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Sample records for glutamate synthase activity

  1. AMPK Activation Affects Glutamate Metabolism in Astrocytes

    DEFF Research Database (Denmark)

    Voss, Caroline Marie; Pajęcka, Kamilla; Stridh, Malin H

    2015-01-01

    acid (TCA) cycle was studied using high-performance liquid chromatography analysis supplemented with gas chromatography-mass spectrometry technology. It was found that AMPK activation had profound effects on the pathways involved in glutamate metabolism since the entrance of the glutamate carbon...... on glutamate metabolism in astrocytes was studied using primary cultures of these cells from mouse cerebral cortex during incubation in media containing 2.5 mM glucose and 100 µM [U-(13)C]glutamate. The metabolism of glutamate including a detailed analysis of its metabolic pathways involving the tricarboxylic...... skeleton into the TCA cycle was reduced. On the other hand, glutamate uptake into the astrocytes as well as its conversion to glutamine catalyzed by glutamine synthetase was not affected by AMPK activation. Interestingly, synthesis and release of citrate, which are hallmarks of astrocytic function, were...

  2. Glutamate mediated astrocytic filtering of neuronal activity.

    Directory of Open Access Journals (Sweden)

    Gilad Wallach

    2014-12-01

    Full Text Available Neuron-astrocyte communication is an important regulatory mechanism in various brain functions but its complexity and role are yet to be fully understood. In particular, the temporal pattern of astrocyte response to neuronal firing has not been fully characterized. Here, we used neuron-astrocyte cultures on multi-electrode arrays coupled to Ca2+ imaging and explored the range of neuronal stimulation frequencies while keeping constant the amount of stimulation. Our results reveal that astrocytes specifically respond to the frequency of neuronal stimulation by intracellular Ca2+ transients, with a clear onset of astrocytic activation at neuron firing rates around 3-5 Hz. The cell-to-cell heterogeneity of the astrocyte Ca2+ response was however large and increasing with stimulation frequency. Astrocytic activation by neurons was abolished with antagonists of type I metabotropic glutamate receptor, validating the glutamate-dependence of this neuron-to-astrocyte pathway. Using a realistic biophysical model of glutamate-based intracellular calcium signaling in astrocytes, we suggest that the stepwise response is due to the supralinear dynamics of intracellular IP3 and that the heterogeneity of the responses may be due to the heterogeneity of the astrocyte-to-astrocyte couplings via gap junction channels. Therefore our results present astrocyte intracellular Ca2+ activity as a nonlinear integrator of glutamate-dependent neuronal activity.

  3. Glutamate Mediated Astrocytic Filtering of Neuronal Activity

    Science.gov (United States)

    Herzog, Nitzan; De Pittà, Maurizio; Jacob, Eshel Ben; Berry, Hugues; Hanein, Yael

    2014-01-01

    Neuron-astrocyte communication is an important regulatory mechanism in various brain functions but its complexity and role are yet to be fully understood. In particular, the temporal pattern of astrocyte response to neuronal firing has not been fully characterized. Here, we used neuron-astrocyte cultures on multi-electrode arrays coupled to Ca2+ imaging and explored the range of neuronal stimulation frequencies while keeping constant the amount of stimulation. Our results reveal that astrocytes specifically respond to the frequency of neuronal stimulation by intracellular Ca2+ transients, with a clear onset of astrocytic activation at neuron firing rates around 3-5 Hz. The cell-to-cell heterogeneity of the astrocyte Ca2+ response was however large and increasing with stimulation frequency. Astrocytic activation by neurons was abolished with antagonists of type I metabotropic glutamate receptor, validating the glutamate-dependence of this neuron-to-astrocyte pathway. Using a realistic biophysical model of glutamate-based intracellular calcium signaling in astrocytes, we suggest that the stepwise response is due to the supralinear dynamics of intracellular IP3 and that the heterogeneity of the responses may be due to the heterogeneity of the astrocyte-to-astrocyte couplings via gap junction channels. Therefore our results present astrocyte intracellular Ca2+ activity as a nonlinear integrator of glutamate-dependent neuronal activity. PMID:25521344

  4. Role of a Highly Conserved and Catalytically Important Glutamate-49 in the Enterococcus faecalis Acetolactate Synthase

    International Nuclear Information System (INIS)

    Lee, Miyoung; Lee, Sangchoon; Cho, Junehaeng; Ryu, Seong Eon; Yoon, Moonyoung; Koo, Bonsung

    2013-01-01

    Acetolactate synthase (ALS) is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylation of pyruvate and then condenses the hydroxyethyl moiety with another molecule of pyruvate to give 2-acetolactate (AL). AL is a key metabolic intermediate in various metabolic pathways of microorganisms. In addition, AL can be converted to acetoin, an important physiological metabolite that is excreted by many microorganisms. There are two types of ALSs reported in the literature, anabolic aceto-hydroxyacid synthase (AHAS) and catabolic ALSs (cALS). The anabolic AHAS is primarily found in plants, fungi, and bacteria, is involved in the biosynthesis of branched-chain amino acids (BCAAs), and contains flavin adenine dinucleotide (FAD), whereas the cALS is found only in some bacteria and is involved in the butanediol fermentation pathway. Both of the enzymes are ThDP-dependent and require a divalent metal ion for catalytic activity. Despite the similarities of the reactions catalyzed, the cALS can be distinguished from anabolic AHAS by a low optimal pH of about 6.0, FAD-independent functionality, a genetic location within the butanediol operon, and lack of a regulatory subunit. It is noteworthy that the structural and functional features of AHAS have been extensively studied, in contrast to those of cALS, for which only limited information is available. To date, the only crystal structure of cALS reported is from Klebsiella pneumonia, which revealed that the overall structure of K. pneumonia ALS is similar to that of AHAS except for the FAD binding region found in AHAS

  5. Influence of UV-A or UV-B light and of the nitrogen source on the induction of ferredoxin-dependent glutamate synthase in etiolated tomato cotyledons

    International Nuclear Information System (INIS)

    Migge, A.; Carrayol, E.; Hirel, B.; Lohmann, M.; Meya, G.; Becker, T.W.

    1998-01-01

    The influence of ultraviolet A (UV-A) or B (UV-B) light and of the nitrogen source on the induction of ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was examined in etiolated cotyledons of tomato (Lycopersicon escu- lentum L.). The Fd-GOGAT activity increased upon illumination of etiolated tomato cotyledons with UV-A or UV-B light. This stimulation of Fd-GOGAT activity was correlated with an increase in both the Fd-GOGAT transcript level and the Fd-GOGAT protein abundance. These results suggest that UV-A or UV-B light stimulates the de novo synthesis of Fd-GOGAT in etiolated tomato cotyledons. Both UV-A and UV-B light failed to influence the activity of NADH-GOGAT (EC 1.4.1.14) in etiolated tomato cotyledons. Taken together, our data indicate that the tomato genes encoding Fd- or NADH-dependent glutamate synthase are regulated differently by UV-A or UV-B light. No difference with respect to both the Fd-GOGAT transcript and protein abundance was found between cotyledons of tomato seedlings grown with either nitrate or ammonium as the sole N-source in the dark or in white light. In addition, the increase in the Fd-GOGAT protein pool induced by white light in etiolated nitrate-grown tomato seedling cotyledons was similar to that induced by white light in etiolated ammonium-grown tomato seedling cotyledons. These results show that the tomato Fd-GOGAT protein level does not depend strongly on the nature of the nitrogen source and that there appears to be no major stimulatory effect on the Fd-GOGAT protein pool produced by nitrate during the illumination of etiolated tomato cotyledons

  6. Glutamic acid as anticancer agent: An overview.

    Science.gov (United States)

    Dutta, Satyajit; Ray, Supratim; Nagarajan, K

    2013-10-01

    The objective of the article is to highlight various roles of glutamic acid like endogenic anticancer agent, conjugates to anticancer agents, and derivatives of glutamic acid as possible anticancer agents. Besides these emphases are given especially for two endogenous derivatives of glutamic acid such as glutamine and glutamate. Glutamine is a derivative of glutamic acid and is formed in the body from glutamic acid and ammonia in an energy requiring reaction catalyzed by glutamine synthase. It also possesses anticancer activity. So the transportation and metabolism of glutamine are also discussed for better understanding the role of glutamic acid. Glutamates are the carboxylate anions and salts of glutamic acid. Here the roles of various enzymes required for the metabolism of glutamates are also discussed.

  7. Glycogen synthase activation by sugars in isolated hepatocytes.

    Science.gov (United States)

    Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

    1988-07-01

    We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

  8. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci

    Directory of Open Access Journals (Sweden)

    Guillermo Hugo Peralta

    Full Text Available ABSTRACT Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor.

  9. Modulation of hyaluronan synthase activity in cellular membrane fractions

    OpenAIRE

    Vigetti, Davide; Genasetti, A; Karousou, Evgenia; Viola, Manuela; Clerici, M; Bartolini, B; Moretto, Paola; DE LUCA, Giancarlo; Hascall, Vc; Passi, Alberto

    2009-01-01

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in euk...

  10. GNC and CGA1 Modulate Chlorophyll Biosynthesis and Glutamate Synthase (GLU1/Fd-GOGAT) Expression in Arabidopsis

    Science.gov (United States)

    Hudson, Darryl; Guevara, David; Yaish, Mahmoud W.; Hannam, Carol; Long, Nykoll; Clarke, Joseph D.; Bi, Yong-Mei; Rothstein, Steven J.

    2011-01-01

    Chloroplast development is an important determinant of plant productivity and is controlled by environmental factors including amounts of light and nitrogen as well as internal phytohormones including cytokinins and gibberellins (GA). The paralog GATA transcription factors GNC and CGA1/GNL up-regulated by light, nitrogen and cytokinin while also being repressed by GA signaling. Modifying the expression of these genes has previously been shown to influence chlorophyll content in Arabidopsis while also altering aspects of germination, elongation growth and flowering time. In this work, we also use transgenic lines to demonstrate that GNC and CGA1 exhibit a partially redundant control over chlorophyll biosynthesis. We provide novel evidence that GNC and CGA1 influence both chloroplast number and leaf starch in proportion to their transcript level. GNC and CGA1 were found to modify the expression of chloroplast localized GLUTAMATE SYNTHASE (GLU1/Fd-GOGAT), which is the primary factor controlling nitrogen assimilation in green tissue. Altering GNC and CGA1 expression was also found to modulate the expression of important chlorophyll biosynthesis genes (GUN4, HEMA1, PORB, and PORC). As previously demonstrated, the CGA1 transgenic plants demonstrated significantly altered timing to a number of developmental events including germination, leaf production, flowering time and senescence. In contrast, the GNC transgenic lines we analyzed maintain relatively normal growth phenotypes outside of differences in chloroplast development. Despite some evidence for partial divergence, results indicate that regulation of both GNC and CGA1 by light, nitrogen, cytokinin, and GA acts to modulate nitrogen assimilation, chloroplast development and starch production. Understanding the mechanisms controlling these processes is important for agricultural biotechnology. PMID:22102866

  11. The metabotropic glutamate receptors: structure, activation mechanism and pharmacology.

    Science.gov (United States)

    Pin, Jean-Philippe; Acher, Francine

    2002-06-01

    The metabotropic glutamate receptors are G-protein coupled receptors (GPCR) involved in the regulation of many synapses, including most glutamatergic fast excitatory synapses. Eight subtypes have been identified that can be classified into three groups. The molecular characterization of these receptors revealed proteins much more complex than any other GPCRs. They are composed of a Venus Flytrap (VFT) module where glutamate binds, connected to a heptahelical domain responsible for G-protein coupling. Recent data including the structure of the VFT module determined with and without glutamate, indicate that these receptors function as dimers. Moreover a number of intracellular proteins can regulate their targeting and transduction mechanism. Such structural features of mGlu receptors offer multiple possibilities for synthetic compounds to modulate their activity. In addition to agonists and competitive antagonists acting at the glutamate binding site, a number of non-competitive antagonists with inverse agonist activity, and positive allosteric modulators have been discovered. These later compounds share specific properties that make them good candidates for therapeutic applications. First, their non-amino acid structure makes them pass more easily the blood brain barrier. Second, they are much more selective than any other compound identified so far, being the first subtype selective molecules. Third, for the negative modulators, their non competitive mechanism of action makes them relatively unaffected by high concentrations of glutamate that may be present in disease states (e.g. stroke, epilepsy, neuropathic pain, etc.). Fourth, like the benzodiazepines acting at the GABA(A) receptors, the positive modulators offer a new way to increase the activity of these receptors in vivo, with a low risk of inducing their desensitization. The present review article focuses on the specific structural features of these receptors and highlights the various possibilities these

  12. Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

    2014-08-01

    Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

    Science.gov (United States)

    Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

    2016-12-21

    Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

  14. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    Insulin resistance in skeletal muscle is a major hallmark of type 2 diabetes and an early detectable abnormality in the development of this disease. The cellular mechanisms of insulin resistance include impaired insulin-mediated muscle glycogen synthesis and increased intramyocellular lipid content......, whereas impaired insulin activation of muscle glycogen synthase represents a consistent, molecular defect found in both type 2 diabetic and high-risk individuals. Despite several studies of the insulin signaling pathway believed to mediate dephosphorylation and hence activation of glycogen synthase......, the molecular mechanisms responsible for this defect remain unknown. Recently, the use of phospho-specific antibodies in human diabetic muscle has revealed hyperphosphorylation of glycogen synthase at sites not regulated by the classical insulin signaling pathway. In addition, novel approaches such as gene...

  15. Fast inhibition of glutamate-activated currents by caffeine.

    Directory of Open Access Journals (Sweden)

    Nicholas P Vyleta

    Full Text Available BACKGROUND: Caffeine stimulates calcium-induced calcium release (CICR in many cell types. In neurons, caffeine stimulates CICR presynaptically and thus modulates neurotransmitter release. METHODOLOGY/PRINCIPAL FINDINGS: Using the whole-cell patch-clamp technique we found that caffeine (20 mM reversibly increased the frequency and decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs in neocortical neurons. The increase in mEPSC frequency is consistent with a presynaptic mechanism. Caffeine also reduced exogenously applied glutamate-activated currents, confirming a separate postsynaptic action. This inhibition developed in tens of milliseconds, consistent with block of channel currents. Caffeine (20 mM did not reduce currents activated by exogenous NMDA, indicating that caffeine block is specific to non-NMDA type glutamate receptors. CONCLUSIONS/SIGNIFICANCE: Caffeine-induced inhibition of mEPSC amplitude occurs through postsynaptic block of non-NMDA type ionotropic glutamate receptors. Caffeine thus has both pre and postsynaptic sites of action at excitatory synapses.

  16. Nitric oxide facilitates active avoidance learning via enhancement of glutamate levels in the hippocampal dentate gyrus.

    Science.gov (United States)

    Wang, Shi; Pan, De-Xi; Wang, Dan; Wan, Peng; Qiu, De-Lai; Jin, Qing-Hua

    2014-09-01

    The hippocampus is a key structure for learning and memory in mammals, and long-term potentiation (LTP) is an important cellular mechanism responsible for learning and memory. Despite a number of studies indicating that nitric oxide (NO) is involved in the formation and maintenance of LTP as a retrograde messenger, few studies have used neurotransmitter release as a visual indicator in awake animals to explore the role of NO in learning-dependent long-term enhancement of synaptic efficiency. Therefore, in the present study, the effects of l-NMMA (a NO synthase inhibitor) and SNP (a NO donor) on extracellular glutamate (Glu) concentrations and amplitudes of field excitatory postsynaptic potential (fEPSP) were measured in the hippocampal dentate gyrus (DG) region during the acquisition and extinction of active-avoidance behavior in freely-moving conscious rats. In the control group, the extracellular concentration of Glu in the DG was significantly increased during the acquisition of active-avoidance behavior and gradually returned to baseline levels following extinction training. In the experimental group, the change in Glu concentration was significantly reduced by local microinjection of l-NMMA, as was the acquisition of the active-avoidance behavior. In contrast, the change in Glu concentration was significantly enhanced by SNP, and the acquisition of the active-avoidance behavior was significantly accelerated. Furthermore, in all groups, the changes in extracellular Glu were accompanied by corresponding changes in fEPSP amplitude and active-avoidance behavior. Our results suggest that NO in the hippocampal DG facilitates active avoidance learning via enhancements of glutamate levels and synaptic efficiency in rats. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Phytochelatin synthase activity as a marker of metal pollution

    Energy Technology Data Exchange (ETDEWEB)

    Zitka, Ondrej; Krystofova, Olga; Sobrova, Pavlina [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Adam, Vojtech [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Zehnalek, Josef; Beklova, Miroslava [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Kizek, Rene, E-mail: kizek@sci.muni.cz [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic)

    2011-08-30

    Highlights: {yields} New tool for determination of phytochelatin synthase activity. {yields} The optimization of experimental condition for determination of the enzyme activity. {yields} First evaluation of K{sub m} for the enzyme. {yields} The effects of cadmium (II) not only on the activity of the enzyme but also on K{sub m}. -- Abstract: The synthesis of phytochelatins is catalyzed by {gamma}-Glu-Cys dipeptidyl transpeptidase called phytochelatin synthase (PCS). Aim of this study was to suggest a new tool for determination of phytochelatin synthase activity in the tobacco BY-2 cells treated with different concentrations of the Cd(II). After the optimization steps, an experiment on BY-2 cells exposed to different concentrations of Cd(NO{sub 3}){sub 2} for 3 days was performed. At the end of the experiment, cells were harvested and homogenized. Reduced glutathione and cadmium (II) ions were added to the cell suspension supernatant. These mixtures were incubated at 35 {sup o}C for 30 min and analysed using high performance liquid chromatography coupled with electrochemical detector (HPLC-ED). The results revealed that PCS activity rises markedly with increasing concentration of cadmium (II) ions. The lowest concentration of the toxic metal ions caused almost three fold increase in PCS activity as compared to control samples. The activity of PCS (270 fkat) in treated cells was more than seven times higher in comparison to control ones. K{sub m} for PCS was estimated as 2.3 mM.

  18. Phytochelatin synthase activity as a marker of metal pollution

    International Nuclear Information System (INIS)

    Zitka, Ondrej; Krystofova, Olga; Sobrova, Pavlina; Adam, Vojtech; Zehnalek, Josef; Beklova, Miroslava; Kizek, Rene

    2011-01-01

    Highlights: → New tool for determination of phytochelatin synthase activity. → The optimization of experimental condition for determination of the enzyme activity. → First evaluation of K m for the enzyme. → The effects of cadmium (II) not only on the activity of the enzyme but also on K m . -- Abstract: The synthesis of phytochelatins is catalyzed by γ-Glu-Cys dipeptidyl transpeptidase called phytochelatin synthase (PCS). Aim of this study was to suggest a new tool for determination of phytochelatin synthase activity in the tobacco BY-2 cells treated with different concentrations of the Cd(II). After the optimization steps, an experiment on BY-2 cells exposed to different concentrations of Cd(NO 3 ) 2 for 3 days was performed. At the end of the experiment, cells were harvested and homogenized. Reduced glutathione and cadmium (II) ions were added to the cell suspension supernatant. These mixtures were incubated at 35 o C for 30 min and analysed using high performance liquid chromatography coupled with electrochemical detector (HPLC-ED). The results revealed that PCS activity rises markedly with increasing concentration of cadmium (II) ions. The lowest concentration of the toxic metal ions caused almost three fold increase in PCS activity as compared to control samples. The activity of PCS (270 fkat) in treated cells was more than seven times higher in comparison to control ones. K m for PCS was estimated as 2.3 mM.

  19. Aspartate and glutamate mimetic structures in biologically active compounds.

    Science.gov (United States)

    Stefanic, Peter; Dolenc, Marija Sollner

    2004-04-01

    Glutamate and aspartate are frequently recognized as key structural elements for the biological activity of natural peptides and synthetic compounds. The acidic side-chain functionality of both the amino acids provides the basis for the ionic interaction and subsequent molecular recognition by specific receptor sites that results in the regulation of physiological or pathophysiological processes in the organism. In the development of new biologically active compounds that possess the ability to modulate these processes, compounds offering the same type of interactions are being designed. Thus, using a peptidomimetic design approach, glutamate and aspartate mimetics are incorporated into the structure of final biologically active compounds. This review covers different bioisosteric replacements of carboxylic acid alone, as well as mimetics of the whole amino acid structure. Amino acid analogs presented include those with different distances between anionic moieties, and analogs with additional functional groups that result in conformational restriction or alternative interaction sites. The article also provides an overview of different cyclic structures, including various cycloalkane, bicyclic and heterocyclic analogs, that lead to conformational restriction. Higher di- and tripeptide mimetics in which carboxylic acid functionality is incorporated into larger molecules are also reviewed. In addition to the mimetic structures presented, emphasis in this article is placed on their steric and electronic properties. These mimetics constitute a useful pool of fragments in the design of new biologically active compounds, particularly in the field of RGD mimetics and excitatory amino acid agonists and antagonists.

  20. Glutamate transporter activity promotes enhanced Na+/K+-ATPase -mediated extracellular K+ management during neuronal activity

    DEFF Research Database (Denmark)

    Larsen, Brian R; Holm, Rikke; Vilsen, Bente

    2016-01-01

    , in addition, Na+ /K+ -ATPase-mediated K+ clearance could be governed by astrocytic [Na+ ]i . During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+ -coupled glutamate transporters, thereby elevating [Na+ ]i . It thus remains unresolved whether...... the different Na+ /K+ -ATPase isoforms are controlled by [K+ ]o or [Na+ ]i during neuronal activity. Hippocampal slice recordings of stimulus-induced [K+ ]o transients with ion-sensitive microelectrodes revealed reduced Na+ /K+ -ATPase-mediated K+ management upon parallel inhibition of the glutamate transporter......+ affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na+ /K+ -ATPase-dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+ /K+ -ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na+ ]i...

  1. Intracellular synthesis of glutamic acid in Bacillus methylotrophicus SK19.001, a glutamate-independent poly(γ-glutamic acid)-producing strain.

    Science.gov (United States)

    Peng, Yingyun; Zhang, Tao; Mu, Wanmeng; Miao, Ming; Jiang, Bo

    2016-01-15

    Bacillus methylotrophicus SK19.001 is a glutamate-independent strain that produces poly(γ-glutamic acid) (γ-PGA), a polymer of D- and L-glutamic acids that possesses applications in food, the environment, agriculture, etc. This study was undertaken to explore the synthetic pathway of intracellular L- and D-glutamic acid in SK19.001 by investigating the effects of tricarboxylic acid cycle intermediates and different amino acids as metabolic precursors on the production of γ-PGA and analyzing the activities of the enzymes involved in the synthesis of L- and D-glutamate. Tricarboxylic acid cycle intermediates and amino acids could participate in the synthesis of γ-PGA via independent pathways in SK19.001. L-Aspartate aminotransferase, L-glutaminase and L-glutamate synthase were the enzymatic sources of L-glutamate. Glutamate racemase was responsible for the formation of D-glutamate for the synthesis of γ-PGA, and the synthetase had stereoselectivity for glutamate substrate. The enzymatic sources of L-glutamate were investigated for the first time in the glutamate-independent γ-PGA-producing strain, and multiple enzymatic sources of L-glutamate were verified in SK19.001, which will benefit efforts to improve production of γ-PGA with metabolic engineering strategies. © 2015 Society of Chemical Industry.

  2. The primary defect in glycogen synthase activity is not based on increased glycogen synthase kinase-3a activity in diabetic myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael; Brusgaard, Klaus; Handberg, Aa.

    2004-01-01

    The mechanism responsible for the diminished activation of glycogen synthase (GS) in diabetic myotubes remains unclear, but may involve increased activity and/or expression of glycogen synthase kinase-3 (GSK-3). In myotubes established from type 2 diabetic and healthy control subjects we determined...

  3. Modulation of hyaluronan synthase activity in cellular membrane fractions.

    Science.gov (United States)

    Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

    2009-10-30

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

  4. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal......NOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age...

  5. Structures of the N-acetyltransferase domain of Xylella fastidiosa N-acetyl-L-glutamate synthase/kinase with and without a His tag bound to N-acetyl-L-glutamate.

    Science.gov (United States)

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

    2015-01-01

    Structures of the catalytic N-acetyltransferase (NAT) domain of the bifunctional N-acetyl-L-glutamate synthase/kinase (NAGS/K) from Xylella fastidiosa bound to N-acetyl-L-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4 Å resolution, respectively. The NAT domain with an N-terminal His tag crystallized in space group P4(1)2(1)2, with unit-cell parameters a=b=51.72, c=242.31 Å. Two subunits form a molecular dimer in the asymmetric unit, which contains ∼41% solvent. The NAT domain without an N-terminal His tag crystallized in space group P21, with unit-cell parameters a=63.48, b=122.34, c=75.88 Å, β=107.6°. Eight subunits, which form four molecular dimers, were identified in the asymmetric unit, which contains ∼38% solvent. The structures with and without the N-terminal His tag provide an opportunity to evaluate how the His tag affects structure and function. Furthermore, multiple subunits in different packing environments allow an assessment of the plasticity of the NAG binding site, which might be relevant to substrate binding and product release. The dimeric structure of the X. fastidiosa N-acetytransferase (xfNAT) domain is very similar to that of human N-acetyltransferase (hNAT), reinforcing the notion that mammalian NAGS is evolutionally derived from bifunctional bacterial NAGS/K.

  6. Reduced ceramide synthase 2 activity causes progressive myoclonic epilepsy

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Olsen, Anne S B; Neess, Ditte

    2014-01-01

    between genes involved in SL metabolism and epilepsy. METHODS: We used quantitative real-time PCR, Western blotting, and enzymatic assays to determine the mRNA, protein, and activity levels of ceramide synthase 2 (CERS2) in fiibroblasts isolated from parental control subjects and from a patient diagnosed...... with progressive myoclonic epilepsy (PME). Mass spectrometry and fluorescence microscopy were used to examine the effects of reduced CERS2 activity on cellular lipid composition and plasma membrane functions. RESULTS: We identify a novel 27 kb heterozygous deletion including the CERS2 gene in a proband diagnosed...... with PME. Compared to parental controls, levels of CERS2 mRNA, protein, and activity were reduced by ˜50% in fibroblasts isolated from this proband, resulting in significantly reduced levels of ceramides and sphingomyelins containing the very long-chain fatty acids C24:0 and C26:0. The change in SL...

  7. Intestinal nitric oxide synthase activity changes during experimental colon obstruction.

    Science.gov (United States)

    Palásthy, Zsolt; Kaszaki, József; Lázár, György; Nagy, Sándor; Boros, Mihály

    2006-08-01

    The experiments in this study were designed to follow the time course of nitric oxide (NO) synthesis in the large bowel during acute mechanical ileus. Occlusion of the mid-transverse colon was maintained for 420 min in anesthetized dogs. Strain-gauge transducers were used to analyze motility changes on the hepatic and lienal flexures, respectively. Constitutive NO synthase (cNOS) and inducible NOS (iNOS) activities were determined in tissue biopsies, and plasma nitrite/nitrate (NOx) level was measured in the portal blood. Following completion of the baseline studies, the animals were treated with either 7-nitroindazole (7-NI, selective neuronal NOS inhibitor), or N-nitro-L-arginine (NNA, non-selective NOS inhibitor). In the sham-operated group the cNOS activities differed significantly in the oral and aboral tissue samples (oral: 102.9; versus aboral: 62.1 fmol/mg protein/min). The obstruction elicited a significant increase in portal NOx and elevated tissue inducible NO synthase (iNOS) activity. NNA treatment decreased the motility index in both intestinal segments for 60 min, but 120 min later the motility index was significantly elevated (2.5-fold increase in the oral part, and 1.8-fold enhancement in the aboral segment, respectively). Treatment with 7-NI decreased the cNOS activity in the oral and aboral parts by approximately 40% and 70%, respectively, and suppressed the motility increase in the aboral colon segment. The motility of the colon was either significantly increased or decreased, depending on the type and selectivity of the NOS inhibitor compounds applied. NO of neuronal origin is a transmitter that stimulates peristaltic activity; but an increased iNOS/nNOS ratio significantly moderates the obstruction-induced motility increase.

  8. Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

    Science.gov (United States)

    Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

    2014-10-01

    Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

  9. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity

    DEFF Research Database (Denmark)

    Yang, Ting; Gao, Liping; Hu, Hao

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first path-way-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate...

  10. Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells

    NARCIS (Netherlands)

    Verschuur, A. C.; van Gennip, A. H.; Muller, E. J.; Voûte, P. A.; Vreken, P.; van Kuilenburg, A. B.

    1999-01-01

    Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood

  11. Glutamic acid as anticancer agent: An overview

    OpenAIRE

    Dutta, Satyajit; Ray, Supratim; Nagarajan, K.

    2013-01-01

    The objective of the article is to highlight various roles of glutamic acid like endogenic anticancer agent, conjugates to anticancer agents, and derivatives of glutamic acid as possible anticancer agents. Besides these emphases are given especially for two endogenous derivatives of glutamic acid such as glutamine and glutamate. Glutamine is a derivative of glutamic acid and is formed in the body from glutamic acid and ammonia in an energy requiring reaction catalyzed by glutamine synthase. I...

  12. Potentiation of insulin release in response to amino acid methyl esters correlates to activation of islet glutamate dehydrogenase activity

    DEFF Research Database (Denmark)

    Kofod, Hans; Lernmark, A; Hedeskov, C J

    1986-01-01

    Column perifusion of mouse pancreatic islets was used to study the ability of amino acids and their methyl esters to influence insulin release and activate islet glutamate dehydrogenase activity. In the absence of L-glutamine, L-serine and the methyl ester of L-phenylalanine, but neither L...... glutamate dehydrogenase activity showed that only the two methyl esters of L-phenylalanine and L-serine activated the enzyme. It is concluded that the mechanism by which methyl esters of amino acids potentiate insulin release is most likely to be mediated by the activation of pancreatic beta-cell glutamate...

  13. Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae.

    Science.gov (United States)

    Fam, Rachel R S; Hiong, Kum C; Choo, Celine Y L; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2018-05-20

    Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Altered medial temporal activation related to local glutamate levels in subjects with prodromal signs of psychosis.

    Science.gov (United States)

    Valli, Isabel; Stone, James; Mechelli, Andrea; Bhattacharyya, Sagnik; Raffin, Marie; Allen, Paul; Fusar-Poli, Paolo; Lythgoe, David; O'Gorman, Ruth; Seal, Marc; McGuire, Philip

    2011-01-01

    Both medial temporal cortical dysfunction and perturbed glutamatergic neurotransmission are regarded as fundamental pathophysiological features of psychosis. However, although animal models of psychosis suggest that these two abnormalities are interrelated, their relationship in humans has yet to be investigated. We used a combination of functional magnetic resonance imaging and magnetic resonance spectroscopy to investigate the relationship between medial temporal activation during an episodic memory task and local glutamate levels in 22 individuals with an at-risk mental state for psychosis and 14 healthy volunteers. We observed a significant between-group difference in the coupling of medial temporal activation with local glutamate levels. In control subjects, medial temporal activation during episodic encoding was positively associated with medial temporal glutamate. However, in the clinical population, medial temporal activation was reduced, and the relationship with glutamate was absent. In individuals at high risk of psychosis, medial temporal dysfunction seemed related to a loss of the normal relationship with local glutamate levels. This study provides the first evidence that links medial temporal dysfunction with the central glutamate system in humans and is consistent with evidence that drugs that modulate glutamatergic transmission might be useful in the treatment of psychosis. Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  15. Enantiospecific (+)- and (-)-germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif.

    Science.gov (United States)

    Prosser, Ian; Altug, Iris G; Phillips, Andy L; König, Wilfried A; Bouwmeester, Harro J; Beale, Michael H

    2004-12-15

    The naturally occurring, volatile sesquiterpene hydrocarbon germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant-insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-germacrene D synthase and Sc19 as (-)-germacrene D synthase. Thus, the enantiomers of germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D,E), Sc11 is unusual in that this motif occurs as (303)NDTYD. Mutagenesis of this motif to (303)DDTYD gave rise to an enzyme that fully retained (+)-germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.

  16. Glutamate decarboxylase activity in rat brain during experimental epileptic seizures induced by pilocarpine

    Energy Technology Data Exchange (ETDEWEB)

    Netopilova, M; Drsata, J [Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, 50005 Hradec Kralove (Czech Republic); Haugvicova, R; Kubova, H; Mares, P [Institute of Physiology, Czech Academy of Sciences, 14220 Prague (Czech Republic)

    1998-07-01

    Glutamate decarboxylase (GAD) activity was studied rat brain parts in a pilocarpine model of epileptic seizures. An increased enzyme activity was found in hippocampus a cerebellum during the acute phase of seizures, while the cortex and cerebellum showed increased GAD activity in the chronic phase of the process. Systematic administration of pilocarpine to rats induces status epilepticus. The aim of this research was to find out if seizures induced by pilocarpine are connected changes in glutamate decarboxylase activity, the enzyme that catalyzes synthesis of inhibitory neurotransmitter GABA. GAD was assayed by means of radiometric method using {sup 14}C-carboxyl-labelled glutamate and measurement of {sup 14}CO{sub 2} radioactivity. Obtained results suggest that pilocarpine seizures are connected with changes of GAD activity in individual parts of rat brain. (authors)

  17. Glutamate decarboxylase activity in rat brain during experimental epileptic seizures induced by pilocarpine

    International Nuclear Information System (INIS)

    Netopilova, M.; Drsata, J.; Haugvicova, R.; Kubova, H.; Mares, P.

    1998-01-01

    Glutamate decarboxylase (GAD) activity was studied rat brain parts in a pilocarpine model of epileptic seizures. An increased enzyme activity was found in hippocampus a cerebellum during the acute phase of seizures, while the cortex and cerebellum showed increased GAD activity in the chronic phase of the process. Systematic administration of pilocarpine to rats induces status epilepticus. The aim of this research was to find out if seizures induced by pilocarpine are connected changes in glutamate decarboxylase activity, the enzyme that catalyzes synthesis of inhibitory neurotransmitter GABA. GAD was assayed by means of radiometric method using 14 C-carboxyl-labelled glutamate and measurement of 14 CO 2 radioactivity. Obtained results suggest that pilocarpine seizures are connected with changes of GAD activity in individual parts of rat brain. (authors)

  18. Vulnerability to glutamate toxicity of dopaminergic neurons is dependent on endogenous dopamine and MAPK activation.

    Science.gov (United States)

    Izumi, Yasuhiko; Yamamoto, Noriyuki; Matsuo, Takaaki; Wakita, Seiko; Takeuchi, Hiroki; Kume, Toshiaki; Katsuki, Hiroshi; Sawada, Hideyuki; Akaike, Akinori

    2009-07-01

    Dopaminergic neurons are more vulnerable than other types of neurons in cases of Parkinson disease and ischemic brain disease. An increasing amount of evidence suggests that endogenous dopamine plays a role in the vulnerability of dopaminergic neurons. Although glutamate toxicity contributes to the pathogenesis of these disorders, the sensitivity of dopaminergic neurons to glutamate toxicity has not been clarified. In this study, we demonstrated that dopaminergic neurons were preferentially affected by glutamate toxicity in rat mesencephalic cultures. Glutamate toxicity in dopaminergic neurons was blocked by inhibiting extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase, and p38 MAPK. Furthermore, depletion of dopamine by alpha-methyl-dl-p-tyrosine methyl ester (alpha-MT), an inhibitor of tyrosine hydroxylase (TH), protected dopaminergic neurons from the neurotoxicity. Exposure to glutamate facilitated phosphoryration of TH at Ser31 by ERK, which contributes to the increased TH activity. Inhibition of ERK had no additive effect on the protection offered by alpha-MT, whereas alpha-MT and c-jun N-terminal kinase or p38 MAPK inhibitors had additive effects and yielded full protection. These data suggest that endogenous dopamine is responsible for the vulnerability to glutamate toxicity of dopaminergic neurons and one of the mechanisms may be an enhancement of dopamine synthesis mediated by ERK.

  19. Prostaglandin endoperoxide H synthases: peroxidase hydroperoxide specificity and cyclooxygenase activation.

    Science.gov (United States)

    Liu, Jiayan; Seibold, Steve A; Rieke, Caroline J; Song, Inseok; Cukier, Robert I; Smith, William L

    2007-06-22

    The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.

  20. Platelet-derived growth factor (PDGF) stimulates glycogen synthase activity in 3T3 cells

    International Nuclear Information System (INIS)

    Chan, C.P.; Bowen-Pope, D.F.; Ross, R.; Krebs, E.G.

    1986-01-01

    Hormonal regulation of glycogen synthase, an enzyme that can be phosphorylated on multiple sites, is often associated with changes in its phosphorylation state. Enzyme activation is conventionally monitored by determining the synthase activity ratio [(activity in the absence of glucose 6-P)/(activity in the presence of glucose 6-P)]. Insulin causes an activation of glycogen synthase with a concomitant decrease in its phosphate content. In a previous report, the authors showed that epidermal growth factor (EGF) increases the glycogen synthase activity ratio in Swiss 3T3 cells. The time and dose-dependency of this response was similar to that of insulin. Their recent results indicate that PDGF also stimulates glycogen synthase activity. Enzyme activation was maximal after 30 min. of incubation with PDGF; the time course observed was very similar to that with insulin and EGF. At 1 ng/ml (0.03nM), PDGF caused a maximal stimulation of 4-fold in synthase activity ratio. Half-maximal stimulation was observed at 0.2 ng/ml (6 pM). The time course of changes in enzyme activity ratio closely followed that of 125 I-PDGF binding. The authors data suggest that PDGF, as well as EFG and insulin, may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms

  1. Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats

    Science.gov (United States)

    Acosta, Gabriela Beatriz; Fernández, María Alejandra; Roselló, Diego Martín; Tomaro, María Luján; Balestrasse, Karina; Lemberg, Abraham

    2009-01-01

    AIM: To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. METHODS: Male Wistar rats were divided into sham-operated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. RESULTS: We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, significantly and persistent increase in this excitatory neurotransmitter activity. CONCLUSION: The presence of moderate ammonia blood levels may add to the toxicity of excitotoxic glutamate in the brain, which causes alterations in brain function. Portal vein stricture that causes portal hypertension modifies the normal function in some brain regions. PMID:19533812

  2. SCREENING OF 6-PYRUVOYL-TETRAHYDROPTERIN SYNTHASE ACTIVITY DEFICIENCY AMONG HYPERP HENYLALANINEMIC PATIENTS

    Directory of Open Access Journals (Sweden)

    DURDI QUJEQ

    1999-10-01

    Full Text Available A deficiency of the phenylalanine hydroxylase activity or its cofactor tetrahydrobiopterin may"nlead to hyperphenylalamnemia and as a result, loss of IQ, poor school performance, and"nbehavior problems occurs. Deficiency in 6-pyruvoyl-tetrahydropterin synthase activity is the"nmajor cause of tetrahydrobiopterin deficient phenylketonuria. In this study, blood specimens"nfrom 165 healthy volunteers and 127 children with phenylketonuria were used to determine"nthe 6-pyruvoyl-tetrahydropterin synthase activity. It was found that the activity of 6-"npyruvoyl- tetrahydropterin synthase was decreased in comparison with control [23.46 +/-"n2.94, (mean +/- SD, mmol/ ml/h, n=I27 vs. 127.63 +/- 4.52, n=165, p<0.05]. Results of"nthis study indicate that examination of 6-pyruvoyl-tetrahydropterin synthase activity is helpful"nand may lead to the diagnosis cause of hyperphenylalaninemia.

  3. Solubilization, partial purification, and reconstitution of glutamate- and N-methyl-D-aspartate-activated cation channels from brain synaptic membranes

    International Nuclear Information System (INIS)

    Ly, A.M.; Michaelis, E.K.

    1991-01-01

    L-Glutamate-activated cation channel proteins from rat brain synaptic membranes were solubilized, partially purified, and reconstituted into liposomes. Optimal conditions for solubilization and reconstitution included treatment of the membranes with nonionic detergents in the presence of neutral phospholipids plus glycerol. Quench-flow procedures were developed to characterize the rapid kinetics of ion flux induced by receptor agonists. [ 14 C]Methylamine, a cation that permeates through the open channel of both vertebrate and invertebrate glutamate receptors, was used to measure the activity of glutamate receptor-ion channel complexes in reconstituted liposomes. L-Glutamate caused an increase in the rate of [ 14 C]methylamine influx into liposomes reconstituted with either solubilized membrane proteins or partially purified glutamate-binding proteins. Of the major glutamate receptor agonists, only N-methyl-D-aspartate activated cation fluxes in liposomes reconstituted with glutamate-binding proteins. In liposomes reconstituted with glutamate-binding proteins, N-methyl-D-aspartate- or glutamate-induced influx of NA + led to a transient increase in the influx of the lipid-permeable anion probe S 14 CN - . These results indicate the functional reconstitution of N-methyl-D-aspartate-sensitive glutamate receptors and the role of the ∼69-kDa protein in the function of these ion channels

  4. Traumatic Brain Injury Increases Cortical Glutamate Network Activity by Compromising GABAergic Control.

    Science.gov (United States)

    Cantu, David; Walker, Kendall; Andresen, Lauren; Taylor-Weiner, Amaro; Hampton, David; Tesco, Giuseppina; Dulla, Chris G

    2015-08-01

    Traumatic brain injury (TBI) is a major risk factor for developing pharmaco-resistant epilepsy. Although disruptions in brain circuitry are associated with TBI, the precise mechanisms by which brain injury leads to epileptiform network activity is unknown. Using controlled cortical impact (CCI) as a model of TBI, we examined how cortical excitability and glutamatergic signaling was altered following injury. We optically mapped cortical glutamate signaling using FRET-based glutamate biosensors, while simultaneously recording cortical field potentials in acute brain slices 2-4 weeks following CCI. Cortical electrical stimulation evoked polyphasic, epileptiform field potentials and disrupted the input-output relationship in deep layers of CCI-injured cortex. High-speed glutamate biosensor imaging showed that glutamate signaling was significantly increased in the injured cortex. Elevated glutamate responses correlated with epileptiform activity, were highest directly adjacent to the injury, and spread via deep cortical layers. Immunoreactivity for markers of GABAergic interneurons were significantly decreased throughout CCI cortex. Lastly, spontaneous inhibitory postsynaptic current frequency decreased and spontaneous excitatory postsynaptic current increased after CCI injury. Our results suggest that specific cortical neuronal microcircuits may initiate and facilitate the spread of epileptiform activity following TBI. Increased glutamatergic signaling due to loss of GABAergic control may provide a mechanism by which TBI can give rise to post-traumatic epilepsy. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Bifunctional activity of deoxyhypusine synthase/hydroxylase from Trichomonas vaginalis.

    Science.gov (United States)

    Quintas-Granados, Laura Itzel; Carvajal Gamez, Bertha Isabel; Villalpando, Jose Luis; Ortega-Lopez, Jaime; Arroyo, Rossana; Azuara-Liceaga, Elisa; Álvarez-Sánchez, María Elizbeth

    2016-04-01

    The Trichomonas vaginalis genome analysis suggested the presence of a putative deoxyhypusine synthase (TvDHS) that catalyzes the posttranslational modification of eIF-5A. Herein, we expressed and purified the recombinant TvDHS (rTvDHS) protein (43 kDa) and the recombinant TveIF-5A (rTveIF-5A) precursor protein (46 kDa). A 41 kDa band of the native TvDHS was recognized by western blot analysis in T. vaginalis total protein extract by a mouse polyclonal anti-rTvDHS antibody. The enzymatic activity of rTvDHS was determined by in vitro rTveIF-5A precursor modification. The modification reaction was performed by using ((3)H)-spermidine, and the biochemical analysis showed that rTvDHS exhibited Km value of 0.6 μM. The rTvDHS activity was inhibited by the spermidine analog, N″-guanyl-1,7-diamino-heptane (GC7). Native gel electrophoresis analysis showed two bands corresponding to an rTvDHS-rTveIF-5A complex and an intermediate form of rTveIF-5A. The two forms were subsequently separated by ion exchange chromatography to identify the hypusine residue by MS/MS analysis. Moreover, mutations in TvDHS showed that the putative HE motif present in this enzyme is involved in the hydroxylation of TveIF-5A. We observed that only hypusine-containing TveIF-5A was bound to an RNA hairpin ERE structure from the cox-2 gene, which contains the AAAUGUCACAC consensus sequence. Interestingly, 2DE-WB assays, using parasites that were grown in DAB-culture conditions and transferred to exogenous putrescine, showed the new isoform of TveIF-5A. In summary, our results indicate that T. vaginalis contains an active TvDHS capable of modifying the precursor TveIF-5A protein, which subsequently exhibits RNA binding activity. Copyright © 2015. Published by Elsevier B.V.

  6. Studies on the Active Site of Deacetoxycephalosporin C Synthase

    NARCIS (Netherlands)

    Lloyd, Matthew D.; Lee, Hwei-Jen; Harlos, Karl; Zhang, Zhi-Hong; Baldwin, Jack E.; Schofield, Christopher J.; Charnock, John M.; Garner, C. David; Hara, Takane; Terwisscha van Scheltinga, Anke C.; Valegård, Karin; Viklund, Jenny A.C.; Hajdu, Janos; Andersson, Inger; Danielsson, Åke; Bhikhabhai, Rama

    1999-01-01

    The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25% of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of

  7. Α-amino-β-fluorocyclopropanecarboxylic acids as a new tool for drug development: synthesis of glutamic acid analogs and agonist activity towards metabotropic glutamate receptor 4.

    Science.gov (United States)

    Lemonnier, Gérald; Lion, Cédric; Quirion, Jean-Charles; Pin, Jean-Philippe; Goudet, Cyril; Jubault, Philippe

    2012-08-01

    Herein we describe the diastereoselective synthesis of glutamic acid analogs and the evaluation of their agonist activity towards metabotropic glutamate receptor subtype 4 (mGluR4). These analogs are based on a monofluorinated cyclopropane core substituted with an α-aminoacid function. The potential of this new building block as a tool for the development of a novel class of drugs is demonstrated with racemic analog 11a that displayed the best agonist activity with an EC50 of 340 nM. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

    Science.gov (United States)

    Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-08-01

    Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.

  9. Activation of β-adrenoceptor facilitates active avoidance learning through enhancement of glutamate levels in the hippocampal dentate gyrus.

    Science.gov (United States)

    Lv, Jing; Feng, Hao; Chen, Ling; Wang, Wei-Yao; Yue, Xue-Ling; Jin, Qing-Hua

    2017-10-18

    Long-term potentiation (LTP) is widely accepted as the best studied model for neurophysiological mechanisms that could underlie learning and memory formation. Despite a number of studies indicating that β-adrenoceptors in the hippocampal dentate gyrus (DG) is involved in the modulation of learning and memory as well as LTP, few studies have used glutamate release as a visual indicator in awake animals to explore the role of β-adrenoceptors in learning-dependent LTP. Therefore, in the present study, the effects of propranolol (an antagonist of β-adrenoceptor) and isoproterenol (an agonist of β-adrenoceptor) on extracellular concentrations of glutamate and amplitudes of field excitatory postsynaptic potential were measured in the DG region during active avoidance learning in freely moving conscious rats. In the control group, the glutamate level in the DG was significantly increased during the acquisition of active avoidance behavior and returned to basal level following extinction training. In propranolol group, antagonism of β-adrenoceptors in the DG significantly reduced the change in glutamate level, and the acquisition of the active avoidance behavior was significantly inhibited. In contrast, the change in glutamate level was significantly enhanced by isoproterenol, and the acquisition of the active avoidance behavior was significantly accelerated. Furthermore, in all groups, the changes in glutamate level were accompanied by corresponding changes in field excitatory postsynaptic potential amplitude and active avoidance behavior. Our results suggest that activation of β-adrenoceptors in the hippocampal DG facilitates active avoidance learning by modulations of glutamate level and synaptic efficiency in rats.

  10. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  11. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

    Science.gov (United States)

    Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

    2003-01-01

    Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  12. A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.

    Directory of Open Access Journals (Sweden)

    Maiko Furubayashi

    Full Text Available Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

  13. A new metabotropic glutamate receptor agonist with in vivo anti-allodynic activity

    DEFF Research Database (Denmark)

    Stanley, Nathan J; Hutchinson, Mark R; Kvist, Trine

    2010-01-01

    -substituted carboxycyclopropylglycines, utilizing novel synthetic chemistry. The reaction between substituted 1,2-dioxines and an aminophosphonate furnished the cyclopropane core in a single step with all required stereochemistry of pendant groups. In vitro binding assays at metabotropic glutamate receptors revealed selective activity...

  14. NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with μ-Opioid Agonist Activity.

    Science.gov (United States)

    Renton, Paul; Green, Brenda; Maddaford, Shawn; Rakhit, Suman; Andrews, John S

    2012-03-08

    A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the μ-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the μ-opioid GPCR was predicated on the modulatory role of nitric oxide on μ-opioid receptor function. Structure-activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 μM), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent μ-opioid binding affinity, K i = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 μM). This work represents a novel approach in the development of new analgesics for the treatment of pain.

  15. Determination of glutamate dehydrogenase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry)

    NARCIS (Netherlands)

    Botman, Dennis; Tigchelaar, Wikky; van Noorden, Cornelis J. F.

    2014-01-01

    Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)(+) to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we

  16. Quantum chemical study of agonist-receptor vibrational interactions for activation of the glutamate receptor.

    Science.gov (United States)

    Kubo, M; Odai, K; Sugimoto, T; Ito, E

    2001-06-01

    To understand the mechanism of activation of a receptor by its agonist, the excitation and relaxation processes of the vibrational states of the receptor should be examined. As a first approach to this problem, we calculated the normal vibrational modes of agonists (glutamate and kainate) and an antagonist (6-cyano-7-nitroquinoxaline-2,3-dione: CNQX) of the glutamate receptor, and then investigated the vibrational interactions between kainate and the binding site of glutamate receptor subunit GluR2 by use of a semiempirical molecular orbital method (MOPAC2000-PM3). We found that two local vibrational modes of kainate, which were also observed in glutamate but not in CNQX, interacted through hydrogen bonds with the vibrational modes of GluR2: (i) the bending vibration of the amine group of kainate, interacting with the stretching vibration of the carboxyl group of Glu705 of GluR2, and (ii) the symmetric stretching vibration of the carboxyl group of kainate, interacting with the bending vibration of the guanidinium group of Arg485. We also found collective modes with low frequency at the binding site of GluR2 in the kainate-bound state. The vibrational energy supplied by an agonist may flow from the high-frequency local modes to the low-frequency collective modes in a receptor, resulting in receptor activation.

  17. Homocysteine threshold value based on cystathionine beta synthase and paraoxonase 1 activities in mice.

    Science.gov (United States)

    Hamelet, J; Aït-Yahya-Graison, E; Matulewicz, E; Noll, C; Badel-Chagnon, A; Camproux, A-C; Demuth, K; Paul, J-L; Delabar, J M; Janel, N

    2007-12-01

    Hyperhomocysteinaemia is a metabolic disorder associated with the development of premature atherosclerosis. Among the determinants which predispose to premature thromboembolic and atherothrombotic events, serum activity of paraoxonase 1, mainly synthesized in the liver, has been shown to be a predictor of cardiovascular disease and to be negatively correlated with serum homocysteine levels in human. Even though treatments of hyperhomocysteinaemic patients ongoing cardiovascular complications are commonly used, it still remains unclear above which homocysteine level a preventive therapy should be started. In order to establish a threshold of plasma homocysteine concentration we have analyzed the hepatic cystathionine beta synthase and paraoxonase 1 activities in a moderate to intermediate murine model of hyperhomocysteinaemia. Using wild type and heterozygous cystathionine beta synthase deficient mice fed a methionine enriched diet or a control diet, we first studied the link between cystathionine beta synthase and paraoxonase 1 activities and plasma homocysteine concentration. Among the animals used in this study, we observed a negative correlation between plasma homocysteine level and cystathionine beta synthase activity (rho=-0.52, P=0.0008) or paraoxonase 1 activity (rho=-0.49, P=0.002). Starting from these results, a homocysteine cut-off value of 15 microm has been found for both cystathionine beta synthase (P=0.0003) and paraoxonase 1 (P=0.0007) activities. Our results suggest that both cystathionine beta synthase and paraoxonase 1 activities are significantly decreased in mice with a plasma homocysteine value greater than 15 microm. In an attempt to set up preventive treatment for cardiovascular disease our results indicate that treatments should be started from 15 microm of plasma homocysteine.

  18. Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine [beta]-synthase

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Kabil, Omer; Smith, Janet L.; Banerjee, Ruma (Michigan-Med)

    2011-08-17

    The catalytic potential for H{sub 2}S biogenesis and homocysteine clearance converge at the active site of cystathionine {beta}-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes {beta}-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H{sub 2}S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 {angstrom}) and an aminoacrylate intermediate (1.55 {angstrom}) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory 'energy-sensing' CBS domains, named after this protein, suggests a mechanism for allosteric activation by S-adenosylmethionine.

  19. Converting S-limonene synthase to pinene or phellandrene synthases reveals the plasticity of the active site.

    Science.gov (United States)

    Xu, Jinkun; Ai, Ying; Wang, Jianhui; Xu, Jingwei; Zhang, Yongkang; Yang, Dong

    2017-05-01

    S-limonene synthase is a model monoterpene synthase that cyclizes geranyl pyrophosphate (GPP) to form S-limonene. It is a relatively specific enzyme as the majority of its products are composed of limonene. In this study, we converted it to pinene or phellandrene synthases after introducing N345A/L423A/S454A or N345I mutations. Further studies on N345 suggest the polarity of this residue plays a critical role in limonene production by stabilizing the terpinyl cation intermediate. If it is mutated to a non-polar residue, further cyclization or hydride shifts occurs so the carbocation migrates towards the pyrophosphate, leading to the production of pinene or phellandrene. On the other hand, mutant enzymes that still possess a polar residue at this position produce limonene as the major product. N345 is not the only polar residue that may stabilize the terpinyl cation because it is not strictly conserved among limonene synthases across species and there are also several other polar residues in this area. These residues could form a "polar pocket" that may collectively play this stabilizing role. Our study provides important insights into the catalytic mechanism of limonene synthases. Furthermore, it also has wider implications on the evolution of terpene synthases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Determination of glutamate dehydrogenase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry).

    Science.gov (United States)

    Botman, Dennis; Tigchelaar, Wikky; Van Noorden, Cornelis J F

    2014-11-01

    Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)(+) to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD(+) or NADP(+) on GDH activity in mouse liver cryostat sections using metabolic mapping. NAD(+)-dependent GDH V(max) was 2.5-fold higher than NADP(+)-dependent V(max), whereas the K(m) was similar, 1.92 mM versus 1.66 mM, when NAD(+) or NADP(+) was used, respectively. With either coenzyme, V(max) was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a K(i) of 12.2 and 3.95 for NAD(+) and NADP(+) used as coenzyme, respectively. NAD(+)- and NADP(+)-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD(+) was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD(+) as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 mM. © The Author(s) 2014.

  1. Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant.

    Science.gov (United States)

    Dormeyer, Miriam; Lübke, Anastasia L; Müller, Peter; Lentes, Sabine; Reuß, Daniel R; Thürmer, Andrea; Stülke, Jörg; Daniel, Rolf; Brantl, Sabine; Commichau, Fabian M

    2017-06-01

    Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. Glutamate and GABA contributions to medial prefrontal cortical activity to emotion: implications for mood disorders.

