Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia.

PubMed

Ficarelli, A; Tassi, F; Restivo, F M

1999-03-01

We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

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

PubMed

Minaeva, Ekaterina; Forchhammer, Karl; Ermilova, Elena

2015-11-01

Glutamine is a metabolite of central importance in nitrogen metabolism of microorganisms and plants. The Chlorella PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine/arginine biosynthesis pathway, N-acetyl-L-glutamate kinase (NAGK) that leads to arginine formation. We provide evidence that glutamine promotes effective growth of C. variabilis strain NC64A. The present study shows that externally supplied glutamine directly influences the internal pool of arginine in NC64A. Glutamine synthetase (GS) catalyzes the ATP-dependent conversion of glutamate and ammonium to glutamine. The results of this study demonstrate that glutamine acts as a negative effector of GS activity. These data emphasize the importance of glutamine-dependent coupling of metabolism and signaling as components of an efficient pathway allowing the maintenance of metabolic homeostasis and sustaining growth of Chlorella. Copyright © 2015 Elsevier GmbH. All rights reserved.

Crystallization of dicalcium phosphate dihydrate with presence of glutamic acid and arginine at 37 °C.

PubMed

Li, Chengfeng; Ge, Xiaolu; Li, Guochang; Bai, Jiahai; Ding, Rui

2014-08-01

The formations of non-metabolic stones, bones and teeth were seriously related to the morphology, size and surface reactivity of dicalcium phosphate dihydrate (DCPD). Herein, a facile biomimetic mineralization method with presence of glutamic acid and arginine was employed to fabricate DCPD with well-defined morphology and adjustable crystallite size. In reaction solution containing more arginine, crystallization of DCPD occurred with faster rate of nucleation and higher density of stacked layers due to the generation of more OH(-) ions after hydrolysis of arginine at 37 °C. With addition of fluorescein or acetone, the consumption of OH(-) ions or desolvation reaction of Ca(2+) ions was modulated, which resulted in the fabrication of DCPD with adjustable crystallite sizes and densities of stacked layers. In comparison with fluorescein-loading DCPD, dicalcium phosphate anhydrate was prepared with enhanced photoluminescence properties due to the reduction of self-quenching effect and regular arrangement of encapsulated fluorescein molecules. With addition of more acetone, DCPD was prepared with smaller crystallite size via antisolvent crystallization. The simulated process with addition of amino acids under 37 °C would shed light on the dynamic process of biomineralization for calcium phosphate compounds. Copyright © 2014 Elsevier B.V. All rights reserved.

Improvement of the ammonia assimilation for enhancing L-arginine production of Corynebacterium crenatum.

PubMed

Guo, Jing; Man, Zaiwei; Rao, Zhiming; Xu, Meijuan; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

2017-03-01

There are four nitrogen atoms in L-arginine molecule and the nitrogen content is 32.1%. By now, metabolic engineering for L-arginine production strain improvement was focused on carbon flux optimization. In previous work, we obtained an L-arginine-producing Corynebacterium crenatum SDNN403 (ARG) through screening and mutation breeding. In this paper, a strain engineering strategy focusing on nitrogen supply and ammonium assimilation for L-arginine production was performed. Firstly, the effects of nitrogen atom donor (L-glutamate, L-glutamine and L-aspartate) addition on L-arginine production of ARG were studied, and the addition of L-glutamine and L-aspartate was beneficial for L-arginine production. Then, the glutamine synthetase gene glnA and aspartase gene aspA from E. coli were overexpressed in ARG for increasing the L-glutamine and L-aspartate synthesis, and the L-arginine production was effectively increased. In addition, the L-glutamate supply re-emerged as a limiting factor for L-arginine biosynthesis. Finally, the glutamate dehydrogenase gene gdh was co-overexpressed for further enhancement of L-arginine production. The final strain could produce 53.2 g l -1 of L-arginine, which was increased by 41.5% compared to ARG in fed-batch fermentation.

Dietary supplementation with arginine and glutamic acid modifies growth performance, carcass traits, and meat quality in growing-finishing pigs.

PubMed

Hu, C J; Jiang, Q Y; Zhang, T; Yin, Y L; Li, F N; Deng, J P; Wu, G Y; Kong, X F

2017-06-01

Sixty Duroc × Large White × Landrace pigs with an average initial BW of 77.1 ± 1.3 kg were used to investigate the effects of dietary supplementation with arginine and glutamic acid on growth performance, carcass traits, and meat quality in growing-finishing pigs. The animals were randomly assigned to 1 of 5 treatment groups (12 pigs/group, male:female ratio 1:1). The pigs in the control group were fed a basal diet (basal diet group), and those in the experimental groups were fed the basal diet supplemented with 2.05% -alanine (isonitrogenous group), 1.0% -arginine (Arg group), 1% glutamic acid + 1.44% -alanine (Glu group), or 1.0% -arginine + 1.0% glutamic acid (Arg+Glu group). After a 60-d period of supplementation, growth performance, carcass traits, and meat quality were evaluated. The results showed no significant differences ( > 0.05) in growth performance and carcass traits of the pigs in the Arg group relative to the basal diet group; however, the longissimus dorsi (LD) muscle and back fat showed a decrease ( < 0.05) in the percentage of SFA. In the Glu group, the final BW, phase 1 (d 1 to 30) and phase 2 (d 31 to 60) ADFI, and average back fat thickness of the pigs decreased ( < 0.05) by 7.14%, 23.43%, 8.03%, and 33.88%, respectively, when compared with the basal diet group. Dietary Arg+Glu supplementation had no effect ( > 0.05) on the final BW, phase 2 ADFI, and average daily weight gain in pigs but decreased ( < 0.05) their phase 1 ADFI, average back fat thickness, and percentage of SFA in the LD muscle and back fat, and increased ( < 0.05) the i.m. fat (IMF) content of the LD and biceps femoris muscles when compared with the basal diet group. Furthermore, a 16% decrease in yellowness (b* value; < 0.05) was observed in the Arg+Glu group pigs when compared with the isonitrogenous group. These findings suggest that dietary supplementation with both Arg and Glu beneficially increases the IMF deposition and improves the meat color and fatty acid

Purification and characterization of moschins, arginine-glutamate-rich proteins with translation-inhibiting activity from brown pumpkin (Cucurbita moschata) seeds.

PubMed

Ng, T B; Parkash, A; Tso, W W

2002-10-01

From fresh brown pumpkin seeds, two proteins with a molecular mass of 12kDa and an N-terminal sequence rich in arginine and glutamate residues were obtained. The protein designated alpha-moschin closely resembled the fruitfly programmed-cell death gene product and the protein designated beta-moschin demonstrated striking similarity to prepro 2S albumin in N-terminal sequence. alpha- and beta-moschins inhibited translation in the rabbit reticulocyte lysate system with an IC(50) of 17 microM and 300nM, respectively.

Characterizing monoclonal antibody formulations in arginine glutamate solutions using 1H NMR spectroscopy

PubMed Central

Kheddo, Priscilla; Cliff, Matthew J.; Uddin, Shahid; van der Walle, Christopher F.; Golovanov, Alexander P.

2016-01-01

ABSTRACT Assessing how excipients affect the self-association of monoclonal antibodies (mAbs) requires informative and direct in situ measurements for highly concentrated solutions, without sample dilution or perturbation. This study explores the application of solution nuclear magnetic resonance (NMR) spectroscopy for characterization of typical mAb behavior in formulations containing arginine glutamate. The data show that the analysis of signal intensities in 1D 1H NMR spectra, when compensated for changes in buffer viscosity, is invaluable for identifying conditions where protein-protein interactions are minimized. NMR-derived molecular translational diffusion rates for concentrated solutions are less useful than transverse relaxation rates as parameters defining optimal formulation. Furthermore, NMR reports on the solution viscosity and mAb aggregation during accelerated stability study assessment, generating data consistent with that acquired by size-exclusion chromatography. The methodology developed here offers NMR spectroscopy as a new tool providing complementary information useful to formulation development of mAbs and other large therapeutic proteins. PMID:27589351

Characterizing monoclonal antibody formulations in arginine glutamate solutions using 1H NMR spectroscopy.

PubMed

Kheddo, Priscilla; Cliff, Matthew J; Uddin, Shahid; van der Walle, Christopher F; Golovanov, Alexander P

2016-10-01

Assessing how excipients affect the self-association of monoclonal antibodies (mAbs) requires informative and direct in situ measurements for highly concentrated solutions, without sample dilution or perturbation. This study explores the application of solution nuclear magnetic resonance (NMR) spectroscopy for characterization of typical mAb behavior in formulations containing arginine glutamate. The data show that the analysis of signal intensities in 1D 1 H NMR spectra, when compensated for changes in buffer viscosity, is invaluable for identifying conditions where protein-protein interactions are minimized. NMR-derived molecular translational diffusion rates for concentrated solutions are less useful than transverse relaxation rates as parameters defining optimal formulation. Furthermore, NMR reports on the solution viscosity and mAb aggregation during accelerated stability study assessment, generating data consistent with that acquired by size-exclusion chromatography. The methodology developed here offers NMR spectroscopy as a new tool providing complementary information useful to formulation development of mAbs and other large therapeutic proteins.

Dietary supplementation with arginine and glutamic acid enhances key lipogenic gene expression in growing pigs.

