gastrointestinal surgery received a primed continuous intravenous infusion of [2-(15)N]glutamine and [ureido-(13)C-(2)H(2)]citrulline. Arterial, portal venous and hepatic venous blood were sampled and portal and hepatic blood flows were measured. Organ specific amino acid uptake (disposal), production and net balance, as well as whole body rates of plasma appearance were calculated according to established methods. The intestines consumed glutamine at a rate that was dependent on glutamine supply. Approximately 13% of glutamine taken up by the intestines was converted to citrulline. Quantitatively glutamine was the only important precursor for intestinal citrulline release. Both glutamine and citrulline were consumed and produced by the liver, but net hepatic flux of both amino acids was not significantly different from zero. Plasma glutamine was the precursor of 80% of plasma citrulline and plasma citrulline in turn was the precursor of 10% of plasma arginine. In conclusion, glutamine is an important precursor for the synthesis of arginine after intestinal conversion to citrulline in humans.

  9. Glutamine: Precursor or nitrogen donor for citrulline synthesis?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although glutamine is considered the main precursor for citrulline synthesis, the current literature does not differentiate between the contribution of glutamine carbon skeleton, versus nonspecific nitrogen (i.e., ammonia) and carbon derived from glutamine oxidation. To elucidate the role of glutami...

  10. Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.

    PubMed

    Willemoës, Martin; Sigurskjold, Bent W

    2002-10-01

    CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself. PMID:12354108

  11. Low-frequency vibrational modes of glutamine

    NASA Astrophysics Data System (ADS)

    Wang, Wei-Ning; Wang, Guo; Zhang, Yan

    2011-12-01

    High-resolution terahertz absorption and Raman spectra of glutamine in the frequency region 0.2 THz-2.8 THz are obtained by using THz time domain spectroscopy and low-frequency Raman spectroscopy. Based on the experimental and the computational results, the vibration modes corresponding to the terahertz absorption and Raman scatting peaks are assigned and further verified by the theoretical calculations. Spectral investigation of the periodic structure of glutamine based on the sophisticated hybrid density functional B3LYP indicates that the vibrational modes come mainly from the inter-molecular hydrogen bond in this frequency region.

  12. Differential inactivation of alfalfa nodule glutamine synthetases by tabtoxinine-. beta. -lactam. [Pseudomonas syringae

    SciTech Connect

    Knight, T.J.; Unkefer, P.J.

    1987-04-01

    The presence of the pathogen Pseudomonas syringae pv. tabaci within the rhizosphere of nodulated alfalfa plants results in an increase in N/sub 2/-fixation potential and growth, but a 40-50% decrease in nodule glutamine synthetase (GS) activity, as compared to nodulated control plants. Tabtoxinine-..beta..-Lactam an exocellular toxin produced by Pseudomonas syringae pv tabaci irreversibly inhibits glutamine synthetase. Partial purification of nodule GS by DEAE-cellulose chromatography reveals two enzyme forms are present (GS/sub n1/ and GS/sub n2/). In vitro inactivation of the two glutamine synthetases associated with the nodule indicates a differential sensitivity to T-..beta..-L. The nodule specific GS/sub n1/ is much less sensitive to T-..beta..-L than the GS/sub n2/ enzyme, which was found to coelute with the root enzyme (GS/sub r/). However, both GS/sub n1/ and GS/sub n2/ are rapidly inactivated by methionine sulfoximine, another irreversible inhibitor of GS.

  13. Bisphosphonic acids as effective inhibitors of Mycobacterium tuberculosis glutamine synthetase.

    PubMed

    Kosikowska, Paulina; Bochno, Marta; Macegoniuk, Katarzyna; Forlani, Giuseppe; Kafarski, Paweł; Berlicki, Łukasz

    2016-12-01

    Inhibition of glutamine synthetase (GS) is one of the most promising strategies for the discovery of novel drugs against tuberculosis. Forty-three bisphosphonic and bis-H-phosphinic acids of various scaffolds, bearing aromatic substituents, were screened against recombinant GS from Mycobacterium tuberculosis. Most of the studied compounds exhibited activities in micromolar range, with N-(3,5-dichlorophenyl)-2-aminoethylidenebisphoshonic acid, N-(3,5-difluorophenyl)-2-aminoethylidene-bisphoshonic acid and N-(3,4-dichlorophenyl)-1-hydroxy-1,1-ethanebisphosphonic acid showing the highest potency with kinetic parameters similar to the reference compound - L-methionine-S-sulfoximine. Moreover, these inhibitors were found to be much more effective against pathogen enzyme than against the human ortholog. Thus, with the bone-targeting properties of the bisphosphonate compounds in mind, this activity/selectivity profile makes these compounds attractive agents for the treatment of bone tuberculosis.

  14. Short-Term Responses of Soil Respiration and C-Cycle Enzyme Activities to Additions of Biochar and Urea in a Calcareous Soil.

    PubMed

    Song, Dali; Xi, Xiangyin; Huang, Shaomin; Liang, Guoqing; Sun, Jingwen; Zhou, Wei; Wang, Xiubin

    2016-01-01

    Biochar (BC) addition to soil is a proposed strategy to enhance soil fertility and crop productivity. However, there is limited knowledge regarding responses of soil respiration and C-cycle enzyme activities to BC and nitrogen (N) additions in a calcareous soil. A 56-day incubation experiment was conducted to investigate the combined effects of BC addition rates (0, 0.5, 1.0, 2.5 and 5.0% by mass) and urea (U) application on soil nutrients, soil respiration and C-cycle enzyme activities in a calcareous soil in the North China Plain. Our results showed soil pH values in both U-only and U plus BC treatments significantly decreased within the first 14 days and then stabilized, and CO2emission rate in all U plus BC soils decreased exponentially, while there was no significant difference in the contents of soil total organic carbon (TOC), dissolved organic carbon (DOC), total nitrogen (TN), and C/N ratio in each treatment over time. At each incubation time, soil pH, electrical conductivity (EC), TOC, TN, C/N ratio, DOC and cumulative CO2 emission significantly increased with increasing BC addition rate, while soil potential activities of the four hydrolytic enzymes increased first and then decreased with increasing BC addition rate, with the largest values in the U + 1.0%BC treatment. However, phenol oxidase activity in all U plus BC soils showed a decreasing trend with the increase of BC addition rate. Our results suggest that U plus BC application at a rate of 1% promotes increases in hydrolytic enzymes, does not highly increase C/N and C mineralization, and can improve in soil fertility. PMID:27589265

  15. Short-Term Responses of Soil Respiration and C-Cycle Enzyme Activities to Additions of Biochar and Urea in a Calcareous Soil

    PubMed Central

    Song, Dali; Xi, Xiangyin; Huang, Shaomin; Liang, Guoqing; Sun, Jingwen; Zhou, Wei; Wang, Xiubin

    2016-01-01

    Biochar (BC) addition to soil is a proposed strategy to enhance soil fertility and crop productivity. However, there is limited knowledge regarding responses of soil respiration and C-cycle enzyme activities to BC and nitrogen (N) additions in a calcareous soil. A 56-day incubation experiment was conducted to investigate the combined effects of BC addition rates (0, 0.5, 1.0, 2.5 and 5.0% by mass) and urea (U) application on soil nutrients, soil respiration and C-cycle enzyme activities in a calcareous soil in the North China Plain. Our results showed soil pH values in both U-only and U plus BC treatments significantly decreased within the first 14 days and then stabilized, and CO2emission rate in all U plus BC soils decreased exponentially, while there was no significant difference in the contents of soil total organic carbon (TOC), dissolved organic carbon (DOC), total nitrogen (TN), and C/N ratio in each treatment over time. At each incubation time, soil pH, electrical conductivity (EC), TOC, TN, C/N ratio, DOC and cumulative CO2 emission significantly increased with increasing BC addition rate, while soil potential activities of the four hydrolytic enzymes increased first and then decreased with increasing BC addition rate, with the largest values in the U + 1.0%BC treatment. However, phenol oxidase activity in all U plus BC soils showed a decreasing trend with the increase of BC addition rate. Our results suggest that U plus BC application at a rate of 1% promotes increases in hydrolytic enzymes, does not highly increase C/N and C mineralization, and can improve in soil fertility. PMID:27589265

  16. Short-Term Responses of Soil Respiration and C-Cycle Enzyme Activities to Additions of Biochar and Urea in a Calcareous Soil.

    PubMed

    Song, Dali; Xi, Xiangyin; Huang, Shaomin; Liang, Guoqing; Sun, Jingwen; Zhou, Wei; Wang, Xiubin

    2016-01-01

    Biochar (BC) addition to soil is a proposed strategy to enhance soil fertility and crop productivity. However, there is limited knowledge regarding responses of soil respiration and C-cycle enzyme activities to BC and nitrogen (N) additions in a calcareous soil. A 56-day incubation experiment was conducted to investigate the combined effects of BC addition rates (0, 0.5, 1.0, 2.5 and 5.0% by mass) and urea (U) application on soil nutrients, soil respiration and C-cycle enzyme activities in a calcareous soil in the North China Plain. Our results showed soil pH values in both U-only and U plus BC treatments significantly decreased within the first 14 days and then stabilized, and CO2emission rate in all U plus BC soils decreased exponentially, while there was no significant difference in the contents of soil total organic carbon (TOC), dissolved organic carbon (DOC), total nitrogen (TN), and C/N ratio in each treatment over time. At each incubation time, soil pH, electrical conductivity (EC), TOC, TN, C/N ratio, DOC and cumulative CO2 emission significantly increased with increasing BC addition rate, while soil potential activities of the four hydrolytic enzymes increased first and then decreased with increasing BC addition rate, with the largest values in the U + 1.0%BC treatment. However, phenol oxidase activity in all U plus BC soils showed a decreasing trend with the increase of BC addition rate. Our results suggest that U plus BC application at a rate of 1% promotes increases in hydrolytic enzymes, does not highly increase C/N and C mineralization, and can improve in soil fertility.

  17. Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations.

    PubMed

    Moreira, Cátia; Ramos, Maria J; Fernandes, Pedro Alexandrino

    2016-06-27

    This paper is devoted to the understanding of the reaction mechanism of mycobacterium tuberculosis glutamine synthetase (mtGS) with atomic detail, using computational quantum mechanics/molecular mechanics (QM/MM) methods at the ONIOM M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory. The complete reaction undergoes a three-step mechanism: the spontaneous transfer of phosphate from ATP to glutamate upon ammonium binding (ammonium quickly loses a proton to Asp54), the attack of ammonia on phosphorylated glutamate (yielding protonated glutamine), and the deprotonation of glutamine by the leaving phosphate. This exothermic reaction has an activation free energy of 21.5 kcal mol(-1) , which is consistent with that described for Escherichia coli glutamine synthetase (15-17 kcal mol(-1) ). The participating active site residues have been identified and their role and energy contributions clarified. This study provides an insightful atomic description of the biosynthetic reaction that takes place in this enzyme, opening doors for more accurate studies for developing new anti-tuberculosis therapies.

  18. Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations.

    PubMed

    Moreira, Cátia; Ramos, Maria J; Fernandes, Pedro Alexandrino

    2016-06-27

    This paper is devoted to the understanding of the reaction mechanism of mycobacterium tuberculosis glutamine synthetase (mtGS) with atomic detail, using computational quantum mechanics/molecular mechanics (QM/MM) methods at the ONIOM M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory. The complete reaction undergoes a three-step mechanism: the spontaneous transfer of phosphate from ATP to glutamate upon ammonium binding (ammonium quickly loses a proton to Asp54), the attack of ammonia on phosphorylated glutamate (yielding protonated glutamine), and the deprotonation of glutamine by the leaving phosphate. This exothermic reaction has an activation free energy of 21.5 kcal mol(-1) , which is consistent with that described for Escherichia coli glutamine synthetase (15-17 kcal mol(-1) ). The participating active site residues have been identified and their role and energy contributions clarified. This study provides an insightful atomic description of the biosynthetic reaction that takes place in this enzyme, opening doors for more accurate studies for developing new anti-tuberculosis therapies. PMID:27225077

  19. Chronic social isolation decreases glutamate and glutamine levels and induces oxidative stress in the rat hippocampus.

    PubMed

    Shao, Yuan; Yan, Gen; Xuan, Yinghua; Peng, Hui; Huang, Qing-Jun; Wu, Renhua; Xu, Haiyun

    2015-04-01

    Social isolation (SI) rearing of rodents is a developmental manipulation, which is commonly compared with the psychological stressors in humans as it produces several behavioral outcomes similar to those observed in humans with early life stress. To explain the SI-induced behavioral outcomes, animal studies have been performed to examine the dopaminergic and glutamatergic systems in the brain. In this study, we measured possible changes in levels of glutamate and glutamine of SI-rats using proton magnetic resonance spectroscopy. We also assessed the oxidative stress parameters in certain brain regions to see if glutamate and/or glutamine changes, if any, are associated with oxidative stress. SI rearing for 8 weeks decreased the activities of antioxidant enzymes catalase, glutathione peroxidase, superoxide dismutase, and the total antioxidant capacity, but increased levels of hydrogen peroxide, in certain brain regions, of which prefrontal cortex and hippocampus were most vulnerable. It also decreased levels of glutamate, glutamine, N-acetyl-l-aspartate (NAA), and phosphocreatine in the dorsal hippocampus, but not in the cerebral cortex. Decreased phosphocreatine and NAA indicate energy metabolism deficit in brain cells; the latter also suggests the neuronal viability was inhibited. Decreased glutamate and glutamine may suggest the neuron-glial integrity was implicated by chronic SI. These neurochemical and biochemical changes may contribute to the SI-induced behavioral abnormalities including a high level of anxiety, social interaction deficit, and impaired spatial working memory shown in this study.

  20. Determining the N and O isotope effects of microbial nitrite reduction: the global N cycle implications of an enzyme-dependent isotope effect

    NASA Astrophysics Data System (ADS)

    Martin, T. S.; Casciotti, K. L.

    2014-12-01

    The marine nitrogen (N) cycle is a dynamic system of critical importance, since nitrogen is the limiting nutrient in over half of the world's oceans. Denitrification and anammox, the main N loss processes from the ocean, have different effects on carbon cycling and greenhouse gas emission. Understanding the balance between the two processes is vital to understanding the role of the N cycle in global climate change. One approach for investigating these processes is by using stable isotope analysis to estimate the relative magnitudes of N fluxes, particularly for biologically mediated processes. In order to make the most of the currently available isotope analysis techniques, it is necessary to know the isotope effects for each processes occurring in the environment. Nitrite reduction is an important step in denitrification. Previous work had begun to explore the N isotope effects for nitrite reduction, but no oxygen (O) isotope effect has been measured. Additionally, no consideration has been given to the type of nitrite reductase carrying out the reaction. There are two main types of respiratory nitrite reductase, one that is Cu-based and another that is Fe-based. We performed batch culture experiments with denitrifier strains possessing either a Cu-type or Fe-type nitrite reductase. Both N and O isotope effects for nitrite reduction were determined for each of these experiments by measuring the NO2- concentration, as well as the N and O isotopes of nitrite and applying a Rayleigh fractionation model. Both the N and O isotope effects were found to be significantly different between the two types of enzymes. This enzyme-linked difference in isotope effects emphasizes the importance of microbial community composition within the global N cycle.

  1. Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

    PubMed

    Howard, Thomas P; Lloyd, Julie C; Raines, Christine A

    2011-07-01

    In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.

  2. The activity of Krebs cycle enzymes in the visual analyzer of rats in the norm and under stress.

    PubMed

    Lutsenko, N S; Yakushev, V S

    1993-01-01

    Higher activity of the NAD-dependent dehydrogenases of the tricarboxylic acid cycle (TAC) is observed in the optic retina, and of FAD-dependent dehydrogenases in the occipital lobes of the brain, in the visual analyzer of intact rats. The influence of stress using Desiderato's method induces a compensatory increase in the activity of succinate dehydrogenase. Acute stress induces a change in the regulation of the activity of the TAC dehydrogenases, assessed on the basis of the reaction to functional load. The animals' remaining in the dark following stress promotes the restoration of the activity of the TAC cycle to the normal level. PMID:8413911

  3. Phylogenetically-based variation in the regulation of the Calvin cycle enzymes, phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase, in algae.

    PubMed

    Maberly, Stephen C; Courcelle, Carine; Groben, Rene; Gontero, Brigitte

    2010-03-01

    Aquatic photosynthesis is responsible for about half of the global production and is undertaken by a huge phylogenetic diversity of algae that are poorly studied. The diversity of redox-regulation of phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was investigated in a wide range of algal groups under standard conditions. Redox-regulation of PRK was greatest in chlorophytes, low or absent in a red alga and most chromalveolates, and linked to the number of amino acids between two regulatory cysteine residues. GAPDH regulation was not strongly-related to the different forms of this enzyme and was less variable than for PRK. Addition of recombinant CP12, a protein that forms a complex with PRK and GAPDH, to crude extracts inhibited GAPDH and PRK inversely in the Plantae, but in most chromalveolates had little effect on GAPDH and inhibited or stimulated PRK depending on the species. Patterns of enzyme regulation were used to produce a phylogenetic tree in which cryptophytes and haptophytes, at the base of the chromalveolates, formed a distinct clade. A second clade comprised only chromalveolates. A third clade comprised a mixture of Plantae, an excavate and three chromalveolates: a marine diatom and two others (a xanthophyte and eustigmatophyte) that are distinguished by a low content of chlorophyll c and a lack of fucoxanthin. Regulation of both enzymes was greater in freshwater than in marine taxa, possibly because most freshwaters are more dynamic than oceans. This work highlights the importance of understanding enzyme regulation in diverse algae if their ecology and productivity is to be understood.

  4. Utilization and synthesis of glutamine in the lens

    SciTech Connect

    Jernigan, H.M. Jr.; Geller, A.M.

    1987-05-01

    Previous studies with rat and calf lenses have shown that the primary source of lenticular glutamate for a wide variety of pathways is glutamine, which is more readily transported from the surrounding fluids than glutamate. In contrast, recent studies showed that monkey lenses utilize glutamate more rapidly than glutamine, although glutamine appears to enter monkey lenses more rapidly than glutamine. Further studies were initiated to elucidate the balance between transport, deamidation, and synthesis of glutamine in the lens. Glutamine synthesis in intact rat lenses was barely detectable; however, homogenates of adult rat lenses converted measurable amounts of (/sup 3/H)-glutamate to glutamine. The identity of the product was confirmed using glutaminase. Glutamine deamidation in intact rhesus monkey lenses incubated in TC-199 medium containing 2 mM glutamine was only 0.19 ..mu..mol g/sup -1/ hr/sup -1/, compared with 1.96 ..mu..mol g/sup -1/ hr/sup -1/ for rat lenses under identical conditions. However, under optimal assay conditions, glutaminase activity in monkey lens homogenates was similar to that of rat lens homogenates. Thus, the rate of synthesis and deamidation of glutamine by intact lenses is apparently limited factors other than total lenticular glutamine synthetase and glutaminase activity.

  5. No effect of glutamine ingestion on indices of oxidative metabolism in stable COPD.

    PubMed

    Marwood, Simon; Jack, Sandy; Patel, Mukhtar; Walker, Paul; Bowtell, Joanna; Calverley, Peter

    2011-06-30

    COPD patients have reduced muscle glutamate which may contribute to an impaired response of oxidative metabolism to exercise. We hypothesised that prior glutamine supplementation would enhance V(O2) peak, V(O2) at lactate threshold and speed pulmonary oxygen uptake kinetics in COPD. 13 patients (9 males, age 66±5 years, mean±SD) with severe COPD (mean FEV(1) 0.88±0.23l, 33±7% predicted) performed on separate days ramp cycle-ergometry (5-10 W min(-1)) to volitional exhaustion and subsequently square-wave transitions to 80% estimated lactate threshold (LT) following consumption of either placebo (CON) or 0.125 g kg bm(-1) of glutamine (GLN) in 5 ml kg bm(-1) placebo. Oral glutamine had no effect on peak or V(O2) at LT, {V(O2) peak: CON=0.70±0.1 l min(-1) vs. GLN=0.73±0.2 l min(-1); LT: CON=0.57±0.1 l min(-1) vs. GLN=0.54±0.1 lmin(-1)} or V(O2) kinetics {tau: CON=68±22 s vs. GLN=68±16 s}. Ingestion of glutamine before exercise did not improve indices of oxidative metabolism in this patient group. PMID:21419239

  6. Inhibition of human glutamine synthetase by L-methionine-S,R-sulfoximine-relevance to the treatment of neurological diseases.

    PubMed

    Jeitner, Thomas M; Cooper, Arthur J L

    2014-12-01

    At high concentrations, the glutamine synthetase inhibitor L-methionine-S,R-sulfoximine (MSO) is a convulsant, especially in dogs. Nevertheless, sub-convulsive doses of MSO are neuroprotective in rodent models of hyperammonemia, acute liver disease, and amyotrophic lateral sclerosis and suggest MSO may be clinically useful. Previous work has also shown that much lower doses of MSO are required to produce convulsions in dogs than in primates. Evidence from the mid-20th century suggests that humans are also less sensitive. In the present work, the inhibition of recombinant human glutamine synthetase by MSO is shown to be biphasic-an initial reversible competitive inhibition (K i 1.19 mM) is followed by rapid irreversible inactivation. This K i value for the human enzyme accounts, in part, for relative insensitivity of primates to MSO and suggests that this inhibitor could be used to safely inhibit glutamine synthetase activity in humans.

  7. Germinating Peanut (Arachis hypogaea L.) Seedlings Attenuated Selenite-Induced Toxicity by Activating the Antioxidant Enzymes and Mediating the Ascorbate-Glutathione Cycle.

    PubMed

    Wang, Guang; Zhang, Hong; Lai, Furao; Wu, Hui

    2016-02-17

    Selenite can enhance the selenium nutrition level of crops, but excessive selenite may be toxic to plant growth. To elucidate the mechanisms underlying the role of selenite in production and detoxification of oxidative toxicity, peanut seedlings were developed with sodium selenite (0, 3, and 6 mg/L). The effects of selenite on antioxidant capacity, transcript levels of antioxidant enzyme genes, and enzyme activities in hypocotyl were investigated. The CuZn-SOD, GSH-Px, GST, and APX gene expression levels and their enzyme activities in selenite treatments were 1.0-3.6-fold of the control. Selenite also significantly increased the glutathione and ascorbate concentrations by mediating the ascorbate-glutathione cycle, and the selenite-induced hydrogen peroxide may act as a second messenger in the signaling pathways. This work has revealed a complex antioxidative response to selenite in peanut seedling. Understanding these mechanisms may help future research in increasing selenite tolerance and selenium accumulation in peanut and other crops.

  8. Glutamine: precursor or nitrogen donor for citrulline synthesis?

    PubMed

    Marini, Juan C; Didelija, Inka Cajo; Castillo, Leticia; Lee, Brendan

    2010-07-01

    Although glutamine is considered the main precursor for citrulline synthesis, the current literature does not differentiate between the contribution of glutamine carbon skeleton vs. nonspecific nitrogen (i.e., ammonia) and carbon derived from glutamine oxidation. To elucidate the role of glutamine and nonspecific nitrogen in the synthesis of citrulline, l-[2-(15)N]- and l-[5-(15)N]glutamine and (15)N-ammonium acetate were infused intragastrically in mice. The amino group of glutamine labeled the three nitrogen groups of citrulline almost equally. The amido group and ammonium acetate labeled the ureido and amino groups of citrulline, but not the delta-nitrogen. D(5)-glutamine also infused in this arm of the study, which traces the carbon skeleton of glutamine, was utilized poorly, accounting for only 0.2-0.4% of the circulating citrulline. Dietary glutamine nitrogen (both N groups) incorporation was 25-fold higher than the incorporation of its carbon skeleton into citrulline. To investigate the relative contributions of the carbon skeleton and nonspecific carbon of glutamine, arginine, and proline to citrulline synthesis, U-(13)C(n) tracers of these amino acids were infused intragastrically. Dietary arginine was the main precursor for citrulline synthesis, accounting for approximately 40% of the circulating citrulline. Proline contribution was minor (3.4%), and glutamine was negligible (0.4%). However, the glutamine tracer resulted in a higher enrichment in the ureido group, indicating incorporation of nonspecific carbon from glutamine oxidation into carbamylphosphate used for citrulline synthesis. In conclusion, dietary glutamine is a poor carbon skeleton precursor for the synthesis of citrulline, although it contributes both nonspecific nitrogen and carbon to citrulline synthesis.

  9. In Situ Association of Calvin Cycle Enzymes, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase, Ferredoxin-NADP+ Reductase, and Nitrite Reductase with Thylakoid and Pyrenoid Membranes of Chlamydomonas reinhardtii Chloroplasts as Revealed by Immunoelectron Microscopy.

    PubMed Central

    Suss, K. H.; Prokhorenko, I.; Adler, K.

    1995-01-01

    The in situ localization of the chloroplast enzymes ribulose-1,5-bisphosphate carboxylase (Rubisco), Rubisco activase, ribose-5-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase was studied by immunoelectron microscopy in Chlamydomonas reinhardtii. Immunogold labeling revealed that, despite Rubisco in the pyrenoid matrix, Calvin cycle enzymes, Rubisco activase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase are associated predominantly with chloroplast thylakoid membranes and the inner surface of the pyrenoid membrane. This is in accord with previous enzyme localization studies in higher plants (K.H. Suss, C. Arkona, R. Manteuffel, K. Adler [1993] Proc Natl Acad Sci USA 90: 5514-5518). Pyrenoid tubules do not contain these enzymes. The pyrenoid matrix consists of Rubisco but is devoid of the other photosynthetic enzymes investigated. Evidence for the occurrence of two Rubisco forms differing in their spatial localization has also been obtained: Rubisco form I appears to be membrane associated like other Calvin cycle components, whereas Rubisco form II is confined to the pyrenoid matrix. It is proposed that enzyme form I represents an active Rubisco when assembled into Calvin cycle enzyme complexes, whereas Rubisco form II may be part of a CO2-concentrating mechanism. Pyrenoidal Calvin cycle complexes are thought to be highly active in CO2 fixation and important for the synthesis of starch around the pyrenoid. PMID:12228443

  10. In Situ Association of Calvin Cycle Enzymes, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase, Ferredoxin-NADP+ Reductase, and Nitrite Reductase with Thylakoid and Pyrenoid Membranes of Chlamydomonas reinhardtii Chloroplasts as Revealed by Immunoelectron Microscopy.

    PubMed

    Suss, K. H.; Prokhorenko, I.; Adler, K.

    1995-04-01

    The in situ localization of the chloroplast enzymes ribulose-1,5-bisphosphate carboxylase (Rubisco), Rubisco activase, ribose-5-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase was studied by immunoelectron microscopy in Chlamydomonas reinhardtii. Immunogold labeling revealed that, despite Rubisco in the pyrenoid matrix, Calvin cycle enzymes, Rubisco activase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase are associated predominantly with chloroplast thylakoid membranes and the inner surface of the pyrenoid membrane. This is in accord with previous enzyme localization studies in higher plants (K.H. Suss, C. Arkona, R. Manteuffel, K. Adler [1993] Proc Natl Acad Sci USA 90: 5514-5518). Pyrenoid tubules do not contain these enzymes. The pyrenoid matrix consists of Rubisco but is devoid of the other photosynthetic enzymes investigated. Evidence for the occurrence of two Rubisco forms differing in their spatial localization has also been obtained: Rubisco form I appears to be membrane associated like other Calvin cycle components, whereas Rubisco form II is confined to the pyrenoid matrix. It is proposed that enzyme form I represents an active Rubisco when assembled into Calvin cycle enzyme complexes, whereas Rubisco form II may be part of a CO2-concentrating mechanism. Pyrenoidal Calvin cycle complexes are thought to be highly active in CO2 fixation and important for the synthesis of starch around the pyrenoid.

  11. Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

    PubMed

    Fiebig, David; Schmelz, Stefan; Zindel, Stephan; Ehret, Vera; Beck, Jan; Ebenig, Aileen; Ehret, Marina; Fröls, Sabrina; Pfeifer, Felicitas; Kolmar, Harald; Fuchsbauer, Hans-Lothar; Scrima, Andrea

    2016-09-23

    Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency. PMID:27493205

  12. A Novel [15N] Glutamine Flux using LC-MS/MS-SRM for Determination of Nucleosides and Nucleobases

    PubMed Central

    Jin, Feng; Bhowmik, Salil Kumar; Putluri, Vasanta; Gu, Franklin; Gohlke, Jie; Von Rundstedt, Friedrich Carl; Dasgupta, Subhamoy; Krishnapuram, Rashmi; O’Malley, Bert W.; Sreekumar, Arun; Putluri, Nagireddy

    2016-01-01

    The growth of cancer cells relies more on increased proliferation and autonomy compared to non-malignant cells. The rate of de novo nucleotide biosynthesis correlates with cell proliferation rates. In part, glutamine is needed to sustain high rates of cellular proliferation as a key nitrogen donor in purine and pyrimidine nucleotide biosynthesis. In addition, glutamine serves as an essential substrate for key enzymes involved in the de novo synthesis of purine and pyrimidine nucleotides. Here, we developed a novel liquid chromatography (LC-MS) to quantify glutamine-derived [15N] nitrogen flux into nucleosides and nucleobases (purines and pyrimidines). For this, DNA from 5637 bladder cancer cell line cultured in 15N labelled glutamine and then enzymatically hydrolyzed by sequential digestion. Subsequently, DNA hydrolysates were separated by LC-MS and Selected Reaction Monitoring (SRM) was employed to identify the nucleobases and nucleosides. Thus, high sensitivity and reproducibility of the method make it a valuable tool to identify the nitrogen flux primarily derived from glutamine and can be further adaptable for high throughput analysis of large set of DNA in a clinical setting. PMID:27158554

  13. Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

    PubMed

    Fiebig, David; Schmelz, Stefan; Zindel, Stephan; Ehret, Vera; Beck, Jan; Ebenig, Aileen; Ehret, Marina; Fröls, Sabrina; Pfeifer, Felicitas; Kolmar, Harald; Fuchsbauer, Hans-Lothar; Scrima, Andrea

    2016-09-23

    Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

  14. The 3' untranslated region of the two cytosolic glutamine synthetase (GS(1)) genes in alfalfa (Medicago sativa) regulates transcript stability in response to glutamine.

    PubMed

    Simon, Bindu; Sengupta-Gopalan, Champa

    2010-10-01

    Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia with glutamate to produce glutamine. The GS enzyme is located either in the chloroplast (GS(2)) or in the cytoplasm (GS(1)). GS(1) is encoded by a small gene family and the members exhibit differential expression pattern mostly attributed to transcriptional regulation. Based on our recent finding that a soybean GS(1) gene, Gmglnβ ( 1 ) is subject to its 3'UTR-mediated post-transcriptional regulation as a transgene in alfalfa (Medicago sativa) we have raised the question of whether the 3'UTR-mediated transcript destabilization is a more universal phenomenon. Gene constructs consisting of the CaMV35S promoter driving the reporter gene, GUS, followed by the 3'UTRs of the two alfalfa GS(1) genes, MsGSa and MsGSb, were introduced into alfalfa and tobacco. The analysis of these transformants suggests that while both the 3'UTRs promote transcript turnover, the MsGSb 3'UTR is more effective than the MsGSa 3'UTR. However, both the 3'UTRs along with Gmglnβ ( 1 ) 3'UTR respond to nitrate as a trigger in transcript turnover. More detailed analysis points to glutamine rather than nitrate as the mediator of transcript turnover. Our data suggests that the 3'UTR-mediated regulation of GS(1) genes at the level of transcript turnover is probably universal and is used for fine-tuning the expression in keeping with the availability of the substrates.