    Science.gov (United States)

    Stan, Ana D; Schirda, Claudiu V; Bertocci, Michele A; Bebko, Genna M; Kronhaus, Dina M; Aslam, Haris A; LaBarbara, Eduard J; Tanase, Costin; Lockovich, Jeanette C; Pollock, Myrna H; Stiffler, Richelle S; Phillips, Mary L

    2014-09-30

    The dorsomedial prefrontal cortex (MdPFC) and anterior cingulate cortices (ACC) play a critical role in implicit emotion regulation; however the understanding of the specific neurotransmitters that mediate such role is lacking. In this study, we examined relationships between MdPFC concentrations of two neurotransmitters, glutamate and γ-amino butyric acid (GABA), and BOLD activity in ACC during performance of an implicit facial emotion-processing task. Twenty healthy volunteers, aged 20-35 years, were scanned while performing an implicit facial emotion-processing task, whereby presented facial expressions changed from neutral to one of the four emotions: happy, anger, fear, or sad. Glutamate concentrations were measured before and after the emotion-processing task in right MdPFC using magnetic resonance spectroscopy (MRS). GABA concentrations were measured in bilateral MdPFC after the emotion-processing task. Multiple regression models were run to determine the relative contribution of glutamate and GABA concentration, age, and gender to BOLD signal in ACC to each of the four emotions. Multiple regression analyses revealed a significant negative correlation between MdPFC GABA concentration and BOLD signal in subgenual ACC (psad versus shape contrast. For the anger versus shape contrast, there was a significant negative correlation between age and BOLD signal in pregenual ACC (p<0.05, corrected) and a positive correlation between MdPFC glutamate concentration (pre-task) and BOLD signal in pregenual ACC (p<0.05, corrected). Our findings are the first to provide insight into relationships between MdPFC neurotransmitter concentrations and ACC BOLD signal, and could further understanding of molecular mechanisms underlying emotion processing in healthy and mood-disordered individuals. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons

    DEFF Research Database (Denmark)

    Bak, Lasse Kristoffer; Johansen, Maja L.; Schousboe, Arne

    2012-01-01

    Synthesis of neuronal glutamate from a-ketoglutarate for neurotransmission necessitates an amino group nitrogen donor; however, it is not clear which amino acid(s) serves this role. Thus, the ability of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, to act as amino...... group nitrogen donors for synthesis of vesicular neurotransmitter glutamate was investigated in cultured mouse cerebellar (primarily glutamatergic) neurons. The cultures were superfused in the presence of (15) N-labeled BCAAs, and synaptic activity was induced by pulses of N-methyl-D-aspartate (300 µ......]valine was able to maintain the amount of vesicular glutamate during synaptic activity. This indicates that, among the BCAAs, only valine supports the increased need for synthesis of vesicular glutamate. © 2012 Wiley Periodicals, Inc....

  4. Structure-activity relationship of daptomycin analogues with substitution at (2S, 3R) 3-methyl glutamic acid position.

    Science.gov (United States)

    Lin, Du'an; Lam, Hiu Yung; Han, Wenbo; Cotroneo, Nicole; Pandya, Bhaumik A; Li, Xuechen

    2017-02-01

    Daptomycin is a highly effective lipopeptide antibiotic against Gram-positive pathogens. The presence of (2S, 3R) 3-methyl glutamic acid (mGlu) in daptomycin has been found to be important to the antibacterial activity. However the role of (2S, 3R) mGlu is yet to be revealed. Herein, we reported the syntheses of three daptomycin analogues with (2S, 3R) mGlu substituted by (2S, 3R) methyl glutamine (mGln), dimethyl glutamic acid and (2S, 3R) ethyl glutamic acid (eGlu), respectively, and their antibacterial activities. The detailed synthesis of dimethyl glutamic acid was also reported. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Naphthazarin protects against glutamate-induced neuronal death via activation of the Nrf2/ARE pathway.

    Science.gov (United States)

    Son, Tae Gen; Kawamoto, Elisa M; Yu, Qian-Sheng; Greig, Nigel H; Mattson, Mark P; Camandola, Simonetta

    2013-04-19

    Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal and glial cultures, and protects neurons against glutamate-induced excitotoxicity. Published by Elsevier Inc.

  6. High specific activity N-Acetyl-3H-α-Aspartyl- L-Glutamic at micro mole scale

    International Nuclear Information System (INIS)

    Suarez, C.

    1984-01-01

    High specific activity N-Acetyl-3 H - α -Aspartyl-I-Glutamic acid at micro mole scale in prepared acetylating L- α -Aspartyl-L-glutamic with 3 H -acetic anhydride in re distilled toluene. The product le purified through cationic and anionic columns. The radiochemical purity as determined by thin-layer chromatography is greater then 99% at the time preparation. (Author) 5 refs

  7. The effect of high pressure on the intracellular trehalose synthase activity of Thermus aquaticus.

    Science.gov (United States)

    Dong, Yongsheng; Ma, Lei; Duan, Yuanliang

    2016-01-01

    To understand the effect of high pressure on the intracellular trehalose synthase activity, Thermus aquaticus (T. aquaticus) in the logarithmic growth phase was treated with high-pressure air, and its intracellular trehalose synthase (TSase) activity was determined. Our results indicated that pressure is a factor strongly affecting the cell growth. High pressure significantly attenuated the growth rate of T. aquaticus and shortened the duration of stationary phase. However, after 2 h of culture under 1.0 MPa pressure, the activity of intracellular TSase in T. aquaticus reached its maximum value, indicating that pressure can significantly increase the activity of intracellular TSase in T. aquaticus. Thus the present study provides an important guide for the enzymatic production of trehalose.

  8. An active site mutant of Escherichia coli cyclopropane fatty acid synthase forms new non-natural fatty acids providing insights on the mechanism of the enzymatic reaction.

    Science.gov (United States)

    E, Guangqi; Drujon, Thierry; Correia, Isabelle; Ploux, Olivier; Guianvarc'h, Dominique

    2013-12-01

    We have produced and purified an active site mutant of the Escherichia coli cyclopropane fatty acid synthase (CFAS) by replacing the strictly conserved G236 within cyclopropane synthases, by a glutamate residue, which corresponds to E146 of the homologous mycolic acid methyltransferase, Hma, producing hydroxymethyl mycolic acids. The G236E CFAS mutant had less than 1% of the in vitro activity of the wild type enzyme. We expressed the G236E CFAS mutant in an E. coli (DE3) strain in which the chromosomal cfa gene had been deleted. After extraction of phospholipids and conversion into the corresponding fatty acid methyl esters (FAMEs), we observed the formation of cyclopropanated FAMEs suggesting that the mutant retained some of the normal activity in vivo. However, we also observed the formation of new C17 methyl-branched unsaturated FAMEs whose structures were determined using GC/MS and NMR analyses. The double bond was located at different positions 8, 9 or 10, and the methyl group at position 10 or 9. Thus, this new FAMEs are likely arising from a 16:1 acyl chain of a phospholipid that had been transformed by the G236E CFAS mutant in vivo. The reaction catalyzed by this G236E CFAS mutant thus starts by the methylation of the unsaturated acyl chain at position 10 or 9 yielding a carbocation at position 9 or 10 respectively. It follows then two competing steps, a normal cyclopropanation or hydride shift/elimination events giving different combinations of alkenes. This study not only provides further evidence that cyclopropane synthases (CSs) form a carbocationic intermediate but also opens the way to CSs engineering for the synthesis of non-natural fatty acids. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  9. Constitutive nitric oxide synthase (cNOS activity in Langerhans islets from streptozotocin diabetic rats

    Directory of Open Access Journals (Sweden)

    Fonovich de Schroeder T.M.

    1998-01-01

    Full Text Available Nitric oxide synthase activity was measured in Langerhans islets isolated from control and streptozotocin diabetic rats. The activity of the enzyme was linear up to 150 µg of protein from control rats and was optimal at 0.1 µM calcium, when it was measured after 45 min of incubation at 37oC in the presence of 200 µM arginine. Specific activity of the enzyme was 25 x 10-4 nmol [3H]citrulline 45 min-1 mg protein-1. Streptozotocin diabetic rats exhibited less enzyme activity both in total pancreas homogenate and in isolated Langerhans islets when compared to control animals. Nitric oxide synthase activity measured in control and diabetic rats 15 days after the last streptozotocin injection in the second group of animals corresponded only to a constitutive enzyme since it was not inhibited by aminoguanidine in any of the mentioned groups. Hyperglycemia in diabetic rats may be the consequence of impaired insulin release caused at least in part by reduced positive modulation mediated by constitutive nitric oxide synthase activity, which was dramatically reduced in islets severely damaged after streptozotocin treatment.

  10. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L. (Michigan)

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  11. Fatty Acid Synthase Activity as a Target for c-Met Driven Prostate Cancer

    Science.gov (United States)

    2013-07-01

    cancer potentially due to increased fecal fat excretion. In addition, several families of plant-derived flavonoid compounds including...Apoptosis by Flavonoids Is Associated with Their Ability to Inhibit Fatty Acid Synthase Activity. J. Biol. Chem., 2005. 280(7): p. 5636-5645. 156... flavonoids , represent a source of relatively nontoxic, orally available and affordable compounds that are known to affect a number of different

  12. Glial activation in nitrous oxide toxicity is related to oxidative stress and glutamate excitotoxicity

    Directory of Open Access Journals (Sweden)

    Sandeep Kumar Singh

    2017-12-01

    Full Text Available Myelin disorders can be due to diverse mechanisms such as autoimmune, parainfectious, metabolic or toxic. The prototype of immune mediated demyelination is multiple sclerosis. To understand the underlying mechanism of cell damage in vitamin b12 deficiency, a number of animal models have been used which include total gastrectomy (TGX, cobalamine deficient diet and N2O exposure (Tredici G, et al., 1998;Scalabrino G, 2001. Six adult wistar male rats were exposed to N2O oxygen mixture in 1:1 ratio at a rate of 2 L/min for 120 min for 60 days. The control rats received only oxygen and room air. At the end of exposure, spontaneous locomotor activity (total distance travelled, time resting, time moving, number of rearing, stereotypic count and grip strength. Plasma glutathione (GSH, total antioxidant capacity (TAC and serum malonodialdehyde (MDA and serum homocysteine (Hcy were measured by spectrophotometer. Glutamate in the cerebral cortex and cerebellum was measured by colorimetry. Immunohistochemistry for GFAP expression in brain and spinal cord was done and quantified using image J software. The N2O exposed rats had significant reduction in total distance travelled, time moving, number of rearing and increased time resting compared to the controls. Hcy, glutamate and MDA levels were increased, and GSH and TAC decreased in N2O exposed group compared to the controls. GFAP was more expressed in N2O exposed group, and its expression was higher in spinal cord compared to brain. The GFAP expression correlated with neurobehavioral changes, oxidative stress and glutamate level.N2O toxicity results in GFAP expression suggesting astrocytic reaction, which is mediated by oxidative stress and excitotoxicity.

  13. Naphthazarin protects against glutamate-induced neuronal death via activation of the Nrf2/ARE pathway

    Energy Technology Data Exchange (ETDEWEB)

    Son, Tae Gen; Kawamoto, Elisa M.; Yu, Qian-Sheng; Greig, Nigel H. [Laboratory of Neurosciences, National Institute on Aging, Intramural Research Program, 251 Bayview Blvd., Baltimore, MD 21224 (United States); Mattson, Mark P. [Laboratory of Neurosciences, National Institute on Aging, Intramural Research Program, 251 Bayview Blvd., Baltimore, MD 21224 (United States); Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD (United States); Camandola, Simonetta, E-mail: camandolasi@mail.nih.gov [Laboratory of Neurosciences, National Institute on Aging, Intramural Research Program, 251 Bayview Blvd., Baltimore, MD 21224 (United States)

    2013-04-19

    Highlights: •Naphthazarin activates the Nrf2/ARE pathway. •Naphthazarin induces Nrf2-driven genes in neurons and astrocytes. •Naphthazarin protects neurons against excitotoxicity. -- Abstract: Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal and glial cultures, and protects neurons against glutamate-induced excitotoxicity.

  14. Naphthazarin protects against glutamate-induced neuronal death via activation of the Nrf2/ARE pathway

    International Nuclear Information System (INIS)

    Son, Tae Gen; Kawamoto, Elisa M.; Yu, Qian-Sheng; Greig, Nigel H.; Mattson, Mark P.; Camandola, Simonetta

    2013-01-01

    Highlights: •Naphthazarin activates the Nrf2/ARE pathway. •Naphthazarin induces Nrf2-driven genes in neurons and astrocytes. •Naphthazarin protects neurons against excitotoxicity. -- Abstract: Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal and glial cultures, and protects neurons against glutamate-induced excitotoxicity

  15. Methylglyoxal Induces Changes in the Glyoxalase System and Impairs Glutamate Uptake Activity in Primary Astrocytes.

    Science.gov (United States)

    Hansen, Fernanda; Galland, Fabiana; Lirio, Franciane; de Souza, Daniela Fraga; Da Ré, Carollina; Pacheco, Rafaela Ferreira; Vizuete, Adriana Fernanda; Quincozes-Santos, André; Leite, Marina Concli; Gonçalves, Carlos-Alberto

    2017-01-01

    The impairment of astrocyte functions is associated with diabetes mellitus and other neurodegenerative diseases. Astrocytes have been proposed to be essential cells for neuroprotection against elevated levels of methylglyoxal (MG), a highly reactive aldehyde derived from the glycolytic pathway. MG exposure impairs primary astrocyte viability, as evaluated by different assays, and these cells respond to MG elevation by increasing glyoxalase 1 activity and glutathione levels, which improve cell viability and survival. However, C6 glioma cells have shown strong signs of resistance against MG, without significant changes in the glyoxalase system. Results for aminoguanidine coincubation support the idea that MG toxicity is mediated by glycation. We found a significant decrease in glutamate uptake by astrocytes, without changes in the expression of the major transporters. Carbenoxolone, a nonspecific inhibitor of gap junctions, prevented the cytotoxicity induced by MG in astrocyte cultures. Thus, our data reinforce the idea that astrocyte viability depends on gap junctions and that the impairment induced by MG involves glutamate excitotoxicity. The astrocyte susceptibility to MG emphasizes the importance of this compound in neurodegenerative diseases, where the neuronal damage induced by MG may be aggravated by the commitment of the cells charged with MG clearance.

  16. Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation.

    Science.gov (United States)

    Zhou, Haoming; Wang, Han; Ni, Ming; Yue, Shi; Xia, Yongxiang; Busuttil, Ronald W; Kupiec-Weglinski, Jerzy W; Lu, Ling; Wang, Xuehao; Zhai, Yuan

    2018-02-13

    Glycogen synthase kinase 3β (Gsk3β [Gsk3b]) is a ubiquitously expressed kinase with distinctive functions in different types of cells. Although its roles in regulating innate immune activation and ischaemia and reperfusion injuries (IRIs) have been well documented, the underlying mechanisms remain ambiguous, in part because of the lack of cell-specific tools in vivo. We created a myeloid-specific Gsk3b knockout (KO) strain to study the function of Gsk3β in macrophages in a murine liver partial warm ischaemia model. Compared with controls, myeloid Gsk3b KO mice were protected from IRI, with diminished proinflammatory but enhanced anti-inflammatory immune responses in livers. In bone marrow-derived macrophages, Gsk3β deficiency resulted in an early reduction of Tnf gene transcription but sustained increase of Il10 gene transcription on Toll-like receptor 4 stimulation in vitro. These effects were associated with enhanced AMP-activated protein kinase (AMPK) activation, which led to an accelerated and higher level of induction of the novel innate immune negative regulator small heterodimer partner (SHP [Nr0b2]). The regulatory function of Gsk3β on AMPK activation and SHP induction was confirmed in wild-type bone marrow-derived macrophages with a Gsk3 inhibitor. Furthermore, we found that this immune regulatory mechanism was independent of Gsk3β Ser9 phosphorylation and the phosphoinositide 3-kinase-Akt signalling pathway. In vivo, myeloid Gsk3β deficiency facilitated SHP upregulation by ischaemia-reperfusion in liver macrophages. Treatment of Gsk3b KO mice with either AMPK inhibitor or SHP small interfering RNA before the onset of liver ischaemia restored liver proinflammatory immune activation and IRI in these otherwise protected hosts. Additionally, pharmacological activation of AMPK protected wild-type mice from liver IRI, with reduced proinflammatory immune activation. Inhibition of the AMPK-SHP pathway by liver ischaemia was demonstrated in tumour resection

  17. TRPA1 activation by lidocaine in nerve terminals results in glutamate release increase

    International Nuclear Information System (INIS)

    Piao, L.-H.; Fujita, Tsugumi; Jiang, C.-Y.; Liu Tao; Yue, H.-Y.; Nakatsuka, Terumasa; Kumamoto, Eiichi

    2009-01-01

    We examined the effects of local anesthetics lidocaine and procaine on glutamatergic spontaneous excitatory transmission in substantia gelatinosa (SG) neurons in adult rat spinal cord slices with whole-cell patch-clamp techniques. Bath-applied lidocaine (1-5 mM) dose-dependently and reversibly increased the frequency but not the amplitude of spontaneous excitatory postsynaptic current (sEPSC) in SG neurons. Lidocaine activity was unaffected by the Na + -channel blocker, tetrodotoxin, and the TRPV1 antagonist, capsazepine, but was inhibited by the TRP antagonist, ruthenium red. In the same neuron, the TRPA1 agonist, allyl isothiocyanate, and lidocaine both increased sEPSC frequency. In contrast, procaine did not produce presynaptic enhancement. These results indicate that lidocaine activates TRPA1 in nerve terminals presynaptic to SG neurons to increase the spontaneous release of L-glutamate.

  18. Cyclic GMP-AMP Synthase is Activated by Double-stranded DNA-Induced Oligomerization

    OpenAIRE

    Li, Xin; Shu, Chang; Yi, Guanghui; Chaton, Catherine T.; Shelton, Catherine L.; Diao, Jiasheng; Zuo, Xiaobing; Kao, C Cheng; Herr, Andrew B.; Li, Pingwei

    2013-01-01

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide 2′,5′ cGAMP that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and ...

  19. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

    International Nuclear Information System (INIS)

    Yip, Wing-Kin; Dong, Jian-Guo; Yang, S.F.; Kenny, J.W.; Thompson, G.A.

    1990-01-01

    The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB 3 H 4 or Ado[ 14 C]Met. Peptide sequencing of both 3 H- and 14 C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the 3 H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the 14 C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado [ 14 C]Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6

  20. Induction of an Olfactory Memory by the Activation of a Metabotropic Glutamate Receptor

    Science.gov (United States)

    Kaba, Hideto; Hayashi, Yasunori; Higuchi, Takashi; Nakanishi, Shigetada

    1994-07-01

    Female mice form an olfactory memory of male pheromones at mating; exposure to the pheromones of a strange male after that mating will block pregnancy. The formation of this memory is mediated by the accessory olfactory system, in which an increase in norepinephrine after mating reduces inhibitory transmission of γ-aminobutyric acid from the granule cells to the mitral cells. This study shows that the activation of mGluR2, a metabotropic glutamate receptor that suppresses the γ-aminobutyric acid inhibition of the mitral cells, permits the formation of a specific olfactory memory without the occurrence of mating by infusion of mGluR2 agonists into the female's accessory olfactory bulb. This memory faithfully reflects the memory formed at mating.

  1. Altered medial temporal activation related to local glutamate levels in subjects with prodromal signs of psychosis.

    OpenAIRE

    Valli, I; Stone, J; Mechelli, A; Bhattacharyya, S; Raffin, M; Allen, P; Fusar-Poli, P; Lythgoe, D; O'Gorman, R; Seal, M; McGuire, P

    2011-01-01

    In individuals at high risk of psychosis, medial temporal dysfunction seemed related to a loss of the normal relationship with local glutamate levels. This study provides the first evidence that links medial temporal dysfunction with the central glutamate system in humans and is consistent with evidence that drugs that modulate glutamatergic transmission might be useful in the treatment of psychosis.

  2. Zinc affects differently growth, photosynthesis, antioxidant enzyme activities and phytochelatin synthase expression of four marine diatoms.

    Science.gov (United States)

    Nguyen-Deroche, Thi Le Nhung; Caruso, Aurore; Le, Thi Trung; Bui, Trang Viet; Schoefs, Benoît; Tremblin, Gérard; Morant-Manceau, Annick

    2012-01-01

    Zinc-supplementation (20 μM) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses.

  3. Zinc Affects Differently Growth, Photosynthesis, Antioxidant Enzyme Activities and Phytochelatin Synthase Expression of Four Marine Diatoms

    Directory of Open Access Journals (Sweden)

    Thi Le Nhung Nguyen-Deroche

    2012-01-01

    Full Text Available Zinc-supplementation (20 μM effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase, and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa. Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses.

  4. The neuroprotective action of pyrroloquinoline quinone against glutamate-induced apoptosis in hippocampal neurons is mediated through the activation of PI3K/Akt pathway

    International Nuclear Information System (INIS)

    Zhang Qi; Shen Mi; Ding Mei; Shen Dingding; Ding Fei

    2011-01-01

    Pyrroloquinoline quinone (PQQ), a cofactor in several enzyme-catalyzed redox reactions, possesses a potential capability of scavenging reactive oxygen species (ROS) and inhibiting cell apoptosis. In this study, we investigated the effects of PQQ on glutamate-induced cell death in primary cultured hippocampal neurons and the possible underlying mechanisms. We found that glutamate-induced apoptosis in cultured hippocampal neurons was significantly attenuated by the ensuing PQQ treatment, which also inhibited the glutamate-induced increase in Ca2+ influx, caspase-3 activity, and ROS production, and reversed the glutamate-induced decrease in Bcl-2/Bax ratio. The examination of signaling pathways revealed that PQQ treatment activated the phosphorylation of Akt and suppressed the glutamate-induced phosphorylation of c-Jun N-terminal protein kinase (JNK). And inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt cascade by LY294002 and wortmannin significantly blocked the protective effects of PQQ, and alleviated the increase in Bcl-2/Bax ratio. Taken together, our results indicated that PQQ could protect primary cultured hippocampal neurons against glutamate-induced cell damage by scavenging ROS, reducing Ca2+ influx, and caspase-3 activity, and suggested that PQQ-activated PI3K/Akt signaling might be responsible for its neuroprotective action through modulation of glutamate-induced imbalance between Bcl-2 and Bax. - Research Highlights: →PQQ attenuated glutamate-induced cell apoptosis of cultured hippocampal neurons. →PQQ inhibited glutamate-induced Ca 2+ influx and caspase-3 activity. →PQQ reduced glutamate-induced increase in ROS production. →PQQ affected phosphorylation of Akt and JNK signalings after glutamate injury. →PI3K/Akt was required for neuroprotection of PQQ by modulating Bcl-2/Bax ratio.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    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...

  6. Trichothecenes induce accumulation of glucosylceramide in neural cells by interfering with lactosylceramide synthase activity

    International Nuclear Information System (INIS)

    Kralj, Ana; Gurgui, Mihaela; Koenig, Gabriele M.; Echten-Deckert, Gerhild van

    2007-01-01

    Trichothecenes are sesquiterpenoid metabolites produced by several fungal strains that impair human and animal health. Since sphingolipids were connected with fungal toxicity the aim of the present study was to test the influence of fungal metabolites on sphingolipid metabolism in neural cells. The crude extract of fungal strain Spicellum roseum induced accumulation of glucosylceramide (GlcCer), and simultaneous reduction of the formation of lactosylceramide (LacCer) and complex gangliosides in primary cultured neurons. Following a bioassay-guided fractionation of the respective fungal extract we could demonstrate that the two isolated trichothecene derivatives, 8-deoxy-trichothecin (8-dT) and trichodermol (Td-ol) were responsible for this effect. Thus, incubation of primary cultured neurons as well as of neuroblastoma B104 cells for 24 h with 30 μM of either of the two fungal metabolites resulted in uncoupling of sphingolipid biosynthesis at the level of LacCer. For the observed reduction of LacCer synthase activity by about 90% cell integrity was crucial in both cell types. In neuroblastoma cells the amount of LacCer synthase mRNA was reduced in the presence of trichothecenes, whereas in primary cultured neurons this was not the case, suggesting a post-transcriptional mechanism of action in the latter cell type. The data also show that the compounds did not interfere with the translocation of GlcCer in neuroblastoma cells. Collectively, our results demonstrate that trichodermol and 8-deoxy-trichothecin inhibit LacCer synthase activity in a cell-type-specific manner

  7. Influence of glutamate-evoked pain and sustained elevated muscle activity on blood oxygenation in the human masseter muscle.

    Science.gov (United States)

    Suzuki, Shunichi; Arima, Taro; Kitagawa, Yoshimasa; Svensson, Peter; Castrillon, Eduardo

    2017-12-01

    This study aimed to investigate the effect of glutamate-evoked masseter muscle pain on intramuscular oxygenation during rest and sustained elevated muscle activity (SEMA). Seventeen healthy individuals participated in two sessions in which they were injected with glutamate and saline in random order. Each session was divided into three, 10-min periods. During the first (period 1) and the last (period 3) 10-min periods, participants performed five intercalated 1-min bouts of masseter SEMA with 1-min periods of 'rest'. At onset of the second 10-min period, glutamate (0.5 ml, 1 M; Ajinomoto, Tokyo, Japan) or isotonic saline (0.5 ml; 0.9%) was injected into the masseter muscle and the participants kept the muscle relaxed in a resting position for 10 min (period 2). The hemodynamic characteristics of the masseter muscle were recorded simultaneously during the experiment by a laser blood-oxygenation monitor. The results demonstrated that glutamate injections caused significant levels of self-reported pain in the masseter muscle; however, this nociceptive input did not have robust effects on intramuscular oxygenation during rest or SEMA tasks. Interestingly, these findings suggest an uncoupling between acute nociceptive activity and hemodynamic parameters in both resting and low-level active jaw muscles. Further studies are needed to explore the pathophysiological significance of blood-flow changes for persistent jaw-muscle pain conditions. © 2017 Eur J Oral Sci.

  8. The relationship between skeletal muscle mitochondrial citrate synthase activity and whole body oxygen uptake adaptations in response to exercise training

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Andersen, Nynne Bjerre; Dela, Flemming

    2014-01-01

    Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity and chan......Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity...

  9. Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus.

    Science.gov (United States)

    Taupin, P; Ben-Ari, Y; Roisin, M P

    1994-05-02

    Using discontinuous density gradient centrifugation in isotonic Percoll sucrose, we have characterized two subcellular fractions (PII and PIII) enriched in mossy fiber synaptosomes and two others (SII and SIII) enriched in small synaptosomes. These synaptosomal fractions were compared with those obtained from adult hippocampus irradiated at neonatal stage to destroy granule cells and their mossy fibers. Synaptosomes were viable as judged by their ability to release aspartate, glutamate and GABA upon K+ depolarization. After irradiation, compared to the control values, the release of glutamate and GABA was decreased by 57 and 74% in the PIII fraction, but not in the other fractions and the content of glutamate, aspartate and GABA was also decreased in PIII fraction by 62, 44 and 52% respectively. These results suggest that mossy fiber (MF) synaptosomes contain and release glutamate and GABA. Measurement of the GABA synthesizing enzyme, glutamate decarboxylase, exhibited no significant difference after irradiation, suggesting that GABA is not synthesized by this enzyme in mossy fibers.

  10. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  11. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

    Directory of Open Access Journals (Sweden)

    Carmen Gherasim

    Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

  12. Expression Patterns, Activities and Carbohydrate-Metabolizing Regulation of Sucrose Phosphate Synthase, Sucrose Synthase and Neutral Invertase in Pineapple Fruit during Development and Ripening

    Science.gov (United States)

    Zhang, Xiu-Mei; Wang, Wei; Du, Li-Qing; Xie, Jiang-Hui; Yao, Yan-Li; Sun, Guang-Ming

    2012-01-01

    Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion. PMID:22949808

  13. Glycogen synthase kinase 3 alpha phosphorylates and regulates the osteogenic activity of Osterix.

    Science.gov (United States)

    Li, Hongyan; Jeong, Hyung Min; Choi, You Hee; Lee, Sung Ho; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl

    2013-05-10

    Osteoblast-specific transcription factor Osterix is a zinc-finger transcription factor that required for osteoblast differentiation and new bone formation. The function of Osterix can be modulated by post-translational modification. Glycogen synthase kinase 3 alpha (GSK3α) is a multifunctional serine/threonine protein kinase that plays a role in the Wnt signaling pathways and is implicated in the control of several regulatory proteins and transcription factors. In the present study, we investigated how GSK3α regulates Osterix during osteoblast differentiation. Wide type GSK3α up-regulated the protein level, protein stability and transcriptional activity of Osterix. These results suggest that GSK3α regulates osteogenic activity of Osterix. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Dual regulation of muscle glycogen synthase during exercise by activation and compartmentalization

    DEFF Research Database (Denmark)

    Prats, Clara; Helge, Jørn W; Nordby, Pernille

    2009-01-01

    Glycogen synthase (GS) is considered the rate-limiting enzyme in glycogenesis but still today there is a lack of understanding on its regulation. We have previously shown phosphorylation-dependent GS intracellular redistribution at the start of glycogen re-synthesis in rabbit skeletal muscle (Prats......, C., Cadefau, J. A., Cussó, R., Qvortrup, K., Nielsen, J. N., Wojtaszewki, J. F., Wojtaszewki, J. F., Hardie, D. G., Stewart, G., Hansen, B. F., and Ploug, T. (2005) J. Biol. Chem. 280, 23165-23172). In the present study we investigate the regulation of human muscle GS activity by glycogen, exercise......, and insulin. Using immunocytochemistry we investigate the existence and relevance of GS intracellular compartmentalization during exercise and during glycogen re-synthesis. The results show that GS intrinsic activity is strongly dependent on glycogen levels and that such regulation involves associated...

  15. Pronounced between-subject and circadian variability in thymidylate synthase and dihydropyrimidine dehydrogenase enzyme activity in human volunteers

    NARCIS (Netherlands)

    Jacobs, Bart A W; Deenen, Maarten J; Pluim, Dick; van Hasselt, J G Coen; Krähenbühl, Martin D; van Geel, Robin M J M; de Vries, Niels; Rosing, Hilde; Meulendijks, Didier; Burylo, Artur M; Cats, Annemieke; Beijnen, Jos H; Huitema, Alwin D R; Schellens, Jan H M

    AIMS: The enzymatic activity of dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are important for the tolerability and efficacy of the fluoropyrimidine drugs. In the present study, we explored between-subject variability (BSV) and circadian rhythmicity in DPD and TS activity in

  16. Imidazopyridine-Based Fatty Acid Synthase Inhibitors That Show Anti-HCV Activity and in Vivo Target Modulation.

    Science.gov (United States)

    Oslob, Johan D; Johnson, Russell J; Cai, Haiying; Feng, Shirley Q; Hu, Lily; Kosaka, Yuko; Lai, Julie; Sivaraja, Mohanram; Tep, Samnang; Yang, Hanbiao; Zaharia, Cristiana A; Evanchik, Marc J; McDowell, Robert S

    2013-01-10

    Potent imidazopyridine-based inhibitors of fatty acid synthase (FASN) are described. The compounds are shown to have antiviral (HCV replicon) activities that track with their biochemical activities. The most potent analogue (compound 19) also inhibits rat FASN and inhibits de novo palmitate synthesis in vitro (cell-based) as well as in vivo.

  17. Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization

    NARCIS (Netherlands)

    Dong, L.; Miettinen, K.; Verstappen, F.W.A.; Voster, A.; Jongsma, M.A.; Memelink, J.; Krol, van der S.; Bouwmeester, H.J.

    2013-01-01

    Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and

  18. A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase

    DEFF Research Database (Denmark)

    Maile, C A; Hingst, Janne Rasmuss; Mahalingan, K K

    2017-01-01

    BACKGROUND: Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. METHODS: Equine muscle biochemical...... had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6...

  19. Glutamate oxidation in astrocytes: Roles of glutamate dehydrogenase and aminotransferases

    DEFF Research Database (Denmark)

    McKenna, Mary C; Stridh, Malin H; McNair, Laura Frendrup

    2016-01-01

    to the presynaptic neurons as the nonexcitatory amino acid glutamine. The cycle was initially thought to function with a 1:1 ratio between glutamate released and glutamine taken up by neurons. However, studies of glutamate metabolism in astrocytes have shown that a considerable proportion of glutamate undergoes...... the enzymes that mediate this conversion. Methods include pharmacological tools such as the transaminase inhibitor aminooxyacetic acid, studies using GDH knockout mice, and siRNA-mediated knockdown of GDH in astrocytes. Studies in brain slices incubated with [15N]glutamate demonstrated activity of GDH......The cellular distribution of transporters and enzymes related to glutamate metabolism led to the concept of the glutamate–glutamine cycle. Glutamate is released as a neurotransmitter and taken up primarily by astrocytes ensheathing the synapses. The glutamate carbon skeleton is transferred back...

  20. Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

    Directory of Open Access Journals (Sweden)

    Ines Lakhal-Naouar

    Full Text Available BACKGROUND: Gene expression analysis in Leishmania donovani (Ld identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS, that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid. However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver. CONCLUSION/SIGNIFICANCE: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a

  1. Glutamate receptor agonists

    DEFF Research Database (Denmark)

    Vogensen, Stine Byskov; Greenwood, Jeremy R; Bunch, Lennart

    2011-01-01

    The neurotransmitter (S)-glutamate [(S)-Glu] is responsible for most of the excitatory neurotransmission in the central nervous system. The effect of (S)-Glu is mediated by both ionotropic and metabotropic receptors. Glutamate receptor agonists are generally a-amino acids with one or more...... stereogenic centers due to strict requirements in the agonist binding pocket of the activated state of the receptor. By contrast, there are many examples of achiral competitive antagonists. The present review addresses how stereochemistry affects the activity of glutamate receptor ligands. The review focuses...... mainly on agonists and discusses stereochemical and conformational considerations as well as biostructural knowledge of the agonist binding pockets, which is useful in the design of glutamate receptor agonists. Examples are chosen to demonstrate how stereochemistry not only determines how the agonist...

  2. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    KAUST Repository

    Beke-Somfai, Tamás

    2010-01-26

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in βTP and, thus, promote ATP. © 2009 American Chemical Society.

  3. Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia.

    Science.gov (United States)

    Podstolski, Andrzej; Havkin-Frenkel, Daphna; Malinowski, Jacek; Blount, Jack W; Kourteva, Galina; Dixon, Richard A

    2002-11-01

    Tissue cultures of the vanilla orchid, Vanilla planifolia, produce the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillin precursors such as 4-hydroxybenzaldehyde. A constitutively expressed enzyme activity catalyzing chain shortening of a hydroxycinnamic acid, believed to be the first reaction specific for formation of vanilla flavor compounds, was identified in these cultures. The enzyme converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement. Several forms of this 4-hydroxybenzaldehyde synthase (4HBS) were resolved and partially purified by a combination of hydrophobic interaction, ion exchange and gel filtration chromatography. These forms appear to be interconvertible. The unusual properties of the 4HBS, and its appearance in different protein fractions, raise questions as to its physiological role in vanillin biosynthesis in vivo.

  4. Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia.

    Science.gov (United States)

    Motta-Mejia, Carolina; Kandzija, Neva; Zhang, Wei; Mhlomi, Vuyane; Cerdeira, Ana Sofia; Burdujan, Alexandra; Tannetta, Dionne; Dragovic, Rebecca; Sargent, Ian L; Redman, Christopher W; Kishore, Uday; Vatish, Manu

    2017-08-01

    Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N   G -nitro-l-arginine methyl ester (eNOS inhibitor; P preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P preeclampsia women had lower STBEV-eNOS expression compared with that from NP women ( P preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease. © 2017 The Authors.

  5. Platelet-activating factor and group I metabotropic glutamate receptors interact for full development and maintenance of long-term potentiation in the rat medial vestibular nuclei.

    Science.gov (United States)

    Grassi, S; Francescangeli, E; Goracci, G; Pettorossi, V E

    1999-01-01

    In rat brainstem slices, we investigated the interaction between platelet-activating factor and group I metabotropic glutamate receptors in mediating long-term potentiation within the medial vestibular nuclei. We analysed the N1 field potential wave evoked in the ventral portion of the medial vestibular nuclei by primary vestibular afferent stimulation. The group I metabotropic glutamate receptor antagonist, (R,S)-1-aminoindan-1,5-dicarboxylic acid, prevented long-term potentiation induced by a platelet-activating factor analogue [1-O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine], as well as the full development of potentiation, induced by high-frequency stimulation under the blocking agent for synaptosomal platelet-activating factor receptors (ginkolide B), at drug washout. However, potentiation directly induced by the group I glutamate metabotropic receptor agonist, (R,S)-3,5-dihydroxyphenylglycine, was reduced by ginkolide B. These findings suggest that platelet-activating factor, whether exogenous or released following potentiation induction, exerts its effect through presynaptic group I metabotropic glutamate receptors, mediating the increase of glutamate release. In addition, we found that this mechanism, which led to full potentiation through presynaptic group I metabotropic glutamate receptor activation, was inactivated soon after application of potentiation-inducing stimulus. In fact, the long-lasting block of the platelet-activating factor and metabotropic glutamate receptors prevented the full potentiation development and the induced potentiation progressively declined to null. Moreover, ginkolide B, given when high-frequency-dependent potentiation was established, only reduced it within 5 min after potentiation induction. We conclude that to fully develop vestibular long-term potentiation requires presynaptic events. Platelet-activating factor, released after the activation of postsynaptic mechanisms which induce potentiation, is necessary

  6. Inhibition of muscle glycogen synthase activity and non-oxidative glucose disposal during hypoglycaemia in normal man

    DEFF Research Database (Denmark)

    Ørskov, Lotte; Bak, Jens Friis; Abildgaard, Ulrik

    1996-01-01

    The purpose of the present study was to evaluate the role of muscle glycogen synthase activity in the reduction of glucose uptake during hypoglycaemia. Six healthy young men were examined twice; during 120 min of hyperinsulinaemic (1.5 mU.kg-1. min-1) euglycaemia followed by: 1)240 min of graded ...

  7. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hoseok Choi

    2016-04-01

    Full Text Available Assaying the glycogen synthase kinase-3 (GSK3 activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  8. A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

    Science.gov (United States)

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

    2013-11-13

    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

  9. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Science.gov (United States)

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-01-01

    Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts. PMID:27092510

  10. Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology.

    Science.gov (United States)

    Albin, Stephanie D; Davis, Graeme W

    2004-08-04

    Here, we show that postsynaptic p21-activated kinase (Pak) signaling diverges into two genetically separable pathways at the Drosophila neuromuscular junction. One pathway controls glutamate receptor abundance. Pak signaling within this pathway is specified by a required interaction with the adaptor protein Dreadlocks (Dock). We demonstrate that Dock is localized to the synapse via an Src homology 2-mediated protein interaction. Dock is not necessary for Pak localization but is necessary to restrict Pak signaling to control glutamate receptor abundance. A second genetically separable function of Pak kinase signaling controls muscle membrane specialization through the regulation of synaptic Discs-large. In this pathway, Dock is dispensable. We present a model in which divergent Pak signaling is able to coordinate two different features of postsynaptic maturation, receptor abundance, and muscle membrane specialization.

  11. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid.

    Directory of Open Access Journals (Sweden)

    Yan Huang

    Full Text Available Glutamate decarboxylase (GAD catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA. In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C. Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C, superior thermostability (2.8-fold greater than that of GAD-C, and higher kcat/Km (1.6-fold higher than that of GAD-C. Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA.

  12. The relationship between skeletal muscle mitochondrial citrate synthase activity and whole body oxygen uptake adaptations in response to exercise training

    DEFF Research Database (Denmark)

    Vigelsø, Andreas; Andersen, Nynne B; Dela, Flemming

    2014-01-01

    Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity and chan......Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity...... and changes in CS activity is often assumed. However, this relationship and absolute values of CS and maximal oxygen uptake (V.O2max) has never been assessed across different studies. A systematic PubMed search on literature published from 1983 to 2013 was performed. The search profile included: citrate...... and CS activity. 70 publications with 97 intervention groups were included. There was a positive (r = 0.45) correlation (P

  13. Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization.

    Science.gov (United States)

    Li, Xin; Shu, Chang; Yi, Guanghui; Chaton, Catherine T; Shelton, Catherine L; Diao, Jiasheng; Zuo, Xiaobing; Kao, C Cheng; Herr, Andrew B; Li, Pingwei

    2013-12-12

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide, 2',5' cGAMP, that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and IFN-β reporter assays of cGAS mutants demonstrated that interactions at both DNA binding sites are essential for cGAS activation. Mutagenesis and DNA binding studies showed that the two sites bind dsDNA cooperatively and that site B plays a critical role in DNA binding. The structure of mouse cGAS bound to dsDNA and 2',5' cGAMP provided insight into the catalytic mechanism of cGAS. These results demonstrated that cGAS is activated by dsDNA-induced oligomerization. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Design, Synthesis, and Herbicidal Activity of Pyrimidine-Biphenyl Hybrids as Novel Acetohydroxyacid Synthase Inhibitors.

    Science.gov (United States)

    Li, Ke-Jian; Qu, Ren-Yu; Liu, Yu-Chao; Yang, Jing-Fang; Devendar, Ponnam; Chen, Qiong; Niu, Cong-Wei; Xi, Zhen; Yang, Guang-Fu

    2018-04-18

    The issue of weed resistance to acetohydroxyacid synthase (EC 2.2.1.6, AHAS) inhibitors has become one of the largest obstacles for the application of this class of herbicides. In a continuing effort to discover novel AHAS inhibitors to overcome weed resistance, a series of pyrimidine-biphenyl hybrids (4aa-bb and 5aa-ah) were designed and synthesized via a scaffold hopping strategy. Among these derivatives, compounds 4aa ( K i = 0.09 μM) and 4bb ( K i = 0.02 μM) displayed higher inhibitory activities against Arabidopsis thaliana AHAS than those of the controls bispyribac ( K i = 0.54 μM) and flumetsulam ( K i = 0.38 μM). Remarkably, compounds 4aa, 4bb, 5ah, and 5ag exhibited excellent postemergence herbicidal activity and a broad spectrum of weed control at application rates of 37.5-150 g of active ingredient (ai)/ha. Furthermore, 4aa and 4bb showed higher herbicidal activity against AHAS inhibitor-resistant Descurainia sophia, Ammannia arenaria, and the corresponding sensitive weeds than that of bispyribac at 0.94-0.235 g ai/ha. Therefore, the pyrimidine-biphenyl motif and lead compounds 4aa and 4bb have great potential for the discovery of novel AHAS inhibitors to combat AHAS-inhibiting herbicide-resistant weeds.

  15. Electrochemical Studies of the Inhibition and Activation Effects of Al (III on the Activity of Bovine Liver Glutamate Dehydrogenase

    Directory of Open Access Journals (Sweden)

    Shuping Bi

    2005-04-01

    Full Text Available Since the study of Al3+ ion on the enzyme activity by using of electrochemical techniques was rarely found in available literatures, the differential-pulse polarography (DPP technique was applied to study the effects of Al3+ ion on the glutamate dehydrogenase (GDH activity in the catalytical reaction of α-KG +NADH+NH4 + ⇔ L-Glu+NAD++H2O by monitoring the DPP reduction current of NAD+. At the plant and animal physiologically relevant pH values (pH=6.5 and 7.5, the GDH enzyme activities were strongly depended on the concentrations of the metal ion in the assay mixture solutions. In the lower Al (III concentration solutions (80μM, the inhibition effects of Al (III were shown again. The cyclic voltammetry of NAD+ and NAD+-GDH in the presence of Al (III can help to explain some biological phenomena. According to the differential-pulse polarography and cyclic voltammetry experiments, the present research confirmed that the electrochemical technique is a convenient and reliable sensor for accurate determination of enzyme activity in biological and environmental samples.

  16. The glutamate aspartate transporter (GLAST) mediates L-glutamate-stimulated ascorbate-release via swelling-activated anion channels in cultured neonatal rodent astrocytes.

    Science.gov (United States)

    Lane, Darius J R; Lawen, Alfons

    2013-03-01

    Vitamin C (ascorbate) plays important neuroprotective and neuromodulatory roles in the mammalian brain. Astrocytes are crucially involved in brain ascorbate homeostasis and may assist in regenerating extracellular ascorbate from its oxidised forms. Ascorbate accumulated by astrocytes can be released rapidly by a process that is stimulated by the excitatory amino acid, L-glutamate. This process is thought to be neuroprotective against excitotoxicity. Although of potential clinical interest, the mechanism of this stimulated ascorbate-release remains unknown. Here, we report that primary cultures of mouse and rat astrocytes release ascorbate following initial uptake of dehydroascorbate and accumulation of intracellular ascorbate. Ascorbate-release was not due to cellular lysis, as assessed by cellular release of the cytosolic enzyme lactate dehydrogenase, and was stimulated by L-glutamate and L-aspartate, but not the non-excitatory amino acid L-glutamine. This stimulation was due to glutamate-induced cellular swelling, as it was both attenuated by hypertonic and emulated by hypotonic media. Glutamate-stimulated ascorbate-release was also sensitive to inhibitors of volume-sensitive anion channels, suggesting that the latter may provide the conduit for ascorbate efflux. Glutamate-stimulated ascorbate-release was not recapitulated by selective agonists of either ionotropic or group I metabotropic glutamate receptors, but was completely blocked by either of two compounds, TFB-TBOA and UCPH-101, which non-selectively and selectively inhibit the glial Na(+)-dependent excitatory amino acid transporter, GLAST, respectively. These results suggest that an impairment of astrocytic ascorbate-release may exacerbate neuronal dysfunction in neurodegenerative disorders and acute brain injury in which excitotoxicity and/or GLAST deregulation have been implicated.

  17. New analogues of ACPD with selective activity for group II metabotropic glutamate receptors

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Madsen, U; Mikiciuk-Olasik, E

    1997-01-01

    In this study we have determined the pharmacology of a series of 1-aminocyclopentane-1,3-dicarboxylic acid (1,3-ACPD) analogues at cloned metabotropic glutamic acid (mGlu) receptors. The new analogues comprise the four possible stereoisomers of 1-amino-1-carboxycyclopentane-3-acetic acid (1,3-hom...

  18. Stereostructure-activity studies on agonists at the AMPA and kainate subtypes of ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Johansen, Tommy N; Greenwood, Jeremy R; Frydenvang, Karla Andrea

    2003-01-01

    (S)-Glutamic acid (Glu), the major excitatory neurotransmitter in the central nervous system, operates through ionotropic as well as metabotropic receptors and is considered to be involved in certain neurological disorders and degenerative brain diseases that are currently without any satisfactory...

  19. Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Masahiro Myojo

    Full Text Available Because endothelial nitric oxide synthase (eNOS has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177 in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172 and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP. Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

  20. Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity in antiviral immunity.

    Science.gov (United States)

    Xia, Pengyan; Ye, Buqing; Wang, Shuo; Zhu, Xiaoxiao; Du, Ying; Xiong, Zhen; Tian, Yong; Fan, Zusen

    2016-04-01

    Cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA during viral infection and catalyzes synthesis of the dinucleotide cGAMP, which activates the adaptor STING to initiate antiviral responses. Here we found that deficiency in the carboxypeptidase CCP5 or CCP6 led to susceptibility to DNA viruses. CCP5 and CCP6 were required for activation of the transcription factor IRF3 and interferons. Polyglutamylation of cGAS by the enzyme TTLL6 impeded its DNA-binding ability, whereas TTLL4-mediated monoglutamylation of cGAS blocked its synthase activity. Conversely, CCP6 removed the polyglutamylation of cGAS, whereas CCP5 hydrolyzed the monoglutamylation of cGAS, which together led to the activation of cGAS. Therefore, glutamylation and deglutamylation of cGAS tightly modulate immune responses to infection with DNA viruses.

  1. Glial and Neuronal Glutamate Transporters Differ in the Na+ Requirements for Activation of the Substrate-Independent Anion Conductance

    Directory of Open Access Journals (Sweden)

    Christopher B. Divito

    2017-05-01

    Full Text Available Excitatory amino acid transporters (EAATs are secondary active transporters of L-glutamate and L- or D-aspartate. These carriers also mediate a thermodynamically uncoupled anion conductance that is gated by Na+ and substrate binding. The activation of the anion channel by binding of Na+ alone, however, has only been demonstrated for mammalian EAAC1 (EAAT3 and EAAT4. To date, no difference has been observed for the substrate dependence of anion channel gating between the glial, EAAT1 and EAAT2, and the neuronal isoforms EAAT3, EAAT4 and EAAT5. Here we describe a difference in the Na+-dependence of anion channel gating between glial and neuronal isoforms. Chloride flux through transporters without glutamate binding has previously been described as substrate-independent or “leak” channel activity. Choline or N-methyl-D-glucamine replacement of external Na+ ions significantly reduced or abolished substrate-independent EAAT channel activity in EAAT3 and EAAT4 yet has no effect on EAAT1 or EAAT2. The interaction of Na+ with the neuronal carrier isoforms was concentration dependent, consistent with previous data. The presence of substrate and Na+-independent open states in the glial EAAT isoforms is a novel finding in the field of EAAT function. Our results reveal an important divergence in anion channel function between glial and neuronal glutamate transporters and highlight new potential roles for the EAAT-associated anion channel activity based on transporter expression and localization in the central nervous system.

  2. Soluble ectodomain of neuroligin 1 decreases synaptic activity by activating metabotropic glutamate receptor 2

    DEFF Research Database (Denmark)

    Gjørlund, Michelle D.; Carlsen, Eva Maria Meier; Kønig, Andreas Bay

    2017-01-01

    Synaptic cell adhesion molecules represent important targets for neuronal activity-dependent proteolysis. Postsynaptic neuroligins (NLs) form trans-synaptic complexes with presynaptic neurexins (NXs). Both NXs and NLs are cleaved from the cell surface by metalloproteases in an activity-dependent ...

  3. UDP-[14C]glucose-labelable polypeptides from pea: Possible components of glucan synthase I activity

    International Nuclear Information System (INIS)

    Ray, P.M.; Dhugga, K.S.; Gallaghar, S.R.

    1989-01-01

    A membrane-bound polypeptide doublet of about 40 kD can be rapidly labeled with UDP-[ 14 C]glucose under the assay conditions for glucan synthase I (GS-I). Label seems covalently bound, and chases when unlabeled UDPG is added; it might represent a covalent intermediate in polysaccharide synthesis. Labeling and GS-I activity show several common features: they co-sediment with Golgi membranes in sucrose gradients; they depend similarly on Mg 2+ or Mn 2+ (not Ca 2+ ); they decrease dramatically from stem apex to base, and are higher in epidermis than internal tissue; they show similar sensitivities to several inhibitors. But the doublet still labels after polysaccharide-synthesizing activity has been destroyed by Triton X-100. The doublet polypeptides might be glucosyl tranferases whose ability to transfer glucose units to a glucan chain is detergent-sensitive, but to accept glucose from UDPG is not; or they might be detergent-insensitive primary glucose acceptors, from which a distinct, detergent-sensitive transferase(s) move(s) these units to glucan chains

  4. Protective effects of agmatine on lipopolysaccharide-injured microglia and inducible nitric oxide synthase activity.

    Science.gov (United States)

    Ahn, Soo Kyung; Hong, Samin; Park, Yu Mi; Choi, Ja Yong; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

    2012-12-17

    Proinflammatory factors released from activated microglia contribute to maintaining homeostasis against various noxious stimuli in the central nervous system. If excessive, however, they may initiate a pathologic neuroinflammatory process. In this investigation, we evaluated whether agmatine, a primary polyamine known to protect neurons, reduces lipopolysaccharide (LPS)-induced damage to microglia in vitro and in vivo. For in vitro study, BV2-immortalized murine microglia were exposed to LPS with agmatine treatment. After 24hours, cell viability and the amount of nitrite generated were determined. For in vivo study, LPS was microinjected into the corpus callosum of adult male albino mice. Agmatine was intraperitoneally administered at the time of injury. Brains were evaluated 24hours after LPS microinjection to check for immunoreactivity with a microglial marker of ionized calcium binding adaptor molecule 1 (Iba1) and inducible nitric oxide synthase (iNOS). Using western blot analysis, protein expression of iNOS as well as that of the proinflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, was determined. Agmatine significantly reduced the LPS-induced BV2 microglial cytotoxicity from over 80% to less than 60% (pAgmatine also decreased the activities of microglia and iNOS induced by LPS microinjection into corpus callosum. Our findings reveal that agmatine attenuates LPS-induced microglial damage and suggest that agmatine may serve as a novel therapeutic strategy for neuroinflammatory diseases. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Nitric Oxide Synthase Type III Overexpression By Gene Therapy Exerts Antitumoral Activity In Mouse Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Raúl González

    2015-08-01

    Full Text Available Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO synthase type III (NOS-3 overexpression induces cell death in hepatoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. Hepa 1-6 cells were used for in vitro and in vivo experiments. The first generation adenovirus was designed to overexpress NOS-3 (or GFP and luciferase cDNA under the regulation of murine alpha-fetoprotein (AFP and Rous Sarcoma Virus (RSV promoters, respectively. Both adenoviruses were administered through the tail vein two weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-Luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8 activity in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by l-NAME or p53 siRNA. The tail vein infusion of AFP-NOS- 3/RSV-Luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

  6. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    KAUST Repository

    Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

    2010-01-01

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations

  7. Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases.

    Science.gov (United States)

    Gao, Daxing; Li, Tuo; Li, Xiao-Dong; Chen, Xiang; Li, Quan-Zhen; Wight-Carter, Mary; Chen, Zhijian J

    2015-10-20

    TREX1 is an exonuclease that digests DNA in the cytoplasm. Loss-of-function mutations of TREX1 are linked to Aicardi-Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE) in humans. Trex1(-/-) mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of interferon (IFN)-induced genes (ISGs). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates the IFN pathway. Upon binding to DNA, cGAS is activated to catalyze the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce IFNs and other cytokines. Here we show that genetic ablation of cGas in Trex1(-/-) mice eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the phenotypes of Trex1(-/-) mice. Similarly, deletion of cGas in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescued the lethal autoimmune phenotypes of the DNaseII(-/-) mice. Through quantitative mass spectrometry, we found that cGAMP accumulated in mouse tissues deficient in Trex1 or DNaseII and that this accumulation was dependent on cGAS. These results demonstrate that cGAS activation causes the autoimmune diseases in Trex1(-/-) and DNaseII(-/-) mice and suggest that inhibition of cGAS may lead to prevention and treatment of some human autoimmune diseases caused by self-DNA.

  8. PhaM is the physiological activator of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha.

    Science.gov (United States)

    Pfeiffer, Daniel; Jendrossek, Dieter

    2014-01-01

    Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.

  9. Modeling of glutamate-induced dynamical patterns

    DEFF Research Database (Denmark)

    Faurby-Bentzen, Christian Krefeld; Zhabotinsky, A.M.; Laugesen, Jakob Lund

    2009-01-01

    Based on established physiological mechanisms, the paper presents a detailed computer model, which supports the hypothesis that temporal lobe epilepsy may be caused by failure of glutamate reuptake from the extracellular space. The elevated glutamate concentration causes an increased activation...

  10. Sequential inter- and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1

    Czech Academy of Sciences Publication Activity Database

    Hlaváčková, Veronika; Zabel, U.; Franková, Daniela; Batz, J.; Hoffmann, C.; Prezeau, L.; Pin, J. P.; Blahoš, Jaroslav; Lohse, M. J.

    2012-01-01

    Roč. 5, č. 237 (2012), ra59 ISSN 1937-9145 R&D Projects: GA ČR GA303/08/1591; GA MŠk(CZ) LC06063; GA ČR GAP303/12/2408 Institutional research plan: CEZ:AV0Z50520514 Keywords : G-protein coupled receptor * metabotropic glutamate receptor 1 * class C GPCR Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.648, year: 2012

  11. Inhibition of nitric oxide synthase expression in activated microglia and peroxynitrite scavenging activity by Opuntia ficus indica var. saboten.

    Science.gov (United States)

    Lee, Ming Hong; Kim, Jae Yeon; Yoon, Jeong Hoon; Lim, Hyo Jin; Kim, Tae Hee; Jin, Changbae; Kwak, Wie-Jong; Han, Chang-Kyun; Ryu, Jae-Ha

    2006-09-01

    Activated microglia by neuronal injury or inflammatory stimulation overproduce nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) such as superoxide anion, resulting in neurodegenerative diseases. The toxic peroxynitrite (ONOO-), the reaction product of NO and superoxide anion further contributes to oxidative neurotoxicity. A butanol fraction obtained from 50% ethanol extracts of Opuntia ficus indica var. saboten (Cactaceae) stem (SK OFB901) and its hydrolysis product (SK OFB901H) inhibited the production of NO in LPS-activated microglia in a dose dependent manner (IC50 15.9, 4.2 microg/mL, respectively). They also suppressed the expression of protein and mRNA of iNOS in LPS-activated microglial cells at higher than 30 microg/mL as observed by western blot analysis and RT-PCR experiment. They also inhibited the degradation of I-kappaB-alpha in activated microglia. Moreover, they showed strong activity of peroxynitrite scavenging in a cell free bioassay system. These results imply that Opuntia ficus indica may have neuroprotective activity through the inhibition of NO production by activated microglial cells and peroxynitrite scavenging activity. Copyright (c) 2006 John Wiley & Sons, Ltd.