PubMed

Hu, C J; Jiang, Q Y; Zhang, T; Yin, Y L; Li, F N; Su, J Y; Wu, G Y; Kong, X F

2017-12-01

Our previous study showed dietary supplementation with Arg and Glu increased intramuscular fat deposition and decreased back fat thickness in pigs, suggesting that the genes involved in lipid metabolism might be regulated differently in muscle and s.c. adipose (SA) tissues. Sixty Duroc × Large White × Landrace pigs with an average initial BW of 77.1 ± 1.3 kg were randomly assigned to 1 of 5 treatment groups (castrated male to female ratio = 1:1). Pigs in the control group were fed a basic diet, and those in experimental groups were fed the basic diet supplemented with 2.05% alanine (isonitrogenous group), 1.00% arginine (Arg group), 1.00% glutamic acid + 1.44% alanine (Glu group), or 1.00% arginine + 1.00% glutamic acid (Arg+Glu group). Fatty acid percentages and mRNA expression levels of the genes involved in lipid metabolism in muscle and SA tissues were examined. The percentages of C14:0 and C16:0 in the SA tissue of Glu group pigs and C14:0 in the longissimus dorsi (LD) muscle of Glu and Arg+Glu groups decreased ( < 0.05) compared to the basic diet group. The Arg+Glu group showed the highest ( < 0.05) hormone-sensitive lipase expression level in SA tissue and higher ( < 0.05) mRNA levels of in the LD muscle than the basic diet and isonitrogenous groups. Additionally, the mRNA level of fatty acid synthase in the Arg+Glu group was more upregulated ( < 0.05) than that of the Arg group. An increase in the mRNA level of in the biceps femoris muscle was also observed in the Arg+Glu group ( < 0.05) compared with the basic diet and isonitrogenous groups. Collectively, these findings suggest that dietary supplementation with Arg and Glu upregulates the expression of genes involved in adipogenesis in muscle tissues and lipolysis in SA tissues.

Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena.

PubMed

Burnat, Mireia; Flores, Enrique

2014-10-01

Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [(14) C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The ∆alr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [(14) C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the ∆alr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

A glutamate is the essential proton transfer gate during the catalytic cycle of the [NiFe] hydrogenase.

PubMed

Dementin, Sébastien; Burlat, Bénédicte; De Lacey, Antonio L; Pardo, Alejandro; Adryanczyk-Perrier, Géraldine; Guigliarelli, Bruno; Fernandez, Victor M; Rousset, Marc

2004-03-12

Kinetic, EPR, and Fourier transform infrared spectroscopic analysis of Desulfovibrio fructosovorans [NiFe] hydrogenase mutants targeted to Glu-25 indicated that this amino acid participates in proton transfer between the active site and the protein surface during the catalytic cycle. Replacement of that glutamic residue by a glutamine did not modify the spectroscopic properties of the enzyme but cancelled the catalytic activity except the para-H(2)/ortho-H(2) conversion. This mutation impaired the fast proton transfer from the active site that allows high turnover numbers for the oxidation of hydrogen. Replacement of the glutamic residue by the shorter aspartic acid slowed down this proton transfer, causing a significant decrease of H(2) oxidation and hydrogen isotope exchange activities, but did not change the para-H(2)/ortho-H(2) conversion activity. The spectroscopic properties of this mutant were totally different, especially in the reduced state in which a non-photosensitive nickel EPR spectrum was obtained.

Protein arginine methylation: a prominent modification and its demethylation.

PubMed

Wesche, Juste; Kühn, Sarah; Kessler, Benedikt M; Salton, Maayan; Wolf, Alexander

2017-09-01

Arginine methylation of histones is one mechanism of epigenetic regulation in eukaryotic cells. Methylarginines can also be found in non-histone proteins involved in various different processes in a cell. An enzyme family of nine protein arginine methyltransferases catalyses the addition of methyl groups on arginines of histone and non-histone proteins, resulting in either mono- or dimethylated-arginine residues. The reversibility of histone modifications is an essential feature of epigenetic regulation to respond to changes in environmental factors, signalling events, or metabolic alterations. Prominent histone modifications like lysine acetylation and lysine methylation are reversible. Enzyme family pairs have been identified, with each pair of lysine acetyltransferases/deacetylases and lysine methyltransferases/demethylases operating complementarily to generate or erase lysine modifications. Several analyses also indicate a reversible nature of arginine methylation, but the enzymes facilitating direct removal of methyl moieties from arginine residues in proteins have been discussed controversially. Differing reports have been seen for initially characterized putative candidates, like peptidyl arginine deiminase 4 or Jumonji-domain containing protein 6. Here, we review the most recent cellular, biochemical, and mass spectrometry work on arginine methylation and its reversible nature with a special focus on putative arginine demethylases, including the enzyme superfamily of Fe(II) and 2-oxoglutarate-dependent oxygenases.

Nitric Oxide Mediates Glutamate-Linked Enhancement of cGMP Levels in the Cerebellum

NASA Astrophysics Data System (ADS)

Bredt, David S.; Snyder, Solomon H.

1989-11-01

Nitric oxide, which mediates influences of numerous neurotransmitters and modulators on vascular smooth muscle and leukocytes, can be formed in the brain from arginine by an enzymatic activity that stoichiometrically generates citrulline. We show that glutamate and related amino acids, such as N-methyl-D-aspartate, markedly stimulate arginine-citrulline transformation in cerebellar slices stoichiometrically with enhancement of cGMP levels. Nω-monomethyl-L-arginine blocks the augmentation both of citrulline and cGMP with identical potencies. Arginine competitively reverses both effects of Nω-monomethyl-L-arginine with the same potencies. Hemoglobin, which complexes nitric oxide, prevents the stimulation by N-methyl-D-aspartate of cGMP levels, and superoxide dismutase, which elevates nitric oxide levels, increases cGMP formation. These data establish that nitric oxide mediates the stimulation by glutamate of cGMP formation.

Arginine Transcriptional Response Does Not Require Inositol Phosphate Synthesis*

PubMed Central

Bosch, Daniel; Saiardi, Adolfo

2012-01-01

Inositol phosphates are key signaling molecules affecting a large variety of cellular processes. Inositol-polyphosphate multikinase (IPMK) is a central component of the inositol phosphate biosynthetic routes, playing essential roles during development. IPMK phosphorylates inositol 1,4,5-trisphosphate to inositol tetrakisphosphate and subsequently to inositol pentakisphosphate and has also been described to function as a lipid kinase. Recently, a catalytically inactive mammalian IPMK was reported to be involved in nutrient signaling by way of mammalian target of rapamycin and AMP-activated protein kinase. In yeast, the IPMK homologue, Arg82, is the sole inositol-trisphosphate kinase. Arg82 has been extensively studied as part of the transcriptional complex regulating nitrogen sensing, in particular arginine metabolism. Whether this role requires Arg82 catalytic activity has long been a matter of contention. In this study, we developed a novel method for the real time study of promoter strength in vivo and used it to demonstrate that catalytically inactive Arg82 fully restored the arginine-dependent transcriptional response. We also showed that expression in yeast of catalytically active, but structurally very different, mammalian or plant IPMK homologue failed to restore arginine regulation. Our work indicates that inositol phosphates do not regulate arginine-dependent gene expression. PMID:22992733

Effects of acute phencyclidine administration on arginine metabolism in the hippocampus and prefrontal cortex in rats.

PubMed

Knox, Logan T; Jing, Yu; Collie, Nicola D; Zhang, Hu; Liu, Ping

2014-06-01

Phencyclidine (PCP), a non-competitive N-methyl-d-aspartate glutamate receptor antagonist, induces schizophrenic symptoms in healthy individuals, and altered arginine metabolism has been implicated in schizophrenia. The present study investigated the effects of a single subcutaneous injection of PCP (2, 5 or 10 mg/kg) on arginine metabolism in the sub-regions of the hippocampus and prefrontal cortex in male young adult Sprague-Dawley rats. Animals' general behaviour was assessed in the open field apparatus 30 min after the treatment, and the brain tissues were collected at the time point of 60 min post-treatment. Behaviourally, PCP resulted in reduced exploratory activity in a dose-dependent manner, and severe stereotype behaviour and ataxia at the highest dose. Neurochemically, PCP significantly altered the nitric oxide synthase and arginase activities, the l-arginine, agmatine, spermine, glutamate and GABA levels, and the glutamine/glutamate and glutamate/GABA ratios in a dose-dependent and/or region-specific manner. Cluster analyses showed that l-arginine and its main metabolites l-citrulline, l-ornithine and agmatine formed distinct groups, which changed as a function of PCP mainly in the hippocampus. Multiple regression analysis revealed significant neurochemical-behavioural correlations. These results demonstrate, for the first time, that a single acute administration of PCP affects animals' behaviour and arginine metabolism in the brain. Copyright © 2014 Elsevier Ltd. All rights reserved.

[NO donors transform neuronal response to glutamate].

PubMed

D'iakonova, T L

1998-10-01

Electrophysiological experiments on three identified neurones were performed. Two NO donors, sodium nitroprusside (SNP) and sodium nitrite, as well as NO synthase inhibitor, were used. In each neurone, bath application of glutamate caused hyperpolarization and suppression of firing. Combined application of glutamate and SNP resulted in that the same cells responded to identical glutamate solutions with depolarization and excitation. Application of N-monomethyl-L-arginin (NMMA) arrested the glutamate-induced firing and depolarization. The findings suggest involvement of NO in the mechanism of transformation of glutamate-induced inhibition into excitation and a mediation of the latter by the N-methyl-D-aspartate-like receptors in the Helix brain.

Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase.

PubMed

Paradisi, Francesca; Dean, Jonathan L E; Geoghegan, Kieran F; Engel, Paul C

2005-03-08

A mutant (D165N) of clostridial glutamate dehydrogenase (GDH) in which the catalytic Asp is replaced by Asn surprisingly showed a residual 2% of wild-type activity when purified after expression in Escherichia coli at 37 degrees C. This low-level activity also displayed Michaelis constants for substrates that were remarkably similar to those of the wild-type enzyme. Expression at 8 degrees C gave a mutant enzyme preparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubation at 37 degrees C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggested that the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptides derived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDH freshly prepared at 8 degrees C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-week incubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainly occurred in sample workup rather than during the 2-week incubation; (iv) that there is no significant deamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) that when the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears that this deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literature search indicates that this finding is not unique and that Asn may not be a suitable mutational replacement in the assessment of putative catalytic Asp residues by site-directed mutagenesis.

Metabolic engineering of Corynebacterium glutamicum for L-arginine production.