  15. Studies of protein-protein interaction using countercurrent distribution in aqueous two-phase systems. Partition behaviour of six Calvin-cycle enzymes from a crude spinach (Spinacia oleracea) chloroplast extract.

    PubMed Central

    Persson, L O; Johansson, G

    1989-01-01

    The partition behaviour of six enzymes of the Calvin cycle in extracts of chloroplasts from spinach (Spinacia oleracea) between two aqueous phases has been studied by countercurrent distribution. The enzymes showed distribution patterns which indicate heterogeneity and the presence of two or three fractions of most of the enzymes. When two of the enzymes, phosphoglycerate kinase and fructose-bisphosphate aldolase, were partitioned in both purified and partially purified form, they behaved like homogeneous substances. These results indicate that countercurrent distribution of crude extracts in aqueous two-phase systems is a useful method to study protein-protein interaction. PMID:2730589

  16. Do salt cravings in children with autistic disorders reveal low blood sodium depleting brain taurine and glutamine?

    PubMed

    Good, Peter

    2011-12-01

    Because boys are four times more likely than girls to develop autism, the role of male hormones (androgens) has received considerable scrutiny. Some researchers implicate arginine vasopressin, an androgen-dependent hormone from the pituitary gland that elicits male behavior. Elevated vasopressin is also the most common cause of low blood sodium (hyponatremia)--most serious in the brains of children. Hyponatremia causes astrocytes to swell, then release the amino acids taurine and glutamine and their water to compensate. Taurin--the brain osmolyte/inhibitory neurotransmitter that suppresses vasopressin--was the amino acid most wasted or depleted in urine of autistic children. Glutamine is a critical metabolic fuel in brain neurons, astrocytes, endothelial cells, and the intestines, especially during hypoglycemia. Because glutamine is not thought to cross the blood-brain barrier significantly, the implications of low blood glutamine in these children are not recognized. Yet children with high brain glutamine from urea cycle disorders are rarely diagnosed with autistic disorders. Other common events in autistic children that release vasopressin are gastrointestinal inflammation, hypoglycemia, and stress. Signs of hyponatremia in these children are salt cravings reported online and anecdotally, deep yellow urine revealing concentration, and relief of autistic behavior by fluid/salt diets. Several interventions offer promise: (a) taurine to suppress vasopressin and replenish astrocytes; (b) glutamine as fuel for intestines and brain; (c) arginine to spare glutamine, detoxify ammonia, and increase brain blood flow; and (d) oral rehydration salts to compensate dilutional hyponatremia. This hypothesis appears eminently testable: Does your child crave salt? Is his urine deep yellow?

  17. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Max, Stephen R.; Konagaya, Masaaki; Konagaya, Yoko; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa

    1986-01-01

    The regulation of glutamine synthetase by glucocorticoids in rat skeletal muscles was studied. Administration of dexamethasone strikingly enhanced glutamine synthetase activity in plantaris and soleus muscles. The dexamethasone-mediated induction of glutamine synthetase activity was blocked to a significant extent by orally administered RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves dramatically increased levels of glutamine synthetase mRNA. The induction of glutamine synthetase was selective in that glutaminase activity of soleus and plantaris muscles was not increased by dexamethasone. Furthermore, dexamethasone treatment resulted in only a small increase in glutamine synthetase activity in the heart. Accordingly, there was only a slight change in glutamine synthetase mRNA level in this tissue. Thus, glucocorticoids regulate glutamine synthetase gene expression in rat muscles at the transcriptional level via interaction with intracellular glutamine production by muscle and to mechanisms underlying glucocorticoid-induced muscle atrophy.

  18. Effect of dietary glutamine on growth performance, non-specific immunity, expression of cytokine genes, phosphorylation of target of rapamycin (TOR), and anti-oxidative system in spleen and head kidney of Jian carp (Cyprinus carpio var. Jian).

    PubMed

    Hu, Kai; Zhang, Jing-Xiu; Feng, Lin; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Jiang, Jun; Zhou, Xiao-Qiu

    2015-06-01

    This study was designed to investigate the effects of dietary glutamine on the growth performance, cytokines, target of rapamycin (TOR), and antioxidant-related parameters in the spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed the basal (control) and glutamine-supplemented (12.0 g glutamine kg(-1) diet) diets for 6 weeks. Results indicated that the dietary glutamine supplementation improved the growth performance, spleen protein content, serum complement 3 content, and lysozyme activity in fish. In the spleen, glutamine down-regulated the expression of the interleukin 1 and interleukin 10 genes, and increased the level of phosphorylation of TOR protein. In the head kidney, glutamine down-regulated the tumor necrosis factor α and interleukin 10 gene expressions, phosphorylated and total TOR protein levels, while up-regulated the transforming growth factor β2 gene expression. Furthermore, the protein carbonyl content was decreased in the spleen of fish fed glutamine-supplemented diet; conversely, the anti-hydroxyl radical capacity and glutathione content in the spleen were increased by glutamine. However, diet supplemented with glutamine did not affect the lipid peroxidation, anti-superoxide anion capacity, and antioxidant enzyme activities in the spleen. Moreover, all of these antioxidant parameters in the head kidney were not affected by glutamine. Results from the present experiment showed the importance of dietary supplementation of glutamine in benefaction of the growth performance and several components of the innate immune system, and the deferential role in cytokine gene expression, TOR kinase activity, and antioxidant status between the spleen and head kidney of juvenile Jian carp.

  19. Structure of Escherichia coli Ribose-5-Phosphate Isomerase: A Ubiquitous Enzyme of the Pentose Phosphate Pathway and the Calvin Cycle

    PubMed Central

    Zhang, Rong-guang; Andersson, C. Evalena; Savchenko, Alexei; Skarina, Tatiana; Evdokimova, Elena; Beasley, Steven; Arrowsmith, Cheryl H.; Edwards, Aled M.; Joachimiak, Andrzej; Mowbray, Sherry L.

    2009-01-01

    Summary Ribose-5-phosphate isomerase A (RpiA; EC 5.3.1.6) interconverts ribose-5-phosphate and ribulose-5-phosphate. This enzyme plays essential roles in carbohydrate anabolism and catabolism; it is ubiquitous and highly conserved. The structure of RpiA from Escherichia coli was solved by multiwavelength anomalous diffraction (MAD) phasing, and refined to 1.5 Å resolution (R factor 22.4%, Rfree 23.7%). RpiA exhibits an α/β/(α/β)/β/α fold, some portions of which are similar to proteins of the alcohol dehydrogenase family. The two subunits of the dimer in the asymmetric unit have different conformations, representing the opening/closing of a cleft. Active site residues were identified in the cleft using sequence conservation, as well as the structure of a complex with the inhibitor arabinose-5-phosphate at 1.25 Å resolution. A mechanism for acid-base catalysis is proposed. PMID:12517338

  20. Structure of escherichia coli ribose-5-phosphate isomerase : a ubiquitous enzyme of the pentose phosphate pathway and the Calvin cycle.

    SciTech Connect

    Zhang, R.; Andersson, C. E.; Savchenko, A.; Skarina, T.; Evdokimova, E.; Beasley, S.; Arrowsmith, C. H.; Edwards, A.; Joachimiak, A.; Mowbray, S. L.; Biosciences Division; Uppsala Univ.; Univ. Health Network; Univ. of Toronto; Swedish Univ. of Agricultural Sciences

    2003-01-01

    Ribose-5-phosphate isomerase A (RpiA; EC 5.3.1.6) interconverts ribose-5-phosphate and ribulose-5-phosphate. This enzyme plays essential roles in carbohydrate anabolism and catabolism; it is ubiquitous and highly conserved. The structure of RpiA from Escherichia coli was solved by multiwavelength anomalous diffraction (MAD) phasing, and refined to 1.5 Angstroms resolution (R factor 22.4%, R{sub free} 23.7%). RpiA exhibits an {alpha}/{beta}/({alpha}/{beta})/{beta}/{alpha} fold, some portions of which are similar to proteins of the alcohol dehydrogenase family. The two subunits of the dimer in the asymmetric unit have different conformations, representing the opening/closing of a cleft. Active site residues were identified in the cleft using sequence conservation, as well as the structure of a complex with the inhibitor arabinose-5-phosphate at 1.25 A resolution. A mechanism for acid-base catalysis is proposed.

  1. Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates.

    PubMed

    Kusevic, Denis; Kudithipudi, Srikanth; Jeltsch, Albert

    2016-03-18

    Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln(185) of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX3R motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the +1 and a preference for Arg at the +7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1. PMID:26797129

  2. Rotational Study of Natural Amino Acid Glutamine

    NASA Astrophysics Data System (ADS)

    Varela, Marcelino; Cabezas, Carlos; Alonso, José L.

    2014-06-01

    Recent improvements in laser ablation molecular beam Fourier transform microwave spectroscopy (LA-MB-FTMW) have allowed the investigation of glutamine (COOH-CH(NH2)-CH2-CH2-CONH2), a natural amino acid with a long polar side chain. One dominant structure has been detected in the rotational spectrum. The nuclear quadrupole hyperfine structure of two 14N nuclei has been totally resolved allowing the conclusive identification of the observed species.

  3. Carbon Nanotubes Influence the Enzyme Activity of Biogeochemical Cycles of Carbon, Nitrogen, Phosphorus and the Pathogenesis of Plants in Annual Agroecosystems

    NASA Astrophysics Data System (ADS)

    Vaishlya, O. B.; Osipov, N. N.; Guseva, N. V.

    2015-09-01

    We conducted pre-sowing seed treatment of spring wheat carbon nanotubes modified with thionyl chloride, ethylene diamine, azobenzole, and dodecylamine. CNTs did not disrupt the structure of the crop, but the activity of extracellular enzymes in the rhizosphere of plants in the flowering stage changed: laccase works more poorly in the variant of the CNTs with the amino groups exochitinase and phosphatase activity increased in the case of chlorinated CNTs, OH and COOH groups on the surface of the nanotubes twice accelerate work β-glucosidase. The changes observed in the biogeochemical cycles in the rhizosphere are a possible cause of the effect of nanotubes on the development of epidemic diseases of wheat.

  4. Effect of sub-chronic selenium toxicosis on lipid peroxidation, glutathione redox cycle and antioxidant enzymes in calves.

    PubMed

    Kaur, Rajdeep; Sharma, Sucheta; Rampal, Satyavan

    2003-08-01

    The present investigation reports the effect of sodium selenite-induced sub-chronic toxicity in crossbred cow calves on various antioxidant enzymes. Sodium selenite (0.25 mg/kg for 16 w) resulted in characteristic signs of sub-chronic selenosis, ie alopecia, cracking and enlargement of hooves, interdigital lesions, ring formation on the coronet region, and gangrene at tip of the tail. The sodium selenite resulted in significant rise of blood selenium levels and concurrent increase in erythrocytic glutathione peroxidase (GPx) activity. Blood selenium levels and GPx activity had a high positive correlation (r = 0.97). Blood glutathione levels were lowered from 211.1 +/- 13.4 to 95.56 +/- 11.8 microg/ml. Selenosis caused oxidative stress as evidenced by a 3-fold increase in lipid peroxidation: activities of glutathione-S-transferase, glutathione reductase, superoxide dismutase and catalase were significantly increased. These findings support the hypothesis that the pro-oxidant attributes of selenium play important roles in its toxicity. PMID:12882488

  5. Targeting Glutamine Induces Apoptosis: A Cancer Therapy Approach

    PubMed Central

    Chen, Lian; Cui, Hengmin

    2015-01-01

    Glutamine metabolism has been proved to be dysregulated in many cancer cells, and is essential for proliferation of most cancer cells, which makes glutamine an appealing target for cancer therapy. In order to be well used by cells, glutamine must be transported to cells by specific transporters and converted to glutamate by glutaminase. There are currently several drugs that target glutaminase under development or clinical trials. Also, glutamine metabolism restriction has been proved to be effective in inhibiting tumor growth both in vivo and vitro through inducing apoptosis, growth arrest and/or autophagy. Here, we review recent researches about glutamine metabolism in cancer, and cell death induced by targeting glutamine, and their potential roles in cancer therapy. PMID:26402672

  6. Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis.

    PubMed Central

    Martin, G; Haehnel, W; Böger, P

    1997-01-01

    In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH. GS inactivation was completed within 30 min. Both the inactivated GS and the active enzyme were isolated. No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide. Mass spectrometry revealed that the molecular masses of active and inactive GS are equal. While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract. This result indicates that the active site was affected. From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration. GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen. In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2. The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen. Accordingly, the inactivating fraction was inhibited by catalase and EDTA. This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli. We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation. PMID:9006027

  7. Involvement of the stringent response in degradation of glutamine phosphoribosylpyrophosphate amidotransferase in Bacillus subtilis.

    PubMed Central

    Ruppen, M E; Switzer, R L

    1983-01-01

    Glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of purine biosynthesis, has previously been shown to be rapidly inactivated and degraded in Bacillus subtilis cells at the end of growth. The loss of enzyme activity appears to involve the oxidation of an iron-sulfur cluster in the enzyme. The degradation of the inactive enzyme involves some elements of the stringent response because it is inhibited in relA and relC mutants. Intracellular pools of guanosine tetra- and pentaphosphate were measured by an improved extraction procedure in cells that had been manipulated in various ways to induce or inhibit amidotransferase degradation. The results are consistent with the hypothesis that one or both of these nucleotides stimulates the synthesis of a protein involved in degradation. An elevated level of these nucleotides was not required for the continued degradation of amidotransferase once it had begun. PMID:6408067

  8. The freshwater Amazonian stingray, Potamotrygon motoro, up-regulates glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15 per thousand) water.

    PubMed

    Ip, Y K; Loong, A M; Ching, B; Tham, G H Y; Wong, W P; Chew, S F

    2009-12-01

    This study aimed to examine whether the stenohaline freshwater stingray, Potamotrygon motoro, which lacks a functional ornithine-urea cycle, would up-regulate glutamine synthetase (GS) activity and protein abundance, and accumulate glutamine during a progressive transfer from freshwater to brackish (15 per thousand) water with daily feeding. Our results revealed that, similar to other freshwater teleosts, P. motoro performed hyperosmotic regulation, with very low urea concentrations in plasma and tissues, in freshwater. In 15 per thousand water, it was non-ureotelic and non-ureoosmotic, acting mainly as an osmoconformer with its plasma osmolality, [Na+] and [Cl-] comparable to those of the external medium. There were significant increases in the content of several free amino acids (FAAs), including glutamate, glutamine and glycine, in muscle and liver, but not in plasma, indicating that FAAs could contribute in part to cell volume regulation. Furthermore, exposure of P. motoro to 15 per thousand water led to up-regulation of GS activity and protein abundance in both liver and muscle. Thus, our results indicate for the first time that, despite the inability to synthesize urea and the lack of functional carbamoyl phosphate synthetase III (CPS III) which uses glutamine as a substrate, P. motoro retained the capacity to up-regulate the activity and protein expression of GS in response to salinity stress. Potamotrygon motoro was not nitrogen (N) limited when exposed to 15 per thousand water with feeding, and there were no significant changes in the amination and deamination activities of hepatic glutamate dehydrogenase. In contrast, P. motoro became N limited when exposed to 10 per thousand water with fasting and could not survive well in 15 per thousand water without food.

  9. Crystal Structure of Reduced and of Oxidized Peroxiredoxin IV Enzyme Reveals a Stable Oxidized Decamer and a Non-disulfide-bonded Intermediate in the Catalytic Cycle*

    PubMed Central

    Cao, Zhenbo; Tavender, Timothy J.; Roszak, Aleksander W.; Cogdell, Richard J.; Bulleid, Neil J.

    2011-01-01

    Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue. PMID:21994946

  10. Over-expressing the C3 photosynthesis cycle enzyme Sedoheptulose-1-7 Bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO2 fumigation (FACE)

    PubMed Central

    2011-01-01

    Background Biochemical models predict that photosynthesis in C3 plants is most frequently limited by the slower of two processes, the maximum capacity of the enzyme Rubisco to carboxylate RuBP (Vc,max), or the regeneration of RuBP via electron transport (J). At current atmospheric [CO2] levels Rubisco is not saturated; consequently, elevating [CO2] increases the velocity of carboxylation and inhibits the competing oxygenation reaction which is also catalyzed by Rubisco. In the future, leaf photosynthesis (A) should be increasingly limited by RuBP regeneration, as [CO2] is predicted to exceed 550 ppm by 2050. The C3 cycle enzyme sedoheptulose-1,7 bisphosphatase (SBPase, EC 3.1.3.17) has been shown to exert strong metabolic control over RuBP regeneration at light saturation. Results We tested the hypothesis that tobacco transformed to overexpressing SBPase will exhibit greater stimulation of A than wild type (WT) tobacco when grown under field conditions at elevated [CO2] (585 ppm) under fully open air fumigation. Growth under elevated [CO2] stimulated instantaneous A and the diurnal photosynthetic integral (A') more in transformants than WT. There was evidence of photosynthetic acclimation to elevated [CO2] via downregulation of Vc,max in both WT and transformants. Nevertheless, greater carbon assimilation and electron transport rates (J and Jmax) for transformants led to greater yield increases than WT at elevated [CO2] compared to ambient grown plants. Conclusion These results provide proof of concept that increasing content and activity of a single photosynthesis enzyme can enhance carbon assimilation and yield of C3 crops grown at [CO2] expected by the middle of the 21st century. PMID:21884586

  11. Fluorescence biosensing strategy based on mercury ion-mediated DNA conformational switch and nicking enzyme-assisted cycling amplification for highly sensitive detection of carbamate pesticide.

    PubMed

    Wang, Xiuzhong; Hou, Ting; Dong, Shanshan; Liu, Xiaojuan; Li, Feng

    2016-03-15

    Pesticides are of great importance in agricultural and biological fields, but pesticide residues may harm the environment and human health. A highly sensitive fluorescent biosensor for the detection of carbamate pesticide has been developed based on acetylcholinesterase (AChE)-catalyzed hydrolysis product triggered Hg(2+) release coupled with subsequent nicking enzyme-induced cleavage of a duplex DNA for cycling amplification. In this protocol, two DNA probes, an unmodified single-stranded helper DNA probe 1 (HP1) and a quencher-fluorophore probe (QFP) are ingeniously designed. HP1 can be folded into hairpin configuration through T-Hg(2+)-T base pair formation. QFP, labeled with FAM and BHQ1 at its two terminals, contains the recognition sequence and the cleavage site of the nicking enzyme. In the presence of carbamate pesticide, the activity of AChE is inhibited, and the amount of the product containing the thiol group generated by the hydrolysis reaction of acetylthiocholine chloride (ACh) decreases, resulting in the release of a low concentration of Hg(2+). The number of HP1 that can be selectively unfolded would be reduced and the subsequent nicking enzyme-assisted cleavage processes would be affected, resulting in decreased fluorescence signals. The fluorescence intensity further decreases with the increase of the pesticide concentration. Therefore, the pesticide content can be easily obtained by monitoring the fluorescence signal change, which is inversely proportional to the logarithm of the pesticide concentration. The detection limit of aldicarb, the model analyte, is 3.3 μgL(-1), which is much lower than the Chinese National Standards or those previously reported. The as-proposed method has also been applied to detect carbamate pesticide residues in fresh ginger and artificial lake water samples with satisfactory results, which demonstrates that the method has great potential for practical application in biological or food safety field.

  12. Molecular characterization of the Calvin cycle enzyme phosphoribulokinase in the stramenopile alga Vaucheria litorea and the plastid hosting mollusc Elysia chlorotica.

    PubMed

    Rumpho, Mary E; Pochareddy, Sirisha; Worful, Jared M; Summer, Elizabeth J; Bhattacharya, Debashish; Pelletreau, Karen N; Tyler, Mary S; Lee, Jungho; Manhart, James R; Soule, Kara M

    2009-11-01

    Phosphoribulokinase (PRK), a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction (Calvin) cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc (sea slug) Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclear-encoded proteins that are directed to complex (i.e. four membrane-bound) algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V. litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V. litorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.

  13. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

    PubMed

    Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J

    2016-03-17

    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation. PMID:26990986

  14. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

    PubMed

    Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J

    2016-03-17

    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

  15. [DYNAMICS OF GLUTAMINE SYNTHASE ACTIVITY IN RAT BRAIN IN PRENATAL HYPOXIA MODEL].

    PubMed

    Khairova, V R; Safarov, M I

    2015-01-01

    Prenatal ontogenesis is a period of high sensitivity to stressful impact, so any stressor can lead to changes of physiological, biochemical indicators, behavioral and cognitive functions. The most common and clinically significant stress factor, which the embryo may be exposed during embryonic development, is hypoxia. In this case pathological changes in the central nervous system depend on the duration and severity of hypoxic exposure, individual tolerance and the stage of prenatal development, at each of which in the brain take place the basic histogenetic processes. By activating energetically disadvantageous anaerobic glycolysis hypoxia leads to excess of glutamate emission and cell apoptosis. Glutamine synthase is a basic enzyme that regulates metabolism of glutamate, catalyzing conversion of glutamate to glutamine with ammonia detoxification. The aim of the presented work was to reveal changes in the activity of one of the key enzyme of glutamate metabolism- glutamine synthetase in the brain of offspring of white rats undergone to hypoxia at different stages of prenatal ontogenesis. Hypoxia was created by placing female rats at stages of the pregnancy, corresponding to progestation period of organogenesis and fetal period of prenatal development, in the hypobaric chamber with exposure to 5% oxygen and 95% nitrogen gas mixture during 30 minutes per day. The offspring obtained from females of control and experimental groups were used for biochemical determinations in the age of 1 and 3 month. It has been established that hypoxia exposed to pregnant females during embryonic organogenesis causes significant changes in enzyme activity, particularly pronounced in the cerebral cortex and cerebellum, as compared with progestational and fetal hypoxia. Enzyme activity decreased in a greater degree in one-month-old rats undergone to prenatal hypoxia, than three- month-old animals. Thus, stress during intensive processes of proliferation and migration of cells of the

  16. [Effects of applying different kind fertilizers on enzyme activities related to carbon, nitrogen, and phosphorus cycles in reddish paddy soil].

    PubMed

    Xu, Li-Li; Wang, Qiu-Bing; Zhang, Xin-Yu; Sun, Xiao-Min; Dai, Xiao-Qin; Yang, Feng-Ting; Bu, Jin-Feng; Wang, Hui-min

    2013-04-01

    Based on the long-term fixed position experimental data from Qianyanzhou Ecological Experiment Station, Chinese Academy of Sciences in 1998, this paper analyzed the effects of applying different kind fertilizers (straw, ST; pig manure, OM; and chemical fertilizer, NPK) on the nutrients (C, N, and P) status and the activities of related enzymes ( beta-1,4-glucosidase, betaG; beta-1,4-N-acetylglucosaminidase, NAG; L-leucine aminopeptidase, LAP; and acid phosphatase, AP) in reddish paddy soil. With the application of OM, the activities of soil betaG, NAG, and LAP increased significantly, as compared with other treatments, and were 1.4, 2. 6, and 1.9 times higher than the control (CK) , respectively. Applying OM also improved the ratio of soil organic carbon to total nitrogen (C/N), but decreased the soil betaG/(NAG+LAP) ratio, suggesting that pig manure could benefit the degradation of soil cellulose and the accumulation of soil organic carbon. Applying NPK increased the activities of soil betaG, NAG, and LAP, but decreased the AP activity, with a decrement of 34% as compared with CK. Under the application of NPK, the soilbetaG/AP and (NAG+ LAP)/AP ratios increased, but the ratios of soil organic carbon to total phosphorus (C/P) and of soil total nitrogen to total phosphorus (N/P) decreased, indicating that chemical fertilizers could induce the accumulation of soil inorganic phosphorus, and inhibit the microbial functions of degrading polysaccharides and phosphate phospholipids.

  17. Combined, Functional Genomic-Biochemical Approach to Intermediary Metabolism: Interaction of Acivicin, a Glutamine Amidotransferase Inhibitor, with Escherichia coli K-12

    PubMed Central

    Smulski, Dana R.; Huang, Lixuan L.; McCluskey, Michael P.; Reeve, Mary Jane Gladnick; Vollmer, Amy C.; Van Dyk, Tina K.; LaRossa, Robert A.

    2001-01-01

    Acivicin, a modified amino acid natural product, is a glutamine analog. Thus, it might interfere with metabolism by hindering glutamine transport, formation, or usage in processes such as transamidation and translation. This molecule prevented the growth of Escherichia coli in minimal medium unless the medium was supplemented with a purine or histidine, suggesting that the HisHF enzyme, a glutamine amidotransferase, was the target of acivicin action. This enzyme, purified from E. coli, was inhibited by low concentrations of acivicin. Acivicin inhibition was overcome by the presence of three distinct genetic regions when harbored on multicopy plasmids. Comprehensive transcript profiling using DNA microarrays indicated that histidine biosynthesis was the predominant process blocked by acivicin. The response to acivicin, however, was quite complex, suggesting that acivicin inhibition resonated through more than a single cellular process. PMID:11344143

  18. The DNA-mismatch repair enzyme hMSH2 modulates UV-B-induced cell cycle arrest and apoptosis in melanoma cells.

    PubMed

    Seifert, Markus; Scherer, Stefan J; Edelmann, Wilfried; Böhm, Markus; Meineke, Viktor; Löbrich, Markus; Tilgen, Wolfgang; Reichrath, Jörg

    2008-01-01

    The mechanisms by which the post-replicative DNA mismatch repair (MMR) enzyme MSH2 is involved in the complex response mechanisms to UV damage are yet to be clarified. Here, we show increased levels of MSH2 mRNA in malignant melanoma, metastases of melanoma, and melanoma cell (MeWo) lines as compared with melanocytic nevi or primary cultured benign melanocytes. UV-B treatment modulated MSH2 expression and silencing of MSH2 gene expression using small interfering RNA technology regulated UV-B-induced cell cycle arrest and apoptosis in human MeWo. We show that MSH2-deficient non-malignant mouse fibroblasts (MEF-/-) are partially resistant against UV-B-induced apoptosis and show reduced S-Phase accumulation. In addition, we show that an Msh2 point mutation (MEFGA) that affects MMR does not affect UV-B-induced apoptosis. In conclusion, we demonstrate that MSH2 modulates in human melanocytes both UV-B-induced cell cycle regulation and apoptosis, most likely via independent, uncoupled mechanisms.

  19. Legacy effects overwhelm the short-term effects of exotic plant invasion and restoration on soil microbial community structure, enzyme activities, and nitrogen cycling.

    PubMed

    Elgersma, Kenneth J; Ehrenfeld, Joan G; Yu, Shen; Vor, Torsten

    2011-11-01

    Plant invasions can have substantial consequences for the soil ecosystem, altering microbial community structure and nutrient cycling. However, relatively little is known about what drives these changes, making it difficult to predict the effects of future invasions. In addition, because most studies compare soils from uninvaded areas to long-established dense invasions, little is known about the temporal dependence of invasion impacts. We experimentally manipulated forest understory vegetation in replicated sites dominated either by exotic Japanese barberry (Berberis thunbergii), native Viburnums, or native Vacciniums, so that each vegetation type was present in each site-type. We compared the short-term effect of vegetation changes to the lingering legacy effects of the previous vegetation type by measuring soil microbial community structure (phospholipid fatty acids) and function (extracellular enzymes and nitrogen mineralization). We also replaced the aboveground litter in half of each plot with an inert substitute to determine if changes in the soil microbial community were driven by aboveground or belowground plant inputs. We found that after 2 years, the microbial community structure and function was largely determined by the legacy effect of the previous vegetation type, and was not affected by the current vegetation. Aboveground litter removal had only weak effects, suggesting that changes in the soil microbial community and nutrient cycling were driven largely by belowground processes. These results suggest that changes in the soil following either invasion or restoration do not occur quickly, but rather exhibit long-lasting legacy effects from previous belowground plant inputs. PMID:21618010

  20. 1,25-Dihydroxyvitamin D inhibits glutamine metabolism in Harvey-ras transformed MCF10A human breast epithelial cell.

    PubMed

    Zhou, Xuanzhu; Zheng, Wei; Nagana Gowda, G A; Raftery, Daniel; Donkin, Shawn S; Bequette, Brian; Teegarden, Dorothy

    2016-10-01

    Breast cancer is the second most common cancer among women in the US. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is proposed to inhibit cellular processes and to prevent breast cancer. The current studies investigated the effect of 1,25(OH)2D on glutamine metabolism during cancer progression employing Harvey-ras oncogene transformed MCF10A human breast epithelial cells (MCF10A-ras). Treatment with 1,25(OH)2D significantly reduced intracellular glutamine and glutamate levels measured by nuclear magnetic resonance (NMR) by 23±2% each. Further, 1,25(OH)2D treatment reduced glutamine and glutamate flux, determined by [U-(13)C5] glutamine tracer kinetics, into the TCA cycle by 31±0.2% and 17±0.4%, respectively. The relative levels of mRNA and protein abundance of the major glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was significantly decreased by 1,25(OH)2D treatment in both MCF10A-ras cells and MCF10A which overexpress ErbB2 (HER-2/neu). Consistent with these results, glutamine uptake was reduced by 1,25(OH)2D treatment and the impact was eliminated with the SLC1A5 inhibitor L-γ-Glutamyl-p-nitroanilide (GPNA). A consensus sequence to the vitamin D responsive element (VDRE) was identified in silico in the SLC1A5 gene promoter, and site-directed mutagenesis analyses with reporter gene studies demonstrate a functional negative VDRE in the promoter of the SLC1A5 gene. siRNA-SLC1A5 transfection in MCF10A-ras cells significantly reduced SLC1A5 mRNA expression as well as decreased viable cell number similar to 1,25(OH)2D treatment. SLC1A5 knockdown also induced an increase in apoptotic cells in MCF10A-ras cells. These results suggest 1,25(OH)2D alters glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through down-regulation of SLC1A5 transcript abundance. Thus, 1,25(OH)2D down-regulation of the glutamine transporter, SLC1A5, may facilitate vitamin D prevention of breast

  1. Competition between ammonia derived from internal glutamine hydrolysis and hydroxylamine present in the solution for incorporation into UTP as catalysed by Lactococcus lactis CTP synthase.

    PubMed

    Willemoës, Martin

    2004-04-01

    CTP synthase catalyses the reaction: glutamine+UTP+ATP --> glutamate+CTP+ADP+P(i). The reaction is greatly stimulated by the allosteric binding of GTP. In addition to glutamine that is hydrolysed by the enzyme to ammonia and glutamate, CTP synthase will also utilise external sources of amino donors such as NH(4)Cl. This reaction is no longer dependent on allosteric activation by GTP. Hydroxylamine is also a substrate for Lactococcus lactis CTP synthase and results in the formation of N4-OH CTP. This product has the feature that it absorbs at 300nm where CTP absorption was shown to be greatly reduced and enabled the determination of N4-OH CTP formation in the presence of CTP synthesis derived from glutamine hydrolysis. Differences in initial rates determined for the hydroxylamine dependent reaction at 291nm in the presence and absence of glutamine and GTP were ascribed to simultaneous CTP and N4-OH CTP synthesis in the presence of these compounds. A characterisation of the apparent inhibition by GTP and glutamine of N4-OH CTP synthesis determined at 300nm showed that glutamine dependent CTP synthesis occurs at a rate of about 60% of that in the absence of hydroxylamine. GTP dependent inhibition of the ammonium chloride dependent reaction of L. lactis CTP synthase by the glutamine analog glutamate gamma-semialdehyde showed a partial inhibition with a maximum inhibition of about 60%. These results are interpreted in terms of a "half of the sites" mechanism for glutamine hydrolysis on CTP synthase.