  12. Lysine and arginine reduce the effects of cerebral ischemic insults and inhibit glutamate-induced neuronal activity in rats

    Directory of Open Access Journals (Sweden)

    Takashi Kondoh

    2010-06-01

    Full Text Available Intravenous administration of arginine was shown to be protective against cerebral ischemic insults via nitric oxide production and possibly via additional mechanisms. The present study aimed at evaluating the neuroprotective effects of oral administration of lysine (a basic amino acid, arginine, and their combination on ischemic insults (cerebral edema and infarction and hemispheric brain swelling induced by transient middle cerebral artery occlusion/reperfusion in rats. Magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining were performed two days after ischemia induction. In control animals, the major edematous areas were observed in the cerebral cortex and striatum. The volumes associated with cortical edema were significantly reduced by lysine (2.0 g/kg, arginine (0.6 g/kg, or their combined administration (0.6 g/kg each. Protective effects of these amino acids on infarction were comparable to the inhibitory effects on edema formation. Interestingly, these amino acids, even at low dose (0.6 g/kg, were effective to reduce hemispheric brain swelling. Additionally, the effects of in vivo microiontophoretic (juxtaneuronal applications of these amino acids on glutamate-evoked neuronal activity in the ventromedial hypothalamus were investigated in awake rats. Glutamate-induced neuronal activity was robustly inhibited by microiontophoretic applications of lysine or arginine onto neuronal membranes. Taken together, our results demonstrate the neuroprotective effects of oral ingestion of lysine and arginine against ischemic insults (cerebral edema and infarction, especially in the cerebral cortex, and suggest that suppression of glutamate-induced neuronal activity might be the primary mechanism associated with these neuroprotective effects.

  13. Inhibition of platelet activation by lachrymatory factor synthase (LFS)-silenced (tearless) onion juice.

    Science.gov (United States)

    Thomson, Susan J; Rippon, Paula; Butts, Chrissie; Olsen, Sarah; Shaw, Martin; Joyce, Nigel I; Eady, Colin C

    2013-11-06

    Onion and garlic are renowned for their roles as functional foods. The health benefits of garlic are attributed to di-2-propenyl thiosulfinate (allicin), a sulfur compound found in disrupted garlic but not found in disrupted onion. Recently, onions have been grown with repressed lachrymatory factor synthase (LFS) activity, which causes these onions to produce increased amounts of di-1-propenyl thiosulfinate, an isomer of allicin. This investigation into the key health attributes of LFS-silenced (tearless) onions demonstrates that they have some attributes more similar to garlic and that this is likely due to the production of novel thiosulfinate or metabolites. The key finding was that collagen-induced in vitro platelet aggregation was significantly reduced by tearless onion extract over normal onion extract. Thiosulfinate or derived compounds were shown not to be responsible for the observed changes in the inflammatory response of AGS (stomach adenocarcinoma) cells to tumor necrosis factor alpha (TNFα) when pretreated with model onion juices. A preliminary rat feeding trial indicated that the tearless onions may also play a key role in reducing weight gain.

  14. It takes two to tango: defining an essential second active site in pyridoxal 5'-phosphate synthase.

    Directory of Open Access Journals (Sweden)

    Cyril Moccand

    Full Text Available The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2 that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5'-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5'-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.

  15. Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

    Directory of Open Access Journals (Sweden)

    Lu Frances Fangjia

    2012-11-01

    Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  16. Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase.

    Directory of Open Access Journals (Sweden)

    C F Reboul

    Full Text Available Dihydrodipicolinate synthase (DHDPS is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA. These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.

  17. Activation of presynaptic oxytocin receptors enhances glutamate release in the ventral hippocampus of prenatally restraint stressed rats.

    Science.gov (United States)

    Mairesse, Jérôme; Gatta, Eleonora; Reynaert, Marie-Line; Marrocco, Jordan; Morley-Fletcher, Sara; Soichot, Marion; Deruyter, Lucie; Camp, Gilles Van; Bouwalerh, Hammou; Fagioli, Francesca; Pittaluga, Anna; Allorge, Delphine; Nicoletti, Ferdinando; Maccari, Stefania

    2015-12-01

    Oxytocin receptors are known to modulate synaptic transmission and network activity in the hippocampus, but their precise function has been only partially elucidated. Here, we have found that activation of presynaptic oxytocin receptor with the potent agonist, carbetocin, enhanced depolarization-evoked glutamate release in the ventral hippocampus with no effect on GABA release. This evidence paved the way for examining the effect of carbetocin treatment in "prenatally restraint stressed" (PRS) rats, i.e., the offspring of dams exposed to repeated episodes of restraint stress during pregnancy. Adult PRS rats exhibit an anxious/depressive-like phenotype associated with an abnormal glucocorticoid feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis, and, remarkably, with a reduced depolarization-evoked glutamate release in the ventral hippocampus. Chronic systemic treatment with carbetocin (1mg/kg, i.p., once a day for 2-3 weeks) in PRS rats corrected the defect in glutamate release, anxiety- and depressive-like behavior, and abnormalities in social behavior, in the HPA response to stress, and in the expression of stress-related genes in the hippocampus and amygdala. Of note, carbetocin treatment had no effect on these behavioral and neuroendocrine parameters in prenatally unstressed (control) rats, with the exception of a reduced expression of the oxytocin receptor gene in the amygdala. These findings disclose a novel function of oxytocin receptors in the hippocampus, and encourage the use of oxytocin receptor agonists in the treatment of stress-related psychiatric disorders in adult life. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Nicotinic receptor activation contrasts pathophysiological bursting and neurodegeneration evoked by glutamate uptake block on rat hypoglossal motoneurons.

    Science.gov (United States)

    Corsini, Silvia; Tortora, Maria; Nistri, Andrea

    2016-11-15

    Impaired uptake of glutamate builds up the extracellular level of this excitatory transmitter to trigger rhythmic neuronal bursting and delayed cell death in the brainstem motor nucleus hypoglossus. This process is the expression of the excitotoxicity that underlies motoneuron degeneration in diseases such as amyotrophic lateral sclerosis affecting bulbar motoneurons. In a model of motoneuron excitotoxicity produced by pharmacological block of glutamate uptake in vitro, rhythmic bursting is suppressed by activation of neuronal nicotinic receptors with their conventional agonist nicotine. Emergence of bursting is facilitated by nicotinic receptor antagonists. Following excitotoxicity, nicotinic receptor activity decreases mitochondrial energy dysfunction, endoplasmic reticulum stress and production of toxic radicals. Globally, these phenomena synergize to provide motoneuron protection. Nicotinic receptors may represent a novel target to contrast pathological overactivity of brainstem motoneurons and therefore to prevent their metabolic distress and death. Excitotoxicity is thought to be one of the early processes in the onset of amyotrophic lateral sclerosis (ALS) because high levels of glutamate have been detected in the cerebrospinal fluid of such patients due to dysfunctional uptake of this transmitter that gradually damages brainstem and spinal motoneurons. To explore potential mechanisms to arrest ALS onset, we used an established in vitro model of rat brainstem slice preparation in which excitotoxicity is induced by the glutamate uptake blocker dl-threo-β-benzyloxyaspartate (TBOA). Because certain brain neurons may be neuroprotected via activation of nicotinic acetylcholine receptors (nAChRs) by nicotine, we investigated if nicotine could arrest excitotoxic damage to highly ALS-vulnerable hypoglossal motoneurons (HMs). On 50% of patch-clamped HMs, TBOA induced intense network bursts that were inhibited by 1-10 μm nicotine, whereas nAChR antagonists

  19. N-Acetyl-cysteine causes analgesia by reinforcing the endogenous activation of type-2 metabotropic glutamate receptors

    Directory of Open Access Journals (Sweden)

    Bernabucci Matteo

    2012-10-01

    Full Text Available Abstract Background Pharmacological activation of type-2 metabotropic glutamate receptors (mGlu2 receptors causes analgesia in experimental models of inflammatory and neuropathic pain. Presynaptic mGlu2 receptors are activated by the glutamate released from astrocytes by means of the cystine/glutamate antiporter (System xc- or Sxc-. We examined the analgesic activity of the Sxc- activator, N-acetyl-cysteine (NAC, in mice developing inflammatory or neuropathic pain. Results A single injection of NAC (100 mg/kg, i.p. reduced nocifensive behavior in the second phase of the formalin test. NAC-induced analgesia was abrogated by the Sxc- inhibitor, sulphasalazine (8 mg/kg, i.p. or by the mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p.. NAC still caused analgesia in mGlu3−/− mice, but was inactive in mGlu2−/− mice. In wild-type mice, NAC retained the analgesic activity in the formalin test when injected daily for 7 days, indicating the lack of tolerance. Both single and repeated injections of NAC also caused analgesia in the complete Freund’s adjuvant (CFA model of chronic inflammatory pain, and, again, analgesia was abolished by LY341495. Data obtained in mice developing neuropathic pain in response to chronic constriction injury (CCI of the sciatic nerve were divergent. In this model, a single injection of NAC caused analgesia that was reversed by LY341495, whereas repeated injections of NAC were ineffective. Thus, tolerance to NAC-induced analgesia developed in the CCI model, but not in models of inflammatory pain. The CFA and CCI models differed with respect to the expression levels of xCT (the catalytic subunit of Sxc- and activator of G-protein signaling type-3 (AGS3 in the dorsal portion of the lumbar spinal cord. CFA-treated mice showed no change in either protein, whereas CCI mice showed an ipislateral reduction in xCT levels and a bilateral increase in AGS3 levels in the spinal cord. Conclusions These data demonstrate that

  20. Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare

    DEFF Research Database (Denmark)

    Zerbe, Philipp; Chiang, Angela; Dullat, Harpreet

    2014-01-01

    Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five...... different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3...

  1. Glutamine-Elicited Secretion of Glucagon-Like Peptide 1 Is Governed by an Activated Glutamate Dehydrogenase.

    Science.gov (United States)

    Andersson, Lotta E; Shcherbina, Liliya; Al-Majdoub, Mahmoud; Vishnu, Neelanjan; Arroyo, Claudia Balderas; Aste Carrara, Jonathan; Wollheim, Claes B; Fex, Malin; Mulder, Hindrik; Wierup, Nils; Spégel, Peter

    2018-03-01

    Glucagon-like peptide 1 (GLP-1), secreted from intestinal L cells, glucose dependently stimulates insulin secretion from β-cells. This glucose dependence prevents hypoglycemia, rendering GLP-1 analogs a useful and safe treatment modality in type 2 diabetes. Although the amino acid glutamine is a potent elicitor of GLP-1 secretion, the responsible mechanism remains unclear. We investigated how GLP-1 secretion is metabolically coupled in L cells (GLUTag) and in vivo in mice using the insulin-secreting cell line INS-1 832/13 as reference. A membrane-permeable glutamate analog (dimethylglutamate [DMG]), acting downstream of electrogenic transporters, elicited similar alterations in metabolism as glutamine in both cell lines. Both DMG and glutamine alone elicited GLP-1 secretion in GLUTag cells and in vivo, whereas activation of glutamate dehydrogenase (GDH) was required to stimulate insulin secretion from INS-1 832/13 cells. Pharmacological inhibition in vivo of GDH blocked secretion of GLP-1 in response to DMG. In conclusion, our results suggest that nonelectrogenic nutrient uptake and metabolism play an important role in L cell stimulus-secretion coupling. Metabolism of glutamine and related analogs by GDH in the L cell may explain why GLP-1 secretion, but not that of insulin, is activated by these secretagogues in vivo. © 2017 by the American Diabetes Association.

  2. Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A

    International Nuclear Information System (INIS)

    Miziorko, H.M.; Behnke, C.E.

    1985-01-01

    3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1- 14 C]-3-Chloropropionyl-CoA binds covalently to the enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of the enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [ 14 C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue

  3. Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

    Czech Academy of Sciences Publication Activity Database

    Benoni, Roberto; De Bei, O.; Paredi, G.; Hayes, C. S.; Franko, N.; Mozzarelli, A.; Bettati, S.; Campanini, B.

    2017-01-01

    Roč. 591, č. 9 (2017), s. 1212-1224 ISSN 0014-5793 Institutional support: RVO:61388963 Keywords : cysteine synthase * protein - protein interaction * serine acetyltransferase Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.623, year: 2016

  4. Heme A synthase in bacteria depends on one pair of cysteinyls for activity.

    Science.gov (United States)

    Lewin, Anna; Hederstedt, Lars

    2016-02-01

    Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Curcumin attenuates glutamate neurotoxicity in the hippocampus by suppression of ER stress-associated TXNIP/NLRP3 inflammasome activation in a manner dependent on AMPK

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying; Li, Jia; Li, Shanshan; Li, Yi; Wang, Xiangxiang; Liu, Baolin [Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, 639, Longmian Road, Nanjing 211198 (China); Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Research, China Pharmaceutical University, 639, Longmian Road, Nanjing 211198 (China); Fu, Qiang, E-mail: fuqiang@cpu.edu.cn [Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, 639, Longmian Road, Nanjing 211198 (China); Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Research, China Pharmaceutical University, 639, Longmian Road, Nanjing 211198 (China); Ma, Shiping, E-mail: spma@cpu.edu.cn [Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, 639, Longmian Road, Nanjing 211198 (China); Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Research, China Pharmaceutical University, 639, Longmian Road, Nanjing 211198 (China)

    2015-07-01

    Curcumin is a natural polyphenolic compound in Curcuma longa with beneficial effects on neuronal protection. This study aims to investigate the action of curcumin in the hippocampus subjected to glutamate neurotoxicity. Glutamate stimulation induced reactive oxygen species (ROS), endoplasmic reticulum stress (ER stress) and TXNIP/NLRP3 inflammasome activation, leading to damage in the hippocampus. Curcumin treatment in the hippocampus or SH-SY5Y cells inhibited IRE1α and PERK phosphorylation with suppression of intracellular ROS production. Curcumin increased AMPK activity and knockdown of AMPKα with specific siRNA abrogated its inhibitory effects on IRE1α and PERK phosphorylation, indicating that AMPK activity was essential for the suppression of ER stress. As a result, curcumin reduced TXNIP expression and inhibited NLRP3 inflammasome activation by downregulation of NLRP3 and cleaved caspase-1 induction, and thus reduced IL-1β secretion. Specific fluorescent probe and flow cytometry analysis showed that curcumin prevented mitochondrial malfunction and protected cell survival from glutamate neurotoxicity. Moreover, oral administration of curcumin reduced brain infarct volume and attenuated neuronal damage in rats subjected to middle cerebral artery occlusion. Immunohistochemistry showed that curcumin inhibited p-IRE1α, p-PERK and NLRP3 expression in hippocampus CA1 region. Together, these results showed that curcumin attenuated glutamate neurotoxicity by inhibiting ER stress-associated TXNIP/NLRP3 inflammasome activation via the regulation of AMPK, and thereby protected the hippocampus from ischemic insult. - Highlights: • Curcumin attenuates glutamate neurotoxicity in the hippocampus. • Curcumin suppresses ER stress in glutamate-induced hippocampus slices. • Curcumin inhibits TXNIP/NLRP3 inflammasome activation. • Regulation of AMPK by curcumin contributes to suppressing ER stress.

  6. Curcumin attenuates glutamate neurotoxicity in the hippocampus by suppression of ER stress-associated TXNIP/NLRP3 inflammasome activation in a manner dependent on AMPK

    International Nuclear Information System (INIS)

    Li, Ying; Li, Jia; Li, Shanshan; Li, Yi; Wang, Xiangxiang; Liu, Baolin; Fu, Qiang; Ma, Shiping

    2015-01-01

    Curcumin is a natural polyphenolic compound in Curcuma longa with beneficial effects on neuronal protection. This study aims to investigate the action of curcumin in the hippocampus subjected to glutamate neurotoxicity. Glutamate stimulation induced reactive oxygen species (ROS), endoplasmic reticulum stress (ER stress) and TXNIP/NLRP3 inflammasome activation, leading to damage in the hippocampus. Curcumin treatment in the hippocampus or SH-SY5Y cells inhibited IRE1α and PERK phosphorylation with suppression of intracellular ROS production. Curcumin increased AMPK activity and knockdown of AMPKα with specific siRNA abrogated its inhibitory effects on IRE1α and PERK phosphorylation, indicating that AMPK activity was essential for the suppression of ER stress. As a result, curcumin reduced TXNIP expression and inhibited NLRP3 inflammasome activation by downregulation of NLRP3 and cleaved caspase-1 induction, and thus reduced IL-1β secretion. Specific fluorescent probe and flow cytometry analysis showed that curcumin prevented mitochondrial malfunction and protected cell survival from glutamate neurotoxicity. Moreover, oral administration of curcumin reduced brain infarct volume and attenuated neuronal damage in rats subjected to middle cerebral artery occlusion. Immunohistochemistry showed that curcumin inhibited p-IRE1α, p-PERK and NLRP3 expression in hippocampus CA1 region. Together, these results showed that curcumin attenuated glutamate neurotoxicity by inhibiting ER stress-associated TXNIP/NLRP3 inflammasome activation via the regulation of AMPK, and thereby protected the hippocampus from ischemic insult. - Highlights: • Curcumin attenuates glutamate neurotoxicity in the hippocampus. • Curcumin suppresses ER stress in glutamate-induced hippocampus slices. • Curcumin inhibits TXNIP/NLRP3 inflammasome activation. • Regulation of AMPK by curcumin contributes to suppressing ER stress

  7. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    International Nuclear Information System (INIS)

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-01-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca 2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32 P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32 P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

  8. Crystal structure of salt-tolerant glutaminase from Micrococcus luteus K-3 in the presence and absence of its product L-glutamate and its activator Tris.

    Science.gov (United States)

    Yoshimune, Kazuaki; Shirakihara, Yasuo; Wakayama, Mamoru; Yumoto, Isao

    2010-02-01

    Glutaminase from Micrococcus luteus K-3 [Micrococcus glutaminase (Mglu); 456 amino acid residues (aa); 48 kDa] is a salt-tolerant enzyme. Our previous study determined the structure of its major 42-kDa fragment. Here, using new crystallization conditions, we determined the structures of the intact enzyme in the presence and absence of its product L-glutamate and its activator Tris, which activates the enzyme by sixfold. With the exception of a 'lid' part (26-29 aa) and a few other short stretches, the structures were all very similar over the entire polypeptide chain. However, the presence of the ligands significantly reduced the length of the disordered regions: 41 aa in the unliganded structure (N), 21 aa for L-glutamate (G), 8 aa for Tris (T) and 6 aa for both L-glutamate and Tris (TG). L-glutamate was identified in both the G and TG structures, whereas Tris was only identified in the TG structure. Comparison of the glutamate-binding site between Mglu and salt-labile glutaminase (YbgJ) from Bacillus subtilis showed significantly smaller structural changes of the protein part in Mglu. A comparison of the substrate-binding pocket of Mglu, which is highly specific for L-glutamine, with that of Erwinia carotovora asparaginase, which has substrates other than L-glutamine, shows that Mglu has a larger substrate-binding pocket that prevents the binding of L-asparagine with proper interactions.

  9. Interaction of the D-isomer of 4-methylene glutamate (4-MG) with an active site thiol group of γ-glutamylcysteine synthetase (γ-GCS)

    International Nuclear Information System (INIS)

    Simondsen, R.P.; Meister, A.

    1986-01-01

    γ-GCS has an SH-group at or close to the glutamate binding site. During efforts to find a covalently bound inhibitor, the authors examined interaction of the enzyme with 4-MG with the thought that a glutamate analog with an α,β-unsaturated moiety might bind to the glutamate site and react with the active site thiol. 4-MG is not a significant substrate, but inhibits in the usual assay. Preincubation of the enzyme with DL-4-MG inactivated markedly and to about the same extent as found after preincubation with half the concentration of D-4-MG (prepared by action of glutamate decarboxylase on DL-4-MG); L-4-MG did not inactivate. Inactivation by 4-MG was decreased in the presence of L-glutamate. Inactivation by 4-MG was prevented by prior treatment of the enzyme with cystamine, which forms a disulfide with the active site thiol. After inactivation of the enzyme with 4-[2- 14 C]MG followed by separation of the enzyme by gel filtration, 0.9 mole of label was found per mole of enzyme, amino acid analysis after acid hydrolysis of the labeled enzyme gave labeled products that include the expected adduct formed by reaction of cysteine with 4-MG

  10. Long-term activation of group I metabotropic glutamate receptors increases functional TRPV1-expressing neurons in mouse dorsal root ganglia

    Directory of Open Access Journals (Sweden)

    Takayoshi eMasuoka

    2016-03-01

    Full Text Available Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1 in dorsal root ganglion (DRG neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC, a transient receptor potential ankyrin type 1 (TRPA1 agonist. Increase in the proportion was suppressed by phospholipase C, protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia.

  11. Modulation of seizure activity in mice by metabotropic glutamate receptor ligands

    DEFF Research Database (Denmark)

    Dalby, Nils Ole; Thomsen, C

    1996-01-01

    The anticonvulsant properties of ligands at metabotropic glutamate receptors (mGluRs) were examined in different seizure models by use of intracerebroventricular infusion. The mGluR1a antagonist/mGluR2 agonist, (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] dose-dependently antagonized...... pentylenetetrazol- and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-2-carboxylate (DMCM)-induced clonic convulsions in mice with ED50 values of 400 and 180 nmol/mice, respectively. A modest increase in electrical seizure threshold was observed in mice injected with (S)-4C3HPG. No effect on seizures induced...... by systemic administration of N-methyl-D-aspartate was observed by prior intracerebroventricular infusion of (S)-4C3HPG. The more selective (but less potent) mGluR1a antagonist, (S)-4-carboxyphenylglycine, was a weak anticonvulsant in similar seizure models with the exception of convulsions induced...

  12. Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity.

    Science.gov (United States)

    Gasanov, Sardar E; Kim, Aleksandr A; Yaguzhinsky, Lev S; Dagda, Ruben K

    2018-02-01

    Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1 H NMR and 31 P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F 0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F 0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F 0 sector, and thereby increase ATP synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. HIV-1 Myristoylated Nef Treatment of Murine Microglial Cells Activates Inducible Nitric Oxide Synthase, NO2 Production and Neurotoxic Activity.

    Directory of Open Access Journals (Sweden)

    Giorgio Mangino

    Full Text Available The potential role of the human immunodeficiency virus-1 (HIV-1 accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef treatment has been shown to activate NF-κB, MAP kinases and interferon responsive factor 3 (IRF-3, thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT-1, STAT-2 and STAT-3 through the production of proinflammatory factors.We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells.These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells.

  14. Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis)

    Science.gov (United States)

    Zak, Megan A.; Regish, Amy M.; McCormick, Stephen; Manzon, Richard G.

    2017-01-01

    Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19 °C) or below (8 °C) the thermal optimum (13 °C) and exposure to exogenous thyroid hormone (60 µg T4/g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation.

  15. Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis).

    Science.gov (United States)

    Zak, Megan A; Regish, Amy M; McCormick, Stephen D; Manzon, Richard G

    2017-06-01

    Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19°C) or below (8°C) the thermal optimum (13°C) and exposure to exogenous thyroid hormone (60µg T 4 /g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Improved production of poly-γ-glutamic acid by Bacillus subtilis D7 isolated from Doenjang, a Korean traditional fermented food, and its antioxidant activity.

    Science.gov (United States)

    Lee, Na-Ri; Lee, Sang-Mee; Cho, Kwang-Sik; Jeong, Seong-Yun; Hwang, Dae-Youn; Kim, Dong-Seob; Hong, Chang-Oh; Son, Hong-Joo

    2014-06-01

    The objectives of this study was to improve poly-γ-glutamic acid (γ-PGA) production by Bacillus subtilis D7 isolated from a Korean traditional fermented food and to assess its antioxidant activity for applications in the cosmetics and pharmaceutical industries. Strain D7 produced γ-PGA in the absence of L-glutamic acid, indicating L-glutamic acid-independent production. However, the addition of L-glutamic acid increased γ-PGA production. Several tricarboxylic acid cycle intermediates and amino acids could serve as the metabolic precursors for γ-PGA production, and the addition of pyruvic acid and D-glutamic acid to culture medium improved the yield of γ-PGA markedly. The maximum yield of γ-PGA obtained was 24.93 ± 0.64 g/l in improved medium, which was about 5.4-fold higher than the yield obtained in basal medium. γ-PGA was found to have 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (46.8 ± 1.5 %), hydroxyl radical scavenging activity (52.0 ± 1.8 %), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) radical scavenging activity (42.1 ± 1.8 %), nitric oxide scavenging activity (35.1 ± 1.3 %), reducing power (0.304 ± 0.008), and metal chelating activity (91.3 ± 3.5 %). These results indicate that γ-PGA has a potential use in the food, cosmetics, and biomedical industries for the development of novel products with radical scavenging activity. As far as we are aware, this is the first report to describe the antioxidant activityof γ-PGA produced by bacteria.

  17. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    OpenAIRE

    Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

    2003-01-01

    Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and...

  18. New procedures to measure synthase and phosphatase activities of bis-phosphoglycerate mutase. Interest for development of therapeutic drugs

    International Nuclear Information System (INIS)

    Ravel, P.; Garel, M.C.; Toullec, D.

    1997-01-01

    In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentrations is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bis-phosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allows us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phospho-glycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM. (author)

  19. Role of carglumic acid in the treatment of acute hyperammonemia due to N-acetylglutamate synthase deficiency

    Directory of Open Access Journals (Sweden)

    Häberle J

    2011-08-01

    Full Text Available Johannes HäberleKinderspital Zürich, Abteilung Stoffwechsel, Zürich, SwitzerlandAbstract: N-acetylglutamate synthase (NAGS deficiency is a rare inborn error of metabolism affecting ammonia detoxification in the urea cycle. The product of NAGS is N-acetylglutamate which is the absolutely required allosteric activator of the first urea cycle enzyme carbamoylphosphate synthetase 1. In defects of NAGS, the urea cycle function can be severely affected resulting in fatal hyperammonemia in neonatal patients or at any later stage in life. NAGS deficiency can be treated with a structural analog of N-acetylglutamate, N-carbamyl-L-glutamate, which is available for enteral use as a licensed drug. Since NAGS deficiency is an extremely rare disorder, reports on the use of N-carbamyl-L-glutamate are mainly based on single patients. According to these, the drug is very effective in treating acute hyperammonemia by avoiding the need for detoxification during the acute metabolic decompensation. Also during long-term treatment, N-carbamyl-L-glutamate is effective in maintaining normal plasma ammonia levels and avoiding the need for additional drug therapy or protein-restricted diet. Open questions remain which concern the optimal dosage in acute and long-term use of N-carbamyl-L-glutamate and potential additional disorders in which the drug might also be effective in treating acute hyperammonemia. This review focuses on the role of N-carbamyl-L-glutamate for the treatment of acute hyperammonemia due to primary NAGS deficiency but will briefly discuss the current knowledge on the role of N-carbamyl-L-glutamate for treatment of secondary NAGS deficiencies.Keywords: carglumic acid, carbamylglutamate, N-carbamyl-L-glutamate, N-acetylglutamate synthase deficiency, NAGS deficiency, hyperammonemia

  20. Clinicopathological and biological significance of aberrant activation of glycogen synthase kinase-3 in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Fu Y

    2014-06-01

    Full Text Available Yunfeng Fu,1 Xinyu Wang,1 Xiaodong Cheng,1 Feng Ye,2 Xing Xie,1,2 Weiguo Lu1,2 1Department of Gynecologic Oncology, Women's Hospital, School of Medicine, Zhejiang University, 2Women's Reproduction and Health Laboratory of Zhejiang Province, Hangzhou, People's Republic of China Background: Glycogen synthase kinase-3 (GSK-3 plays an important role in human cancer. The aim of this study is to evaluate the clinicopathological significance of expression of GSK-3α/β and pGSK-3α/βTyr279/216 in patients with epithelial ovarian cancer and to investigate whether GSK-3 inhibition can influence cell viability and tumor growth of ovarian cancer. Methods: Immunohistochemistry was used to examine expression of GSK-3α/β and pGSK-3α/βTyr279/216 in 71 human epithelial ovarian cancer tissues and correlations between protein expression, and clinicopathological factors were analyzed. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay following exposure of ovarian carcinoma cells to pharmacological inhibitors of GSK-3 or GSK-3 small interfering RNA. In vivo validation of tumor growth inhibition was performed with xenograft mice. Results: The expression levels of GSK-3α/β and pGSK-3α/βTyr279/216 in ovarian cancers were significantly higher than those in benign tumors. High expression of GSK-3α/β was more likely to be found in patients with advanced International Federation of Gynecology and Obstetrics (FIGO stages and high serum cancer antigen 125. Higher expression of pGSK-3α/βTyr279/216 was associated with advanced FIGO stages, residual tumor mass, high serum cancer antigen 125, and poor chemoresponse. Worse overall survival was revealed by Kaplan–Meier survival curves in patients with high expression of GSK-3α/β or pGSK-3α/βTyr279/216. Multivariate analysis indicated that FIGO stage, GSK-3α/β expression, and pGSK-3α/βTyr279/216 expression were independent prognostic factors for overall

  1. Aspergillus fumigatus Trehalose-Regulatory Subunit Homolog Moonlights To Mediate Cell Wall Homeostasis through Modulation of Chitin Synthase Activity

    Directory of Open Access Journals (Sweden)

    Arsa Thammahong

    2017-04-01

    Full Text Available Trehalose biosynthesis is found in fungi but not humans. Proteins involved in trehalose biosynthesis are essential for fungal pathogen virulence in humans and plants through multiple mechanisms. Loss of canonical trehalose biosynthesis genes in the human pathogen Aspergillus fumigatus significantly alters cell wall structure and integrity, though the mechanistic link between these virulence-associated pathways remains enigmatic. Here we characterize genes, called tslA and tslB, which encode proteins that contain domains similar to those corresponding to trehalose-6-phosphate phosphatase but lack critical catalytic residues for phosphatase activity. Loss of tslA reduces trehalose content in both conidia and mycelia, impairs cell wall integrity, and significantly alters cell wall structure. To gain mechanistic insights into the role that TslA plays in cell wall homeostasis, immunoprecipitation assays coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS were used to reveal a direct interaction between TslA and CsmA, a type V chitin synthase enzyme. TslA regulates not only chitin synthase activity but also CsmA sub-cellular localization. Loss of TslA impacts the immunopathogenesis of murine invasive pulmonary aspergillosis through altering cytokine production and immune cell recruitment. In conclusion, our data provide a novel model whereby proteins in the trehalose pathway play a direct role in fungal cell wall homeostasis and consequently impact fungus-host interactions.

  2. Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

    Directory of Open Access Journals (Sweden)

    Helena H. Chowdhury

    2014-10-01

    Full Text Available Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

  3. Functional Comparison of the Two Bacillus anthracis Glutamate Racemases▿

    OpenAIRE

    Dodd, Dylan; Reese, Joseph G.; Louer, Craig R.; Ballard, Jimmy D.; Spies, M. Ashley; Blanke, Steven R.

    2007-01-01

    Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because l-glutamate stereoisomerization to d-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and rac...

  4. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph, E-mail: kappock@purdue.edu [Purdue University, 175 South University Street, West Lafayette, IN 47907-2063 (United States)

    2015-09-23

    Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

  5. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    International Nuclear Information System (INIS)

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

    2015-01-01

    Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS

  6. Cyclic GMP-AMP Synthase is a Cytosolic DNA Sensor that Activates the Type-I Interferon Pathway

    Science.gov (United States)

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J.

    2013-01-01

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). Cytosolic DNA induces IFN through the production of cyclic-GMP-AMP (cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP. PMID:23258413

  7. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

    Science.gov (United States)

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J

    2013-02-15

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

  8. Epinephrine-stimulated glycogen breakdown activates glycogen synthase and increases insulin-stimulated glucose uptake in epitrochlearis muscles

    DEFF Research Database (Denmark)

    Kolnes, Anders J; Birk, Jesper Bratz; Eilertsen, Einar

    2015-01-01

    Adrenaline increases glycogen synthase (GS) phosphorylation and decreases GS activity but also stimulates glycogen breakdown and low glycogen content normally activates GS. To test the hypothesis that glycogen content directly regulates GS phosphorylation, glycogen breakdown was stimulated...... in condition with decreased GS activation. Saline or adrenaline (0.02mg/100g rat) was injected subcutaneously in Wistar rats (~130 g) with low (24 h fasted), normal (normal diet) and high glycogen content (fasted-refed) and epitrochlearis muscles were removed after 3 h and incubated ex vivo eliminating...... adrenaline action. Adrenaline injection reduced glycogen content in epitrochlearis muscles with high (120.7±17.8 vs 204.6±14.5 mmol•kg(-1); pglycogen (89.5±7.6 vs 152.6±8.1 mmol•kg(-1); pglycogen (90.0±5.0 vs 102.8±7.8 mmol•kg(-1); p=0...

  9. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    International Nuclear Information System (INIS)

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-01-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3β (GSK-3β) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-α (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity

  10. Physicochemical properties of cross-linked poly-gamma-glutamic acid and its flocculating activity against kaolin suspension

    International Nuclear Information System (INIS)

    Taniguchi, M.; Kato, K.; Shimauchi, A.; Ping, X.; Fujita, K.; Tanaka, T.; Tarui, Y.; Hirasawa, E.

    2005-01-01

    Cross-linked poly-Gamma-glutamic acid (C-L Gamma-PGA) was prepared with Gamma-PGA irradiated with Gamma-PGA at various kGy values. The physicochemical properties including viscosity and water absorption capacity were compared between C-L Gamma-PGA and several typical flocculating agents. The viscosity of C-L Gamma-PGA increased with the dose of Gamma-lrradiatio, although the water absorption capacity of C-L Gamma-PGA did not, which showed a maximum of 1005.6 ml/g at 20 kGy. Flocculating activity against kaolin suspension was not observed for any of the test compounds when used singly. However, the activity of C-L Gamma-PGA markedly increased following the addition of polyaluminum chloride. The activity increased with temperature up to 80deg C and remained at 80 deg C of heat pretreatment for 1 h, but did not at more than 50 deg C of beat pretreatment for 24 h. The activity was also observed within a pH range of 4.5-10.0. Roth the water absorption capacity and flocculating activity of C-L Gamma-PGA decreased in parallel with increasing NaCl concentration, suggesting that the hocculating activity of C-L Gamma-PGA was associated with its water absorption capacity, rather than viscosity. An investigation of the effects of various cations on the flocculating activity of C-L Gamma-PGA showed that only trivalent catlons had a synergistic effect. The mechanism of C-L Gamma-PGA flocculating activity is discussed based on the results of preliminary experiments

  11. Methylglyoxal and carboxyethyllysine reduce glutamate uptake and S100B secretion in the hippocampus independently of RAGE activation.

    Science.gov (United States)

    Hansen, Fernanda; Battú, Cíntia Eickhoff; Dutra, Márcio Ferreira; Galland, Fabiana; Lirio, Franciane; Broetto, Núbia; Nardin, Patrícia; Gonçalves, Carlos-Alberto

    2016-02-01

    Diabetes is a metabolic disease characterized by high fasting-glucose levels. Diabetic complications have been associated with hyperglycemia and high levels of reactive compounds, such as methylglyoxal (MG) and advanced glycation endproducts (AGEs) formation derived from glucose. Diabetic patients have a higher risk of developing neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. Herein, we examined the effect of high glucose, MG and carboxyethyllysine (CEL), a MG-derived AGE of lysine, on oxidative, metabolic and astrocyte-specific parameters in acute hippocampal slices, and investigated some of the mechanisms that could mediate these effects. Glucose, MG and CEL did not alter reactive oxygen species (ROS) formation, glucose uptake or glutamine synthetase activity. However, glutamate uptake and S100B secretion were decreased after MG and CEL exposure. RAGE activation and glycation reactions, examined by aminoguanidine and L-lysine co-incubation, did not mediate these changes. Acute MG and CEL exposure, but not glucose, were able to induce similar effects on hippocampal slices, suggesting that conditions of high glucose concentrations are primarily toxic by elevating the rates of these glycation compounds, such as MG, and by generation of protein cross-links. Alterations in the secretion of S100B and the glutamatergic activity mediated by MG and AGEs can contribute to the brain dysfunction observed in diabetic patients.

  12. High titers of autoantibodies to glutamate decarboxylase in Type 1 Diabetes Patients: Epitope Analysis and Inhibition of Enzyme Activity

    Science.gov (United States)

    Hampe, Christiane S.; Maitland, Murray E.; Gilliam, Lisa K.; Thi Phan, Thanh-H.; Sweet, Ian R.; Radtke, Jared R.; Bota, Vasile; Ransom, Bruce R.; Hirsch, Irl B.

    2014-01-01

    Objective Autoantibodies to glutamate decarboxylase (GAD65Ab) are found in patients with autoimmune neurological disorders and patients with type 1 diabetes. The correct diagnosis of GAD65Ab-associated neurological disorders is often delayed by the variability of symptoms and a lack of diagnostic markers. We hypothesize that the frequency of neurological disorders with high GAD65Ab titers is significantly higher than currently recognized. Methods We analyzed GAD65Ab titer, inhibition of GAD65 enzyme activity, and pattern of GAD65Ab epitopes in a cohort of type 1 diabetes patients (n=100) and correlated our findings with neurological symptoms and diseases. Results Fourty-three percent (43/100) of the patients had detectable GAD65Ab titers (median=400 U/ml, range: 142–250,000U/ml). The GAD65Ab titers in 10 type 1 diabetes patients exceeded the 90th percentile of the cohort (2,000–250,000 U/ml). Sera of these 10 patients were analyzed for their GAD65Ab epitope specificity and their ability to inhibit GAD65 enzyme activity in vitro. GAD65Ab of five patients inhibited the enzyme activity significantly (by 34–55%). Three of these patients complained of muscle stiffness and pain, which was documented in two of these patients. Conclusions Based on our findings we suggest that neurological disorders with high GAD65Ab titers are more frequent in type 1 diabetes patients than currently recognized. PMID:23512385

  13. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    Energy Technology Data Exchange (ETDEWEB)

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette, E-mail: annette.rompel@univie.ac.at [Universität Wien, Althanstrasse 14, 1090 Wien (Austria)

    2015-05-22

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P2{sub 1}2{sub 1}2{sub 1} and P12{sub 1}1 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3{sub 1}21. The crystals of latent cgAUS1 belonged to space group P12{sub 1}1 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  14. Cobalt-vitamin B12 deficiency and the activity of methylmalonyl CoA mutase and methionine synthase in cattle.

    Science.gov (United States)

    Kennedy, D G; Young, P B; Kennedy, S; Scott, J M; Molloy, A M; Weir, D G; Price, J

    1995-01-01

    Cobalt deficiency was induced in cattle by feeding two groups of animals either a basal diet that was very low in Co (12.9-17.6 micrograms Co per kg), or the same diet supplemented with cobalt, for a total of 64 weeks. Vitamin B12 deficiency was induced, as judged by hepatic concentrations of vitamin B12 and plasma concentrations of MMA. However, the activity of holo-methylmalonyl CoA mutase was significantly reduced only in brain. This was reflected in very minor alterations in the tissue concentrations of branched chain- and odd numbered-fatty acids. The activity of holo-methionine synthase was significantly reduced in liver and brain, but there were no consequent alterations in the concentrations of phosphatidyl choline and phosphatidyl ethanolamine. This study confirms that cattle are less susceptible to the effects of cobalt deficiency than sheep, and concludes that prolonged cobalt deficiency had little significant effect on tissue metabolism.

  15. The alpha2-5'AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading

    DEFF Research Database (Denmark)

    Jørgensen, Sebastian B; Nielsen, Jakob N.; Birk, Jesper Bratz

    2004-01-01

    The 5'AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-beta-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). ...

  16. Poly(γ-glutamic acid)-coated lipoplexes loaded with Doxorubicin for enhancing the antitumor activity against liver tumors

    Science.gov (United States)

    Qi, Na; Tang, Bo; Liu, Guang; Liang, Xingsi

    2017-05-01

    The study was to develop poly-γ-glutamic acid (γ-PGA)-coated Doxorubicin (Dox) lipoplexes that enhance the antitumor activity against liver tumors. γ-PGA-coated lipoplexes were performed by electrostatistically attracting to the surface of cationic charge liposomes with anionic γ-PGA. With the increasing of γ-PGA concentration, the particle size of γ-PGA-coated Dox lipoplexes slightly increased, the zeta potential from positive shifted to negative, and the entrapment efficiency (EE) were no significant change. The release rate of γ-PGA-coated Dox lipoplexes slightly increased at acidic pH, the accelerated Dox release might be attributed to greater drug delivery to tumor cells, resulting in a higher antitumor activity. Especially, γ-PGA-coated Dox lipoplexes exhibited higher cellular uptake, significant in vitro cytotoxicity in HepG2 cells, and improved in vivo antitumor efficacy toward HepG2 hepatoma-xenografted nude models in comparison with Dox liposomes and free Dox solution. In addition, the analysis results via flow cytometry showed that γ-PGA-coated Dox lipoplexes induce S phase cell cycle arrest and significantly increased apoptosis rate of HepG2 cells. In conclusion, the presence of γ-PGA on the surface of Dox lipoplexes enhanced antitumor effects of liver tumors.

  17. Multitracer Stable Isotope Quantification of Arginase and Nitric Oxide Synthase Activity in a Mouse Model of Pseudomonas Lung Infection

    Directory of Open Access Journals (Sweden)

    Hartmut Grasemann

    2014-01-01

    Full Text Available Cystic fibrosis airways are deficient for L-arginine, a substrate for nitric oxide synthases (NOSs and arginases. The rationale for this study was to quantify NOS and arginase activity in the mouse lung. Anesthetized unventilated mice received a primed constant stable isotope intravenous infusion containing labeled L-arginine, ornithine, and citrulline. The isotopic enrichment of each of the infused isotopomers and its product amino acids were measured in plasma and organ homogenates using liquid chromatography-tandem mass spectrometry. The effect of infection was studied three days after direct tracheal instillation of Pseudomonas-coated agar beads. In the infusion model, lung infection resulted in a significant (28-fold increase in NOS activity in lung but not in trachea, kidney, liver, or plasma. Absolute rates of arginase activity in solid tissues could not be calculated in this model. In an isolated lung perfusion model used for comparison increased NOS activity in infected lungs was confirmed (28.5-fold and lung arginase activity was increased 9.7-fold. The activity of L-arginine metabolizing enzymes can be measured using stable isotope conversion in the mouse. Accumulation of L-ornithine in the whole mouse model hindered the exact quantification of arginase activity in the lung, a problem that was overcome utilizing an isolated lung perfusion model.

  18. Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

    Science.gov (United States)

    Nakagawa, Hidehiko; Seike, Suguru; Sugimoto, Masatoshi; Ieda, Naoya; Kawaguchi, Mitsuyasu; Suzuki, Takayoshi; Miyata, Naoki

    2015-12-01

    Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Lithium chloride ameliorates learning and memory ability and inhibits glycogen synthase kinase-3 beta activity in a mouse model of fragile X syndrome

    Institute of Scientific and Technical Information of China (English)

    Shengqiang Chen; Xuegang Luo; Quan Yang; Weiwen Sun; Kaiyi Cao; Xi Chen; Yueling Huang; Lijun Dai; Yonghong Yi

    2011-01-01

    In the present study, Fmr1 knockout mice (KO mice) were used as the model for fragile X syndrome. The results of step-through and step-down tests demonstrated that Fmr1 KO mice had shorter latencies and more error counts, indicating a learning and memory disorder. After treatment with 30, 60, 90, 120, or 200 mg/kg lithium chloride, the learning and memory abilities of the Fmr1 KO mice were significantly ameliorated, in particular, the 200 mg/kg lithium chloride treatment had the most significant effect. Western blot analysis showed that lithium chloride significantly enhanced the expression of phosphorylated glycogen synthase kinase 3 beta, an inactive form of glycogen synthase kinase 3 beta, in the cerebral cortex and hippocampus of the Fmr1 KO mice. These results indicated that lithium chloride improved learning and memory in the Fmr1 KO mice, possibly by inhibiting glycogen synthase kinase 3 beta activity.

  20. A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules

    Directory of Open Access Journals (Sweden)

    Hildgund Schrempf

    2010-09-01

    Full Text Available A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein, has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes.

  1. Chitinase and chitin synthase 1: counterbalancing activities in cell separation of Saccharomyces cerevisiae.

    Science.gov (United States)

    Cabib, E; Silverman, S J; Shaw, J A

    1992-01-01

    Previous results [E. Cabib, A. Sburlati, B. Bowers & S. J. Silverman (1989) Journal of Cell Biology 108, 1665-1672] strongly suggested that the lysis observed in daughter cells of Saccharomyces cerevisiae defective in chitin synthase 1 (Chs1) was caused by a chitinase that partially degrades the chitin septum in the process of cell separation. Consequently, it was proposed that in wild-type cells, Chs1 acts as a repair enzyme by replenishing chitin during cytokinesis. The chitinase requirement for lysis has been confirmed in two different ways: (a) demethylallosamidin, a more powerful chitinase inhibitor than the previously used allosamidin, is also a much better protector against lysis and (b) disruption of the chitinase gene in chs1 cells eliminates lysis. Reintroduction of a normal chitinase gene, by transformation of those cells with a suitable plasmid, restores lysis. The percentage of lysed cells in strains lacking Chs1 was not increased by elevating the chitinase level with high-copy-number plasmids carrying the hydrolase gene. Furthermore, the degree of lysis varied in different chs1 strains; lysis was abolished in chs1 mutants containing the scs1 suppressor. These results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting.

  2. Developing a Highly Active Blood Anticoagulant—a Heparin Complex with Glutamic Acid—by Simulating Chemical Equilibria Based on pH-Metric Data

    Science.gov (United States)

    Nikolaeva, L. S.; Semenov, A. N.

    2018-02-01

    The anticoagulant activity of high-molecular-weight heparin is increased by developing a new highly active heparin complex with glutamate using the thermodynamic model of chemical equilibria based on pH-metric data. The anticoagulant activity of the developed complexes is estimated in the pH range of blood plasma according to the drop in the calculated equilibrium Ca2+ concentration associated with the formation of mixed ligand complexes of Ca2+ ions, heparin (Na4hep), and glutamate (H2Glu). A thermodynamic model is calculated by mathematically modelling chemical equilibria in the CaCl2-Na4hep-H2Glu-H2O-NaCl system in the pH range of 2.30 ≤ pH ≤ 10.50 in diluted saline that acts as a background electrolyte (0.154 M NaCl) at 37°C and initial concentrations of the main components of ν × 10-3 M, where n ≤ 4. The thermodynamic model is used to determine the main complex of the monomeric unit of heparin with glutamate (HhepGlu5-) and the most stable mixed ligand complex of Ca2+ with heparin and glutamate (Ca2hepGlu2-) in the pH range of blood plasma (6.80 ≤ pH ≤ 7.40). It is concluded that the Ca2hepGlu2- complex reduces the Ca2+ concentration 107 times more than the Ca2+ complex with pure heparin. The anticoagulant effect of the developed HhepGlu5- complex is confirmed in vitro and in vivo via coagulation tests on the blood plasma of laboratory rats. Additional antithrombotic properties of the developed complex are identified. The new highly active anticoagulant, HhepGlu5- complex with additional antithrombotic properties, is patented.

  3. Introduction to the Glutamate-Glutamine Cycle

    DEFF Research Database (Denmark)

    Sonnewald, Ursula; Schousboe, Arne

    2016-01-01

    . This is metabolically impossible unless it is assumed that at least two distinct pools of these amino acids exist. This combined with the finding that the enzyme synthesizing glutamine from glutamate was expressed in astrocytes but not in neurons formed the basis of the notion that a cycle must exist in which glutamate......The term 'glutamate-glutamine cycle' was coined several decades ago based on the observation that using certain (14)C-labeled precursors for studies of brain metabolism the specific radioactivity of glutamine generated from glutamate was higher than that of glutamate, its immediate precursor...... released from neurons is transported into astrocytes, converted to glutamine which is subsequently returned to neurons and converted to glutamate by an enzyme the activity of which is much higher in neurons than in astrocytes. Originally this cycle was supposed to function in a stoichiometric fashion...

  4. Introduction to the Glutamate-Glutamine Cycle

    DEFF Research Database (Denmark)

    Sonnewald, Ursula; Schousboe, Arne

    2016-01-01

    released from neurons is transported into astrocytes, converted to glutamine which is subsequently returned to neurons and converted to glutamate by an enzyme the activity of which is much higher in neurons than in astrocytes. Originally this cycle was supposed to function in a stoichiometric fashion......The term 'glutamate-glutamine cycle' was coined several decades ago based on the observation that using certain (14)C-labeled precursors for studies of brain metabolism the specific radioactivity of glutamine generated from glutamate was higher than that of glutamate, its immediate precursor....... This is metabolically impossible unless it is assumed that at least two distinct pools of these amino acids exist. This combined with the finding that the enzyme synthesizing glutamine from glutamate was expressed in astrocytes but not in neurons formed the basis of the notion that a cycle must exist in which glutamate...

  5. Role of thyroid hormones in the normal and glucocorticosteroid hormone-induced evolution of carbamoyl-phosphate synthase (ammonia) activity in axolotl liver

    NARCIS (Netherlands)

    Lamers, W. H.; Vink, C.; Charles, R.

    1978-01-01

    1. In axolotl liver, the activity of carbamoyl-phosphate synthase (ammonia), expressed per mg liver protein, decreases to a minimum at 5 months of age, then increases to a maximum at 8 months of age which is followed by a decrease again. The initial decrease between 3 and 5 months of age appears to

  6. C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism.

    Science.gov (United States)

    Landree, Leslie E; Hanlon, Andrea L; Strong, David W; Rumbaugh, Gavin; Miller, Ian M; Thupari, Jagan N; Connolly, Erin C; Huganir, Richard L; Richardson, Christine; Witters, Lee A; Kuhajda, Francis P; Ronnett, Gabriele V

    2004-01-30

    C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.

  7. Low-Dose Ribavirin Treatments Attenuate Neuroinflammatory Activation of BV-2 Cells by Interfering with Inducible Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    Iva Bozic

    2015-01-01

    Full Text Available Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 μM modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 μM to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 μM ribavirin promoted LPS induced apoptosis. We determined that 1 μM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 μM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation.

  8. Low-Dose Ribavirin Treatments Attenuate Neuroinflammatory Activation of BV-2 Cells by Interfering with Inducible Nitric Oxide Synthase

    Science.gov (United States)

    Bozic, Iva; Savic, Danijela; Jovanovic, Marija; Bjelobaba, Ivana; Laketa, Danijela; Nedeljkovic, Nadezda; Stojiljkovic, Mirjana; Pekovic, Sanja; Lavrnja, Irena

    2015-01-01

    Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 μM) modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 μM) to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS) stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 μM ribavirin promoted LPS induced apoptosis. We determined that 1 μM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 μM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation. PMID:26413464

  9. Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3 Leads to Increase of the Fatty Acid Biotransformation Activity.

    Directory of Open Access Journals (Sweden)

    Ji-Min Woo

    Full Text Available The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid-induced stress. The metabolic and genomic responses of E. coli BL21(DE3 and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3. Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3 expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1 into n-heptanoic acid (5 and 11-hydroxyundec-9-enoic acid (4. This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

  10. Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity.

    Science.gov (United States)

    Woo, Ji-Min; Kim, Ji-Won; Song, Ji-Won; Blank, Lars M; Park, Jin-Byung

    The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

  11. Glufosinate aerogenic exposure induces glutamate and IL-1 receptor dependent lung inflammation.

    Science.gov (United States)

    Maillet, Isabelle; Perche, Olivier; Pâris, Arnaud; Richard, Olivier; Gombault, Aurélie; Herzine, Ameziane; Pichon, Jacques; Huaux, Francois; Mortaud, Stéphane; Ryffel, Bernhard; Quesniaux, Valérie F J; Montécot-Dubourg, Céline

    2016-11-01

    Glufosinate-ammonium (GLA), the active component of an herbicide, is known to cause neurotoxicity. GLA shares structural analogy with glutamate. It is a powerful inhibitor of glutamine synthetase (GS) and may bind to glutamate receptors. Since these potentials targets of GLA are present in lung and immune cells, we asked whether airway exposure to GLA may cause lung inflammation in mice. A single GLA exposure (1 mg/kg) induced seizures and inflammatory cell recruitment in the broncho-alveolar space, and increased myeloperoxidase (MPO), inducible NO synthase (iNOS), interstitial inflammation and disruption of alveolar septae within 6-24 h. Interleukin 1β (IL-1β) was increased and lung inflammation depended on IL-1 receptor 1 (IL-1R1). We demonstrate that glutamate receptor pathway is central, since the N-methyl-D-aspartate (NMDA) receptor inhibitor MK-801 prevented GLA-induced lung inflammation. Chronic exposure (0.2 mg/kg 3× per week for 4 weeks) caused moderate lung inflammation and enhanced airway hyperreactivity with significant increased airway resistance. In conclusion, GLA aerosol exposure causes glutamate signalling and IL-1R-dependent pulmonary inflammation with airway hyperreactivity in mice. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  12. [Effect of L-arginine and the nitric oxide synthase blocker L-NNA on calcium capacity in rat liver mitochondria with differing resistance to hypoxia].

    Science.gov (United States)

    Kurhaliuk, N M; Ikkert, O V; Vovkanych, L S; Horyn', O V; Hal'kiv, M O; Hordiĭ, S K

    2001-01-01

    The effect of L-arginine and blockator of nitric oxide synthase L-NNA on processes of calcium mitochondrial capacity in liver with different resistance to hypoxia in the experiments with Wistar rats has been studied using the followrng substrates of energy support: succinic, alpha-ketoglutaric acids, alpha-ketolutarate and inhibitor succinatedehydrogenase malonate. As well we used substrates mixtures combination providing for activation of aminotransferase mechanism: glutamate and piruvate, glutamate and malate. It has been shown that L-arginine injection increases calcium mitochondrial capacity of low resistant rats using as substrates the succinate and alpha-ketoglutarate to control meanings of high resistance rats. Effects of donors nitric oxide on this processes limit NO-synthase inhibitor L-NNA.

  13. Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Zhichao Zhang

    2018-05-01

    Full Text Available Glioblastoma multiforme (GBM is the most lethal glioma variant in the adult brain and among the deadliest of human cancers. Increasing evidence has shown that metabotropic glutamate receptor subtype 4 (mGluR4 expression may play roles in regulating the growth of neural stem cells as well as several cancer cell lines. Here, we investigated the effects of mGluR4 on the growth and apoptosis of the LN229 GBM cell line. Involvement of Gli-1, one of the key transcription factors in the sonic Hedgehog (SHH signaling pathway, was further explored. In this study, mGluR4 was activated using selective agonist VU0155041; and gene-targeted siRNAs were used to generate loss of function of mGluR4 and Gli-1 in LN229 cells. The results demonstrated that LN229 cells expressed mGluR4 and the agonist VU0155041 decreased cell viability in a dose- and time-dependent manner. Activation of mGluR4 inhibited cyclin D1 expression, activated pro-caspase-8/9/3, and disrupted the balance of Bcl-2/Bax expression, which indicated cell cycle arrest and apoptosis of LN229 cells, respectively. Furthermore, Gli-1 expression was reduced by mGluR4 activation in LN229 cells, and downregulation of Gli-1 expression by gene-targeted siRNA resulted in both inhibition of cell proliferation and promotion of apoptosis. Moreover, VU0155041 treatment substantially blocked SHH-induced cyclin D1 expression and cell proliferation, while increasing TUNEL-positive cells and the activation of apoptosis-related proteins. We concluded that activation of mGluR4 expressed in LN229 cells could inhibit GBM cell growth by decreasing cell proliferation and promoting apoptosis. Further suppression of intracellular Gli-1 expression might be involved in the action of mGluR4 on cancer cells. Our study suggested a novel role of mGluR4, which might serve as a potential drug target for control of GBM cell growth.

  14. Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

    KAUST Repository

    Beke-Somfai, T.; Lincoln, P.; Norden, B.

    2013-01-01

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

  15. Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

    International Nuclear Information System (INIS)

    Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

    1986-01-01

    Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with [ 3 H]-NaBH 4 , and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P

  16. Effects of Intracerebroventricularly (ICV) Injected Ghrelin on Cardiac Inducible Nitric Oxide Synthase Activity/Expression in Obese Rats.