PubMed

Park, Seok Hyun; Kim, Hyun Uk; Kim, Tae Yong; Park, Jun Seok; Kim, Suok-Su; Lee, Sang Yup

2014-08-05

L-arginine is an important amino acid for diverse industrial and health product applications. Here we report the development of metabolically engineered Corynebacterium glutamicum ATCC 21831 for the production of L-arginine. Random mutagenesis is first performed to increase the tolerance of C. glutamicum to L-arginine analogues, followed by systems metabolic engineering for further strain improvement, involving removal of regulatory repressors of arginine operon, optimization of NADPH level, disruption of L-glutamate exporter to increase L-arginine precursor and flux optimization of rate-limiting L-arginine biosynthetic reactions. Fed-batch fermentation of the final strain in 5 l and large-scale 1,500 l bioreactors allows production of 92.5 and 81.2 g l(-1) of L-arginine with the yields of 0.40 and 0.35 g L-arginine per gram carbon source (glucose plus sucrose), respectively. The systems metabolic engineering strategy described here will be useful for engineering Corynebacteria strains for the industrial production of L-arginine and related products.

A cyanase is transcriptionally regulated by arginine and involved in cyanate decomposition in Sordaria macrospora.

PubMed

Elleuche, Skander; Pöggeler, Stefanie

2008-11-01

Cyanase degrades toxic cyanate to NH3 and CO2 in a bicarbonate-dependent reaction. High concentrations of cyanate are fairly toxic to organisms. Here, we characterize a eukaryotic cyanase for the first time. We have isolated the cyn1 gene encoding a cyanase from the filamentous ascomycete Sordaria macrospora and functionally characterized the cyn1 product after heterologous expression in Escherichia coli. Site-directed mutagenesis confirmed a predicted catalytic centre of three conserved amino-acids. A Deltacyn1 knockout in S. macrospora was totally devoid of cyanase activity and showed an increased sensitivity to exogenously supplied cyanate in an arginine-depleted medium, defects in ascospore germination, but no other obvious morphological phenotype. By means of real-time PCR we have demonstrated that the transcriptional level of cyn1 is markedly elevated in the presence of cyanate and down-regulated by addition of arginine. The putative functions of cyanase in fungi are discussed.

Comparative Monomethylarginine Proteomics Suggests that Protein Arginine Methyltransferase 1 (PRMT1) is a Significant Contributor to Arginine Monomethylation in Toxoplasma gondii

PubMed Central

Yakubu, Rama R.; Silmon de Monerri, Natalie C.; Nieves, Edward; Kim, Kami; Weiss, Louis M.

2017-01-01

Arginine methylation is a common posttranslational modification found on nuclear and cytoplasmic proteins that has roles in transcriptional regulation, RNA metabolism and DNA repair. The protozoan parasite Toxoplasma gondii has a complex life cycle requiring transcriptional plasticity and has unique transcriptional regulatory pathways. Arginine methylation may play an important part in transcriptional regulation and splicing biology in this organism. The T. gondii genome contains five putative protein arginine methyltransferases (PRMTs), of which PRMT1 is important for cell division and growth. In order to better understand the function(s) of the posttranslational modification monomethyl arginine (MMA) in T. gondii, we performed a proteomic analysis of MMA proteins using affinity purification employing anti-MMA specific antibodies followed by mass spectrometry. The arginine monomethylome of T. gondii contains a large number of RNA binding proteins and multiple ApiAP2 transcription factors, suggesting a role for arginine methylation in RNA biology and transcriptional regulation. Surprisingly, 90% of proteins that are arginine monomethylated were detected as being phosphorylated in a previous phosphoproteomics study which raises the possibility of interplay between MMA and phosphorylation in this organism. Supporting this, a number of kinases are also arginine methylated. Because PRMT1 is thought to be a major PRMT in T. gondii, an organism which lacks a MMA-specific PRMT, we applied comparative proteomics to understand how PRMT1 might contribute to the MMA proteome in T. gondii. We identified numerous putative PRMT1 substrates, which include RNA binding proteins, transcriptional regulators (e.g. AP2 transcription factors), and kinases. Together, these data highlight the importance of MMA and PRMT1 in arginine methylation in T. gondii, as a potential regulator of a large number of processes including RNA biology and transcription. PMID:28143887

A Comparison of Two Single-Stranded DNA Binding Models by Mutational Analysis of APOBEC3G

PubMed Central

Shindo, Keisuke; Li, Ming; Gross, Phillip J.; Brown, William L.; Harjes, Elena; Lu, Yongjian; Matsuo, Hiroshi; Harris, Reuben S.

2012-01-01

APOBEC3G is the best known of several DNA cytosine deaminases that function to inhibit the replication of parasitic genetic elements including the lentivirus HIV. Several high-resolution structures of the APOBEC3G catalytic domain have been generated, but none reveal how this enzyme binds to substrate single-stranded DNA. Here, we constructed a panel of APOBEC3G amino acid substitution mutants and performed a series of biochemical, genetic, and structural assays to distinguish between “Brim” and “Kink” models for single-strand DNA binding. Each model predicts distinct sets of interactions between surface arginines and negatively charged phosphates in the DNA backbone. Concordant with both models, changing the conserved arginine at position 313 to glutamate abolished both catalytic and restriction activities. In support of the Brim model, arginine to glutamate substitutions at positions 213, 215, and 320 also compromised these APOBEC3G activities. Arginine to glutamate substitutions at Kink model residues 374 and 376 had smaller effects. These observations were supported by A3G catalytic domain-ssDNA chemical shift perturbation experiments. The overall data set is most consistent with the Brim model for single-stranded DNA binding by APOBEC3G. PMID:24832226

Arginine metabolism and nutrition in growth, health and disease

USDA-ARS?s Scientific Manuscript database

L-Arginine (Arg) is synthesised from glutamine, glutamate, and proline via the intestinal-renal axis in humans and most other mammals (including pigs, sheep, and rats). Arg degradation occurs via multiple pathways that are initiated by arginase, nitric-oxide synthase, Arg:glycine amidinotransferase,...

Occurrence, Functions and Biological Significance of Arginine-Rich Proteins.

PubMed

Chandana, Thimmegowda; Venkatesh, Yeldur P

2016-01-01

Arginine, the most basic among the 20 amino acids, occurs less frequently than lysine in proteins despite being coded by six codons. Only a few important proteins of biological significance have been found to be abundant in arginine. It has been established that these arginine-rich proteins have been assigned important roles in the biological systems. Arginine-rich cationic proteins are known to stabilize macromolecular structures by establishing appropriate interactions (salt bridges, hydrogen bonds and cation-π interactions). These proteins are also known to be the key members of many regulatory pathways such as gene expression, chromatin stability, expurgation of introns from naïve mRNA, mRNA splicing, membrane-penetrating activity and pathogenesis-related defense, to name a few. Further, arginine occurs in various combinations with other amino acids (serine, lysine, proline, tryptophan, valine, glycine and glutamic acid) which diversify the potential functions of arginine-rich proteins. Arginine-rich proteins known till date from dietary sources have been described in terms of their structure and functional properties. A variety of activities such as bactericidal, membrane-penetrating, antimicrobial, anti-hypertensive, pro-angiogenic and others have been reported for arginine-rich proteins. This review attempts to collate the occurrence, functions and the biological significance of this unique class of proteins rich in arginine.

An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation.

PubMed

Friedt, Jenna; Leavens, Fern M V; Mercier, Evan; Wieden, Hans-Joachim; Kothe, Ute

2014-04-01

Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB's catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.

An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation

PubMed Central

Friedt, Jenna; Leavens, Fern M. V.; Mercier, Evan; Wieden, Hans-Joachim; Kothe, Ute

2014-01-01

Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism. PMID:24371284

Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli: An in silico study.

PubMed

Tavakoli, Yasaman; Esmaeili, Abolghasem; Saber, Hossein

2016-10-01

Glutamate decarboxylase (GAD) is an enzyme that converts l-glutamate to gamma amino butyric acid (GABA) that is a widely used drug to treat mental disorders like Alzheimer's disease. In this study for the first time point mutation was performed virtually in the active site of the E. coli GAD in order to increase thermal stability and catalytic activity of the enzyme. Energy minimization and addition of water box were performed using GROMACS 5.4.6 package. PoPMuSiC 2.1 web server was used to predict potential spots for point mutation and Modeller software was used to perform point mutation on three dimensional model. Molegro virtual docker software was used for cavity detection and stimulated docking study. Results indicate that performing mutation separately at positions 164, 302, 304, 393, 396, 398 and 410 increase binding affinity to substrate. The enzyme is predicted to be more thermo- stable in all 7 mutants based on ΔΔG value. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4.

PubMed

Boulanger, Marie-Chloé; Miranda, Tina Branscombe; Clarke, Steven; Di Fruscio, Marco; Suter, Beat; Lasko, Paul; Richard, Stéphane

2004-04-15

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

Metabolism of arginine by aging and 7 day old pumpkin seedlings.

PubMed

Splittstoesser, W E

1969-03-01

The metabolism of arginine by etiolated pumpkin (Cucurbita moschata) seedlings was studied over various time and age intervals by injecting arginine-U-(14)C into the cotyledons. At most, 25% of the (14)C was transported from the cotyledon to the axis tissue and the amount of this transport decreased with increasing age of the seedlings. The cotyledons of 25 day old plants contained 60% of the administered (14)C as unmetabolized arginine. Little (14)C was in sugars and it appeared that arginine was the primary translocation product. Time course studies showed that arginine was extensively metabolized and the labeling patterns suggest that different pathways were in operation in the axis and cotyledons. The amount of arginine incorporated into cotyledonary protein show that synthesis and turnover were occurring at rapid rate. Only 25% of the label incorporated into protein by 1.5 hr remained after 96 hr. The label in protein was stable in the axis tissue. By 96 hr 50% of the administered label occurred as (14)CO(2) and it appeared that arginine was metabolized, through glutamate, by the citrio acid cycle in the cotyledons. The experiments showed that an extensive conversion of arginine carbon into other amino acids did not occur.

A novel method for the functionalization of aminoacids L-glycine, L-glutamic acid and L-arginine on maghemite/magnetite nanoparticles

NASA Astrophysics Data System (ADS)

Bruno, A. J.; Correa, J. R.; Peláez-Abellán, E.; Urones-Garrote, E.