  2. Highly sensitive and selective microRNA detection based on DNA-bio-bar-code and enzyme-assisted strand cycle exponential signal amplification.

    PubMed

    Dong, Haifeng; Meng, Xiangdan; Dai, Wenhao; Cao, Yu; Lu, Huiting; Zhou, Shufeng; Zhang, Xueji

    2015-04-21

    Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-assisted strand cycle for exponential signal amplification was designed. The DNA-BCA system contains a locked nucleic acid (LNA) modified DNA probe for improving hybridization efficiency, while a signal reported molecular beacon (MB) with an endonuclease recognition site was designed for strand cycle amplification. In the presence of target miRNA, the oligonucleotides functionalized magnetic nanoprobe (MNP-DNA) and gold nanoprobe (AuNP-DNA) with numerous reported probes (RP) can hybridize with target miRNA, respectively, to form a sandwich structure. After sandwich structures were separated from the solution by the magnetic field, the RP were released under high temperature to recognize the MB and cleaved the hairpin DNA to induce the dissociation of RP. The dissociated RP then triggered the next strand cycle to produce exponential fluorescent signal amplification for miRNA detection. Under optimized conditions, the exponential signal amplification system shows a good linear range of 6 orders of magnitude (from 0.3 pM to 3 aM) with limit of detection (LOD) down to 52.5 zM, while the sandwich structure renders the system with high selectivity. Meanwhile, the feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in HeLa lysates. Given the high-performance for miRNA analysis, the strategy has a promising application in biological detection and in clinical diagnosis.

  3. Chloroplast and Cytoplasmic Enzymes

    PubMed Central

    Anderson, Louise E.; Advani, Vimal R.

    1970-01-01

    Three pea (Pisum sativum) leaf chloroplast enzymes—triose phosphate isomerase, glyceric acid 3-phosphate kinase, and fructose 1,6-diphosphate aldolase—have been separated from the corresponding cytoplasmic enzymes by isoelectric focusing. These three enzymes of the reductive pentose phosphate cycle are therefore distinct proteins, not identical with the analogous enzymes of the Embden-Meyerhof-Parnas pathway. PMID:16657347

  4. Crystal structures capture three states in the catalytic cycle of a pyridoxal phosphate (PLP) synthase.

    PubMed

    Smith, Amber Marie; Brown, William Clay; Harms, Etti; Smith, Janet L

    2015-02-27

    PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5'-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate. PMID:25568319

  5. Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

    NASA Astrophysics Data System (ADS)

    Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C.; Sylvestersen, Kathrine B.; Nelson, Christopher J.; Nielsen, Michael L.; Kouzarides, Tony

    2014-01-01

    Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

  6. Structural analysis of the wheat genes encoding NADH-dependent glutamine-2-oxoglutarate amidotransferases genes and correlation with grain protein content

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrogen uptake and the efficient absorption and metabolism of nitrogen are essential elements in attempts to breed improved cereal cultivars for grain or silage production. One of the enzymes related to nitrogen metabolism is glutamine-2-oxoglutarate amidotransferase (GOGAT). Together with glutami...

  7. Targeting glutamine metabolism in multiple myeloma enhances BIM binding to BCL-2 eliciting synthetic lethality to venetoclax

    PubMed Central

    Bajpai, R; Matulis, SM; Wei, C; Nooka, AK; Von Hollen, HE; Lonial, S; Boise, LH; Shanmugam, M

    2016-01-01

    Multiple myeloma (MM) is a plasma cell malignancy that is largely incurable due to development of resistance to therapy-elicited cell death. Nutrients are intricately connected to maintenance of cellular viability in part by inhibition of apoptosis. We were interested to determine if examination of metabolic regulation of BCL-2 proteins may provide insight on alternative routes to engage apoptosis. MM cells are reliant on glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. We and others have demonstrated that glucose maintains levels of key resistance-promoting BCL-2 family member, myeloid cell leukemic factor 1 (MCL-1). Cells continuing to survive in the absence of glucose or glutamine were found to maintain expression of MCL-1 but importantly induce pro-apoptotic BIM expression. One potential mechanism for continued survival despite induction of BIM could be due to binding and sequestration of BIM to alternate pro-survival BCL-2 members. Our investigation revealed that cells surviving glutamine withdrawal in particular, enhance expression and binding of BIM to BCL-2, consequently sensitizing these cells to the BH3 mimetic venetoclax. Glutamine deprivation-driven sensitization to venetoclax can be reversed by metabolic supplementation with TCA cycle intermediate α-ketoglutarate. Inhibition of glucose metabolism with the GLUT4 inhibitor ritonavir elicits variable cytotoxicity in MM that is marginally enhanced with venetoclax treatment, however, targeting glutamine metabolism with 6-diazo-5-oxo-l-norleucine uniformly sensitized MM cell lines and relapse/refractory patient samples to venetoclax. Our studies reveal a potent therapeutic strategy of metabolically driven synthetic lethality involving targeting glutamine metabolism for sensitization to venetoclax in MM. PMID:26640142

  8. Targeting glutamine metabolism in multiple myeloma enhances BIM binding to BCL-2 eliciting synthetic lethality to venetoclax.

    PubMed

    Bajpai, R; Matulis, S M; Wei, C; Nooka, A K; Von Hollen, H E; Lonial, S; Boise, L H; Shanmugam, M

    2016-07-28

    Multiple myeloma (MM) is a plasma cell malignancy that is largely incurable due to development of resistance to therapy-elicited cell death. Nutrients are intricately connected to maintenance of cellular viability in part by inhibition of apoptosis. We were interested to determine if examination of metabolic regulation of BCL-2 proteins may provide insight on alternative routes to engage apoptosis. MM cells are reliant on glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. We and others have demonstrated that glucose maintains levels of key resistance-promoting BCL-2 family member, myeloid cell leukemic factor 1 (MCL-1). Cells continuing to survive in the absence of glucose or glutamine were found to maintain expression of MCL-1 but importantly induce pro-apoptotic BIM expression. One potential mechanism for continued survival despite induction of BIM could be due to binding and sequestration of BIM to alternate pro-survival BCL-2 members. Our investigation revealed that cells surviving glutamine withdrawal in particular, enhance expression and binding of BIM to BCL-2, consequently sensitizing these cells to the BH3 mimetic venetoclax. Glutamine deprivation-driven sensitization to venetoclax can be reversed by metabolic supplementation with TCA cycle intermediate α-ketoglutarate. Inhibition of glucose metabolism with the GLUT4 inhibitor ritonavir elicits variable cytotoxicity in MM that is marginally enhanced with venetoclax treatment, however, targeting glutamine metabolism with 6-diazo-5-oxo-l-norleucine uniformly sensitized MM cell lines and relapse/refractory patient samples to venetoclax. Our studies reveal a potent therapeutic strategy of metabolically driven synthetic lethality involving targeting glutamine metabolism for sensitization to venetoclax in MM.

  9. Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase.

    PubMed

    Pérez-Delgado, Carmen M; García-Calderón, Margarita; Márquez, Antonio J; Betti, Marco

    2015-01-01

    It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4(+) accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4(+) when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4(+). Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4(+) when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.

  10. Comparative aspects of tissue glutamine and proline metabolism.

    PubMed

    Bertolo, Robert F; Burrin, Douglas G

    2008-10-01

    The cellular metabolism of glutamine and proline are closely interrelated, because they can be interconverted with glutamate and ornithine via the mitochondrial pathway involving pyrroline-5-carboxylate (P5C). In adults, glutamine and proline are converted via P5C to citrulline in the gut, then citrulline is converted to arginine in the kidney. In neonates, arginine is a semiindispensable amino acid and is synthesized from proline completely in the gut; because of low P5C synthase activity, glutamine is not an important precursor for neonatal arginine synthesis. Thus, splanchnic metabolism of glutamine and proline is important, because both amino acids serve as key precursors for arginine synthesis with some developmental differences. Studies investigating splanchnic extraction demonstrate that about two-thirds of dietary glutamine and almost all dietary glutamate are extracted on first pass and the vast majority is oxidized in the gut. This capacity to extract glutamine and glutamate appears to be very large, so diets high in glutamine or glutamate probably have little impact on circulating concentrations and consequent potential toxicity. In contrast, it appears that very little proline is extracted by the gut and liver, at least in the neonate, which may result in hyperprolinemia and potential toxicity. Therefore, the upper limits of safe dietary intake for glutamine and proline, and other amino acids, appear to be substantially different depending on the extent of first-pass splanchnic extraction and irreversible catabolism.

  11. Corticosteroids increase glutamine utilization in human splanchnic bed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutamine is the most abundant amino acid in the body and is extensively taken up in gut and liver in healthy humans. To determine whether glucocorticosteroids alter splanchnic glutamine metabolism, the effect of prednisone was assessed in healthy volunteers using isotope tracer methods. Two groups ...

  12. Exogenous Glutamine in Respiratory Diseases: Myth or Reality?

    PubMed Central

    Oliveira, Gisele P.; de Abreu, Marcelo Gama; Pelosi, Paolo; Rocco, Patricia R. M.

    2016-01-01

    Several respiratory diseases feature increased inflammatory response and catabolic activity, which are associated with glutamine depletion; thus, the benefits of exogenous glutamine administration have been evaluated in clinical trials and models of different respiratory diseases. Recent reviews and meta-analyses have focused on the effects and mechanisms of action of glutamine in a general population of critical care patients or in different models of injury. However, little information is available about the role of glutamine in respiratory diseases. The aim of the present review is to discuss the evidence of glutamine depletion in cystic fibrosis (CF), asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), and lung cancer, as well as the results of exogenous glutamine administration in experimental and clinical studies. Exogenous glutamine administration might be beneficial in ARDS, asthma, and during lung cancer treatment, thus representing a potential therapeutic tool in these conditions. Further experimental and large randomized clinical trials focusing on the development and progression of respiratory diseases are necessary to elucidate the effects and possible therapeutic role of glutamine in this setting. PMID:26861387

  13. Exogenous Glutamine in Respiratory Diseases: Myth or Reality?

    PubMed

    Oliveira, Gisele P; de Abreu, Marcelo Gama; Pelosi, Paolo; Rocco, Patricia R M

    2016-02-04

    Several respiratory diseases feature increased inflammatory response and catabolic activity, which are associated with glutamine depletion; thus, the benefits of exogenous glutamine administration have been evaluated in clinical trials and models of different respiratory diseases. Recent reviews and meta-analyses have focused on the effects and mechanisms of action of glutamine in a general population of critical care patients or in different models of injury. However, little information is available about the role of glutamine in respiratory diseases. The aim of the present review is to discuss the evidence of glutamine depletion in cystic fibrosis (CF), asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), and lung cancer, as well as the results of exogenous glutamine administration in experimental and clinical studies. Exogenous glutamine administration might be beneficial in ARDS, asthma, and during lung cancer treatment, thus representing a potential therapeutic tool in these conditions. Further experimental and large randomized clinical trials focusing on the development and progression of respiratory diseases are necessary to elucidate the effects and possible therapeutic role of glutamine in this setting.

  14. Phosphorus cycling in the Sargasso Sea: Investigation using the oxygen isotopic composition of phosphate, enzyme-labeled fluorescence, and turnover times

    NASA Astrophysics Data System (ADS)

    McLaughlin, Karen; Sohm, Jill A.; Cutter, Gregory A.; Lomas, Michael W.; Paytan, Adina

    2013-04-01

    Dissolved inorganic phosphorus (DIP) concentrations in surface water of vast areas of the ocean are extremely low (<10 nM) and phosphorus (P) availability could limit primary productivity in these regions. We explore the use of oxygen isotopic signature of dissolved phosphate (δ18OPO4) to investigate biogeochemical cycling of P in the Sargasso Sea, Atlantic Ocean. Additional techniques for studying P dynamics including 33P-based DIP turnover time estimates and percent of cells expressing alkaline phosphatase (AP) activity as measured by enzyme-labeling fluorescence are also used. In surface waters, δ18OPO4 values were lower than equilibrium by 3-6‰, indicative of dissolved organic phosphorous (DOP) remineralization by extracellular enzymes. An isotope mass balance model using a variety of possible combinations of enzymatic pathways and substrates indicates that DOP remineralization in the euphotic zone can account for a large proportion on P utilized by phytoplankton (as much as 82%). Relatively short DIP turnover times (4-8 h) and high expression of AP (38-77% of the cells labeled) are consistent with extensive DOP utilization and low DIP availability in the euphotoc zone. In deep water where DOP utilization rates are lower, δ18OPO4 values approach isotopic equilibrium and DIP turnover times are longer. Our data suggests that in the euphotic zone of the Sargasso Sea, DOP may be appreciably remineralized and utilized by phytoplankton and bacteria to supplement cellular requirements. A substantial fraction of photosynthesis in this region is supported by DOP uptake.

  15. Influence of sodium chloride on the regulation of Krebs cycle intermediates and enzymes of respiratory chain in mungbean (Vigna radiata L. Wilczek) seedlings.

    PubMed

    Saha, Papiya; Kunda, Pranamita; Biswas, Asok K

    2012-11-01

    The effect of common salt (NaCl) on ion contents, Krebs cycle intermediates and its regulatory enzymes was investigated in growing mungbean (Vigna radiata L. Wilczek, B 105) seedlings. Sodium and chloride ion contents increased in both root and shoot whereas potassium ion content decreased in shoot of test seedlings with increasing concentrations of NaCl. Organic acids like pyruvate and citrate levels increased whereas malate level decreased under stress in both roots and shoots. Salt stress also variedly affected the activities of different enzymes of respiratory chain. The activity of pyruvate dehydrogenase (E.C. 1.2.4.1) decreased in 50 mM NaCl but increased in 100 mM and 150 mM concentrations, in both root and shoot samples. Succinate dehydrogenase (E.C. 1.3.5.1) activity was reduced in root whereas stimulated in shoot under increasing concentrations of salt. The activity of isocitrate dehydrogenase (E.C. 1.1.1.41) and malate dehydrogenase (E.C. 1.1.1.37) decreased in both root and shoot samples under salt stress. On the contrary, pretreatment of mungbean seeds with sublethal dose of NaCl was able to overcome the adverse effects of stress imposed by NaCl to variable extents with significant alterations of all the tested parameters, resulting in better growth and efficient respiration in mungbean seedlings. Thus, plants can acclimate to lethal level of salinity by pretreatment of seeds with sublethal level of NaCl, which serves to improve their health and production under saline condition, but the sublethal concentration of NaCl should be carefully chosen.

  16. Prompt and easy activation by specific thioredoxins of calvin cycle enzymes of Arabidopsis thaliana associated in the GAPDH/CP12/PRK supramolecular complex.

    PubMed

    Marri, Lucia; Zaffagnini, Mirko; Collin, Valérie; Issakidis-Bourguet, Emmanuelle; Lemaire, Stéphane D; Pupillo, Paolo; Sparla, Francesca; Miginiac-Maslow, Myroslawa; Trost, Paolo

    2009-03-01

    The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins (TRXs). We have investigated the interactions of eight different chloroplast thioredoxin isoforms (TRX f1, m1, m2, m3, m4, y1, y2, x) with GAPDH (A(4), B(4), and B(8) isoforms), PRK and CP12 (isoform 2), all from Arabidopsis thaliana. In the complex, both A(4)-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs m1 and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B(4)- and B(8)-GAPDH were absolutely specific for TRX f1. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A(4)-GAPDH in the complex was much faster (and less demanding in terms of reducing potential) than activation of free oxidized B(4)- or B(8)-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH (B-containing isoforms) would be more suited to conditions of very active photosynthesis.

  17. Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities.

    PubMed

    Alvarez, Maricel; Huygens, Dries; Olivares, Erick; Saavedra, Isabel; Alberdi, Miren; Valenzuela, Eduardo

    2009-08-01

    Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs. PMID:19470091

  18. Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities.

    PubMed

    Alvarez, Maricel; Huygens, Dries; Olivares, Erick; Saavedra, Isabel; Alberdi, Miren; Valenzuela, Eduardo

    2009-08-01

    Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs.

  19. L-glutamine is a key parameter in the immunosuppression phenomenon

    SciTech Connect

    Hammami, Ines; Chen, Jingkui; Bronte, Vincenzo; DeCrescenzo, Gregory; Jolicoeur, Mario

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer The absence of L-Gln inhibited iNOS activity, but not ARG1 one. Black-Right-Pointing-Pointer MSC-1 cells were able to inhibit Jurkat cell growth, but not their viability. Black-Right-Pointing-Pointer Absence of L-Gln down-regulated central carbon metabolism and L-Arg recycling. Black-Right-Pointing-Pointer Absence of L-Gln deteriorated cell bioenergetic status. Black-Right-Pointing-Pointer L-Gln is crucial for iNOS-mediated immunosuppression activity. -- Abstract: Suppression of tumour-specific T-cell functions by myeloid-derived suppressor cells (MDSCs) is a dominant mechanism of tumour escape. MDSCs express two enzymes, i.e. inducible nitric oxide synthase (iNOS) and arginase (ARG1), which metabolize the semi-essential amino acid L-arginine (L-Arg) whose bioavailability is crucial for T-cell proliferation and functions. Recently, we showed that glutaminolysis supports MDSC maturation process by ensuring the supply of intermediates and energy. In this work, we used an immortalized cell line derived from mouse MDSCs (MSC-1 cell line) to further investigate the role of L-glutamine (L-Gln) in the maintenance of MDSC immunosuppressive activity. Culturing MSC-1 cells in L-Gln-limited medium inhibited iNOS activity, while ARG1 was not affected. MSC-1 cells inhibited Jukat cell growth without any noticeable effect on their viability. The characterization of MSC-1 cell metabolic profile revealed that L-Gln is an important precursor of lactate production via the NADP{sup +}-dependent malic enzyme, which co-produces NADPH. Moreover, the TCA cycle activity was down-regulated in the absence of L-Gln and the cell bioenergetic status was deteriorated accordingly. This strongly suggests that iNOS activity, but not that of ARG1, is related to an enhanced central carbon metabolism and a high bioenergetic status. Taken altogether, our results suggest that the control of glutaminolysis fluxes may represent a valuable target for immunotherapy.

  20. Cloning of prophenoloxidase from hemocytes of the blue crab, Callinectes sapidus and its expression and enzyme activity during the molt cycle.

    PubMed

    Alvarez, Javier V; Chung, J Sook

    2013-11-01

    The arthropods cuticle undergoes dramatic morphological and biochemical changes from being soft to hardness through each molting process. Prophenoloxidase (PPO) known as a key enzyme in the arthropod innate immune system involved in the melanization reaction, has been related with the initial shell-hardening process, specifically in the sclerotization of the protein matrix in the new cuticle. Since hemocytes have been reported as the main PPO source in arthropods, the transport of hemocyte PPO into the newly laid, soft cuticle has been proposed for shell-hardening occurring during and immediately after ecdysis. In order to define the role of hemocyte PPO in the shell-hardening of crustaceans, the full-length cDNA sequence (2806 nt) of hemocytes PPO of the blue crab Callinectes sapidus (CasPPO-hemo) is isolated using degenerate PCR and 5'-3' RACE. CasPPO-hemo encodes a putative PPO (672 aa) showing three hemocyanin domains: N, M, and C in order and two copper binding sites (CuA & CuB). The sequence analysis identifies the putative CasPPO-hemo as zymogen which requires the cleavage at the N-terminus for its activation. Hemocyte extract (CasHLS) contains the PO, the activity of which depends on the in vitro activation of trypsin. The expression levels of CasPPO-hemo are kept constant during the molt cycle. The increase in the number of hemocytes at early premolt correlates with the elevated PO activity, while at late premolt, the increment in hemocyte numbers does not reflect on the PO activity. The functional importance of the changes in the levels of CasHLS-PO activity during molt cycle is discussed in relation to cuticle hardening process.

  1. Urea cycle defects and hyperammonemia: effects on functional imaging.

    PubMed

    Gropman, Andrea L; Prust, Morgan; Breeden, Andrew; Fricke, Stanley; VanMeter, John

    2013-06-01

    The urea-cycle disorders (UCDs) are a group of congenital enzyme and carrier deficiencies predisposing to hyperammonemia (HA). HA causes changes in the central nervous system (CNS) including alterations of neurotransmitter function, cell volume, and energy deprivation ultimately leading to cerebral edema. Neuropathological findings of UCDs primarily reflect changes in astrocyte morphology. Neurological features accompanying acute HA include changes in behavior and consciousness in the short term, and potential for impairments in memory and executive function as long-term effects. Plasma measures of ammonia and glutamine, although useful for clinical monitoring, prove poor markers of CNS function. Multimodal neuroimaging has potential to investigate impact on cognitive function by interrogating neural networks, connectivity and biochemistry. As neuroimaging methods become increasingly sophisticated, they will play a critical role in clinical monitoring and treatment of metabolic disease. We describe our findings in UCDs; with focus on Ornithine Transcarbamylase deficiency (OTCD) the only X linked UCD.

  2. Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy

    PubMed Central

    Swamy, Mahima; Pathak, Shalini; Grzes, Katarzyna M.; Damerow, Sebastian; Sinclair, Linda V.; van Aalten, Daan M. F.; Cantrell, Doreen A.

    2016-01-01

    Sustained glucose and glutamine transport are essential for activated T lymphocytes to support ATP and macromolecule biosynthesis. We now show that glutamine and glucose also fuel an indispensible dynamic regulation of intracellular protein O-GlcNAcylation at key stages of T cell development, transformation and differentiation. Glucose and glutamine are precursors of UDP-GlcNAc, a substrate for cellular glycosyltransferases. Immune activated T cells contained higher concentrations of UDP-GlcNAc and increased intracellular protein O-GlcNAcylation controlled by the enzyme O-GlcNAc glycosyltransferase as compared to naïve cells. We identified Notch, the T cell antigen receptor and c-Myc as key controllers of T cell protein O-GlcNAcylation, via regulation of glucose and glutamine transport. Loss of O-GlcNAc transferase blocked T cell progenitor renewal, malignant transformation, and peripheral T cell clonal expansion. Nutrient-dependent signaling pathways regulated by O-GlcNAc glycosyltransferase are thus fundamental for T cell biology. PMID:27111141

  3. UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation.

    PubMed

    Vozza, Angelo; Parisi, Giovanni; De Leonardis, Francesco; Lasorsa, Francesco M; Castegna, Alessandra; Amorese, Daniela; Marmo, Raffaele; Calcagnile, Valeria M; Palmieri, Luigi; Ricquier, Daniel; Paradies, Eleonora; Scarcia, Pasquale; Palmieri, Ferdinando; Bouillaud, Frédéric; Fiermonte, Giuseppe

    2014-01-21

    Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.

  4. Glutamine: a precursor of glutathione and its effect on liver

    PubMed Central

    Yu, Jian-Chun; Jiang, Zhu-Ming; Li, De-Min

    1999-01-01

    AIM To investigate the relationship between alanyl-glutamine (ALA-GLN) and glutathione (GSH) biosynthesis in hepatic protection. METHODS Twenty male Wistar rats were randomly divided into two groups: one receiving standard parenteral nutrition (STD) and the other supplemented with or without ALA-GLN for 7 days. The blood and liver tissue samples were examined after 5-fluorouracil (5-FU) was injected peritoneally. RESULTS The concentration measurements were significantly highe r in ALA-GLN group than in STD group in serum GLN (687 μmol/ L ± 50 μmol/L vs 505 μmol/L ± 39 μmol/L,P < 0.05), serum GSH (14 μmol/L ± 5 μmol/L vs 7 μmol/L ± 3 μmol/L, P < 0.01) and in liver GSH content (6.9 μmol/g ± 2.5 μmol/g vs 4.4 μmol/ g ± 1.6 μmol/g liver tissue, P < 0.05). Rats in ALA-GLN group had lesser elevations in hepatic enzymes after 5-FU administration. CONCLUSION The supplemented nutrition ALA-GLN can protect the liver function through increasing the glutathione biosynthesis and pre-serving the glutathione stores in hepatic tissue. PMID:11819414

  5. Systematic Underutilization of Glutamine In Thermophile Proteins

    NASA Technical Reports Server (NTRS)

    Liang, Shoudan; Weber, Arthur; Biegel, Bryan A. (Technical Monitor)

    2002-01-01

    Rapid racemization above 100 C of L-amino acids to Domino acids, as well as deamidation, is probably a hazard for high temperature life. For example, the half-life of some asparaginyl peptides can be as short as 10 minutes at 100 C. High temperature organisms could protect themselves by reducing usage of amino acids that are easily racemized/deamidazed, by having a rapid rate of protein turnover which requires energy, or by adapting special cis-peptide conformations. We have searched eight completely sequenced thermophile genomes, and compare them to mesophile genomes, in order to identify underutilized amino acids. To our surprise, asparagine, the most unstable amino acid to deamidation, is used at about the same level in thermophile proteins in comparison to mesophiles whereas it is the second most unstable amino acid, glutamine, that is underutilized in all of eight thermophile species. Glutamines are present at 2% level in a typical thermophile protein, instead of 4% in mesophile. We argue that it is easier to protect asparagines from deamidation by cis-peptide conformations. We discuss statistical as well as structural evidence in support of our conclusions.

  6. Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

    NASA Technical Reports Server (NTRS)

    Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

    1987-01-01

    The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

  7. Preparation and characterization of electrospun nanofibers containing glutamine.

    PubMed

    Tort, Serdar; Acartürk, Füsun

    2016-11-01

    Oral mucositis is a painful inflammation of mucous membranes commonly after chemotherapy or radiotherapy. The aim of this study was to develop mucoadhesive nanofibers containing glutamine via electrospinning and to characterize them for the treatment of oral mucositis. Different mucoadhesive polymers were tried for preparing nanofibers and sodium alginate nanofibers were chosen after the characterization studies. Glutamine-loaded nanofibers were produced and characterized. Glutamine loaded onto nanofibers was confirmed by differantial scanning calorimetry and fourier transform infrared spectroscopy analyses. As a result, scanning electron microscopy observations showed that the glutamine loaded nanofibers had average diameter of 160nm. Glutamine amount was found to be 0.452mg/cm(2). Work of mucoadhesion, tensile strength and elongation at break values of the glutamine loaded nanofibers were found to be 0.165mJ/cm(2), 2.61mPa and 6.62% respectively. In vitro dissolution tests showed that more than 85% of the drug was diffused from the nanofibers at the end of 4h. Stability studies showed that there was no significant changes at 4 and 25°C/65% relative humidity storage conditions. Therefore, these results demonstrate that glutamine loaded nanofibers could have potential as an oromucosal drug delivery system for the treatment oral mucositis. PMID:27516332

  8. Preparation and characterization of electrospun nanofibers containing glutamine.

    PubMed

    Tort, Serdar; Acartürk, Füsun

    2016-11-01

    Oral mucositis is a painful inflammation of mucous membranes commonly after chemotherapy or radiotherapy. The aim of this study was to develop mucoadhesive nanofibers containing glutamine via electrospinning and to characterize them for the treatment of oral mucositis. Different mucoadhesive polymers were tried for preparing nanofibers and sodium alginate nanofibers were chosen after the characterization studies. Glutamine-loaded nanofibers were produced and characterized. Glutamine loaded onto nanofibers was confirmed by differantial scanning calorimetry and fourier transform infrared spectroscopy analyses. As a result, scanning electron microscopy observations showed that the glutamine loaded nanofibers had average diameter of 160nm. Glutamine amount was found to be 0.452mg/cm(2). Work of mucoadhesion, tensile strength and elongation at break values of the glutamine loaded nanofibers were found to be 0.165mJ/cm(2), 2.61mPa and 6.62% respectively. In vitro dissolution tests showed that more than 85% of the drug was diffused from the nanofibers at the end of 4h. Stability studies showed that there was no significant changes at 4 and 25°C/65% relative humidity storage conditions. Therefore, these results demonstrate that glutamine loaded nanofibers could have potential as an oromucosal drug delivery system for the treatment oral mucositis.

  9. Crystal Structure Analysis of Human Glutamine : Fructose 6-Phosphate Amidotransferase, a Key Regulator in Type 2 Diabetes

    NASA Astrophysics Data System (ADS)

    Nakaishi, Yuichiro; Bando, Masahiko

    Glutamine : fructose 6-phosphate amidotransferase (GFAT) is a rate-limiting enzyme in the hexoamine biosythetic pathway and plays an important role in type 2 diabetes. We now report the first structures of the isomerase domain of the human GFAT in the presence of cyclic glucose 6-phosphate and linear glucosamine 6-phosphate. The C-terminal tail including the active site displays a rigid conformation, similar to the corresponding Escherichia coli enzyme. The diversity of the CF helix near the active site suggests the helix is a major target for drug design. Our study provides insights into the development of therapeutic drugs for type 2 diabetes.

  10. Low-Cost Optical Lifetime Assisted Ratiometric Glutamine Sensor Based on Glutamine Binding Protein

    PubMed Central

    Lam, Hung; Kostov, Yordan; Rao, Govind; Tolosa, Leah

    2008-01-01

    Here we report a reagentless fluorescence sensing technique for glutamine in the submicromolar range based on the glutamine binding protein (QBP). The S179C mutant is labeled with the short-lived acrylodan (lifetime<5ns) and the long-lived tris(dibenzoylmethane) mono(5-amino-1,10-phenanthroline)europium(III) (lifetime>300µs) at the –SH and the N-terminal positions, respectively. In the presence of glutamine the fluorescence of acrylodan is quenched, while the fluorescence of europium complex remains constant. In this report we describe an innovative technique, the so called lifetime assisted ratiometric sensing to discriminate the two fluorescence signals using minimal optics and power requirements. This method exploits the large difference between the fluorescence lifetimes of the two fluorophores to isolate the individual fluorescence from each other by alternating the modulation frequency of the excitation light between 300 Hz and 10 kHz. The result is a ratiometric optical method that does not require expensive and highly attenuating band pass filters for each of the dyes, but only one long pass filter for both. Thus, the signal to noise ratio is enhanced, and at the same time, the optical setup is simplified. The end product is a simple sensing device suitable for low-cost applications such as point-of-care diagnostics or in-the-field analysis. PMID:18786501

  11. Enzyme-free surface plasmon resonance aptasensor for amplified detection of adenosine via target-triggering strand displacement cycle and Au nanoparticles.