    Science.gov (United States)

    Sudar Milovanovic, E; Jovanovic, A; Misirkic-Marjanovic, M; Vucicevic, Lj; Janjetovic, K; Isenovic, E R

    2015-11-01

    The aim of this study was to examine the effects of ghrelin on regulation of cardiac inducible nitric oxide synthase (iNOS) activity/expression in high fat (HF), obese rats.For this study, male Wistar rats fed with HF diet (30% fat) for 4 weeks were injected every 24 h for 5 days intracerebroventricularly (ICV) with ghrelin (0.3 nmol/5 µl) or with an equal volume of phosphate buffered saline (PBS). Control rats were ICV injected with an equal volume of PBS. Glucose, insulin and nitric oxide (NO) concentrations were measured in serum, while arginase activity and citrulline concentrations were measured in heart lysate. Protein iNOS and regulatory subunit of nuclear factor-κB (NFκB-p65), phosphorylation of enzymes protein kinase B (Akt) at Ser(473), and extracellular signal-regulated kinases 1/2 (ERK1/2) at Tyr(202)/Tyr(204) were determined in heart lysate by Western blot. For gene expression of iNOS qRT-PCR was used.Results show significantly (parginase activity (pactivity of cardiac iNOS via Akt phosphorylation followed by NFκB activation in HF rats. © Georg Thieme Verlag KG Stuttgart · New York.

  17. (−-Epigallocatechin 3-Gallate Synthetic Analogues Inhibit Fatty Acid Synthase and Show Anticancer Activity in Triple Negative Breast Cancer

    Directory of Open Access Journals (Sweden)

    Joan Crous-Masó

    2018-05-01

    Full Text Available (−-Epigallocatechin 3-gallate (EGCG is a natural polyphenol from green tea with reported anticancer activity and capacity to inhibit the lipogenic enzyme fatty acid synthase (FASN, which is overexpressed in several human carcinomas. To improve the pharmacological profile of EGCG, we previously developed a family of EGCG derivatives and the lead compounds G28, G37 and G56 were characterized in HER2-positive breast cancer cells overexpressing FASN. Here, diesters G28, G37 and G56 and two G28 derivatives, monoesters M1 and M2, were synthesized and assessed in vitro for their cytotoxic, FASN inhibition and apoptotic activities in MDA-MB-231 triple-negative breast cancer (TNBC cells. All compounds displayed moderate to high cytotoxicity and significantly blocked FASN activity, monoesters M1 and M2 being more potent inhibitors than diesters. Interestingly, G28, M1, and M2 also diminished FASN protein expression levels, but only monoesters M1 and M2 induced apoptosis. Our results indicate that FASN inhibition by such polyphenolic compounds could be a new strategy in TNBC treatment, and highlight the potential anticancer activities of monoesters. Thus, G28, G37, G56, and most importantly M1 and M2, are anticancer candidates (alone or in combination to be further characterized in vitro and in vivo.

  18. Structural and mechanistic analysis of engineered trichodiene synthase enzymes from Trichoderma harzianum: towards higher catalytic activities empowering sustainable agriculture.

    Science.gov (United States)

    Kumari, Indu; Chaudhary, Nitika; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

    2016-06-01

    Trichoderma spp. are well-known bioagents for the plant growth promotion and pathogen suppression. The beneficial activities of the fungus Trichoderma spp. are attributed to their ability to produce and secrete certain secondary metabolites such as trichodermin that belongs to trichothecene family of molecules. The initial steps of trichodermin biosynthetic pathway in Trichoderma are similar to the trichothecenes from Fusarium sporotrichioides. Trichodiene synthase (TS) encoded by tri5 gene in Trichoderma catalyses the conversion of farnesyl pyrophosphate to trichodiene as reported earlier. In this study, we have carried out a comprehensive comparative sequence and structural analysis of the TS, which revealed the conserved residues involved in catalytic activity of the protein. In silico, modelled tertiary structure of TS protein showed stable structural behaviour during simulations. Two single-substitution mutants, i.e. D109E, D248Y and one double-substitution mutant (D109E and D248Y) of TS with potentially higher activities are screened out. The mutant proteins showed more stability than the wild type, an increased number of electrostatic interactions and better binding energies with the ligand, which further elucidates the amino acid residues involved in the reaction mechanism. These results will lead to devise strategies for higher TS activity to ultimately enhance the trichodermin production by Trichoderma spp. for its better exploitation in the sustainable agricultural practices.

  19. Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

    Science.gov (United States)

    Kitta, Ryo; Kuwamoto, Marina; Yamahama, Yumi; Mase, Keisuke; Sawada, Hiroshi

    2016-12-01

    To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori. © 2016 Japanese Society of Developmental Biologists.

  20. (-)-Epigallocatechin 3-Gallate Synthetic Analogues Inhibit Fatty Acid Synthase and Show Anticancer Activity in Triple Negative Breast Cancer.

    Science.gov (United States)

    Crous-Masó, Joan; Palomeras, Sònia; Relat, Joana; Camó, Cristina; Martínez-Garza, Úrsula; Planas, Marta; Feliu, Lidia; Puig, Teresa

    2018-05-11

    (-)-Epigallocatechin 3-gallate (EGCG) is a natural polyphenol from green tea with reported anticancer activity and capacity to inhibit the lipogenic enzyme fatty acid synthase (FASN), which is overexpressed in several human carcinomas. To improve the pharmacological profile of EGCG, we previously developed a family of EGCG derivatives and the lead compounds G28, G37 and G56 were characterized in HER2-positive breast cancer cells overexpressing FASN. Here, diesters G28, G37 and G56 and two G28 derivatives, monoesters M1 and M2, were synthesized and assessed in vitro for their cytotoxic, FASN inhibition and apoptotic activities in MDA-MB-231 triple-negative breast cancer (TNBC) cells. All compounds displayed moderate to high cytotoxicity and significantly blocked FASN activity, monoesters M1 and M2 being more potent inhibitors than diesters. Interestingly, G28, M1, and M2 also diminished FASN protein expression levels, but only monoesters M1 and M2 induced apoptosis. Our results indicate that FASN inhibition by such polyphenolic compounds could be a new strategy in TNBC treatment, and highlight the potential anticancer activities of monoesters. Thus, G28, G37, G56, and most importantly M1 and M2, are anticancer candidates (alone or in combination) to be further characterized in vitro and in vivo.

  1. Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

    KAUST Repository

    Beke-Somfai, T.

    2013-01-23

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

  2. Essential histidyl residues at the active site(s) of sucrose-phosphate synthase from Prosopis juliflora.

    Science.gov (United States)

    Sinha, A K; Pathre, U V; Sane, P V

    1998-11-10

    Chemical modification of sucrose-phosphate synthase (EC 2.4.1.14) from Prosopis juliflora by diethyl pyrocarbonate (DEP) and photo-oxidation in the presence of rose bengal (RB) which modify the histidyl residues of the protein resulted in the inactivation of the enzyme activity. This inactivation was dependent on the concentration of the modifying reagent and the time of incubation and followed pseudo-first order kinetics. For both the reagents, the inactivation was maximum at pH 7.5, which is consistent with the involvement and presence of histidine residues at the active site of the enzyme. Substrates, UDPG and F6P protected the enzyme against the inactivation by the modifying reagents suggesting that the histidine residues may be involved in the binding of these substrates and are essential for the catalytic activity. Specificity of DEP was indicated by an increase in absorbance at 240 nm along with concomitant inactivation of the enzyme and reactivation of the modified enzyme by hydroxylamine. These results strongly suggest the presence of histidine residue(s) at or near the active site of the enzyme.

  3. Morphological changes of the filamentous fungus Mucor mucedo and inhibition of chitin synthase activity induced by anethole.

    Science.gov (United States)

    Yutani, Masahiro; Hashimoto, Yukie; Ogita, Akira; Kubo, Isao; Tanaka, Toshio; Fujita, Ken-ichi

    2011-11-01

    trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum with antimicrobial activity relatively weaker than those of well-known antibiotics, and significantly enhances the antifungal activity of polygodial and dodecanol against the baker's yeast Saccharomyces cerevisiae and human pathogenic yeast Candida albicans. However, the antifungal mechanism of anethole is unresolved. Anethole demonstrated antifungal activity against the filamentous fungus, Mucor mucedo IFO 7684, accompanied by hyphal morphological changes such as swollen hyphae at the tips. Its minimum growth inhibitory concentration was 0.625 mM. A hyperosmotic condition (1.2 M sorbitol) restricted the induction of morphological changes, while hypoosmotic treatment (distilled water) induced bursting of hyphal tips and leakage of cytoplasmic constituents. Furthermore, anethole dose-dependently inhibited chitin synthase (CHS) activity in permeabilized hyphae in an uncompetitive manner. These results suggest that the morphological changes of M. mucedo could be explained by the fragility of cell walls caused by CHS inhibition. Copyright © 2011 John Wiley & Sons, Ltd.

  4. Glutamate acid decarboxylase 1 promotes metastasis of human oral cancer by β-catenin translocation and MMP7 activation

    International Nuclear Information System (INIS)

    Kimura, Ryota; Tanzawa, Hideki; Uzawa, Katsuhiro; Kasamatsu, Atsushi; Koyama, Tomoyoshi; Fukumoto, Chonji; Kouzu, Yukinao; Higo, Morihiro; Endo-Sakamoto, Yosuke; Ogawara, Katsunori; Shiiba, Masashi

    2013-01-01

    Glutamate decarboxylase 1 (GAD1), a rate-limiting enzyme in the production of γ-aminobutyric acid (GABA), is found in the GABAergic neurons of the central nervous system. Little is known about the relevance of GAD1 to oral squamous cell carcinoma (OSCC). We investigated the expression status of GAD1 and its functional mechanisms in OSCCs. We evaluated GAD1 mRNA and protein expressions in OSCC-derived cells using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. To assess the critical functions of GAD1, i.e., cellular proliferation, invasiveness, and migration, OSCC-derived cells were treated with the shRNA and specific GAD1 inhibitor, 3-mercaptopropionic acid (3-MPA). GAD1 expression in 80 patients with primary OSCCs was analyzed and compared to the clinicopathological behaviors of OSCC. qRT-PCR and immunoblotting analyses detected frequent up-regulation of GAD1 in OSCC-derived cells compared to human normal oral keratinocytes. Suppression of nuclear localization of β-catenin and MMP7 secretion was observed in GAD1 knockdown and 3-MPA-treated cells. We also found low cellular invasiveness and migratory abilities in GAD1 knockdown and 3-MPA-treated cells. In the clinical samples, GAD1 expression in the primary OSCCs was significantly (P < 0.05) higher than in normal counterparts and was correlated significantly (P < 0.05) with regional lymph node metastasis. Our data showed that up-regulation of GAD1 was a characteristic event in OSCCs and that GAD1 was correlated with cellular invasiveness and migration by regulating β-catenin translocation and MMP7 activation. GAD1 might play an important role in controlling tumoral invasiveness and metastasis in oral cancer

  5. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China); Zhang, Qunye, E-mail: wz.zhangqy@sdu.edu.cn [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, Shandong (China); Li, Guorong, E-mail: grli@sdnu.edu.cn [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China)

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

  6. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    International Nuclear Information System (INIS)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi; Zhang, Qunye; Li, Guorong

    2015-01-01

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation

  7. Control activity of yeast geranylgeranyl diphosphate synthase from dimer interface through H-bonds and hydrophobic interaction.

    Science.gov (United States)

    Chang, Chih-Kang; Teng, Kuo-Hsun; Lin, Sheng-Wei; Chang, Tao-Hsin; Liang, Po-Huang

    2013-04-23

    Previously we showed that yeast geranylgeranyl diphosphate synthase (GGPPS) becomes an inactive monomer when the first N-terminal helix involved in dimerization is deleted. This raises questions regarding why dimerization is required for GGPPS activity and which amino acids in the dimer interface are essential for dimerization-mediated activity. According to the GGPPS crystal structure, three amino acids (N101, N104, and Y105) located in the helix F of one subunit are near the active site of the other subunit. As presented here, when these residues were replaced individually with Ala caused insignificant activity changes, N101A/Y105A and N101A/N104A but not N104A/Y105A showed remarkably decreased k(cat) values (200-250-fold). The triple mutant N101A/N104A/Y105A displayed no detectable activity, although dimer was retained in these mutants. Because N101 and Y105 form H-bonds with H139 and R140 in the other subunit, respectively, we generated H139A/R140A double mutant and found it was inactive and became monomeric. Therefore, the multiple mutations apparently influence the integrity of the catalytic site due to the missing H-bonding network. Moreover, Met111, also on the highly conserved helix F, was necessary for dimer formation and enzyme activity. When Met111 was replaced with Glu, the negative-charged repulsion converted half of the dimer into a monomer. In conclusion, the H-bonds mainly through N101 for maintaining substrate binding stability and the hydrophobic interaction of M111 in dimer interface are essential for activity of yeast GGPPS.

  8. Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia-reperfusion in mice

    Directory of Open Access Journals (Sweden)

    Shailendra P. Singh

    2015-08-01

    Full Text Available Glycogen synthase kinase-3β (GSK3β is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3β expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-β1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-β1 treatment increased GSK3β expression and GSK3 inhibition abolished TGF-β1-induced SMAD3 activation and α-smooth muscle actin (α-SMA expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3β stimulated α-SMA expression even in the absence of TGF-β1 treatment. These results suggest that TGF-β regulates GSK3β, which in turn is important for TGF-β–SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-β signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury.

  9. The Influence of Hyperoxia On Heat Shock Proteins Expression and Nitric Oxide Synthase Activity – the Review

    Directory of Open Access Journals (Sweden)

    Szyller Jakub

    2017-03-01

    Full Text Available Any stay in an environment with an increased oxygen content (a higher oxygen partial pressure, pO2 and an increased pressure (hyperbaric conditions leads to an intensification of oxidative stress. Reactive oxygen species (ROS damage the molecules of proteins, nucleic acids, cause lipid oxidation and are engaged in the development of numerous diseases, including diseases of the circulatory system, neurodegenerative diseases, etc. There are certain mechanisms of protection against unfavourable effects of oxidative stress. Enzymatic and non-enzymatic systems belong to them. The latter include, among others, heat shock proteins (HSP. Their precise role and mechanism of action have been a subject of intensive research conducted in recent years. Hyperoxia and hyperbaria also have an effect on the expression and activity of nitrogen oxide synthase (NOS. Its product - nitrogen oxide (NO can react with reactive oxygen species and contribute to the development of nitrosative stress. NOS occurs as isoforms in various tissues and exhibit different reactions to the discussed factors. The authors have prepared a brief review of research determining the effect of hyperoxia and hyperbaria on HSP expression and NOS activity.

  10. Synthesis of alkyl-ether glycerophospholipids in rat glomerular mesangial cells: evidence for alkyldihydroxyacetone phosphate synthase activity

    International Nuclear Information System (INIS)

    Zanglis, A.; Lianos, E.A.

    1987-01-01

    We studied the ability of rat glomerular mesangial cells and their microsomal fractions to incorporate 1-[ 14 C]hexadecanol to glycerophospholipids via an O-alkyl ether linkage and assessed the presence and activity of the required enzyme: alkyl-dihydroxy acetone phosphate synthase. Suspensions of cultured mesangial cells incorporated 1-[ 14 C]hexadecanol to the phosphatidyl ethanolamine and phosphatidyl choline lipid pools, via a bond resistant to acid and base hydrolysis. When cell homogenates or microsomal fractions were incubated with palmitoyl-DHAP and 1-[ 14 C]hexadecanol, alkyl-DHAP and 1-O-alkyl glycerol were formed (alkyl:hexadecyl). The activity of the enzyme responsible for the O-alkyl product formation was calculated to be 2.5 +/- 0.3 and 544 +/- 50 pmoles/min/mg protein for mesangial cell homogenates and mesangial cell microsomes, respectively. These observations provide evidence that mesangial cells may elaborate either linked lipid precursors de novo for the biosynthesis of O-alkyl glycerophospholipids

  11. Maintaining glycogen synthase kinase-3 activity is critical for mTOR kinase inhibitors to inhibit cancer cell growth.

    Science.gov (United States)

    Koo, Junghui; Yue, Ping; Gal, Anthony A; Khuri, Fadlo R; Sun, Shi-Yong

    2014-05-01

    mTOR kinase inhibitors that target both mTORC1 and mTORC2 are being evaluated in cancer clinical trials. Here, we report that glycogen synthase kinase-3 (GSK3) is a critical determinant for the therapeutic response to this class of experimental drugs. Pharmacologic inhibition of GSK3 antagonized their suppressive effects on the growth of cancer cells similarly to genetic attenuation of GSK3. Conversely, expression of a constitutively activated form of GSK3β sensitized cancer cells to mTOR inhibition. Consistent with these findings, higher basal levels of GSK3 activity in a panel of human lung cancer cell lines correlated with more efficacious responses. Mechanistic investigations showed that mTOR kinase inhibitors reduced cyclin D1 levels in a GSK3β-dependent manner, independent of their effects on suppressing mTORC1 signaling and cap binding. Notably, selective inhibition of mTORC2 triggered proteasome-mediated cyclin D1 degradation, suggesting that mTORC2 blockade is responsible for GSK3-dependent reduction of cyclin D1. Silencing expression of the ubiquitin E3 ligase FBX4 rescued this reduction, implicating FBX4 in mediating this effect of mTOR inhibition. Together, our findings define a novel mechanism by which mTORC2 promotes cell growth, with potential implications for understanding the clinical action of mTOR kinase inhibitors. ©2014 AACR.

  12. High specific activity N-Acetyl-3{sup H}-{alpha}-Aspartyl- L-Glutamic at micro mole scale; Sintesis de N-Acetil-3{sup H}- {alpha} -Aspartil-Glutamico a escala de Micromoles

    Energy Technology Data Exchange (ETDEWEB)

    Suarez, C

    1984-07-01

    High specific activity N-Acetyl-3{sup H}- {alpha} -Aspartyl-I-Glutamic acid at micro mole scale in prepared acetylating L- {alpha} -Aspartyl-L-glutamic with 3{sup H}-acetic anhydride in re distilled toluene. The product le purified through cationic and anionic columns. The radiochemical purity as determined by thin-layer chromatography is greater then 99% at the time preparation. (Author) 5 refs.

  13. Carbon monoxide releasing molecule induces endothelial nitric oxide synthase activation through a calcium and phosphatidylinositol 3-kinase/Akt mechanism.

    Science.gov (United States)

    Yang, Po-Min; Huang, Yu-Ting; Zhang, Yu-Qi; Hsieh, Chia-Wen; Wung, Being-Sun

    2016-12-01

    The production of nitric oxide (NO) by endothelial NO synthase (eNOS) plays a major role in maintaining vascular homeostasis. This study elucidated the potential role of carbon monoxide (CO)-releasing molecules (CORMs) in NO production and explored the underlying mechanisms in endothelial cells. We observed that 25μM CORM-2 could increase NO production and stimulate an increase in the intracellular Ca 2+ level. Furthermore, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetra acetic acid caused CORM-2-induced NO production, which was abolished by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetraacetoxy-methyl ester (BAPTA-AM), indicating that intracellular Ca 2+ release plays a major role in eNOS activation. The inhibition of the IP3 receptor diminished the CORM-2-induced intracellular Ca 2+ increase and NO production. Furthermore, CORM-2 induced eNOS Ser 1179 phosphorylation and eNOS dimerization, but it did not alter eNOS expression. CORM-2 (25μM) also prolonged Akt phosphorylation, lasting for at least 12h. Pretreatment with phosphatidylinositol 3-kinase inhibitors (wortmannin or LY294002) inhibited the increases in NO production and phosphorylation but did not affect eNOS dimerization. CORM-2-induced eNOS Ser 1179 phosphorylation was intracellularly calcium-dependent, because pretreatment with an intracellular Ca 2+ chelator (BAPTA-AM) inhibited this process. Although CORM-2 increases intracellular reactive oxygen species (ROS), pretreatment with antioxidant enzyme catalase and N-acetyl-cysteine did not abolish the CORM-2-induced eNOS activity or phosphorylation, signifying that ROS is not involved in this activity. Hence, CORM-2 enhances eNOS activation through intracellular calcium release, Akt phosphorylation, and eNOS dimerization. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase activity shows enhanced growth when grown under N2-fixing conditions.

    Science.gov (United States)

    Gebril, Sayed; Seger, Mark; Villanueva, Fabiola Muro; Ortega, Jose Luis; Bagga, Suman; Sengupta-Gopalan, Champa

    2015-10-01

    Overexpression of SPS in alfalfa is accompanied by early flowering, increased plant growth and an increase in elemental N and protein content when grown under N2-fixing conditions. Sucrose phosphate synthase (SPS; EC 2.3.1.14) is the key enzyme in the synthesis of sucrose in plants. The outcome of overexpression of SPS in different plants using transgenic approaches has been quite varied, but the general consensus is that increased SPS activity is associated with the production of new sinks and increased sink strength. In legumes, the root nodule is a strong C sink and in this study our objective was to see how increasing SPS activity in a legume would affect nodule number and function. Here we have transformed alfalfa (Medicago sativa, cv. Regen SY), with a maize SPS gene driven by the constitutive CaMV35S promoter. Our results showed that overexpression of SPS in alfalfa, is accompanied by an increase in nodule number and mass and an overall increase in nitrogenase activity at the whole plant level. The nodules exhibited an increase in the level of key enzymes contributing to N assimilation including glutamine synthetase and asparagine synthetase. Moreover, the stems of the transformants showed higher level of the transport amino acids, Asx, indicating increased export of N from the nodules. The transformants exhibited a dramatic increase in growth both of the shoots and roots, and earlier flowering time, leading to increased yields. Moreover, the transformants showed an increase in elemental N and protein content. The overall conclusion is that increased SPS activity improves the N status and plant performance, suggesting that the availability of more C in the form of sucrose enhances N acquisition and assimilation in the nodules.

  15. Low concentrations of salicylic acid delay methyl jasmonate-induced leaf senescence by up-regulating nitric oxide synthase activity.

    Science.gov (United States)

    Ji, Yingbin; Liu, Jian; Xing, Da

    2016-09-01

    In plants, extensive efforts have been devoted to understanding the crosstalk between salicylic acid (SA) and jasmonic acid (JA) signaling in pathogen defenses, but this crosstalk has scarcely been addressed during senescence. In this study, the effect of SA application on methyl jasmonate (MeJA)-induced leaf senescence was assessed. We found that low concentrations of SA (1-50 μM) played a delayed role against the senescence promoted by MeJA. Furthermore, low concentrations of SA enhanced plant antioxidant defenses and restricted reactive oxygen species (ROS) accumulation in MeJA-treated leaves. When applied simultaneously with MeJA, low concentrations of SA triggered a nitric oxide (NO) burst, and the elevated NO levels were linked to the nitric oxide associated 1 (NOA1)-dependent pathway via nitric oxide synthase (NOS) activity. The ability of SA to up-regulate plant antioxidant defenses, reduce ROS accumulation, and suppress leaf senescence was lost in NO-deficient Atnoa1 plants. In a converse manner, exogenous addition of NO donors increased the plant antioxidant capacity and lowered the ROS levels in MeJA-treated leaves. Taken together, the results indicate that SA at low concentrations counteracts MeJA-induced leaf senescence through NOA1-dependent NO signaling and strengthening of the antioxidant defense. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    Energy Technology Data Exchange (ETDEWEB)

    Bian, Yong, E-mail: drbiany@126.com [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China); Yu, Yun [College of Pharmacy, Nanjing University of Chinese Medicine, 210023 (China); Wang, Shanshan; Li, Lin [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China)

    2015-08-07

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression.

  17. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    International Nuclear Information System (INIS)

    Bian, Yong; Yu, Yun; Wang, Shanshan; Li, Lin

    2015-01-01

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression

  18. The effect of anandamide on uterine nitric oxide synthase activity depends on the presence of the blastocyst.

    Directory of Open Access Journals (Sweden)

    Micaela S Sordelli

    2011-04-01

    Full Text Available Nitric oxide production, catalyzed by nitric oxide synthase (NOS, should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1 h(-1 compared to days 4 (0.34±0.03 and 5 (0.35±0.02 of pregnancy and to day 6 implantation sites (0.33±0.01. This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA, an endocannabinoid, binds to cannabinoid receptors type 1 (CB1 and type 2 (CB2, and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04 and URB-597 (1.08±0.09 vs 0.83±0.06 inhibited NOS activity in the absence of a blastocyst (pseudopregnancy through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05. While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02, a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01. Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These

  19. Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

    Science.gov (United States)

    Dong, Lemeng; Miettinen, Karel; Goedbloed, Miriam; Verstappen, Francel W A; Voster, Alessandra; Jongsma, Maarten A; Memelink, Johan; van der Krol, Sander; Bouwmeester, Harro J

    2013-11-01

    Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Comparative study of enzyme activity and heme reactivity in Drosophila melanogaster and Homo sapiens cystathionine β-synthases.

    Science.gov (United States)

    Su, Yang; Majtan, Tomas; Freeman, Katherine M; Linck, Rachel; Ponter, Sarah; Kraus, Jan P; Burstyn, Judith N

    2013-01-29

    Cystathionine β-synthase (CBS) is the first and rate-limiting enzyme in the transsulfuration pathway, which is critical for the synthesis of cysteine from methionine in eukaryotes. CBS uses coenzyme pyridoxal 5'-phosphate (PLP) for catalysis, and S-adenosylmethionine regulates the activity of human CBS, but not yeast CBS. Human and fruit fly CBS contain heme; however, the role for heme is not clear. This paper reports biochemical and spectroscopic characterization of CBS from fruit fly Drosophila melanogaster (DmCBS) and the CO/NO gas binding reactions of DmCBS and human CBS. Like CBS enzymes from lower organisms (e.g., yeast), DmCBS is intrinsically highly active and is not regulated by AdoMet. The DmCBS heme coordination environment, the reactivity, and the accompanying effects on enzyme activity are similar to those of human CBS. The DmCBS heme bears histidine and cysteine axial ligands, and the enzyme becomes inactive when the cysteine ligand is replaced. The Fe(II) heme in DmCBS is less stable than that in human CBS, undergoing more facile reoxidation and ligand exchange. In both CBS proteins, the overall stability of the protein is correlated with the heme oxidation state. Human and DmCBS Fe(II) hemes react relatively slowly with CO and NO, and the rate of the CO binding reaction is faster at low pH than at high pH. Together, the results suggest that heme incorporation and AdoMet regulation in CBS are not correlated, possibly providing two independent means for regulating the enzyme.

  1. Glycogen synthase kinase-3β activity and cognitive functioning in patients with bipolar I disorder

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Miskowiak, Kamilla Woznica; Jacoby, Anne Sophie

    2018-01-01

    but the relation between GSK-3 activity, cognition and lithium treatment is unknown. We therefore investigated the possible association between GSK-3 activity and cognition and whether lithium treatment moderates this association in patients with BD. In a prospective 6-12 month follow-up study, GSK- 3β activity...... in peripheral blood mononuclear cells was measured concurrently with cognitive performance assessed using a comprehensive test battery in 27 patients with BD-I in early and late remission following a manic or mixed episode. The GSK-3β activity, measured as serine-9 phosphorylated GSK-3β (pGSK-3β) and the GSK-3β...... ratio (serine-9-pGSK-3β /total GSK-3β), was negatively associated with sustained attention (p = 0.009 and p = 0.042, respectively), but not with other cognitive domains or global cognition. A crossover interaction between lithium treatment and the GSK activity was observed, indicating that lower pGSK-3β...

  2. Improvement in antioxidant activity, angiotensin-converting enzyme inhibitory activity and in vitro cellular properties of fermented pepino milk by Lactobacillus strains containing the glutamate decarboxylase gene.

    Science.gov (United States)

    Chiu, Tsai-Hsin; Tsai, Shwu-Jene; Wu, Tsung-Yen; Fu, Szu-Chieh; Hwang, Yi-Ting

    2013-03-15

    The purpose of this study was to evaluate the functional potential of fermented pepino extract (PE) milk by Lactobacillus strains containing the glutamate decarboxylase (GAD) gene. Three Lactobacillus strains were selected, including L. brevis BCRC 12310, L. casei BCRC 14082 and L. salivarius subsp. salivarius BCRC 14759. The contents of free amino acids, total phenolics content, total carotenoids and the associated functional and antioxidant abilities were analyzed, including angiotensin-converting enzyme (ACE) inhibition activity, 1,1-diphenyl-2-picylhydrazyl (DPPH) radical-scavenging ability and oxygen radical absorbance capacity (ORAC). Cell proliferation of fermented PE milk was also evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compared to the unfermented PE, fermented PE milk from Lactobacillus strains with the GAD gene showed higher levels of total phenolics, γ-aminobutyric acid, ACE inhibitory activity, DPPH, and ORAC. The viability of human promyelocytic leukemia cells (HL-60) determined by the MTT method decreased significantly when the cells were incubated with the PE and the fermented PE milk extracts. The consumption of fermented PE milk from Lactobacillus strains with the GAD gene is expected to benefit health. Further application as a health food is worthy of investigation. © 2012 Society of Chemical Industry. © 2012 Society of Chemical Industry.

  3. A radiometric microassay for glutamic acid decarboxylase

    International Nuclear Information System (INIS)

    Maderdrut, J.L.; North Carolina Univ., Chapel Hill

    1979-01-01

    A simple method for purifying L-[ 3 H] glutamic acid and incubation conditions suitable for estimating L-glutamic acid decarboxylase activity are described. Routine and recycled cation-exchange procedure for separating γ-aminobutyric acid from L-glutamate are outlined and compared. Recycling increases the sensitivity of the cation-exchange method by 6-7 fold. L-Glutamate decarboxylase activity can be measured reliably in samples of embryonic neural tissue having wet-weights of approximately 1 μg. The cation-exchange method is compared with the anion-exchange and CO 2 -trapping methods. L-Glutamate decarboxylase activity has been detected in the lumbar spinal cord of the chick embryo at Day 21/4 (stage 14) using the cation-exchange method. This is 5-6 days earlier than L-glutamate decarboxylase activity has been detected in embryonic neural tissue by previous investigators. L-Glutamate decarboxylase is present in the lumbar spinal cord at least as early as the birth of the first lumbar spinal cord neurons and at least 1-2 days before the initiation of synaptogenesis. (author)

  4. Radiometric microassay for glutamic acid decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Maderdrut, J L [North Carolina Dept. of Mental Health, Raleigh (USA); North Carolina Univ., Chapel Hill (USA). School of Medicine)

    1979-01-01

    A simple method for purifying L-(/sup 3/H) glutamic acid and incubation conditions suitable for estimating L-glutamic acid decarboxylase activity are described. Routine and recycled cation-exchange procedure for separating ..gamma..-aminobutyric acid from L-glutamate are outlined and compared. Recycling increases the sensitivity of the cation-exchange method by 6-7 fold. L-Glutamate decarboxylase activity can be measured reliably in samples of embryonic neural tissue having wet-weights of approximately 1 ..mu..g. The cation-exchange method is compared with the anion-exchange and CO/sub 2/-trapping methods. L-Glutamate decarboxylase activity has been detected in the lumbar spinal cord of the chick embryo at Day 21/4 (stage 14) using the cation-exchange method. This is 5-6 days earlier than L-glutamate decarboxylase activity has been detected in embryonic neural tissue by previous investigators. L-Glutamate decarboxylase is present in the lumbar spinal cord at least as early as the birth of the first lumbar spinal cord neurons and at least 1-2 days before the initiation of synaptogenesis.

  5. Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia

    OpenAIRE

    Gregus, Ann M.; Dumlao, Darren S.; Wei, Spencer C.; Norris, Paul C.; Catella, Laura C.; Meyerstein, Flore G.; Buczynski, Matthew W.; Steinauer, Joanne J.; Fitzsimmons, Bethany L.; Yaksh, Tony L.; Dennis, Edward A.

    2013-01-01

    Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presenc...

  6. Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2+2a) phosphorylation

    DEFF Research Database (Denmark)

    Friedrichsen, Martin; Birk, Jesper Bratz; Richter, Erik

    2013-01-01

    Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. We previously demonstrated that phosphorylation of Threonine-308 on Akt (p......Akt-T308), Akt2 activity, and GS activity in muscle were positivity associated with insulin sensitivity. Now, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. 181 non-diabetic twins were examined...

  7. Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine.

    Directory of Open Access Journals (Sweden)

    Janny A Villa-Pulgarín

    2017-08-01

    Full Text Available Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.The present study shows that the

  8. A stable blue-light-derived signal modulates ultraviolet-light-induced activation of the chalcone-synthase gene in cultured parsley cells

    International Nuclear Information System (INIS)

    Ohl, S.; Hahlbrock, K.; Schäfer, E.

    1989-01-01

    Run-off transcription assays were used to demonstrate that both the ultraviolet (UV)-B and blue-light receptors control transcription rates for chalcone-synthase mRNA in the course of light-induced flavonoid synthesis in parsley (Petroselinum crispum Miller (A.W. Hill)) cell-suspension cultures. Blue and red light alone, presumably acting via a blue-light receptor and active phytochrome (far-red absorbing form) respectively, can induce accumulation of chalcone-synthase mRNA. The extent of the response is however considerably smaller than that obtained when these wavebands are applied in combination with UV light. A preirradiation with blue light strongly increases the response to a subsequent UV pulse and this modulating effect of blue light is stable for at least 20 h. The modulating effect is abolished by a UV induction but can be reestablished by a second irradiation with blue light. (author)

  9. Huperzine A Alleviates Oxidative Glutamate Toxicity in Hippocampal HT22 Cells via Activating BDNF/TrkB-Dependent PI3K/Akt/mTOR Signaling Pathway.

    Science.gov (United States)

    Mao, Xiao-Yuan; Zhou, Hong-Hao; Li, Xi; Liu, Zhao-Qian

    2016-08-01

    Oxidative glutamate toxicity is involved in diverse neurological disorders including epilepsy and ischemic stroke. Our present work aimed to assess protective effects of huperzine A (HupA) against oxidative glutamate toxicity in a mouse-derived hippocampal HT22 cells and explore its potential mechanisms. Cell survival and cell injury were analyzed by MTT method and LDH release assay, respectively. The production of ROS was measured by detection kits. Protein expressions of BDNF, phosphor-TrkB (p-TrkB), TrkB, phosphor-Akt (p-Akt), Akt, phosphor-mTOR (p-mTOR), mTOR, phosphor-p70s6 (p-p70s6) kinase, p70s6 kinase, Bcl-2, Bax, and β-actin were assayed via Western blot analysis. Enzyme-linked immunosorbent assay was employed to measure the contents of nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Our findings illustrated 10 μM HupA for 24 h significantly protected HT22 from cellular damage and suppressed the generation of ROS. Additionally, after treating with LY294002 or wortmannin [the selective inhibitors of phosphatidylinositol 3 kinase (PI3K)], HupA dramatically prevented the down-regulations of p-Akt, p-mTOR, and p-p70s6 kinase in HT22 cells under oxidative toxicity. Furthermore, it was observed that the protein levels of BDNF and p-TrkB were evidently enhanced after co-treatment with HupA and glutamate in HT22 cells. The elevations of p-Akt and p-mTOR were abrogated under toxic conditions after blockade of TrkB by TrkB IgG. Cellular apoptosis was significantly suppressed (decreased caspase-3 activity and enhanced Bcl-2 protein level) after HupA treatment. It was concluded that HupA attenuated oxidative glutamate toxicity in murine hippocampal HT22 cells via activating BDNF/TrkB-dependent PI3K/Akt/mTOR signaling pathway.

  10. Feedback-induced glutamate spillover enhances negative feedback from horizontal cells to cones

    NARCIS (Netherlands)

    Vroman, Rozan; Kamermans, Maarten

    2015-01-01

    In the retina, horizontal cells feed back negatively to cone photoreceptors. Glutamate released from cones can spill over to neighbouring cones. Here we show that cone glutamate release induced by negative feedback can also spill over to neighbouring cones. This glutamate activates the glutamate

  11. Impairments in cognition and neural precursor cell proliferation in mice expressing constitutively active glycogen synthase kinase-3

    Directory of Open Access Journals (Sweden)

    Marta ePardo

    2015-03-01

    Full Text Available ABSTRACTBrain glycogen synthase kinase-3 (GSK3 is hyperactive in several neurological conditions that involve impairments in both cognition and neurogenesis. This raises the hypotheses that hyperactive GSK3 may directly contribute to impaired cognition, and that this may be related to deficiencies in neural precursor cells (NPC. To study the effects of hyperactive GSK3 in the absence of disease influences, we compared adult hippocampal NPC proliferation and performance in three cognitive tasks in male and female wild-type mice and GSK3 knockin mice, which express constitutively active GSK3. NPC proliferation was ~40% deficient in both male and female GSK3 knockin mice compared with wild-type mice. Environmental enrichment (EE increased NPC proliferation in male, but not female, GSK3 knockin mice and wild-type mice. Male and female GSK3 knockin mice exhibited impairments in novel object recognition, temporal order memory, and coordinate spatial processing compared with gender-matched wild-type mice. EE restored impaired novel object recognition and temporal ordering in both sexes of GSK3 knockin mice, indicating that this repair was not dependent on NPC proliferation, which was not increased by EE in female GSK3 knockin mice. Acute 1 hr pretreatment with the GSK3 inhibitor TDZD-8 also improved novel object recognition and temporal ordering in male and female GSK3 knockin mice. These findings demonstrate that hyperactive GSK3 is sufficient to impair adult hippocampal NPC proliferation and to impair performance in three cognitive tasks in both male and female mice, but these changes in NPC proliferation do not directly regulate novel object recognition and temporal ordering tasks.

  12. Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity

    NARCIS (Netherlands)

    Chavaroche, A.A.E.; Broek, van den L.A.M.; Springer, J.; Boeriu, C.; Eggink, G.

    2011-01-01

    Heparosan synthase catalyzes the polymerization of heparosan [-4GlcUAß1-4GlcNAca1-]n by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its

  13. The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells

    International Nuclear Information System (INIS)

    Ribeiro, Mariana P.C.; Nunes-Correia, Isabel; Santos, Armanda E.; Custódio, José B.A.

    2014-01-01

    Recent reports suggest that N-methyl-D-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination. - Highlights: • MK-801 and memantine decrease melanoma cell proliferation. • The combination of MK-801 with antiestrogens inhibits melanoma cell proliferation. • These combinations greatly enhance the effects of the compounds individually. • MK-801 combined with tamoxifen active metabolites induces cell cycle arrest in G1. • The combination of MK-801 and antiestrogens is an innovative strategy for melanoma

  14. The time-dependent effect of provinolsTM on brain NO synthase activity in L-NAME-induced hypertension

    Czech Academy of Sciences Publication Activity Database

    Jendeková, L.; Kojšová, S.; Andriantsitohaina, R.; Pecháňová, Olga

    2006-01-01

    Roč. 55, č. S1 (2006), S31-S37 ISSN 0862-8408 Grant - others:VEGA(SK) 2/6148/26; VEGA(SK) 1/342906 Institutional research plan: CEZ:AV0Z50110509 Keywords : red wine polyphenols * oxidative damage * nitric oxide synthase Subject RIV: ED - Physiology Impact factor: 2.093, year: 2006

  15. Cooperative functioning between phenylalanine ammonia lyase and isochorishmate synthase activities contributes to salicylic acid biosynthesis in soybean

    Science.gov (United States)

    Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL), or the isochorishmate synthase (ICS) catalyzed steps. The ICS pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We...

  16. A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex

    International Nuclear Information System (INIS)

    Grum, Daniel; Boom, Johannes van den; Neumann, Daniel; Matena, Anja; Link, Nina M.; Mueller, Jonathan W.

    2010-01-01

    3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Foerster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.

  17. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  18. Glutamate and Neurodegenerative Disease

    Science.gov (United States)

    Schaeffer, Eric; Duplantier, Allen

    As the main excitatory neurotransmitter in the mammalian central nervous system, glutamate is critically involved in most aspects of CNS function. Given this critical role, it is not surprising that glutamatergic dysfunction is associated with many CNS disorders. In this chapter, we review the literature that links aberrant glutamate neurotransmission with CNS pathology, with a focus on neurodegenerative diseases. The biology and pharmacology of the various glutamate receptor families are discussed, along with data which links these receptors with neurodegenerative conditions. In addition, we review progress that has been made in developing small molecule modulators of glutamate receptors and transporters, and describe how these compounds have helped us understand the complex pharmacology of glutamate in normal CNS function, as well as their potential for the treatment of neurodegenerative diseases.

  19. The Influence of Glutamate on Axonal Compound Action Potential In Vitro.

    Science.gov (United States)

    Abouelela, Ahmed; Wieraszko, Andrzej

    2016-01-01

    Background  Our previous experiments demonstrated modulation of the amplitude of the axonal compound action potential (CAP) by electrical stimulation. To verify assumption that glutamate released from axons could be involved in this phenomenon, the modification of the axonal CAP induced by glutamate was investigated. Objectives  The major objective of this research is to verify the hypothesis that axonal activity would trigger the release of glutamate, which in turn would interact with specific axonal receptors modifying the amplitude of the action potential. Methods  Segments of the sciatic nerve were exposed to exogenous glutamate in vitro, and CAP was recorded before and after glutamate application. In some experiments, the release of radioactive glutamate analog from the sciatic nerve exposed to exogenous glutamate was also evaluated. Results  The glutamate-induced increase in CAP was blocked by different glutamate receptor antagonists. The effect of glutamate was not observed in Ca-free medium, and was blocked by antagonists of calcium channels. Exogenous glutamate, applied to the segments of sciatic nerve, induced the release of radioactive glutamate analog, demonstrating glutamate-induced glutamate release. Immunohistochemical examination revealed that axolemma contains components necessary for glutamatergic neurotransmission. Conclusion  The proteins of the axonal membrane can under the influence of electrical stimulation or exogenous glutamate change membrane permeability and ionic conductance, leading to a change in the amplitude of CAP. We suggest that increased axonal activity leads to the release of glutamate that results in changes in the amplitude of CAPs.

  20. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    International Nuclear Information System (INIS)

    Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-01-01

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-α-stimulated monocytes to endothelial cells and suppressed the TNF-α induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-α-induced nuclear factor-κB activation, which was attenuated by pretreatment with N G -nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: ► Puerarin induced the phosphorylation of eNOS and the production of NO. ► Puerarin activated eNOS through ER-dependent PI3-kinase and Ca 2+ -dependent AMPK. ► Puerarin-induced NO was involved in the inhibition of NF-kB activation. ► Puerarin may help for prevention of vascular dysfunction and diabetes.

  1. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  2. In SilicoModel-driven Assessment of the Effects of Brain-derived Neurotrophic Factor Deficiency on Glutamate and Gamma-Aminobutyric Acid: Implications for Understanding Schizophrenia Pathophysiology.

    Science.gov (United States)

    Agrawal, Rimjhim; Kalmady, Sunil Vasu; Venkatasubramanian, Ganesan

    2017-05-31

    Deficient brain-derived neurotrophic factor (BDNF) is one of the important mechanisms underlying the neuroplasticity abnormalities in schizophrenia. Aberration in BDNF signaling pathways directly or circuitously influences neurotransmitters like glutamate and gamma-aminobutyric acid (GABA). For the first time, this study attempts to construct and simulate the BDNF-neurotransmitter network in order to assess the effects of BDNF deficiency on glutamate and GABA. Using CellDesigner, we modeled BDNF interactions with calcium influx via N-methyl-D-aspartate receptor (NMDAR)- Calmodulin activation; synthesis of GABA via cell cycle regulators protein kinase B, glycogen synthase kinase and β-catenin; transportation of glutamate and GABA. Steady state stability, perturbation time-course simulation and sensitivity analysis were performed in COPASI after assigning the kinetic functions, optimizing the unknown parameters using random search and genetic algorithm. Study observations suggest that increased glutamate in hippocampus, similar to that seen in schizophrenia, could potentially be contributed by indirect pathway originated from BDNF. Deficient BDNF could suppress Glutamate decarboxylase 67-mediated GABA synthesis. Further, deficient BDNF corresponded to impaired transport via vesicular glutamate transporter, thereby further increasing the intracellular glutamate in GABAergic and glutamatergic cells. BDNF also altered calcium dependent neuroplasticity via NMDAR modulation. Sensitivity analysis showed that Calmodulin, cAMP response element-binding protein (CREB) and CREB regulated transcription coactivator-1 played significant role in this network. The study presents in silico quantitative model of biochemical network constituting the key signaling molecules implicated in schizophrenia pathogenesis. It provides mechanistic insights into putative contribution of deficient BNDF towards alterations in neurotransmitters and neuroplasticity that are consistent with current

  3. Activation of Group II Metabotropic Glutamate Receptors Increases Proliferation but does not Influence Neuronal Differentiation of a Human Neural Stem Cell Line

    DEFF Research Database (Denmark)

    Dindler, Anne; Blaabjerg, Morten; Kamand, Morad

    2018-01-01

    of pharmacological activation and inhibition of mGluR2/3 on proliferation, differentiation and viability of a human neural stem cell line. Immunofluorescence staining revealed the presence of mGluR2/3 receptors on both proliferating and differentiating stem cells, including cells differentiated into β-tubulin III....... Western blot analysis revealed that the active, dimeric form of mGluR2/3 was mainly present on the proliferating cells, which may explain our findings. The present study emphasises the importance of glutamate and mGluRs on regulation of human neural stem cells and suggests a significant role of mGluR2....../3 during cell proliferation. This article is protected by copyright. All rights reserved....

  4. Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

    International Nuclear Information System (INIS)

    Ida, Tomoyo; Suzuki, Hideyuki; Fukuyama, Keiichi; Hiratake, Jun; Wada, Kei

    2014-01-01

    The binding modes of acivicin, a classical and an electrophilic active-site-directed glutamate analogue, to bacterial γ-glutamyltranspeptidases were found to be diverse. γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalently through its C3 atom with sp 2 hybridization to Thr403 O γ , the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs

  5. Synaptically evoked glutamate transporter currents in Spinal Dorsal Horn Astrocytes

    Directory of Open Access Journals (Sweden)

    Dougherty Patrick M

    2009-07-01

    Full Text Available Abstract Background Removing and sequestering synaptically released glutamate from the extracellular space is carried out by specific plasma membrane transporters that are primarily located in astrocytes. Glial glutamate transporter function can be monitored by recording the currents that are produced by co-transportation of Na+ ions with the uptake of glutamate. The goal of this study was to characterize glutamate transporter function in astrocytes of the spinal cord dorsal horn in real time by recording synaptically evoked glutamate transporter currents. Results Whole-cell patch clamp recordings were obtained from astrocytes in the spinal substantia gelatinosa (SG area in spinal slices of young adult rats. Glutamate transporter currents were evoked in these cells by electrical stimulation at the spinal dorsal root entry zone in the presence of bicuculline, strychnine, DNQX and D-AP5. Transporter currents were abolished when synaptic transmission was blocked by TTX or Cd2+. Pharmacological studies identified two subtypes of glutamate transporters in spinal astrocytes, GLAST and GLT-1. Glutamate transporter currents were graded with stimulus intensity, reaching peak responses at 4 to 5 times activation threshold, but were reduced following low-frequency (0.1 – 1 Hz repetitive stimulation. Conclusion These results suggest that glutamate transporters of spinal astrocytes could be activated by synaptic activation, and recording glutamate transporter currents may provide a means of examining the real time physiological responses of glial cells in spinal sensory processing, sensitization, hyperalgesia and chronic pain.

  6. Fear potentiated startle increases phospholipase D (PLD) expression/activity and PLD-linked metabotropic glutamate receptor mediated post-tetanic potentiation in rat amygdala.

    Science.gov (United States)

    Krishnan, Balaji; Scott, Michael T; Pollandt, Sebastian; Schroeder, Bradley; Kurosky, Alexander; Shinnick-Gallagher, Patricia

    2016-02-01

    Long-term memory (LTM) of fear stores activity dependent modifications that include changes in amygdala signaling. Previously, we identified an enhanced probability of release of glutamate mediated signaling to be important in rat fear potentiated startle (FPS), a well-established translational behavioral measure of fear. Here, we investigated short- and long-term synaptic plasticity in FPS involving metabotropic glutamate receptors (mGluRs) and associated downstream proteomic changes in the thalamic-lateral amygdala pathway (Th-LA). Aldolase A, an inhibitor of phospholipase D (PLD), expression was reduced, concurrent with significantly elevated PLD protein expression. Blocking the PLD-mGluR signaling significantly reduced PLD activity. While transmitter release probability increased in FPS, PLD-mGluR agonist and antagonist actions were occluded. In the unpaired group (UNP), blocking the PLD-mGluR increased while activating the receptor decreased transmitter release probability, consistent with decreased synaptic potentials during tetanic stimulation. FPS Post-tetanic potentiation (PTP) immediately following long-term potentiation (LTP) induction was significantly increased. Blocking PLD-mGluR signaling prevented PTP and reduced cumulative PTP probability but not LTP maintenance in both groups. These effects are similar to those mediated through mGluR7, which is co-immunoprecipitated with PLD in FPS. Lastly, blocking mGluR-PLD in the rat amygdala was sufficient to prevent behavioral expression of fear memory. Thus, our study in the Th-LA pathway provides the first evidence for PLD as an important target of mGluR signaling in amygdala fear-associated memory. Importantly, the PLD-mGluR provides a novel therapeutic target for treating maladaptive fear memories in posttraumatic stress and anxiety disorders. Published by Elsevier Inc.

  7. Antiepileptic activity of total triterpenes isolated from Poria cocos is mediated by suppression of aspartic and glutamic acids in the brain.

    Science.gov (United States)

    Gao, Yanqiong; Yan, Hua; Jin, Ruirui; Lei, Peng

    2016-11-01

    Triterpenes from Poria cocos Wolf (Polyporaceae) have been used to treat various diseases in traditional Chinese medicine. However, the antiepileptic effects and mechanism are not fully understood. The objective of this study is to investigate the antiepileptic properties of total triterpenes (TTP) from the whole P. cocos. The ethanol extract TTP was identified by HPLC fingerprint analysis. Male ICR mice were gavaged (i.g.) with TTP (5, 20, 80 or 160 mg/kg) or reference drugs twice a day for 7 d. Antiepileptic activities of TTP were evaluated by maximal electroshock (MES)- and pentylenetetrazole (PTZ)-induced seizures in mice for 30 and 60 min, respectively. Locomotor activity and Rota-rod tests were performed for 60 min and 5 min, respectively. The levels of glutamic acid (Glu), aspartic acid (Asp), γ-aminobutyric acid (GABA) and glycine (Gly) in convulsive mice were estimated. The chronic epileptic model of Wistar rats was built to measure expressions of glutamate decarboxylase 65 (GAD65) and GABA A in rat brain after TTP treatment. The LC 50 of TTP (i.g.) was above 6 g/kg. TTP (5-160 mg/kg) protected mice against MES- and PTZ-induced convulsions at 65.0% and 62.5%, respectively, but have no effect on rota-rod treadmill; TTP (20-160 mg/kg) significantly reduced the locomotor activities, shortened the onset of pentobarbital sodium-induced sleep; TTP decreased Glu and Asp levels in convulsive mice, but increased the GAD65 and GABA A expressions in chronic epileptic rats at doses usage. TTP extracted from P. cocos possessed potential antiepileptic properties and is a candidate for further antiepileptic drug development.

  8. In-silico docking based design and synthesis of [1H,3H] imidazo[4,5-b] pyridines as lumazine synthase inhibitors for their effective antimicrobial activity.

    Science.gov (United States)

    Harer, Sunil L; Bhatia, Manish S

    2014-10-01

    The imidazopyridine moiety is important pharmacophore that has proven to be useful for a number of biologically relevant targets, also reported to display antibacterial, antifungal, antiviral properties. Riboflavin biosynthesis involving catalytic step of Lumazine synthase is absent in animals and human, but present in microorganism, one of marked advantage of this study. Still, this path is not exploited as antiinfective target. Here, we proposed different interactions between [1H,3H] imidazo[4,5-b] pyridine test ligands and target protein Lumazine synthase (protein Data Bank 2C92), one-step synthesis of title compounds and further evaluation of them for in vitro antimicrobial activity. Active pocket of the target protein involved in the interaction with the test ligands molecules was found using Biopredicta tools in VLifeMDS 4.3 Suite. In-silico docking suggests H-bonding, hydrophobic interaction, charge interaction, aromatic interaction, and Vanderwaal forces responsible for stabilizing enzyme-inhibitor complex. Disc diffusion assay method was used for in vitro antimicrobial screening. Investigation of possible interaction between test ligands and target lumazine synthase of Mycobacterium tuberculosis suggested 1i and 2f as best fit candidates showing hydrogen bonding, hydrophobic, aromatic and Vanderwaal's forces. Among all derivatives 1g, 1j, 1k, 1l, 2a, 2c, 2d, 2e, 2h, and 2j exhibited potent activities against bacteria and fungi compared to the standard Ciprofloxacin and Fluconazole, respectively. The superiority of 1H imidazo [4,5-b] pyridine compounds having R' = Cl >No2 > NH2 at the phenyl/aliphatic moiety resident on the imidazopyridine, whereas leading 3H imidazo[4,5-b] pyridine compounds containing R/Ar = Cl > No2 > NH2> OCH3 substituents on the 2(nd) position of imidazole.

  9. In-silico docking based design and synthesis of [1H,3H] imidazo[4,5-b] pyridines as lumazine synthase inhibitors for their effective antimicrobial activity

    Directory of Open Access Journals (Sweden)

    Sunil L Harer

    2014-01-01

    Full Text Available Purpose: The imidazopyridine moiety is important pharmacophore that has proven to be useful for a number of biologically relevant targets, also reported to display antibacterial, antifungal, antiviral properties. Riboflavin biosynthesis involving catalytic step of Lumazine synthase is absent in animals and human, but present in microorganism, one of marked advantage of this study. Still, this path is not exploited as antiinfective target. Here, we proposed different interactions between [1H,3H] imidazo[4,5-b] pyridine test ligands and target protein Lumazine synthase (protein Data Bank 2C92, one-step synthesis of title compounds and further evaluation of them for in vitro antimicrobial activity. Materials and Methods: Active pocket of the target protein involved in the interaction with the test ligands molecules was found using Biopredicta tools in VLifeMDS 4.3 Suite. In-silico docking suggests H-bonding, hydrophobic interaction, charge interaction, aromatic interaction, and Vanderwaal forces responsible for stabilizing enzyme-inhibitor complex. Disc diffusion assay method was used for in vitro antimicrobial screening. Results and Discussion: Investigation of possible interaction between test ligands and target lumazine synthase of Mycobacterium tuberculosis suggested 1i and 2f as best fit candidates showing hydrogen bonding, hydrophobic, aromatic and Vanderwaal′s forces. Among all derivatives 1g, 1j, 1k, 1l, 2a, 2c, 2d, 2e, 2h, and 2j exhibited potent activities against bacteria and fungi compared to the standard Ciprofloxacin and Fluconazole, respectively. The superiority of 1H imidazo [4,5-b] pyridine compounds having R′ = Cl >No 2 > NH 2 at the phenyl/aliphatic moiety resident on the imidazopyridine, whereas leading 3H imidazo[4,5-b] pyridine compounds containing R/Ar = Cl > No 2 > NH 2> OCH 3 substituents on the 2 nd position of imidazole.

  10. [Study on the seasonal variations of the active components in Acer truncatum leaves and the inhibitory ability on fatty acid synthase].

    Science.gov (United States)

    Fan, Yuan-Jie; Ye, Yan-Bin; Gao, Wen; Zhang, Feng; Zhang, Ying-Xia

    2010-11-01

    To study the dynamic variations of the contents of total polyphenols, flvonoids and chlorogenic acid from Acer truncatum leaves in different months, and their inhibitory activities on fatty acid synthase. Spectrophotometry was used to determine the contents of total polyphenols, flavonoids and chlorogenic acid in extracts and the extracts' inhibitory effects were also investigated. All Leaves picked from May to November have inhibitory effect. But the contents of polyphenols in leaves of July appeared to be higher than other months', and consequently exhibited stronger inhibition against FAS. A positive correlation between the content of polyphenols in leaves extract and the inhibitory efficacy on FAS could be established.

  11. Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii

    DEFF Research Database (Denmark)

    Kadziola, Anders; Jepsen, Clemens H; Johansson, Eva

    2005-01-01

    The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and...