2018-06-01

Nanoparticles of maghemite/magnetite functionalized with L-glycine, L-glutamic acid and L-arginine were synthesized by a novel method. The novel procedure consists in an alternative of that reported by Massart for the precipitation of magnetite in which the aminoacid is added in the carboxylate form. The amounts of aminoacid in the initial molar concentrations were 35%, 45% and 65% with respect to the ferrophase. The obtained nanoparticles were characterized by several techniques: X-ray diffraction (XRD), Fourier transform IR spectroscopy (FT-IR), transmission electron microscopy (TEM), Electron energy-loss spectroscopy (EELS) and magnetometry. The IR spectroscopy confirmed that the selected aminoacids were functionalized on the surface of iron oxide. XRD and EELS confirm that iron oxide consists of a maghemite-magnetite intermediate phase with an average particle size about 6 nm, which was measured by transmission electron microscopy. The superparamagnetic character of the nanoparticles was evaluated by magnetometry.

The CASTOR proteins are arginine sensors for the mTORC1 pathway

PubMed Central

Chantranupong, Lynne; Scaria, Sonia M.; Saxton, Robert A.; Gygi, Melanie P.; Shen, Kuang; Wyant, Gregory A.; Wang, Tim; Harper, J. Wade; Gygi, Steven P.; Sabatini, David M.

2016-01-01

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 μM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway. PMID:26972053

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4.

PubMed Central

Boulanger, Marie-Chloé; Miranda, Tina Branscombe; Clarke, Steven; Di Fruscio, Marco; Suter, Beat; Lasko, Paul; Richard, Stéphane

2004-01-01

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation. PMID:14705965

Altered brain arginine metabolism in schizophrenia

PubMed Central

Liu, P; Jing, Y; Collie, N D; Dean, B; Bilkey, D K; Zhang, H

2016-01-01

Previous research implicates altered metabolism of l-arginine, a versatile amino acid with a number of bioactive metabolites, in the pathogenesis of schizophrenia. The present study, for we believe the first time, systematically compared the metabolic profile of l-arginine in the frontal cortex (Brodmann's area 8) obtained post-mortem from schizophrenic individuals and age- and gender-matched non-psychiatric controls (n=20 per group). The enzyme assays revealed no change in total nitric oxide synthase (NOS) activity, but significantly increased arginase activity in the schizophrenia group. Western blot showed reduced endothelial NOS protein expression and increased arginase II protein level in the disease group. High-performance liquid chromatography and liquid chromatography/mass spectrometric assays confirmed significantly reduced levels of γ-aminobutyric acid (GABA), but increased agmatine concentration and glutamate/GABA ratio in the schizophrenia cases. Regression analysis indicated positive correlations between arginase activity and the age of disease onset and between l-ornithine level and the duration of illness. Moreover, cluster analyses revealed that l-arginine and its main metabolites l-citrulline, l-ornithine and agmatine formed distinct groups, which were altered in the schizophrenia group. The present study provides further evidence of altered brain arginine metabolism in schizophrenia, which enhances our understanding of the pathogenesis of schizophrenia and may lead to the future development of novel preventions and/or therapeutics for the disease. PMID:27529679

Altered brain arginine metabolism in schizophrenia.

PubMed

Liu, P; Jing, Y; Collie, N D; Dean, B; Bilkey, D K; Zhang, H

2016-08-16

Previous research implicates altered metabolism of l-arginine, a versatile amino acid with a number of bioactive metabolites, in the pathogenesis of schizophrenia. The present study, for we believe the first time, systematically compared the metabolic profile of l-arginine in the frontal cortex (Brodmann's area 8) obtained post-mortem from schizophrenic individuals and age- and gender-matched non-psychiatric controls (n=20 per group). The enzyme assays revealed no change in total nitric oxide synthase (NOS) activity, but significantly increased arginase activity in the schizophrenia group. Western blot showed reduced endothelial NOS protein expression and increased arginase II protein level in the disease group. High-performance liquid chromatography and liquid chromatography/mass spectrometric assays confirmed significantly reduced levels of γ-aminobutyric acid (GABA), but increased agmatine concentration and glutamate/GABA ratio in the schizophrenia cases. Regression analysis indicated positive correlations between arginase activity and the age of disease onset and between l-ornithine level and the duration of illness. Moreover, cluster analyses revealed that l-arginine and its main metabolites l-citrulline, l-ornithine and agmatine formed distinct groups, which were altered in the schizophrenia group. The present study provides further evidence of altered brain arginine metabolism in schizophrenia, which enhances our understanding of the pathogenesis of schizophrenia and may lead to the future development of novel preventions and/or therapeutics for the disease.

A glycine-to-glutamate substitution abolishes alanine:glyoxylate aminotransferase catalytic activity in a subset of patients with primary hyperoxaluria type 1.

PubMed

Purdue, P E; Lumb, M J; Allsop, J; Minatogawa, Y; Danpure, C J

1992-05-01

We have synthesized and sequenced alanine:glyoxylate aminotransferase (AGT; HGMW-approved symbol for the gene--AGXT) cDNA from the liver of a primary hyperoxaluria type 1 (PH1) patient who had normal levels of hepatic peroxisomal immunoreactive AGT protein, but no AGT catalytic activity. This revealed the presence of a single point mutation (G----A at cDNA nucleotide 367), which is predicted to cause a glycine-to-glutamate substitution at residue 82 of the AGT protein. This mutation is located in exon 2 of the AGT gene and leads to the loss of an AvaI restriction site. Exon 2-specific PCR followed by AvaI digestion showed that this patient was homozygous for this mutation. In addition, three other PH1 patients, one related to and two unrelated to, but with enzymological phenotype similar to that of the first patient, were also shown to be homozygous for the mutation. However, one other phenotypically similar PH1 patient was shown to lack this mutation. The mechanism by which the glycine-to-glutamate substitution at residue 82 causes loss of catalytic activity remains to be resolved. However, the protein sequence in this region is highly conserved between different mammals, and the substitution at residue 82 is predicted to cause significant local structural alterations.

Important roles for the arginine family of amino acids in swine nutrition and production

USDA-ARS?s Scientific Manuscript database

Arginine, glutamine, glutamate, proline, aspartate, asparagine, ornithine, and citrulline are interconvertible via complex interorgan metabolism in most mammals (including the pig). The major sites for their metabolism are the small intestine, kidneys, and liver, with cortisol being a key regulatory...

Protein arginine methyltransferase 7 has a novel homodimer-like structure formed by tandem repeats.

PubMed

Hasegawa, Morio; Toma-Fukai, Sachiko; Kim, Jun-Dal; Fukamizu, Akiyoshi; Shimizu, Toshiyuki

2014-05-21

Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Here, we describe the crystal structure of Caenorhabditis elegans PRMT7 in complex with its reaction product S-adenosyl-L-homocysteine. The structural data indicated that PRMT7 harbors two tandem repeated PRMT core domains that form a novel homodimer-like structure. S-adenosyl-L-homocysteine bound to the N-terminal catalytic site only; the C-terminal catalytic site is occupied by a loop that inhibits cofactor binding. Mutagenesis demonstrated that only the N-terminal catalytic site of PRMT7 is responsible for cofactor binding. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The story of protein arginine methylation: characterization, regulation, and function.

PubMed

Peng, Chao; Wong, Catherine Cl

2017-02-01

Arginine methylation is an important post-translational modification (PTM) in cells, which is catalyzed by a group of protein arginine methyltransferases (PRMTs). It plays significant roles in diverse cellular processes and various diseases. Misregulation and aberrant expression of PRMTs can provide potential biomarkers and therapeutic targets for drug discovery. Areas covered: Herein, we review the arginine methylation literature and summarize the methodologies for the characterization of this modification, as well as describe the recent insights into arginine methyltransferases and their biological functions in diseases. Expert commentary: Benefits from the enzyme-based large-scale screening approach, the novel affinity enrichment strategies, arginine methylated protein family is the focus of attention. Although a number of arginine methyltransferases and related substrates are identified, the catalytic mechanism of different types of PRMTs remains unclear and few related demethylases are characterized. Novel functional studies continuously reveal the importance of this modification in the cell cycle and diseases. A deeper understanding of arginine methylated proteins, modification sites, and their mechanisms of regulation is needed to explore their role in life processes, especially their relationship with diseases, thus accelerating the generation of potent, selective, cell-penetrant drug candidates.

Arginine-Ornithine Antiporter ArcD Controls Arginine Metabolism and Interspecies Biofilm Development of Streptococcus gordonii*♦

PubMed Central

Sakanaka, Akito; Kuboniwa, Masae; Takeuchi, Hiroki; Hashino, Ei; Amano, Atsuo

2015-01-01

Arginine is utilized by the oral inhabitant Streptococcus gordonii as a substrate of the arginine deiminase system (ADS), eventually producing ATP and NH3, the latter of which is responsible for microbial resistance to pH stress. S. gordonii expresses a putative arginine-ornithine antiporter (ArcD) whose function has not been investigated despite relevance to the ADS and potential influence on inter-bacterial communication with periodontal pathogens that utilize amino acids as a main energy source. Here, we generated an S. gordonii ΔarcD mutant to explore the role of ArcD in physiological homeostasis and bacterial cross-feeding. First, we confirmed that S. gordonii ArcD plays crucial roles for mediating arginine uptake and promoting bacterial growth, particularly under arginine-limited conditions. Next, metabolomic profiling and transcriptional analysis of the ΔarcD mutant revealed that deletion of this gene caused intracellular accumulation of ornithine leading to malfunction of the ADS and suppression of de novo arginine biosynthesis. The mutant strain also showed increased susceptibility to low pH stress due to reduced production of ammonia. Finally, accumulation of Fusobacterium nucleatum was found to be significantly decreased in biofilm formed by the ΔarcD mutant as compared with the wild-type strain, although ornithine supplementation restored fusobacterium biovolume in dual-species biofilms with the ΔarcD mutant and also enhanced single species biofilm development by F. nucleatum. Our results are the first direct evidence showing that S. gordonii ArcD modulates not only alkali and energy production but also interspecies interaction with F. nucleatum, thus initiating a middle stage of periodontopathic biofilm formation, by metabolic cross-feeding. PMID:26085091

Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif.