    PubMed

    Yao, Gui-Hong; Liang, Ru-Ping; Huang, Chun-Fang; Zhang, Li; Qiu, Jian-Ding

    2015-04-29

    Herein, we combine the advantage of aptamer technique with the amplifying effect of an enzyme-free signal-amplification and Au nanoparticles (NPs) to design a sensitive surface plasmon resonance (SPR) aptasensor for detecting small molecules. This detection system consists of aptamer, detection probe (c-DNA1) partially hybridizing to the aptamer strand, Au NPs-linked hairpin DNA (Au-H-DNA1), and thiolated hairpin DNA (H-DNA2) previously immobilized on SPR gold chip. In the absence of target, the H-DNA1 possessing hairpin structure cannot hybridize with H-DNA2 and thereby Au NPs will not be captured on the SPR gold chip surface. Upon addition of target, the detection probe c-DNA1 is forced to dissociate from the c-DNA1/aptamer duplex by the specific recognition of the target to its aptamer. The released c-DNA1 hybridizes with Au-H-DNA1 and opens the hairpin structure, which accelerate the hybridization between Au-H-DNA1 and H-DNA2, leading to the displacement of the c-DNA1 through a branch migration process. The released c-DNA1 then hybridizes with another Au-H-DNA1 probe, and the cycle starts anew, resulting in the continuous immobilization of Au-H-DNA1 probes on the SPR chip, generating a significant change of SPR signal due to the electronic coupling interaction between the localized surface plasma of the Au NPs and the surface plasma wave. With the use of adenosine as a proof-of-principle analyte, this sensing platform can detect adenosine specifically with a detection limit as low as 0.21 pM, providing a simple, sensitive and selective protocol for small target molecules detection. PMID:25847158

  12. Neurological implications of urea cycle disorders.

    PubMed

    Gropman, A L; Summar, M; Leonard, J V

    2007-11-01

    The urea cycle disorders constitute a group of rare congenital disorders caused by a deficiency of the enzymes or transport proteins required to remove ammonia from the body. Via a series of biochemical steps, nitrogen, the waste product of protein metabolism, is removed from the blood and converted into urea. A consequence of these disorders is hyperammonaemia, resulting in central nervous system dysfunction with mental status changes, brain oedema, seizures, coma, and potentially death. Both acute and chronic hyperammonaemia result in alterations of neurotransmitter systems. In acute hyperammonaemia, activation of the NMDA receptor leads to excitotoxic cell death, changes in energy metabolism and alterations in protein expression of the astrocyte that affect volume regulation and contribute to oedema. Neuropathological evaluation demonstrates alterations in the astrocyte morphology. Imaging studies, in particular (1)H MRS, can reveal markers of impaired metabolism such as elevations of glutamine and reduction of myoinositol. In contrast, chronic hyperammonaemia leads to adaptive responses in the NMDA receptor and impairments in the glutamate-nitric oxide-cGMP pathway, leading to alterations in cognition and learning. Therapy of acute hyperammonaemia has relied on ammonia-lowering agents but in recent years there has been considerable interest in neuroprotective strategies. Recent studies have suggested restoration of learning abilities by pharmacological manipulation of brain cGMP with phosphodiesterase inhibitors. Thus, both strategies are intriguing areas for potential investigation in human urea cycle disorders.

  13. Cross-linking with diimidates of glutamine synthetase from Bacillus stearothermophilus.

    PubMed

    Sekiguchi, T; Oshiro, S; Goingo, E M; Nosoh, Y

    1979-08-01

    Glutamine synthetase [EC 6.3.2.1] from Bacillus stearothermophilus was modified with diethyl malonimidate (DEM), dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). DMA modified most epsilon-amino groups. On modification with DMA, formation of 3 to 4 cross-links/subunit resulted in a large increase in thermostability. The activity, allosteric properties and fluorescence spectrum of the enzyme were not changed on cross-linking. The SDS-polyacrylamide gel electrophoretic profiles of DEM-, DMA-, and DMS-modified enzymes suggested that the interaction berween six subunits in each of the two hexagonal rings of the protein are heterologous and are different from those between the piled subunits on different rings. PMID:39071

  14. Plasma glutamine concentration in spinal cord injured patients.

    PubMed

    Rogeri, P S; Costa Rosa, L F B P

    2005-09-23

    Glutamine, a non-essential amino acid, is the most important source of energy for macrophages and lymphocytes. Reduction in its plasma concentration is related with loss of immune function, as leukocyte proliferation and cytokine production. It is well known that glutamine is largely produced by the skeletal muscle which is severely compromised as a consequence of the paralysis due to the damage of the spinal cord. In spinal cord injury (SCI) patients, infections, such as pneumonia and sepsis in general, are a major cause of morbidity and mortality. In comparison with the control group, a 54% decrease in plasma glutamine concentration was observed as well as a decrease in the production of TNF and IL-1 by peripheral blood mononuclear cells cultivated for 48 h in SCI patients. Therefore, we propose that a decrease in plasma glutamine concentration is an important contributor to the immunosuppression seen in SCI patients. PMID:16024049

  15. Role of glucocorticoids in increased muscle glutamine production in starvation

    NASA Technical Reports Server (NTRS)

    Tischler, Marc E.; Henriksen, Erik J.; Cook, Paul H.

    1988-01-01

    The role of glucocorticoids in the synthesis of muscle glutamine during starvation was investigated in adrenalectomized fasted rats injected with cortisol (1 mg/100 g body weight). It was found that administration of cortisol in vivo increased (compared to nontreated starved adrenalectomized controls) the glutamine/glutamate ratio and the activity of glutamine synthetase in the diaphragm and the extensor digitorum muscles, and that these effects were abolished by prior treatment with actinomycin D or proflavine. The results obtained in in vitro experiments, using fresh-frozen soleus, extensor digitorum longus, and diaphragm muscle preparations, supported the in vivo indications of the cortisol-enhanced glutamine synthesis and protein turnover in starved adrenalectomized animals.

  16. Identification of the reactive cysteinyl residue and ATP binding site in Bacillus cereus glutamine synthetase by chemical modification.

    PubMed

    Nakano, Y; Itoh, M; Tanaka, E; Kimura, K

    1990-02-01

    Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.

  17. Ethylene-Induced Increase in Glutamine Synthetase Activity and mRNA Levels in Hevea brasiliensis Latex Cells.

    PubMed Central

    Pujade-Renaud, V.; Clement, A.; Perrot-Rechenmann, C.; Prevot, J. C.; Chrestin, H.; Jacob, J. L.; Guern, J.

    1994-01-01

    Ethylene, used as a stimulant of latex production in Hevea brasiliensis, significantly activates the regenerating metabolism within the laticiferous cells. In this context, attention was focused on glutamine synthetase (GS; EC 6.3.1.2), a key enzyme in nitrogen metabolism. A specific and significant activation of the cytosolic glutamine synthetase (GScyt) in the laticiferous cells after ethylene treatment parallels the increase of latex yield. A marked accumulation of the corresponding mRNA was found, but in contrast, a slight and variable increase of the polypeptide level is at the limit of detection by western blotting. The GS response to ethylene might be mediated by ammonia that increases in latex cytosol following ethylene treatment. The physiological significance for such a regulation by ethylene of the GScyt is discussed in terms of the nitrogen requirement for protein synthesis associated with latex regeneration. PMID:12232192

  18. From Krebs to clinic: glutamine metabolism to cancer therapy.

    PubMed

    Altman, Brian J; Stine, Zachary E; Dang, Chi V

    2016-10-01

    The resurgence of research into cancer metabolism has recently broadened interests beyond glucose and the Warburg effect to other nutrients, including glutamine. Because oncogenic alterations of metabolism render cancer cells addicted to nutrients, pathways involved in glycolysis or glutaminolysis could be exploited for therapeutic purposes. In this Review, we provide an updated overview of glutamine metabolism and its involvement in tumorigenesis in vitro and in vivo, and explore the recent potential applications of basic science discoveries in the clinical setting. PMID:27492215

  19. From Krebs to clinic: glutamine metabolism to cancer therapy.

    PubMed

    Altman, Brian J; Stine, Zachary E; Dang, Chi V

    2016-10-01

    The resurgence of research into cancer metabolism has recently broadened interests beyond glucose and the Warburg effect to other nutrients, including glutamine. Because oncogenic alterations of metabolism render cancer cells addicted to nutrients, pathways involved in glycolysis or glutaminolysis could be exploited for therapeutic purposes. In this Review, we provide an updated overview of glutamine metabolism and its involvement in tumorigenesis in vitro and in vivo, and explore the recent potential applications of basic science discoveries in the clinical setting.

  20. A second glutamine synthetase gene with expression in the gills of the gulf toadfish (opsanus beta)

    SciTech Connect

    Walsh, Patrick J.; Mayer, Gregory D.; Medina, Monica; Bernstein, Matthew L.; Barimo, John F.; Mommsen, Thomas P.

    2003-05-08

    Enzyme and molecular biology approaches were used to more completely characterize the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in a variety of tissues of the gulf toadfish (Opsanus beta) subjected to unconfined (ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that while weight-specific GSase activities rank in the order of brain > liver > stomach {approx} kidney > intestine > gill> heart/spleen > muscle, when tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle > stomach and intestine with minor contributions from the remaining tissues. Additionally, during confinement stress, GSase activity only increases significantly in liver (5-fold) and muscle (2-fold), tissues which previously showed significant expression of the other enzymes of urea synthesis. RT PCR and RACE PCR revealed the presence of a second GSas e cDNA from gill tissue that appears to share relatively low nucleotide and amino acid sequence similarity ({approx}73 percent) with the original GSase cloned from liver, and furthermore lacks a mitochondrial leader targeting sequence. RT PCR and restriction digestion experiments demonstrated that mRNA from the original ''liver'' GSase is expressed in all tissues examined (liver, gill, stomach, intestine, kidney, brain and muscle), whereas the new ''gill'' form shows expression primarily in the gill. Enzyme activities of gill GSase also exhibit a different subcellular compartmentation with apparent exclusive expression in the soluble compartment, whereas other tissues expressing the ''liver'' form show both cytoplasmic and mitochondrial activities. Finally, phylogenetic analysis of a number of GSases demonstrates that the toadfish gill GSase has a greater affinity for a clade that includes the Xenopus GSase genes and one of two Fugu GSase genes, than it has for a clade

  1. N-acetyl-L-glutamate and the urea cycle in gulf toadfish (Opsanus beta) and other fish.

    PubMed

    Julsrud, E A; Walsh, P J; Anderson, P M

    1998-02-01

    Carbamoyl phosphate synthetase I (CPSase I) catalyzes the first reaction of the urea cycle in mammalian ureotelic species. The positive allosteric cofactor N-acetyl-L-glutamate (AGA) is required for CPSase I activity and is important for regulation of the urea cycle. A similar enzyme, CPSase III, catalyzes this reaction in fish; CPSase III differs from CPSase I in that it utilizes glutamine as the nitrogen-donating substrate instead of ammonia. AGA also stimulates the CPSase III-catalyzed reaction, but is not absolutely required for activity if the glutamine concentration is high. There has been no report of the presence or function of AGA in fish. Here we report that AGA is present in those species and tissues of fish that have significant levels of CPSase III and urea cycle activity; the levels of AGA were higher in liver of adult gulf toadfish (Opsanus beta) and spiny dogfish shark (Squalus acanthias), both of which have high CPSase III activity, than in bass (Micropterus salmoides) or trout (Oncorhynchus mykiss), which have much lower or no CPSase III activity, respectively. In the toadfish the levels of AGA in liver and muscle tissue were considerably higher in the fed than in the fasting state, as is observed in mammalian species; in liver, but not in muscle, the level of AGA increased when the toadfish were confined (stressed), which has been shown to induce a ureotelic response. Toadfish muscle had CPSase III and ornithine carbamoyltransferase activities; the increase in AGA concentration in muscle when fed suggests that the presence of these first two enzymes of the urea cycle in muscle may be physiologically significant. The results indicate that the fish investigated have physiologically significant levels of AGA and that the levels correlate with parameters related to urea cycle activity. PMID:9466820

  2. Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

    PubMed

    Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

    2015-11-01

    Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

  3. The ubiquitin conjugating enzyme UbcH10 competes with UbcH3 for binding to the SCF complex, a ubiquitin ligase involved in cell cycle progression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ubiquitylation, which regulates most biological pathways, occurs through an enzymatic cascade involving a ubiquitin (ub) activating enzyme (E1), a ub conjugating enzyme (E2) and a ub ligase (E3). UbcH3 is the E2 that interacts with SCF (Skp1/Cul1/F-box protein) complex and ubiquitylates many protein...

  4. ACTIVITIES OF AMMONIA ASSIMILATION ENZYMES AS INDICATORS OF THE RELATIVE SUPPLY OF NITROGEN SUBSTRATES FOR MARINE BACTERIOPLANKTON IN SUB-TROPICAL COASTAL WATER

    EPA Science Inventory

    The supply of nitrogen substrates available for bacterial production in seawater was determined using the activities of ammonia assimilation enzymes, glutamine synthetase (GS) and glutamate dehydrogenase (GDH). Expression of GS and GDH by bacteria in pure culture is generally ind...

  5. Expression of genes encoding enzymes involved in the one carbon cycle in rat placenta is determined by maternal micronutrients (folic acid, vitamin B12) and omega-3 fatty acids.

    PubMed

    Khot, Vinita; Kale, Anvita; Joshi, Asmita; Chavan-Gautam, Preeti; Joshi, Sadhana

    2014-01-01

    We have reported that folic acid, vitamin B12, and omega-3 fatty acids are interlinked in the one carbon cycle and have implications for fetal programming. Our earlier studies demonstrate that an imbalance in maternal micronutrients influence long chain polyunsaturated fatty acid metabolism and global methylation in rat placenta. We hypothesize that these changes are mediated through micronutrient dependent regulation of enzymes in one carbon cycle. Pregnant dams were assigned to six dietary groups with varying folic acid and vitamin B12 levels. Vitamin B12 deficient groups were supplemented with omega-3 fatty acid. Placental mRNA levels of enzymes, levels of phospholipids, and glutathione were determined. Results suggest that maternal micronutrient imbalance (excess folic acid with vitamin B12 deficiency) leads to lower mRNA levels of methylene tetrahydrofolate reductase (MTHFR) and methionine synthase , but higher cystathionine b-synthase (CBS) and Phosphatidylethanolamine-N-methyltransferase (PEMT) as compared to control. Omega-3 supplementation normalized CBS and MTHFR mRNA levels. Increased placental phosphatidylethanolamine (PE), phosphatidylcholine (PC), in the same group was also observed. Our data suggests that adverse effects of a maternal micronutrient imbalanced diet may be due to differential regulation of key genes encoding enzymes in one carbon cycle and omega-3 supplementation may ameliorate most of these changes.

  6. Expression of glutamine synthetase in Tegillarca granosa (Bivalvia, Arcidae) hemocytes stimulated by Vibrio parahaemolyticus and lipopolysaccharides.

    PubMed

    Bao, Y B; Li, L; Ye, M X; Dong, Y H; Jin, W X; Lin, Z H

    2013-01-01

    The blood cockle, Tegillarca granosa, is a widely consumed clam in the Indo-Pacific region. Glutamine synthetase (GS) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. We identified the GS of T. granosa (Tg-GS) from hemocytes by 3'- and 5'-rapid amplification of cDNA ends (RACE)-PCR. The full-length cDNA consisted of 1762 bp, with a 1104-bp open reading frame encoding 367 amino acids. Sequence comparison showed that Tg-GS has homology to GS of other organisms, with 79.78% identity with GS from the Pacific oyster Crassostrea gigas, 71.98% identity with GS from the zebrafish Danio rerio, and 68.96% identity with human Homo sapiens GS. A C-beta-Grasp domain and an N-catalytic domain were identified in Tg-GS, indicating that Tg-GS should be classified as a new member of the GS family. A quantitative RT-PCR assay was used to detect mRNA expression of Tg-GS in five different tissues. Higher levels of mRNA expression of GS were detected in the tissues of hemocytes and the mantle. Up-regulation of GS by challenge with the bacteria Vibrio parahaemolyticus and with bacterial wall lipopolysaccharides showed that GS plays a role in anti-bacterial immunity. We conclude that pathogen infection significantly induces expression level of Tg- GS, and that activation of GS influences the immune response of T. granosa by increasing glutamine concentration. PMID:23661439

  7. Effects of glutamine and hyperoxia on pulmonary oxygen uptake and muscle deoxygenation kinetics.

    PubMed

    Marwood, Simon; Bowtell, Joanna L

    2007-01-01

    The aim of the present study was to determine whether glutamine ingestion, which has been shown to enhance the exercise-induced increase in the tricarboxylic acid intermediate (TCAi) pool size, resulted in augmentation of the rate of increase in oxidative metabolism at the onset of exercise. In addition, the potential interaction with oxygen availability was investigated by completing exercise in both normoxic and hyperoxic conditions. Eight male cyclists cycled for 6 min at 70% VO2max following consumption of a drink (5 ml kg body mass(-1)) containing a placebo or 0.125 g kg body mass(-1) of glutamine in normoxic (CON and GLN respectively) and hyperoxic (HYP and HPG respectively) conditions. Breath-by-breath pulmonary oxygen uptake and continuous, non-invasive muscle deoxygenation (via near infrared spectroscopy: NIRS) data were collected throughout exercise. The time constant of the phase II component of pulmonary oxygen uptake kinetics was unchanged between trials (CON: 21.5 +/- 3.0 vs. GLN: 18.2 +/- 1.3 vs. HYP: 18.9 +/- 2.0 vs. HPG: 18.6 +/- 1.2 s). There was also no alteration of the kinetics of relative muscle deoxygenation as measured via NIRS (CON: 5.9 +/- 0.7 vs. GLN: 7.3 +/- 0.8 vs. HYP: 6.5 +/- 0.9 vs. HPG: 5.2 +/- 0.4 s). Conversely, the mean response time of pulmonary oxygen uptake kinetics was faster (CON: 33.4 +/- 1.2 vs. GLN: 29.8 +/- 2.3 vs. HYP: 33.2 +/- 2.6 vs. HPG: 31.6 +/- 2.6 s) and the time at which muscle deoxygenation increased above pre-exercise values was earlier (CON: 9.6 +/- 0.9 vs. GLN: 8.7 +/- 1.1 vs. HYP: 8.5 +/- 0.8 vs. HPG: 8.4 +/- 0.7 s) following glutamine ingestion. In normoxic conditions, plasma lactate concentration was lower following glutamine ingestion compared to placebo. Whilst the results of the present study provide some support for the present hypothesis, the lack of any alteration in the time constant of pulmonary oxygen uptake and muscle deoxygenation kinetics suggest that the normal exercise induced expansion of

  8. Allosteric regulation of the state of adenylylation of glutamine synthetase in permeabilized cell preparations of Rhodopseudomonas sphaeroides.

    PubMed

    Wang, X; Song, H Y

    1989-08-01

    Following a freeze-thaw cycle, and the treatment of Rhodopseudomonas sphaeroides with the nonionic detergent Lubrol PX, the permeabilized cell suspensions can be assayed directly both for the intracellular levels of glutamine synthetase and the state of adenylylation (i.e. the average number n of adenylylated subunits/dodecameric molecules). It seems that all components of the bicycle system are retained if cells grown with low concentrations of ammonia as the sole nitrogen source are used. The value of n was dependent upon the concentration of substrates (ATP, Pi) and allosteric effectors (ATP, glutamine and alpha-ketoglutarate) of adenylytransferase. The value of n affected by UTP, the specific substrate of the uridylyltransferase shows first the evidence that the bicycle cascade control system studied in Escherichia coli may exist in this phototrophic bacterium. PMID:2575389

  9. Glutamine Synthetase of Nicotiana plumbaginifolia1

    PubMed Central

    Tingey, Scott V.; Coruzzi, Gloria M.

    1987-01-01

    We have characterized the distinct forms of glutamine synthetase (GS) which are present in leaves and roots of Nicotiana plumbaginifolia. Mature leaves contain a single GS polypeptide (44 kilodaltons in size) which is localized to the stroma of intact chloroplasts. In contrast, the GS polypeptide in roots is distinct in size (38 kilodaltons) and charge. A lectin stain of leaf soluble protein indicates that the size difference of these mature GS polypeptides is not the result of posttranslational glycosylation. cDNA clones encoding a GS mRNA of N. plumbaginifolia were characterized and used as molecular probes to examine GS transcripts in leaves and roots. GS mRNA hybrid-selected from leaves or roots translated in vitro into distinct GS primary translation products (49 or 38 kilodaltons). The 49 kilodalton GS primary translation product, specific to leaf poly(A)RNA is proposed to be a precursor to the mature 44 kilodalton chloroplast stromal GS polypeptide. The 38 kilodalton GS primary translation product encoded by root GS mRNA, corresponds in size to the polypeptide encoded by the GS cDNA clones characterized. Southern blot analysis of nuclear DNA indicates that there are several different genomic fragments encoding GS in N. plumbaginifolia. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 Fig. 6 Fig. 7 PMID:16665445

  10. Glutamine attenuates post-traumatic glutathione depletion in human muscle.

    PubMed

    Fläring, U B; Rooyackers, O E; Wernerman, J; Hammarqvist, F

    2003-03-01

    Glutathione is quantitatively the most important endogenous scavenger system. Glutathione depletion in skeletal muscle is pronounced following major trauma and sepsis in intensive care unit patients. Also, following elective surgery, glutathione depletion occurs in parallel with a progressive decline in muscle glutamine concentration. The present study was designed to test the hypothesis that glutamine supplementation may counteract glutathione depletion in a human trauma model. A homogeneous group of patients (n = 17) undergoing a standardized surgical procedure were prospectively randomly allocated to receive glutamine (0.56 g x day(-1) x kg(-1)) or placebo as part of isonitrogenous and isocaloric nutrition. Percutaneous muscle biopsies and blood samples were taken pre-operatively and at 24 and 72 h after surgery. The concentrations of muscle glutathione and related amino acids were determined in muscle tissue and plasma. In the control (unsupplemented) subjects, total muscle glutathione had decreased by 47+/-8% and 37+/-11% and reduced glutathione had decreased by 53+/-10% and 45+/-16% respectively at 24 and 72 h after surgery (P < 0.05). In contrast, in the glutamine-supplemented group, no significant post-operative decreases in total or reduced glutathione were seen following surgery. Muscle free glutamine had decreased at 72 h after surgery in both groups, by 41.4+/-14.8% (P < 0.05) in the glutamine-supplemented group and by 46.0+/-14.3% (P < 0.05) in the control group. In conclusion, the present study demonstrates that intravenous glutamine supplementation attenuates glutathione depletion in skeletal muscle in humans following standardized surgical trauma.

  11. Arginine deiminase resistance in melanoma cells is associated with metabolic reprogramming, glucose dependence, and glutamine addiction.

    PubMed

    Long, Yan; Tsai, Wen-Bin; Wangpaichitr, Medhi; Tsukamoto, Takashi; Savaraj, Niramol; Feun, Lynn G; Kuo, Macus Tien

    2013-11-01

    Many malignant human tumors, including melanomas, are auxotrophic for arginine due to reduced expression of argininosuccinate synthetase-1 (ASS1), the rate-limiting enzyme for arginine biosynthesis. Pegylated arginine deiminase (ADI-PEG20), which degrades extracellular arginine, resulting in arginine deprivation, has shown favorable results in clinical trials for treating arginine-auxotrophic tumors. Drug resistance is the major obstacle for effective ADI-PEG20 usage. To elucidate mechanisms of resistance, we established several ADI-PEG20-resistant (ADI(R)) variants from A2058 and SK-Mel-2 melanoma cells. Compared with the parental lines, these ADI(R) variants showed the following characteristics: (i) all ADI(R) cell lines showed elevated ASS1 expression, resulting from the constitutive binding of the transcription factor c-Myc on the ASS1 promoter, suggesting that elevated ASS1 is the major mechanism of resistance; (ii) the ADI(R) cell lines exhibited enhanced AKT signaling and were preferentially sensitive to PI3K/AKT inhibitors, but reduced mTOR signaling, and were preferentially resistant to mTOR inhibitor; (iii) these variants showed enhanced expression of glucose transporter-1 and lactate dehydrogenase-A, reduced expression of pyruvate dehydrogenase, and elevated sensitivity to the glycolytic inhibitors 2-deoxy-glucose and 3-bromopyruvate, consistent with the enhanced glycolytic pathway (the Warburg effect); (iv) the resistant cells showed higher glutamine dehydrogenase and glutaminase expression and were preferentially vulnerable to glutamine inhibitors. We showed that c-Myc, not elevated ASS1 expression, is involved in upregulation of many of these enzymes because knockdown of c-Myc reduced their expression, whereas overexpressed ASS1 by transfection reduced their expression. This study identified multiple targets for overcoming ADI-PEG resistance in cancer chemotherapy using recombinant arginine-degrading enzymes.

  12. Oxidative Turnover of Soybean Root Glutamine Synthetase. In Vitro and in Vivo Studies1

    PubMed Central

    Ortega, Jose Luis; Roche, Dominique; Sengupta-Gopalan, Champa

    1999-01-01

    Glutamine synthetase (GS) is the key enzyme in ammonia assimilation and catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine. GS in plants is an octameric enzyme. Recent work from our laboratory suggests that GS activity in plants may be regulated at the level of protein turnover (S.J. Temple, T.J. Knight, P.J. Unkefer, C. Sengupta-Gopalan [1993] Mol Gen Genet 236: 315–325; S.J. Temple, S. Kunjibettu, D. Roche, C. Sengupta-Gopalan [1996] Plant Physiol 112: 1723–1733; S.J. Temple, C. Sengupta-Gopalan [1997] In C.H. Foyer, W.P. Quick, eds, A Molecular Approach to Primary Metabolism in Higher Plants. Taylor & Francis, London, pp 155–177). Oxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria. By incubating soybean (Glycine max) root extract enriched in GS in a metal-catalyzed oxidation system to produce the ·OH radical, we have shown that GS is oxidized and that oxidized GS is inactive and more susceptible to degradation than nonoxidized GS. Histidine and cysteine protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the GS active site and that cysteine and histidine residues are the site of modification. Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative inactivation. The roots of plants fed ammonium nitrate showed a 3-fold increase in the level of GS polypeptides and activity compared with plants not fed ammonium nitrate but without a corresponding increase in the GS transcript level. This would suggest either translational or posttranslational control of GS levels. PMID:10198108

  13. Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis.

    PubMed

    Bearne, S L; Hekmat, O; Macdonnell, J E

    2001-05-15

    Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate gamma-semialdehyde. Glutamate gamma-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate gamma-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (K(is) 0.16+/-0.03 mM; K(ii) 0.4+/-0.1 mM) and a competitive inhibitor with respect to ammonia (K(i) 0.39+/-0.06 mM) in the presence of GTP at pH 8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate gamma-semialdehyde. Although glutamate gamma-semialdehyde exists in solution primarily in its cyclic form, Delta(1)-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Delta(1)-pyrroline-5-carboxylate (L-proline, L-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate gamma-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis. PMID:11336655

  14. Enteral Glutamine Administration in Critically Ill Nonseptic Patients Does Not Trigger Arginine Synthesis.

    PubMed

    Vermeulen, Mechteld A R; Brinkmann, Saskia J H; Buijs, Nikki; Beishuizen, Albertus; Bet, Pierre M; Houdijk, Alexander P J; van Goudoever, Johannes B; van Leeuwen, Paul A M

    2016-01-01

    Glutamine supplementation in specific groups of critically ill patients results in favourable clinical outcome. Enhancement of citrulline and arginine synthesis by glutamine could serve as a potential mechanism. However, while receiving optimal enteral nutrition, uptake and enteral metabolism of glutamine in critically ill patients remain unknown. Therefore we investigated the effect of a therapeutically relevant dose of L-glutamine on synthesis of L-citrulline and subsequent L-arginine in this group. Ten versus ten critically ill patients receiving full enteral nutrition, or isocaloric isonitrogenous enteral nutrition including 0.5 g/kg L-alanyl-L-glutamine, were studied using stable isotopes. A cross-over design using intravenous and enteral tracers enabled splanchnic extraction (SE) calculations. Endogenous rate of appearance and SE of glutamine citrulline and arginine was not different (SE controls versus alanyl-glutamine: glutamine 48 and 48%, citrulline 33 versus 45%, and arginine 45 versus 42%). Turnover from glutamine to citrulline and arginine was not higher in glutamine-administered patients. In critically ill nonseptic patients receiving adequate nutrition and a relevant dose of glutamine there was no extra citrulline or arginine synthesis and glutamine SE was not increased. This suggests that for arginine synthesis enhancement there is no need for an additional dose of glutamine when this population is adequately fed. This trial is registered with NTR2285.

  15. Enteral Glutamine Administration in Critically Ill Nonseptic Patients Does Not Trigger Arginine Synthesis

    PubMed Central

    Vermeulen, Mechteld A. R.; Brinkmann, Saskia J. H.; Buijs, Nikki; Beishuizen, Albertus; Bet, Pierre M.; Houdijk, Alexander P. J.; van Goudoever, Johannes B.; van Leeuwen, Paul A. M.

    2016-01-01

    Glutamine supplementation in specific groups of critically ill patients results in favourable clinical outcome. Enhancement of citrulline and arginine synthesis by glutamine could serve as a potential mechanism. However, while receiving optimal enteral nutrition, uptake and enteral metabolism of glutamine in critically ill patients remain unknown. Therefore we investigated the effect of a therapeutically relevant dose of L-glutamine on synthesis of L-citrulline and subsequent L-arginine in this group. Ten versus ten critically ill patients receiving full enteral nutrition, or isocaloric isonitrogenous enteral nutrition including 0.5 g/kg L-alanyl-L-glutamine, were studied using stable isotopes. A cross-over design using intravenous and enteral tracers enabled splanchnic extraction (SE) calculations. Endogenous rate of appearance and SE of glutamine citrulline and arginine was not different (SE controls versus alanyl-glutamine: glutamine 48 and 48%, citrulline 33 versus 45%, and arginine 45 versus 42%). Turnover from glutamine to citrulline and arginine was not higher in glutamine-administered patients. In critically ill nonseptic patients receiving adequate nutrition and a relevant dose of glutamine there was no extra citrulline or arginine synthesis and glutamine SE was not increased. This suggests that for arginine synthesis enhancement there is no need for an additional dose of glutamine when this population is adequately fed. This trial is registered with NTR2285. PMID:27200186

  16. Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism.

    PubMed

    You, Di; Yin, Bin-Cheng; Li, Zhi-Hai; Zhou, Ying; Yu, Wen-Bang; Zuo, Peng; Ye, Bang-Ce

    2016-06-14

    In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR-DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes. PMID:27247389

  17. Pb2+ exposure induced microsatellite instability in Pisum sativum in a locus related with glutamine metabolism.

    PubMed

    Rodriguez, E; Azevedo, R; Moreira, H; Souto, L; Santos, Conceição

    2013-01-01

    Lead (Pb) is a toxic element, but its putative mutagenic effects in plant cells, using molecular markers, remain to unveil. To evaluate if Pb induces mutagenicity, Pisum sativum L. seedlings were exposed to Pb(2+) (up to 2000 mg L(-1)) for 28 days and the instability of microsatellites (or Simple Sequence Repeats, SSR) was analyzed in leaves and roots. The analysis of eight selected microsatellites (SSR1-SSR8) demonstrated that only at the highest dosage microsatellite instability (MSI) occurred, at a frequency of 4.2%. Changes were detected in one microsatellite (SSR6) that is inserted in the locus for glutamine synthetase. SSR6 products of roots exposed to the highest concentration of Pb were 3 bp larger than those of the control. Our data demonstrate that: (a) SSR technique is sensitive to detect Pb-induced mutagenicity in plants. MSI instability is Pb dose dependent and organ dependent (roots are more sensitive); (b) the Pb-sensitive SSR6 is inserted in the glutamine synthetase locus, with still unknown relation with functional changes of this enzyme; (c) Pb levels inducing MSI are much above the maximum admitted levels in some European Union countries for agricultural purpose waters. In conclusion, we propose here the potential use of SSR to evaluate Pb(2+)-induced mutagenicity, in combination with other genetic markers.

  18. Supramolecular Probes for Assessing Glutamine Uptake Enable Semi-Quantitative Metabolic Models in Single Cells

    PubMed Central

    Xue, Min; Wei, Wei; Su, Yapeng; Johnson, Dazy; Heath, James R.

    2016-01-01

    We describe a supramolecular surface competition assay for quantifying glutamine uptake from single cells. Cy3-labeled cyclodextrins were immobilized on a glass surface as a supramolecular host/FRET donor, and adamantane-BHQ2 conjugates were employed as the guest/quencher. An adamantane-labeled glutamine analog was selected through screening a library of compounds and validated by cell uptake experiments. When integrated onto a single cell barcode chip (SCBC) with a multiplex panel of 15 other metabolites, associated metabolic enzymes, and phosphoproteins, the resultant data provided input for a steady state model that describes energy potential in single cells, and correlates that potential with receptor tyrosine kinase signaling. We utilize this integrated assay to interrogate a dose-dependent response of model brain cancer cells to EGFR inhibition. We find that low dose (1 μM erlotinib) drugging actually increases cellular energy potential even as glucose uptake and phosphoprotein signaling is repressed. We also identify new interactions between phosphoprotein signaling and cellular energy processes that may help explain the facile resistance exhibited by certain cancer patients to EGFR inhibitors. PMID:26916347

  19. Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies

    PubMed Central

    Frieg, Benedikt; Görg, Boris; Homeyer, Nadine; Keitel, Verena; Häussinger, Dieter; Gohlke, Holger

    2016-01-01

    Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically. PMID:26836257

  20. Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies.