  12. N-acetylglutamate synthase deficiency: an insight into the genetics, epidemiology, pathophysiology, and treatment

    Directory of Open Access Journals (Sweden)

    Caldovic L

    2011-08-01

    Full Text Available Nicholas Ah Mew, Ljubica CaldovicCenter for Genetic Medicine Research, Children’s Research Institute, Children’s National Medical Center, Washington DC, USAAbstract: The conversion of ammonia into urea by the human liver requires the coordinated function of the 6 enzymes and 2 transporters of the urea cycle. The initial and rate-limiting enzyme of the urea cycle, carbamylphosphate synthetase 1 (CPS1, requires an allosteric activator, N-acetylglutamate (NAG. The formation of this unique cofactor from glutamate and acetyl Coenzyme-A is catalyzed by N-acetylglutamate synthase (NAGS. An absence of NAG as a consequence of NAGS deficiency may compromise flux through CPS1 and result in hyperammonemia. The NAGS gene encodes a 528-amino acid protein, consisting of a C-terminal catalytic domain, a variable segment, and an N-terminal mitochondrial targeting signal. Only 22 mutations in the NAGS gene have been reported to date, mostly in the catalytic domain. NAGS is primarily expressed in the liver and intestine. However, it is also surprisingly expressed in testis, stomach and spleen, and during early embryonic development at levels not concordant with the expression of other urea cycle enzymes, CPS1, or ornithine transcarbamylase. The purpose of NAGS expression in these tissues, and its significance to NAGS deficiency is as yet unknown. Inherited NAGS deficiency is the rarest of the urea cycle disorders, and we review the currently reported 34 cases. Treatment of NAGS deficiency with N-carbamyglutamate, a stable analog of NAG, can restore deficient urea cycle function and normalize blood ammonia in affected patients.Keywords: urea cycle, urea cycle disorder, N-acetyl-L-glutamate, N-acetylglutamate synthase, hyperammonemia, N-carbamyl-L-glutamate

  13. Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones.

    Science.gov (United States)

    Crossthwaite, Andrew J; Valli, Haseeb; Williams, Robert J

    2004-03-01

    Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.

  14. Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of Arabidopsis roots to L-glutamate.

    Science.gov (United States)

    López-Bucio, Jesús Salvador; Raya-González, Javier; Ravelo-Ortega, Gustavo; Ruiz-Herrera, León Francisco; Ramos-Vega, Maricela; León, Patricia; López-Bucio, José; Guevara-García, Ángel Arturo

    2018-03-01

    The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.

  15. Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase.

    Science.gov (United States)

    Talwar, Pooja Saigal; Gupta, Ravi; Maurya, Arun Kumar; Deswal, Renu

    2012-11-01

    Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 μM) and l-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased K(m) (∼13%) and almost two fold increase in V(max). PMA (438 nM), a kinase activator, activated BjNOSla ∼1.9 fold while its inactive analog 4αPDD was ineffective. Calcium and PMA activated the enzyme to ∼3 folds. Interestingly, 1,2-DG6 (2.5 μM) and PS (1 μM) with calcium activated the enzyme activity to ∼7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (∼90%) and calphostin-C (∼40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7-46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  16. [Inhibition of glycogen synthase kinase 3b activity regulates Toll-like receptor 4-mediated liver inflammation].

    Science.gov (United States)

    Ren, Feng; Zhang, Hai-yan; Piao, Zheng-fu; Zheng, Su-jun; Chen, Yu; Chen, De-xi; Duan, Zhong-ping

    2012-09-01

    To determine the mechanism underlying the therapeutic activities of glycogen synthase kinase 3b (GSK3b) against hepatic ischemia-reperfusion (H-IR) injury by investigating the inhibitive effects of GSK3b on inflammation mediated by Toll-like receptor 4 (TLR4). C57BL/6 male mice were subjected to 90 min of warm liver cephalad lobe ischemia, followed by reperfusion for various lengths of time. The mice were divided into three groups: the H-IR untreated model (control group), and the H-IR inflammation-induced models that received an intraperitoneal injection of purified lipopolysaccharide (LPS) endotoxin alone (inflammation group) or with pretreatment of the SB216763 GSK3b-specific inhibitor (intervention group). To create a parallel isolated cell system for detailed investigations of macrophages, marrow-derived stem cells were isolated from femurs of the H-IR control group of mice and used to derive primary macrophages. The cells were then divided into the same three groups as the whole mouse system: control, LPS-induced inflammation model, and inflammation model with SB216763 intervention. Differential expressions of inflammation-related proteins and genes were detected by Western blotting and real-time quantitative PCR, respectively. The phosphorylation levels of ERK, JNK and p38 MAPK were induced in liver at 1 h after reperfusion, but then steadily decreased and returned to baseline levels by 4 h after reperfusion. In addition, the phosphorylation levels of ERK and JNK were induced in macrophages at 15 min after LPS stimulation, while the phosphorylation level of p38 MAPK was induced at 1 h; SB216763 pretreatment suppressed the LPS-stimulated ERK, JNK and p38 phosphorylation in macrophages. In the mouse model, GSK3b activity was found to promote the gene expression of anti-inflammatory cytokine IL-10 (control: 0.21 ± 0.08, inflammation: 0.83 ± 0.21, intervention: 1.76 ± 0.67; F = 3.16, P = 0.027) but to significantly inhibit the gene expression of pro

  17. Fluorescence studies on the aggregation behaviors of collagen modified with NHS-activated poly(γ-glutamic acid).

    Science.gov (United States)

    Zhang, Min; Yang, Junhui; Yang, Qili; Huang, Liulian; Wu, Hui; Chen, Lihui; Ding, Cuicui

    2018-06-01

    The poly(γ-glutamic acid)-NHS (γ-PGA-NHS) esters were used to endow collagen with both of excellent water-solubility and thermal stability via cross-linking reaction between γ-PGA-NHS and collagen. In the present work, the effect of γ-PGA-NHS on the aggregation of collagen molecules was studied by fluorescence techniques. The fluorescence emission spectra of pyrene in collagen solutions and the intrinsic fluorescence emission spectra of collagen suggested different effects of γ-PGA-NHS on collagen molecules: inhibiting aggregation below critical aggregation concentration (CAC) and promoting aggregation above CAC. The two-dimensional (2D) fluorescence correlation spectra indicated that the intermolecular hydrogen bonding and cross-linking between γ-PGA-NHS and collagen would influence the aggregation of collagen molecules. By the ultra-sensitive differential scanning calorimeter (VP-DSC), it was found that the main denaturational transition temperature (T m2 ) of modified collagen increased, while its calorimetric enthalpy changes (ΔH 2 ) decreased compared to those of native collagen, further indicating that the modification of γ-PGA-NHS influenced the aggregation of collagen molecules. The study provide useful information for the utilizing and or the processing of water-soluble collagen in aqueous solution in the fields such as cosmetics, health care products, tissue engineering and biomedical materials, etc. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Plasmodium falciparum avoids change in erythrocytic surface expression of phagocytosis markers during inhibition of nitric oxide synthase activity

    DEFF Research Database (Denmark)

    Hempel, Casper; Kohnke, Hannes; Maretty, Lasse

    2014-01-01

    Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS...... increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion...

  19. Analogies between respiration and a light-driven proton pump as sources of energy for active glutamate transport in Halobacterium halobium

    Science.gov (United States)

    Belliveau, J. W.; Lanyi, J. K.

    1977-01-01

    Halobacterium halobium is known to contain sheets of bacteriorhodopsin, a pigment which upon exposure to light undergoes cyclic protonation and deprotonation, resulting in net H(+) translocation. In this paper, experiments were conducted to test H. halobium cell envelope vesicles for respiration-induced glutamate uptake. It is shown that glutamate transport in H. halobium cell envelope vesicles can occur as a result of respiration, as well as light acting on bacteriorhodopsin. Glutamate transport can be energized by the oxidation of dimethyl phenylenediamine, and the properties of the transport system are entirely analogous to those observed with illumination as the source of energy. In the case of respiration-dependent glutamate transport, the transportation is also driven by a Na(+) gradient, thereby confirming the existence of a single glutamate transport system independent of the source of energy. The analogy observed is indirect evidence that the cytochrome oxidase of H. halobium functions as a H(+) pump.

  20. Glutamate decarboxylase and. gamma. -aminobutyric acid transaminase activity in brain structures during action of high concentrated sulfide gas on a background of hypo- and hypercalcemia

    Energy Technology Data Exchange (ETDEWEB)

    Kadyrov, G.K.; Aliyev, A.M.

    Activity of the following enzymes was studied on the background of hypo- and hypercalcemia and exposure to high concentration of sulfide gas: glutamate decarboxylase (GDC) and {gamma}-aminobutyric acid transaminase (GABA-T). These enzymes regulate metabolism of GABA. The results showed that a 3.5 hr exposure to sulfide gas at a concentration of 0.3 mg/1 led to significantly increased activity of GDC in cerebral hemispheres, cerebellum and in brain stem. Activity of GABA-T dropped correspondingly. On the background of hypercalcemia induced by im. injection of 10% calcium gluconate (0.6 m1/200 g body weight of experimental rats) the negative effect caused by the exposure to sulfide gas was diminished. Under conditions of hypocalcemia (im. injection of 10 mg/200 g body weight of sodium oxalate), exposure to sulfide gas led to a significantly decreased activity of GDC and GABA-T in the hemispheres and in the brain stem, but in the cerebellum the activity of GDC increased sharply while that of GABA-T decreased correspondingly. 8 refs.

  1. Characterization of glutamate decarboxylase from Lactobacillus plantarum and its C-terminal function for the pH dependence of activity.

    Science.gov (United States)

    Shin, Sun-Mi; Kim, Hana; Joo, Yunhye; Lee, Sang-Jae; Lee, Yong-Jik; Lee, Sang Jun; Lee, Dong-Woo

    2014-12-17

    The gadB gene encoding glutamate decarboxylase (GAD) from Lactobacillus plantarum was cloned and expressed in Escherichia coli. The recombinant enzyme exhibited maximal activity at 40 °C and pH 5.0. The 3D model structure of L. plantarum GAD proposed that its C-terminal region (Ile454-Thr468) may play an important role in the pH dependence of catalysis. Accordingly, C-terminally truncated (Δ3 and Δ11 residues) mutants were generated and their enzyme activities compared with that of the wild-type enzyme at different pH values. Unlike the wild-type GAD, the mutants showed pronounced catalytic activity in a broad pH range of 4.0-8.0, suggesting that the C-terminal region is involved in the pH dependence of GAD activity. Therefore, this study may provide effective target regions for engineering pH dependence of GAD activity, thereby meeting industrial demands for the production of γ-aminobutyrate in a broad range of pH values.

  2. TCA cycle activity in Staphylococcus aureus is essential for iron-regulated synthesis of staphyloferrin A, but not staphyloferrin B: the benefit of a second citrate synthase.

    Science.gov (United States)

    Sheldon, Jessica R; Marolda, Cristina L; Heinrichs, David E

    2014-05-01

    Staphylococcus aureus elaborates two citrate-containing siderophores, staphyloferrin A (SA) and staphyloferrin B (SB), that enhance growth under iron-restriction, yet, paradoxically, expression of the TCA cycle citrate synthase, CitZ, is downregulated during iron starvation. Iron starvation does, however, result in expression of SbnG, recently identified as a novel citrate synthase that is encoded from within the iron-regulated SB biosynthetic locus, suggesting an important role for SbnG in staphyloferrin production. We demonstrate that during growth of S. aureus in iron-restricted media containing glucose, SB is produced but, in contrast, SA production is severely repressed; accordingly, SB-deficient mutants grow poorly in these media. Hypothesizing that reduced TCA cycle activity hinders SA production, we show that a citZ mutant is capable of SB synthesis, but not SA synthesis, providing evidence that SbnG does not generate citrate for incorporation into SA. A citZ sbnG mutant synthesizes neither staphyloferrin, is severely compromised for growth in iron-restricted media, and is significantly more impaired for virulence than either of the single-deletion mutants. We propose that SB is the more important of the two siderophores for S. aureus insofar as it is synthesized, and supports iron-restricted growth, without need of TCA cycle activity. © 2014 John Wiley & Sons Ltd.

  3. Hydrogen-rich saline protects retina against glutamate-induced excitotoxic injury in guinea pig.

    Science.gov (United States)

    Wei, Lihua; Ge, Li; Qin, Shucun; Shi, Yunzhi; Du, Changqing; Du, Hui; Liu, Liwei; Yu, Yang; Sun, Xuejun

    2012-01-01

    Molecular hydrogen (H(2)) is an efficient antioxidant that can selectively reduce hydroxyl radicals and inhibit oxidative stress-induced injuries. We investigated the protective effects and mechanism of hydrogen-rich saline in a glutamate-induced retinal injury model. Retinal excitotoxicity was induced in healthy guinea pigs by injecting glutamate into the vitreous cavity. After 30 min, hydrogen-rich saline was injected into the vitreous cavity, the peritoneal cavity or both. Seven days later, the retinal stress response was evaluated by examining the stress biomarkers, inducible nitric-oxide synthase (iNOS) and glucose-regulated protein 78 (GRP78). The impaired glutamate uptake was assessed by the expression of the excitatory amino acid transporter 1(EAAT-1). The retinal histopathological changes were investigated, focusing on the thicknesses of the entire retina and its inner layer, the number of cells in the retinal ganglion cell layer (GCL) and the ultrastructure of the retinal ganglion cells (RGCs) and glial cells. Compared with the glutamate-induced injury group, the hydrogen-rich saline treatment reduced the loss of cells in the GCL and thinning of the retina and attenuated cellular morphological damage. These improvements were greatest in animals that received H(2) injections into both the vitreous and the peritoneal cavities. The hydrogen-rich saline also inhibited the expression of glial fibrillary acidic protein (GFAP) in Müller cells, CD11b in microglia, and iNOS and GRP78 in glial cells. Moreover, the hydrogen-rich saline increased the expression of EAAT-1. In conclusion, the administration of hydrogen-rich saline through the intravitreal or/and intraperitoneal routes could reduce the retinal excitotoxic injury and promote retinal recovery. This result likely occurs by inhibiting the activation of glial cells, decreasing the production of the iNOS and GRP78 and promoting glutamate clearance. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Detection of the enzymatically-active polyhydroxyalkanoate synthase subunit gene, phaC, in cyanobacteria via colony PCR.

    Science.gov (United States)

    Lane, Courtney E; Benton, Michael G

    2015-12-01

    A colony PCR-based assay was developed to rapidly determine if a cyanobacterium of interest contains the requisite genetic material, the PHA synthase PhaC subunit, to produce polyhydroxyalkanoates (PHAs). The test is both high throughput and robust, owing to an extensive sequence analysis of cyanobacteria PHA synthases. The assay uses a single detection primer set and a single reaction condition across multiple cyanobacteria strains to produce an easily detectable positive result - amplification via PCR as evidenced by a band in electrophoresis. In order to demonstrate the potential of the presence of phaC as an indicator of a cyanobacteria's PHA accumulation capabilities, the ability to produce PHA was assessed for five cyanobacteria with a traditional in vivo PHA granule staining using an oxazine dye. The confirmed in vivo staining results were then compared to the PCR-based assay results and found to be in agreement. The colony PCR assay was capable of successfully detecting the phaC gene in all six of the diverse cyanobacteria tested which possessed the gene, while exhibiting no undesired product formation across the nine total cyanobacteria strains tested. The colony PCR quick prep provides sufficient usable DNA template such that this assay could be readily expanded to assess multiple genes of interest simultaneously. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

    DEFF Research Database (Denmark)

    Aschenbach, W G; Suzuki, Y; Breeden, K

    2001-01-01

    In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase...... that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol....... Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction...

  6. Anti-herpes simplex virus 1 and immunomodulatory activities of a poly-γ- glutamic acid from Bacillus horneckiae strain APA of shallow vent origin.

    Science.gov (United States)

    Marino-Merlo, Francesca; Papaianni, Emanuela; Maugeri, Teresa L; Zammuto, Vincenzo; Spanò, Antonio; Nicolaus, Barbara; Poli, Annarita; Di Donato, Paola; Mosca, Claudia; Mastino, Antonio; Gugliandolo, Concetta

    2017-10-01

    Herpes simplex virus type 1 (HSV-1) is responsible of common and widespread viral infections in humans through the world, and of rare, but extremely severe, clinical syndromes in the central nervous system. The emergence of resistant strains to drugs actually in use encourages the searching for novel antiviral compounds, including those of natural origin. In this study, the recently described poly-γ-glutamic acid (γ-PGA-APA), produced by the marine thermotolerant Bacillus horneckiae strain APA, and previously shown to possess biological and antiviral activity, was evaluated for its anti-HSV-1 and immunomodulatory properties. The biopolymer hindered the HSV-1 infection in the very early phase of virus replication. In addition, the γ-PGA-APA was shown to exert low cytotoxicity and noticeable immunomodulatory activities towards TNF-α and IL-1β gene expression. Moreover, the capacity to positively modulate the transcriptional activity of the cytokine genes was paired with increased level of activation of the transcription factor NF-kB by γ-PGA-APA. Overall, as non-cytotoxic biopolymer able to contribute in the antiviral defense against HSV-1, γ-PGA-APA could lead to the development of novel natural drugs for alternative therapies.

  7. Old Yellow Enzyme from Trypanosoma cruzi Exhibits In Vivo Prostaglandin F2α Synthase Activity and Has a Key Role in Parasite Infection and Drug Susceptibility

    Directory of Open Access Journals (Sweden)

    Florencia Díaz-Viraqué

    2018-03-01

    Full Text Available The discovery that trypanosomatids, unicellular organisms of the order Kinetoplastida, are capable of synthesizing prostaglandins raised questions about the role of these molecules during parasitic infections. Multiple studies indicate that prostaglandins could be related to the infection processes and pathogenesis in trypanosomatids. This work aimed to unveil the role of the prostaglandin F2α synthase TcOYE in the establishment of Trypanosoma cruzi infection, the causative agent of Chagas disease. This chronic disease affects several million people in Latin America causing high morbidity and mortality. Here, we propose a prokaryotic evolutionary origin for TcOYE, and then we used in vitro and in vivo experiments to show that T. cruzi prostaglandin F2α synthase plays an important role in modulating the infection process. TcOYE overexpressing parasites were less able to complete the infective cycle in cell culture infections and increased cardiac tissue parasitic load in infected mice. Additionally, parasites overexpressing the enzyme increased PGF2α synthesis from arachidonic acid. Finally, an increase in benznidazole and nifurtimox susceptibility in TcOYE overexpressing parasites showed its participation in activating the currently anti-chagasic drugs, which added to its observed ability to confer resistance to hydrogen peroxide, highlights the relevance of this enzyme in multiple events including host–parasite interaction.

  8. AJS1669, a novel small-molecule muscle glycogen synthase activator, improves glucose metabolism and reduces body fat mass in mice

    Science.gov (United States)

    Nakano, Kazuhiro; Takeshita, Sen; Kawasaki, Noriko; Miyanaga, Wataru; Okamatsu, Yoriko; Dohi, Mizuki; Nakagawa, Tadakiyo

    2017-01-01

    Impaired glycogen synthesis and turnover are common in insulin resistance and type 2 diabetes. As glycogen synthase (GS) is a key enzyme involved in the synthetic process, it presents a promising therapeutic target for the treatment of type 2 diabetes. In the present study, we identified a novel, potent and orally available GS activator AJS1669 {sodium 2-[[5-[[4-(4,5-difluoro-2-methylsulfanyl-phenyl) phenoxy] methyl]furan-2-carbonyl]-(2-furylmethyl)amino] acetate}. In vitro, we performed a glycogen synthase 1 (GYS1) activation assay for screening GS activators and identified that the activity of AJS1669 was further potentiated in the presence of glucose-6-phosphate (G6P). In vivo, we used ob/ob mice to evaluate the novel anti-diabetic effects of AJS1669 by measuring basal blood glucose levels, glucose tolerance and body fat mass index. Repeated administration of AJS1669 over 4 weeks reduced blood glucose and hemoglobin A1c (HbA1c) levels in ob/ob mice. AJS1669 also improved glucose tolerance in a dose-dependent manner, and decreased body fat mass. The mRNA levels of genes involved in mitochondrial fatty acid oxidation and mitochondrial biogenesis were elevated in skeletal muscle tissue following AJS1669 treatment. Hepatic tissue of treated mice also exhibited elevated expression of genes associated with fatty acid oxidation. In contrast to ob/ob mice, in C57Bl/6 mice AJS1669 administration did not alter body weight or reduce glucose levels. These results demonstrate that pharmacological agents that activate GYS1, the main GS subtype found in skeletal muscle, have potential for use as novel treatments for diabetes that improve glucose metabolism in skeletal muscle. PMID:28290602

  9. Changes in D-aspartic acid and D-glutamic acid levels in the tissues and physiological fluids of mice with various D-aspartate oxidase activities.

    Science.gov (United States)

    Han, Hai; Miyoshi, Yurika; Koga, Reiko; Mita, Masashi; Konno, Ryuichi; Hamase, Kenji

    2015-12-10

    D-Aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are currently paid attention as modulators of neuronal transmission and hormonal secretion. These two D-amino acids are metabolized only by D-aspartate oxidase (DDO) in mammals. Therefore, in order to design and develop new drugs controlling the D-Asp and D-Glu amounts via regulation of the DDO activities, changes in these acidic D-amino acid amounts in various tissues are expected to be clarified in model animals having various DDO activities. In the present study, the amounts of Asp and Glu enantiomers in 6 brain tissues, 11 peripheral tissues and 2 physiological fluids of DDO(+/+), DDO(+/-) and DDO(-/-) mice were determined using a sensitive and selective two-dimensional HPLC system. As a result, the amounts of D-Asp were drastically increased with the decrease in the DDO activity in all the tested tissues and physiological fluids. On the other hand, the amounts of D-Glu were almost the same among the 3 strains of mice. The present results are useful for designing new drug candidates, such as DDO inhibitors, and further studies are expected. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. A novel approach in acidic disinfection through inhibition of acid resistance mechanisms; Maleic acid-mediated inhibition of glutamate decarboxylase activity enhances acid sensitivity of Listeria monocytogenes.

    Science.gov (United States)

    Paudyal, Ranju; Barnes, Ruth H; Karatzas, Kimon Andreas G

    2018-02-01

    Here it is demonstrated a novel approach in disinfection regimes where specific molecular acid resistance systems are inhibited aiming to eliminate microorganisms under acidic conditions. Despite the importance of the Glutamate Decarboxylase (GAD) system for survival of Listeria monocytogenes and other pathogens under acidic conditions, its potential inhibition by specific compounds that could lead to its elimination from foods or food preparation premises has not been studied. The effects of maleic acid on the acid resistance of L. monocytogenes were investigated and found that it has a higher antimicrobial activity under acidic conditions than other organic acids, while this could not be explained by its pKa or Ka values. The effects were found to be more pronounced on strains with higher GAD activity. Maleic acid affected the extracellular GABA levels while it did not affect the intracellular ones. Maleic acid had a major impact mainly on GadD2 activity as also shown in cell lysates. Furthermore, it was demonstrated that maleic acid is able to partly remove biofilms of L. monocytogenes. Maleic acid is able to inhibit the GAD of L. monocytogenes significantly enhancing its sensitivity to acidic conditions and together with its ability to remove biofilms, make a good candidate for disinfection regimes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Introduction to the Glutamate-Glutamine Cycle.

    Science.gov (United States)

    Sonnewald, Ursula; Schousboe, Arne

    2016-01-01

    The term 'glutamate-glutamine cycle' was coined several decades ago based on the observation that using certain 14 C-labeled precursors for studies of brain metabolism the specific radioactivity of glutamine generated from glutamate was higher than that of glutamate, its immediate precursor. This is metabolically impossible unless it is assumed that at least two distinct pools of these amino acids exist. This combined with the finding that the enzyme synthesizing glutamine from glutamate was expressed in astrocytes but not in neurons formed the basis of the notion that a cycle must exist in which glutamate released from neurons is transported into astrocytes, converted to glutamine which is subsequently returned to neurons and converted to glutamate by an enzyme the activity of which is much higher in neurons than in astrocytes. Originally this cycle was supposed to function in a stoichiometric fashion but more recent research has seriously questioned this.This volume of Advances in Neurobiology is intended to provide a detailed discussion of recent developments in research aimed at delineating the functional roles of the cycle taking into account that in order for this system to work there must be a tight coupling between metabolism of glutamate in astrocytes, transfer of glutamine to neurons and de novo synthesis of glutamine in astrocytes. To understand this, knowledge about the activity and regulation of the enzymes and transporters involved in these processes is required and as can be seen from the table of contents these issues will be dealt with in detail in the individual chapters of the book.

  12. Metabolome analysis reveals the effect of carbon catabolite control on the poly(γ-glutamic acid) biosynthesis of Bacillus licheniformis ATCC 9945.

    Science.gov (United States)

    Mitsunaga, Hitoshi; Meissner, Lena; Palmen, Thomas; Bamba, Takeshi; Büchs, Jochen; Fukusaki, Eiichiro

    2016-04-01

    Poly(γ-glutamic acid) (PGA) is a polymer composed of L- and/or D-glutamic acids that is produced by Bacillus sp. Because the polymer has various features as water soluble, edible, non-toxic and so on, it has attracted attention as a candidate for many applications such as foods, cosmetics and so on. However, although it is well known that the intracellular metabolism of Bacillus sp. is mainly regulated by catabolite control, the effect of the catabolite control on the PGA producing Bacillus sp. is largely unknown. This study is the first report of metabolome analysis on the PGA producing Bacillus sp. that reveals the effect of carbon catabolite control on the metabolism of PGA producing Bacillus licheniformis ATCC 9945. Results showed that the cells cultivated in glycerol-containing medium showed higher PGA production than the cells in glucose-containing medium. Furthermore, metabolome analysis revealed that the activators of CcpA and CodY, global regulatory proteins of the intracellular metabolism, accumulated in the cells cultivated in glycerol-containing and glucose-containing medium, respectively, with CodY apparently inhibiting PGA production. Moreover, the cells seemed to produce glutamate from citrate and ammonium using glutamine synthetase/glutamate synthase. Pulsed addition of di-ammonium hydrogen citrate, as suggested by the metabolome result, was able to achieve the highest value so far for PGA production in B. licheniformis. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Deletion of glutamate dehydrogenase 1 (Glud1) in the central nervous system affects glutamate handling without altering synaptic transmission

    DEFF Research Database (Denmark)

    Frigerio, Francesca; Karaca, Melis; De Roo, Mathias

    2012-01-01

    Glutamate dehydrogenase (GDH), encoded by GLUD1, participates in the breakdown and synthesis of glutamate, the main excitatory neurotransmitter. In the CNS, besides its primary signaling function, glutamate is also at the crossroad of metabolic and neurotransmitter pathways. Importance of brain GDH...... was questioned here by generation of CNS-specific GDH-null mice (CnsGlud1(-/-)); which were viable, fertile and without apparent behavioral problems. GDH immunoreactivity as well as enzymatic activity were absent in Cns-Glud1(-/-) brains. Immunohistochemical analyses on brain sections revealed that the pyramidal...... oxidative catabolism of glutamate in astrocytes, showing that GDH is required for Krebs cycle pathway. As revealed by NMR studies, brain glutamate levels remained unchanged, whereas glutamine levels were increased. This pattern was favored by up-regulation of astrocyte-type glutamate and glutamine...

  14. Studies of gene expression and activity of hexokinase, phosphofructokinase and glycogen synthase in human skeletal muscle in states of altered insulin-stimulated glucose metabolism

    DEFF Research Database (Denmark)

    Vestergaard, H

    1999-01-01

    been reported to increase the basal concentration of muscle GS mRNA in NIDDM patients to a level similar to that seen in control subjects although insulin-stimulated glucose disposal rates remain reduced in NIDDM patients. In the insulin resistant states examined so far, basal and insulin-stimulated......When whole body insulin-stimulated glucose disposal rate is measured in man applying the euglycaemic, hyperinsulinaemic clamp technique it has been shown that approximately 75% of glucose is taken up by skeletal muscle. After the initial transport step, glucose is rapidly phosphorylated to glucose...... critical roles in glucose oxidation/glycolysis and glucose storage, respectively. Glucose transporters and glycogen synthase activities are directly and acutely stimulated by insulin whereas the activities of hexokinases and phosphofructokinase may primarily be allosterically regulated. The aim...

  15. Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

    Science.gov (United States)

    Tsuda, Hirohisa; Shiraki, Mari; Inoue, Eri; Saito, Terumi

    2016-08-20

    It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination. Copyright © 2016 Elsevier GmbH. All rights reserved.

  16. The cellulose synthase companion proteins act non-redundantly with CELLULOSE SYNTHASE INTERACTING1/POM2 and CELLULOSE SYNTHASE 6

    OpenAIRE

    Endler, Anne; Schneider, Rene; Kesten, Christopher; Lampugnani, Edwin R.; Persson, Staffan

    2016-01-01

    Cellulose is a cell wall constituent that is essential for plant growth and development, and an important raw material for a range of industrial applications. Cellulose is synthesized at the plasma membrane by massive cellulose synthase (CesA) complexes that track along cortical microtubules in elongating cells of Arabidopsis through the activity of the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1). In a recent study we identified another family of proteins that also are associated with the ...

  17. Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

    Energy Technology Data Exchange (ETDEWEB)

    Ida, Tomoyo [Osaka University, Toyonaka, Osaka 560-0043 (Japan); Suzuki, Hideyuki [Kyoto Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Fukuyama, Keiichi [Osaka University, Toyonaka, Osaka 560-0043 (Japan); Hiratake, Jun [Kyoto University, Uji, Kyoto 611-0011 (Japan); Wada, Kei, E-mail: keiwada@med.miyazaki-u.ac.jp [University of Miyazaki, Miyazaki 889-1692 (Japan); Osaka University, Toyonaka, Osaka 560-0043 (Japan)

    2014-02-01

    The binding modes of acivicin, a classical and an electrophilic active-site-directed glutamate analogue, to bacterial γ-glutamyltranspeptidases were found to be diverse. γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalently through its C3 atom with sp{sup 2} hybridization to Thr403 O{sup γ}, the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs.

  18. Prolactin-induced neuroprotection against glutamate excitotoxicity is mediated by the reduction of [Ca2+]i overload and NF-κB activation

    Science.gov (United States)

    Rivero-Segura, Nadia A.; Flores-Soto, Edgar; García de la Cadena, Selene; Coronado-Mares, Isabel; Gomez-Verjan, Juan C.; Ferreira, Diana G.; Cabrera-Reyes, Erika Alejandra; Lopes, Luísa V.; Massieu, Lourdes

    2017-01-01

    Prolactin (PRL) is a peptidic hormone that displays pleiotropic functions in the organism including different actions in the brain. PRL exerts a neuroprotective effect against excitotoxicity produced by glutamate (Glu) or kainic acid in both in vitro and in vivo models. It is well known that Glu excitotoxicity causes cell death through apoptotic or necrotic pathways due to intracellular calcium ([Ca2+] i) overload. Therefore, the aim of the present study was to assess the molecular mechanisms by which PRL maintains cellular viability of primary cultures of rat hippocampal neurons exposed to Glu excitotoxicity. We determined cell viability by monitoring mitochondrial activity and using fluorescent markers for viable and dead cells. The intracellular calcium level was determined by a fluorometric assay and proteins involved in the apoptotic pathway were determined by immunoblot. Our results demonstrated that PRL afforded neuroprotection against Glu excitotoxicity, as evidenced by a decrease in propidium iodide staining and by the decrease of the LDH activity. In addition, the MTT assay shows that PRL maintains normal mitochondrial activity even in neurons exposed to Glu. Furthermore, the Glu-induced intracellular [Ca2+]i overload was attenuated by PRL. These data correlate with the reduction found in the level of active caspase-3 and the pro-apoptotic ratio (Bax/Bcl-2). Concomitantly, PRL elicited the nuclear translocation of the transcriptional factor NF-κB, which was detected by immunofluorescence and confocal microscopy. To our knowledge, this is the first report demonstrating that PRL prevents Glu excitotoxicity by a mechanism involving the restoration of the intracellular calcium homeostasis and mitochondrial activity, as well as an anti-apoptotic action possibly mediated by the activity of NF-κB. Overall, the current results suggest that PRL could be of potential therapeutic advantage in the treatment of neurodegenerative diseases. PMID:28475602

  19. Altered cytochrome P450 activities and expression levels in the liver and intestines of the monosodium glutamate-induced mouse model of human obesity.

    Science.gov (United States)

    Tomankova, Veronika; Liskova, Barbora; Skalova, Lenka; Bartikova, Hana; Bousova, Iva; Jourova, Lenka; Anzenbacher, Pavel; Ulrichova, Jitka; Anzenbacherova, Eva

    2015-07-15

    Cytochromes P450 (CYPs) are enzymes present from bacteria to man involved in metabolism of endogenous and exogenous compounds incl. drugs. Our objective was to assess whether obesity leads to changes in activities and expression of CYPs in the mouse liver, small intestine and colon. An obese mouse model with repeated injection of monosodium glutamate (MSG) to newborns was used. Controls were treated with saline. All mice were sacrificed at 8 months. In the liver and intestines, levels of CYP mRNA and proteins were analyzed using RT-PCR and Western blotting. Activities of CYP enzymes were measured with specific substrates of human orthologous forms. At the end of the experiment, body weight, plasma insulin and leptin levels as well as the specific content of hepatic CYP enzymes were increased in obese mice. Among CYP enzymes, hepatic CYP2A5 activity, protein and mRNA expression increased most significantly in obese animals. Higher activities and protein levels of hepatic CYP2E1 and 3A in the obese mice were also found. No or a weak effect on CYPs 2C and 2D was observed. In the small intestine and colon, no changes of CYP enzymes were detected except for increased expression of CYP2E1 and decreased expression of CYP3A mRNAs in the colon of the obese mice. Results of our study suggest that the specific content and activities of some liver CYP enzymes (especially CYP2A5) can be increased in obese mice. Higher activity of CYP2A5 (CYP2A6 human ortholog) could lead to altered metabolism of drug substrates of this enzyme (valproic acid, nicotine, methoxyflurane). Copyright © 2015 Elsevier Inc. All rights reserved.

  20. probing the cob(ii)alamin conductor hypothesis with glutamate ...

    African Journals Online (AJOL)

    dell

    Glutamate mutase activity was also demonstrated upon incubation of GlmS and E with 3',5'- ... overproduced in E.coli (Huhta et al. 2001,. Huhta et ..... Biochemistry. 37: 9704-9715. Buckel W 2001 Unusual enzymes involved in five pathways of glutamate fermentation. Appl. Microbiol. Biotechnol. 57: 263-273. Buckel W and ...

  1. Some Properties of Glutamate Dehydrogenase from the Marine Red ...

    African Journals Online (AJOL)

    Keywords: ammonia assimilation, glutamate dehydrogenase, GDH, Gracilaria sordida, red alga, enzyme activity. Glutamate dehydrogenases (GDH, EC ... Anabolic functions could be assimilation of ammonia released during photorespiration and synthesis of N-rich transport compounds. Western Indian Ocean Journal of ...

  2. Piperine Inhibits the Activities of Platelet Cytosolic Phospholipase A2 and Thromboxane A2 Synthase without Affecting Cyclooxygenase-1 Activity: Different Mechanisms of Action Are Involved in the Inhibition of Platelet Aggregation and Macrophage Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Dong Ju Son

    2014-08-01

    Full Text Available PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum and long pepper (Piper longum, was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2, COX-1, COX-2, and thromboxane A2 (TXA2 synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PGE2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms.

  3. Glutamate receptor ligands

    DEFF Research Database (Denmark)

    Guldbrandt, Mette; Johansen, Tommy N; Frydenvang, Karla Andrea

    2002-01-01

    Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA...

  4. Monosodium Glutamate Toxicity

    African Journals Online (AJOL)

    Dr Olaleye

    The brain is reportedly sensitive to monosodium glutamate (MSG) toxicity via oxidative stress. Sida acuta leaf ethanolic .... wherein the right hemisphere, was preserved for histology and fixed in 10% ... Biochemical Assays: The left hemisphere of the brain samples was ...... development in male and female rats. Exp Physiol.

  5. The possible interaction of dopamine system in nucleus accumbens shell and glutamate system of prelimbic region on locomotor activity in rat

    Directory of Open Access Journals (Sweden)

    Hatam Ahmadi

    2013-06-01

    Full Text Available Background: Nucleus accumbens (NAc and prefrontal cortex (PFC dopaminergic and glutamatergic systems are involved in regulating of locomotor activity behaviors. This study has investigated the interaction of NAc shell dopaminergic system and prelimbic glutamatergic systems in regulating locomotor activity and related parameters. Methods: The aim of this study was the effect the drugs injection interaction in the brain of male Wistar rats on locomotor activity and related parameters, in the order of this purpose, open field apparatus that automatically recorded locomotor activity was employed. Unilateral intra-cerebral injection of drugs was done. Results: Unilateral intra-prelimbic injection of D-AP7 (N-methyl-D-aspartic acid= NMDA receptor antagonist; 0.25, 0.5 and 1μg/μl did not alter locomotor activity behaviors. However, infusion of NMDA (0.9μg/μl in this region increased locomotor activity (P<0.01, whereas decreased rearing (P<0.01 and grooming (P<0.01 which was blocked by D-AP7 (0.25μg/μl (P<0.01. Moreover, unilateral infusion of SCH23390 (dopamine D1 receptor antagonist; 0.25, 0.5 and 1μg/μl into the left NAc shell did not alter locomotor activity. However, injection of SKF38393 (dopamine D1 receptor agonist; 4μg/μl into the left NAc shell increased locomotor activity (P<0.05 which was blocked by SCH23390 (0.25μg/μl (P<0.01. Furthermore, the subthreshold dose infusion of SCH23390 (0.25μg/μl into the left NAc shell reduced the effect of intra- prelimbic NMDA on locomotor activity (P<0.01. In addition, intra-NAc shell administration of the subthreshold dose of SKF38393 (1μg/μl potentiated the middle dose (P<0.05, whereas decreased the higher dose of intra-left prelimbic NMDA response (P<0.05 on locomotor activity. Conclusion: The results suggested a modulatory effect of the NAc shell dopaminergic system on increased locomotor activity by activating glutamate system in prelimbic.

  6. Chronic SO2 inhalation above environmental standard impairs neuronal behavior and represses glutamate receptor gene expression and memory-related kinase activation via neuroinflammation in rats.

    Science.gov (United States)

    Yao, Gaoyi; Yue, Huifeng; Yun, Yang; Sang, Nan

    2015-02-01

    Sulfur dioxide (SO2), as a ubiquitous air pollutant implicated in the genesis of pulmonary disease, is now being considered to be involved in neurotoxicity and increased risk for hospitalization of brain disorders. However, comparatively little is known about the impact of chronically SO2 inhalation on neuronal function. In the present study, by exposing male Wistar rats to SO2 at 3.50 and 7.00 mg/m(3) (approximately 1225 and 2450 ppb, 4.08-8.16 (24h average concentration) times higher than the EPA standard for environmental air concentrations) or filtered air for 90 days, we investigated the impact of chronic SO2 inhalation on performance in Morris water maze, and probed the accompanying neurobiological effects, including activity-regulated cytoskeletal associated gene (Arc) and glutamate receptor gene expression, memory-related kinase level and inflammatory cytokine release in the hippocampus. Here, we found that SO2 exposure reduced the number of target zone crossings and time spent in the target quadrant during the test session in the spatial memory retention of the Morris water maze. Following the neuro-functional abnormality, we detected that SO2 inhalation reduced the expression of Arc and glutamate receptor subunits (GluR1, GluR2, NR1, NR2A, and NR2B) with a concentration-dependent property in comparison to controls. Additionally, the expression of memory kinases was attenuated statistically in the animals receiving the higher concentration, including protein kinase A (PKA), protein kinase C (PKC) and calcium/calmodulin-dependent protein kinaseIIα (CaMKIIα). And the inflammatory cytokine release was increased in rats exposed to SO2. Taken together, our results suggest that long-term exposure to SO2 air pollution at concentrations above the environmental standard in rats impaired spatial learning and memory, and indicate a close link between the neurobiological changes highlighted in the brain and the behavioral disturbances. Copyright © 2014 Elsevier Inc

  7. Involvement of Salicylic Acid on Antioxidant and Anticancer Properties, Anthocyanin Production and Chalcone Synthase Activity in Ginger (Zingiber officinale Roscoe Varieties

    Directory of Open Access Journals (Sweden)

    Ehsan Karimi

    2012-11-01

    Full Text Available The effect of foliar application of salicylic acid (SA at different concentrations (10−3 M and 10−5 M was investigated on the production of secondary metabolites (flavonoids, chalcone synthase (CHS activity, antioxidant activity and anticancer activity (against breast cancer cell lines MCF-7 and MDA-MB-231 in two varieties of Malaysian ginger, namely Halia Bentong and Halia Bara. The results of high performance liquid chromatography (HPLC analysis showed that application of SA induced the synthesis of anthocyanin and fisetin in both varieties. Anthocyanin and fisetin were not detected in the control plants. Accordingly, the concentrations of some flavonoids (rutin and apigenin decreased significantly in plants treated with different concentrations of SA. The present study showed that SA enhanced the chalcone synthase (CHS enzyme activity (involving flavonoid synthesis and recorded the highest activity value of 5.77 nkat /mg protein in Halia Bara with the 10−5 M SA treatment. As the SA concentration was decreased from 10−3 M to 10−5 M, the free radical scavenging power (FRAP increased about 23% in Halia Bentong and 10.6% in Halia Bara. At a concentration of 350 μg mL−1, the DPPH antioxidant activity recorded the highest value of 58.30%–72.90% with the 10−5 M SA treatment followed by the 10−3 M SA (52.14%–63.66% treatment. The lowest value was recorded in the untreated control plants (42.5%–46.7%. These results indicate that SA can act not only as an inducer but also as an inhibitor of secondary metabolites. Meanwhile, the highest anticancer activity against MCF-7 and MDA-MB-231 cell lines was observed for H. Bara extracts treated with 10−5 M SA with values of 61.53 and 59.88%, respectively. The results suggest that the high anticancer activity in these varieties may be related to the high concentration of potent anticancer components including fisetin and anthocyanin. The results thus indicate that the synthesis of

  8. Chronic glutamate toxicity in neurodegenerative diseases-what is the evidence?

    Directory of Open Access Journals (Sweden)

    Pamela eMaher

    2015-12-01

    Full Text Available Together with aspartate, glutamate is the major excitatory neurotransmitter in the brain. Glutamate binds and activates both ligand-gated ion channels (ionotropic glutamate receptors and a class of G-protein coupled receptors (metabotropic glutamate receptors. Although the intracellular glutamate concentration in the brain is in the millimolar range, the extracellular glutamate concentration is kept in the low micromolar range by the action of excitatory amino acid transporters that import glutamate and aspartate into astrocytes and neurons. Excess extracellular glutamate may lead to excitotoxicity in vitro and in vivo in acute insults like ischemic stroke via the overactivation of ionotropic glutamate receptors. In addition, chronic excitotoxicity has been hypothesized to play a role in numerous neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer’s disease and Huntington’s disease. Based on this hypothesis, a good deal of effort has been devoted to develop and test drugs that either inhibit glutamate receptors or decrease extracellular glutamate. In this review, we provide an overview of the different pathways that are thought to lead to an over-activation of the glutamatergic system and glutamate toxicity in neurodegeneration. In addition, we summarize the available experimental evidence for glutamate toxicity in animal models of neurodegenerative diseases.

  9. The synthesis and characterization of poly(γ-glutamic acid)-coated magnetite nanoparticles and their effects on antibacterial activity and cytotoxicity

    International Nuclear Information System (INIS)

    Stephen Inbaraj, B; Kao, T H; Tsai, T Y; Chiu, C P; Kumar, R; Chen, B H

    2011-01-01

    Magnetite nanoparticles (MNPs) modified with sodium and calcium salts of poly(γ-glutamic acid) (NaPGA and CaPGA) were synthesized by the coprecipitation method, followed by characterization and evaluation of their antibacterial and cytotoxic effects. Superparamagnetic MNPs are particularly attractive for magnetic driving as well as bacterial biofilm and cell targeting in in vivo applications. Characterization of synthesized MNPs by the Fourier transform infrared spectra and magnetization curves confirmed the PGA coating on MNPs. The mean diameter of NaPGA- and CaPGA-coated MNPs as determined by transmission electron microscopy was 11.8 and 14 nm, respectively, while the x-ray diffraction pattern revealed the as-synthesized MNPs to be pure magnetite. Based on agar dilution assay, both NaPGA- and CaPGA-coated MNPs showed a lower minimum inhibitory concentration in Salmonella enteritidis SE 01 than the commercial antibiotics linezolid and cefaclor, but the former was effective against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 10832, whereas the latter was effective against Escherichia coli O157:H7 TWC 01. An in vitro cytotoxicity study in human skin fibroblast cells as measured by MTT assay implied the as-synthesized MNPs to be nontoxic. This outcome demonstrated that both γ-PGA-modified MNPs are cytocompatible and possess antibacterial activity in vitro, and thereby should be useful in in vivo studies for biomedical applications.

  10. Determination of minor, trace and toxic elements in chewing tobacco products by instrumental neutron activation analysis and identification of glutamic acid

    International Nuclear Information System (INIS)

    Garg, A.N.; Paul Choudhury, R.; Acharya, R.; Reddy, A.V.R.

    2012-01-01

    Tobacco smoking/chewing has been a cause of concern because of it being related with oral cancer. It causes stimulation and ill physiological effects. Ten different brands of spit tobacco, eight gutkaas and five paan masalas have been analyzed for seven minor (Al, Na, K, Ca, Cl, Mg, and P) and 17 trace (As, Ba, Br, Co, Cr, Cs, Cu, Eu, Fe, Hg, La, Mn, Rb, Sb, Sc, Th, and Zn) elements by instrumental neutron activation analysis. Also Ni and Pb were determined by atomic absorption spectrophotometry. Concentration of Cd was below detection limit ( -1 ) in the tobacco samples. Mg, generally added as MgCO 3 to prevent caking, is present as minor constituent in spit tobacco and gutkaas but is below detection limit ( -1 ) in paan masalas. Most elemental concentrations vary in a wide range depending on the nature of chewing tobacco. Spit tobacco has been found to be more enriched in essential elements (Ca, K, Na, P, Mn, and Rb), whereas gutkaas contain higher concentrations of Fe, Cr, Cu, and Zn. Paan masalas contain lower contents of other elements but higher content of Hg. Gutkaas also contain higher amounts of As and Pb. Further glutamic acid has been separated from tobacco leaves and characterized as it might bind with some elements. (author)

  11. Structure-Activity Relationship Study of Ionotropic Glutamate Receptor Antagonist (2S,3R)-3-(3-Carboxyphenyl)pyrrolidine-2-carboxylic Acid

    DEFF Research Database (Denmark)

    Krogsgaard-Larsen, Niels; Storgaard, Morten; Møller, Charlotte

    2015-01-01

    Herein we describe the first structure-activity relationship study of the broad-range iGluR antagonist (2S,3R)-3-(3-carboxyphenyl)pyrrolidine-2-carboxylic acid (1) by exploring the pharmacological effect of substituents in the 4, 4', or 5' positions and the bioisosteric substitution of the distal...... carboxylic acid for a phosphonic acid moiety. Of particular interest is a hydroxyl group in the 4' position 2a which induced a preference in binding affinity for homomeric GluK3 over GluK1 (Ki = 0.87 and 4.8 μM, respectively). Two X-ray structures of ligand binding domains were obtained: 2e in GluA2-LBD...... and 2f in GluK1-LBD, both at 1.9 Å resolution. Compound 2e induces a D1-D2 domain opening in GluA2-LBD of 17.3-18.8° and 2f a domain opening in GluK1-LBD of 17.0-17.5° relative to the structures with glutamate. The pyrrolidine-2-carboxylate moiety of 2e and 2f shows a similar binding mode as kainate...

  12. The synthesis and characterization of poly({gamma}-glutamic acid)-coated magnetite nanoparticles and their effects on antibacterial activity and cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Stephen Inbaraj, B; Kao, T H; Tsai, T Y; Chiu, C P; Kumar, R; Chen, B H, E-mail: 002622@mail.fju.edu.tw [Department of Food Science, Fu Jen University, Taipei 242, Taiwan (China)

    2011-02-18

    Magnetite nanoparticles (MNPs) modified with sodium and calcium salts of poly({gamma}-glutamic acid) (NaPGA and CaPGA) were synthesized by the coprecipitation method, followed by characterization and evaluation of their antibacterial and cytotoxic effects. Superparamagnetic MNPs are particularly attractive for magnetic driving as well as bacterial biofilm and cell targeting in in vivo applications. Characterization of synthesized MNPs by the Fourier transform infrared spectra and magnetization curves confirmed the PGA coating on MNPs. The mean diameter of NaPGA- and CaPGA-coated MNPs as determined by transmission electron microscopy was 11.8 and 14 nm, respectively, while the x-ray diffraction pattern revealed the as-synthesized MNPs to be pure magnetite. Based on agar dilution assay, both NaPGA- and CaPGA-coated MNPs showed a lower minimum inhibitory concentration in Salmonella enteritidis SE 01 than the commercial antibiotics linezolid and cefaclor, but the former was effective against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 10832, whereas the latter was effective against Escherichia coli O157:H7 TWC 01. An in vitro cytotoxicity study in human skin fibroblast cells as measured by MTT assay implied the as-synthesized MNPs to be nontoxic. This outcome demonstrated that both {gamma}-PGA-modified MNPs are cytocompatible and possess antibacterial activity in vitro, and thereby should be useful in in vivo studies for biomedical applications.

  13. Transplantation of N-Acetyl Aspartyl-Glutamate Synthetase-Activated Neural Stem Cells after Experimental Traumatic Brain Injury Significantly Improves Neurological Recovery

    Directory of Open Access Journals (Sweden)

    Mingfeng Li

    2013-12-01

    Full Text Available Background/Aims: Neural stem cells (NSCs hold considerable potential as a therapeutic tool for repair of the damaged nervous system. In the current study, we examined whether transplanted N-acetyl aspartyl-glutamate synthetase (NAAGS-activated NSCs (NAAGS/NSCs further improve neurological recovery following traumatic brain injury (TBI in Sprague-Dawley rats. Methods: Animals received TBI and stereotactic injection of NSCs, NAAGS/NSCs or phosphate buffered saline without cells (control into the injured cortex. NAAGS protein expression was detected through western blot analysis. Dialysate NAAG levels were analyzed with radioimmunoassay. Cell apoptosis was detected via TUNEL staining. The expression levels of specific pro-inflammatory cytokines were detected with enzyme-linked immunosorbent assay. Results: Groups with transplanted NSCs and NAAGS/NSCs displayed significant recovery of the motor behavior, compared to the control group. At 14 and 21 days post-transplantation, the motor behavior in NAAGS/NSC group was significantly improved than that in NSC group (pConclusion: Our results collectively demonstrate that NAAGS/NSCs provide a more powerful autoplastic therapy for the injured nervous system.

  14. Glutamate receptor antibodies in neurological diseases: anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies, anti-NMDA-NR2A/B antibodies, anti-mGluR1 antibodies or anti-mGluR5 antibodies are present in subpopulations of patients with either: epilepsy, encephalitis, cerebellar ataxia, systemic lupus erythematosus (SLE) and neuropsychiatric SLE, Sjogren's syndrome, schizophrenia, mania or stroke. These autoimmune anti-glutamate receptor antibodies can bind neurons in few brain regions, activate glutamate receptors, decrease glutamate receptor's expression, impair glutamate-induced signaling and function, activate blood brain barrier endothelial cells, kill neurons, damage the brain, induce behavioral/psychiatric/cognitive abnormalities and ataxia in animal models, and can be removed or silenced in some patients by immunotherapy.

    Science.gov (United States)

    Levite, Mia

    2014-08-01

    Glutamate is the major excitatory neurotransmitter of the Central Nervous System (CNS), and it is crucially needed for numerous key neuronal functions. Yet, excess glutamate causes massive neuronal death and brain damage by excitotoxicity--detrimental over activation of glutamate receptors. Glutamate-mediated excitotoxicity is the main pathological process taking place in many types of acute and chronic CNS diseases and injuries. In recent years, it became clear that not only excess glutamate can cause massive brain damage, but that several types of anti-glutamate receptor antibodies, that are present in the serum and CSF of subpopulations of patients with a kaleidoscope of human neurological diseases, can undoubtedly do so too, by inducing several very potent pathological effects in the CNS. Collectively, the family of anti-glutamate receptor autoimmune antibodies seem to be the most widespread, potent, dangerous and interesting anti-brain autoimmune antibodies discovered up to now. This impression stems from taking together the presence of various types of anti-glutamate receptor antibodies in a kaleidoscope of human neurological and autoimmune diseases, their high levels in the CNS due to intrathecal production, their multiple pathological effects in the brain, and the unique and diverse mechanisms of action by which they can affect glutamate receptors, signaling and effects, and subsequently impair neuronal signaling and induce brain damage. The two main families of autoimmune anti-glutamate receptor antibodies that were already found in patients with neurological and/or autoimmune diseases, and that were already shown to be detrimental to the CNS, include the antibodies directed against ionotorpic glutamate receptors: the anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies and anti-NMDA-NR2 antibodies, and the antibodies directed against Metabotropic glutamate receptors: the anti-mGluR1 antibodies and the anti-mGluR5 antibodies. Each type of these anti-glutamate

  15. Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.

    LENUS (Irish Health Repository)

    Krijt, Jakub

    2011-02-01

    Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS\\/MS. The median enzyme activity in control plasma samples was 404 nmol\\/h\\/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol\\/ho\\/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol\\/hour\\/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5\\'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS\\/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

  16. Amperometric L-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Bo [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China); Zhang, Shu [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100 (China); Lang, Qiaolin [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); Song, Jianxia; Han, Lihui [Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100 (China); Liu, Aihua, E-mail: liuah@qibebt.ac.cn [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China)

    2015-07-16

    Highlights: • E. coli surface-dispalyed Gldh exhibiting excellent enzyme activity and stability. • Sensitive amperometric biosensor for glutamate using Gldh-bacteria and MWNTs. • The glutamate biosensor exhibited high specificity and stability. - Abstract: A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP{sup +}-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP{sup +} involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current–time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM–1 mM and 2–10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N = 3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection.

  17. Feedback-induced glutamate spillover enhances negative feedback from horizontal cells to cones

    NARCIS (Netherlands)

    Vroman, Rozan; Kamermans, M.

    2015-01-01

    KEY POINTS: In the retina, horizontal cells feed back negatively to cone photoreceptors. Glutamate released from cones can spill over to neighbouring cones. Here we show that cone glutamate release induced by negative feedback can also spill over to neighbouring cones. This glutamate activates the

  18. The Effect of Nigella Sativa Extract on Alpha-ketoglutarate Activity and Histopathologic Changes on Rat Liver Induced by Monosodium Glutamate

    Directory of Open Access Journals (Sweden)

    Ala Sh Emhemed Eshami

    2015-09-01

    Full Text Available Monosodium glutamate (MSG is a commonly used food additive and found in most soups, fish, and processed meat. The use of MSG in food is growing. However, the fear of consuming MSG has increased in the last few years due to the adverse reactions and toxicity in the liver. Nigella sativa (NS is used as traditional medicine for the treatment of many diseases. It has been extensively investigated in recent years due to its notable pharmacological properties such as inhibit oxidative stress. The present study was undertaken to investigate the effect of different doses of Nigella Sativa on alpha KGDH activity and liver histology of MSG-induced rats. The animals (n=30 were grouped into A (control, B (treated with MSG 1g/kg.bw , C (treated with MSG 1g/kg.bw and NS 0.1 g/kg.bw, D (treated with MSG 1g/kg.bw and NS 0.2 g/kg.bw, E (treated with MSG 1g/kg.bw and NS 0.4 g/kg.bw and F (given a daily NS extract 0.2 g/kg.bw. Alpha KGDH activity was investigated using ELISA method and liver histopathology by light microscope. The MSG treatment increased Alpha KGDH activity and disturbed liver architecture, hemorrhage in the central veins, areas of necrosis, vacuolation and increased inflammatory cells infiltration. The condition was normalized by treatment NS on dose 0.2 and 0.4 g/kg.bw. The findings showed that the administration of MSG increases alpha KGDH and induces damage in liver tissue. Nigella sativa extract can reduce alpha KGDH and prevent liver damage caused by MSG.

  19. Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-Parkin pathway and modulate cellular drug response.