PubMed

Hurd, Matthew C; Kwon, Moonhyuk; Ro, Dae-Kyun

2017-08-26

Lippia dulcis (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of L. dulcis leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (LdTPS3 and LdTPS9) were isolated from L. dulcis leaf cDNA. To elucidate their catalytic functions, E. coli-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). In vitro enzyme assays showed that LdTPS3 encodes bornyl diphosphate synthase (thus named LdBPPS) while LdTPS9 encodes linalool synthase. Interestingly, the N-terminus of LdBPPS possesses two arginine-rich (RRX 8 W) motifs, and enzyme assays showed that the presence of both RRX 8 W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX 8 W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and L. dulcis. The discovery of LdBPPS opens up the possibility of engineering L. dulcis to remove the undesirable product, camphor. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of Detergents on Catalytic Activity of Human Endometase/Matrilysin-2, a Putative Cancer Biomarker†

PubMed Central

Park, Hyun I.; Lee, Seakwoo; Ullah, Asad; Cao, Qiang; Sang, Qing-Xiang Amy

2009-01-01

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration-dependent exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matirilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 (~90 μM). Their IC50 values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was mixed-type as determined by Dixon’s plot, however, that of endometase was non-competitive with a Ki value of 240 μM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block substrate binding site to impede catalysis. Under physiological conditions lipid or membrane microenvironment may regulate enzymatic activity. PMID:19818727

Glutamine and arginine improve permeability and tight junction protein expression in methotrexate-treated Caco-2 cells.

PubMed

Beutheu, Stéphanie; Ghouzali, Ibtissem; Galas, Ludovic; Déchelotte, Pierre; Coëffier, Moïse

2013-10-01

Chemotherapy induces an increase of intestinal permeability that is partially related to an alteration of tight junction proteins, occludin and zonula occludens-1 (ZO-1). Protective effects of glutamine on intestinal barrier function have been previously shown but the effects of other amino acids remained poorly documented. Thus, we aimed to evaluate the effects of nine amino acids on intestinal permeability during methotrexate (MTX) treatment in Caco-2 cells. Caco-2 cells were incubated in culture medium supplemented with glutamine, arginine, glutamate, leucine, taurine, citrulline, glycine, histidine or cysteine during 24 h and then treated with MTX (100 ng/ml). The dose of each amino acid was 16.6 fold the physiological plasma concentrations. Barrier function was assessed by transepithelial electrical resistance (TEER), FITC-dextran paracellular flux, occludin and ZO-1 expression and localization. Signaling pathways were also studied. Only glutamine, glutamate, arginine and leucine reversed the decrease of TEER observed after MTX treatment (P < 0.05). Interestingly, the addition of 6-diazo-5-oxo-1-norleucine, an inhibitor of glutaminase, blunted the effect of glutamine on MTX-treated cells (P < 0.05). Glutamine and arginine combination restored TEER and FITC-dextran flux to a similar extent than glutamine alone. In addition, pretreatment of Caco-2 cells with glutamine and arginine, alone or combined, differently limited the decrease of ZO-1 and occludin expression (P < 0.05) and the alteration of their cellular distribution, through c-Jun N-terminal kinase (JNK), Extracellular signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) pathways. Glutamine prevented MTX-induced barrier disruption in Caco-2 cells. Arginine also had protective effects but in a lesser extent. The effect of glutamine and arginine should be evaluated in vivo. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

The role of the arginine metabolome in pain: implications for sickle cell disease.

PubMed

Bakshi, Nitya; Morris, Claudia R

2016-01-01

Sickle cell disease (SCD) is the most common hemoglobinopathy in the US, affecting approximately 100,000 individuals in the US and millions worldwide. Pain is the hallmark of SCD, and a subset of patients experience pain virtually all of the time. Of interest, the arginine metabolome is associated with several pain mechanisms highlighted in this review. Since SCD is an arginine deficiency syndrome, the contribution of the arginine metabolome to acute and chronic pain in SCD is a topic in need of further attention. Normal arginine metabolism is impaired in SCD through various mechanisms that contribute to endothelial dysfunction, vaso-occlusion, pulmonary complications, risk of leg ulcers, and early mortality. Arginine is a semiessential amino acid that serves as a substrate for protein synthesis and is the precursor to nitric oxide (NO), polyamines, proline, glutamate, creatine, and agmatine. Since arginine is involved in multiple metabolic processes, a deficiency of this amino acid has the potential to disrupt many cellular and organ functions. NO is a potent vasodilator that is depleted in SCD and may contribute to vaso-occlusive pain. As the obligate substrate for NO production, arginine also plays a mechanistic role in SCD-related pain, although its contribution to pain pathways likely extends beyond NO. Low global arginine bioavailability is associated with pain severity in both adults and children with SCD as well as other non-SCD pain syndromes. Preliminary clinical studies of arginine therapy in SCD demonstrate efficacy in treating acute vaso-occlusive pain, as well as leg ulcers and pulmonary hypertension. Restoration of arginine bioavailability through exogenous supplementation of arginine is, therefore, a promising therapeutic target. Phase II clinical trials of arginine therapy for sickle-related pain are underway and a Phase III randomized controlled trial is anticipated in the near future.

Probing the catalytic roles of n2-site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis.

PubMed Central

Witmer, M. R.; Palmieri-Young, D.; Villafranca, J. J.

1994-01-01

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS. PMID:7849593

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis.

PubMed

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2014-01-01

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.

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

PubMed

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.

Crystal structure of a chimaeric bacterial glutamate dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oliveira, Tânia; Sharkey, Michael A.; Engel, Paul C.

2016-05-23

Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P) +as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD +versusNADP +, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies,more » as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP +cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.« less

Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels.

PubMed

Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L

2013-03-05

AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl(-) lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels

PubMed Central

Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L.

2014-01-01

SUMMARY AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine and phenylalanine which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate and serine. AvGluR1 LBD crystal structures reveal a novel scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, while in the alanine, methionine and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl− lowers affinity for these ligands, but not for glutamate, aspartate or for phenylalanine which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure based studies on iGluR-ligand interactions. PMID:23434404

Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahmad, Md. Faiz; Kaushal, Prem Singh; Wan, Qun

2012-11-01

Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 ({alpha}) that contains the catalytic site and RR2 ({beta}) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-typemore » enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-({beta},{gamma}-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.« less

The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy

PubMed Central

Kötting, Carsten; Kallenbach, Angela; Suveyzdis, Yan; Wittinghofer, Alfred; Gerwert, Klaus

2008-01-01

Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved “arginine finger” of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras·RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-I region of Ras from the nonsignaling “off” to the signaling “on” state with a rate of 3 s−1. The next step is the movement of the “arginine finger” into the active site of Ras with a rate of k2 = 0.8 s−1. Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound Pi intermediate forms. The switch-I reversal to the “off” state, the release of Pi, and the movement of arginine back into an aqueous environment is observed simultaneously with k3 = 0.1 s−1, the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the “arginine finger” movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a Pi-intermediate. PMID:18434546

Recent advances in arginine metabolism: roles and regulation of the arginases

PubMed Central

Morris, Sidney M

2009-01-01

As arginine can serve as precursor to a wide range of compounds, including nitric oxide, creatine, urea, polyamines, proline, glutamate and agmatine, there is considerable interest in elucidating mechanisms underlying regulation of its metabolism. It is now becoming apparent that the two isoforms of arginase in mammals play key roles in regulation of most aspects of arginine metabolism in health and disease. In particular, work over the past several years has focused on the roles and regulation of the arginases in vascular disease, pulmonary disease, infectious disease, immune cell function and cancer. As most of these topics have been considered in recent review articles, this review will focus more closely on results of recent studies on expression of the arginases in endothelial and vascular smooth muscle cells, post-translational modulation of arginase activity and applications of arginase inhibitors in vivo. PMID:19508396

Lack of a synergistic effect of arginine-glutamic acid on the physical stability of spray-dried bovine serum albumin.

PubMed

Reslan, Mouhamad; Demir, Yusuf K; Trout, Bernhardt L; Chan, Hak-Kim; Kayser, Veysel

2017-09-01

Improving the physical stability of spray-dried proteins is essential for enabling pulmonary delivery of biotherapeutics as a noninvasive alternative to injections. Recently, a novel combination of two amino acids - l-arginine (l-Arg) and l-glutamic acid (l-Glu), has been reported to have synergistic protein-stabilizing effects on various protein solutions. Using spray-dried bovine serum albumin (BSA) reconstituted in solution as a model protein, we investigated the synergistic effect of these amino acids on the physical stability of proteins. Five BSA solutions were prepared: (1) BSA with no amino acids (control); (2) with 50 mM l-Arg; (3) with 200 mM l-Arg, (4) with 50 mM l-Glu and (5) with 25:25 mM of Arg:Glu. All solutions were spray-dried and accelerated studies at high temperatures were performed. Following accelerated studies, monomer BSA loss was measured using SE-HPLC. We found that l-Arg significantly improved the physical stability of spray-dried BSA even at low concentrations, however, when combined with l-Glu, was ineffective at reducing monomer BSA loss. Our findings demonstrate the limitations in using Arg-Glu for the stabilization of spray-dried BSA. Furthermore, we found that a low concentration of l-Glu enhanced monomer BSA loss. These findings may have significant implications on the design of future biotherapeutic formulations.

Proteomic Identification and Analysis of Arginine-Methylated Proteins of Plasmodium falciparum at Asexual Blood Stages.