    PubMed

    Frieg, Benedikt; Görg, Boris; Homeyer, Nadine; Keitel, Verena; Häussinger, Dieter; Gohlke, Holger

    2016-02-01

    Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.

  1. Glutamine--from conditionally essential to totally dispensable?

    PubMed

    Wernerman, Jan

    2014-01-01

    Recently a large multicentre randomised controlled trial in critically ill patients reported harm to the patients given supplementary glutamine. In the original publication, no explanation was offered for why this result was obtained; a large number of studies have reported beneficial effects or no effect, but never before reported harm. These results have been commented upon in a number of communications. Now some of the authors of the multicentre randomised controlled trial present a review and meta-analysis of glutamine supplementation, and the discrepancy of results is suggested to relate to intravenous administration to patients of supplementary glutamine via parenteral nutrition or a combination of enteral and parenteral nutrition in contrast to enteral administration of supplementation or a combination of enteral and parenteral supplementation. To explain results by epidemiological means only, by combining results into a meta-analysis, is perhaps not the best way to explain mechanisms behind results. Meta-analyses are primarily hypothesis generating. Launching treatment without a solid mechanistic explanation is always risky. Glutamine supplementation of the critically ill comes into that category. Now we will all have to do our homework and try to understand whether supplementation or omission of glutamine for patients fed parenterally is a good idea or not. PMID:25042856

  2. Glutamine Metabolism Regulates the Pluripotency Transcription Factor OCT4

    PubMed Central

    Marsboom, Glenn; Zhang, Guo-Fang; Pohl-Avila, Nicole; Zhang, Yanmin; Yuan, Yang; Kang, Hojin; Hao, Bo; Brunengraber, Henri; Malik, Asrar B.; Rehman, Jalees

    2016-01-01

    SUMMARY The molecular mechanisms underlying the regulation of pluripotency by cellular metabolism in human embryonic stem cells (hESCs) are not fully understood. We found that high levels of glutamine metabolism are essential to prevent degradation of OCT4, a key transcription factor regulating hESC pluripotency. Glutamine withdrawal depletes the endogenous anti-oxidant glutathione, which results in the oxidation of OCT4 cysteine residues required for its DNA binding and enhanced OCT4 degradation. The emergence of the OCT4lo cell population following glutamine withdrawal did not result in greater propensity for cell death. Instead, glutamine withdrawal during vascular differentiation of hESCs generated cells with greater angiogenic capacity, thus indicating that modulating glutamine metabolism enhances the differentiation and functional maturation of cells. These findings demonstrate that the pluripotency transcription factor OCT4 can serve as a metabolic-redox sensor in hESCs and that metabolic cues can act in concert with growth factor signaling to orchestrate stem cell differentiation. PMID:27346346

  3. Glutamine synthetase in liver of the American alligator, Alligator mississippiensis.

    PubMed

    Smith, D D; Campbell, J W

    1987-01-01

    Glutamine synthetase was shown to be localized in liver mitochondria of the American alligator, Alligator mississippiensis, by immunofluorescent staining of frozen liver sections and by the detection of enzymatic activity and immunoreactive protein in the mitochondrial fraction following subcellular fractionation of liver tissue by differential centrifugation. The primary translation product of alligator liver glutamine synthetase mRNA was shown to have an Mr = 45,000 which is similar if not identical in size to that of the mature subunit. This mRNA was found to be heterogeneous in size with a major form corresponding to 2.8-3.0 kb and a lesser form corresponding to around 2 kb. Both are in excess of the size required to code for the glutamine synthetase subunit. The synthesis and presumably the mitochondrial import of glutamine synthetase in alligator liver are thus very similar to the same processes in avian liver. Despite the excretion of a high percentage of nitrogen as ammonia, the demonstration of a mitochondrial glutamine synthetase indicates the alligator has the typical avian-type uricotelic ammonia-detoxification system in liver. This suggests that the transition to uricotelism occurred in the sauropsid line of evolution and has persisted through both the lepidosaurian (snakes, lizards) and archosaurian (dinosaurs, crocodilians, birds) lines.

  4. Hypoxic regulation of glutamine metabolism through HIF1 and SIAH2 supports lipid synthesis that is necessary for tumor growth.

    PubMed

    Sun, Ramon C; Denko, Nicholas C

    2014-02-01

    Recent reports have identified a phenomenon by which hypoxia shifts glutamine metabolism from oxidation to reductive carboxylation. We now identify the mechanism by which HIF-1 activation results in a dramatic reduction in the activity of the key mitochondrial enzyme complex α ketoglutarate dehydrogenase (αKGDH). HIF-1 activation promotes SIAH2 targeted ubiquitination and proteolysis of the 48 kDa splice variant of the E1 subunit of the αKGDH complex (OGDH2). Knockdown of SIAH2 or mutation of the ubiquitinated lysine residue on OGDH2 (336KA) reverses the hypoxic drop in αKGDH activity, stimulates glutamine oxidation, and reduces glutamine-dependent lipid synthesis. 336KA OGDH2-expressing cells require exogenous lipids or citrate for growth in hypoxia in vitro and fail to grow as model tumors in immunodeficient mice. Reversal of hypoxic mitochondrial function may provide a target for the development of next-generation anticancer agents targeting tumor metabolism. PMID:24506869

  5. Plasma glutamine responses to high-intensity exercise before and after endurance training.

    PubMed

    Kargotich, Stephen; Goodman, Carmél; Dawson, Brian; Morton, Alan R; Keast, David; Joske, David J L

    2005-01-01

    Glutamine responses to strenuous interval exercise were examined before and after 6 weeks of endurance training. Glutamine measures were obtained before and after the interval exercise sessions and training in untrained males assigned to training (T; n = 10) or control (C; n = 10) groups. Before training, C and T group glutamine progressively decreased (p < 0.05) by 18% and 16%, respectively, by 150-min postinterval exercise. Over the training period C group glutamine did not change, while T group values increased (p < 0.05) by 14%. After training, glutamine again decreased (p < 0.05) by similar percentages (C = 16% and T = 15%) by 150-min postinterval exercise, but the T group recorded higher (p < 0.05) resting and postexercise glutamine concentrations than the C group. Training induced increases in glutamine may prevent the decline in glutamine levels following strenuous exercise falling below a threshold where immune function might be acutely compromised. PMID:16440504

  6. Glutamine Supplementation in Intensive Care Patients

    PubMed Central

    Oldani, Massimo; Sandini, Marta; Nespoli, Luca; Coppola, Sara; Bernasconi, Davide Paolo; Gianotti, Luca

    2015-01-01

    Abstract The role of glutamine (GLN) supplementation in critically ill patients is controversial. Our aim was to analyze its potential effect in patients admitted to intensive care unit (ICU). We performed a systematic literature review through Medline, Embase, Pubmed, Scopus, Ovid, ISI Web of Science, and the Cochrane-Controlled Trials Register searching for randomized clinical trials (RCTs) published from 1983 to 2014 and comparing GLN supplementation to no supplementation in patients admitted to ICU. A random-effect meta-analysis for each outcome (hospital and ICU mortality and rate of infections) of interest was carried out. The effect size was estimated by the risk ratio (RR). Thirty RCTs were analyzed with a total of 3696 patients, 1825 (49.4%) receiving GLN and 1859 (50.6%) no GLN (control groups). Hospital mortality rate was 27.6% in the GLN patients and 28.6% in controls with an RR of 0.93 (95% CI = 0.81–1.07; P = 0.325, I2 = 10.7%). ICU mortality was 18.0 % in the patients receiving GLN and 17.6% in controls with an RR of 1.01 (95% CI = 0.86–1.19; P = 0.932, I2 = 0%). The incidence of infections was 39.7% in GLN group versus 41.7% in controls. The effect of GLN was not significant (RR = 0.88; 95% CI = 0.76–1.03; P = 0.108, I2 = 56.1%). These results do not allow to recommend GLN supplementation in a generic population of critically ills. Further RCTs are needed to explore the effect of GLN in more specific cohort of patients. PMID:26252319

  7. A Role for Glutamine Synthetase in the Remobilization of Leaf Nitrogen during Natural Senescence in Rice Leaves.

    PubMed

    Kamachi, K; Yamaya, T; Mae, T; Ojima, K

    1991-06-01

    Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of corresponding mRNAs were determined in leaves of hydroponically grown rice (Oryza sativa) plants during natural senescence. The plants were grown in the greenhouse for 105 days at which time the thirteenth leaf was fully expanded. This was counted as zero time for senescence of the twelfth leaf. The twelfth leaf blade on the main stem was analyzed over a time period of -7 days (98 days after germination) to +42 days (147 days after germination). Total GS activity declined to less than a quarter of its initial level during the senescence for 35 days and this decline was mainly caused by a decrease in the amount of GS2 polypeptide. Immunoblotting analyses showed that contents of other chloroplastic enzymes, such as ribulose-1,5-bisphosphate carboxylase/oxygenase and Fd-glutamate synthase, declined in parallel with GS2. In contrast, the GS1 polypeptide remained constant throughout the senescence period. Translatable mRNA for GS1 increased about fourfold during the senescence for 35 days. During senescence, there was a marked decrease in content of glutamate (to about one-sixth of the zero time value); glutamate is the major form of free amino acid in rice leaves. Glutamine, the major transported amino acid, increased about threefold compared to the early phase of the harvest in the senescing rice leaf blades. These observations suggest that GS1 in senescing leaf blades is responsible for the synthesis of glutamine, which is then transferred to the growing tissues in rice plants. PMID:16668201

  8. The Influence of Manganese and Glutamine Intake on Antioxidants and Neurotransmitter Amino Acids Levels in Rats' Brain.

    PubMed

    Szpetnar, Maria; Luchowska-Kocot, Dorota; Boguszewska-Czubara, Anna; Kurzepa, Jacek

    2016-08-01

    Depending on the concentration, Mn can exert protective or toxic effect. Potential mechanism for manganese neurotoxicity is manganese-induced oxidative stress. Glutamine supplementation could reduce manganese-induced neurotoxicity and is able to influence the neurotransmission processes. The aim of this study was to investigate whether the long term administration of manganese (alone or in combination with glutamine) in dose and time dependent manner could affect the selected parameters of oxidative-antioxidative status (superoxide dismutase and glutathione peroxidase activities, concentrations of vitamin C and malonic dialdehyde) and concentrations of excitatory (Asp, Glu) and inhibitory amino acids (GABA, Gly) in the brain of rats. The experiments were carried out on 2-months-old albino male rats randomly divided into 6 group: Mn300 and Mn500-received solution of MnCl2 to drink (dose 300 and 500 mg/L, respectively), Gln group-solution of glutamine (4 g/L), Mn300-Gln and Mn500-Gln groups-solution of Mn at 300 and 500 mg/L and Gln at 4 g/L dose. The control group (C) received deionized water. Half of the animals were euthanized after three and the other half-after 6 weeks of experiment. The exposure of rats to Mn in drinking water contributes to diminishing of the antioxidant enzymes activity and the increase in level of lipid peroxidation. Glutamine in the diet admittedly increases SOD and GPx activity, but it is unable to restore the intracellular redox balance. The most significant differences in the examined amino acids levels in comparison to both control and Gln group were observed in the group of rats receiving Mn at 500 mg/L dose alone or with Gln. It seems that Gln is amino acid which could improve antioxidant status and affect the concentrations of the neurotransmitters. PMID:27161372

  9. An in silico model of enterocytic glutamine to citrulline conversion pathway.

    PubMed

    Bensaci, J; Curis, E; Nicolis, I; de Bandt, J-P; Bénazeth, S

    2012-10-01

    Enterocyte is one of the main sites of amino acids metabolism and particularly of the citrulline biosynthesis. Working at the cellular scale and applying ordinary differential equations (ODEs) formalism, we have built a mathematical model of the enterocytic glutamine to citrulline conversion in the fasting state. This model enables us to test different physiopathological scenarios of enzyme activity loss. Results from two different approaches were compared: a standard approach (KA) based on the Michaelis-Menten assumptions and an association-dissociation approach (VH) based on the kinetic mass action law. For both approaches, ODEs system was numerically solved using Mathematica™. In both cases, the model correctly predicts the physiological plasma citrulline steady-state, but the two approaches present clear differences for metabolites of enzymes having a complex mechanism, challenging the validity of the KA approach in such cases. When physiopathological scenarios of enzyme activity loss are simulated, both approaches predict a very sharp transition from the physiological citrulline plasma level to the lack of its production: the concentration profiles of these simulations show a clear threshold of which characteristics vary with the involved enzyme. Moreover, amongst all enzymes included in the model, the ornithine aminotransferase (OAT) shows the highest sensitivity in the system whatever the approach used. This model points out the limits of the Michaelis-Menten approach to model complex enzyme mechanisms. It highlights the key role of OAT in the studied citrulline synthesis pathway and also suggests an order of magnitude about the optimal ratio of enzyme concentrations in this pathway. PMID:22399052

  10. Have we enough glutamine and how does it work? A clinician's view.

    PubMed

    Soeters, P B; Grecu, I

    2012-01-01

    There is a gap between the scientific basis of the claim that in several disease states glutamine is lacking and the widespread belief that supplementation of glutamine to the nutritional regimen is beneficial in severely ill patients. Glutamine shortage exists when consuming tissues, playing a crucial role in the response to trauma and disease, receive insufficient amounts of glutamine. In these tissues (immune system, wound), glutamine is only partly oxidized but has more specific roles as nontoxic nitrogen carrier, precursor of several crucial metabolites required for cell proliferation and for maintenance of the redox potential, and as osmolyte. In inflammatory states, glutamine concentrations in plasma and tissues are decreased due to many disease-related factors, precluding its use as a reliable indicator of shortage. Isotope studies have yielded equivocal results, precluding their use as a reliable indicator of glutamine shortage or adequacy. The increase in the net release of glutamine from peripheral tissues to central tissues (immune system, liver, spleen, wound) in inflammatory states provides a better basis for the necessity to supplement the organism with extra glutamine in these conditions. Glutamine supplementation was beneficial in a few studies in burn or trauma patients. The clinical benefit of parenteral glutamine supplementation in patients with severe inflammation has been demonstrated more convincingly. The amounts of glutamine supplemented approximate the amounts released by peripheral tissues and utilized by central organs operative in host defense and are therefore in the physiological range.

  11. On the existence of 'bis (L-glutamine) potassium nitrate' crystal

    NASA Astrophysics Data System (ADS)

    Srinivasan, Bikshandarkoil R.; Shyama, Soorambail K.; Naik, Suvidha G.; Jyai, Rita N.

    2015-02-01

    The slow evaporation of an aqueous solution containing L-glutamine and potassium nitrate in 2:1 mol ratio results in the fractional crystallization of L-glutamine and not the formation of a so called bis (L-glutamine) potassium nitrate as reported recently by Hanumantharao and Kalainathan (2012).

  12. Physical mapping of the human glutamine:fructose-6-phosphate amidotransferase gene (GFPT) to chromosome 2p13

    SciTech Connect

    Whitmore, T.E.; Mudri, S.L.; McKnight, G.L.

    1995-03-20

    Diabetic hyperglycemia influences insulin resistance through a process termed glucose toxicity. Implicated as a source of the mediators of this toxicity is an increased intracellular glucose metabolism through the hexosamine pathway. The hexosamine pathway itself is controlled by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT), which is the first enzyme of the pathway. It has been shown that there is a close correlation between the glucose-mediated reduction of GFAT activity and the onset of insulin desensitization of the glucose transport system, a condition associated with insulin-resistant states of non-insulin-dependent diabetes mellitus and obesity. To gain a better understanding of the molecular regulation of GFAT and its role in the induction of insulin resistance, we previously isolated and cloned the cDNA for the human form of this enzyme and expressed the functional protein in Escherichia coli. 9 refs., 1 fig.

  13. Dietary supplementation with glutamine and γ-aminobutyric acid improves growth performance and serum parameters in 22- to 35-day-old broilers exposed to hot environment.

    PubMed

    Hu, H; Bai, X; Shah, A A; Wen, A Y; Hua, J L; Che, C Y; He, S J; Jiang, J P; Cai, Z H; Dai, S F

    2016-04-01

    This study was designed using 360 21-day-old chicks to determine the influences of diet supplementation with glutamine (5 g/kg), γ-aminobutyric acid (GABA, 100 mg/kg) or their combinations on performance and serum parameters exposed to cycling high temperatures. From 22 to 35 days, the experimental groups (2 × 2) were subjected to circular heat stress by exposing them to 30-34 °C cycling, while the positive control group was exposed to 23 °C constant. The blood of broilers was collected to detect serum parameters on days 28 and 35. Compared with the positive control group, the cycling high temperature decreased (p < 0.05) the feed consumption, weight gain and serum total protein (TP), glucose, thyroxine (T4), insulin, alkaline phosphatase (ALP), glutamine, GABA and glutamate levels, while increased (p < 0.05) the serum triglyceride (TG), corticosterone (CS), glucagon (GN), creatine kinase (CK), glutamic oxaloacetic transaminase (GOT), nitric oxide synthase (NOS), glutamate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) levels during 22-35 days. However, dietary glutamine (5 g/kg) increased (p < 0.05) the feed consumption, weight gain and serum levels of glutamine, TP, insulin and ALP, but decreased (p < 0.05) the serum TG, CK, GOT, NOS and GPT levels. Diet supplemented with GABA also increased (p < 0.05) weight gain and the serum levels of TP, T4, ALP, GABA and glutamine. In addition, the significant interactions (p < 0.05) between glutamine and GABA were found in the feed consumption, weight gain and the serum ALP, CK, LDH, GABA, T3 and T4 levels of heat-stressed chickens. This research indicated that dietary glutamine and GABA improved the antistress ability in performance and serum parameters of broilers under hot environment. PMID:25980810

  14. Localization of transglutaminase-reactive glutamine residues in bovine osteopontin.

    PubMed Central

    Sørensen, E S; Rasmussen, L K; Møller, L; Jensen, P H; Højrup, P; Petersen, T E

    1994-01-01

    Here we report the identification of two transglutaminase-reactive glutamines (Gln-34 and Gln-36) in bovine osteopontin (OPN). Sequence alignment revealed that these glutamines are conserved in all known OPN sequences, indicating a functional importance of this region of the protein. Furthermore, immunological analysis of bovine bone demonstrated that OPN is present in high-molecular-mass complexes in vivo. These findings support the functional aspects of a transglutaminase-catalysed cross-linking of OPN in facilitating cellular attachment and tissue calcification. Images Figure 4 PMID:7998923

  15. A rare glutamine derivative from the flower buds of daylily.

    PubMed

    Matsumoto, Takahiro; Nakamura, Seikou; Ohta, Tomoe; Fujimoto, Katsuyoshi; Yoshikawa, Masayuki; Ogawa, Keiko; Matsuda, Hisashi

    2014-06-01

    A rare glutamine derivative, hemerocallisamine I (1), was isolated from the methanolic extract of the flower buds of daylily, together with a new pyrrole alkaloid hemerocallisamine II (2) and a new γ-lactam derivative, hemerocallisamine III (3). The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. For hemerocallisamine I (1), the absolute configuration was determined by Mo-Kα X-ray crystallographic analysis. This is the first report of a glutamine derivative with a pyrrole ring from natural plants. PMID:24835438

  16. How to understand the results of studies of glutamine supplementation.

    PubMed

    Wernerman, Jan

    2015-11-03

    The lack of understanding of the mechanisms behind possible beneficial and possible harmful effects of glutamine supplementation makes the design of interventional studies of glutamine supplementations difficult, perhaps even hazardous. What is the interventional target, and how might it relate to outcomes? Taking one step further and aggregating results from interventional studies into meta-analyses does not diminish the difficulties. Therefore, conducting basic research seems to be a better idea than groping in the dark and exposing patients to potential harm in this darkness.

  17. Computational studies of ammonia channel function in glutamine 5'-phosphoribosylpyrophosphate amidotransferase.

    PubMed

    Wang, Xiang S; Roitberg, Adrian E; Richards, Nigel G J

    2009-12-29

    Glutamine 5'-phosphoribosylpyrophosphate amidotransferase (GPATase) catalyzes the synthesis of 5'-phosphoribosylamine in a reaction that involves the translocation of ammonia along an intramolecular tunnel linking the two active sites of the enzyme. We now report a locally enhanced sampling (LES) strategy for modeling ammonia transfer between the active sites of Escherichia coli GPATase in its active conformation. Our calculations demonstrate that the ammonia channel in GPATase is best regarded as a "pipe" through which ammonia travels in the absence of an external "driving" potential. This combined LES/PMF computational approach, which offers a straightforward alternative to steered molecular dynamics simulations in studies of substrate channeling, also provides new insights into the molecular basis of the reduced ammonia transfer efficiency exhibited by the L415A GPATase mutant.

  18. Barley chloroplast glutamine synthetase activity is not affected by CO sub 2 -concentration

    SciTech Connect

    Avila, C.; Forde, B.; Wallsgrove, R. )

    1990-05-01

    It has been reported that when photorespiration is suppressed by raising the concentration of CO{sub 2}, the expression of the chloroplast glutamine synthetase (GS2) gene in pea leaves is reduced (Plant Cell, 1, 241). We have examined this effect in barley (Hordeum vulgare), and confirm that plants grown continuously in 0.8% CO{sub 2}, or transferred to such conditions after growth in air, appear to have a reduced GS2 mRNA abundance. However, we were unable to detect any significant difference in the extractable GS2 activity, or any change in amount of GS2 protein (judged by Western blots). Whatever controls are operating on gS2 mRNA expression in response to changes is external CO{sub 2}, they do not affect the activity or amount of the enzyme in barley.

  19. Neuronal Cell Death Induced by Mechanical Percussion Trauma in Cultured Neurons is not Preceded by Alterations in Glucose, Lactate and Glutamine Metabolism.

    PubMed

    Jayakumar, A R; Bak, L K; Rama Rao, K V; Waagepetersen, H S; Schousboe, A; Norenberg, M D

    2016-02-01

    Traumatic brain injury (TBI) is a devastating neurological disorder that usually presents in acute and chronic forms. Brain edema and associated increased intracranial pressure in the early phase following TBI are major consequences of acute trauma. On the other hand, neuronal injury, leading to neurobehavioral and cognitive impairments, that usually develop months to years after single or repetitive episodes of head trauma, are major consequences of chronic TBI. The molecular mechanisms responsible for TBI-induced injury, however, are unclear. Recent studies have suggested that early mitochondrial dysfunction and subsequent energy failure play a role in the pathogenesis of TBI. We therefore examined whether oxidative metabolism of (13)C-labeled glucose, lactate or glutamine is altered early following in vitro mechanical percussion-induced trauma (5 atm) to neurons (4-24 h), and whether such events contribute to the development of neuronal injury. Cell viability was assayed using the release of the cytoplasmic enzyme lactate dehydrogenase (LDH), together with fluorescence-based cell staining (calcein and ethidium homodimer-1 for live and dead cells, respectively). Trauma had no effect on the LDH release in neurons from 1 to 18 h. However, a significant increase in LDH release was detected at 24 h after trauma. Similar findings were identified when traumatized neurons were stained with fluorescent markers. Additionally (13)C-labeling of glutamate showed a small, but statistically significant decrease at 14 h after trauma. However, trauma had no effect on the cycling ratio of the TCA cycle at any time-period examined. These findings indicate that trauma does not cause a disturbance in oxidative metabolism of any of the substrates used for neurons. Accordingly, such metabolic disturbance does not appear to contribute to the neuronal death in the early stages following trauma. PMID:26729365

  20. Overproduction of peroxide-scavenging enzymes in Escherichia coli suppresses spontaneous mutagenesis and sensitivity to redox-cycling agents in oxyR-mutants.

    PubMed Central

    Greenberg, J T; Demple, B

    1988-01-01

    Mutations that suppressed the H2O2 sensitivity of Escherichia coli oxyR- strains caused elevated levels of one three enzymes that destroy organic and hydrogen peroxides: catalase-hydroperoxidase I (the katG gene product), catalase-hydroperoxidase II (controlled by katEF) or alkyl hydroperoxide reductase (specified by the ahp genes). The continuous high-level expression of any one of these enzymes also conferred resistance in an oxyR deletion mutant against other compounds such as N-ethylmaleimide and the superoxide-generator menadione. Overproduction of alkyl hydroperoxide reductase, but not of the catalases, gave resistance to the organic oxidant cumene hydroperoxide. The E. coli delta oxyR strains also exhibited a strongly elevated frequency of spontaneous mutagenesis, as reported for such mutants in Salmonella typhimurium. This mutagenesis was greatly diminished by the individual overexpression of these scavenging enzymes. All of these phenotypes--enzyme overproduction, resistance to oxidants and suppression of spontaneous mutagenesis--remained linked upon transduction of the mutant katG or ahp genes. Peroxides thus appear to mediate the toxicity of a variety of redox agents, and are produced in sufficient quantity during normal metabolism to cause a substantial increase in 'spontaneous' mutations in cells that lack adequate antioxidant defenses. Images PMID:2847922

  1. Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation.

    PubMed

    Sontakke, Pallavi; Koczula, Katarzyna M; Jaques, Jennifer; Wierenga, Albertus T J; Brouwers-Vos, Annet Z; Pruis, Maurien; Günther, Ulrich L; Vellenga, Edo; Schuringa, Jan Jacob

    2016-01-01

    The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells. PMID:27055152

  2. Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation

    PubMed Central

    Sontakke, Pallavi; Koczula, Katarzyna M.; Jaques, Jennifer; Wierenga, Albertus T. J.; Brouwers-Vos, Annet Z.; Pruis, Maurien; Günther, Ulrich L.; Vellenga, Edo; Schuringa, Jan Jacob

    2016-01-01

    The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells. PMID:27055152

  3. [Effects of different long-term fertilization on the activities of enzymes related to carbon, nitrogen, and phosphorus cycles in a red soil].

    PubMed

    Fan, Miao-zhen; Yin, Chang; Fan, Fen-liang; Song, A-lin; Wang, Bo-ren; Li, Dong-chu; Liang, Yong-chao

    2015-03-01

    Using a microplate fluorimetric assay method, five fertilization treatments, i.e. no-fertilizer control (CK) , sole application of nitrogen (N), balanced application of nitrogen, phosphorus, and potassium fertilizer (NPK), application of pig manure (M), and combination of pig manure with balanced chemical fertilizer (MNPK) were selected to investigate the effects of different long-term fertilization regimes on the activity of five enzymes (β-1, 4-glucosidase, βG; cellobiohydrolase, CBH; β-1, 4-xylosidase, βX; β-1, 4-N-acetylglucosaminidase, NAG; acid phosphatase, AP) in a red soil sampled from Qiyang, Hunnan Province. The results showed that compared with CK treatment, N treatment had no impact on βG, βX, CBH, and NAG activities but reduced AP activity, while NPK, M and MNPK treatments increased the activities of all the five enzymes. Correlation analysis indicated that all the five enzyme activities were positively correlated with the content of nitrate (r=0.465-0.733) , the content of available phosphorus (r=0.612-0.947) , soil respiration (r=0.781-0.949) and crop yield (r=0.735-0.960), while βG, CBH and AP were positively correlated with pH (r= 0.707-0.809), only AP was significantly correlated with dissolvable organic carbon (r = -0.480). These results suggested that the activities of the measured enzymes could be used as indicators of red soil fertility under different fertilization regimes, but the five enzymes tested provided limited information on the degree of acidification induced by application of mineral nitrogen. PMID:26211066

  4. [Effects of different long-term fertilization on the activities of enzymes related to carbon, nitrogen, and phosphorus cycles in a red soil].

    PubMed

    Fan, Miao-zhen; Yin, Chang; Fan, Fen-liang; Song, A-lin; Wang, Bo-ren; Li, Dong-chu; Liang, Yong-chao

    2015-03-01

    Using a microplate fluorimetric assay method, five fertilization treatments, i.e. no-fertilizer control (CK) , sole application of nitrogen (N), balanced application of nitrogen, phosphorus, and potassium fertilizer (NPK), application of pig manure (M), and combination of pig manure with balanced chemical fertilizer (MNPK) were selected to investigate the effects of different long-term fertilization regimes on the activity of five enzymes (β-1, 4-glucosidase, βG; cellobiohydrolase, CBH; β-1, 4-xylosidase, βX; β-1, 4-N-acetylglucosaminidase, NAG; acid phosphatase, AP) in a red soil sampled from Qiyang, Hunnan Province. The results showed that compared with CK treatment, N treatment had no impact on βG, βX, CBH, and NAG activities but reduced AP activity, while NPK, M and MNPK treatments increased the activities of all the five enzymes. Correlation analysis indicated that all the five enzyme activities were positively correlated with the content of nitrate (r=0.465-0.733) , the content of available phosphorus (r=0.612-0.947) , soil respiration (r=0.781-0.949) and crop yield (r=0.735-0.960), while βG, CBH and AP were positively correlated with pH (r= 0.707-0.809), only AP was significantly correlated with dissolvable organic carbon (r = -0.480). These results suggested that the activities of the measured enzymes could be used as indicators of red soil fertility under different fertilization regimes, but the five enzymes tested provided limited information on the degree of acidification induced by application of mineral nitrogen.

  5. Knockout mutants as a tool to identify the subunit composition of Arabidopsis glutamine synthetase isoforms.

    PubMed

    Dragićević, Milan; Todorović, Slađana; Bogdanović, Milica; Filipović, Biljana; Mišić, Danijela; Simonović, Ana

    2014-06-01

    Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, which catalyzes the formation of glutamine from ammonia and glutamate. Plant GS isoforms are multimeric enzymes, recently shown to be decamers. The Arabidopsis genome encodes five cytosolic (GS1) proteins labeled as GLN1;1 through GLN1;5 and one chloroplastic (GS2) isoform, GLN2;0. However, as many as 11 GS activity bands were resolved from different Arabidopsis tissues by Native PAGE and activity staining. Western analysis showed that all 11 isoforms are composed exclusively of 40 kDa GS1 subunits. Of five GS1 genes, only GLN1;1, GLN1;2 and GLN1;3 transcripts accumulated to significant levels in vegetative tissues, indicating that only subunits encoded by these three genes produce the 11-band zymogram. Even though the GS2 gene also had significant expression, the corresponding activity was not detected, probably due to inactivation. To resolve the subunit composition of 11 active GS1 isoforms, homozygous knockout mutants deficient in the expression of different GS1 genes were selected from the progeny of T-DNA insertional SALK and SAIL lines. Comparison of GS isoenzyme patterns of the selected GS1 knockout mutants indicated that all of the detected isoforms consist of varying proportions of GLN1;1, GLN1;2 and GLN1;3 subunits, and that GLN1;1 and GLN1;3, as well as GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 proteins combine in all proportions to form active homo- and heterodecamers.