    Science.gov (United States)

    Zhang, Conggang; Liu, Zeyu; Bunker, Eric; Ramirez, Adrian; Lee, Schuyler; Peng, Yinghua; Tan, Aik-Choon; Eckhardt, S Gail; Chapnick, Douglas A; Liu, Xuedong

    2017-09-08

    Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m -chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Mechanical unloading of the failing human heart fails to activate the protein kinase B/Akt/glycogen synthase kinase-3beta survival pathway.

    Science.gov (United States)

    Razeghi, Peter; Bruckner, Brian A; Sharma, Saumya; Youker, Keith A; Frazier, O H; Taegtmeyer, Heinrich

    2003-01-01

    Left ventricular assist device (LVAD) support of the failing human heart improves myocyte function and increases cell survival. One potential mechanism underlying this phenomenon is activation of the protein kinase B (PKB)/Akt/glycogen synthase kinase-3beta (GSK-3beta) survival pathway. Left ventricular tissue was obtained both at the time of implantation and explantation of the LVAD (n = 11). Six patients were diagnosed with idiopathic dilated cardiomyopathy, 4 patients with ischemic cardiomyopathy and 1 patient with peripartum cardiomyopathy. The mean duration of LVAD support was 205 +/- 35 days. Myocyte diameter and phosphorylation of ERK were used as indices for reverse remodeling. Transcript levels of genes required for the activation of PKB/Akt (insulin-like growth factor-1, insulin receptor substrate-1) were measured by quantitative RT-PCR. In addition, we measured the relative activity of PKB/Akt and GSK-3beta, and assayed for molecular and histological indices of PKB/Akt activation (cyclooxygenase mRNA levels and glycogen levels). Myocyte diameter and phosphorylation of ERK decreased with LVAD support. In contrast, none of the components of the PKB/Akt/GSK-3beta pathway changed significantly with mechanical unloading. The PKB/Akt/GSK-3beta pathway is not activated during LVAD support. Other signaling pathways must be responsible for the improvement of cellular function and cell survival during LVAD support. Copyright 2003 S. Karger AG, Basel

  1. Nitric oxide synthase-I containing cortical interneurons co-express antioxidative enzymes and anti-apoptotic Bcl-2 following focal ischemia: evidence for direct and indirect mechanisms towards their resistance to neuropathology.

    Science.gov (United States)

    Bidmon, H J; Emde, B; Kowalski, T; Schmitt, M; Mayer, B; Kato, K; Asayama, K; Witte, O W; Zilles, K

    2001-09-01

    Neuronal nitric oxide-I is constitutively expressed in approximately 2% of cortical interneurons and is co-localized with gamma-amino butric acid, somatostatin or neuropeptide Y. These interneurons additionally express high amounts of glutamate receptors which mediate the glutamate-induced hyperexcitation following cerebral injury, under these conditions nitric oxide production increases contributing to a potentiation of oxidative stress. However, perilesional nitric oxide synthase-I containing neurons are known to be resistant to ischemic and excitotoxic injury. In vitro studies show that nitrosonium and nitroxyl ions inactivate N-methyl-D-aspartate receptors, resulting in neuroprotection. The question remains of how these cells are protected against their own high intracellular nitric oxide production after activation. In this study, we investigated immunocytochemically nitric oxide synthase-I containing cortical neurons in rats after unilateral, cortical photothrombosis. In this model of focal ischemia, perilesional, constitutively nitric oxide synthase-I containing neurons survived and co-expressed antioxidative enzymes, such as manganese- and copper-zinc-dependent superoxide dismutases, heme oxygenase-2 and cytosolic glutathione peroxidase. This enhanced antioxidant expression was accompanied by a strong perinuclear presence of the antiapoptotic Bcl-2 protein. No colocalization was detectable with upregulated heme oxygenase-1 in glia and the superoxide and prostaglandin G(2)-producing cyclooxygenase-2 in neurons. These results suggest that nitric oxide synthase-I containing interneurons are protected against intracellular oxidative damage and apoptosis by Bcl-2 and several potent antioxidative enzymes. Since nitric oxide synthase-I positive neurons do not express superoxide-producing enzymes such as cyclooxygenase-1, xanthine oxidase and cyclooxygenase-2 in response to injury, this may additionally contribute to their resistance by reducing their internal

  2. Effects of lodoxamide (LOD), disodium cromoglycate (DSCG) and N-acetyl-aspartyl-glutamate sodium salt (NAAGA) on ocular active anaphylaxis.

    Science.gov (United States)

    Goldschmidt, P; Luyckx, J

    1996-04-01

    LOD, DSCG and NAAGA eye-drops were evaluated on experimentally-induced ocular active anaphylaxis in guinea pigs. Twelve animals per group were sensitized with egg albumin i.p. and challenged on the surface of the eye 14 days later. Two days before challenge, animals were treated with LOD, DSCG or NAAGA 4 times a day. Permeability indexes were calculated after intracardiac injection of Evans Blue. No effect on ocular active anaphylaxis was found with LOD nor with DSCG. NAAGA was able to significantly reduce blood-eye permeability indexes.

  3. The cAMP effectors PKA and Epac activate endothelial NO synthase through PI3K/Akt pathway in human endothelial cells.

    Science.gov (United States)

    García-Morales, Verónica; Luaces-Regueira, María; Campos-Toimil, Manuel

    2017-12-01

    3',5'-Cyclic adenosine monophosphate (cAMP) exerts an endothelium-dependent vasorelaxant action by stimulating endothelial NO synthase (eNOS) activity, and the subsequent NO release, through cAMP protein kinase (PKA) and exchange protein directly activated by cAMP (Epac) activation in endothelial cells. Here, we have investigated the mechanism by which the cAMP-Epac/PKA pathway activates eNOS. cAMP-elevating agents (forskolin and dibutyryl-cAMP) and the joint activation of PKA (6-Bnz-cAMP) and Epac (8-pCPT-2'-O-Me-cAMP) increased cytoplasmic Ca 2+ concentration ([Ca 2+ ] c ) in ≤30% of fura-2-loaded isolated human umbilical vein endothelial cells (HUVEC). However, these drugs did not modify [Ca 2+ ] c in fluo-4-loaded HUVEC monolayers. In DAF-2-loaded HUVEC monolayers, forskolin, PKA and Epac activators significantly increased NO release, and the forskolin effect was reduced by inhibition of PKA (Rp-cAMPs), Epac (ESI-09), eNOS (L-NAME) or phosphoinositide 3-kinase (PI3K; LY-294,002). On the other hand, inhibition of CaMKII (KN-93), AMPK (Compound C), or total absence of Ca 2+ , was without effect. In Western blot experiments, Serine 1177 phosphorylated-eNOS was significantly increased in HUVEC by cAMP-elevating agents and PKA or Epac activators. In isolated rat aortic rings LY-294,002, but not KN-93 or Compound C, significantly reduced the vasorelaxant effects of forskolin in the presence of endothelium. Our results suggest that Epac and PKA activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway, and independently of AMPK or CaMKII activation or [Ca 2+ ] c increase. This action explains, in part, the endothelium-dependent vasorelaxant effect of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Hopperton, Kathryn E., E-mail: kathryn.hopperton@mail.utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Duncan, Robin E., E-mail: robin.duncan@uwaterloo.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Bazinet, Richard P., E-mail: richard.bazinet@utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Archer, Michael C., E-mail: m.archer@utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada)

    2014-01-15

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from {sup 14}C-labeled acetate to those supplied exogenously as {sup 14}C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. - Highlights: • Fatty acid synthase (FASN) is over-expressed in cancer but its function is unknown. • We compare

  5. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

    International Nuclear Information System (INIS)

    Hopperton, Kathryn E.; Duncan, Robin E.; Bazinet, Richard P.; Archer, Michael C.

    2014-01-01

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from 14 C-labeled acetate to those supplied exogenously as 14 C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. - Highlights: • Fatty acid synthase (FASN) is over-expressed in cancer but its function is unknown. • We compare utilization of

  6. Cyclic GMP-AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria.

    Science.gov (United States)

    Gallego-Marin, Carolina; Schrum, Jacob E; Andrade, Warrison A; Shaffer, Scott A; Giraldo, Lina F; Lasso, Alvaro M; Kurt-Jones, Evelyn A; Fitzgerald, Katherine A; Golenbock, Douglas T

    2018-01-15

    Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human monocytes binds and activates cyclic GMP-AMP synthase (cGAS). Activated cGAS synthesizes 2'3'-cGAMP, which we subsequently can detect using liquid chromatography-tandem mass spectrometry. 2'3'-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-β induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN in response to malaria parasites. Copyright © 2018 by The American Association of Immunologists, Inc.

  7. Depolarization by K+ and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: vesicular versus non-vesicular release of GABA

    DEFF Research Database (Denmark)

    Belhage, B; Hansen, Gert Helge; Schousboe, A

    1993-01-01

    differences in the mode of action of the two depolarizing stimuli were reflected in the properties of the increase in [Ca++]i elicited by 55 mM K+ and 100 microM glutamate, respectively. The K(+)-induced increase in [Ca++]i was reduced by both verapamil and Ca(++)-free media whereas the corresponding...... neurotransmitter glutamate (100 microM). Both depolarizing stimuli exerted prompt increases in the release of preloaded [3H]GABA as well as in [Ca++]i. However, the basic properties of transmitter release and the increase in [Ca++]i under a variety of conditions were different during stimulation with K...... was also reduced by organic (verapamil) and inorganic (Co++) Ca++ channel blockers but was insensitive to the GABA transport inhibitor SKF 89976A. In contrast, the second phase was less sensitive to nocodazole and Ca++ channel antagonists but could be inhibited by SKF 89976A. The glutamate-induced [3H...

  8. Induction of human microsomal prostaglandin E synthase 1 by activated oncogene RhoA GTPase in A549 human epithelial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hye Jin [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Lee, Dong-Hyung [Department of Obstetrics and Gynecology, Medical Research Institute, Pusan National University, Busan (Korea, Republic of); Park, Seong-Hwan; Kim, Juil; Do, Kee Hun [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); An, Tae Jin; Ahn, Young Sup; Park, Chung Berm [Department of Herbal Crop Research, NIHHS, RDA, Eumseong (Korea, Republic of); Moon, Yuseok, E-mail: moon@pnu.edu [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Medical Research Institute and Research Institute for Basic Sciences, Pusan National University, Busan (Korea, Republic of)

    2011-09-30

    Highlights: {yields} As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. {yields} RhoA activation increases PGE{sub 2} levels and its metabolic enzyme mPGES-1. {yields} RhoA-activated NF-{kappa}B and EGR-1 are positively involved in mPGES-1 induction. -- Abstract: Oncogenic RhoA GTPase has been investigated as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Among the various targets of RhoA-linked signals, pro-inflammatory prostaglandin E{sub 2} (PGE{sub 2}), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE{sub 2} levels and gene expression of the rate-limiting PGE{sub 2} producing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase 1 (mPGES-1). In particular, human mPGES-1 was induced by RhoA via transcriptional activation in control and interleukin (IL)-1{beta}-activated cancer cells. To address the involvement of potent signaling pathways in RhoA-activated mPGES-1 induction, various signaling inhibitors were screened for their effects on mPGES-1 promoter activity. RhoA activation enhanced basal and IL-1{beta}-mediated phosphorylated nuclear factor-{kappa}B and extracellular signal-regulated kinase1/2 proteins, all of which were positively involved in RhoA-induced gene expression of mPGES-1. As one potent down-stream transcription factor of ERK1/2 signals, early growth response gene 1 product also mediated RhoA-induced gene expression of mPGES-1 by enhancing transcriptional activity. Since oncogene-triggered PGE{sub 2} production is a critical modulator of epithelial tumor cells, RhoA-associated mPGES-1 represents a promising chemo-preventive or therapeutic target for epithelial inflammation and its associated cancers.

  9. Activation of Phosphatidylinositol-Linked Dopamine Receptors Induces a Facilitation of Glutamate-Mediated Synaptic Transmission in the Lateral Entorhinal Cortex.

    Directory of Open Access Journals (Sweden)

    Iulia Glovaci

    Full Text Available The lateral entorhinal cortex receives strong inputs from midbrain dopamine neurons that can modulate its sensory and mnemonic function. We have previously demonstrated that 1 µM dopamine facilitates synaptic transmission in layer II entorhinal cortex cells via activation of D1-like receptors, increased cAMP-PKA activity, and a resulting enhancement of AMPA-receptor mediated currents. The present study assessed the contribution of phosphatidylinositol (PI-linked D1 receptors to the dopaminergic facilitation of transmission in layer II of the rat entorhinal cortex, and the involvement of phospholipase C activity and release of calcium from internal stores. Whole-cell patch-clamp recordings of glutamate-mediated evoked excitatory postsynaptic currents were obtained from pyramidal and fan cells. Activation of D1-like receptors using SKF38393, SKF83959, or 1 µM dopamine induced a reversible facilitation of EPSCs which was abolished by loading cells with either the phospholipase C inhibitor U-73122 or the Ca2+ chelator BAPTA. Neither the L-type voltage-gated Ca2+ channel blocker nifedipine, nor the L/N-type channel blocker cilnidipine, blocked the facilitation of synaptic currents. However, the facilitation was blocked by blocking Ca2+ release from internal stores via inositol 1,4,5-trisphosphate (InsP3 receptors or ryanodine receptors. Follow-up studies demonstrated that inhibiting CaMKII activity with KN-93 failed to block the facilitation, but that application of the protein kinase C inhibitor PKC(19-36 completely blocked the dopamine-induced facilitation. Overall, in addition to our previous report indicating a role for the cAMP-PKA pathway in dopamine-induced facilitation of synaptic transmission, we demonstrate here that the dopaminergic facilitation of synaptic responses in layer II entorhinal neurons also relies on a signaling cascade dependent on PI-linked D1 receptors, PLC, release of Ca2+ from internal stores, and PKC activation which is

  10. Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity.

    Science.gov (United States)

    Fattorini, L; Veloccia, A; Della Rovere, F; D'Angeli, S; Falasca, G; Altamura, M M

    2017-07-11

    Indole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control adventitious root (AR) formation in planta. Adventitious roots are also crucial for propagation via cuttings. However, IBA role(s) is/are still far to be elucidated. In Arabidopsis thaliana stem cuttings, 10 μM IBA is more AR-inductive than 10 μM IAA, and, in thin cell layers (TCLs), IBA induces ARs when combined with 0.1 μM kinetin (Kin). It is unknown whether arabidopsis TCLs produce ARs under IBA alone (10 μM) or IAA alone (10 μM), and whether they contain endogenous IAA/IBA at culture onset, possibly interfering with the exogenous IBA/IAA input. Moreover, it is unknown whether an IBA-to-IAA conversion is active in TCLs, and positively affects AR formation, possibly through the activity of the nitric oxide (NO) deriving from the conversion process. Revealed undetectable levels of both auxins at culture onset, showing that arabidopsis TCLs were optimal for investigating AR-formation under the total control of exogenous auxins. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants was analyzed under different treatments. It was shown that ARs are better induced by IBA than IAA and IBA + Kin. IBA induced IAA-efflux (PIN1) and IAA-influx (AUX1/LAX3) genes, IAA-influx carriers activities, and expression of ANTHRANILATE SYNTHASE -alpha1 (ASA1), a gene involved in IAA-biosynthesis. ASA1 and ANTHRANILATE SYNTHASE -beta1 (ASB1), the other subunit of the same enzyme, positively affected AR-formation in the presence of exogenous IBA, because the AR-response in the TCLs of their mutant wei2wei7 was highly reduced. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA-conversion, was also strongly reduced. Nitric oxide, an IAA downstream signal and a by-product of IBA-to-IAA conversion, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter explants. Altogether, results showed that IBA induced

  11. Blood Glutamate Scavenging: Insight into Neuroprotection

    Directory of Open Access Journals (Sweden)

    Alexander Zlotnik

    2012-08-01

    Full Text Available Brain insults are characterized by a multitude of complex processes, of which glutamate release plays a major role. Deleterious excess of glutamate in the brain’s extracellular fluids stimulates glutamate receptors, which in turn lead to cell swelling, apoptosis, and neuronal death. These exacerbate neurological outcome. Approaches aimed at antagonizing the astrocytic and glial glutamate receptors have failed to demonstrate clinical benefit. Alternatively, eliminating excess glutamate from brain interstitial fluids by making use of the naturally occurring brain-to-blood glutamate efflux has been shown to be effective in various animal studies. This is facilitated by gradient driven transport across brain capillary endothelial glutamate transporters. Blood glutamate scavengers enhance this naturally occurring mechanism by reducing the blood glutamate concentration, thus increasing the rate at which excess glutamate is cleared. Blood glutamate scavenging is achieved by several mechanisms including: catalyzation of the enzymatic process involved in glutamate metabolism, redistribution of glutamate into tissue, and acute stress response. Regardless of the mechanism involved, decreased blood glutamate concentration is associated with improved neurological outcome. This review focuses on the physiological, mechanistic and clinical roles of blood glutamate scavenging, particularly in the context of acute and chronic CNS injury. We discuss the details of brain-to-blood glutamate efflux, auto-regulation mechanisms of blood glutamate, natural and exogenous blood glutamate scavenging systems, and redistribution of glutamate. We then propose different applied methodologies to reduce blood and brain glutamate concentrations and discuss the neuroprotective role of blood glutamate scavenging.

  12. Development of a biomarker for Geobacter activity and strain composition: Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI)

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, M.J.; Callister, S.J.; Miletto, M.; Williams, K.H.; Nicora, C.D.; Lovley, D.R.; Long, P.E.; Lipton, M.S.

    2010-02-15

    Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

  13. Differential Activity of the Oral Glucan Synthase Inhibitor SCY-078 against Wild-Type and Echinocandin-Resistant Strains of Candida Species.

    Science.gov (United States)

    Pfaller, Michael A; Messer, Shawn A; Rhomberg, Paul R; Borroto-Esoda, Katyna; Castanheira, Mariana

    2017-08-01

    SCY-078 (formerly MK-3118) is a novel orally active inhibitor of fungal β-(1,3)-glucan synthase (GS). SCY-078 is a derivative of enfumafungin and is structurally distinct from the echinocandin class of antifungal agents. We evaluated the in vitro activity of this compound against wild-type (WT) and echinocandin-resistant isolates containing mutations in the FKS genes of Candida spp. Against 36 Candida spp. FKS mutants tested, 30 (83.3%) were non-WT to 1 or more echinocandins, and only 9 (25.0%) were non-WT (MIC, >WT-upper limit) to SCY-078. Among C. glabrata isolates carrying FKS alterations, 84.0% were non-WT to the echinocandins versus only 24.0% for SCY-078. In contrast to the echinocandin comparators, the activity of SCY-078 was minimally affected by the presence of FKS mutations, suggesting that this agent is useful in the treatment of Candida infections due to echinocandin-resistant strains. Copyright © 2017 American Society for Microbiology.

  14. The role of nitric oxide in aspirin induced thrombolysis in vitro and the purification of aspirin activated nitric oxide synthase from human blood platelets.

    Science.gov (United States)

    Karmohapatra, Soumendra K; Chakraborty, Kushal; Kahn, Nighat N; Sinha, Asru K

    2007-11-01

    Aspirin, a well-known inhibitor of platelet aggregation, is extensively used for the prevention/treatment of coronary artery disease. The beneficial and antithrombotic effects of the compound are related to the inhibition of platelet cyclooxygenase. It is currently believed that aspirin has no effect on the formed thrombus, which results in coronary artery disease. It was found that the exposure of platelets to 4.0 microM aspirin either in vitro or in vivo resulted in fibrinolysis of the formed "clot" produced by the recalcification of platelet-rich plasma due to the production of NO in these cells by the compound. The lysis of clot in the presence of aspirin was found to be related to the fibrinolysis with simultaneous appearance of fibrin degradation products due to the generation of serine proteinase activity by NO in the assay mixture. The aspirin activated nitric oxide synthase that catalyzed the synthesis of NO in platelets was solubilized by Triton X-100 treatment and purified to homogeneity by chromatography on DEAE cellulose and Sephadex G-50 columns. The enzyme was found to be a single chain polypeptide with M.W. 19 kDa. The treatment of human plasminogen with NO was found to directly activate the zymogen to plasmin with the production of preactivation peptide in the absence of cofactors, or cells without the formation of cyclic GMP in the assay mixture. (c) 2007 Wiley-Liss, Inc.

  15. Low sulfide levels and a high degree of cystathionine β-synthase (CBS) activation by S-adenosylmethionine (SAM) in the long-lived naked mole-rat.

    Science.gov (United States)

    Dziegelewska, Maja; Holtze, Susanne; Vole, Christiane; Wachter, Ulrich; Menzel, Uwe; Morhart, Michaela; Groth, Marco; Szafranski, Karol; Sahm, Arne; Sponholz, Christoph; Dammann, Philip; Huse, Klaus; Hildebrandt, Thomas; Platzer, Matthias

    2016-08-01

    Hydrogen sulfide (H2S) is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS), an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Low sulfide levels and a high degree of cystathionine β-synthase (CBS activation by S-adenosylmethionine (SAM in the long-lived naked mole-rat

    Directory of Open Access Journals (Sweden)

    Maja Dziegelewska

    2016-08-01

    Full Text Available Hydrogen sulfide (H2S is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS, an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration.

  17. Defective insulin signaling pathway and increased glycogen synthase kinase-3 activity in the brain of diabetic mice: parallels with Alzheimer's disease and correction by insulin.

    Science.gov (United States)

    Jolivalt, C G; Lee, C A; Beiswenger, K K; Smith, J L; Orlov, M; Torrance, M A; Masliah, E

    2008-11-15

    We have evaluated the effect of peripheral insulin deficiency on brain insulin pathway activity in a mouse model of type 1 diabetes, the parallels with Alzheimer's disease (AD), and the effect of treatment with insulin. Nine weeks of insulin-deficient diabetes significantly impaired the learning capacity of mice, significantly reduced insulin-degrading enzyme protein expression, and significantly reduced phosphorylation of the insulin-receptor and AKT. Phosphorylation of glycogen synthase kinase-3 (GSK3) was also significantly decreased, indicating increased GSK3 activity. This evidence of reduced insulin signaling was associated with a concomitant increase in tau phosphorylation and amyloid beta protein levels. Changes in phosphorylation levels of insulin receptor, GSK3, and tau were not observed in the brain of db/db mice, a model of type 2 diabetes, after a similar duration (8 weeks) of diabetes. Treatment with insulin from onset of diabetes partially restored the phosphorylation of insulin receptor and of GSK3, partially reduced the level of phosphorylated tau in the brain, and partially improved learning ability in insulin-deficient diabetic mice. Our data indicate that mice with systemic insulin deficiency display evidence of reduced insulin signaling pathway activity in the brain that is associated with biochemical and behavioral features of AD and that it can be corrected by insulin treatment.

  18. Near infrared radiation protects against oxygen-glucose deprivation-induced neurotoxicity by down-regulating neuronal nitric oxide synthase (nNOS) activity in vitro.

    Science.gov (United States)

    Yu, Zhanyang; Li, Zhaoyu; Liu, Ning; Jizhang, Yunneng; McCarthy, Thomas J; Tedford, Clark E; Lo, Eng H; Wang, Xiaoying

    2015-06-01

    Near infrared radiation (NIR) has been shown to be neuroprotective against neurological diseases including stroke and brain trauma, but the underlying mechanisms remain poorly understood. In the current study we aimed to investigate the hypothesis that NIR may protect neurons by attenuating oxygen-glucose deprivation (OGD)-induced nitric oxide (NO) production and modulating cell survival/death signaling. Primary mouse cortical neurons were subjected to 4 h OGD and NIR was applied at 2 h reoxygenation. OGD significantly increased NO level in primary neurons compared to normal control, which was significantly ameliorated by NIR at 5 and 30 min post-NIR. Neither OGD nor NIR significantly changed neuronal nitric oxide synthase (nNOS) mRNA or total protein levels compared to control groups. However, OGD significantly increased nNOS activity compared to normal control, and this effect was significantly diminished by NIR. Moreover, NIR significantly ameliorated the neuronal death induced by S-Nitroso-N-acetyl-DL-penicillamine (SNAP), a NO donor. Finally, NIR significantly rescued OGD-induced suppression of p-Akt and Bcl-2 expression, and attenuated OGD-induced upregulation of Bax, BAD and caspase-3 activation. These results suggest NIR may protect against OGD at least partially through reducing NO production by down-regulating nNOS activity, and modulating cell survival/death signaling.

  19. ATP induces NO production in hippocampal neurons by P2X(7 receptor activation independent of glutamate signaling.

    Directory of Open Access Journals (Sweden)

    Juan Francisco Codocedo

    Full Text Available To assess the putative role of adenosine triphosphate (ATP upon nitric oxide (NO production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2'(3'-O-(4-Benzoylbenzoyl ATP (Bz-ATP elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG or by N(ω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV, but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.

  20. Disruption of ATCSLD5 results in reduced growth, reduced xylan and homogalacturonan synthase activity and altered xylan occurrence in Arabidopsis

    DEFF Research Database (Denmark)

    Bernal Giraldo, Adriana Jimena; Jensen, Jacob Krüger; Harholt, Jesper

    2007-01-01

    Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs...... are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module...

  1. Memory Trace Reactivation and Behavioral Response during Retrieval Are Differentially Modulated by Amygdalar Glutamate Receptors Activity: Interaction between Amygdala and Insular Cortex

    Science.gov (United States)

    Osorio-Gómez, Daniel; Guzmán-Ramos, Kioko; Bermúdez-Rattoni, Federico

    2017-01-01

    The insular cortex (IC) is required for conditioned taste aversion (CTA) retrieval. However, it remains unknown which cortical neurotransmitters levels are modified upon CTA retrieval. Using in vivo microdialysis, we observed that there were clear elevations in extracellular glutamate, norepinephrine, and dopamine in and around the center of the…

  2. Depolarization by K+ and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: vesicular versus non-vesicular release of GABA

    DEFF Research Database (Denmark)

    Belhage, B; Hansen, G H; Schousboe, A

    1993-01-01

    was also reduced by organic (verapamil) and inorganic (Co++) Ca++ channel blockers but was insensitive to the GABA transport inhibitor SKF 89976A. In contrast, the second phase was less sensitive to nocodazole and Ca++ channel antagonists but could be inhibited by SKF 89976A. The glutamate-induced [3H...

  3. Glucose dependence of glycogen synthase activity regulation by GSK3 and MEK/ERK inhibitors and angiotensin-(1-7) action on these pathways in cultured human myotubes.

    Science.gov (United States)

    Montori-Grau, Marta; Tarrats, Núria; Osorio-Conles, Oscar; Orozco, Anna; Serrano-Marco, Lucía; Vázquez-Carrera, Manuel; Gómez-Foix, Anna M

    2013-05-01

    Glycogen synthase (GS) is activated by glucose/glycogen depletion in skeletal muscle cells, but the contributing signaling pathways, including the chief GS regulator GSK3, have not been fully defined. The MEK/ERK pathway is known to regulate GSK3 and respond to glucose. The aim of this study was to elucidate the GSK3 and MEK/ERK pathway contribution to GS activation by glucose deprivation in cultured human myotubes. Moreover, we tested the glucose-dependence of GSK3 and MEK/ERK effects on GS and angiotensin (1-7) actions on these pathways. We show that glucose deprivation activated GS, but did not change phospho-GS (Ser640/1), GSK3β activity or activity-activating phosphorylation of ERK1/2. We then treated glucose-replete and -depleted cells with SB415286, U0126, LY294 and rapamycin to inhibit GSK3, MEK1/2, PI3K and mTOR, respectively. SB415286 activated GS and decreased the relative phospho-GS (Ser640/1) level, more in glucose-depleted than -replete cells. U0126 activated GS and reduced the phospho-GS (Ser640/1) content significantly in glucose-depleted cells, while GSK3β activity tended to increase. LY294 inactivated GS in glucose-depleted cells only, without affecting relative phospho-GS (Ser640/1) level. Rapamycin had no effect on GS activation. Angiotensin-(1-7) raised phospho-ERK1/2 but not phospho-GSK3β (Ser9) content, while it inactivated GS and increased GS phosphorylation on Ser640/1, in glucose-replete cells. In glucose-depleted cells, angiotensin-(1-7) effects on ERK1/2 and GS were reverted, while relative phospho-GSK3β (Ser9) content decreased. In conclusion, activation of GS by glucose deprivation is not due to GS Ser640/1 dephosphorylation, GSK3β or ERK1/2 regulation in cultured myotubes. However, glucose depletion enhances GS activation/Ser640/1 dephosphorylation due to both GSK3 and MEK/ERK inhibition. Angiotensin-(1-7) inactivates GS in glucose-replete cells in association with ERK1/2 activation, not with GSK3 regulation, and glucose

  4. Alkylation of acetohydroxyacid synthase I from Escherichia coli K-12 by 3-bromopyruvate: evidence for a single active site catalyzing acetolactate and acetohydroxybutyrate synthesis.

    Science.gov (United States)

    Silverman, P M; Eoyang, L

    1987-01-01

    Acetohydroxyacid synthase I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate. Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate. Inactivation was accompanied by incorporation of radioactivity from 3-bromo[2-14C]pyruvate into acid-insoluble material. More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a sodium dodecyl sulfate-polyacrylamide gel; less than 5% coelectrophoresed with the 11.2-kilodalton IlvN subunit. The stoichiometry of incorporation at nearly complete inactivation was 1 mol of 14C per mol of IlvB polypeptide. These data indicate that bromopyruvate inactivates AHAS I by alkylating an amino acid at or near a single active site located in the IlvB subunit of the enzyme. We confirmed that this alkylation inactivated both AHAS reactions normally catalyzed by AHAS I. These results provide the first direct evidence that AHAS I catalyzes both acetohydroxybutyrate and acetolactate synthesis from the same active site. Images PMID:3294793

  5. Phylogenetic diversity of culturable endophytic fungi in Dongxiang wild rice (Oryza rufipogon Griff), detection of polyketide synthase gene and their antagonistic activity analysis.

    Science.gov (United States)

    Wang, Ya; Gao, Bo Liang; Li, Xi Xi; Zhang, Zhi Bin; Yan, Ri Ming; Yang, Hui Lin; Zhu, Du

    2015-11-01

    The biodiversity of plant endophytic fungi is enormous, numerous competent endophytic fungi are capable of providing different forms of fitness benefits to host plants and also could produce a wide array of bioactive natural products, which make them a largely unexplored source of novel compounds with potential bioactivity. In this study, we provided a first insights into revealing the diversity of culturable endophytic fungi in Dongxiang wild rice (Oryza rufipogon Griff.) from China using rDNA-ITS phylogenetic analysis. Here, the potential of fungi in producing bioactive natural products was estimated based on the beta-ketosynthase detected in the polyketide synthase (PKS) gene cluster and on the bioassay of antagonistic activity against two rice phytopathogens Thanatephorus cucumeris and Xanthomonas oryzae. A total of 229 endophytic fungal strains were validated in 19 genera. Among the 24 representative strains, 13 strains displayedantagonistic activity against the phytopathogens. Furthermore, PKS genes were detected in 9 strains, indicating their potential for synthesising PKS compounds. Our study confirms the phylogenetic diversity of endophytic fungi in O. rufipogon G. and highlights that endophytic fungi are not only promising resources of biocontrol agents against phytopathogens of rice plants, but also of bioactive natural products and defensive secondary metabolites. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  6. Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.

    Science.gov (United States)

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L; Zillinger, Thomas; Serganov, Artem A; Liu, Yizhou; Jones, Roger A; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J

    2013-05-23

    Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2',5')pA and A(3',5')pG phosphodiester linkages, designated c[G(2',5')pA(3',5')p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2',5')pA(3',5')p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5'-pppG(2',5')pA prior to cyclization to c[G(2',5')pA(3',5')p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2',5')pA(3',5')p] as a founding member of a family of metazoan 2',5'-containing cyclic heterodinucleotide second messengers distinct from bacterial 3',5' cyclic dinucleotides. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Protective Effects of Kaempferol against Myocardial Ischemia/Reperfusion Injury in Isolated Rat Heart via Antioxidant Activity and Inhibition of Glycogen Synthase Kinase-3β

    Science.gov (United States)

    Zhou, Mingjie; Ren, Huanhuan; Wang, Wenjuan; Zheng, Qiusheng; Wang, Dong

    2015-01-01

    Objective. This study aimed to evaluate the protective effect of kaempferol against myocardial ischemia/reperfusion (I/R) injury in rats. Method. Left ventricular developed pressure (LVDP) and its maximum up/down rate (±dp/dt max) were recorded as myocardial function. Infarct size was detected with 2,3,5-triphenyltetrazolium chloride staining. Cardiomyocyte apoptosis was determined using terminal deoxynucleotidyl nick-end labeling (TUNEL). The levels of creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione/glutathione disulfide (GSH/GSSG) ratio, and tumor necrosis factor-alpha (TNF-α) were determined using enzyme linked immunosorbent assay (ELISA). Moreover, total glycogen synthase kinase-3β (GSK-3β), phospho-GSK-3β (P-GSK-3β), precaspase-3, cleaved caspase-3, and cytoplasm cytochrome C were assayed using Western blot analysis. Results. Pretreatment with kaempferol significantly improved the recovery of LVDP and ±dp/dt max, as well as increased the levels of SOD and P-GSK-3β and GSH/GSSG ratio. However, the pretreatment reduced myocardial infarct size and TUNEL-positive cell rate, as well as decreased the levels of cleaved caspase-3, cytoplasm cytochrome C, CK, LDH, MDA, and TNF-α. Conclusion. These results suggested that kaempferol provides cardioprotection via antioxidant activity and inhibition of GSK-3β activity in rats with I/R. PMID:26265983

  8. Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity.

    Science.gov (United States)

    Gunasekera, Bhagya; Abou Diwan, Charbel; Altawallbeh, Ghaith; Kalil, Haitham; Maher, Shaimaa; Xu, Song; Bayachou, Mekki

    2018-03-07

    Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate l-arginine. In this work, we report on the modulation of enzyme loading in layer-by-layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.

  9. A 31 bp VNTR in the cystathionine beta-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels.

    NARCIS (Netherlands)

    Lievers, K.J.; Kluijtmans, L.A.J.; Heil, S.G.; Boers, G.H.J.; Verhoef, P.; Oppenraaij-Emmerzaal, D. van; Heijer, M. den; Trijbels, J.M.F.; Blom, H.J.

    2001-01-01

    Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinaemia, an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine beta-synthase (CBS) deficiency has been excluded as a major

  10. A 31 bp VNTR in the cystathionine beta-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels

    NARCIS (Netherlands)

    Lievers, K.J.; Kluijtmans, L.A.; Heil, S.G.; Boers, G.H.J.; Verhoef, P.; Oppenraay-Emmerzaal, van D.; Heijer, den M.; Trijbels, F.J.M.; Blom, H.J.

    2001-01-01

    Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinaemia, an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine -synthase (CBS) deficiency has been excluded as a major

  11. Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis.

    Science.gov (United States)

    Zhang, Nannan; Mao, Zejing; Luo, Ling; Wan, Xia; Huang, Fenghong; Gong, Yangmin

    2017-01-01

    Triacylglycerols (TAGs) and wax esters (WEs) are important neutral lipids which serve as energy reservoir in some plants and microorganisms. In recent years, these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production because of the increased interest on developing renewable and environmentally benign alternatives for fossil fuels. In bacteria, the final step in TAG and WE biosynthetic pathway is catalyzed by wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT). This bifunctional WS/DGAT enzyme is also a key enzyme in biotechnological production of liquid WE via engineering of plants and microorganisms. To date, knowledge about this class of biologically and biotechnologically important enzymes is mainly from biochemical characterization of WS/DGATs from Arabidopsis, jojoba and some bacteria that can synthesize both TAGs and WEs intracellularly, whereas little is known about WS/DGATs from eukaryotic microorganisms. Here, we report the identification and characterization of two bifunctional WS/DGAT enzymes (designated TrWSD4 and TrWSD5) from the marine protist Thraustochytrium roseum . Both TrWSD4 and TrWSD5 comprise a WS-like acyl-CoA acyltransferase domain and the recombinant proteins purified from Escherichia coli Rosetta (DE3) have substantial WS and lower DGAT activity. They exhibit WS activity towards various-chain-length saturated and polyunsaturated acyl-CoAs and fatty alcohols ranging from C 10 to C 18 . TrWSD4 displays WS activity with the lowest K m value of 0.14 μM and the highest k cat / K m value of 1.46 × 10 5  M -1  s -1 for lauroyl-CoA (C 12:0 ) in the presence of 100 μM hexadecanol, while TrWSD5 exhibits WS activity with the lowest K m value of 0.96 μM and the highest k cat / K m value of 9.83 × 10 4  M -1  s -1 for decanoyl-CoA (C 10:0 ) under the same reaction condition. Both WS/DGAT enzymes have the highest WS activity at 37 and 47

  12. KB-R7943 reduces 4-aminopyridine-induced epileptiform activity in adult rats after neuronal damage induced by neonatal monosodium glutamate treatment.

    Science.gov (United States)

    Hernandez-Ojeda, Mariana; Ureña-Guerrero, Monica E; Gutierrez-Barajas, Paola E; Cardenas-Castillo, Jazmin A; Camins, Antoni; Beas-Zarate, Carlos

    2017-05-09

    Neonatal monosodium glutamate (MSG) treatment triggers excitotoxicity and induces a degenerative process that affects several brain regions in a way that could lead to epileptogenesis. Na + /Ca 2+ exchangers (NCX1-3) are implicated in Ca 2+ brain homeostasis; normally, they extrude Ca 2+ to control cell inflammation, but after damage and in epilepsy, they introduce Ca 2+ by acting in the reverse mode, amplifying the damage. Changes in NCX3 expression in the hippocampus have been reported immediately after neonatal MSG treatment. In this study, the expression level of NCX1-3 in the entorhinal cortex (EC) and hippocampus (Hp); and the effects of blockade of NCXs on the seizures induced by 4-Aminopyridine (4-AP) were analysed in adult rats after neonatal MSG treatment. KB-R7943 was applied as NCXs blocker, but is more selective to NCX3 in reverse mode. Neonatal MSG treatment was applied to newborn male rats at postnatal days (PD) 1, 3, 5, and 7 (4 g/kg of body weight, s.c.). Western blot analysis was performed on total protein extracts from the EC and Hp to estimate the expression level of NCX1-3 proteins in relative way to the expression of β-actin, as constitutive protein. Electrographic activity of the EC and Hp were acquired before and after intracerebroventricular (i.c.v.) infusion of 4-AP (3 nmol) and KB-R7943 (62.5 pmol), alone or in combination. All experiments were performed at PD60. Behavioural alterations were also recorder. Neonatal MSG treatment significantly increased the expression of NCX3 protein in both studied regions, and NCX1 protein only in the EC. The 4-AP-induced epileptiform activity was significantly higher in MSG-treated rats than in controls, and KB-R7943 co-administered with 4-AP reduced the epileptiform activity in more prominent way in MSG-treated rats than in controls. The long-term effects of neonatal MSG treatment include increases on functional expression of NCXs (mainly of NCX3) in the EC and Hp, which seems to contribute to

  13. Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia.

    Science.gov (United States)

    Gregus, Ann M; Dumlao, Darren S; Wei, Spencer C; Norris, Paul C; Catella, Laura C; Meyerstein, Flore G; Buczynski, Matthew W; Steinauer, Joanne J; Fitzsimmons, Bethany L; Yaksh, Tony L; Dennis, Edward A

    2013-05-01

    Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs. vehicle-treated and mock-transfected controls. We detected the following primary intrinsic activities: 12-LOX (Alox12, Alox15), 15-LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12-LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12-LOX activity when coexpressed with Alox12b or supplemented with 12-HpETE. CDC potently inhibited HXS and 12-LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan-evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3-selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3-mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12-LOX/HXS inhibitors.

  14. Glycogen synthase kinase 3β regulation of nuclear factor of activated T-cells isoform c1 in the vascular smooth muscle cell response to injury

    International Nuclear Information System (INIS)

    Chow Winsion; Hou Guangpei; Bendeck, Michelle P.

    2008-01-01

    The migration and proliferation of vascular smooth muscle cells (vSMCs) are critical events in neointima formation during atherosclerosis and restenosis. The transcription factor nuclear factor of activated T-cells-isoform c1 (NFATc1) is regulated by atherogenic cytokines, and has been implicated in the migratory and proliferative responses of vSMCs through the regulation of gene expression. In T-cells, calcineurin de-phosphorylates NFATc1, leading to its nuclear import, while glycogen synthase kinase 3 β (GSK3β) phosphorylates NFATc1 and promotes its nuclear export. However, the relationship between NFATc1 and GSK3β has not been studied during SMC migration and proliferation. We investigated this by scrape wounding vSMCs in vitro, and studying wound repair. NFATc1 protein was transiently increased, reaching a peak at 8 h after wounding. Cell fractionation and immunocytochemistry revealed that NFATc1 accumulation in the nucleus was maximal at 4 h after injury, and this was coincident with a significant 9 fold increase in transcriptional activity. Silencing NFATc1 expression with siRNA or inhibition of NFAT with cyclosporin A (CsA) attenuated wound closure by vSMCs. Phospho-GSK3β (inactive) increased to a peak at 30 min after injury, preceding the nuclear accumulation of NFATc1. Overexpression of a constitutively active mutant of GSK3β delayed the nuclear accumulation of NFATc1, caused a 50% decrease in NFAT transcriptional activity, and attenuated vSMC wound repair. We conclude that NFATc1 promotes the vSMC response to injury, and that inhibition of GSK3β is required for the activation of NFAT during wound repair

  15. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    Science.gov (United States)

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-04

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  16. Dysfunctional TCA-Cycle Metabolism in Glutamate Dehydrogenase Deficient Astrocytes.

    Science.gov (United States)

    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. © 2015 Wiley Periodicals, Inc.

  17. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity.

    Science.gov (United States)

    Hopperton, Kathryn E; Duncan, Robin E; Bazinet, Richard P; Archer, Michael C

    2014-01-15

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from (14)C-labeled acetate to those supplied exogenously as (14)C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2-3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  19. Dietary supplementation with apple juice concentrate alleviates the compensatory increase in glutathione synthase transcription and activity that accompanies dietary- and genetically-induced oxidative stress.

    Science.gov (United States)

    Tchantchou, F; Graves, M; Ortiz, D; Rogers, E; Shea, T B

    2004-01-01

    Increased oxidative stress, which can arise from dietary, environmental and/or genetic sources, contributes to the decline in cognitive performance during normal aging and in neurodegenerative conditions such as Alzheimer's disease. Supplementation with fruits and vegetables that are high in antioxidant potential can compensate for dietary and/or genetic deficiencies that promote increased oxidative stress. We have recently demonstrated that apple juice concentrate (AJC) prevents the increase in oxidative damage to brain tissue and decline in cognitive performance observed when transgenic mice lacking apolipoprotein E (ApoE-/-) are maintained on a vitamin-deficient diet and challenged with excess iron (included in the diet as a pro-oxidant). However, the mechanism by which AJC provided neuroprotection was not conclusively determined. Herein, we demonstrate that supplementation with AJC also prevents the compensatory increases in glutathione synthase transcription and activity that otherwise accompany maintenance of ApoE-/- mice on this vitamin-free diet in the presence of iron. Inclusion of the equivalent composition and concentration of sugars of AJC did not prevent these increases. These findings provide further evidence that the antioxidant potential of AJC can compensate for dietary and genetic deficiencies that otherwise promote neurodegeneration.

  20. Attenuation of ischemia-reperfusion injury by sevoflurane postconditioning involves protein kinase B and glycogen synthase kinase 3 beta activation in isolated rat hearts.

    Science.gov (United States)

    Fang, Neng-Xin; Yao, Yun-Tai; Shi, Chun-Xia; Li, Li-Huan

    2010-12-01

    Volatile anesthetic ischemic postconditioning reduces infarct size following ischemia/reperfusion. Whether phosphorylation of protein kinase B (PKB/Akt) and glycogen synthase kinase 3 beta (GSK3β) is causal for cardioprotection by postconditioning is controversial. We therefore investigated the impact of PKB/Akt and GSK3β in isolated perfused rat hearts subjected to 40 min of ischemia followed by 1 h of reperfusion. 2.0% sevoflurane (1.0 minimum alveolar concentration) was administered at the onset of reperfusion in 15 min as postconditioning. Western blot analysis was used to determine phosphorylation of PKB/Akt and its downstream target GSK3β after 1 h of reperfusion. Mitochondrial and cytosolic content of cytochrome C checked by western blot served as a marker for mitochondrial permeability transition pore opening. Sevoflurane postconditioning significantly improved functional cardiac recovery and decreased infarct size in isolated rat hearts. Compared with unprotected hearts, sevoflurane postconditioning-induced phosphorylation of PKB/Akt and GSK3β were significantly increased. Increase of cytochrome C in mitochondria and decrease of it in cytosol is significant when compared with unprotected ones which have reversal effects on cytochrome C. The current study presents evidence that sevoflurane-induced cardioprotection at the onset of reperfusion are partly through activation of PKB/Akt and GSK3β.

  1. Changes in H(+)-ATP Synthase Activity, Proton Electrochemical Gradient, and pH in Pea Chloroplast Can Be Connected with Variation Potential.

    Science.gov (United States)

    Sukhov, Vladimir; Surova, Lyubov; Morozova, Ekaterina; Sherstneva, Oksana; Vodeneev, Vladimir

    2016-01-01

    Local stimulation induces generation and propagation of electrical signals, including the variation potential (VP) and action potential, in plants. Burning-induced VP changes the physiological state of plants; specifically, it inactivates photosynthesis. However, the mechanisms that decrease photosynthesis are poorly understood. We investigated these mechanisms by measuring VP-connected systemic changes in CO2 assimilation, parameters of light reactions of photosynthesis, electrochromic pigment absorbance shifts, and light scattering. We reveal that inactivation of photosynthesis in the pea, including inactivation of dark and light reactions, was connected with the VP. Inactivation of dark reactions decreased the rate constant of the fast relaxation of the electrochromic pigment absorbance shift, which reflected a decrease in the H(+)-ATP synthase activity. This decrease likely contributed to the acidification of the chloroplast lumen, which developed after VP induction. However, VP-connected decrease of the proton motive force across the thylakoid membrane, possibly, reflected a decreased pH in the stroma. This decrease may be another mechanism of chloroplast lumen acidification. Overall, stroma acidification can decrease electron flow through photosystem I, and lumen acidification induces growth of fluorescence non-photochemical quenching and decreases electron flow through photosystem II, i.e., pH decreases in the stroma and lumen, possibly, contribute to the VP-induced inactivation of light reactions of photosynthesis.

  2. Maintained activity of glycogen synthase kinase-3β despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    International Nuclear Information System (INIS)

    Lim, Yong-Whan; Yoon, Seung-Yong; Choi, Jung-Eun; Kim, Sang-Min; Lee, Hui-Sun; Choe, Han; Lee, Seung-Chul; Kim, Dong-Hou

    2010-01-01

    Glycogen synthase kinase-3β (GSK3β) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3β. However, the inactive form of GSK3β which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3β substrates, such as β-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3β at serine-9 and other substrates including tau, β-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3β inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3β may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3β inhibitors could be a valuable drug candidate in AD.

  3. TNF-Mediated Restriction of Arginase 1 Expression in Myeloid Cells Triggers Type 2 NO Synthase Activity at the Site of Infection

    Directory of Open Access Journals (Sweden)

    Ulrike Schleicher

    2016-05-01

    Full Text Available Neutralization or deletion of tumor necrosis factor (TNF causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase 1 (Arg1 expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO synthase (NOS2 was detected in inflammatory dendritic cells or macrophages, some of which co-expressed Arg1. In TNF-deficient mice, Arg1 was hyperexpressed, causing an impaired production of NO in situ. A similar phenotype was seen in L. major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating T cell response (Th2, Treg was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2, which is critical for pathogen control.

  4. An aureobasidin A resistance gene isolated from Aspergillus is a homolog of yeast AUR1, a gene responsible for inositol phosphorylceramide (IPC) synthase activity.

    Science.gov (United States)

    Kuroda, M; Hashida-Okado, T; Yasumoto, R; Gomi, K; Kato, I; Takesako, K

    1999-03-01

    The AUR1 gene of Saccharomyces cerevisiae, mutations in which confer resistance to the antibiotic aureobasidin A, is necessary for inositol phosphorylceramide (IPC) synthase activity. We report the molecular cloning and characterization of the Aspergillus nidulans aurA gene, which is homologous to AUR1. A single point mutation in the aurA gene of A. nidulans confers a high level of resistance to aureobasidin A. The A. nidulans aurA gene was used to identify its homologs in other Aspergillus species, including A. fumigatus, A. niger, and A. oryzae. The deduced amino acid sequence of an aurA homolog from the pathogenic fungus A. fumigatus showed 87% identity to that of A. nidulans. The AurA proteins of A. nidulans and A. fumigatus shared common characteristics in primary structure, including sequence, hydropathy profile, and N-glycosylation sites, with their S. cerevisiae, Schizosaccharomyces pombe, and Candida albicans counterparts. These results suggest that the aureobasidin resistance gene is conserved evolutionarily in various fungi.

  5. Glycogen synthase kinase-3beta and the p25 activator of cyclin dependent kinase 5 increase pausing of mitochondria in neurons.

    Science.gov (United States)

    Morel, M; Authelet, M; Dedecker, R; Brion, J P

    2010-06-02

    The complex bi-directional axoplasmic transport of mitochondria is essential for proper metabolic functioning of neurons and is controlled by phosphorylation. We have investigated by time-lapse imaging the effects of increased expression of glycogen synthase kinase-3beta (GSK-3beta) and of the p25 activator of cyclin dependent kinase 5 on mitochondria movements in mammalian cortical neurons and in PC12 cells. Both GSK-3beta and p25 increased the stationary behaviour of mitochondria in PC12 and in neurons, decreased their anterograde transport but did not affect the intrinsic velocities of mitochondria. The microtubule-associated tau proteins were more phosphorylated in GSK-3beta and p25 transfected neurons, but ultrastructural observation showed that these cells still contained microtubules and nocodazole treatment further reduced residual mitochondria movements in GSK-3beta or p25 transfected neurons, indicating that microtubule disruption was not the primary cause of increased mitochondrial stationary behaviour in GSK-3beta or p25 transfected neurons. Our results suggest that increased expression of GSK-3beta and p25 acted rather by decreasing the frequency of mitochondrial movements driven by molecular motors and that GSK-3beta and p25 might regulate these transports by controlling the time that mitochondria spend pausing, rather than their velocities. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  7. Glutamate mechanisms underlying opiate memories

    NARCIS (Netherlands)

    Peters, J.; de Vries, T.J.

    2012-01-01

    As the major excitatory neurotransmitter in the brain, glutamate plays an undisputable integral role in opiate addiction. This relates, in part, to the fact that addiction is a disorder of learning and memory, and glutamate is required for most types of memory formation. As opiate addiction

  8. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 Synergistically Activate Transcription of Fatty-acid Synthase Gene (FASN)*S⃞

    Science.gov (United States)

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F.; Hur, Man-Wook

    2008-01-01

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation. PMID:18682402

  9. Identification of 2-aminothiazole-4-carboxylate derivatives active against Mycobacterium tuberculosis H37Rv and the beta-ketoacyl-ACP synthase mtFabH.

    Directory of Open Access Journals (Sweden)

    Qosay Al-Balas

    Full Text Available BACKGROUND: Tuberculosis (TB is a disease which kills two million people every year and infects approximately over one-third of the world's population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration. METHODOLOGY/PRINCIPAL FINDINGS: Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM's novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H(37R(v and, dissociatively, against the beta-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H(37R(v with an MIC of 0.06 microg/ml (240 nM, but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido-5-(3-chlorophenylthiazole-4-carboxylate inhibited mtFabH with an IC(50 of 0.95+/-0.05 microg/ml (2.43+/-0.13 microM but was not active against the whole cell organism. CONCLUSIONS/SIGNIFICANCE: These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents.

  10. Alterations of nitric-oxide synthase and xanthine-oxidase activities of human keratinocytes by ultraviolet-B radiation -potential role for peroxynitrite in skin inflammation

    Energy Technology Data Exchange (ETDEWEB)

    Deliconstantinos, G.; Villiotou, V.; Stavrides, J.C. [Athens Univ. (Greece). School of Medicine

    1996-06-28

    In the present study, we demonstrated that NO synthase (cNOS) and xanthine oxidase (XO) of human keratinocytes can be activated to release NO, superoxide (O-2(-)) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation. We defined that this photo induced response may be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human keratinocytes with UVB (290-320 nm) radiation (up to 200 mJ/cm(2)) resulted in a dose-dependent increase in NO and ONOO-release that was inhibited by N-monomethyl-L-arginine (L-NMMA). NO and ONOO- release from keratinocytes was accompanied by an increase in intracellular cGMP levels. Treatment of human keratinocyte cytosol with various doses of UVB (up to 100 mJ/cm(2)) resulted in an increase in XO activity that was inhibited by oxypurinol. In in vivo experiments, when experimental animals were subjected to UVB radiation, a protection factor (PF) of 6.5 {+-} 1.8 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA) (2%) and L-NMMA (2%) was applied to their skin. The present study indicates that UVB radiation acts as a potent stimulator of cNOS and XO activities in human keratinocytes. NO and ONOO- may exert cytotoxic effects in keratinocytes themselves, as well as in their neighbouring endothelial and smooth muscle cells. This may be a major part of the integrated response leading to erythema production and the inflammation process. (UK).

  11. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus1[OPEN

    Science.gov (United States)

    Zeng, Wei; Picard, Kelsey L.; Song, Lili; Wu, Ai-Min; Farion, Isabela M.; Zhao, Jia; Ford, Kris; Bacic, Antony

    2016-01-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana. We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. PMID:26951434

  12. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.

    Science.gov (United States)

    Zeng, Wei; Lampugnani, Edwin R; Picard, Kelsey L; Song, Lili; Wu, Ai-Min; Farion, Isabela M; Zhao, Jia; Ford, Kris; Doblin, Monika S; Bacic, Antony

    2016-05-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  13. Activation of neuronal nitric oxide synthase in cerebellum of chronic hepatic encephalopathy rats is associated with up-regulation of NADPH-producing pathway.

    Science.gov (United States)

    Singh, Santosh; Trigun, Surendra K

    2010-09-01

    Cerebellum-associated functions get affected during mild hepatic encephalopathy (MHE) in patients with chronic liver failure (CLF). Involvement of nitrosative and antioxidant factors in the pathogenesis of chronic hepatic encephalopathy is an evolving concept and needs to be defined in a true CLF animal model. This article describes profiles of NADPH-dependent neuronal nitric oxide synthase (nNOS) and those of glutathione peroxidase and glutathione reductase (GR) vis-a-vis regulation of NADPH-producing pathway in the cerebellum of CLF rats induced by administration of thioacetamide (100 mg kg⁻¹ b.w., i.p.) up to 10 days and confirming MHE on Morris water maze tests. Significant increases in the expression of nNOS protein and nitric oxide (NOx) level coincided with a similar increment in NADPH-diaphorase activity in the cerebellum of CLF rats. Glutathione peroxidase and GR utilize NADPH to regenerate reduced glutathione (GSH) in the cells. Both these enzymes and GSH level were found to be static and thus suggested efficient turnover of GSH in the cerebellum of MHE rats. Relative levels of glucose-6-phosphate dehydrogenase (G6PD) vs. phosphofructokinase 2 (PFK2) determine the rate of pentose phosphate pathway (PPP) responsible to synthesize NADPH. The cerebellum of CLF rats showed overactivation of G6PD with a significant decline in the expression of PFK2 and thus suggested activation of PPP in the cerebellum during MHE. It is concluded that concordant activations of PPP and nNOS in cerebellum of MHE rats could be associated with the implication of NOx in the pathogenesis of MHE.

  14. Alterations of nitric-oxide synthase and xanthine-oxidase activities of human keratinocytes by ultraviolet-B radiation -potential role for peroxynitrite in skin inflammation

    International Nuclear Information System (INIS)

    Deliconstantinos, G.; Villiotou, V.; Stavrides, J.C.