PubMed

Zeeshan, Mohammad; Kaur, Inderjeet; Joy, Joseph; Saini, Ekta; Paul, Gourab; Kaushik, Abhinav; Dabral, Surbhi; Mohmmed, Asif; Gupta, Dinesh; Malhotra, Pawan

2017-02-03

Plasmodium falciparum undergoes a tightly regulated developmental process in human erythrocytes, and recent studies suggest an important regulatory role of post-translational modifications (PTMs). As compared with Plasmodium phosphoproteome, little is known about other PTMs in the parasite. In the present study, we performed a global analysis of asexual blood stages of Plasmodium falciparum to identify arginine-methylated proteins. Using two different methyl arginine-specific antibodies, we immunoprecipitated the arginine-methylated proteins from the stage-specific parasite lysates and identified 843 putative arginine-methylated proteins by LC-MS/MS. Motif analysis of the protein sequences unveiled that the methylation sites are associated with the previously known methylation motifs such as GRx/RGx, RxG, GxxR, or WxxxR. We identified Plasmodium homologues of known arginine-methylated proteins in trypanosomes, yeast, and human. Hydrophilic interaction liquid chromatography (HILIC) was performed on the immunoprecipitates from the trophozoite stage to enrich arginine-methylated peptides. Mass spectrometry analysis of immunoprecipitated and HILIC fractions identified 55 arginine-methylated peptides having 62 methylated arginine sites. Functional classification revealed that the arginine-methylated proteins are involved in RNA metabolism, protein synthesis, intracellular protein trafficking, proteolysis, protein folding, chromatin organization, hemoglobin metabolic process, and several other functions. Summarily, the findings suggest that protein methylation of arginine residues is a widespread phenomenon in Plasmodium, and the PTM may play an important regulatory role in a diverse set of biological pathways, including host-pathogen interactions.

The central active site arginine in sulfite oxidizing enzymes alters kinetic properties by controlling electron transfer and redox interactions.

PubMed

Hsiao, Ju-Chun; McGrath, Aaron P; Kielmann, Linda; Kalimuthu, Palraj; Darain, Farzana; Bernhardt, Paul V; Harmer, Jeffrey; Lee, Mihwa; Meyers, Kimberley; Maher, Megan J; Kappler, Ulrike

2018-01-01

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated. We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (Mo VI/V potential lowered by ca. 60-80mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorT WT , precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in K Msulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations. Copyright © 2017 Elsevier B.V. All rights reserved.

Non-Targeted Metabolomics Analysis of Golden Retriever Muscular Dystrophy-Affected Muscles Reveals Alterations in Arginine and Proline Metabolism, and Elevations in Glutamic and Oleic Acid In Vivo

PubMed Central

Abdullah, Muhammad; Kornegay, Joe N.; Honcoop, Aubree; Parry, Traci L.; Balog-Alvarez, Cynthia J.; Muehlbauer, Michael J.; Newgard, Christopher B.; Patterson, Cam

2017-01-01

Background: Like Duchenne muscular dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog model of DMD is characterized by muscle necrosis, progressive paralysis, and pseudohypertrophy in specific skeletal muscles. This severe GRMD phenotype includes moderate atrophy of the biceps femoris (BF) as compared to unaffected normal dogs, while the long digital extensor (LDE), which functions to flex the tibiotarsal joint and serves as a digital extensor, undergoes the most pronounced atrophy. A recent microarray analysis of GRMD identified alterations in genes associated with lipid metabolism and energy production. Methods: We, therefore, undertook a non-targeted metabolomics analysis of the milder/earlier stage disease GRMD BF muscle versus the more severe/chronic LDE using GC-MS to identify underlying metabolic defects specific for affected GRMD skeletal muscle. Results: Untargeted metabolomics analysis of moderately-affected GRMD muscle (BF) identified eight significantly altered metabolites, including significantly decreased stearamide (0.23-fold of controls, p = 2.89 × 10−3), carnosine (0.40-fold of controls, p = 1.88 × 10−2), fumaric acid (0.40-fold of controls, p = 7.40 × 10−4), lactamide (0.33-fold of controls, p = 4.84 × 10−2), myoinositol-2-phosphate (0.45-fold of controls, p = 3.66 × 10−2), and significantly increased oleic acid (1.77-fold of controls, p = 9.27 × 10−2), glutamic acid (2.48-fold of controls, p = 2.63 × 10−2), and proline (1.73-fold of controls, p = 3.01 × 10−2). Pathway enrichment analysis identified significant enrichment for arginine/proline metabolism (p = 5.88 × 10−4, FDR 4.7 × 10−2), where alterations in L-glutamic acid, proline, and carnosine were found. Additionally, multiple Krebs cycle intermediates were significantly decreased (e.g., malic acid, fumaric acid, citric/isocitric acid, and succinic acid), suggesting that altered energy metabolism may be underlying the observed GRMD BF muscle

Non-Targeted Metabolomics Analysis of Golden Retriever Muscular Dystrophy-Affected Muscles Reveals Alterations in Arginine and Proline Metabolism, and Elevations in Glutamic and Oleic Acid In Vivo.

PubMed

Abdullah, Muhammad; Kornegay, Joe N; Honcoop, Aubree; Parry, Traci L; Balog-Alvarez, Cynthia J; O'Neal, Sara K; Bain, James R; Muehlbauer, Michael J; Newgard, Christopher B; Patterson, Cam; Willis, Monte S

2017-07-29

Like Duchenne muscular dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog model of DMD is characterized by muscle necrosis, progressive paralysis, and pseudohypertrophy in specific skeletal muscles. This severe GRMD phenotype includes moderate atrophy of the biceps femoris (BF) as compared to unaffected normal dogs, while the long digital extensor (LDE), which functions to flex the tibiotarsal joint and serves as a digital extensor, undergoes the most pronounced atrophy. A recent microarray analysis of GRMD identified alterations in genes associated with lipid metabolism and energy production. We, therefore, undertook a non-targeted metabolomics analysis of the milder/earlier stage disease GRMD BF muscle versus the more severe/chronic LDE using GC-MS to identify underlying metabolic defects specific for affected GRMD skeletal muscle. Untargeted metabolomics analysis of moderately-affected GRMD muscle (BF) identified eight significantly altered metabolites, including significantly decreased stearamide (0.23-fold of controls, p = 2.89 × 10 -3 ), carnosine (0.40-fold of controls, p = 1.88 × 10 -2 ), fumaric acid (0.40-fold of controls, p = 7.40 × 10 -4 ), lactamide (0.33-fold of controls, p = 4.84 × 10 -2 ), myoinositol-2-phosphate (0.45-fold of controls, p = 3.66 × 10 -2 ), and significantly increased oleic acid (1.77-fold of controls, p = 9.27 × 10 -2 ), glutamic acid (2.48-fold of controls, p = 2.63 × 10 -2 ), and proline (1.73-fold of controls, p = 3.01 × 10 -2 ). Pathway enrichment analysis identified significant enrichment for arginine/proline metabolism (p = 5.88 × 10 -4 , FDR 4.7 × 10 -2 ), where alterations in L-glutamic acid, proline, and carnosine were found. Additionally, multiple Krebs cycle intermediates were significantly decreased (e.g., malic acid, fumaric acid, citric/isocitric acid, and succinic acid), suggesting that altered energy metabolism may be underlying the observed GRMD BF muscle dysfunction. In contrast, two

GABA and glutamate immunoreactivity in tentacles of the sea anemone Phymactis papillosa (LESSON 1830).

PubMed

Delgado, Luz M; Couve, Eduardo; Schmachtenberg, Oliver

2010-07-01

Sea anemones have a structurally simple nervous system that controls behaviors like feeding, locomotion, aggression, and defense. Specific chemical and tactile stimuli are transduced by ectodermal sensory cells and transmitted via a neural network to cnidocytes and epithelio-muscular cells, but the nature of the neurotransmitters operating in these processes is still under discussion. Previous studies demonstrated an important role of peptidergic transmission in cnidarians, but during the last decade the contribution of conventional neurotransmitters became increasingly evident. Here, we used immunohistochemistry on light and electron microscopical preparations to investigate the localization of glutamate and GABA in tentacle cross-sections of the sea anemone Phymactis papillosa. Our results demonstrate strong glutamate immunoreactivity in the nerve plexus, while GABA labeling was most prominent in the underlying epithelio-muscular layer. Immunoreactivity for both molecules was also found in glandular epithelial cells, and putative sensory cells were GABA positive. Under electron microscopy, both glutamate and GABA immunogold labeling was found in putative neural processes within the neural plexus. These data support a function of glutamate and GABA as signaling molecules in the nervous system of sea anemones.

Effect of dietary arginine on growth, intestinal enzyme activities and gene expression in muscle, hepatopancreas and intestine of juvenile Jian carp (Cyprinus carpio var. Jian).

PubMed

Chen, Gangfu; Feng, Lin; Kuang, Shengyao; Liu, Yang; Jiang, Jun; Hu, Kai; Jiang, Weidan; Li, Shuhong; Tang, Ling; Zhou, Xiaoqiu

2012-07-01

The present study was conducted to test the hypothesis that dietary arginine promotes digestion and absorption capacity, and, thus, enhances fish growth. This improvement might be related to the target of rapamycin (TOR) and eIF4E-binding protein (4E-BP). A total of 1200 juvenile Jian carp, Cyprinus carpio var. Jian, with an average initial weight of 6.33 (SE 0.03) g, were fed with diets containing graded concentrations of arginine, namely, 9.8 (control), 12.7, 16.1, 18.5, 21.9 and 24.5 g arginine/kg diet for 9 weeks. An real-time quantitative PCR analysis was performed to determine the relative expression of TOR and 4E-BP in fish muscle, hepatopancreas and intestine. Dietary arginine increased (P < 0.05): (1) glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities in muscle and hepatopancreas; (2) intestine and hepatopancreas protein content, folds height, and trypsin, chymotrypsin, lipase, Na⁺/K⁺-ATPase, alkaline phosphatase, γ-glutamyl transpeptidase and creatine kinase activities in intestine; (3) Lactobacillus counts; (4) relative expression of TOR in the muscle, hepatopancreas and distal intestine (DI); (5) relative expression of 4E-BP in proximal intestine (PI) and mid-intestine (MI), as compared with the control group. In contrast, dietary arginine reduced (P < 0.05): (1) plasma ammonia content; (2) Aeromonas hydrophila and Escherichia coli counts; (3) relative expression of TOR in PI and MI; (4) relative expression of 4E-BP in the muscle, hepatopancreas and DI. The arginine requirement estimated by specific growth rate using quadratic regression analysis was found to be 18.0 g/kg diet. These results indicate that arginine improved fish growth, digestive and absorptive ability and regulated the expression of TOR and 4E-BP genes.