  6. Effects of glutamine on wound healing.

    PubMed

    Kesici, Ugur; Kesici, Sevgi; Ulusoy, Hulya; Yucesan, Fulya; Turkmen, Aygen U; Besir, Ahmet; Tuna, Verda

    2015-06-01

    Studies reporting the need for replacing amino acids such as glutamine (Gln), hydroxymethyl butyrate (HMB) and arginine (Arg) to accelerate wound healing are available in the literature. The primary objective of this study was to present the effects of Gln on tissue hydroxyproline (OHP) levels in wound healing. This study was conducted on 30 female Sprague Dawley rats with a mean weight of 230 ± 20 g. Secondary wounds were formed by excising 2 × 1 cm skin subcutaneous tissue on the back of the rats. The rats were divided into three equal groups. Group C (Control): the group received 1 ml/day isotonic solution by gastric gavage after secondary wound was formed. Group A (Abound): the group received 0·3 g/kg/day/ml Gln, 0·052 g/kg/day/ml HMB and 0·3 g/kg/day/ml Arg by gastric gavage after secondary wound was formed. Group R (Resource): the group received 0·3 g/kg/day/ml Gln by gastric gavage after secondary wound was formed. The OHP levels of the tissues obtained from the upper half region on the 8th day and the lower half region on the 21st day from the same rats in the groups were examined. Statistical analysis was performed using the statistics program SPSS version 17.0. No statistically significant differences were reported with regard to the OHP measurements on the 8th and 21st days (8th day: F = 0·068, P = 0·935 > 0·05; 21st day: F = 0·018, P = 0·983 > 0·05). The increase in mean OHP levels on the 8th and 21st days within each group was found to be statistically significant (F = 1146·34, P = 0·000 < 0·001). We conclude that in adults who eat healthy food, who do not have any factor that can affect wound healing negatively and who do not have large tissue loss at critical level, Gln, Arg and HMB support would not be required to accelerate secondary wound healing.

  7. Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus.

    PubMed Central

    Montanini, Barbara; Betti, Marco; Márquez, Antonio J; Balestrini, Raffaella; Bonfante, Paola; Ottonello, Simone

    2003-01-01

    The nucleotide sequences reported in this paper have been submitted to the GenBank(R)/EBI Nucleotide Sequence Databases with accession numbers AF462037 (glutamine synthetase) and AF462032 (glutamate synthase). Nitrogen retrieval and assimilation by symbiotic ectomycorrhizal fungi is thought to play a central role in the mutualistic interaction between these organisms and their plant hosts. Here we report on the molecular characterization of the key N-assimilation enzyme glutamine synthetase from the mycorrhizal ascomycete Tuber borchii (TbGS). TbGS displayed a strong positive co-operativity ( n =1.7+/-0.29) and an unusually high S(0.5) value (54+/-16 mM; S(0.5) is the substrate concentration value at which v =(1/2) V (max)) for glutamate, and a correspondingly low sensitivity towards inhibition by the glutamate analogue herbicide phosphinothricin. The TbGS mRNA, which is encoded by a single-copy gene in the Tuber genome, was up-regulated in N-starved mycelia and returned to basal levels upon resupplementation of various forms of N, the most effective of which was nitrate. Both responses were accompanied by parallel variations of TbGS protein amount and glutamine synthetase activity, thus indicating that TbGS levels are primarily controlled at the pre-translational level. As revealed by a comparative analysis of the TbGS mRNA and of the mRNAs for the metabolically related enzymes glutamate dehydrogenase and glutamate synthase, TbGS is not only the sole messenger that positively responds to N starvation, but also the most abundant under N-limiting conditions. A similar, but even more discriminating expression pattern, with practically undetectable glutamate dehydrogenase mRNA levels, was observed in fruitbodies. The TbGS mRNA was also found to be expressed in symbiosis-engaged hyphae, with distinctively higher hybridization signals in hyphae that were penetrating among and within root cells. PMID:12683951

  8. Effect of intravenous amino acids on glutamine and protein kinetics in low-birth-weight preterm infants during the immediate neonatal period.

    PubMed

    Kadrofske, Mark M; Parimi, Prabhu S; Gruca, Lourdes L; Kalhan, Satish C

    2006-04-01

    Glutamine may be a conditionally essential amino acid in low-birth-weight (LBW) preterm neonates. Exogenously administered amino acids, by providing anaplerotic carbon into the tricarboxylic acid cycle, could result in greater cataplerotic efflux and glutamine de novo synthesis. The effect of dose and duration of amino acid infusion on glutamine and nitrogen (N) kinetics was examined in LBW infants in the period immediately after birth. Preterm neonates (<32 weeks gestation, birth weights 809-1,755 g) were randomized to initially receive either 480 or 960 micromol x kg(-1) x h(-1) of an intravenous amino acid solution for 19-24 hours, followed by a higher or lower amino acid load for either 5 h or 24 h. Glutamine de novo synthesis, leucine N, phenylalanine, and urea kinetics were determined using stable isotopic tracers. An increase in amino acid infusion from 480 to 960 micromol x kg(-1) x h(-1) for 5 h resulted in decreased glutamine de novo synthesis in every neonate (384.4 +/- 38.0 to 368.9 +/- 38.2 micromol x kg(-1) x h(-1), P < 0.01) and a lower whole body rate of proteolysis (P < 0.001) and urea synthesis (P < 0.001). However, when the increased amino acid infusion was extended for 24 h, glutamine de novo synthesis increased (369.7 +/- 92.6 to 483.4 +/- 97.5 micromol x kg(-1) x h(-1), P < 0.001), whole body rate of proteolysis did not change, and urea production increased. Decreasing the amino acid load resulted in a decrease in glutamine rate of appearance (R(a)) and leucine N R(a), but had no effect on phenylalanine R(a). Acutely stressed LBW infants responded to an increase in amino acid load by transiently suppressing whole body rate of glutamine synthesis, proteolysis, and oxidation of protein. The mechanisms of this transient effect on whole body protein/nitrogen metabolism remain unknown.

  9. Dysfunctional TCA-Cycle Metabolism in Glutamate Dehydrogenase Deficient Astrocytes.

    PubMed

    Nissen, Jakob D; Pajęcka, Kamilla; Stridh, Malin H; Skytt, Dorte M; Waagepetersen, Helle S

    2015-12-01

    Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids.

  10. Addition of glutamine to essential amino acids and carbohydrate does not enhance anabolism in young human males following exercise.

    PubMed

    Wilkinson, Sarah B; Kim, Paul L; Armstrong, David; Phillips, Stuart M

    2006-10-01

    We examined the effect of a post-exercise oral carbohydrate (CHO, 1 g.kg(-1).h(-1)) and essential amino acid (EAA, 9.25 g) solution containing glutamine (0.3 g/kg BW; GLN trial) versus an isoenergetic CHO-EAA solution without glutamine (control, CON trial) on muscle glycogen resynthesis and whole-body protein turnover following 90 min of cycling at 65% VO2 peak. Over the course of 3 h of recovery, muscle biopsies were taken to measure glycogen resynthesis and mixed muscle protein synthesis (MPS), by incorporation of [ring-2H5] phenylalanine. Infusion of [1-13C] leucine was used to measure whole-body protein turnover. Exercise resulted in a significant decrease in muscle glycogen (p < 0.05) with similar declines in each trial. Glycogen resynthesis following 3 h of recovery indicated no difference in total accumulation or rate of repletion. Leucine oxidation increased 2.5 fold (p < 0.05) during exercise, returned to resting levels immediately post-exercise,and was again elevated at 3 h post-exercise (p < 0.05). Leucine flux, an index of whole-body protein breakdown rate, was reduced during exercise, but increased to resting levels immediately post-exercise, and was further increased at 3 h post-exercise (p < 0.05), but only during the CON trial. Exercise resulted in a marked suppression of whole-body protein synthesis (50% of rest; p < 0.05), which was restored post-exercise; however, the addition of glutamine did not affect whole-body protein synthesis post-exercise. The rate of MPS was not different between trials. The addition of glutamine to a CHO + EAA beverage had no effect on post-exercise muscle glycogen resynthesis or muscle protein synthesis, but may suppress a rise in whole-body proteolysis during the later stages of recovery. PMID:17111006

  11. A metabolic perturbation by U0126 identifies a role for glutamine in resveratrol-induced cell death

    PubMed Central

    Kim, Jayoung; Lisanti, Michael P.; Di Vizio, Dolores

    2011-01-01

    Recent evidence has identified substantial overlap between metabolic and oncogenic biochemical pathways, suggesting novel approaches to cancer intervention. For example, cholesterol lowering statins and the antidiabetes medication metformin both act as chemopreventive agents in prostate and other cancers. The natural compound resveratrol has similar properties: increasing insulin sensitivity, suppressing adipogenesis, and inducing apoptotic death of cancer cells in vitro. However, in vivo tumor xenografts acquire resistance to resveratrol by an unknown mechanism, while mouse models of metabolic disorders respond more consistently to the compound. Here we demonstrate that castration-resistant human prostate cancer C4-2 cells are more sensitive to resveratrol-induced apoptosis than isogenic androgen-dependent LNCaP cells. The MEK inhibitor U0126 antagonized resveratrol-induced apoptosis in C4-2 cells, but this effect was not seen with other MEK inhibitors. U0126 was found to inhibit mitochondrial function and shift cells to aerobic glycolysis independently of MEK. Mitochondrial activity of U0126 arose through decomposition, producing both mitochondrial fluorescence and cyanide, a known inhibitor of complex IV. Applying U0126 mitochondrial inhibition to C4-2 cell apoptosis, we tested the possibility that glutamine supplementation of citric acid cycle intermediate α-ketoglutarate may be involved. Suppression of the conversion of glutamate to α-ketoglutarate antagonized resveratrol-induced death in C4-2 cells. A similar effect was also seen by reducing extracellular glutamine concentration in the culture medium, suggesting that resveratrol-induced death is dependent on glutamine metabolism, a process frequently dysregulated in cancer. Further work on resveratrol and metabolism in cancer is warranted to ascertain if the glutamine dependence has clinical implications. PMID:22108021

  12. [Cytochemical study of different stages in the life cycle of Toxoplasma gondii. VII. Oxidation--reduction enzymes in the cyst forms].

    PubMed

    Beyer, T V; Siim, J C; Hutchison, U M

    1977-11-01

    Dehydrogenases of glycolysis, Kreb's cycle, and pentose-phosphate shunt were detected in cystozoites of Toxoplasma gondii strain SS-119 with various degrees of activity. A mixed oxidative metabolism may be postulated on this stage of the toxoplasma life cycle. Besides, the activity of cytochrome oxidase was detected in cystozoites; the addition of cytochrome c to the incubation medium significantly intensified the reaction intensity. Of interest seems the observation of a layer of higher enzymatic activity in the host brain tissue in the immediate neighbourhood with the cyst body. This may be regarded as the host cells' (or tissue') response to the presence of the parasite's alien body.

  13. Extracellular enzyme activity and microbial diversity measured on seafloor exposed basalts from Loihi seamount indicate the importance of basalts to global biogeochemical cycling.

    PubMed

    Jacobson Meyers, Myrna E; Sylvan, Jason B; Edwards, Katrina J

    2014-08-01

    Seafloor basalts are widely distributed and host diverse prokaryotic communities, but no data exist concerning the metabolic rates of the resident microbial communities. We present here potential extracellular enzyme activities of leucine aminopeptidase (LAP) and alkaline phosphatase (AP) measured on basalt samples from different locations on Loihi Seamount, HI, coupled with analysis of prokaryotic biomass and pyrosequencing of the bacterial 16S rRNA gene. The community maximum potential enzyme activity (Vmax) of LAP ranged from 0.47 to 0.90 nmol (g rock)(-1) h(-1); the Vmax for AP was 28 to 60 nmol (g rock)(-1) h(-1). The Km of LAP ranged from 26 to 33 μM, while the Km for AP was 2 to 7 μM. Bacterial communities on Loihi basalts were comprised primarily of Alpha-, Delta-, andGammaproteobacteria, Bacteroidetes, and Planctomycetes. The putative ability to produce LAP is evenly distributed across the most commonly detected bacterial orders, but the ability to produce AP is likely dominated by bacteria in the orders Xanthomonadales, Flavobacteriales, and Planctomycetales. The enzyme activities on Loihi basalts were compared to those of other marine environments that have been studied and were found to be similar in magnitude to those from continental shelf sediments and orders of magnitude higher than any measured in the water column, demonstrating that the potential for exposed basalts to transform organic matter is substantial. We propose that microbial communities on basaltic rock play a significant, quantifiable role in benthic biogeochemical processes. PMID:24907315

  14. Glutamine, arginine, and leucine signaling in the intestine.

    PubMed

    Marc Rhoads, J; Wu, Guoyao

    2009-05-01

    Glutamine and leucine are abundant constituents of plant and animal proteins, whereas the content of arginine in foods and physiological fluids varies greatly. Besides their role in protein synthesis, these three amino acids individually activate signaling pathway to promote protein synthesis and possibly inhibit autophagy-mediated protein degradation in intestinal epithelial cells. In addition, glutamine and arginine stimulate the mitogen-activated protein kinase and mammalian target of rapamycin (mTOR)/p70 (s6) kinase pathways, respectively, to enhance mucosal cell migration and restitution. Moreover, through the nitric oxide-dependent cGMP signaling cascade, arginine regulates multiple physiological events in the intestine that are beneficial for cell homeostasis and survival. Available evidence from both in vitro and in vivo animal studies shows that glutamine and arginine promote cell proliferation and exert differential cytoprotective effects in response to nutrient deprivation, oxidative injury, stress, and immunological challenge. Additionally, when nitric oxide is available, leucine increases the migration of intestinal cells. Therefore, through cellular signaling mechanisms, arginine, glutamine, and leucine play crucial roles in intestinal growth, integrity, and function.

  15. Regional tumour glutamine supply affects chromatin and cell identity.

    PubMed

    Højfeldt, Jonas W; Helin, Kristian

    2016-09-28

    Limited perfusion of solid tumours produces a nutrient-deprived tumour core microenvironment. Low glutamine levels in the tumour core are now shown to lead to reduced levels of α-ketoglutarate and decreased histone demethylase activity, thereby promoting a less differentiated and more therapy-resistant state of the tumour cells.

  16. Regional tumour glutamine supply affects chromatin and cell identity.

    PubMed

    Højfeldt, Jonas W; Helin, Kristian

    2016-09-28

    Limited perfusion of solid tumours produces a nutrient-deprived tumour core microenvironment. Low glutamine levels in the tumour core are now shown to lead to reduced levels of α-ketoglutarate and decreased histone demethylase activity, thereby promoting a less differentiated and more therapy-resistant state of the tumour cells. PMID:27684506

  17. Glutamine: precursor or nitrogen donor for citrulline synthesis?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutamine (Gln) is considered the main precursor for citrulline (Cit) synthesis, but no attempts have been made to differentiate the contribution of Gln carbon (Gln-C) skeleton vs. the nonspecific contribution through NH3 and CO2. To study the contribution of dietary Gln-N to the synthesis of Cit, t...

  18. Glutamine transport. From energy supply to sensing and beyond.

    PubMed

    Scalise, Mariafrancesca; Pochini, Lorena; Galluccio, Michele; Indiveri, Cesare

    2016-08-01

    Glutamine is the most abundant amino acid in plasma and is actively involved in many biosynthetic and regulatory processes. It can be synthesized endogenously but becomes "conditionally essential" in physiological or pathological conditions of high proliferation rate. To accomplish its functions glutamine has to be absorbed and distributed in the whole body. This job is efficiently carried out by a network of membrane transporters that differ in transport mechanisms and energetics, belonging to families SLC1, 6, 7, 38, and possibly, 25. Some of the transporters are involved in glutamine traffic across different membranes for metabolic purposes; others are involved in specific signaling functions through mTOR. Structure/function relationships and regulatory aspects of glutamine transporters are still at infancy. In the while, insights in involvement of these transporters in cell redox control, cancer metabolism and drug interactions are arising, stimulating basic research to uncover molecular mechanisms of transport and regulation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26951943

  19. Dosing and efficacy of glutamine supplementation in human exercise and sport training.

    PubMed

    Gleeson, Michael

    2008-10-01

    Some athletes can have high intakes of l-glutamine because of their high energy and protein intakes and also because they consume protein supplements, protein hydrolysates, and free amino acids. Prolonged exercise and periods of heavy training are associated with a decrease in the plasma glutamine concentration and this has been suggested to be a potential cause of the exercise-induced immune impairment and increased susceptibility to infection in athletes. However, several recent glutamine feeding intervention studies indicate that although the plasma glutamine concentration can be kept constant during and after prolonged strenuous exercise, the glutamine supplementation does not prevent the postexercise changes in several aspects of immune function. Although glutamine is essential for lymphocyte proliferation, the plasma glutamine concentration does not fall sufficiently low after exercise to compromise the rate of proliferation. Acute intakes of glutamine of approximately 20-30 g seem to be without ill effect in healthy adult humans and no harm was reported in 1 study in which athletes consumed 28 g glutamine every day for 14 d. Doses of up to 0.65 g/kg body mass of glutamine (in solution or as a suspension) have been reported to be tolerated by patients and did not result in abnormal plasma ammonia levels. However, the suggested reasons for taking glutamine supplements (support for immune system, increased glycogen synthesis, anticatabolic effect) have received little support from well-controlled scientific studies in healthy, well-nourished humans.

  20. Dosing and efficacy of glutamine supplementation in human exercise and sport training.

    PubMed

    Gleeson, Michael

    2008-10-01

    Some athletes can have high intakes of l-glutamine because of their high energy and protein intakes and also because they consume protein supplements, protein hydrolysates, and free amino acids. Prolonged exercise and periods of heavy training are associated with a decrease in the plasma glutamine concentration and this has been suggested to be a potential cause of the exercise-induced immune impairment and increased susceptibility to infection in athletes. However, several recent glutamine feeding intervention studies indicate that although the plasma glutamine concentration can be kept constant during and after prolonged strenuous exercise, the glutamine supplementation does not prevent the postexercise changes in several aspects of immune function. Although glutamine is essential for lymphocyte proliferation, the plasma glutamine concentration does not fall sufficiently low after exercise to compromise the rate of proliferation. Acute intakes of glutamine of approximately 20-30 g seem to be without ill effect in healthy adult humans and no harm was reported in 1 study in which athletes consumed 28 g glutamine every day for 14 d. Doses of up to 0.65 g/kg body mass of glutamine (in solution or as a suspension) have been reported to be tolerated by patients and did not result in abnormal plasma ammonia levels. However, the suggested reasons for taking glutamine supplements (support for immune system, increased glycogen synthesis, anticatabolic effect) have received little support from well-controlled scientific studies in healthy, well-nourished humans. PMID:18806122

  1. Chloride-dependent acceleration of cell cycle via modulation of Rb and cdc2 in osteoblastic cells

    SciTech Connect

    Maki, Masahiro; Miyazaki, Hiroaki; Nakajima, Ken-ichi; Yamane, Junko; Niisato, Naomi; Morihara, Toru; Kubo, Toshikazu; Marunaka, Yoshinori

    2007-10-05

    In the present study, we investigated if Cl{sup -} regulates the proliferation of the MC3T3-E1 osteoblastic cells. The proliferation of MC3T3-E1 osteoblastic cells was diminished by lowering the extracellular Cl{sup -} concentration ([Cl{sup -}]{sub o}) in the culture medium. The lowered in [Cl{sup -}]{sub o} increased the periods of the G{sub 0}/G{sub 1} and the G{sub 2}/M phases in cell cycle. We further studied the effects of [Cl{sup -}]{sub o} on the key enzymes, Rb and cdc2, playing key roles in checking points of the G{sub 0}/G{sub 1} and the G{sub 2}/M phases in cell cycle. The lowered in [Cl{sup -}]{sub o} diminished the active forms of enzymes, Rb and cdc2. We further found that the action of lowered [Cl{sup -}]{sub o} on the cell proliferation, the cell cycle, Rb and cdc2 was abolished by the presence of 2 mM glutamine, but not by that of pyruvate as another Krebs cycle substrate. Taken together, these observations indicate here for the first time that Cl{sup -} modulates Rb and cdc2, enhancing the proliferation of the MC3T3-E1 osteoblastic cells.

  2. Glutamine as a feedback inhibitor of the Rhodopseudomonas sphaeroides nitrogenase system.

    PubMed Central

    Jones, B L; Monty, K J

    1979-01-01

    In whole cells of Rhodopseudomonas sphaeroides, nitrogen fixation, as measured by hydrogen production and acetylene reduction, was totally inhibited by micromolar concentrations of ammonia. This inhibition could not be duplicated by glutamate or glutamine alone. The inhibition by ammonia was abolished by methionine sulfoximine, a glutamine synthetase inhibitor. Inhibition by glutamine was complete in the presence of methionine sulfone, a preferential inhibitor of glutamate synthase, presumably by permitting a rise in the glutamine pool. The results indicated that the level of the glutamine pool controlled the activity of nitrogenase. None of these effects could be duplicated with cell-free nitrogenase, indicating there is probably a mediator which responds to the glutamine pool and inhibits nitrogenase, rather than glutamine itself being a direct inhibitor. PMID:314444

  3. Proline metabolism and cancer: emerging links to glutamine and collagen

    PubMed Central

    Phang, James M.; Liu, Wei; Hancock, Chad N.; Fischer, Joseph W.

    2015-01-01

    Purpose of review Proline metabolism impacts a number of regulatory targets in both animals and plants and is especially important in cancer. Glutamine, a related amino acid, is considered second in importance only to glucose as a substrate for tumors. But proline and glutamine are interconvertible and linked in their metabolism. In animals, proline and glutamine have specific regulatory functions and their respective physiologic sources. A comparison of the metabolism of proline and glutamine would help us understand the importance of these two nonessential amino acids in cancer metabolism. Recent findings The regulatory functions of proline metabolism proposed 3 decades ago have found relevance in many areas. For cancer, these functions play a role in apoptosis, autophagy and in response to nutrient and oxygen deprivation. Importantly, proline-derived reactive oxygen species served as a driving signal for reprogramming. This model has been applied by others to metabolic regulation for the insulin-prosurvival axis, induction of adipose triglyceride lipase for lipid metabolism and regulation of embryonic stem cell development. Of special interest, modulatory proteins such as parkinson protein 7 and oral cancer overexpressed 1 interact with pyrroline-5-carboxylate reductase, a critical component of the proline regulatory axis. Although the interconvertibility of proline and glutamine has been long established, recent findings showed that the proto-oncogene, cellular myelocytomatosis oncogene, upregulates glutamine utilization (glutaminase) and routes glutamate to proline biosynthesis (pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductases). Additionally, collagen, which contains large amounts of proline, may be metabolized to serve as a reservoir for proline. This metabolic relationship as well as the new regulatory targets of proline metabolism invites an elucidation of the differential effects of these nonessential amino acids and their production

  4. Occurrence of Only One Form of Glutamine Synthetase in the Green Alga Monoraphidium braunii.

    PubMed Central

    Garcia-Fernandez, J. M.; Lopez-Ruiz, A.; Toribio, F.; Roldan, J. M.; Diez, J.

    1994-01-01

    Anion-exchange chromatography of crude extracts from the green alga Monoraphidium braunii yielded two glutamine synthetase (GS) activities. The ratio of activities was markedly different when crude extracts were subjected to various processing conditions but was not influenced by environmental factors of cell cultures. However, high performance liquid chromatography anion-exchange chromatograms showed only one GS if the crude extracts were processed immediately after cell disruption. Moreover, standard chromatography of crude extracts obtained in the absence of dithioerythritol, a reductant generally used in disruption buffers, yielded a single activity peak. Enzyme samples from the two activities obtained in the presence of dithioerythritol were purified for physicochemical characterization and antibody production. Both enzyme samples exhibited similar reactions to different inactivating agents and were undistinguishable by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Additionally, the two GS preparations showed absolute antigenic identity as demonstrated by immunodiffusion and immunoblotting experiments. Immunocytochemistry of M. braunii cryosections evidenced a chloroplast-specific distribution of the enzyme, which rules out the existence of a cytoplasmic counterpart. All these results support the proposal that M. braunii possesses only one form of GS. PMID:12232093

  5. Extracellular Enzyme Activity and Microbial Diversity Measured on Seafloor Exposed Basalts from Loihi Seamount Indicate the Importance of Basalts to Global Biogeochemical Cycling

    PubMed Central

    Sylvan, Jason B.; Edwards, Katrina J.

    2014-01-01

    Seafloor basalts are widely distributed and host diverse prokaryotic communities, but no data exist concerning the metabolic rates of the resident microbial communities. We present here potential extracellular enzyme activities of leucine aminopeptidase (LAP) and alkaline phosphatase (AP) measured on basalt samples from different locations on Loihi Seamount, HI, coupled with analysis of prokaryotic biomass and pyrosequencing of the bacterial 16S rRNA gene. The community maximum potential enzyme activity (Vmax) of LAP ranged from 0.47 to 0.90 nmol (g rock)−1 h−1; the Vmax for AP was 28 to 60 nmol (g rock)−1 h−1. The Km of LAP ranged from 26 to 33 μM, while the Km for AP was 2 to 7 μM. Bacterial communities on Loihi basalts were comprised primarily of Alpha-, Delta-, andGammaproteobacteria, Bacteroidetes, and Planctomycetes. The putative ability to produce LAP is evenly distributed across the most commonly detected bacterial orders, but the ability to produce AP is likely dominated by bacteria in the orders Xanthomonadales, Flavobacteriales, and Planctomycetales. The enzyme activities on Loihi basalts were compared to those of other marine environments that have been studied and were found to be similar in magnitude to those from continental shelf sediments and orders of magnitude higher than any measured in the water column, demonstrating that the potential for exposed basalts to transform organic matter is substantial. We propose that microbial communities on basaltic rock play a significant, quantifiable role in benthic biogeochemical processes. PMID:24907315

  6. Inhibition of glutamine synthetase in the central nucleus of the amygdala induces anhedonic behavior and recurrent seizures in a rat model of mesial temporal lobe epilepsy.

    PubMed

    Gruenbaum, Shaun E; Wang, Helen; Zaveri, Hitten P; Tang, Amber B; Lee, Tih-Shih W; Eid, Tore; Dhaher, Roni

    2015-10-01

    The prevalence of depression and suicide is increased in patients with mesial temporal lobe epilepsy (MTLE); however, the underlying mechanism remains unknown. Anhedonia, a core symptom of depression that is predictive of suicide, is common in patients with MTLE. Glutamine synthetase, an astrocytic enzyme that metabolizes glutamate and ammonia to glutamine, is reduced in the amygdala in patients with epilepsy and depression and in suicide victims. Here, we sought to develop a novel model of anhedonia in MTLE by testing the hypothesis that deficiency in glutamine synthetase in the central nucleus of the amygdala (CeA) leads to epilepsy and comorbid anhedonia. Nineteen male Sprague-Dawley rats were implanted with an osmotic pump infusing either the glutamine synthetase inhibitor methionine sulfoximine [MSO (n=12)] or phosphate buffered saline [PBS (n=7)] into the right CeA. Seizure activity was monitored by video-intracranial electroencephalogram (EEG) recordings for 21days after the onset of MSO infusion. Sucrose preference, a measure of anhedonia, was assessed after 21days. Methionine sulfoximine-infused rats exhibited recurrent seizures during the monitoring period and showed decreased sucrose preference over days when compared with PBS-infused rats (p<0.01). Water consumption did not differ between the PBS-treated group and the MSO-treated group. Neurons were lost in the CeA, but not the medial amygdala, lateral amygdala, basolateral amygdala, or the hilus of the dentate gyrus, in the MSO-treated rats. The results suggest that decreased glutamine synthetase activity in the CeA is a possible common cause of anhedonia and seizures in TLE. We propose that the MSO CeA model can be used for mechanistic studies that will lead to the development and testing of novel drugs to prevent seizures, depression, and suicide in patients with TLE.

  7. Inhibition of glutamine synthetase in the central nucleus of the amygdala induces anhedonic behavior and recurrent seizures in a rat model of mesial temporal lobe epilepsy

    PubMed Central

    Gruenbaum, Shaun E.; Wang, Helen; Zaveri, Hitten P.; Tang, Amber B.; Lee, Tih-Shih W.; Eid, Tore; Dhaher, Roni

    2015-01-01

    The prevalence of depression and suicide is increased in patients with mesial temporal lobe epilepsy (MTLE); however, the underlying mechanism remains unknown. Anhedonia, a core symptom of depression that is predictive of suicide, is common in patients with MTLE. Glutamine synthetase, an astrocytic enzyme that metabolizes glutamate and ammonia to glutamine, is reduced in the amygdala in patients with epilepsy and depression and in suicide victims. Here, we sought to develop a novel model of anhedonia in MTLE by testing the hypothesis that deficiency in glutamine synthetase in the central nucleus of the amygdala (CeA) leads to epilepsy and comorbid anhedonia. Nineteen male Sprague–Dawley rats were implanted with an osmotic pump infusing either the glutamine synthetase inhibitor methionine sulfoximine [MSO (n = 12)] or phosphate buffered saline [PBS (n = 7)] into the right CeA. Seizure activity was monitored by video-intracranial electroencephalogram (EEG) recordings for 21 days after the onset of MSO infusion. Sucrose preference, a measure of anhedonia, was assessed after 21 days. Methionine sulfoximine-infused rats exhibited recurrent seizures during the monitoring period and showed decreased sucrose preference over days when compared with PBS-infused rats (p < 0.01). Water consumption did not differ between the PBS-treated group and the MSO-treated group. Neurons were lost in the CeA, but not the medial amygdala, lateral amygdala, basolateral amygdala, or the hilus of the dentate gyrus, in the MSO-treated rats. The results suggest that decreased glutamine synthetase activity in the CeA is a possible common cause of anhedonia and seizures in TLE. We propose that the MSO CeA model can be used for mechanistic studies that will lead to the development and testing of novel drugs to prevent seizures, depression, and suicide in patients with TLE. PMID:26262937

  8. Expression of the Saccharomyces cerevisiae DNA repair gene RAD6 that encodes a ubiquitin conjugating enzyme, increases in response to DNA damage and in meiosis but remains constant during the mitotic cell cycle.

    PubMed

    Madura, K; Prakash, S; Prakash, L

    1990-02-25

    The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating (E2) enzyme and is required for the repair of damaged DNA, mutagenesis, and sporulation. Here, we report our studies on the regulation of RAD6 gene expression after UV damage, during the mitotic cell cycle, in meiosis, and following heat shock and starvation. RAD6 mRNA levels became elevated in cells exposed to UV light, and at all UV doses the increase in mRNA levels was rapid and occurred within 30 min after exposure to UV. RAD6 mRNA levels also increased in sporulating MATa/MAT alpha cells, and the period of maximal accumulation of RAD6 mRNA during meiosis is coincident with the time during which recombination occurs. However, RAD6 mRNA levels showed no periodic fluctuation in the mitotic cell cycle, were not elevated upon heat shock, and fell in cells in the stationary phase of growth. These observations suggest that RAD6 activity is required throughout the cell cycle rather than being restricted to a specific stage, and that during meiosis, high levels of RAD6 activity may be needed at a stage coincident with genetic recombination. The observation that RAD6 transcription is not induced by heat and starvation, treatments that activate stress responses, suggests that the primary role of RAD6 is in the repair of damaged DNA rather than in adapting cells to stress situations.