    1996-01-01

    In the present study, we demonstrated that NO synthase (cNOS) and xanthine oxidase (XO) of human keratinocytes can be activated to release NO, superoxide (O-2(-)) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation. We defined that this photo induced response may be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human keratinocytes with UVB (290-320 nm) radiation (up to 200 mJ/cm(2)) resulted in a dose-dependent increase in NO and ONOO-release that was inhibited by N-monomethyl-L-arginine (L-NMMA). NO and ONOO- release from keratinocytes was accompanied by an increase in intracellular cGMP levels. Treatment of human keratinocyte cytosol with various doses of UVB (up to 100 mJ/cm(2)) resulted in an increase in XO activity that was inhibited by oxypurinol. In in vivo experiments, when experimental animals were subjected to UVB radiation, a protection factor (PF) of 6.5 ± 1.8 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA) (2%) and L-NMMA (2%) was applied to their skin. The present study indicates that UVB radiation acts as a potent stimulator of cNOS and XO activities in human keratinocytes. NO and ONOO- may exert cytotoxic effects in keratinocytes themselves, as well as in their neighbouring endothelial and smooth muscle cells. This may be a major part of the integrated response leading to erythema production and the inflammation process. (UK)

  15. The novel oral glucan synthase inhibitor SCY-078 shows in vitro activity against sessile and planktonic Candida spp.

    Science.gov (United States)

    Marcos-Zambrano, Laura Judith; Gómez-Perosanz, Marta; Escribano, Pilar; Bouza, Emilio; Guinea, Jesús

    2017-07-01

    We studied the antifungal activity of SCY-078 (an orally bioavailable 1,3-β -d- glucan synthesis inhibitor), micafungin and fluconazole against the planktonic and sessile forms of 178 Candida and non- Candida isolates causing fungaemia in patients recently admitted to a large European hospital. The in vitro activity of SCY-078, micafungin and fluconazole against the planktonic form of the isolates was assessed using EUCAST EDef 7.3 and CLSI M27-A3. Antibiofilm activity was assessed using the XTT reduction assay. SCY-078 and micafungin showed potent in vitro activity against Candida and non- Candida isolates. The in vitro activity of both drugs was similar, but SYC-078 displayed significantly lower MIC values than micafungin against Candida parapsilosis and non- Candida isolates, whereas micafungin displayed significantly lower MIC values for the remaining species ( P  Candida glabrata , in which the micafungin sessile MIC values were significantly lower ( P  Candida isolates in both sessile and planktonic forms is comparable to that of micafungin. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. The influence of Na+,K+-ATPase on glutamate signaling in neurodegenerative diseases and senescence

    Directory of Open Access Journals (Sweden)

    Paula Fernanda Kinoshita

    2016-06-01

    Full Text Available Decreased Na+,K+-ATPase (NKA activity causes energy deficiency, which is commonly observed in neurodegenerative diseases. The NKA is constituted of three subunits: α, β and γ, with four distinct isoforms of the catalytic α subunit (α1-4. Genetic mutations in the ATP1A2 gene and ATP1A3 gene, encoding the α2 and α3 subunit isoforms, respectively can cause distinct neurological disorders, concurrent to impaired NKA activity. Within the central nervous system (CNS, the α2 isoform is expressed mostly in glial cells and the α3 isoform is neuron-specific. Mutations in ATP1A2 gene can result in familial hemiplegic migraine (FHM2, while mutations in the ATP1A3 gene can cause Rapid-onset dystonia-Parkinsonism (RDP and alternating hemiplegia of childhood (AHC, as well as the cerebellar ataxia, areflexia, pescavus, optic atrophy and sensorineural hearing loss (CAPOS syndrome. Data indicates that the central glutamatergic system is affected by mutations in the α2 isoform, however further investigations are required to establish a connection to mutations in the α3 isoform, especially given the diagnostic confusion and overlap with glutamate transporter disease. The age-related decline in brain α2/3 activity may arise from changes in the cyclic guanosine monophosphate (cGMP and cGMP‐dependent protein kinase (PKG pathway. Glutamate, through nitric oxide synthase (NOS, cGMP and PKG, stimulates brain α2/3 activity, with the glutamatergic N-methyl-D-aspartate (NMDA receptor cascade able to drive an adaptive, neuroprotective response to inflammatory and challenging stimuli, including amyloid‐β. Here we review the NKA, both as an ion pump as well as a receptor that interacts with NMDA, including the role of NKA subunits mutations. Failure of the NKA-associated adaptive response mechanisms may render neurons more susceptible to degeneration over the course of aging.

  17. Changes in Phytochemical Synthesis, Chalcone Synthase Activity and Pharmaceutical Qualities of Sabah Snake Grass (Clinacanthus nutans L. in Relation to Plant Age

    Directory of Open Access Journals (Sweden)

    Ali Ghasemzadeh

    2014-10-01

    Full Text Available In the current study, changes in secondary metabolite synthesis and the pharmaceutical quality of sabah snake grass leaves and buds were considered in relation to plant age (1 month, 6 months, and 1 year old. The activity of the enzyme chalcone synthase (CHS, EC 2.3.1.74 was measured, as it is a key enzyme for flavonoid production. Significant differences in total flavonoid (TF production were observed between the three plant growth periods and the different plant parts. The highest contents of TF (6.32 mg/g dry weight [DW] and total phenolic (TP (18.21 mg/g DW were recorded in 6-month-old buds. Among the flavonoids isolated in this study the most important ones based on concentration were from high to low as follows: catechin > quercetin > kaempferol > luteolin. Production of phenolic acids increased from 1 to 6 months, but after 6 months up to 1 year of age, they decreased significantly. The highest contents of caffeic acid (0.307 mg/g DW and gallic acid (5.96 mg/g DW were recorded in 1-year and 6-month-old buds, respectively. The lowest and highest activity of CHS was recorded in 1-month and 6-month-old buds with values of 3.6 and 9.5 nkat/mg protein, respectively. These results indicate that the increment in flavonoids and phenolic acids in 6-month-old buds can be attributed to an increase in CHS activity. The highest 1,1-diphenyl-2-picrylhydrazyl (DPPH activity was observed in the extract of 1-year-old buds followed by 6-month-old buds, with 50% of free radical scavenging (IC50 values of 64.6 and 73.5 µg/mL, respectively. Interestingly, a ferric reducing antioxidant power (FRAP assay showed a higher activity in 6-month-old buds (488 μM of Fe(II/g than in 1-year-old buds (453 μM of Fe(II/g, in contrast to the DPPH result. Significant correlations (p < 0.05 were observed between CHS enzyme activity and FRAP activity, TF, catechin, and kaempferol content. Extracts of 6-month-old bud exhibited a significant in vitro anticancer activity

  18. Posttranslational Modification Biology of Glutamate Receptors and Drug Addiction

    Directory of Open Access Journals (Sweden)

    Li-Min eMao

    2011-03-01

    Full Text Available Posttranslational covalent modifications of glutamate receptors remain a hot topic. Early studies have established that this family of receptors, including almost all ionotropic and metabotropic glutamate receptor subtypes, undergoes active phosphorylation at serine, threonine, or tyrosine residues on their intracellular domains. Recent evidence identifies several glutamate receptor subtypes to be direct substrates for palmitoylation at cysteine residues. Other modifications such as ubiquitination and sumoylation at lysine residues also occur to certain glutamate receptors. These modifications are dynamic and reversible in nature and are regulatable by changing synaptic inputs. The regulated modifications significantly impact the receptor in many ways, including interrelated changes in biochemistry (synthesis, subunit assembling and protein-protein interactions, subcellular redistribution (trafficking, endocytosis, synaptic delivery and clustering, and physiology, usually associated with changes in synaptic plasticity. Glutamate receptors are enriched in the striatum and cooperate closely with dopamine to regulate striatal signaling. Emerging evidence shows that modification processes of striatal glutamate receptors are sensitive to addictive drugs, such as psychostimulants (cocaine and amphetamines. Altered modifications are believed to be directly linked to enduring receptor/synaptic plasticity and drug-seeking. This review summarizes several major types of modifications of glutamate receptors and analyzes the role of these modifications in striatal signaling and in the pathogenesis of psychostimulant addiction.

  19. Food Application of Newly Developed Handy-type Glutamate Sensor.

    Science.gov (United States)

    Mukai, Yuuka; Oikawa, Tsutomu

    2016-01-01

    Tests on physiological functions of umami have been actively conducted and a need recognized for a high-performance quantification device that is simple and cost-effective, and whose use is not limited to a particular location or user. To address this need, Ajinomoto Co. and Tanita Corp. have jointly been researching and developing a simple device for glutamate measurement. The device uses L-glutamate oxidase immobilized on a hydrogen peroxide electrode. L-glutamate in the sample is converted to α-ketoglutaric acid, which produces hydrogen peroxide. Subsequently, the electrical current from the electrochemical reaction of hydrogen peroxide is measured to determine the L-glutamate concentration. In order to evaluate its basic performance, we used this device to measure the concentration of L-glutamate standard solutions. In a concentration range of 0-1.0%, the difference from the theoretical value was minimal. The coefficient of variation (CV) value of 3 measurements was 4% or less. This shows that the device has a reasonable level of precision and accuracy. The device was also used in trial measurements of L-glutamate concentrations in food. There was a good correlation between the results obtained using the developed device and those obtained with an amino acid analyzer; the correlation coefficient was R=0.997 (n=24). In this review, we demonstrate the use of our device to measure the glutamate concentration in miso soup served daily at a home for elderly people, and other foods and ingredients.

  20. High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase, and arginine decarboxylase activities

    DEFF Research Database (Denmark)

    Volke, A; Wegener, Gregers; Vasar, E

    2006-01-01

    regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here...

  1. Identification of protein phosphatase involvement in the AT-receptor induced activation of endothelial nitric oxide synthase

    DEFF Research Database (Denmark)

    Peluso, A Augusto; Bertelsen, Jesper Bork; Andersen, Kenneth

    2018-01-01

    -antagonist), L-NAME (10µM; eNOS inhibitor), MK-2206 (100nM; Akt-inhibitor) sodium fluoride (1nM; serine/threonine-phosphatase inhibitor) or sodium orthovanadate (10nM; tyrosine-phosphatase inhibitor). NO release was estimated by quantifying DAF-FM fluorescence. The phosphorylation status of activating (e...

  2. Cobalamin-Independent Methionine Synthase (MetE): A Face-to-Face Double Barrel that Evolved by Gene Duplication

    Energy Technology Data Exchange (ETDEWEB)

    Pejcha, Robert; Ludwig, Martha L. (Michigan)

    2010-03-08

    Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy) without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two ({beta}{alpha}){sub 8} barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys){sub 3}Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E {center_dot} Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.

  3. Cobalamin-Independent Methionine Synthase (MetE): A Face-to-Face Double Barrel that Evolved by Gene Duplication

    International Nuclear Information System (INIS)

    Pejcha, Robert; Ludwig, Martha L.

    2005-01-01

    Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy) without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two (βα) 8 barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys) 3 Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E · Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.

  4. Cobalamin-independent methionine synthase (MetE: a face-to-face double barrel that evolved by gene duplication.

    Directory of Open Access Journals (Sweden)

    Robert Pejchal

    2005-02-01

    Full Text Available Cobalamin-independent methionine synthase (MetE catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH, both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two (betaalpha(8 barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys(3Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E.Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.

  5. Metabolic control of vesicular glutamate transport and release.

    Science.gov (United States)

    Juge, Narinobu; Gray, John A; Omote, Hiroshi; Miyaji, Takaaki; Inoue, Tsuyoshi; Hara, Chiaki; Uneyama, Hisayuki; Edwards, Robert H; Nicoll, Roger A; Moriyama, Yoshinori

    2010-10-06

    Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl(-). Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl(-) acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with Cl(-) at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity. Copyright © 2010 Elsevier Inc. All rights reserved.

  6. Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

    Science.gov (United States)

    Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Regulatory role of tumor necrosis factor receptor-associated factor 6 in breast cancer by activating the protein kinase B/glycogen synthase kinase 3β signaling pathway.

    Science.gov (United States)

    Shen, Hongyu; Li, Liangpeng; Yang, Sujin; Wang, Dandan; Zhou, Siying; Chen, Xiu; Tang, Jinhai

    2017-08-01

    Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an endogenous adaptor of innate and adaptive immune responses, and serves a crucial role in tumor necrosis factor receptor and toll‑like/interleukin‑1 receptor signaling. Although studies have demonstrated that TRAF6 has oncogenic activity, its potential contributions to breast cancer in human remains largely uninvestigated. The present study examined the expression levels and function of TRAF6 in breast carcinoma (n=32) and adjacent healthy (n=25) tissue samples. Compared with adjacent healthy tissues, TRAF6 protein expression levels were significantly upregulated in breast cancer tissues. Reverse transcription‑quantitative polymerase chain reaction analysis revealed a significant upregulation of the cellular proliferative marker Ki‑67 and proliferation cell nuclear antigen expression levels in breast carcinoma specimens. Furthermore, protein expression levels of the accessory molecule, transforming growth factor β‑activated kinase 1 (TAK1), were significantly increased in breast cancer patients, as detected by western blot analysis. As determined by MTT assay, TRAF6 exerted profoundly proliferative effects in the MCF‑7 breast cancer cell line; however, these detrimental effects were ameliorated by TAK1 inhibition. Notably, protein kinase B (AKT)/glycogen synthase kinase (GSK)3β phosphorylation levels were markedly upregulated in breast cancer samples, compared with adjacent healthy tissues. In conclusion, an altered TRAF6‑TAK1 axis and its corresponding downstream AKT/GSK3β signaling molecules may contribute to breast cancer progression. Therefore, TRAF6 may represent a potential therapeutic target for the treatment of breast cancer.

  8. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling.

    Science.gov (United States)

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2015-01-15

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation. Copyright © 2015 by The American Association of Immunologists, Inc.

  9. Discovery and characterization of a novel lachrymatory factor synthase in Petiveria alliacea and its influence on alliinase-mediated formation of biologically active organosulfur compounds.

    Science.gov (United States)

    Musah, Rabi A; He, Quan; Kubec, Roman

    2009-11-01

    A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is a heterotetrameric glycoprotein comprised of two alpha-subunits (68.8 kD each), one gamma-subunit (22.5 kD), and one delta-subunit (11.9 kD). The two alpha-subunits are glycosylated and connected by a disulfide bridge. The LFS has an isoelectric point of 5.2. It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-l-cysteine sulfoxide (petiveriin). Depending on its concentration relative to that of P. alliacea alliinase, the LFS sequesters, to varying degrees, the sulfenic acid intermediate formed by alliinase-mediated breakdown of petiveriin. At LFS:alliinase of 5:1, LFS sequesters all of the sulfenic acid formed by alliinase action on petiveriin, and converts it entirely to (Z)-phenylmethanethial S-oxide. However, starting at LFS:alliinase of 5:2, the LFS is unable to sequester all of the sulfenic acid produced by the alliinase, with the result that sulfenic acid that escapes the action of the LFS condenses with loss of water to form S-benzyl phenylmethanethiosulfinate (petivericin). The results show that the LFS and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the LFS acts. The results also show that the LFS modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the LFS and the alliinase. These observations suggest that manipulation of LFS-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules.

  10. Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486.

    Science.gov (United States)

    Kalandadze, Avtandil; Wu, Ying; Robinson, Michael B

    2002-11-29

    Na(+)-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475-2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475-517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of (32)P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475-517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtype-specific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an

  11. Increased killing of SCCVII squamous cell carcinoma cells after the combination of Pc 4 photodynamic therapy and dasatinib is associated with enhanced caspase-3 activity and ceramide synthase 1 upregulation

    Science.gov (United States)

    SEPAROVIC, DUSKA; BREEN, PAUL; BOPPANA, NITHIN B.; VAN BUREN, ERIC; JOSEPH, NICHOLAS; KRAVEKA, JACQUELINE M.; RAHMANIYAN, MEHRDAD; LI, LI; GUDZ, TATYANA I.; BIELAWSKA, ALICJA; BAI, AIPING; BIELAWSKI, JACEK; PIERCE, JASON S.; KORBELIK, MLADEN

    2013-01-01

    Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1-and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of

  12. Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

    Science.gov (United States)

    Brew, Helen; Attwell, David

    1987-06-01

    Glutamate is taken up avidly by glial cells in the central nervous system1. Glutamate uptake may terminate the transmitter action of glutamate released from neurons1, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique2. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.

  13. HAEM SYNTHASE AND COBALT PORPHYRIN SYNTHASE IN VARIOUS MICRO-ORGANISMS.

    Science.gov (United States)

    PORRA, R J; ROSS, B D

    1965-03-01

    1. The preparation of a crude extract of Clostridium tetanomorphum containing cobalt porphyrin synthase but little haem-synthase activity is described. 2. The properties of cobalt porphyrin synthase in the clostridial extracts is compared with the properties of a haem synthase present in crude extracts of the yeast Torulopsis utilis. 3. Cobalt porphyrin synthase in extracts of C. tetanomorphum inserts Co(2+) ions into the following dicarboxylic porphyrins in descending order of rate of insertion: meso-, deutero- and proto-porphyrins. Esterification renders meso- and deutero-porphyrins inactive as substrates. Neither the tetracarboxylic (coproporphyrin III) nor the octacarboxylic (uroporphyrin III) compounds are converted into cobalt porphyrins by the extract, but the non-enzymic incorporation of Co(2+) ions into these two porphyrins is rapid. These extracts are unable to insert Mn(2+), Zn(2+), Mg(2+) or Cu(2+) ions into mesoporphyrin. 4. Crude extracts of T. utilis readily insert both Co(2+) and Fe(2+) ions into deutero-, meso, and proto-porphyrins. Unlike the extracts of C. tetanomorphum, these preparations catalyse the insertion of Co(2+) ions into deuteroporphyrin more rapidly than into mesoporphyrin. This parallels the formation of haems by the T. utilis extract. 5. Cobalt porphyrin synthase is present in the particulate fraction of the extracts of C. tetanomorphum but requires a heat-stable factor present in the soluble fraction. This soluble factor can be replaced by GSH. 6. Cobalt porphyrin synthase in the clostridial extract is inhibited by iodoacetamide and to a smaller extent by p-chloromercuribenzoate and N-ethylmaleimide. The haem synthases of T. utilis and Micrococcus denitrificans are also inhibited by various thiol reagents.

  14. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    cells were quantitated using enzyme immunometric assays. The activity of GSK-3β (serine-9-phosphorylated GSK-3β/total GSK-3β) was lower at baseline compared with follow-up. No significant mean change over time was observed in levels of total GSK-3β and serine-9-phosphorylated GSK-3β. Exploratory......Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...... with emotional lability, subjective mood fluctuations and cognitive function in healthy individuals. Thirty-seven healthy subjects were evaluated with neuropsychological tests and blood samples at baseline and 12-week follow-up. Total GSK-3β and serine-9-phosphorylated GSK-3β in peripheral blood mononuclear...

  15. Inhibition of the Mitochondrial Glutamate Carrier SLC25A22 in Astrocytes Leads to Intracellular Glutamate Accumulation

    Directory of Open Access Journals (Sweden)

    Emmanuelle Goubert

    2017-05-01

    Full Text Available The solute carrier family 25 (SLC25 drives the import of a large diversity of metabolites into mitochondria, a key cellular structure involved in many metabolic functions. Mutations of the mitochondrial glutamate carrier SLC25A22 (also named GC1 have been identified in early epileptic encephalopathy (EEE and migrating partial seizures in infancy (MPSI but the pathophysiological mechanism of GC1 deficiency is still unknown, hampered by the absence of an in vivo model. This carrier is mainly expressed in astrocytes and is the principal gate for glutamate entry into mitochondria. A sufficient supply of energy is essential for the proper function of the brain and mitochondria have a pivotal role in maintaining energy homeostasis. In this work, we wanted to study the consequences of GC1 absence in an in vitro model in order to understand if glutamate catabolism and/or mitochondrial function could be affected. First, short hairpin RNA (shRNA designed to specifically silence GC1 were validated in rat C6 glioma cells. Silencing GC1 in C6 resulted in a reduction of the GC1 mRNA combined with a decrease of the mitochondrial glutamate carrier activity. Then, primary astrocyte cultures were prepared and transfected with shRNA-GC1 or mismatch-RNA (mmRNA constructs using the Neon® Transfection System in order to target a high number of primary astrocytes, more than 64%. Silencing GC1 in primary astrocytes resulted in a reduced nicotinamide adenine dinucleotide (Phosphate (NAD(PH formation upon glutamate stimulation. We also observed that the mitochondrial respiratory chain (MRC was functional after glucose stimulation but not activated by glutamate, resulting in a lower level of cellular adenosine triphosphate (ATP in silenced astrocytes compared to control cells. Moreover, GC1 inactivation resulted in an intracellular glutamate accumulation. Our results show that mitochondrial glutamate transport via GC1 is important in sustaining glutamate homeostasis in

  16. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

    Science.gov (United States)

    Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Jenkins, Jermaine L; Heimiller, Chelsea; Maines, Mahin D

    2016-08-01

    Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner

  17. 4G/5G Variant of Plasminogen Activator Inhibitor-1 Gene and Severe Pregnancy-Induced Hypertension: Subgroup Analyses of Variants of Angiotensinogen and Endothelial Nitric Oxide Synthase

    Science.gov (United States)

    Kobashi, Gen; Ohta, Kaori; Yamada, Hideto; Hata, Akira; Minakami, Hisanori; Sakuragi, Noriaki; Tamashiro, Hiko; Fujimoto, Seiichiro

    2009-01-01

    Background Pregnancy-induced hypertension (PIH) is a common cause of perinatal mortality. It is believed to result from the interaction of several factors, including those related to the blood coagulation system. We performed genotyping and subgroup analyses to determine if the 4G/5G genotypes of the plasminogen activator inhibitor-1 gene (PAI-1) play a role in the pathogenesis of PIH, and to evaluate possible interactions of the PAI-1 polymorphisms with those of the angiotensinogen gene (AGT) and the endothelial nitric oxide synthase gene (NOS3). Methods An association study of PAI-1 polymorphism, and subgroup analyses of common variants of AGT and NOS3, among 128 patients with PIH and 376 healthy pregnant controls. Results No significant differences were found between the cases and controls in the frequencies of allele 4G or the 4G/4G genotype. In subgroup analyses, after adjustment for multiple comparison, a significant association with the AGT TT genotype was found among women with the PAI-1 4G/4G genotype, and an association with the NOS3 GA+AA genotype was found among women with the 5G/5G or 4G/5G genotypes. Conclusions Our findings suggest that there are at least 2 pathways in the pathogenesis of severe PIH. However, with respect to early prediction and prevention of severe PIH, although the PAI-1 4G/4G genotype alone was not a risk factor for severe PIH, the fact that PAI-1 genotypes are associated with varying risks for severe PIH suggests that PAI-1 genotyping of pregnant women, in combination with other tests, may be useful in the development of individualized measures that may prevent severe PIH. PMID:19838007

  18. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

    Energy Technology Data Exchange (ETDEWEB)

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C [UWASH

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  19. Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH.

    Science.gov (United States)

    Broadbent, J R; Oberg, T S; Hughes, J E; Ward, R E; Brighton, C; Welker, D L; Steele, J L

    2014-03-01

    Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid's pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH₄OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.

  20. Effects of tumor necrosis factor alpha antagonist, platelet activating factor antagonist, and nitric oxide synthase inhibitor on experimental otitis media with effusion.

    Science.gov (United States)

    Kim, Dong-Hyun; Park, Yong-Soo; Jeon, Eun-Ju; Yeo, Sang-Won; Chang, Ki-Hong; Lee, Seung Kyun

    2006-08-01

    We studied the inflammatory responses in otitis media with effusion induced by lipopolysaccharide (LPS) in rats, and compared the preventive effects of tumor necrosis factor (TNF) soluble receptor type I (sTNFRI, a TNF-alpha antagonist), platelet activating factor antagonist, and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). We used 2 control groups of Sprague Dawley rats (untreated and saline-treated) and 4 experimental groups, which all received an intratympanic injection of LPS, followed in 3 groups by experimental treatment of the same ear. The LPS group had no additional treatment. The L-NAME group received intraperitoneal injection of L-NAME and was reinjected after 12 hours. The A-85783 group was first given an intraperitoneal injection of A-85783. The sTNFRI group was first given an intratympanic injection of sTNFRI. Twenty-four hours after the initial intratympanic injection of LPS, temporal bones from each group were examined histopathologically and the vascular permeability of the middle ear mucosa was measured by Evans blue vital dye staining. The L-NAME, A-85783, and sTNFRI groups showed significantly reduced capillary permeability, subepithelial edema, and infiltration of inflammatory cells in comparison with the LPS group. There were no differences in capillary permeability, subepithelial edema, or infiltration of inflammatory cells between the A-85783 and sTNFRI groups. The L-NAME group showed no difference in vascular permeability or subepithelial edema in comparison with the A-85783 and sTNFRI groups, but showed more infiltration of inflammatory cells. We conclude that sTNFRI, A-85783, and L-NAME can be proposed as alternative future treatments for otitis media with effusion. However, L-NAME may be the least effective of these agents.

  1. Glycogen synthase kinase 3 beta alters anxiety-, depression-, and addiction-related behaviors and neuronal activity in the nucleus accumbens shell

    Science.gov (United States)

    Crofton, Elizabeth J.; Nenov, Miroslav N.; Zhang, Yafang; Scala, Federico; Page, Sean A.; McCue, David L.; Li, Dingge; Hommel, Jonathan D.; Laezza, Fernanda; Green, Thomas A.

    2017-01-01

    Psychiatric disorders such as anxiety, depression and addiction are often comorbid brain pathologies thought to share common mechanistic biology. As part of the cortico-limbic circuit, the nucleus accumbens shell (NAcSh) plays a fundamental role in integrating information in the circuit, such that modulation of NAcSh circuitry alters anxiety, depression, and addiction-related behaviors. Intracellular kinase cascades in the NAcSh have proven important mediators of behavior. To investigate glycogen-synthase kinase 3 (GSK3) beta signaling in the NAcSh in vivo we knocked down GSK3beta expression with a novel adeno-associated viral vector (AAV2) and assessed changes in anxiety- and depression-like behavior and cocaine self-administration in GSK3beta knockdown rats. GSK3beta knockdown reduced anxiety-like behavior while increasing depression-like behavior and cocaine self-administration. Correlative electrophysiological recordings in acute brain slices were used to assess the effect of AAV-shGSK3beta on spontaneous firing and intrinsic excitability of tonically active interneurons (TANs), cells required for input and output signal integration in the NAcSh and for processing reward-related behaviors. Loose-patch recordings showed that TANs transduced by AAV-shGSK3beta exhibited reduction in tonic firing and increased spike half width. When assessed by whole-cell patch clamp recordings these changes were mirrored by reduction in action potential firing and accompanied by decreased hyperpolarization-induced depolarizing sag potentials, increased action potential current threshold, and decreased maximum rise time. These results suggest that silencing of GSK3beta in the NAcSh increases depression- and addiction-related behavior, possibly by decreasing intrinsic excitability of TANs. However, this study does not rule out contributions from other neuronal sub-types. PMID:28126496

  2. Activation of endothelial nitric oxide synthase by dietary isoflavones: role of NO in Nrf2-mediated antioxidant gene expression.

    Science.gov (United States)

    Mann, Giovanni E; Rowlands, David J; Li, Francois Y L; de Winter, Patricia; Siow, Richard C M

    2007-07-15

    The endothelium plays a key role in the maintenance of vascular homeostasis, and increased oxidative stress in vascular disease leads to reduced nitric oxide bioavailability and impaired endothelium-dependent relaxation of resistance vessels. Although epidemiological evidence suggests that diets containing high amounts of natural antioxidants afford protection against coronary heart disease (CHD), antioxidant supplementation trials have largely reported only marginal health benefits. There is controversy concerning the cardiovascular benefits of prolonged estrogen/progestin or soy isoflavone therapy for postmenopausal women and patients with an increased risk of CHD. Research on the potential health benefits of soy isoflavones and other polyphenols contained in red wine, green and black tea and dark chocolate developed rapidly during the 1990's, and recent clinical trials and studies in animal models and cultured endothelial cells provide important and novel insights into the mechanisms by which dietary polyphenols afford protection against oxidative stress. In this review, we highlight that NO and reactive oxygen radicals may mediate dietary polyphenol induced activation of Nrf2, which in turn triggers antioxidant response element (ARE) driven transcription of phase II detoxifying and antioxidant defense enzymes in vascular cells.

  3. Exogenous glutamate induces short and long-term potentiation in the rat medial vestibular nuclei.

    Science.gov (United States)

    Grassi, S; Frondaroli, A; Pessia, M; Pettorossi, V E

    2001-08-08

    In rat brain stem slices, high concentrations of exogenous glutamate induce long-term potentiation (LTP) of the field potentials evoked in the medial vestibular nuclei (MVN) by vestibular afferent stimulation. At low concentrations, glutamate can also induce short-term potentiation (STP), indicating that LTP and STP are separate events depending on the level of glutamatergic synapse activation. LTP and STP are prevented by blocking NMDA receptors and nitric oxide (NO) synthesis. Conversely, blocking platelet-activating factor (PAF) and group I metabotropic glutamate receptors only prevents the full development of LTP. Moreover, in the presence of blocking agents, glutamate causes transient inhibition, suggesting that when potentiation is impeded, exogenous glutamate can activate presynaptic mechanisms that reduce glutamate release.

  4. Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

    Directory of Open Access Journals (Sweden)

    Mamaghani Shadi

    2009-04-01

    Full Text Available Abstract Background Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. Methods GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. Results GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-XL, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. Conclusion GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard

  5. Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

    International Nuclear Information System (INIS)

    Mamaghani, Shadi; Patel, Satish; Hedley, David W

    2009-01-01

    Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-X L , and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard chemotherapy agent gemcitabine. This lack of synergy might be

  6. Process-driven bacterial community dynamics are key to cured meat colour formation by coagulase-negative staphylococci via nitrate reductase or nitric oxide synthase activities.

    Science.gov (United States)

    Sánchez Mainar, María; Leroy, Frédéric

    2015-11-06

    The cured colour of European raw fermented meats is usually achieved by nitrate-into-nitrite reduction by coagulase-negative staphylococci (CNS), subsequently generating nitric oxide to form the relatively stable nitrosomyoglobin pigment. The present study aimed at comparing this classical curing procedure, based on nitrate reductase activity, with a potential alternative colour formation mechanism, based on nitric oxide synthase (NOS) activity, under different acidification profiles. To this end, meat models with and without added nitrate were fermented with cultures of an acidifying strain (Lactobacillus sakei CTC 494) and either a nitrate-reducing Staphylococcus carnosus strain or a rare NOS-positive CNS strain (Staphylococcus haemolyticus G110), or by relying on the background microbiota. Satisfactory colour was obtained in the models prepared with added nitrate and S. carnosus. In the presence of nitrate but absence of added CNS, however, cured colour was only obtained when L. sakei CTC 494 was also omitted. This was ascribed to the pH dependency of the emerging CNS background microbiota, selecting for nitrate-reducing Staphylococcus equorum strains at mild acidification conditions but for Staphylococcus saprophyticus strains with poor colour formation capability when the pH decrease was more rapid. This reliance of colour formation on the composition of the background microbiota was further explored by a side experiment, demonstrating the heterogeneity in nitrate reduction of a set of 88 CNS strains from different species. Finally, in all batches prepared with S. haemolyticus G110, colour generation failed as the strain was systematically outcompeted by the background microbiota, even when imposing milder acidification profiles. Thus, when aiming at colour formation through CNS metabolism, technological processing can severely interfere with the composition and functionality of the meat-associated CNS communities, for both nitrate reductase and NOS activities

  7. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  8. Laser-scanning astrocyte mapping reveals increased glutamate-responsive domain size and disrupted maturation of glutamate uptake following neonatal cortical freeze-lesion

    Directory of Open Access Journals (Sweden)

    Mortiz eArmbruster

    2014-09-01

    Full Text Available Astrocytic uptake of glutamate shapes extracellular neurotransmitter dynamics, receptor activation, and synaptogenesis. During development, glutamate transport becomes more robust. How neonatal brain insult affects the functional maturation of glutamate transport remains unanswered. Neonatal brain insult can lead to developmental delays, cognitive losses, and epilepsy; the disruption of glutamate transport is known to cause changes in synaptogenesis, receptor activation, and seizure. Using the neonatal freeze-lesion (FL model, we have investigated how insult affects the maturation of astrocytic glutamate transport. As lesioning occurs on the day of birth, a time when astrocytes are still functionally immature, this model is ideal for identifying changes in astrocyte maturation following insult. Reactive astrocytosis, astrocyte proliferation, and in vitro hyperexcitability are known to occur in this model. To probe astrocyte glutamate transport with better spatial precision we have developed a novel technique, Laser Scanning Astrocyte Mapping (LSAM, which combines glutamate transport current (TC recording from astrocytes with laser scanning glutamate photolysis. LSAM allows us to identify the area from which a single astrocyte can transport glutamate and to quantify spatial heterogeneity in the rate of glutamate clearance kinetics within that domain. Using LSAM, we report that cortical astrocytes have an increased glutamate-responsive area following FL and that TCs have faster decay times in distal, as compared to proximal processes. Furthermore, the developmental shift from GLAST- to GLT-1-dominated clearance is disrupted following FL. These findings introduce a novel method to probe astrocyte glutamate uptake and show that neonatal cortical FL disrupts the functional maturation of cortical astrocytes.

  9. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  10. Mouse glutamate carboxypeptidaseII (GCPII) has a similar enzyme activity and inhibition profile but a different tissue distribution to human GCPII

    Czech Academy of Sciences Publication Activity Database

    Knedlík, Tomáš; Vorlová, Barbora; Navrátil, Václav; Tykvart, Jan; Sedlák, František; Vaculín, Š.; Franěk, M.; Šácha, Pavel; Konvalinka, Jan

    2017-01-01

    Roč. 7, č. 9 (2017), s. 1362-1378 ISSN 2211-5463 R&D Projects: GA ČR(CZ) GA16-02938S; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : glutamate carboxypeptidase II * mouse animal model * neuronal disorders * prostate cancer * prostate-specific membrane antigen Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 2.143, year: 2016 http://onlinelibrary.wiley.com/doi/10.1002/2211-5463.12276/full

  11. Nitric oxide synthase, calcitonin gene-related peptide and NK-1 receptor mechanisms are involved in GTN-induced neuronal activation

    DEFF Research Database (Denmark)

    Ramachandran, Roshni; Bhatt, Deepak Kumar; Ploug, Kenneth Beri

    2014-01-01

    BACKGROUND AND AIM: Infusion of glyceryltrinitrate (GTN), a nitric oxide (NO) donor, in awake, freely moving rats closely mimics a universally accepted human model of migraine and responds to sumatriptan treatment. Here we analyse the effect of nitric oxide synthase (NOS) and calcitonin gene-rela...

  12. The diabetic phenotype is conserved in myotubes established from diabetic subjects: evidence for primary defects in glucose transport and glycogen synthase activity

    DEFF Research Database (Denmark)

    Gaster, Michael; Petersen, Ingrid; Højlund, Kurt

    2002-01-01

    The most well-described defect in the pathophysiology of type 2 diabetes is reduced insulin-mediated glycogen synthesis in skeletal muscles. It is unclear whether this defect is primary or acquired secondary to dyslipidemia, hyperinsulinemia, or hyperglycemia. We determined the glycogen synthase...

  13. Serum Glutamate Is a Predictor for the Diagnosis of Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Gheyath Al Gawwam

    2017-01-01

    Full Text Available One neurotransmitter, glutamate, has been implicated in the autoimmune demyelination seen in multiple sclerosis (MS. Glutamate is present in many tissues in the body, so consideration should be given to whether the serum level of glutamate is likely well correlated with the activity of the disease. This research aimed to compare the serum glutamate levels from patients diagnosed with MS with those from an age-matched control population. A review of this data could shed light upon whether the serum testing of glutamate using Enzyme-Linked Immunosorbent Assay (ELISA is a reliable indicator of MS activity. Serum samples were obtained from 55 patients with different patterns of MS and from 25 healthy adults as a control group. The ELISA technique was used to determine the glutamate levels in the serum samples. The mean serum glutamate level for patients with MS was 1.318±0.543 nmol/ml and that of the controls was 0.873±0.341 nmol/ml. The serum glutamate levels showed an area under the curve via the receiver operating characteristics (ROC of 0.738, which was significant (p value = 0.001. The present study is the first to establish a strong connection between the serum glutamate levels and MS patients, where there was statistically significant elevation of serum glutamate in MS patients; hence this elevation might be used as a monitor to help in the diagnosis of MS patients.

  14. Thymidylate synthase genetic polymorphism and plasma total homocysteine level in a group of Turkish patients with rheumatoid arthritis: relationship with disease activity and methotrexate toxicity.

    Science.gov (United States)

    Borman, Pınar; Taşbaş, Özgur; Karabulut, Halil; Tukun, Ajlan; Yorgancıoğlu, Rezan

    2015-01-01

    The polymorphism of thymidylate synthase (TS) gene and homocysteine are reported to have a relationship to methotrexate (MTX) metabolism, with conflicting results. The aim of this study was to determine homocysteine levels and the frequency of TS gene triple repeat (TS3R) and double repeat (TS2R) polymorphisms in a group of Turkish RA patients and evaluate its association with MTX toxicity and disease activity. Sixty-four patients with RA and 31 control subjects with a mean age of 48.7 ± 12.5 and 46.2 ± 13.4 years, were enrolled to the study. Demographic characteristics were obtained and number of patients with MTX-related adverse affects, were recorded in the patient group. The homocysteine levels and TS2R/TS3R polymorphisms of the TS gene were analyzed and the distribution of genotypes according to MTX toxicity and disease activity, were determined. The demographic properties were similar between the patient and control subjects. Folic acid supplementation with a mean dose of 5mg folic acid/week, was present in all patients. Thirty-six of the 64 patients showed adverse effects to MTX treatment. The frequency of TS2R and TS3R polymorphisms were found to be similar in the patient and control groups. TS2R and TS3R gene polymorphisms were found to be similar in patients with and without MTX-related adverse events. The mean homocysteine level was also similar in patients with and without TS gene polymorphism, but was found to be higher (12.45μmol/L vs 10.7μmol/L) in patients with MTX-related side effects than in patients without side effects. The mean level of homocysteine was correlated with levels of ESR in the patient group. In conclusion, homocysteine levels might effect the disease activity and toxicity of MTX but 2R and 3R polymorphisms in the TS gene, were not related with MTX-related toxicity in RA patients receiving folate supplementation. Further studies are needed to illuminate the polymorphisms in other enzymes that might be responsible from the MTX

  15. Glucose replaces glutamate as energy substrate to fuel glutamate uptake in glutamate dehydrogenase-deficient astrocytes

    DEFF Research Database (Denmark)

    Pajęcka, Kamilla; Nissen, Jakob D; Stridh, Malin H

    2015-01-01

    -500 µM) in the presence or in the absence of glucose, the metabolism of these substrates was studied by using tritiated glutamate or 2-deoxyglucose as tracers. In addition, the cellular contents of glutamate and ATP were determined. The astrocytes were able to maintain physiological levels of ATP...... regardless of the expression level of GDH and the incubation condition, indicating a high degree of flexibility with regard to regulatory mechanisms involved in maintaining an adequate energy level in the cells. Glutamate uptake was found to be increased in these cells when exposed to increasing levels...

  16. Circadian Regulation of Glutamate Transporters

    Directory of Open Access Journals (Sweden)

    Donají Chi-Castañeda

    2018-06-01

    Full Text Available L-glutamate is the major excitatory amino acid in the mammalian central nervous system (CNS. This neurotransmitter is essential for higher brain functions such as learning, cognition and memory. A tight regulation of extra-synaptic glutamate levels is needed to prevent a neurotoxic insult. Glutamate removal from the synaptic cleft is carried out by a family of sodium-dependent high-affinity transporters, collectively known as excitatory amino acid transporters. Dysfunction of glutamate transporters is generally involved in acute neuronal injury and neurodegenerative diseases, so characterizing and understanding the mechanisms that lead to the development of these disorders is an important goal in the design of novel treatments for the neurodegenerative diseases. Increasing evidence indicates glutamate transporters are controlled by the circadian system in direct and indirect manners, so in this contribution we focus on the mechanisms of circadian regulation (transcriptional, translational, post-translational and post-transcriptional regulation of glutamate transport in neuronal and glial cells, and their consequence in brain function.

  17. GDH-Dependent Glutamate Oxidation in the Brain Dictates Peripheral Energy Substrate Distribution

    DEFF Research Database (Denmark)

    Karaca, Melis; Frigerio, Francesca; Migrenne, Stephanie

    2015-01-01

    in a central energy-deprivation state with increased ADP/ATP ratios and phospho-AMPK in the hypothalamus. This induced changes in the autonomous nervous system balance, with increased sympathetic activity promoting hepatic glucose production and mobilization of substrates reshaping peripheral energy stores...... glutamate dehydrogenase (GDH) activity. Here, we investigated the significance of glutamate as energy substrate for the brain. Upon glutamate exposure, astrocytes generated ATP in a GDH-dependent way. The observed lack of glutamate oxidation in brain-specific GDH null CnsGlud1(-/-) mice resulted...

  18. Protein kinase A-dependent Neuronal Nitric Oxide Synthase Activation Mediates the Enhancement of Baroreflex Response by Adrenomedullin in the Nucleus Tractus Solitarii of Rats

    Directory of Open Access Journals (Sweden)

    Ho I-Chun

    2011-05-01

    Full Text Available Abstract Background Adrenomedullin (ADM exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA, or neuronal nitric oxide synthase (nNOS. We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS. Methods Intravenous injection of phenylephrine was administered to evoke the BRR before and at 10, 30, and 60 min after microinjection of the test agents into NTS of Sprague-Dawley rats. Western blotting analysis was used to measure the level and phosphorylation of proteins that involved in BRR-enhancing effects of ADM (0.2 pmol in NTS. The colocalization of PKA and nNOS was examined by immunohistochemical staining and observed with a laser confocal microscope. Results We found that ADM-induced enhancement of BRR response was blunted by microinjection of NPLA or Rp-8-Br-cGMP, a selective inhibitor of nNOS or protein kinase G (PKG respectively, into NTS. Western blot analysis further revealed that ADM induced an increase in the protein level of PKG-I which could be attenuated by co-microinjection with the ADM receptor antagonist ADM22-52 or NPLA. Moreover, we observed an increase in phosphorylation at Ser1416 of nNOS at 10, 30, and 60 min after intra-NTS administration of ADM. As such, nNOS/PKG signaling may also account for the enhancing effect of ADM on BRR response. Interestingly, biochemical evidence further showed that ADM-induced increase of nNOS phosphorylation was prevented by co-microinjection with Rp-8-Br-cAMP, a PKA inhibitor. The possibility of PKA-dependent nNOS activation was substantiated by immunohistochemical demonstration of co-localization of PKA and nNOS in putative NTS neurons. Conclusions The novel finding of this study is that the signal transduction cascade that

  19. Crystal structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase of riboflavin biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Calabrese, J.C.; Wawrzak, Z.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    2010-03-05

    3,4-Dihydroxy-2-butanone-4-phosphate synthase catalyzes a commitment step in the biosynthesis of riboflavin. On the enzyme, ribulose 5-phosphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate and formate in steps involving enolization, ketonization, dehydration, skeleton rearrangement, and formate elimination. The enzyme is absent in humans and an attractive target for the discovery of antimicrobials for pathogens incapable of acquiring sufficient riboflavin from their hosts. The homodimer of 23 kDa subunits requires Mg{sup 2+} for activity. The first three-dimensional structure of the enzyme was determined at 1.4 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on Escherichia coli protein crystals containing gold. The protein consists of an {alpha} + {beta} fold having a complex linkage of {beta} strands. Intersubunit contacts are mediated by numerous hydrophobic interactions and three hydrogen bond networks. A proposed active site was identified on the basis of amino acid residues that are conserved among the enzyme from 19 species. There are two well-separated active sites per dimer, each of which comprise residues from both subunits. In addition to three arginines and two threonines, which may be used for recognizing the phosphate group of the substrate, the active site consists of three glutamates, two aspartates, two histidines, and a cysteine which may provide the means for general acid and base catalysis and for coordinating the Mg{sup 2+} cofactor within the active site.

  20. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    Directory of Open Access Journals (Sweden)

    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  1. Effects of pentylenetetrazole and glutamate on metabolism of [U-(13)C]glucose in cultured cerebellar granule neurons.

    Science.gov (United States)

    Eloqayli, Haytham; Qu, Hong; Unsgård, Geirmund; Sletvold, Olav; Hadidi, Hakam; Sonnewald, Ursula

    2002-02-01

    This study was performed to analyze the effects of glutamate and the epileptogenic agent pentylenetetrazole (PTZ) on neuronal glucose metabolism. Cerebellar granule neurons were incubated for 2 h in medium containing 3 mM [U-(13)C]glucose, with and without 0.25 mM glutamate and/or 10 mM PTZ. In the presence of PTZ, decreased glucose consumption with unchanged lactate release was observed, indicating decreased glucose oxidation. PTZ also slowed down tricarboxylic acid (TCA) cycle activity as evidenced by the decreased amounts of labeled aspartate and [1,2-(13)C]glutamate. When glutamate was present, glucose consumption was also decreased. However, the amount of glutamate, derived from [U-(13)C]glucose via the first turn of the TCA cycle, was increased. The decreased amount of [1,2-(13)C]glutamate, derived from the second turn in the TCA cycle, and increased amount of aspartate indicated the dilution of label due to the entrance of unlabeled glutamate into TCA cycle. In the presence of glutamate plus PTZ, the effect of PTZ was enhanced by glutamate. Labeled alanine was detected only in the presence of glutamate plus PTZ, which indicated that oxaloacetate was a better amino acid acceptor than pyruvate. Furthermore, there was also evidence for intracellular compartmentation of oxaloacetate metabolism. Glutamate and PTZ caused similar metabolic changes, however, via different mechanisms. Glutamate substituted for glucose as energy substrate in the TCA cycle, whereas, PTZ appeared to decrease mitochondrial activity.

  2. Immunochemical characterization of the brain glutamate binding protein

    International Nuclear Information System (INIS)

    Roy, S.

    1986-01-01

    A glutamate binding protein (GBP) was purified from bovine and rat brain to near homogeneity. Polyclonal antibodies were raised against this protein. An enzyme-linked-immunosorbent-assay was used to quantify and determine the specificity of the antibody response. The antibodies were shown to strongly react with bovine brain GBP and the analogous protein from rat brain. The antibodies did not show any crossreactivity with the glutamate metabolizing enzymes, glutamate dehydrogenase, glutamine synthetase and glutamyl transpeptidase, however it crossreacted moderately with glutamate decarboxylase. The antibodies were also used to define the possible physiologic activity of GBP in synaptic membranes. The antibodies were shown: (i) to inhibit the excitatory amino-acid stimulation of thiocyanate (SCN)flux, (ii) had no effect on transport of L-Glutamic acid across the synaptic membrane, and (iii) had no effect on the depolarization-induced release of L-glutamate. When the anti-GBP antibodies were used to localize and quantify the GBP distribution in various subcellular fractions and in brain tissue samples, it was found that the hippocampus had the highest immunoreactivity followed by the cerebral cortex, cerebellar cortex and caudate-putamen. The distribution of immunoreactivity in the subcellular fraction were as follows: synaptic membranes > crude mitochondrial fraction > homogenate > myelin. In conclusion these studies suggest that: (a) the rat brain GBP and the bovine brain GBP are immunologically homologous protein, (b) there are no structural similarities between the GBP and the glutamate metabolizing enzymes with the exception of glutamate decarboxylase and (c) the subcellular and regional distribution of the GBP immunoreactivity followed a similar pattern as observed for L-[ 3 H]-binding

  3. Morphine Protects Spinal Cord Astrocytes from Glutamate-Induced Apoptosis via Reducing Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Chao Zhang

    2016-10-01

    Full Text Available Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS. Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca2+ release, thereby inducing endoplasmic reticulum (ER stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca2+ release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca2+ overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca2+ release and ER stress.

  4. Glutamate may be an efferent transmitter that elicits inhibition in mouse taste buds.

    Directory of Open Access Journals (Sweden)

    Yijen A Huang

    Full Text Available Recent studies suggest that l-glutamate may be an efferent transmitter released from axons innervating taste buds. In this report, we determined the types of ionotropic synaptic glutamate receptors present on taste cells and that underlie this postulated efferent transmission. We also studied what effect glutamate exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura 2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings show that a large fraction of Presynaptic (Type III taste bud cells (∼50% respond to 100 µM glutamate, NMDA, or kainic acid (KA with an increase in intracellular Ca(2+. In contrast, Receptor (Type II taste cells rarely (4% responded to 100 µM glutamate. At this concentration and with these compounds, these agonists activate glutamatergic synaptic receptors, not glutamate taste (umami receptors. Moreover, applying glutamate, NMDA, or KA caused taste buds to secrete 5-HT, a Presynaptic taste cell transmitter, but not ATP, a Receptor cell transmitter. Indeed, glutamate-evoked 5-HT release inhibited taste-evoked ATP secretion. The findings are consistent with a role for glutamate in taste buds as an inhibitory efferent transmitter that acts via ionotropic synaptic glutamate receptors.

  5. Glutamate may be an efferent transmitter that elicits inhibition in mouse taste buds.

    Science.gov (United States)

    Huang, Yijen A; Grant, Jeff; Roper, Stephen

    2012-01-01

    Recent studies suggest that l-glutamate may be an efferent transmitter released from axons innervating taste buds. In this report, we determined the types of ionotropic synaptic glutamate receptors present on taste cells and that underlie this postulated efferent transmission. We also studied what effect glutamate exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura 2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings show that a large fraction of Presynaptic (Type III) taste bud cells (∼50%) respond to 100 µM glutamate, NMDA, or kainic acid (KA) with an increase in intracellular Ca(2+). In contrast, Receptor (Type II) taste cells rarely (4%) responded to 100 µM glutamate. At this concentration and with these compounds, these agonists activate glutamatergic synaptic receptors, not glutamate taste (umami) receptors. Moreover, applying glutamate, NMDA, or KA caused taste buds to secrete 5-HT, a Presynaptic taste cell transmitter, but not ATP, a Receptor cell transmitter. Indeed, glutamate-evoked 5-HT release inhibited taste-evoked ATP secretion. The findings are consistent with a role for glutamate in taste buds as an inhibitory efferent transmitter that acts via ionotropic synaptic glutamate receptors.

  6. Enhancing poly-γ-glutamic acid production in Bacillus amyloliquefaciens by introducing the glutamate synthesis features from Corynebacterium glutamicum.

    Science.gov (United States)

    Feng, Jun; Quan, Yufen; Gu, Yanyan; Liu, Fenghong; Huang, Xiaozhong; Shen, Haosheng; Dang, Yulei; Cao, Mingfeng; Gao, Weixia; Lu, Xiaoyun; Wang, Yi; Song, Cunjiang; Wang, Shufang

    2017-05-22

    Poly-γ-glutamic acid (γ-PGA) is a valuable polymer with glutamate as its sole precursor. Enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-PGA production, especially for those glutamate-independent γ-PGA producing strains. Corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate γ-PGA production. In this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent γ-PGA producing Bacillus amyloliquefaciens NK-1 strain. After introducing the energy-saving NADPH-dependent glutamate dehydrogenase (NADPH-GDH) pathway, the NK-1 (pHT315-gdh) strain showed slightly increase (by 9.1%) in γ-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of ɑ-oxoglutarate dehydrogenase complex (ODHC) was introduced into the NK-1 strain, because it was previously shown that the ODHC in C. glutamicum was completely inhibited when glutamate was actively produced. The obtained NK-PO1 (pHT01-xylR) strain showed 66.2% higher γ-PGA production than the NK-1 strain. However, the further combination of these two strategies (introducing both NADPH-GDH pathway and the metabolic toggle switch) did not lead to further increase of γ-PGA production but rather the resultant γ-PGA production was even lower than that in the NK-1 strain. We proposed new metabolic engineering strategies to improve the γ-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in γ-PGA production. The NK-PO1 (pHT01-xylR) strain with the introduction of a metabolic toggle switch for controlling the expression of ODHC showed 66.2% higher

  7. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  8. Evidence that L-glutamate can act as an exogenous signal to modulate root growth and branching in Arabidopsis thaliana.

    Science.gov (United States)

    Walch-Liu, Pia; Liu, Lai-Hua; Remans, Tony; Tester, Mark; Forde, Brian G

    2006-08-01

    The roots of many plant species are known to use inorganic nitrogen, in the form of , as a cue to initiate localized root proliferation within nutrient-rich patches of soil. We report here that, at micromolar concentrations and in a genotype-dependent manner, exogenous l-glutamate is also able to elicit complex changes in Arabidopsis root development. l-Glutamate is perceived specifically at the primary root tip and inhibits mitotic activity in the root apical meristem, but does not interfere with lateral root initiation or outgrowth. Only some time after emergence do lateral roots acquire l-glutamate sensitivity, indicating that their ability to respond to l-glutamate is developmentally regulated. Comparisons between different Arabidopsis ecotypes revealed a remarkable degree of natural variation in l-glutamate sensitivity, with C24 being the most sensitive. The aux1-7 auxin transport mutant had reduced l-glutamate sensitivity, suggesting a possible interaction between l-glutamate and auxin signaling. Surprisingly, two loss-of-function mutants at the AXR1 locus (axr1-3 and axr1-12) were hypersensitive to l-glutamate. A pharmacological approach, using agonists and antagonists of mammalian ionotropic glutamate receptors, was unable to provide evidence of a role for their plant homologs in sensing exogenous glutamate. We discuss the mechanism of l-glutamate sensing and the possible ecological significance of the observed l-glutamate-elicited changes in root architecture.

  9. Dual Effects of TARP γ-2 on Glutamate Efficacy Can Account for AMPA Receptor Autoinactivation

    Directory of Open Access Journals (Sweden)

    Ian D. Coombs

    2017-08-01

    Full Text Available Fast excitatory transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPARs associated with transmembrane AMPAR regulatory proteins (TARPs. At the high glutamate concentrations typically seen during synaptic transmission, TARPs slow receptor desensitization and enhance mean channel conductance. However, their influence on channels gated by low glutamate concentrations, as encountered during delayed transmitter clearance or synaptic spillover, is poorly understood. We report here that TARP γ-2 reduces the ability of low glutamate concentrations to cause AMPAR desensitization and enhances channel gating at low glutamate occupancy. Simulations show that, by shifting the balance between AMPAR activation and desensitization, TARPs can markedly facilitate the transduction of spillover-mediated synaptic signaling. Furthermore, the dual effects of TARPs can account for biphasic steady-state glutamate concentration-response curves—a phenomenon termed “autoinactivation,” previously thought to reflect desensitization-mediated AMPAR/TARP dissociation.

  10. Zinc and glutamate dehydrogenase in putative glutamatergic brain structures.

    Science.gov (United States)

    Wolf, G; Schmidt, W

    1983-01-01

    A certain topographic parallelism between the distribution of histochemically (TIMM staining) identified zinc and putative glutamatergic structures in the rat brain was demonstrated. Glutamate dehydrogenase as a zinc containing protein is in consideration to be an enzyme synthesizing transmitter glutamate. In a low concentration range externally added zinc ions (10(-9) to 10(-7) M) induced an increase in the activity of glutamate dehydrogenase (GDH) originating from rat hippocampal formation, neocortex, and cerebellum up to 142.4%. With rising molarity of Zn(II) in the incubation medium, the enzyme of hippocampal formation and cerebellum showed a biphasic course of activation. Zinc ions of a concentration higher than 10(-6) M caused a strong inhibition of GDH. The effect of Zn(II) on GDH originating from spinal ganglia and liver led only to a decrease of enzyme activity. These results are discussed in connection with a functional correlation between zinc and putatively glutamatergic system.

  11. Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase

    OpenAIRE

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L.; Zillinger, Thomas; Serganov, Artem A.; Liu, Yizhou; Jones, Roger A.; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J.

    2013-01-01

    Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages, designated c[G(2′,5′) pA(3′,5′)p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, res...