Absence of specific binding of several putative neuro-transmitters to human fibroblasts.

PubMed

Berrettini, W H; Nadi, N S; Gershon, E S

1983-01-01

Fibroblasts were examined for specific binding sites of ten putative neurotransmitters to determine whether this tissue could be used in receptor studies of neurologic and psychiatric disorders. Stereospecific saturable binding was not found for any of the ligands: arginine vasopressin, neurotensin, somatostatin, angiotensin II, thyrotropin-releasing hormone (TRH), alpha-bungarotoxin, LSD, dihydromorphine, muscimol and spiperone.

Calcium alpha-ketoglutarate administration to malnourished hemodialysis patients improves plasma arginine concentrations.

PubMed

Riedel, E; Hampl, H; Steudle, V; Nündel, M

1996-01-01

Calcium alpha-ketoglutarate administration to 24 malnourished hemodialysis patients for 1 year leads to a significant increase in plasma concentrations of L-arginine from 53.6 +/- 18.3 (compared to a healthy control group: 87.5 +/- 27.3) to 71.1 +/- 15.9 mumol/l (p < 0.05). Furthermore, concentrations in plasma of proline and histidine, precursors of glutamate biosynthesis, are increased; inorganic phosphate and urea are significantly decreased in hemodialysis patients after 1 year of calcium alpha-ketoglutarate administration.

Catalytic site identification--a web server to identify catalytic site structural matches throughout PDB.

PubMed

Kirshner, Daniel A; Nilmeier, Jerome P; Lightstone, Felice C

2013-07-01

The catalytic site identification web server provides the innovative capability to find structural matches to a user-specified catalytic site among all Protein Data Bank proteins rapidly (in less than a minute). The server also can examine a user-specified protein structure or model to identify structural matches to a library of catalytic sites. Finally, the server provides a database of pre-calculated matches between all Protein Data Bank proteins and the library of catalytic sites. The database has been used to derive a set of hypothesized novel enzymatic function annotations. In all cases, matches and putative binding sites (protein structure and surfaces) can be visualized interactively online. The website can be accessed at http://catsid.llnl.gov.

Expression, purification and preliminary X-ray analysis of the C-terminal domain of an arginine repressor protein from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, George J.; Garen, Craig R.; Cherney, Maia M.

2007-11-01

The C-terminal portion of the arginine repressor protein from M. tuberculosis H37Rv has been crystallized. The complete transcriptional factor regulates arginine biosynthesis by binding operator DNA when arginine is bound at the C-terminal domain. The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 Å. The crystals belong to space group P1 and themore » Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of six molecules per unit cell.« less

Contribution of glutamate decarboxylase in Lactobacillus reuteri to acid resistance and persistence in sourdough fermentation

PubMed Central

2011-01-01

Background Acid stress impacts the persistence of lactobacilli in industrial sourdough fermentations, and in intestinal ecosystems. However, the contribution of glutamate to acid resistance in lactobacilli has not been demonstrated experimentally, and evidence for the contribution of acid resistance to the competitiveness of lactobacilli in sourdough is lacking. It was therefore the aim of this study to investigate the ecological role of glutamate decarboxylase in L. reuteri. Results A gene coding for a putative glutamate decarboxylase, gadB, was identified in the genome of L. reuteri 100-23. Different from the organization of genetic loci coding for glutamate decarboxylase in other lactic acid bacteria, gadB was located adjacent to a putative glutaminase gene, gls3. An isogenic deletion mutant, L. reuteri ∆gadB, was generated by a double crossover method. L. reuteri 100-23 but not L. reuteri ∆gadB converted glutamate to γ-aminobutyrate (GABA) in phosphate butter (pH 2.5). In sourdough, both strains converted glutamine to glutamate but only L. reuteri 100-23 accumulated GABA. Glutamate addition to phosphate buffer, pH 2.5, improved survival of L. reuteri 100-23 100-fold. However, survival of L. reuteri ∆gadB remained essentially unchanged. The disruption of gadB did not affect growth of L. reuteri in mMRS or in sourdough. However, the wild type strain L. reuteri 100-23 displaced L. reuteri ∆gadB after 5 cycles of fermentation in back-slopped sourdough fermentations. Conclusions The conversion of glutamate to GABA by L. reuteri 100-23 contributes to acid resistance and to competitiveness in industrial sourdough fermentations. The organization of the gene cluster for glutamate conversion, and the availability of amino acids in cereals imply that glutamine rather than glutamate functions as the substrate for GABA formation. The exceptional coupling of glutamine deamidation to glutamate decarboxylation in L. reuteri likely reflects adaptation to cereal

Arginine in the salt-induced peptide formation reaction: enantioselectivity facilitated by glycine, L- and D-histidine.

PubMed

Li, Feng; Fitz, Daniel; Fraser, Donald G; Rode, Bernd M

2010-07-01

The salt-induced peptide formation reaction has been proposed as a conceivable preliminary to the prebiotic evolution of peptides. In the present paper, the behaviour of arginine is reported for this reaction together with a discussion of the catalytic effects of glycine, and L- and D-histidine. Importantly, the behaviour of the two histidine enantiomers is different. Both histidine enantiomers perform better than glycine in enhancing the yields of arginine dipeptide with L-histidine being more effective than D-histidine. Yields in the presence of histidine are up to 70 times greater than for arginine solutions alone. This compares with 4.2 times higher in the presence of glycine. This difference is most pronounced in the most concentrated (containing 80 mM arginine) reaction solution where arginine has the lowest reactivity. A distinct preference for dimerisation of L-arginine also appears in the 80 mM cases for catalyses of other amino acids. This phenomenon is different from the behaviour of aliphatic amino acids, which display obvious inherent enantioselectivity for the L-stereomers in the SIPF reaction on their own rather than when catalysed by glycine or histidine.

The PREPL A protein, a new member of the prolyl oligopeptidase family, lacking catalytic activity.

PubMed

Szeltner, Z; Alshafee, I; Juhász, T; Parvari, R; Polgár, L

2005-10-01

The PREPL (previously called KIAA0436) gene encodes a putative serine peptidase from the prolyl oligopeptidase family. A chromosomal deletion involving the PREPL gene leads to a severe syndrome with multiple symptoms. Homology with oligopeptidase B suggested that the enzyme cleaves after an arginine or lysine residue. Several PREPL splice variants have been identified, and a 638-residue variant (PREPL A) was expressed in Escherichia coli and purified. Its secondary structure was similar to that of oligopeptidase B, but differential-scanning calorimetry indicated a higher conformational stability. Dimerization may account for the enhanced stability. Unexpectedly, the PREPL A protein did not cleave peptide substrates containing a P1 basic residue, but did slowly hydrolyse an activated ester substrate, and reacted with diisopropyl fluorophosphate. These results indicated that the catalytic serine is a reactive residue. However, the negligible hydrolytic activity suggests that the function of PREPL A is different from that of the other members of the prolyl oligopeptidase family.

Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells

Treesearch

Andrew F. Page; Rakesh Minocha; Subhash C. Minocha

2012-01-01

Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied...

Catalytic site identification—a web server to identify catalytic site structural matches throughout PDB

PubMed Central

Kirshner, Daniel A.; Nilmeier, Jerome P.; Lightstone, Felice C.

2013-01-01

The catalytic site identification web server provides the innovative capability to find structural matches to a user-specified catalytic site among all Protein Data Bank proteins rapidly (in less than a minute). The server also can examine a user-specified protein structure or model to identify structural matches to a library of catalytic sites. Finally, the server provides a database of pre-calculated matches between all Protein Data Bank proteins and the library of catalytic sites. The database has been used to derive a set of hypothesized novel enzymatic function annotations. In all cases, matches and putative binding sites (protein structure and surfaces) can be visualized interactively online. The website can be accessed at http://catsid.llnl.gov. PMID:23680785

Sequence-structural features and evolutionary relationships of family GH57 α-amylases and their putative α-amylase-like homologues.

PubMed

Janeček, Stefan; Blesák, Karol

2011-08-01

The glycoside hydrolase family 57 (GH57) contains α-amylase and a few other amylolytic specificities. It counts ~400 members from Archaea (1/4) and Bacteria (3/4), mostly of extremophilic prokaryotes. Only 17 GH57 enzymes have been biochemically characterized. The main goal of the present bioinformatics study was to analyze sequences having the clear GH57 α-amylase features. Of the 107 GH57 sequences, 59 were evaluated as α-amylases (containing both GH57 catalytic residues), whereas 48 were assigned as GH57 α-amylase-like proteins (having a substitution in one or both catalytic residues). Forty-eight of 59 α-amylases were from Archaea, but 42 of 48 α-amylase-like proteins were of bacterial origin. The catalytic residues were substituted in most cases in Bacteroides and Prevotella by serine (instead of catalytic nucleophile glutamate) and glutamate (instead of proton donor aspartate). The GH57 α-amylase specificity has thus been evolved and kept enzymatically active mainly in Archaea.

The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes.

PubMed

Köhler, Eleonore S; Sankaranarayanan, Selvakumari; van Ginneken, Christa J; van Dijk, Paul; Vermeulen, Jacqueline L M; Ruijter, Jan M; Lamers, Wouter H; Bruder, Elisabeth

2008-11-10

Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens. Between 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2. The human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants.

The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes

PubMed Central

Köhler, Eleonore S; Sankaranarayanan, Selvakumari; van Ginneken, Christa J; van Dijk, Paul; Vermeulen, Jacqueline LM; Ruijter, Jan M; Lamers, Wouter H; Bruder, Elisabeth

2008-01-01

Background Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens. Results Between 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2. Conclusion The human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants. PMID:19000307

Opposite Associations of Plasma Homoarginine and Ornithine with Arginine in Healthy Children and Adolescents

PubMed Central

JaŸwińska-Kozuba, Aleksandra; Martens-Lobenhoffer, Jens; Kruszelnicka, Olga; Rycaj, Jarosław; Chyrchel, Bernadeta; Surdacki, Andrzej; Bode-Böger, Stefanie M.