  9. The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65.

    PubMed

    Lopes, Vanda S; Gibbs, Daniel; Libby, Richard T; Aleman, Tomas S; Welch, Darcy L; Lillo, Concepción; Jacobson, Samuel G; Radu, Roxana A; Steel, Karen P; Williams, David S

    2011-07-01

    Mutations in the MYO7A gene cause a deaf-blindness disorder, known as Usher syndrome 1B.  In the retina, the majority of MYO7A is in the retinal pigmented epithelium (RPE), where many of the reactions of the visual retinoid cycle take place.  We have observed that the retinas of Myo7a-mutant mice are resistant to acute light damage. In exploring the basis of this resistance, we found that Myo7a-mutant mice have lower levels of RPE65, the RPE isomerase that has a key role in the retinoid cycle.  We show for the first time that RPE65 normally undergoes a light-dependent translocation to become more concentrated in the central region of the RPE cells.  This translocation requires MYO7A, so that, in Myo7a-mutant mice, RPE65 is partly mislocalized in the light.  RPE65 is degraded more quickly in Myo7a-mutant mice, perhaps due to its mislocalization, providing a plausible explanation for its lower levels.  Following a 50-60% photobleach, Myo7a-mutant retinas exhibited increased all-trans-retinyl ester levels during the initial stages of dark recovery, consistent with a deficiency in RPE65 activity.  Lastly, MYO7A and RPE65 were co-immunoprecipitated from RPE cell lysate by antibodies against either of the proteins, and the two proteins were partly colocalized, suggesting a direct or indirect interaction.  Together, the results support a role for MYO7A in the translocation of RPE65, illustrating the involvement of a molecular motor in the spatiotemporal organization of the retinoid cycle in vision. PMID:21493626

  10. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  11. Expression of apical Na(+)-L-glutamine co-transport activity, B(0)-system neutral amino acid co-transporter (B(0)AT1) and angiotensin-converting enzyme 2 along the jejunal crypt-villus axis in young pigs fed a liquid formula.

    PubMed

    Yang, Chengbo; Yang, Xiaojian; Lackeyram, Dale; Rideout, Todd C; Wang, Zirong; Stoll, Barbara; Yin, Yulong; Burrin, Douglas G; Fan, Ming Z

    2016-06-01

    Gut apical amino acid (AA) transport activity is high at birth and during suckling, thus being essential to maintain luminal nutrient-dependent mucosal growth through providing AA as essential metabolic fuel, substrates and nutrient stimuli for cellular growth. Because system-B(0) Na(+)-neutral AA co-transporter (B(0)AT1, encoded by the SLC6A19 gene) plays a dominant role for apical uptake of large neutral AA including L-Gln, we hypothesized that high apical Na(+)-Gln co-transport activity, and B(0)AT1 (SLC6A19) in co-expression with angiotensin-converting enzyme 2 (ACE2) were expressed along the entire small intestinal crypt-villus axis in young animals via unique control mechanisms. Kinetics of Na(+)-Gln co-transport activity in the apical membrane vesicles, prepared from epithelial cells sequentially isolated along the jejunal crypt-villus axis from liquid formula-fed young pigs, were measured with the membrane potential being clamped to zero using thiocyanate. Apical maximal Na(+)-Gln co-transport activity was much higher (p < 0.05) in the upper villus cells than in the middle villus (by 29 %) and the crypt (by 30 %) cells, whereas Na(+)-Gln co-transport affinity was lower (p < 0.05) in the upper villus cells than in the middle villus and the crypt cells. The B(0)AT1 (SLC6A19) mRNA abundance was lower (p < 0.05) in the crypt (by 40-47 %) than in the villus cells. There were no significant differences in B(0)AT1 and ACE2 protein abundances on the apical membrane among the upper villus, the middle villus and the crypt cells. Our study suggests that piglet fast growth is associated with very high intestinal apical Na(+)-neutral AA uptake activities via abundantly co-expressing B(0)AT1 and ACE2 proteins in the apical membrane and by transcribing the B(0)AT1 (SLC6A19) gene in the epithelia along the entire crypt-villus axis. PMID:26984322

  12. Glutamine Provides Effective Protection against Deltamethrin-Induced Acute Hepatotoxicity in Rats But Not Against Nephrotoxicity

    PubMed Central

    Gündüz, Ercan; Ülger, Burak Veli; İbiloğlu, İbrahim; Ekinci, Aysun; Dursun, Recep; Zengin, Yılmaz; İçer, Mustafa; Uslukaya, Ömer; Ekinci, Cenap; Güloğlu, Cahfer

    2015-01-01

    Background The aim of this study was to investigate the protective effects of L-glutamine (GLN) against liver and kidney injury caused by acute toxicity of deltamethrin (DLM). Material/Methods Thirty-two rats were indiscriminately separated into 4 groups with 8 rats each: control group (distilled water; 10 ml/kg, perorally [p.o.]), DLM group (35 mg/kg p.o. one dose.), GLN group (1.5 gr/kg, p.o. single dose.) and DLM (35 mg/kg p.o. one dose.) + GLN group (1.5 gr/kg, p.o. one dose after 4 hours.). Testing for total antioxidant status (TAS), total oxidant status (TOS), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) analyses were performed on tissue samples, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), urea, and creatinine were analyzed on serum samples. Liver and kidney samples were histopathologically analyzed. Results The TOS level in liver was significantly higher in the DLM group than in the control group, and the level in DLM+GLN group was considerably lower than in the DLM group. The TAS level in the DLM+GLN group was considerably higher than in the control and DLM groups. The TAS level in kidney tissues was considerably lower in the DLM group than in controls, but was similar to other groups. Histopathological analyses of liver tissues established a significant difference between DLM and DLM+GLN groups in terms of grade 2 hepatic injury. However, no significant difference was found between DLM and DLM+GLN groups in terms of kidney injury. Conclusions Glutamine leads to significant improvement in deltamethrin-induced acute hepatotoxicity in terms of histopathologic results, tissue oxidative stress parameters, and serum liver function marker enzymes. PMID:25890620

  13. Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways.

    PubMed

    Nikel, Pablo I; Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; de Lorenzo, Víctor

    2015-10-23

    The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using (13)C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats.

  14. Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways.

    PubMed

    Nikel, Pablo I; Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; de Lorenzo, Víctor

    2015-10-23

    The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using (13)C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats. PMID:26350459

  15. Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways*

    PubMed Central

    Nikel, Pablo I.; Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; de Lorenzo, Víctor

    2015-01-01

    The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using 13C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats. PMID:26350459

  16. Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake

    PubMed Central

    Cheng, Zhihui; Lin, Mingqun

    2014-01-01

    ABSTRACT How the obligatory intracellular bacterium Ehrlichia chaffeensis begins to replicate upon entry into human monocytes is poorly understood. Here, we examined the potential role of amino acids in initiating intracellular replication. PutA converts proline to glutamate, and GlnA converts glutamate to glutamine. E. chaffeensis PutA and GlnA complemented Escherichia coli putA and glnA mutants. Methionine sulfoximine, a glutamine synthetase inhibitor, inhibited E. chaffeensis GlnA activity and E. chaffeensis infection of human cells. Incubation of E. chaffeensis with human cells rapidly induced putA and glnA expression that peaked at 24 h postincubation. E. chaffeensis took up proline and glutamine but not glutamate. Pretreatment of E. chaffeensis with a proline transporter inhibitor (protamine), a glutamine transporter inhibitor (histidine), or proline analogs inhibited E. chaffeensis infection, whereas pretreatment with proline or glutamine enhanced infection and upregulated putA and glnA faster than no treatment or glutamate pretreatment. The temporal response of putA and glnA expression was similar to that of NtrY and NtrX, a two-component system, and electrophoretic mobility shift assays showed specific binding of recombinant E. chaffeensis NtrX (rNtrX) to the promoter regions of E. chaffeensis putA and glnA. Furthermore, rNtrX transactivated E. chaffeensis putA and glnA promoter-lacZ fusions in E. coli. Growth-promoting activities of proline and glutamine were also accompanied by rapid degradation of the DNA-binding protein CtrA. Our results suggest that proline and glutamine uptake regulates putA and glnA expression through NtrY/NtrX and facilitates degradation of CtrA to initiate a new cycle of E. chaffeensis growth. PMID:25425236

  17. Synthesis of biobased succinonitrile from glutamic acid and glutamine.

    PubMed

    Lammens, Tijs M; Le Nôtre, Jérôme; Franssen, Maurice C R; Scott, Elinor L; Sanders, Johan P M

    2011-06-20

    Succinonitrile is the precursor of 1,4-diaminobutane, which is used for the industrial production of polyamides. This paper describes the synthesis of biobased succinonitrile from glutamic acid and glutamine, amino acids that are abundantly present in many plant proteins. Synthesis of the intermediate 3-cyanopropanoic amide was achieved from glutamic acid 5-methyl ester in an 86 mol% yield and from glutamine in a 56 mol % yield. 3-Cyanopropanoic acid can be converted into succinonitrile, with a selectivity close to 100% and a 62% conversion, by making use of a palladium(II)-catalyzed equilibrium reaction with acetonitrile. Thus, a new route to produce biobased 1,4-diaminobutane has been discovered. PMID:21557494

  18. Resistance to BRAF inhibitors induces glutamine dependency in melanoma cells

    PubMed Central

    Baenke, Franziska; Chaneton, Barbara; Smith, Matthew; Van Den Broek, Niels; Hogan, Kate; Tang, Haoran; Viros, Amaya; Martin, Matthew; Galbraith, Laura; Girotti, Maria R.; Dhomen, Nathalie; Gottlieb, Eyal; Marais, Richard

    2016-01-01

    BRAF inhibitors can extend progression-free and overall survival in melanoma patients whose tumors harbor mutations in BRAF. However, the majority of patients eventually develop resistance to these drugs. Here we show that BRAF mutant melanoma cells that have developed acquired resistance to BRAF inhibitors display increased oxidative metabolism and increased dependency on mitochondria for survival. Intriguingly, the increased oxidative metabolism is associated with a switch from glucose to glutamine metabolism and an increased dependence on glutamine over glucose for proliferation. We show that the resistant cells are more sensitive to mitochondrial poisons and to inhibitors of glutaminolysis, suggesting that targeting specific metabolic pathways may offer exciting therapeutic opportunities to treat resistant tumors, or to delay emergence of resistance in the first-line setting. PMID:26365896

  19. Characterization of null mutants of the glyoxylate cycle and gluconeogenic enzymes in S. cerevisiae through metabolic network modeling verified by chemostat cultivation.

    PubMed

    Stückrath, I; Lange, H C; Kötter, P; van Gulik, W M; Entian, K-D; Heijnen, J J

    2002-01-01

    Biomass yields for several null mutants in Saccharomyces cerevisiae were successfully predicted with a metabolic network model. Energetic parameters of the model were obtained from growth data in C-limited aerobic chemostat cultures of the corresponding wild-type strain, which exhibited a P/O ratio of 1.46, a non-growth-related maintenance of 56 mmol ATP/C-mol biomass/h, and a growth-related requirement of 655 mmol ATP/C-mol biomass. Biomass yields and carbon uptake rates were modeled for different mutants incapacitated in their glyoxylate cycle and their gluconeogenesis. Biomass yields were calculated for different feed ratios of glucose to ethanol, and decreases for higher ethanol fractions were correctly predicted for mutants with deletions of the malate synthase, the isocitrate lyase, or the phosphoenolpyruvate carboxykinase. The growth of the fructose- 1,6-bisphosphatase deletion mutant was anticipated less accurate, but the tendency was modeled correctly.

  20. Site-directed mutagenesis of Glu-297 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase alters kinetic and structural properties and confers resistance to L-methionine sulfoximine.

    PubMed

    Clemente, M T; Márquez, A J

    1999-07-01

    In this paper we examine the functionality of Glu-297 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase (EC 6.3.1.2). For this purpose, the gln alpha cDNA was recombinantly expressed in Escherichia coli, and site-directed mutants constructed, in which this residue was replaced by alanine. The level of glutamine synthetase transferase catalytic activity in the mutant strain was 70-fold lower while biosynthetic activity remained practically unaffected. Kinetic parameters for both enzyme activities were not greatly altered except for the Km for ammonium in biosynthetic activity, which increased 100-fold. A similar result was reported when mutagenizing Glu-327 from E. coli glutamine synthetase, a residue shown to be present at the active site. This suggests that the Glu residue mutated in the higher-plant enzyme could develop a similar catalytic role to that of bacteria. Another characteristic feature of the mutant protein was its higher resistance to inhibition of the biosynthetic activity by L-methionine sulfoximine, a typical inhibitor of glutamine synthetase. In addition, we show that immunoreactivity of the glutamine synthetase mutant protein, both under native and denaturing conditions, is similar to the wild type, indicating that no deep conformational changes were produced as a consequence of the introduced mutation. However, structural changes in the active site can be predicted from alterations detected in the behaviour of the mutant protein towards affinity chromatography on 2',5'-ADP-Sepharose, as compared to the wild type. Nevertheless, complementation of an E. coli glnA mutation indicated that the E297A mutant enzyme was physiologically functional.

  1. Physiological Studies of Glutamine Synthetases I and III from Synechococcus sp. WH7803 Reveal Differential Regulation.

    PubMed

    Domínguez-Martín, María Agustina; Díez, Jesús; García-Fernández, José M

    2016-01-01

    The marine picocyanobacterium Synechococcus sp. WH7803 possesses two glutamine synthetases (GSs; EC 6.3.1.2), GSI encoded by glnA and GSIII encoded by glnN. This is the first work addressing the physiological regulation of both enzymes in a marine cyanobacterial strain. The increase of GS activity upon nitrogen starvation was similar to that found in other model cyanobacteria. However, an unusual response was found when cells were grown under darkness: the GS activity was unaffected, reflecting adaptation to the environment where they thrive. On the other hand, we found that GSIII did not respond to nitrogen availability, in sharp contrast with the results observed for this enzyme in other cyanobacteria thus far studied. These features suggest that GS activities in Synechococcus sp. WH7803 represent an intermediate step in the evolution of cyanobacteria, in a process of regulatory streamlining where GSI lost the regulation by light, while GSIII lost its responsiveness to nitrogen. This is in good agreement with the phylogeny of Synechococcus sp. WH7803 in the context of the marine cyanobacterial radiation. PMID:27446010

  2. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

    PubMed

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L; Paradiso, Annalisa; Gu, Yong Q; Blanco, Antonio; de Pinto, Maria C; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes.

  3. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

    PubMed

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L; Paradiso, Annalisa; Gu, Yong Q; Blanco, Antonio; de Pinto, Maria C; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes. PMID:27468287

  4. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content

    PubMed Central

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L.; Paradiso, Annalisa; Gu, Yong Q.; Blanco, Antonio; de Pinto, Maria C.; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes. PMID:27468287

  5. Physiological Studies of Glutamine Synthetases I and III from Synechococcus sp. WH7803 Reveal Differential Regulation

    PubMed Central

    Domínguez-Martín, María Agustina; Díez, Jesús; García-Fernández, José M.

    2016-01-01

    The marine picocyanobacterium Synechococcus sp. WH7803 possesses two glutamine synthetases (GSs; EC 6.3.1.2), GSI encoded by glnA and GSIII encoded by glnN. This is the first work addressing the physiological regulation of both enzymes in a marine cyanobacterial strain. The increase of GS activity upon nitrogen starvation was similar to that found in other model cyanobacteria. However, an unusual response was found when cells were grown under darkness: the GS activity was unaffected, reflecting adaptation to the environment where they thrive. On the other hand, we found that GSIII did not respond to nitrogen availability, in sharp contrast with the results observed for this enzyme in other cyanobacteria thus far studied. These features suggest that GS activities in Synechococcus sp. WH7803 represent an intermediate step in the evolution of cyanobacteria, in a process of regulatory streamlining where GSI lost the regulation by light, while GSIII lost its responsiveness to nitrogen. This is in good agreement with the phylogeny of Synechococcus sp. WH7803 in the context of the marine cyanobacterial radiation. PMID:27446010

  6. Rapid bioassay of human interferon by direct enzyme immunoassay of encephalomyocarditis virus in HEp-2 cell monolayers after a single cycle of infection.

    PubMed

    Vlaspolder, F; Donkers, E; Harmsen, T; Kraaijeveld, C A; Snippe, H

    1989-01-01

    Multiplication of encephalomyocarditis virus (EMCV) in human HEp-2 cells, and its suppression by interferon (IFN), was demonstrated by direct enzyme immunoassay (EIA) in cell culture. EMCV was detected in glutaraldehyde fixed HEp-2 cell monolayers, in wells of 96-well plates, with a horse radish peroxidase (HRPO) labelled EMCV specific monoclonal antibody. Multiplication of EMCV (multiplicity of infection: 50) was indicated by a steep rise of absorbance values measured against infected monolayers starting as early as 5 h after infection and reaching relatively high values at 6 and 7 h. The rise in absorbance values did not occur after preincubation of the HEp-2 cells with either Newcastle disease virus-induced IFN, recombinant gamma IFN or recombinant alfa-2a IFN. Absorbance values were inversely dependent on the amount of IFN used. Therefore the EIA was suitable for rapid titration of IFN. The titres of recombinant gamma and alfa-2a IFN determined with EIA proved to be similar to those given by the manufacturers. The described bioassay of human IFN is objective, rapid and easy to perform and suitable for large scale experiments.

  7. The effect of immunonutrition (glutamine, alanine) on fracture healing

    PubMed Central

    Küçükalp, Abdullah; Durak, Kemal; Bayyurt, Sarp; Sönmez, Gürsel; Bilgen, Muhammed S.

    2014-01-01

    Background There have been various studies related to fracture healing. Glutamine is an amino acid with an important role in many cell and organ functions. This study aimed to make a clinical, radiological, and histopathological evaluation of the effects of glutamine on fracture healing. Methods Twenty rabbits were randomly allocated into two groups of control and immunonutrition. A fracture of the fibula was made to the right hind leg. All rabbits received standard food and water. From post-operative first day for 30 days, the study group received an additional 2 ml/kg/day 20% L-alanine L-glutamine solution via a gastric catheter, and the control group received 2 ml/kg/day isotonic via gastric catheter. At the end of 30 days, the rabbits were sacrificed and the fractures were examined clinically, radiologically, and histopathologically in respect to the degree of union. Results Radiological evaluation of the control group determined a mean score of 2.5 according to the orthopaedists and 2.65 according to the radiologists. In the clinical evaluation, the mean score was 1.875 for the control group and 2.0 for the study group. Histopathological evaluation determined a mean score of 8.5 for the control group and 9.0 for the study group. Conclusion One month after orally administered glutamine–alanine, positive effects were observed on fracture healing radiologically, clinically, and histopathologically, although no statistically significant difference was determined.

  8. Antioxidant enzyme systems and the ascorbate-glutathione cycle as contributing factors to cadmium accumulation and tolerance in two oilseed rape cultivars (Brassica napus L.) under moderate cadmium stress.

    PubMed

    Wu, Zhichao; Zhao, Xiaohu; Sun, Xuecheng; Tan, Qiling; Tang, Yafang; Nie, Zhaojun; Qu, Chanjuan; Chen, Zuoxin; Hu, Chengxiao

    2015-11-01

    Oilseed rape (Brassica napus L.) with high tolerance to cadmium (Cd) may be used in the phytoremediation of Cd-contaminated fields. However, the mechanisms responsible for Cd accumulation and tolerance in oilseed rape are still poorly understood. Here, we investigated the physiological and molecular processes involved in Cd tolerance of two oilseed rape cultivars with different Cd accumulation abilities. The total Cd accumulation in cultivar L351 was higher than cultivar L338, particularly with increasing concentrations of Cd exposure. L338 was a more pronounced Cd-sensitive cultivar than L351, while higher activities of antioxidant enzymes (CAT, APX, GR, DHAR) as well as higher contents of GSH and AsA were all observed in L351 under Cd treatments, especially at high levels. No differences were found in SOD activities between the two cultivars under the same Cd treatments, suggesting that SOD was not the key factor in relation to the differences of Cd tolerance and accumulation between them. Gene expression levels of BnFe-SOD, BnCAT, BnAPX, BcGR and BoDHAR in roots of L351 were relatively higher than that in L338 under Cd exposure as well as BnCAT and BcGR in leaves. It is concluded that antioxidant enzymes and the ascorbate-glutathione cycle play important roles in oilseed rape Cd accumulation and tolerance.

  9. Antioxidant enzyme systems and the ascorbate-glutathione cycle as contributing factors to cadmium accumulation and tolerance in two oilseed rape cultivars (Brassica napus L.) under moderate cadmium stress.

    PubMed

    Wu, Zhichao; Zhao, Xiaohu; Sun, Xuecheng; Tan, Qiling; Tang, Yafang; Nie, Zhaojun; Qu, Chanjuan; Chen, Zuoxin; Hu, Chengxiao

    2015-11-01

    Oilseed rape (Brassica napus L.) with high tolerance to cadmium (Cd) may be used in the phytoremediation of Cd-contaminated fields. However, the mechanisms responsible for Cd accumulation and tolerance in oilseed rape are still poorly understood. Here, we investigated the physiological and molecular processes involved in Cd tolerance of two oilseed rape cultivars with different Cd accumulation abilities. The total Cd accumulation in cultivar L351 was higher than cultivar L338, particularly with increasing concentrations of Cd exposure. L338 was a more pronounced Cd-sensitive cultivar than L351, while higher activities of antioxidant enzymes (CAT, APX, GR, DHAR) as well as higher contents of GSH and AsA were all observed in L351 under Cd treatments, especially at high levels. No differences were found in SOD activities between the two cultivars under the same Cd treatments, suggesting that SOD was not the key factor in relation to the differences of Cd tolerance and accumulation between them. Gene expression levels of BnFe-SOD, BnCAT, BnAPX, BcGR and BoDHAR in roots of L351 were relatively higher than that in L338 under Cd exposure as well as BnCAT and BcGR in leaves. It is concluded that antioxidant enzymes and the ascorbate-glutathione cycle play important roles in oilseed rape Cd accumulation and tolerance. PMID:26207887

  10. No benefit of glutamine supplementation on persistent diarrhea in Ugandan children.

    PubMed

    Kamuchaki, Justine M; Kiguli, Sarah; Wobudeya, Eric; Bortolussi, Robert

    2013-05-01

    We evaluated the efficacy of oral glutamine supplementation in children 2 to 60 months of age with persistent diarrhea by 1:1 randomization to standard treatment alone or together with twice daily glutamine. The failure rate was similar in both arms (relative risk: 1.8 [95% confidence interval: 0.8-3.7], P = 0.12). Glutamine supplementation showed no benefit on the outcome of persistent diarrhea.

  11. Effects of xylitol- and/or glutamine-supplemented parenteral nutrition on septic rats.

    PubMed

    Ardawi, M S

    1992-04-01

    1. The effects of parenteral nutrition with or without xylitol and/or glutamine supplementation were studied in septic rats after 4 days of treatment. 2. Septic rats treated with xylitol- and/or glutamine-supplemented parenteral nutrition survived sepsis significantly better than other parenteral nutrition-treated septic rats: the cumulative percentage of deaths over 4 days in septic rats treated with xylitol-glutamine-supplemented parenteral nutrition was 9.5% compared with 54.5% in septic rats given parenteral nutrition without xylitol and glutamine, and 52.4% in septic rats treated with parenteral nutrition supplemented with glucose. 3. Xylitol- and/or glutamine-supplemented parenteral nutrition resulted in improved nitrogen balance in septic rats: the cumulative nitrogen balance over the 4 days of treatment was positive in the rats given xylitol-supplemented parenteral nutrition and more positive when rats were treated with xylitol-glutamine-supplemented parenteral nutrition, as compared with other groups of septic rats. 4. The rate of loss of intracellular glutamine in skeletal muscle was markedly decreased (P less than 0.001) in response to xylitol- and/or glutamine-supplemented parenteral nutrition in septic rats. 5. Hepatic protein and RNA contents were increased in septic rats treated with xylitol- and/or glutamine-supplemented parenteral nutrition. Similarly, protein and RNA contents were markedly increased in muscles of septic rats treated with xylitol- and/or glutamine-supplemented parenteral nutrition. 6. The rates of incorporation of leucine/tyrosine into liver/muscle proteins in vitro were increased and the rate of muscular tyrosine release was decreased in response to xylitol- and/or glutamine-supplemented parenteral nutrition in septic rats. 7. It is concluded that the administration of xylitol- and/or glutamine-supplemented parenteral nutrition is beneficial to septic rats and possibly to septic patients.

  12. Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes.

    PubMed

    Fisher, Susan H; Wray, Lewis V

    2008-01-22

    The Bacillus subtilis GlnR repressor controls gene expression in response to nitrogen availability. Because all GlnR-regulated genes are expressed constitutively in mutants lacking glutamine synthetase (GS), GS is required for repression by GlnR. Feedback-inhibited GS (FBI-GS) was shown to activate GlnR DNA binding with an in vitro electophoretic mobility shift assay (EMSA). The activation of GlnR DNA binding by GS in these experiments depended on the feedback inhibitor glutamine and did not occur with mutant GS proteins defective in regulating GlnR activity in vivo. Although stable GS-GlnR-DNA ternary complexes were not observed in the EMSA experiments, cross-linking experiments showed that a protein-protein interaction occurs between GlnR and FBI-GS. This interaction was reduced in the absence of the feedback inhibitor glutamine and with mutant GS proteins. Because FBI-GS significantly reduced the dissociation rate of the GlnR-DNA complexes, the stability of these complexes is enhanced by FBI-GS. These results argue that FBI-GS acts as a chaperone that activates GlnR DNA binding through a transient protein-protein interaction that stabilizes GlnR-DNA complexes. GS was shown to control the activity of the B. subtilis nitrogen transcription factor TnrA by forming a stable complex between FBI-GS and TnrA that inhibits TnrA DNA binding. Thus, B. subtilis GS is an enzyme with dual catalytic and regulatory functions that uses distinct mechanisms to control the activity of two different transcription factors.

  13. Glutamine treatment attenuates hyperglycemia-induced mitochondrial stress and apoptosis in umbilical vein endothelial cells

    PubMed Central

    Safi, Sher Zaman; Batumalaie, Kalaivani; Mansor, Marzida; Chinna, Karuthan; Mohan, Syam; Kumar, Selva; Karimian, Hamed; Qvist, Rajes; Ashraf, Muhammad Aqeel; Yan, Garcie Ong Siok

    2015-01-01

    OBJECTIVE: The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis, mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilical vein endothelial cells. MATERIALS AND METHODS: Human umbilical vein endothelial cells were grown and subjected to glutamine and insulin to examine the effects of these agents on the hyperglycemic state. Mitochondrial function and the production of inflammatory cytokines were assessed using fluorescence analysis and multiple cytotoxicity assays. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. RESULTS: Glutamine maintains the integrity of the mitochondria by reducing the cell permeability and cytochrome c levels and increasing the mitochondrial membrane potential. The cytochrome c level was significantly (p<0.005) reduced when the cells were treated with glutamine. An apoptosis assay revealed significantly reduced apoptosis (p<0.005) in the glutamine-treated cells. Moreover, glutamine alone or in combination with insulin modulated inflammatory cytokine levels. Interleukin-10, interleukin-6, and vascular endothelial growth factor were up-regulated while tumor necrosis factor-α was down-regulated after treatment with glutamine. CONCLUSIONS: Glutamine, either alone or in combination with insulin, can positively modulate the mitochondrial stress and cell permeability in umbilical vein endothelial cells. Glutamine regulates the expression of inflammatory cytokines and maintains the balance of the mitochondria in a cytoprotective manner. PMID:26247670

  14. [Concentration of monoamines and activity of several enzymes in the arcuate nucleus of the hypothalamus in young and aging rats during the estrous cycle].

    PubMed

    Grantyn', V A

    1976-07-01

    The arcuate nucleus (AN) and the median eminence (ME) of the hypothalamus were investigated in young and ageing female rats. During the estral cycle (EC) the monoamine (MA) content, the monoaminoxidase (MAO), NADP and NAD-diaphorase activities were determined in the AN, and the MA content and the activity of alkaline phosphatase (AP) -- in the ME. In young rats in the proestrus-estrus there was an increase in the activity of the NADP and NAD-diaphorase and of the MA content, but a decrease of the MAO activity. This indicated an intensified function of the nucleus at these stages of the EC. Accumulation of the MA in the ME was noted in the diestrus, while in the proestrus their concentration sharply fell; on the other hand, the activity of the AP was considerably increased. In the ageing rats the dynamics of the indices under study during the EC were largely unchanged. However, the functional activity of the AN proved to increase, and in the ME and elevation of the MA concentration and disturbance of its release from the nerve terminals was seen.

  15. Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways.

    PubMed

    Hutson, Susan M; Islam, Mohammad Mainul; Zaganas, Ioannis

    2011-09-01

    Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5'-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5'-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations. PMID:21621574

  16. L-glutamine supplementations enhance liver glutamine-glutathione axis and heat shock factor-1 expression in endurance-exercise trained rats.

    PubMed

    Petry, Éder Ricardo; Cruzat, Vinicius Fernandes; Heck, Thiago Gomes; Homem de Bittencourt, Paulo Ivo; Tirapegui, Julio

    2015-04-01

    Liver L-glutamine is an important vehicle for the transport of ammonia and intermediary metabolism of amino acids between tissues, particularly under catabolic situations, such as high-intensity exercise. Hence, the aim of this study was to investigate the effects of oral supplementations with L-glutamine in its free or dipeptide forms (with L-alanine) on liver glutamine-glutathione (GSH) axis, and 70 kDa heat shock proteins (HSP70)/heat shock transcription factor 1 (HSF1) expressions. Adult male Wistar rats were 8-week trained (60 min/day, 5 days/week) on a treadmill. During the last 21 days, the animals were daily supplemented with 1 g of L-glutamine/kg body weight per day in either l-alanyl-L-glutamine dipeptide (DIP) form or a solution containing L-glutamine and l-alanine in their free forms (GLN+ALA) or water (controls). Exercise training increased cytosolic and nuclear HSF1 and HSP70 expression, as compared with sedentary animals. However, both DIP and GLN+ALA supplements enhanced HSF1 expression (in both cytosolic and nuclear fractions) in relation to exercised controls. Interestingly, HSF1 rises were not followed by enhanced HSP70 expression. DIP and GLN+ALA supplements increased plasma glutamine concentrations (by 62% and 59%, respectively) and glutamine to glutamate plasma ratio in relation to trained controls. This was in parallel with a decrease in plasma ammonium levels. Supplementations increased liver GSH (by 90%), attenuating the glutathione disulfide (GSSG) to GSH ratio, suggesting a redox state protection. In conclusion, oral administration with DIP and GLN+ALA supplements in endurance-trained rats improve liver glutamine-GSH axis and modulate HSF1 pathway. PMID:25202991

  17. Decreased glutamate, glutamine and citrulline concentrations in plasma and muscle in endotoxemia cannot be reversed by glutamate or glutamine supplementation: a primary intestinal defect?

    PubMed

    Boutry, Claire; Matsumoto, Hideki; Bos, Cécile; Moinard, Christophe; Cynober, Luc; Yin, Yulong; Tomé, Daniel; Blachier, François

    2012-10-01

    Endotoxemia affects intestinal physiology. A decrease of circulating citrulline concentration is considered as a reflection of the intestinal function. Citrulline can be produced in enterocytes notably from glutamate and glutamine. The aim of this work was to determine if glutamate, glutamine and citrulline concentrations in blood, intestine and muscle are decreased by endotoxemia, and if supplementation with glutamate or glutamine can restore normal concentrations. We induced endotoxemia in rats by an intraperitoneal injection of 0.3 mg kg(-1) lipopolysaccharide (LPS). This led to a rapid anorexia, negative nitrogen balance and a transient increase of the circulating level of IL-6 and TNF-α. When compared with the values measured in pair fed (PF) animals, almost all circulating amino acids (AA) including citrulline decreased, suggesting a decrease of intestinal function. However, at D2 after LPS injection, most circulating AA concentrations were closed to the values recorded in the PF group. At that time, among AA, only glutamate, glutamine and citrulline were decreased in gastrocnemius muscle without change in intestinal mucosa. A supplementation with 4% monosodium glutamate (MSG) or an isomolar amount of glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscle. However, MSG supplementation led to an accumulation of glutamate in the intestinal mucosa. In conclusion, endotoxemia rapidly but transiently decreased the circulating concentrations of almost all AA and more durably of glutamate, glutamine and citrulline in muscle. Supplementation with glutamate or glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscles. The implication of a loss of the intestinal capacity for AA absorption and/or metabolism in endotoxemia (as judged from decreased citrulline plasma concentration) for explaining such results are discussed.