  12. Frontal glutamate and reward processing in adolescence and adulthood.

    Science.gov (United States)

    Gleich, Tobias; Lorenz, Robert C; Pöhland, Lydia; Raufelder, Diana; Deserno, Lorenz; Beck, Anne; Heinz, Andreas; Kühn, Simone; Gallinat, Jürgen

    2015-11-01

    The fronto-limbic network interaction, driven by glutamatergic and dopaminergic neurotransmission, represents a core mechanism of motivated behavior and personality traits. Reward seeking behavior undergoes tremendous changes in adolescence paralleled by neurobiological changes of this network including the prefrontal cortex, striatum and amygdala. Since fronto-limbic dysfunctions also underlie major psychiatric diseases beginning in adolescence, this investigation focuses on network characteristics separating adolescents from adults. To investigate differences in network interactions, the brain reward system activity (slot machine task) together with frontal glutamate concentration (anterior cingulate cortex, ACC) was measured in 28 adolescents and 26 adults employing functional magnetic resonance imaging and magnetic resonance spectroscopy, respectively. An inverse coupling of glutamate concentrations in the ACC and activation of the ventral striatum was observed in adolescents. Further, amygdala response in adolescents was negatively correlated with the personality trait impulsivity. For adults, no significant associations of network components or correlations with impulsivity were found. The inverse association between frontal glutamate concentration and striatal activation in adolescents is in line with the triadic model of motivated behavior stressing the important role of frontal top-down inhibition on limbic structures. Our data identified glutamate as the mediating neurotransmitter of this inhibitory process and demonstrates the relevance of glutamate on the reward system and related behavioral traits like impulsivity. This fronto-limbic coupling may represent a vulnerability factor for psychiatric disorders starting in adolescence but not in adulthood.

  13. GABA and glutamate uptake and metabolism in retinal glial (Müller cells

    Directory of Open Access Journals (Sweden)

    Andreas eBringmann

    2013-04-01

    Full Text Available Müller cells, the principal glial cells of the retina, support the synaptic activity by the uptake and metabolization of extracellular neurotransmitters. Müller cells express uptake and exchange systems for various neurotransmitters including glutamate and -aminobutyric acid (GABA. Müller cells remove the bulk of extracellular glutamate in the inner retina and contribute to the glutamate clearance around photoreceptor terminals. By the uptake of glutamate, Müller cells are involved in the shaping and termination of the synaptic activity, particularly in the inner retina. Reactive Müller cells are neuroprotective, e.g., by the clearance of excess extracellular glutamate, but may also contribute to neuronal degeneration by a malfunctioning or even reversal of glial glutamate transporters, or by a downregulation of the key enzyme, glutamine synthetase. This review summarizes the present knowledge about the role of Müller cells in the clearance and metabolization of extracellular glutamate and GABA. Some major pathways of GABA and glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate-glutamine cycle of the retina, in the defense against oxidative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism.

  14. Hydroxychavicol, a Piper betle leaf component, induces apoptosis of CML cells through mitochondrial reactive oxygen species-dependent JNK and endothelial nitric oxide synthase activation and overrides imatinib resistance.

    Science.gov (United States)

    Chakraborty, Jayashree B; Mahato, Sanjit K; Joshi, Kalpana; Shinde, Vaibhav; Rakshit, Srabanti; Biswas, Nabendu; Choudhury Mukherjee, Indrani; Mandal, Labanya; Ganguly, Dipyaman; Chowdhury, Avik A; Chaudhuri, Jaydeep; Paul, Kausik; Pal, Bikas C; Vinayagam, Jayaraman; Pal, Churala; Manna, Anirban; Jaisankar, Parasuraman; Chaudhuri, Utpal; Konar, Aditya; Roy, Siddhartha; Bandyopadhyay, Santu

    2012-01-01

    Alcoholic extract of Piper betle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr-Abl with imatinib resistance phenotype. Hydroxy-chavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti-CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr-Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria-derived reactive oxygen species. Reactive oxygen species-dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase-mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly-adenosine diphosphate-ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr-Abl, without showing significant bodyweight loss. Our data describe the role of JNK-dependent endothelial nitric oxide synthase-mediated nitric oxide for anti-CML activity of HCH and this molecule merits further testing in pre-clinical and clinical settings. © 2011 Japanese Cancer Association.

  15. A new type of Na(+-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

    Directory of Open Access Journals (Sweden)

    Sarah Schulz

    Full Text Available The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F₁F₀-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c₁₁ ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.

  16. The glutamate/GABA-glutamine cycle

    DEFF Research Database (Denmark)

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

    2006-01-01

    Neurons are metabolically handicapped in the sense that they are not able to perform de novo synthesis of neurotransmitter glutamate and gamma-aminobutyric acid (GABA) from glucose. A metabolite shuttle known as the glutamate/GABA-glutamine cycle describes the release of neurotransmitter glutamate...... or GABA from neurons and subsequent uptake into astrocytes. In return, astrocytes release glutamine to be taken up into neurons for use as neurotransmitter precursor. In this review, the basic properties of the glutamate/GABA-glutamine cycle will be discussed, including aspects of transport and metabolism...... of intercellular transfer of ammonia produced in neurons (when glutamine is deamidated to glutamate) and utilized in astrocytes (for amidation of glutamate) when the glutamate/GABA-glutamine cycle is operating. A main objective of this review is to endorse the view that the glutamate/GABA-glutamine cycle must...

  17. Regulation of glycogen synthesis in rat skeletal muscle after glycogen-depleting contractile activity: effects of adrenaline on glycogen synthesis and activation of glycogen synthase and glycogen phosphorylase.

    OpenAIRE

    Franch, J; Aslesen, R; Jensen, J

    1999-01-01

    We investigated the effects of insulin and adrenaline on the rate of glycogen synthesis in skeletal muscles after electrical stimulation in vitro. The contractile activity decreased the glycogen concentration by 62%. After contractile activity, the glycogen stores were fully replenished at a constant and high rate for 3 h when 10 m-i.u./ml insulin was present. In the absence of insulin, only 65% of the initial glycogen stores was replenished. Adrenaline decreased insulin-stimulated glycogen s...

  18. The activity of inducible nitric oxide synthase in rejected skin xenografts is selectively inhibited by a factor produced by grafted cells

    Czech Academy of Sciences Publication Activity Database

    Holáň, Vladimír; Pindjáková, Jana; Zajícová, Alena; Krulová, Magdalena; Železná, Blanka; Matoušek, Petr; Svoboda, Petr

    2005-01-01

    Roč. 12, č. 3 (2005), s. 227-234 ISSN 0908-665X R&D Projects: GA MZd(CZ) NR7816; GA ČR(CZ) GP310/02/D162; GA ČR(CZ) GD310/03/H147; GA MŠk(CZ) ME 300; GA AV ČR KSK5020115 Institutional research plan: CEZ:AV0Z5052915; CEZ:AV0Z50110509 Keywords : inducible nitric oxide synthase production * nitric oxide * suppressive molecule Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.114, year: 2005

  19. Elucidation of the pathways of catabolic glutamate conversion in three thermophilic anaerobic bacteria.

    Science.gov (United States)

    Plugge, C M; van Leeuwen, J M; Hummelen, T; Balk, M; Stams, A J

    2001-07-01

    The glutamate catabolism of three thermophilic syntrophic anaerobes was compared based on the combined use of [(13)C] glutamate NMR measurements and enzyme activity determinations. In some cases the uptake of intermediates from different pathways was studied. The three organisms, Caloramator coolhaasii, Thermanaerovibrio acidaminovorans and strain TGO, had a different stoichiometry of glutamate conversion and were dependent on the presence of a hydrogen scavenger (Methanobacterium thermoautotrophicum Z245) to a different degree for their growth. C. coolhaasii formed acetate, CO(2), NH(4)(+) and H(2) from glutamate. Acetate was found to be formed through the beta-methylaspartate pathway in pure culture as well as in coculture. T. acidaminovorans converted glutamate to acetate, propionate, CO(2), NH(4)(+) and H(2). Most likely, this organism uses the beta-methylaspartate pathway for acetate formation. Propionate formation occurred through a direct oxidation of glutamate via succinyl-CoA and methylmalonyl-CoA. The metabolism of T. acidaminovorans shifted in favour of propionate formation when grown in coculture with the methanogen, but this did not lead to the use of a different glutamate degradation pathway. Strain TGO, an obligate syntrophic glutamate-degrading organism, formed propionate, traces of succinate, CO(2), NH(4)(+) and H(2). Glutamate was converted to propionate oxidatively via the intermediates succinyl-CoA and methylmalonyl-CoA. A minor part of the succinyl-CoA was converted to succinate and excreted.

  20. Effect of parenteral glutamate treatment on the localization of neurotransmitters in the mediobasal hypothalamus

    Energy Technology Data Exchange (ETDEWEB)

    Walaas, I; Fonnum, F

    1978-01-01

    The localization of cholinergic, aminergic and amino acid-ergic neurones in the mediobasal hypothalamus has been studied in normal rat brain and in brains where neurones in nucleus arcuatus were destroyed by repeated administration of 2 mg/g body weight monosodium glutamate to newborn animals. In normal animals acetylcholinesterase staining, choline acetyltransferase and aromatic L-amino acid decarboxylase were concentrated in the median eminence and the arcuate nucleus. Glutamate decarboxylase was concentrated at the boundary between the ventromedial and the arcuate nuclei, with lower activity in the arcuate nucleus and very low activity in the median eminence. Nucleus arcuatus contained an intermediate level of high affinity glutamate uptake. In the lesioned animals, there were significant decreases in choline acetyltransferase, acetylcholinesterase staining and glutamate decarboxylase in the median eminence, whereas choline acetyltransferase activity and acetylcholinesterase staining, but not glutamate decarboxylase activity, were decreased in nucleus arcuatus. Aromatic L-amino acid decarboxylase was unchanged in all regions studied. The high affinity uptakes of glutamate, dopamine and noradrenaline, and the endogenous amino acid levels were also unchanged in the treated animals. The results indicate the existence of acetylcholine- and GABA-containing elements in the tuberoinfundibular tract. They further indicate that the dopamine cells in the arcuate nucleus are less sensitive to the toxic effect of glutamate than other cell types, possibly because they contain less glutamate receptors.

  1. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-α-dependent pathway in human dermal fibroblasts

    International Nuclear Information System (INIS)

    Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2011-01-01

    Highlights: ► Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. ► Adiponectin also increases the phosphorylation of AMPK. ► A pharmacological activator of AMPK increases mRNA levels of PPARα and HAS2. ► Adiponectin-induced HAS2 mRNA expression is blocked by a PPARα antagonist. ► Adiponectin promotes hyaluronan synthesis via an AMPK/PPARα-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1β-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-α (PPARα), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPARα antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPARα-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  2. Thirst is associated with suppression of LHB outputs and active stress coping: Is there a role for a non-canonical vasopressin-glutamate pathway?

    Directory of Open Access Journals (Sweden)

    Limei eZhang

    2016-03-01

    Full Text Available Water-homeostasis is a fundamental physiological process for terrestrial life. In vertebrates, thirst drives water intake, but the neuronal circuits that connect the physiology of water regulation with emotional context are poorly understood. Vasopressin (VP is a prominent messenger in this circuit, as well as L-glutamate. We have investigated the role of a VP circuit and interaction between thirst and motivational behaviors evoked by life-threatening stimuli in rats. We demonstrate a direct pathway from hypothalamic paraventricular VP-expressing, glutamatergic magnocellular neurons to the medial division of lateral habenula (LHbM, a region containing GABAergic neurons. In vivo recording and juxtacellular labeling revealed that GABAergic neurons in the LHbM had locally branching axons, and received VP-positive axon terminal contacts on their dendrites. Water deprivation significantly reduced freezing and immobility behaviors evoked by innate fear and behavioral despair respectively, accompanied by decreased Fos expression in the lateral habenula. Our results reveal a novel VP-expressing hypothalamus to the LHbM circuit that is likely to evoke GABA-mediated inhibition in the LHbM, which promotes escape behavior during stress coping.

  3. 21 CFR 182.1045 - Glutamic acid.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) [Reserved] (c) Limitations, restrictions, or...

  4. Enzymatic production of α-ketoglutaric acid from l-glutamic acid via l-glutamate oxidase.

    Science.gov (United States)

    Niu, Panqing; Dong, Xiaoxiang; Wang, Yuancai; Liu, Liming

    2014-06-10

    In this study, a novel strategy for α-ketoglutaric acid (α-KG) production from l-glutamic acid using recombinant l-glutamate oxidase (LGOX) was developed. First, by analyzing the molecular structure characteristics of l-glutamic acid and α-KG, LGOX was found to be the best catalyst for oxidizing the amino group of l-glutamic acid to a ketonic group without the need for exogenous cofactor. Then the LGOX gene was expressed in Escherichia coli BL21 (DE3) in a soluble and active form, and the recombinant LGOX activity reached to a maximum value of 0.59U/mL at pH 6.5, 30°C. Finally, the maximum α-KG concentration reached 104.7g/L from 110g/L l-glutamic acid in 24h, under the following optimum conditions: 1.5U/mL LGOX, 250U/mL catalase, 3mM MnCl2, 30°C, and pH 6.5. Copyright © 2014. Published by Elsevier B.V.

  5. Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase.

    Directory of Open Access Journals (Sweden)

    Steffen Kawelke

    Full Text Available Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2 and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2 was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.

  6. Co-administration of ethanol and nicotine: the enduring alterations in the rewarding properties of nicotine and glutamate activity within the mesocorticolimbic system of female alcohol-preferring (P) rats.

    Science.gov (United States)

    Deehan, Gerald A; Hauser, Sheketha R; Waeiss, R Aaron; Knight, Christopher P; Toalston, Jamie E; Truitt, William A; McBride, William J; Rodd, Zachary A

    2015-12-01

    The co-abuse of ethanol (EtOH) and nicotine (NIC) increases the likelihood that an individual will relapse to drug use while attempting to maintain abstinence. There is limited research examining the consequences of long-term EtOH and NIC co-abuse. The current experiments determined the enduring effects of chronic EtOH, NIC, or EtOH + NIC intake on the reinforcing properties of NIC and glutamate (GLU) activity within the mesocorticolimbic (MCL) system. Alcohol-preferring (P) rats self-administered EtOH, Sacc + NIC, or EtOH + NIC combined for 10 weeks. The reinforcing properties of 0.1-3.0 μM NIC within the nucleus accumbens shell (AcbSh) were assessed following a 2-3-week drug-free period using intracranial self-administration (ICSA) procedures. The effects of EtOH, Sacc, Sacc + NIC, or EtOH + NIC intake on extracellular levels and clearance of glutamate (GLU) in the medial prefrontal cortex (mPFC) were also determined. Binge intake of EtOH (96-100 mg%) and NIC (21-27 mg/mL) were attained. All groups of P rats self-infused 3.0 μM NIC directly into the AcbSh, whereas only animals in the EtOH + NIC co-abuse group self-infused the 0.3 and 1.0 μM NIC concentrations. Additionally, self-administration of EtOH + NIC, but not EtOH, Sacc or Sacc + NIC, resulted in enduring increases in basal extracellular GLU levels in the mPFC. Overall, the co-abuse of EtOH + NIC produced enduring neuronal alterations within the MCL which enhanced the rewarding properties of NIC in the AcbSh and elevated extracellular GLU levels within the mPFC.

  7. Fluorescence imaging of glutamate release in neurons

    International Nuclear Information System (INIS)

    Wang, Ziqiang; Yeung, Edward S.

    1999-01-01

    A noninvasive detection scheme based on glutamate dehydrogenase (GDH) enzymatic assay combined with microscopy was developed to measure the glutamate release in cultured cells from the central nervous system (CNS). The enzyme reaction is very specific and sensitive. The detection limit with charge-coupled device (CCD) imaging is down to μM levels of glutamate with reasonable response time (∼30 s). The standard glutamate test shows a linear response over 3 orders of magnitude, from μM to 0.1 mM range. The in vitro monitoring of glutamate release from cultured neuron cells demonstrated excellent spatial and temporal resolution. (c) 1999 Society for Applied Spectroscopy

  8. A Novel Corynebacterium glutamicum l-Glutamate Exporter.

    Science.gov (United States)

    Wang, Yu; Cao, Guoqiang; Xu, Deyu; Fan, Liwen; Wu, Xinyang; Ni, Xiaomeng; Zhao, Shuxin; Zheng, Ping; Sun, Jibin; Ma, Yanhe

    2018-03-15

    Besides metabolic pathways and regulatory networks, transport systems are also pivotal for cellular metabolism and hyperproduction of biochemicals using microbial cell factories. The identification and characterization of transporters are therefore of great significance for the understanding and engineering of transport reactions. Herein, a novel l-glutamate exporter, MscCG2, which exists extensively in Corynebacterium glutamicum strains but is distinct from the only known l-glutamate exporter, MscCG, was discovered in an industrial l-glutamate-producing C. glutamicum strain. MscCG2 was predicted to possess three transmembrane helices in the N-terminal region and located in the cytoplasmic membrane, which are typical structural characteristics of the mechanosensitive channel of small conductance. MscCG2 has a low amino acid sequence identity (23%) to MscCG and evolved separately from MscCG with four transmembrane helices. Despite the considerable differences between MscCG2 and MscCG in sequence and structure, gene deletion and complementation confirmed that MscCG2 also functioned as an l-glutamate exporter and an osmotic safety valve in C. glutamicum Besides, transcriptional analysis showed that MscCG2 and MscCG genes were transcribed in similar patterns and not induced by l-glutamate-producing conditions. It was also demonstrated that MscCG2-mediated l-glutamate excretion was activated by biotin limitation or penicillin treatment and that constitutive l-glutamate excretion was triggered by a gain-of-function mutation of MscCG2 (A151V). Discovery of MscCG2 will enrich the understanding of bacterial amino acid transport and provide additional targets for exporter engineering. IMPORTANCE The exchange of matter, energy, and information with surroundings is fundamental for cellular metabolism. Therefore, studying transport systems that are essential for these processes is of great significance. Besides, transport systems of bacterial cells are usually related to

  9. Reduced expression of glutamate transporter EAAT2 and impaired glutamate transport in human primary astrocytes exposed to HIV-1 or gp120

    International Nuclear Information System (INIS)

    Wang Zhuying; Pekarskaya, Olga; Bencheikh, Meryem; Chao Wei; Gelbard, Harris A.; Ghorpade, Anuja; Rothstein, Jeffrey D.; Volsky, David J.

    2003-01-01

    L-Glutamate is the major excitatory neurotransmitter in the brain. Astrocytes maintain low levels of synaptic glutamate by high-affinity uptake and defects in this function may lead to neuronal cell death by excitotoxicity. We tested the effects of HIV-1 and its envelope glycoprotein gp120 upon glutamate uptake and expression of glutamate transporters EAAT1 and EAAT2 in fetal human astrocytes in vitro. Astrocytes isolated from fetal tissues between 16 and 19 weeks of gestation expressed EAAT1 and EAAT2 RNA and proteins as detected by Northern blot analysis and immunoblotting, respectively, and the cells were capable of specific glutamate uptake. Exposure of astrocytes to HIV-1 or gp120 significantly impaired glutamate uptake by the cells, with maximum inhibition within 6 h, followed by gradual decline during 3 days of observation. HIV-1-infected cells showed a 59% reduction in V max for glutamate transport, indicating a reduction in the number of active transporter sites on the cell surface. Impaired glutamate transport after HIV-1 infection or gp120 exposure correlated with a 40-70% decline in steady-state levels of EAAT2 RNA and protein. EAAT1 RNA and protein levels were less affected. Treatment of astrocytes with tumor necrosis factor-α (TNF-α) decreased the expression of both EAAT1 and EAAT2, but neither HIV-1 nor gp120 were found to induce TNF-α production by astrocytes. These findings demonstrate that HIV-1 and gp120 induce transcriptional downmodulation of the EAAT2 transporter gene in human astrocytes and coordinately attenuate glutamate transport by the cells. Reduction of the ability of HIV-1-infected astrocytes to take up glutamate may contribute to the development of neurological disease

  10. Inibição da atividade da citrato sintase cerebral em um modelo animal de sepse Inhibition of brain citrate synthase activity in an animal model of sepsis

    Directory of Open Access Journals (Sweden)

    Giselli Scaini

    2011-06-01

    fisiopatologia desta doença.OBJECTIVE: An extensive body of evidence from experimental studies indicates that sepsis is associated with increased reactive oxygen species production, depletion of antioxidants, and accumulation of markers of oxidative stress. Moreover, mitochondrial dysfunction has been implicated in the pathogenesis of multiple organ dysfunction syndrome (MODS. Citrate synthase is an enzyme localized in the mitochondrial matrix and an important component of the Krebs cycle; consequently, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase activity in the brains of rats submitted to a cecal ligation puncture model of sepsis. METHODS: At several times points (3, 6, 12, 24 and 48 hours after the cecal ligation puncture operation, six rats were killed by decapitation. Their brains were removed, and the hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were dissected and used to determine citrate synthase activity. RESULTS: We found that citrate synthase activity in the prefrontal cortex was inhibited 12, 24 and 48 hours after cecal ligation puncture. In the cerebral cortex, citrate synthase activity was inhibited 3, 12, 24 and 48 hours after cecal ligation puncture. Citrate synthase was not affected in the hippocampus, striatum or cerebellum up to 48 hours after cecal ligation puncture. CONCLUSION: Considering that energy impairment due to mitochondrial dysfunction in sepsis has been well described and that oxidative stress plays a crucial role in sepsis development, we believe that energy impairment may also be involved in these processes. If citrate synthase inhibition also occurs in a sepsis model, it is tempting to speculate that a reduction in brain metabolism may be related to the pathophysiology of this disease.

  11. Monosodium glutamate toxicity: Sida acuta leaf extract ameliorated ...

    African Journals Online (AJOL)

    The brain is reportedly sensitive to monosodium glutamate (MSG) toxicity via oxidative stress. Sida acuta leaf ethanolic extract (SALEE) possesses antioxidant activity which can mitigate this neurotoxicity. The present study investigated the possible protective effect of SALEE on MSG-induced toxicity in rats. Twenty-six ...

  12. Assay of partially purified glutamate dehydrogenase isolated from ...

    African Journals Online (AJOL)

    Glutamate dehydrogenase (E C 1.4.1.1) isolated from the seeds of asparagus beans was partially purified to a factor of 22 by dialysis after fractional precipitation with solid ammonium sulphate at 40 and 60% saturation. A specific activity of 11.78μmol min-1 mg-1 protein was calculated for the partially purified enzyme when ...

  13. Distinct roles of two anaplerotic pathways in glutamate production induced by biotin limitation in Corynebacterium glutamicum.

    Science.gov (United States)

    Sato, Hiroki; Orishimo, Keita; Shirai, Tomokazu; Hirasawa, Takashi; Nagahisa, Keisuke; Shimizu, Hiroshi; Wachi, Masaaki

    2008-07-01

    Corynebacterium glutamicum is a biotin auxotrophic bacterium in which glutamate production is induced under biotin-limited conditions. During glutamate production, anaplerotic reactions catalyzed by phosphoenolpyruvate carboxylase (PEPC) and a biotin-containing enzyme pyruvate carboxylase (PC) are believed to play an important role in supplying oxaloacetate in the tricarboxylic acid cycle. To understand the distinct roles of PEPC and PC on glutamate production by C. glutamicum, we observed glutamate production induced under biotin-limited conditions in the disruptants of the genes encoding PEPC (ppc) and PC (pyc), respectively. The pyc disruptant retained the ability to produce high amounts of glutamate, and lactate was simultaneously produced probably due to the increased intracellular pyruvate levels. On the other hand, the ppc knockout mutant could not produce glutamate. Additionally, glutamate production in the pyc disruptant was enhanced by overexpression of ppc rather than disruption of the lactate dehydrogenase gene (ldh), which is involved in lactate production. Metabolic flux analysis based on the 13C-labeling experiment and measurement of 13C-enrichment in glutamate using nuclear magnetic resonance spectroscopy revealed that the flux for anaplerotic reactions in the pyc disruptant was lower than that in the wild type, concomitantly increasing the flux for lactate formation. Moreover, overexpression of ppc increased this flux in both the pyc disruptant and the wild type. Our results suggest that the PEPC-catalyzed anaplerotic reaction is necessary for glutamate production induced under biotin-limited conditions, because PC is not active during glutamate production, and overexpression of ppc effectively enhances glutamate production under biotin-limited conditions.

  14. The microbe-secreted isopeptide poly-γ-glutamic acid induces stress tolerance in Brassica napus L. seedlings by activating crosstalk between H2O2 and Ca2+

    Science.gov (United States)

    Lei, Peng; Pang, Xiao; Feng, Xiaohai; Li, Sha; Chi, Bo; Wang, Rui; Xu, Zongqi; Xu, Hong

    2017-01-01

    Poly-γ-glutamic acid (γ-PGA) is a microbe-secreted isopeptide that has been shown to promote growth and enhance stress tolerance in crops. However, its site of action and downstream signaling pathways are still unknown. In this study, we investigated γ-PGA-induced tolerance to salt and cold stresses in Brassica napus L. seedlings. Fluorescent labeling of γ-PGA was used to locate the site of its activity in root protoplasts. The relationship between γ-PGA-induced stress tolerance and two signal molecules, H2O2 and Ca2+, as well as the γ-PGA-elicited signaling pathway at the whole plant level, were explored. Fluorescent labeling showed that γ-PGA did not enter the cytoplasm but instead attached to the surface of root protoplasm. Here, it triggered a burst of H2O2 in roots by enhancing the transcription of RbohD and RbohF, and the elicited H2O2 further activated an influx of Ca2+ into root cells. Ca2+ signaling was transmitted via the stem from roots to leaves, where it elicited a fresh burst of H2O2, thus promoting plant growth and enhancing stress tolerance. On the basis of these observation, we propose that γ-PGA mediates stress tolerance in Brassica napus seedlings by activating an H2O2 burst and subsequent crosstalk between H2O2 and Ca2+ signaling. PMID:28198821

  15. The Staphylococcus aureus α-Acetolactate Synthase ALS Confers Resistance to Nitrosative Stress

    Directory of Open Access Journals (Sweden)

    Sandra M. Carvalho

    2017-07-01

    Full Text Available Staphylococcus aureus is a worldwide pathogen that colonizes the human nasal cavity and is a major cause of respiratory and cutaneous infections. In the nasal cavity, S. aureus thrives with high concentrations of nitric oxide (NO produced by the innate immune effectors and has available for growth slow-metabolizing free hexoses, such as galactose. Here, we have used deep sequencing transcriptomic analysis (RNA-Seq and 1H-NMR to uncover how S. aureus grown on galactose, a major carbon source present in the nasopharynx, survives the deleterious action of NO. We observed that, like on glucose, S. aureus withstands high concentrations of NO when using galactose. Data indicate that this resistance is, most likely, achieved through a distinct metabolism that relies on the increased production of amino acids, such as glutamate, threonine, and branched-chain amino acids (BCAAs. Moreover, we found that under NO stress the S. aureus α-acetolactate synthase (ALS enzyme, which converts pyruvate into α-acetolactate, plays an important role. ALS is proposed to prevent intracellular acidification, to promote the production of BCAAs and the activation of the TCA cycle. Additionally, ALS is shown to contribute to the successful infection of murine macrophages. Furthermore, ALS contributes to the resistance of S. aureus to beta-lactam antibiotics such as methicillin and oxacillin.

  16. Group I Metabotropic Glutamate Receptors

    DEFF Research Database (Denmark)

    Erichsen, Julie Ladeby; Blaabjerg, Morten; Bogetofte Thomasen, Helle

    2015-01-01

    differentiated an immortalized, forebrain-derived stem cell line in the presence or absence of glutamate and with addition of either the group I mGluR agonist DHPG or the selective antagonists; MPEP (mGluR5) and LY367385 (mGluR1). Characterization of differentiated cells revealed that both mGluR1 and mGluR5 were...

  17. A Glutamate Homeostat Controls the Presynaptic Inhibition of Neurotransmitter Release

    Directory of Open Access Journals (Sweden)

    Xiling Li

    2018-05-01

    Full Text Available Summary: We have interrogated the synaptic dialog that enables the bi-directional, homeostatic control of presynaptic efficacy at the glutamatergic Drosophila neuromuscular junction (NMJ. We find that homeostatic depression and potentiation use disparate genetic, induction, and expression mechanisms. Specifically, homeostatic potentiation is achieved through reduced CaMKII activity postsynaptically and increased abundance of active zone material presynaptically at one of the two neuronal subtypes innervating the NMJ, while homeostatic depression occurs without alterations in CaMKII activity and is expressed at both neuronal subtypes. Furthermore, homeostatic depression is only induced through excess presynaptic glutamate release and operates with disregard to the postsynaptic response. We propose that two independent homeostats modulate presynaptic efficacy at the Drosophila NMJ: one is an intercellular signaling system that potentiates synaptic strength following diminished postsynaptic excitability, while the other adaptively modulates presynaptic glutamate release through an autocrine mechanism without feedback from the postsynaptic compartment. : Homeostatic mechanisms stabilize synaptic strength, but the signaling systems remain enigmatic. Li et al. suggest the existence of a homeostat operating at the Drosophila neuromuscular junction that responds to excess glutamate through an autocrine mechanism to adaptively inhibit presynaptic neurotransmitter release. This system parallels forms of plasticity at central synapses. Keywords: homeostatic synaptic plasticity, glutamate homeostasis, synaptic depression, Drosophila neuromuscular junction

  18. Peripheral Glutamate Receptors Are Required for Hyperalgesia Induced by Capsaicin

    Directory of Open Access Journals (Sweden)

    You-Hong Jin

    2012-01-01

    Full Text Available Transient receptor potential vanilloid1 (TRPV1 and glutamate receptors (GluRs are located in small diameter primary afferent neurons (nociceptors, and it was speculated that glutamate released in the peripheral tissue in response to activation of TRPV1 might activate nociceptors retrogradely. But, it was not clear which types of GluRs are functioning in the nociceptive sensory transmission. In the present study, we examined the c-Fos expression in spinal cord dorsal horn following injection of drugs associated with glutamate receptors with/without capsaicin into the hindpaw. The subcutaneous injection of capsaicin or glutamate remarkably evoked c-Fos expression in ipsilateral sides of spinal cord dorsal horn. This capsaicin evoked increase of c-Fos expression was significantly prevented by concomitant administration of MK801, CNQX, and CPCCOEt. On the other hand, there were not any significant changes in coinjection of capsaicin and MCCG or MSOP. These results reveal that the activation of iGluRs and group I mGluR in peripheral afferent nerves play an important role in mechanisms whereby capsaicin evokes/maintains nociceptive responses.

  19. The neuroprotective effects of tocotrienol rich fraction and alpha tocopherol against glutamate injury in astrocytes

    Directory of Open Access Journals (Sweden)

    Thilaga Rati Selvaraju

    2014-11-01

    Full Text Available Tocotrienol rich fraction (TRF is an extract of palm oil, which consists of 25% alpha tocopherol (α-TCP and 75% tocotrienols. TRF has been shown to possess potent antioxidant, anti-inflammatory, anticancer, neuroprotection, and cholesterol lowering activities. Glutamate is the main excitatory amino acid neurotransmitter in the central nervous system of mammalian, which can be excitotoxic, and it has been suggested to play a key role in neurodegenerative disorders like Parkinson’s and Alzheimer’s diseases. In this present study, the effects of vitamin E (TRF and α-TCP in protecting astrocytes against glutamate injury were elucidated. Astrocytes induced with 180 mM of glutamate lead to significant cell death. However, glutamate mediated cytotoxicity was diminished via pre and post supplementation of TRF and α-TCP. Hence, vitamin E acted as a potent antioxidant agent in recovering mitochondrial injury due to elevated oxidative stress, and enhanced better survivability upon glutamate toxicity.  

  20. Therapeutic effects of glutamic acid in piglets challenged with deoxynivalenol.

    Science.gov (United States)

    Wu, Miaomiao; Xiao, Hao; Ren, Wenkai; Yin, Jie; Tan, Bie; Liu, Gang; Li, Lili; Nyachoti, Charles Martin; Xiong, Xia; Wu, Guoyao

    2014-01-01

    The mycotoxin deoxynivalenol (DON), one of the most common food contaminants, primarily targets the gastrointestinal tract to affect animal and human health. This study was conducted to examine the protective function of glutamic acid on intestinal injury and oxidative stress caused by DON in piglets. Twenty-eight piglets were assigned randomly into 4 dietary treatments (7 pigs/treatment): 1) uncontaminated control diet (NC), 2) NC+DON at 4 mg/kg (DON), 3) NC+2% glutamic acid (GLU), and 4) NC+2% glutamic acid + DON at 4 mg/kg (DG). At day 15, 30 and 37, blood samples were collected to determine serum concentrations of CAT (catalase), T-AOC (total antioxidant capacity), H2O2 (hydrogen peroxide), NO (nitric oxide), MDA (maleic dialdehyde), DAO (diamine oxidase) and D-lactate. Intestinal morphology, and the activation of Akt/mTOR/4EBP1 signal pathway, as well as the concentrations of H2O2, MDA, and DAO in kidney, liver and small intestine, were analyzed at day 37. Results showed that DON significantly (Pglutamic acid supplementation according to the change of oxidative parameters in blood and tissues. Meanwhile, DON caused obvious intestinal injury from microscopic observations and permeability indicators, which was alleviated by glutamic acid supplementation. Moreover, the inhibition of DON on Akt/mTOR/4EBP1 signal pathway was reduced by glutamic acid supplementation. Collectively, these data suggest that glutamic acid may be a useful nutritional regulator for DON-induced damage manifested as oxidative stress, intestinal injury and signaling inhibition.

  1. Availability of neurotransmitter glutamate is diminished when beta-hydroxybutyrate replaces glucose in cultured neurons.

    Science.gov (United States)

    Lund, Trine M; Risa, Oystein; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S

    2009-07-01

    Ketone bodies serve as alternative energy substrates for the brain in cases of low glucose availability such as during starvation or in patients treated with a ketogenic diet. The ketone bodies are metabolized via a distinct pathway confined to the mitochondria. We have compared metabolism of [2,4-(13)C]beta-hydroxybutyrate to that of [1,6-(13)C]glucose in cultured glutamatergic neurons and investigated the effect of neuronal activity focusing on the aspartate-glutamate homeostasis, an essential component of the excitatory activity in the brain. The amount of (13)C incorporation and cellular content was lower for glutamate and higher for aspartate in the presence of [2,4-(13)C]beta-hydroxybutyrate as opposed to [1,6-(13)C]glucose. Our results suggest that the change in aspartate-glutamate homeostasis is due to a decreased availability of NADH for cytosolic malate dehydrogenase and thus reduced malate-aspartate shuttle activity in neurons using beta-hydroxybutyrate. In the presence of glucose, the glutamate content decreased significantly upon activation of neurotransmitter release, whereas in the presence of only beta-hydroxybutyrate, no decrease in the glutamate content was observed. Thus, the fraction of the glutamate pool available for transmitter release was diminished when metabolizing beta-hydroxybutyrate, which is in line with the hypothesis of formation of transmitter glutamate via an obligatory involvement of the malate-aspartate shuttle.

  2. Transcriptional profiling of striatal neurons in response to single or concurrent activation of dopamine D2, adenosine A(2A) and metabotropic glutamate type 5 receptors: focus on beta-synuclein expression.

    Science.gov (United States)

    Canela, Laia; Selga, Elisabet; García-Martínez, Juan Manuel; Amaral, Olavo B; Fernández-Dueñas, Víctor; Alberch, Jordi; Canela, Enric I; Franco, Rafael; Noé, Véronique; Lluís, Carme; Ciudad, Carlos J; Ciruela, Francisco

    2012-10-25

    G protein-coupled receptor oligomerization is a concept which is changing the understanding of classical pharmacology. Both, oligomerization and functional interaction between adenosine A(2A,) dopamine D(2) and metabotropic glutamate type 5 receptors have been demonstrated in the striatum. However, the transcriptional consequences of receptors co-activation are still unexplored. We aim here to determine the changes in gene expression of striatal primary cultured neurons upon isolated or simultaneous receptor activation. Interestingly, we found that 95 genes of the total analyzed (15,866 transcripts and variants) changed their expression in response to simultaneous stimulation of all three receptors. Among these genes, we focused on the β-synuclein (β-Syn) gene (SCNB). Quantitative PCR verified the magnitude and direction of change in expression of SCNB. Since β-Syn belongs to the homologous synuclein family and may be considered a natural regulator of α-synuclein (α-Syn), it has been proposed that β-Syn might act protectively against α-Syn neuropathology. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Metabotropic glutamate receptor-5 and protein kinase C-epsilon increase in dorsal root ganglion neurons and spinal glial activation in an adolescent rat model of painful neck injury.

    Science.gov (United States)

    Weisshaar, Christine L; Dong, Ling; Bowman, Alex S; Perez, Federico M; Guarino, Benjamin B; Sweitzer, Sarah M; Winkelstein, Beth A

    2010-12-01

    There is growing evidence that neck pain is common in adolescence and is a risk factor for the development of chronic neck pain in adulthood. The cervical facet joint and its capsular ligament is a common source of pain in the neck in adults, but its role in adolescent pain remains unknown. The aim of this study was to define the biomechanics, behavioral sensitivity, and indicators of neuronal and glial activation in an adolescent model of mechanical facet joint injury. A bilateral C6-C7 facet joint distraction was imposed in an adolescent rat and biomechanical metrics were measured during injury. Following injury, forepaw mechanical hyperalgesia was measured, and protein kinase C-epsilon (PKCɛ) and metabotropic glutamate receptor-5 (mGluR5) expression in the dorsal root ganglion and markers of spinal glial activation were assessed. Joint distraction induced significant mechanical hyperalgesia during the 7 days post-injury (p capsule during injury were 32.8 ± 12.9%, which were consistent with the strains associated with comparable degrees of hypersensitivity in the adult rat. These results suggest that adolescents may have a lower tissue tolerance to induce pain and associated nociceptive response than do adults.

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

    Science.gov (United States)

    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.

  5. Relationship between glutamate, GOT and GPT levels in maternal and fetal blood: a potential mechanism for fetal neuroprotection.

    Science.gov (United States)

    Zlotnik, Alexander; Tsesis, Svetlana; Gruenbaum, Benjamin Fredrick; Ohayon, Sharon; Gruenbaum, Shaun Evan; Boyko, Matthew; Sheiner, Eyal; Brotfain, Evgeny; Shapira, Yoram; Teichberg, Vivian Itzhak

    2012-09-01

    Excess glutamate in the brain is thought to be implicated in the pathophysiology of fetal anoxic brain injury, yet little is known about the mechanisms by which glutamate is regulated in the fetal brain. This study examines whether there are differences between maternal and fetal glutamate concentrations, and whether a correlation between them exists. 10 ml of venous blood was extracted from 87 full-term (>37 weeks gestation) pregnant women in active labor. Immediately after delivery of the neonate, 10 ml of blood from the umbilical artery and vein was extracted. Samples were analyzed for levels of glutamate, glutamate-oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT). Fetal blood glutamate concentrations in both the umbilical artery and vein were found to be significantly higher than maternal blood (pGOT levels in the umbilical artery and vein were found to be significantly higher than maternal GOT levels (pGOT or GPT between the umbilical artery and vein. There was an association observed between glutamate levels in maternal blood and glutamate levels in both venous (R=0.32, pGOT, but not GPT levels. An association was observed between maternal and fetal blood glutamate levels. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Agmatine reduces extracellular glutamate during pentylenetetrazole-induced seizures in rat brain: A potential mechanism for the anticonvulsive effects

    OpenAIRE

    Feng, Yangzheng; LeBlanc, Michael H.; Regunathan, Soundar

    2005-01-01

    Glutamate has been implicated in the initiation and spread of seizure activity. Agmatine, an endogenous neuromodulator, is an antagonist of NMDA receptors and has anticonvulsive effects. Whether agmatine regulate glutamate release, as measured by in vivo microdialysis, is not known. In this study, we used pentylenetetrazole (PTZ)-induced seizure model to determine the effect of agmatine on extracellular glutamate in rat brain. We also determined the time course and the amount of agmatine that...

  7. Sexual attraction enhances glutamate transmission in mammalian anterior cingulate cortex

    Directory of Open Access Journals (Sweden)

    Wu Long-Jun

    2009-05-01

    Full Text Available Abstract Functional human brain imaging studies have indicated the essential role of cortical regions, such as the anterior cingulate cortex (ACC, in romantic love and sex. However, the neurobiological basis of how the ACC neurons are activated and engaged in sexual attraction remains unknown. Using transgenic mice in which the expression of green fluorescent protein (GFP is controlled by the promoter of the activity-dependent gene c-fos, we found that ACC pyramidal neurons are activated by sexual attraction. The presynaptic glutamate release to the activated neurons is increased and pharmacological inhibition of neuronal activities in the ACC reduced the interest of male mice to female mice. Our results present direct evidence of the critical role of the ACC in sexual attraction, and long-term increases in glutamate mediated excitatory transmission may contribute to sexual attraction between male and female mice.

  8. Paraventricular Stimulation with Glutamate Elicits Bradycardia and Pituitary Responses

    Science.gov (United States)

    Darlington, Daniel N.; Miyamoto, Michael; Keil, Lanny C.; Dallman, Mary F.

    1989-01-01

    The excitatory neurotransmitter, L-glutamate (0.5 M, pH 7.4), or the organic acid, acetate (0.5 M, pH 7.4), was microinjected (50 nl over 2 min) directly into the paraventricular nuclei (PVN) of pentobarbital sodium-anesthetized rats while arterial blood pressure and heart rate and plasma adrenocorticotropic hormone (ACTH), vasopressin, and oxytocin were measured. Activation of PVN neurons with L-glutamate led to increases in plasma ACTH, vasopressin, and oxytocin and a profound bradycardia (-80 beats/min) with little change in arterial blood pressure. Microinjection of acetate had no effect on the above variables. The decrease in heart rate was shown to be dependent on the concentration of glutamate injected and the volume of injectate. The bradycardia was mediated through the autonomic nervous system because ganglionic blockade (pentolinium tartrate) eliminated the response; atropine and propranolol severely attenuated the bradycardia. The bradycardia was greatest when L-glutamate was microinjected into the caudal PVN. Injections into the rostral PVN or into nuclei surrounding the PVN led to small or nonsignificant decreases in heart rate. Focal electric stimulation (2-50 pA) of the PVN also led to decreases in heart rate and arterial blood pressure. These data suggest that activation of PVN neurons leads to the release of ACTH, vasopressin, and oxytocin from the pituitary and a bradycardia that is mediated by the autonomic nervous system.

  9. Vesicular glutamate release from central axons contributes to myelin damage.

    Science.gov (United States)

    Doyle, Sean; Hansen, Daniel Bloch; Vella, Jasmine; Bond, Peter; Harper, Glenn; Zammit, Christian; Valentino, Mario; Fern, Robert

    2018-03-12

    The axon myelin sheath is prone to injury associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor activation but the source of glutamate in this context is unknown. Myelin damage results in permanent action potential loss and severe functional deficit in the white matter of the CNS, for example in ischemic stroke. Here, we show that in rats and mice, ischemic conditions trigger activation of myelinic NMDA receptors incorporating GluN2C/D subunits following release of axonal vesicular glutamate into the peri-axonal space under the myelin sheath. Glial sources of glutamate such as reverse transport did not contribute significantly to this phenomenon. We demonstrate selective myelin uptake and retention of a GluN2C/D NMDA receptor negative allosteric modulator that shields myelin from ischemic injury. The findings potentially support a rational approach toward a low-impact prophylactic therapy to protect patients at risk of stroke and other forms of excitotoxic injury.

  10. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants.

    Science.gov (United States)

    Degenhardt, Jörg; Köllner, Tobias G; Gershenzon, Jonathan

    2009-01-01

    The multitude of terpene carbon skeletons in plants is formed by enzymes known as terpene synthases. This review covers the monoterpene and sesquiterpene synthases presenting an up-to-date list of enzymes reported and evidence for their ability to form multiple products. The reaction mechanisms of these enzyme classes are described, and information on how terpene synthase proteins mediate catalysis is summarized. Correlations between specific amino acid motifs and terpene synthase function are described, including an analysis of the relationships between active site sequence and cyclization type and a discussion of whether specific protein features might facilitate multiple product formation.

  11. Inducible nitric oxide inhibitors block NMDA antagonist-stimulated motoric behaviors and medial prefrontal cortical glutamate efflux

    Directory of Open Access Journals (Sweden)

    Hadley C Bergstrom

    2015-12-01

    Full Text Available Nitric oxide (NO plays a critical role in the motoric and glutamate releasing action of N-methyl-D-aspartate (NMDA-antagonist stimulants. Earlier studies utilized neuronal nitric oxide synthase inhibitors (nNOS for studying the neurobehavioral effects of noncompetitive NMDA-antagonist stimulants such as dizocilpine (MK-801 and phencyclidine (PCP. This study explores the role of the inducible nitric oxide synthase inhibitors (iNOS aminoguanidine (AG and (--epigallocatechin-3-gallate (EGCG in NMDA-antagonist induced motoric behavior and prefrontal cortical glutamate efflux. Adult male rats were administered a dose range of AG, EGCG or vehicle prior to receiving NMDA antagonists MK-801, PCP or a conventional psychostimulant (cocaine and tested for motoric behavior in an open arena. Glutamate in the medial prefrontal cortex was measured using in vivo microdialysis after a combination of AG or EGCG prior to MK-801. Acute administration of AG or EGCG dose-dependently attenuated the locomotor and ataxic properties of MK-801 and PCP. Both AG and EGCG were unable to block the motoric effects of cocaine, indicating the acute pharmacologic action of AG and EGCG is specific to NMDA antagonism and not generalizable to all stimulant class drugs. AG and EGCG normalized MK-801-stimulated medial prefrontal cortical glutamate efflux. These data demonstrate that AG and EGCG attenuates NMDA antagonist-stimulated motoric behavior and cortical glutamate efflux. Our results suggest that EGCG-like polyphenol nutraceuticals (contained in green tea and chocolate may be clinically useful in protecting against the adverse behavioral dissociative and cortical glutamate stimulating effects of NMDA antagonists. Medications that interfere with NMDA antagonists such as MK-801 and PCP have been proposed as treatments for schizophrenia.

  12. Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes.

    Science.gov (United States)

    Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw; Stridh, Malin H; Zaganas, Ioannis; Skytt, Dorte M; Schousboe, Arne; Bak, Lasse K; Enard, Wolfgang; Pääbo, Svante; Waagepetersen, Helle S

    2017-03-01

    A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO 2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [ 3 H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of 13 C and 14 C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO 2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity. GLIA 2017;65:474-488. © 2016 Wiley Periodicals, Inc.

  13. Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes

    International Nuclear Information System (INIS)

    Chen, J.W.; Cunningham, M.D.; Galton, N.; Michaelis, E.K.

    1988-01-01

    Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an ∼ 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger M/sub r/ protein in synaptic membranes. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 + M, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L[ 3 H]glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes

  14. SIRT1-mediated deacetylation of PGC1α attributes to the protection of curcumin against glutamate excitotoxicity in cortical neurons

    International Nuclear Information System (INIS)

    Jia, Ning; Sun, Qinru; Su, Qian; Chen, Guomin

    2016-01-01

    It is widely accepted that accumulation of extracellular glutamate mediates neuronal injuries in a number of neurological disorders via binding glutamate receptors. However, usage of the glutamate receptor antagonists aimed to prevent glutamate excitotoxicity is still controversial. As a polyphenol natural product, curcumin, has been implied multiple bioactivities. In this study, we explored whether the silent information regulator 1 (SIRT1)-peroxisome proliferator-activated receptor-coactivator 1α (PGC1α) pathway participated in the protection of curcumin against glutamate excitotoxicity. The cultured primary cortical neurons were treated with glutamate to set up a neuronal excitotoxicity model. The MTT and TUNEL methods were employed to measure cell viability and apoptosis, respectively. The mitochondrial function, the expression levels of SIRT1, PGC1α and acetylated PGC1α (ac-PGC1α) were measured to explore the mechanism of curcumin against glutamate excitotoxicity. The results showed that glutamate significantly induced cell death and apoptosis, which was blocked by pretreatment with curcumin. Meanwhile, curcumin preserved mitochondrial function, increased the expression level of SIRT1 and reduced the level of ac-PGC1α in the presence of glutamate. These results suggest that SIRT1-mediated deacetylation of PGC1α attributes to the neuroprotection of curcumin against glutamate excitotoxicity. - Highlights: • Curcumin attenuates glutamate induced cell death and apoptosis in cultured neurons. • Curcumin preserves mitochondrial function in the presence of glutamate. • Curcumin enhanced the expression of SIRT1 in the glutamate rich environment. • SIRT1-mediated deacetylation of PGC1α attributes to the neuroprotection of curcumin.

  15. Transcriptional activation of a geranylgeranyl diphosphate synthase gene, GGPPS2, isolated from Scoparia dulcis by treatment with methyl jasmonate and yeast extract.

    Science.gov (United States)

    Yamamura, Y; Mizuguchi, Y; Taura, F; Kurosaki, F

    2014-10-01

    A cDNA clone, designated SdGGPPS2, was isolated from young seedlings of Scoparia dulcis. The putative amino acid sequence of the translate of the gene showed high homology with geranylgeranyl diphosphate synthase (GGPPS) from various plant sources, and the N-terminal residues exhibited the characteristics of chloroplast targeting sequence. An appreciable increase in the transcriptional level of SdGGPPS2 was observed by exposure of the leaf tissues of S. dulcis to methyl jasmonate, yeast extract or Ca(2+) ionophore A23187. In contrast, SdGGPPS1, a homologous GGPPS gene of the plant, showed no or only negligible change in the expression level upon treatment with these stimuli. The truncated protein heterologously expressed in Escherichia coli in which the putative targeting domain was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to liberate geranylgeranyl diphosphate. These results suggested that SdGGPPS2 plays physiological roles in methyl jasmonate and yeast extract-induced metabolism in the chloroplast of S. dulcis cells.

  16. Fisetin Confers Cardioprotection against Myocardial Ischemia Reperfusion Injury by Suppressing Mitochondrial Oxidative Stress and Mitochondrial Dysfunction and Inhibiting Glycogen Synthase Kinase 3β Activity

    Directory of Open Access Journals (Sweden)

    Karthi Shanmugam

    2018-01-01

    Full Text Available Acute myocardial infarction (AMI is the leading cause of morbidity and mortality worldwide. Timely reperfusion is considered an optimal treatment for AMI. Paradoxically, the procedure of reperfusion can itself cause myocardial tissue injury. Therefore, a strategy to minimize the reperfusion-induced myocardial tissue injury is vital for salvaging the healthy myocardium. Herein, we investigated the cardioprotective effects of fisetin, a natural flavonoid, against ischemia/reperfusion (I/R injury (IRI using a Langendorff isolated heart perfusion system. I/R produced significant myocardial tissue injury, which was characterized by elevated levels of lactate dehydrogenase and creatine kinase in the perfusate and decreased indices of hemodynamic parameters. Furthermore, I/R resulted in elevated oxidative stress, uncoupling of the mitochondrial electron transport chain, increased mitochondrial swelling, a decrease of the mitochondrial membrane potential, and induction of apoptosis. Moreover, IRI was associated with a loss of the mitochondrial structure and decreased mitochondrial biogenesis. However, when the animals were pretreated with fisetin, it significantly attenuated the I/R-induced myocardial tissue injury, blunted the oxidative stress, and restored the structure and function of mitochondria. Mechanistically, the fisetin effects were found to be mediated via inhibition of glycogen synthase kinase 3β (GSK3β, which was confirmed by a biochemical assay and molecular docking studies.

  17. Fisetin Confers Cardioprotection against Myocardial Ischemia Reperfusion Injury by Suppressing Mitochondrial Oxidative Stress and Mitochondrial Dysfunction and Inhibiting Glycogen Synthase Kinase 3β Activity.

    Science.gov (United States)

    Shanmugam, Karthi; Ravindran, Sriram; Kurian, Gino A; Rajesh, Mohanraj

    2018-01-01

    Acute myocardial infarction (AMI) is the leading cause of morbidity and mortality worldwide. Timely reperfusion is considered an optimal treatment for AMI. Paradoxically, the procedure of reperfusion can itself cause myocardial tissue injury. Therefore, a strategy to minimize the reperfusion-induced myocardial tissue injury is vital for salvaging the healthy myocardium. Herein, we investigated the cardioprotective effects of fisetin, a natural flavonoid, against ischemia/reperfusion (I/R) injury (IRI) using a Langendorff isolated heart perfusion system. I/R produced significant myocardial tissue injury, which was characterized by elevated levels of lactate dehydrogenase and creatine kinase in the perfusate and decreased indices of hemodynamic parameters. Furthermore, I/R resulted in elevated oxidative stress, uncoupling of the mitochondrial electron transport chain, increased mitochondrial swelling, a decrease of the mitochondrial membrane potential, and induction of apoptosis. Moreover, IRI was associated with a loss of the mitochondrial structure and decreased mitochondrial biogenesis. However, when the animals were pretreated with fisetin, it significantly attenuated the I/R-induced myocardial tissue injury, blunted the oxidative stress, and restored the structure and function of mitochondria. Mechanistically, the fisetin effects were found to be mediated via inhibition of glycogen synthase kinase 3 β (GSK3 β ), which was confirmed by a biochemical assay and molecular docking studies.

  18. Insulin like growth factor-1 prevents 1-mentyl-4-phenylphyridinium-induced apoptosis in PC12 cells through activation of glycogen synthase kinase-3beta

    International Nuclear Information System (INIS)

    Sun, Xin; Huang, Luqi; Zhang, Min; Sun, Shenggang; Wu, Yan

    2010-01-01

    Dopaminergic neurons are lost mainly through apoptosis in Parkinson's disease. Insulin like growth factor-1 (IGF-1) inhibits apoptosis in a wide variety of tissues. Here we have shown that IGF-1 protects PC12 cells from toxic effects of 1-methyl-4-phenylpyridiniumion (MPP + ). Treatment of PC12 cells with recombinant human IGF-1 significantly decreased apoptosis caused by MPP + as measured by acridine orange/ethidium bromide staining. IGF-1 treatment induced sustained phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) as shown by western blot analysis. The anti-apoptotic effect of IGF-1 was abrogated by LY294002, which indirectly inhibits phosphorylation of GSK-3beta. Lithium chloride (LiCl), a known inhibitor of GSK-3beta, also blocked MPP + -induced apoptosis. Finally, although IGF-1 enhanced phosphorylation of extracellular signal-regulated kinases ERK1 and 2 (ERK1/2), PD98059, a specific inhibitor of ERK1/2, did not alter the survival effect of IGF-1. Thus, our findings indicate that IGF-1 protects PC12 cells exposed to MPP + from apoptosis via the GSK-3beta signaling pathway.

  19. Glutamate and GABA in vestibulo-sympathetic pathway neurons

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    Gay R Holstein

    2016-02-01

    Full Text Available The vestibulo-sympathetic reflex actively modulates blood pressure during changes in posture. This reflex allows humans to stand up and quadrupeds to rear or climb without a precipitous decline in cerebral perfusion. The vestibulo-sympathetic reflex pathway conveys signals from the vestibular end organs to the caudal vestibular nuclei. These cells, in turn, project to pre-sympathetic neurons in the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively. The present study assessed glutamate- and GABA-related immunofluorescence associated with central vestibular neurons of the vestibulo-sympathetic reflex pathway in rats. Retrograde FluoroGold tract tracing was used to label vestibular neurons with projections to RVLM or CVLM, and sinusoidal galvanic vestibular stimulation was employed to activate these pathways. Central vestibular neurons of the vestibulo-sympathetic reflex were identified by co-localization of FluoroGold and cFos protein, which accumulates in some vestibular neurons following galvanic stimulation. Triple-label immunofluorescence was used to co-localize glutamate- or GABA- labeling in the identified vestibulo-sympathetic reflex pathway neurons. Most activated projection neurons displayed intense glutamate immunofluorescence, suggestive of glutamatergic neurotransmission. To support this, anterograde tracer was injected into the caudal vestibular nuclei. Vestibular axons and terminals in RVLM and CVLM co-localized the anterograde tracer and vesicular glutamate transporter-2 signals. Other retrogradely-labeled cFos-positive neurons displayed intense GABA immunofluorescence. Vestibulo-sympathetic reflex pathway neurons of both phenotypes were present in the caudal medial and spinal vestibular nuclei, and projected to both RVLM and CVLM. As a group, however, triple-labeled vestibular cells with intense glutamate immunofluorescence were located more rostrally in the vestibular nuclei than the GABAergic neurons. Only the

  20. Glutamate and GABA in appetite regulation

    Directory of Open Access Journals (Sweden)

    Teresa Cardoso Delgado

    2013-08-01

    Full Text Available Appetite is regulated by a coordinated interplay between gut, adipose tissue and brain. A primary site for the regulation of appetite is the hypothalamus where interaction between