  18. Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes.

    PubMed

    Fulks, R M; Stadtman, E R

    1985-12-13

    When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged by the incorporation of [14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incorporation of [14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogenously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe2+ (or Ni2+) together with ATP (or other nucleoside di- and triphosphates. After 40-60 h dialysis Fe3+ together with NADH (but

  19. Arginine synthesis from enteral glutamine in healthy adults in the fed state.

    PubMed

    Tomlinson, Chris; Rafii, Mahroukh; Ball, Ronald O; Pencharz, Paul

    2011-08-01

    Recent studies have documented transfer of labeled nitrogen from [2-(15)N]glutamine to citrulline and arginine in fasting human adults. Conversely, in neonates and piglets we have shown no synthesis of arginine from [2-(15)N]glutamate, and others have shown in mice that glutamine is a nitrogen, but not a carbon donor, for arginine synthesis. Therefore, we performed a multitracer study to determine whether glutamine is a nitrogen and/or carbon donor for arginine in healthy adult men. Two glutamine tracers, 2-(15)N and 1-(13)C, were given enterally to five healthy men fed a standardized milkshake diet. There was no difference in plasma enrichments between the two glutamine tracers. 1-(13)C isotopomers of citrulline and arginine were synthesized from [1-(13)C]glutamine. Three isotopomers each of citrulline and arginine were synthesized from the [2-(15)N]glutamine tracer: 2-(15)N, 5-(15)N, and 2,5-(15)N(2). Significantly greater enrichment was found of both [5-(15)N]arginine (0.75%) and citrulline (3.98%) compared with [2-(15)N]arginine (0.44%) and [2-(15)N]citrulline (2.62%), indicating the amino NH(2) from glutamine is mostly transferred to arginine and citrulline by transamination. Similarly, the enrichment of the 1-(13)C isotopomers was significantly less than the 2-(15)N isotopomers, suggesting rapid formation of α-ketoglutarate and recycling of the nitrogen label. Our results show that the carbon for 50% of newly synthesized arginine comes from dietary glutamine but that glutamine acts primarily as a nitrogen donor for arginine synthesis. Hence, studies using [2-(15)N]glutamine will overestimate arginine synthesis rates.

  20. Free and protein-bound glutamine have identical splanchnic extraction in healthy human volunteers.

    PubMed

    Boza, J J; Dangin, M; Moënnoz, D; Montigon, F; Vuichoud, J; Jarret, A; Pouteau, E; Gremaud, G; Oguey-Araymon, S; Courtois, D; Woupeyi, A; Finot, P A; Ballèvre, O

    2001-07-01

    The objectives of the present study were to determine the splanchnic extraction of glutamine after ingestion of glutamine-rich protein ((15)N-labeled oat proteins) and to compare it with that of free glutamine and to determine de novo glutamine synthesis before and after glutamine consumption. Eight healthy adults were infused intravenously in the postabsorptive state with L-[1-(13)C]glutamine (3 micromol x kg(-1) x h(-1)) and L-[1-(13)C]lysine (1.5 micromol x kg(-1) x h(-1)) for 8 h. Four hours after the beginning of the infusion, subjects consumed (every 20 min) a liquid formula providing either 2.5 g of protein from (15)N-labeled oat proteins or a mixture of free amino acids that mimicked the oat-amino acid profile and contained L-[2,5-(15)N(2)]glutamine and L-[2-(15)N]lysine. Splanchnic extraction of glutamine reached 62.5 +/- 5.0% and 66.7 +/- 3.9% after administration of (15)N-labeled oat proteins and the mixture of free amino acids, respectively. Lysine splanchnic extraction was also not different (40.9 +/- 11.9% and 34.9 +/- 10.6% for (15)N-labeled oat proteins and free amino acids, respectively). The main conclusion of the present study is that glutamine is equally bioavailable when given enterally as a free amino acid and when protein bound. Therefore, and taking into consideration the drawbacks of free glutamine supplementation of ready-to-use formulas for enteral nutrition, protein sources naturally rich in this amino acid are the best option for providing stable glutamine.

  1. Characteristics of L-glutamine transport in perfused rat skeletal muscle.

    PubMed Central

    Hundal, H S; Rennie, M J; Watt, P W

    1987-01-01

    1. We have investigated glutamine transport in the perfused rat hindlimb using the paired-tracer isotope dilution technique. 2. Uptake of L-glutamine was stereospecific, saturable, sodium dependent, insulin sensitive and pH insensitive in the physiological range. The maximum capacity of transport (Vmax) under normal perfusate conditions at 37 degrees C, 145 mM-Na+ and in the absence of insulin was 1156 +/- 193 nmol min-1 g-1 with transport being half-maximal at a perfusate glutamine concentration of 9.25 +/- 1.15 mM. 3. The kinetics of Na+ dependence strongly suggested co-transport of Na+ and glutamine with a stoichiometry of 1:1; furthermore, Na+ activated the carrier without any change in the concentration of glutamine at which transport was half-maximal, i.e. a 'Vmax effect' rather than a 'Km effect'. 4. The characteristics of glutamine transport, especially its substrate specificity and the pattern of competitive and non-competitive inhibition of glutamine transport by other amino acids, suggest that it is mediated by a carrier or carriers for which asparagine and histidine are also suitable substrates. 5. The characteristics of muscle glutamine transport are related but distinct from those of system N identified in hepatocytes; we suggest that they are sufficiently distinct to justify the identification of a new variant of mammalian amino acid transport systems which may be identified by the symbol Nm. 6. The kinetic characteristics of system Nm are such that glutamine is likely to be the most rapidly exchanging amino acid across the muscle membrane at physiological intra- and extracellular glutamine concentrations. Its hormone and ion sensitivities are likely to be important in the physiological modulation of whole-body glutamine metabolism and also during derangements observed in disease and after injury. PMID:3328779

  2. Functional role of glutamine 28 and arginine 39 in double stranded RNA cleavage by human pancreatic ribonuclease.

    PubMed

    Rehman, Md Tabish; Dey, Punyatirtha; Hassan, Md Imtaiyaz; Ahmad, Faizan; Batra, Janendra K

    2011-03-08

    Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR.

  3. Teaching the Krebs Cycle.

    ERIC Educational Resources Information Center

    Akeroyd, F. Michael

    1983-01-01

    Outlines a simple but rigorous treatment of the Krebs Cycle suitable for A-level Biology students. The importance of the addition of water molecules in various stages of the cycle is stressed as well as the removal of hydrogen atoms by the oxidizing enzymes. (JN)

  4. Modeling the role of covalent enzyme modification in Escherichia coli nitrogen metabolism

    NASA Astrophysics Data System (ADS)

    Kidd, Philip B.; Wingreen, Ned S.

    2010-03-01

    In the bacterium Escherichia coli, the enzyme glutamine synthetase (GS) converts ammonium into the amino acid glutamine. GS is principally active when the cell is experiencing nitrogen limitation, and its activity is regulated by a bicyclic covalent modification cascade. The advantages of this bicyclic-cascade architecture are poorly understood. We analyze a simple model of the GS cascade in comparison to other regulatory schemes and conclude that the bicyclic cascade is suboptimal for maintaining metabolic homeostasis of the free glutamine pool. Instead, we argue that the lag inherent in the covalent modification of GS slows the response to an ammonium shock and thereby allows GS to transiently detoxify the cell, while maintaining homeostasis over longer times.

  5. Modeling the role of covalent enzyme modification in Escherichia coli nitrogen metabolism

    PubMed Central

    Kidd, Philip B

    2013-01-01

    In the bacterium Escherichia coli, the enzyme glutamine synthetase (GS) converts ammonium into the amino acid glutamine. GS is principally active when the cell is experiencing nitrogen limitation, and its activity is regulated by a bicyclic covalent modification cascade. The advantages of this bicyclic-cascade architecture are poorly understood. We analyze a simple model of the GS cascade in comparison to other regulatory schemes and conclude that the bicyclic cascade is suboptimal for maintaining metabolic homeostasis of the free glutamine pool. Instead, we argue that the lag inherent in the covalent modification of GS slows the response to an ammonium shock and thereby allows GS to transiently detoxify the cell, while maintaining homeostasis over longer times. PMID:20057006

  6. Enteral L-Arginine and Glutamine Supplementation for Prevention of NEC in Preterm Neonates.

    PubMed

    El-Shimi, M S; Awad, H A; Abdelwahed, M A; Mohamed, M H; Khafagy, S M; Saleh, G

    2015-01-01

    Objective. Evaluating the efficacy and safety of arginine and glutamine supplementation in decreasing the incidence of NEC among preterm neonates. Methods. Prospective case-control study done on 75 preterm neonates ≤34 weeks, divided equally into L-arginine group receiving enteral L-arginine, glutamine group receiving enteral glutamine, and control group. Serum L-arginine and glutamine levels were measured at time of enrollment (sample 1), after 14 days of enrollment (sample 2), and at time of diagnosis of NEC (sample 3). Results. The incidence of NEC was 9.3%. There was no difference in the frequency of NEC between L-arginine and control groups (P > 0.05). NEC was not detected in glutamine group; L-arginine concentrations were significantly lower in arginine group than control group in both samples while glutamine concentrations were comparable in glutamine and control groups in both samples. No significant difference was found between groups as regards number of septic episodes, duration to reach full oral intake, or duration of hospital stay. Conclusion. Enteral L-arginine supplementation did not seem to reduce the incidence of NEC. Enteral glutamine may have a preventive role against NEC if supplied early to preterm neonates. However, larger studies are needed to confirm these findings. This work is registered in ClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT01263041).

  7. Effects of glutamine supplementation on the immune status in weaning piglets with intrauterine growth retardation.

    PubMed

    Zhong, Xiang; Li, Wei; Huang, Xuexin; Wang, Yuanxiao; Zhang, Lili; Zhou, Yanmin; Hussain, Ahmad; Wang, Tian

    2012-10-01

    Neonates with intrauterine growth retardation (IUGR) often suffer from impaired cellular immunity, and weaning may further aggravate adverse effects of IUGR on development and function of the immune system. In this study, we investigated effects of glutamine supplementation on immune status in the intestines of weaning pigs with IUGR, focusing on molecular mechanisms underlying altered immune response. Piglets with IUGR were weaned at 21 days of age and received orally 1.22 g alanine or 1 g glutamine per kg body weight every 12 h. Weight gain and intestinal weight of weaning piglets were increased by glutamine supplementation. Levels of serum IgG in piglets supplemented with glutamine were increased compared with Control piglets. The production of IL-1 and IL-8 in the serum and jejunum was decreased by glutamine supplementation, whereas the levels of IL-4 in the serum and the concentrations of IL-4 and IL-10 in the jejunum were increased. The expression of heat shock protein 70 (Hsp70) in the jejunum was increased by glutamine supplementation, but the degradation of inhibitor κB and the activity of nuclear factor-κB (NF-κB) were decreased. In conclusion, glutamine supplementation enhanced immune response in weaning piglets with IUGR. The effects of glutamine in IUGR are associated with increased Hsp70 expression and suppression of NF-κB activation.

  8. The clinical role of glutamine supplementation in patients with multiple trauma: a narrative review.

    PubMed

    Al Balushi, R M; Cohen, J; Banks, M; Paratz, J D

    2013-01-01

    Glutamine is considered an essential amino acid during stress and critical illness. Parenteral glutamine supplementation in critically ill patients has been shown to improve survival rate and minimise infectious complications, costs and hospital length-of-stay. However, glutamine supplementation in patients receiving enteral nutrition and the best method of administration are still controversial. The purpose of this article is to provide a narrative review of the current evidence and trials of enteral and parenteral glutamine supplementation in multiple trauma patients. A search in PubMed and EMBASE was conducted and relevant papers that investigated the effect of enteral or parenteral glutamine supplementation in patients with multiple trauma were reviewed. Although recent nutritional guidelines recommend that glutamine supplementation should be considered in these patients, further well-designed trials are required to provide a confirmed conclusion. Due to the inconclusive results of enteral glutamine supplementation trials in patients receiving enteral nutrition, future trials should focus on intravenous glutamine supplementation in patients requiring enteral nutrition and on major clinical outcome measures (e.g. mortality rate, infectious complications).

  9. Enteral L-Arginine and Glutamine Supplementation for Prevention of NEC in Preterm Neonates

    PubMed Central

    El-Shimi, M. S.; Awad, H. A.; Abdelwahed, M. A.; Mohamed, M. H.; Khafagy, S. M.; Saleh, G.

    2015-01-01

    Objective. Evaluating the efficacy and safety of arginine and glutamine supplementation in decreasing the incidence of NEC among preterm neonates. Methods. Prospective case-control study done on 75 preterm neonates ≤34 weeks, divided equally into L-arginine group receiving enteral L-arginine, glutamine group receiving enteral glutamine, and control group. Serum L-arginine and glutamine levels were measured at time of enrollment (sample 1), after 14 days of enrollment (sample 2), and at time of diagnosis of NEC (sample 3). Results. The incidence of NEC was 9.3%. There was no difference in the frequency of NEC between L-arginine and control groups (P > 0.05). NEC was not detected in glutamine group; L-arginine concentrations were significantly lower in arginine group than control group in both samples while glutamine concentrations were comparable in glutamine and control groups in both samples. No significant difference was found between groups as regards number of septic episodes, duration to reach full oral intake, or duration of hospital stay. Conclusion. Enteral L-arginine supplementation did not seem to reduce the incidence of NEC. Enteral glutamine may have a preventive role against NEC if supplied early to preterm neonates. However, larger studies are needed to confirm these findings. This work is registered in ClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT01263041). PMID:25861285

  10. Glutamine randomized studies in early life: the unsolved riddle of experimental and clinical studies.

    PubMed

    Briassouli, Efrossini; Briassoulis, George

    2012-01-01

    Glutamine may have benefits during immaturity or critical illness in early life but its effects on outcome end hardpoints are controversial. Our aim was to review randomized studies on glutamine supplementation in pups, infants, and children examining whether glutamine affects outcome. Experimental work has proposed various mechanisms of glutamine action but none of the randomized studies in early life showed any effect on mortality and only a few showed some effect on inflammatory response, organ function, and a trend for infection control. Although apparently safe in animal models (pups), premature infants, and critically ill children, glutamine supplementation does not reduce mortality or late onset sepsis, and its routine use cannot be recommended in these sensitive populations. Large prospectively stratified trials are needed to better define the crucial interrelations of "glutamine-heat shock proteins-stress response" in critical illness and to identify the specific subgroups of premature neonates and critically ill infants or children who may have a greater need for glutamine and who may eventually benefit from its supplementation. The methodological problems noted in the reviewed randomized experimental and clinical trials should be seriously considered in any future well-designed large blinded randomized controlled trial involving glutamine supplementation in critical illness.

  11. [Cellular immunity changes after total parenteral nutrition enriched with glutamine in patients with sepsis and malnutrition].

    PubMed

    Słotwiński, R; Pertkiewicz, M; Lech, G; Szczygieł, B

    2000-06-01

    The influence of glutamine on human immune system is multidirectional but the exact changes still remain unclear. In this study the effect of total parenteral nutrition (TPN) enriched with glutamine on some selected immunological and nutritional parameters was examined in twelve surgical patients with sepsis and malnutrition. The reason for glutamine supplementation was lack of clinical improvement after standard TPN. All patients received TPN enriched with glutamine for 10 days. Phenotypic analysis of peripheral blood mononuclear subsets (CD4, CD8, CD16, CD56, HLA-DR) were measured before, during (on days 2, 4, 6) glutamine administration and two days after (day 12) glutamine withdrawal. Simultaneously some nutritional parameters were assessed. The number and percentage of CD4, CD16, CD56 mononuclear subsets increased significantly on day 2 and stayed on the same level during observation (with exception in CD4 on day 6, 12 and CD56 on day 4). No significant differences in CD8 and HLA-DR number and percentages were observed after TPN enriched with glutamine. BIA examination revealed on days 2 and 12 significant decrease of total body water and significant increase of body cell mass, intracellular water on day 12. It was correlated with significant higher total lymphocytes count and significantly higher total protein, serum albumin, transferrin, cholesterol and CRP concentration. Results demonstrated that TPN supplemented with glutamine improved rapidly some immunological and nutritional parameters in surgical, malnutrition patients with sepsis.

  12. Glutamine Randomized Studies in Early Life: The Unsolved Riddle of Experimental and Clinical Studies

    PubMed Central

    Briassouli, Efrossini; Briassoulis, George

    2012-01-01

    Glutamine may have benefits during immaturity or critical illness in early life but its effects on outcome end hardpoints are controversial. Our aim was to review randomized studies on glutamine supplementation in pups, infants, and children examining whether glutamine affects outcome. Experimental work has proposed various mechanisms of glutamine action but none of the randomized studies in early life showed any effect on mortality and only a few showed some effect on inflammatory response, organ function, and a trend for infection control. Although apparently safe in animal models (pups), premature infants, and critically ill children, glutamine supplementation does not reduce mortality or late onset sepsis, and its routine use cannot be recommended in these sensitive populations. Large prospectively stratified trials are needed to better define the crucial interrelations of “glutamine-heat shock proteins-stress response” in critical illness and to identify the specific subgroups of premature neonates and critically ill infants or children who may have a greater need for glutamine and who may eventually benefit from its supplementation. The methodological problems noted in the reviewed randomized experimental and clinical trials should be seriously considered in any future well-designed large blinded randomized controlled trial involving glutamine supplementation in critical illness. PMID:23019424

  13. Glutamine Synthetase is Expressed by Primary Sensory Neurons from Chick Embryos In Vitro but not In Vivo: Influence of Skeletal Muscle Extract.

    PubMed

    Barakat-Walter, I.; Droz, B.

    1990-01-01

    Glutamine synthetase (GS) catalyses the ATP-dependent formation of glutamine from glutamate and ammonia. To determine whether dorsal root ganglion (DRG) cells from chick embryos express the enzyme in vivo or in vitro, GS was detected by immunocytochemical reaction either in vibratome sections of DRG or in dissociated DRG cell cultures. The immunocytochemical detection of GS showed that in vivo the DRG taken from chick embryos at day 10 (E10), E14, E18 or from chickens after hatching were free of any GS-positive ganglion cells; in contrast, in neuron-enriched cultures of DRG cells grown in vitro at E10, virtually all the neuronal cells (98.6 +/- 1.0%) express GS at 3, 5 or 7 days of culture. In mixed DRG cell cultures, only 83.6+/-4.6% of the neurons displayed a GS-immunoreactivity. In both culture conditions, neither the presence of horse serum nor the age of the culture appeared to affect the percentage of neurons which displayed a GS-immunoreactivity. After [3H]glutamine uptake, radioautographs revealed that only 80% of the neurons were labelled in neuron-enriched DRG cell cultures while 96% of the neurons were radioactive in mixed DRG cell cultures. Furthermore the most heavily [3H]glutamine-labelled neurons were exclusively found in mixed DRG cell cultures. Combination of both immunocytochemical detection of GS and radioautography after [3H]glutamine uptake showed that strongly GS-immunostained neurons corresponded to poorly radioactive ones and vice versa. When skeletal muscle extract (ME) was added to DRG cell cultures, the number of GS-positive neurons was reduced to 77.5 +/- 2.5% in neuron-enriched cultures or to 43.6 +/- 3.8% in mixed DRG cell cultures; in both types of culture, the intensity of the neuronal immunostaining was depressed. Furthermore, combined action of ME and non-neuronal cells potentiates the enzyme repression exerted separately by ME or non-neuronal cells. Since GS-immunoreactivity is expressed in DRG cells grown in vitro, but not in

  14. Butachlor impact on protein, free amino acid and glutamine contents, and on activity levels of aminotransferases, glutamate dehydrogenase and glutamine synthetase in the fresh water snail, Pila globosa (Swainson).

    PubMed

    Rajyalakshmi, T; Srinivas, T; Swamy, K V; Mohan, P M

    1996-08-01

    Biochemical changes followed in the freshwater snail Pila globosa (Swainson) during exposure to sublethal concentrations of the herbicide butachlor (26.6 ppm) in the ambient medium, at 3,6,12,24 and 48 h intervals, were marked by a significant decrease in total and soluble proteins, and an increase in free amino acids in foot and hepatopancreas up to 12 h before gradually recovering. Aminotransferase activities and glutamine content decreased during the early periods of exposure, while glutamate dehydrogenase activity increased. After an initial elevation, glutamate synthetase activity decreased at later intervals. Maximum effect of butachlor on the enzymes was seen after 12 h exposure. The extent of increase or decrease in different parameters examined varied between the two tissues studied. These changes are discussed in relation to the toxic stress of butachlor.

  15. Effects of supplementation with free glutamine and the dipeptide alanyl-glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise.

    PubMed

    Cruzat, Vinicius Fernandes; Rogero, Marcelo Macedo; Tirapegui, Julio

    2010-01-01

    In this study, we investigated the effect of the supplementation with the dipeptide L-alanyl-L-glutamine (DIP) and a solution containing L-glutamine and L-alanine on plasma levels markers of muscle damage and levels of pro-inflammatory cytokines and glutamine metabolism in rats submitted to prolonged exercise. Rats were submitted to sessions of swim training for 6 weeks. Twenty-one days prior to euthanasia, the animals were supplemented with DIP (n = 8) (1.5 g.kg(-1)), a solution of free L-glutamine (1 g.kg(-1)) and free L-alanine (0.61 g.kg(-1)) (G&A, n = 8) or water (control (CON), n = 8). Animals were killed at rest before (R), after prolonged exercise (PE-2 h of exercise). Plasma concentrations of glutamine, glutamate, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2 (PGE2) and activity of creatine kinase (CK), lactate dehydrogenase (LDH) and muscle concentrations of glutamine and glutamate were measured. The concentrations of plasma TNF-alpha, PGE2 and the activity of CK were lower in the G&A-R and DIP-R groups, compared to the CON-R. Glutamine in plasma (p < 0.04) and soleus muscle (p < 0.001) was higher in the DIP-R and G&A-R groups relative to the CON-R group. G&A-PE and DIP-PE groups exhibited lower concentrations of plasma PGE2 (p < 0.05) and TNF-alpha (p < 0.05), and higher concentrations of glutamine and glutamate in soleus (p < 0.001) and gastrocnemius muscles (p < 0.05) relative to the CON-PE group. We concluded that supplementation with free L-glutamine and the dipeptide LL-alanyl-LL-glutamine represents an effective source of glutamine, which may attenuate inflammation biomarkers after periods of training and plasma levels of CK and the inflammatory response induced by prolonged exercise.

  16. Effects of supplementation with free glutamine and the dipeptide alanyl-glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise.

    PubMed

    Cruzat, Vinicius Fernandes; Rogero, Marcelo Macedo; Tirapegui, Julio

    2010-01-01

    In this study, we investigated the effect of the supplementation with the dipeptide L-alanyl-L-glutamine (DIP) and a solution containing L-glutamine and L-alanine on plasma levels markers of muscle damage and levels of pro-inflammatory cytokines and glutamine metabolism in rats submitted to prolonged exercise. Rats were submitted to sessions of swim training for 6 weeks. Twenty-one days prior to euthanasia, the animals were supplemented with DIP (n = 8) (1.5 g.kg(-1)), a solution of free L-glutamine (1 g.kg(-1)) and free L-alanine (0.61 g.kg(-1)) (G&A, n = 8) or water (control (CON), n = 8). Animals were killed at rest before (R), after prolonged exercise (PE-2 h of exercise). Plasma concentrations of glutamine, glutamate, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2 (PGE2) and activity of creatine kinase (CK), lactate dehydrogenase (LDH) and muscle concentrations of glutamine and glutamate were measured. The concentrations of plasma TNF-alpha, PGE2 and the activity of CK were lower in the G&A-R and DIP-R groups, compared to the CON-R. Glutamine in plasma (p < 0.04) and soleus muscle (p < 0.001) was higher in the DIP-R and G&A-R groups relative to the CON-R group. G&A-PE and DIP-PE groups exhibited lower concentrations of plasma PGE2 (p < 0.05) and TNF-alpha (p < 0.05), and higher concentrations of glutamine and glutamate in soleus (p < 0.001) and gastrocnemius muscles (p < 0.05) relative to the CON-PE group. We concluded that supplementation with free L-glutamine and the dipeptide LL-alanyl-LL-glutamine represents an effective source of glutamine, which may attenuate inflammation biomarkers after periods of training and plasma levels of CK and the inflammatory response induced by prolonged exercise. PMID:19885855

  17. Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism.

    PubMed

    Murray, David S; Chinnam, Nagababu; Tonthat, Nam Ky; Whitfill, Travis; Wray, Lewis V; Fisher, Susan H; Schumacher, Maria A

    2013-12-13

    Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg(62), from an adjacent subunit. Notably, Arg(62) must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.

  18. Oral supplementations with free and dipeptide forms of L-glutamine in endotoxemic mice: effects on muscle glutamine-glutathione axis and heat shock proteins.

    PubMed

    Cruzat, Vinicius F; Pantaleão, Lucas C; Donato, José; de Bittencourt, Paulo Ivo Homem; Tirapegui, Julio

    2014-03-01

    Sepsis is a leading cause of death in intensive care units worldwide. Low availability of glutamine contributes to the catabolic state of sepsis. L-Glutamine supplementation has antioxidant properties and modulates the expression of heat shock proteins (HSPs). This study investigated the effects of oral supplementation with L-glutamine plus L-alanine (GLN+ALA), both in the free form and L-alanyl-L-glutamine dipeptide (DIP), on glutamine-glutathione (GSH) axis and HSPs expression in endotoxemic mice. B6.129F2/J mice were subjected to endotoxemia (lipopolysaccharides from Escherichia coli, 5 mg.kg(-1), LPS group) and orally supplemented for 48 h with either L-glutamine (1 g.kg(-1)) plus L-alanine (0.61 g.kg(-1)) (GLN+ALA-LPS group) or 1.49 g.kg(-1) of DIP (DIP-LPS group). Endotoxemia reduced plasma and muscle glutamine concentrations [relative to CTRL group] which were restored in both GLN+ALA-LPS and DIP-LPS groups (P<.05). In supplemented groups were re-established GSH content and intracellular redox status (GSSG/GSH ratio) in circulating erythrocytes and muscle. Thiobarbituric acid reactive substance was 4-fold in LPS treated mice relative to the untreated CTRL group, and plasma TNF-α and IL-1β levels were attenuated by the supplements. Heat shock proteins 27, 70 and 90 (protein and mRNA) were elevated in the LPS group and were returned to basal levels (relative to CTRL group) in both GLN+ALA-LPS and DIP-LPS groups. Supplementations to endotoxemic mice resulted in up-regulation of GSH reductase, GSH peroxidase and glutamate cysteine ligase mRNA expression in muscle. In conclusion, oral supplementations with GLN+ALA or DIP are effective in reversing the conditions of LPS-induced deleterious impact on glutamine-GSH axis in mice under endotoxemia.

  19. The effect of glutamine supplementation and physical exercise on neutrophil function.

    PubMed

    Lagranha, C J; Levada-Pires, A C; Sellitti, D F; Procopio, J; Curi, R; Pithon-Curi, T C

    2008-04-01

    Glutamine is the most abundant free amino acid in the body. Its primary source is skeletal muscle, from where it is released into the bloodstream and transported to a variety of tissues. Several studies have shown that glutamine is important for rat and human neutrophil function and that these cells utilize glutamine at high rates. Physical exercise has also been shown to induce considerable changes in neutrophil metabolism and function. As neutrophils represent 50-60% of the total circulating leukocyte pool and play a key role in inflammation, both physical exercise and glutamine might be expected to regulate the inflammatory process. In this review, the changes in neutrophil function induced by physical exercise and glutamine supplementation are compared. PMID:17928941

  20. Combined Use of Residual Dipolar Couplings and Solution X-ray Scattering To Rapidly Probe Rigid-Body Conformational Transitions in a Non-phosphorylatable Active-Site Mutant of the 128 kDa Enzyme I Dimer

    SciTech Connect

    Takayama, Yuki; Schwieters, Charles D.; Grishaev, Alexander; Ghirlando, Rodolfo; Clore, G. Marius

    2012-10-23

    The first component of the bacterial phosphotransferase system, enzyme I (EI), is a multidomain 128 kDa dimer that undergoes large rigid-body conformational transitions during the course of its catalytic cycle. Here we investigate the solution structure of a non-phosphorylatable active-site mutant in which the active-site histidine is substituted by glutamine. We show that perturbations in the relative orientations and positions of the domains and subdomains can be rapidly and reliably determined by conjoined rigid-body/torsion angle/Cartesian simulated annealing calculations driven by orientational restraints from residual dipolar couplings and shape and translation information afforded by small- and wide-angle X-ray scattering. Although histidine and glutamine are isosteric, the conformational space available to a Gln side chain is larger than that for the imidazole ring of His. An additional hydrogen bond between the side chain of Gln189 located on the EIN{sup {alpha}/{beta}} subdomain and an aspartate (Asp129) on the EIN{sup {alpha}} subdomain results in a small ({approx}9{sup o}) reorientation of the EIN{sup {alpha}} and EIN{sup {alpha}/{beta}} subdomains that is in turn propagated to a larger reorientation ({approx}26{sup o}) of the EIN domain relative to the EIC dimerization domain, illustrating the positional sensitivity of the EIN domain and its constituent subdomains to small structural perturbations.

  1. Gene-Enzyme Relationships of Aromatic Amino Acid Biosynthesis in Higher Plants

    SciTech Connect

    2002-08-12

    Inhibition studies of amino acids in Nicotiana silvestris suspension cells gave clues to the difficulties for obtaining mutants deficient in post prephenate pathway proteins of aromatic amino acid biosynthesis (prephenate aminotransferase, arogenate dehydrogenase and arogenate dehydratase). Such mutants, if successfully obtained, would allow gene-enzyme relationships of aromatic amino acid proteins to be studied. We found that amino acids were inhibitory toward plant cell growth, and thus were unable to rescue analog resistant mutants. Toxicity of all amino acids toward exponentially dividing Nicotiana silvestris suspension cultured cells was monitored by following growth rates. Except for L-glutamine, all 19 protein amino acids inhibited cell growth. Inhibition of growth progressed to cell deterioration. Electron microscopy showed that amino acids triggered a state of cell shrinkage that eventually degenerated to total cellular disorganization. L-glutamine was not only an effective agent for prevention of amino acid toxicity, but enhanced the final growth yield. L-glutamine also was able to completely reverse inhibition effects in cells that had been in the slowed exponential phase. Two types of inhibition occurred and we have proposed that any amino acid inhibition that can be completely antagonized by L-glutamine be called ''general amino acid inhibition''. ''Specific amino acid inhibition'' resulting from particular pathway imbalances caused by certain exogenous amino acids, can be recognized and studied in the presence of L-glutamine which can abolishes the complication effects of general amino acid inhibition.

  2. Regulation of glutamine carrier proteins by RNF5 determines breast cancer response to ER stress-inducing chemotherapies.

    PubMed

    Jeon, Young Joo; Khelifa, Sihem; Ratnikov, Boris; Scott, David A; Feng, Yongmei; Parisi, Fabio; Ruller, Chelsea; Lau, Eric; Kim, Hyungsoo; Brill, Laurence M; Jiang, Tingting; Rimm, David L; Cardiff, Robert D; Mills, Gordon B; Smith, Jeffrey W; Osterman, Andrei L; Kluger, Yuval; Ronai, Ze'ev A

    2015-03-01

    Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination, an