2013-01-01

Homoarginine, a non-proteinogenic amino acid, is formed when lysine replaces ornithine in reactions catalyzed by hepatic urea cycle enzymes or lysine substitutes for glycine as a substrate of renal arginine:glycine amidinotransferase. Decreased circulating homoarginine and elevated ornithine, a downstream product of arginase, predict adverse cardiovascular outcome. Our aim was to investigate correlates of plasma homoarginine and ornithine and their relations with carotid vascular structure in 40 healthy children and adolescents aged 3–18 years without coexistent diseases or subclinical carotid atherosclerosis. Homoarginine, ornithine, arginine, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) were measured by liquid chromatography-tandem mass spectrometry with stable isotope-labeled internal standards. Intima-media thickness (IMT) and extra-medial thickness (EMT) of common carotid arteries were estimated by B-mode ultrasound. Homoarginine correlated with arginine (r = 0.43, p = 0.005), age (r = 0.42, p = 0.007) and, weakly, with an increased arginine-to-ornithine ratio, a putative measure of lower arginase activity (r = 0.31, p = 0.048). Ornithine correlated inversely with arginine (r = −0.64, p < 0.001). IMT, EMT or their sum were unrelated to any of the biochemical parameters (p > 0.12). Thus, opposite associations of plasma homoarginine and ornithine with arginine may partially result from possible involvement of arginase, an enzyme controlling homoarginine degradation and ornithine synthesis from arginine. Age-dependency of homoarginine levels can reflect developmental changes in homoarginine metabolism. However, neither homoarginine nor ornithine appears to be associated with carotid vascular structure in healthy children and adolescents. PMID:24192823

Regional variations and age-related changes in arginine metabolism in the rat brain stem and spinal cord.

PubMed

Jing, Y; Fleete, M S; Collie, N D; Zhang, H; Liu, P

2013-11-12

Accumulating evidence suggests that the metabolism of l-arginine, a metabolically versatile amino acid, is critically involved in the aging process. The present study compared the activity and protein expression of nitric oxide synthase (NOS) and arginase, and the levels of l-arginine and its eight down-stream metabolites in the brain stem (pons and medulla) and the cervical spinal cord in 3- (young) and 22- (aged) month-old male Sprague-Dawley rats. Total NOS activity was significantly reduced with age in the spinal cord (but not brain stem), and there were no age-related changes in arginase activity in both regions. Western blot revealed decreased protein expression of endothelial NOS, but not neuronal NOS, with age in both regions. Furthermore, there were significantly decreased l-arginine, glutamate, GABA and spermine levels and increased putrescine and spermidine levels with age in both regions. Although the absolute concentrations of l-arginine and six metabolites were significantly different between the brain stem and spinal cord in both age groups, there were similar clusters between l-arginine and its three main metabolites (l-citrulline, l-ornithine and agmatine) in both regions, which changed as a function of age. These findings, for the first time, demonstrate the regional variations and age-related changes in arginine metabolism in the rat brain stem and spinal cord. Future research is required to understand the functional significance of these changes and the underlying mechanisms. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Disposable sensor based on enzyme-free Ni nanowire array electrode to detect glutamate.

PubMed

Jamal, Mamun; Hasan, Maksudul; Mathewson, Alan; Razeeb, Kafil M

2013-02-15

Enzyme free electrochemical sensor platform based on a vertically aligned nickel nanowire array (NiNAE) and Pt coated nickel nanowire array (Pt/NiNAE) have been developed to detect glutamate. Morphological characterisation of Ni electrodes was carried out using scanning and transmission electron microscopy combined with energy dispersive X-ray (SEM-EDX), X-ray diffraction (XRD) and transmission electron microscopy (TEM). Cyclic voltammetry (CV) and amperometry were used to evaluate the catalytic activity of the NiNAE and the Pt/NiNAE for glutamate. It has been found that both NiNAE and Pt/NiNAE electrodes showed remarkably enhanced electrocatalytic activity towards glutamate compared to planar Ni electrodes, and showed higher catalytic activity when compared to other metallic nanostructure electrodes such as gold nanowire array electrodes (AuNAE) and Pt coated gold nanowire array electrode (Pt/AuNAE). The sensitivity of NiNAE and Pt/NiNAE has been found to be 65 and 96 μA mM(-1) cm(-2), respectively, which is approximately 6 to 9 times higher than the state of the art glutamate sensor. Under optimal detection conditions, the as prepared sensors exhibited linear behaviour for glutamate detection in the concentration up to 8mM for both NiNAE and Pt/NiNAE with a limit of detection of 68 and 83 μM, respectively. Experimental results show that the vertically aligned ordered nickel nanowire array electrode (NiNAE) has significant promise for fabricating cost effective, enzyme-less, sensitive, stable and selective sensor platform. Copyright © 2012 Elsevier B.V. All rights reserved.

Expression, Purification And Preliminary X-Ray Analysis of the C-Terminal Domain of An Arginine Repressor Protein From Mycobacterium Tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, G.J.; Garen, C.R.; Cherney, M.M.

2009-06-03

The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 {angstrom}. The crystals belong to space group P1 and the Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of sixmore » molecules per unit cell.« less

Supplemental Citrulline Is More Efficient Than Arginine in Increasing Systemic Arginine Availability in Mice.

PubMed

Agarwal, Umang; Didelija, Inka C; Yuan, Yang; Wang, Xiaoying; Marini, Juan C

2017-04-01

Background: Arginine is considered to be an essential amino acid in various (patho)physiologic conditions of high demand. However, dietary arginine supplementation suffers from various drawbacks, including extensive first-pass extraction. Citrulline supplementation may be a better alternative than arginine, because its only fate in vivo is conversion into arginine. Objective: The goal of the present research was to determine the relative efficiency of arginine and citrulline supplementation to improve arginine availability. Methods: Six-week-old C57BL/6J male mice fitted with gastric catheters were adapted to 1 of 7 experimental diets for 2 wk. The basal diet contained 2.5 g l-arginine/kg, whereas the supplemented diets contained an additional 2.5, 7.5, and 12.5 g/kg diet of either l-arginine or l-citrulline. On the final day, after a 3-h food deprivation, mice were continuously infused intragastrically with an elemental diet similar to the dietary treatment, along with l-[ 13 C 6 ]arginine, to determine the splanchnic first-pass metabolism (FPM) of arginine. In addition, tracers were continuously infused intravenously to determine the fluxes and interconversions between citrulline and arginine. Linear regression slopes were compared to determine the relative efficiency of each supplement. Results: Whereas all the supplemented citrulline (105% ± 7% SEM) appeared in plasma and resulted in a marginal increase of 86% in arginine flux, supplemental arginine underwent an ∼70% FPM, indicating that only 30% of the supplemental arginine entered the peripheral circulation. However, supplemental arginine did not increase arginine flux. Both supplements linearly increased ( P < 0.01) plasma arginine concentration from 109 μmol/L for the basal diet to 159 and 214 μmol/L for the highest arginine and citrulline supplementation levels, respectively. However, supplemental citrulline increased arginine concentrations to a greater extent (35%, P < 0.01). Conclusions: Citrulline

Neuraminidase-mediated haemagglutination of recent human influenza A(H3N2) viruses is determined by arginine 150 flanking the neuraminidase catalytic site.

PubMed

Mögling, Ramona; Richard, Mathilde J; Vliet, Stefan van der; Beek, Ruud van; Schrauwen, Eefje J A; Spronken, Monique I; Rimmelzwaan, Guus F; Fouchier, Ron A M

2017-06-01

Over the last decade, an increasing proportion of circulating human influenza A(H3N2) viruses exhibited haemagglutination activity that was sensitive to neuraminidase inhibitors. This change in haemagglutination as compared to older circulating A(H3N2) viruses prompted an investigation of the underlying molecular basis. Recent human influenza A(H3N2) viruses were found to agglutinate turkey erythrocytes in a manner that could be blocked with either oseltamivir or neuraminidase-specific antisera, indicating that agglutination was driven by neuraminidase, with a low or negligible contribution of haemagglutinin. Using representative virus recombinants it was shown that the haemagglutinin of a recent A(H3N2) virus indeed had decreased activity to agglutinate turkey erythrocytes, while its neuraminidase displayed increased haemagglutinating activity. Viruses with chimeric and mutant neuraminidases were used to identify the amino acid substitution histidine to arginine at position 150 flanking the neuraminidase catalytic site as the determinant of this neuraminidase-mediated haemagglutination. An analysis of publicly available neuraminidase gene sequences showed that viruses with histidine at position 150 were rapidly replaced by viruses with arginine at this position between 2005 and 2008, in agreement with the phenotypic data. As a consequence of neuraminidase-mediated haemagglutination of recent A(H3N2) viruses and poor haemagglutination via haemagglutinin, haemagglutination inhibition assays with A(H3N2) antisera are no longer useful to characterize the antigenic properties of the haemagglutinin of these viruses for vaccine strain selection purposes. Continuous monitoring of the evolution of these viruses and potential consequences for vaccine strain selection remains important.

N-hydroxylamine is not an intermediate in the conversion of L-arginine to an activator of soluble guanylate cyclase in neuroblastoma N1E-115 cells.

PubMed Central

Pou, S; Pou, W S; Rosen, G M; el-Fakahany, E E

1991-01-01

This study evaluates the role of N-hydroxylamine (NH2OH) in activating soluble guanylate cyclase in the mouse neuroblastoma clone N1E-115. It has been proposed that NH2OH is a putative intermediate in the biochemical pathway for the generation of nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) from L-arginine. NH2OH caused a time- and concentration-dependent increase in cyclic GMP formation in intact cells. This response was not dependent on Ca2+. In cytosol preparations the activation of guanylate cyclase by L-arginine was dose-dependent and required Ca2+ and NADPH. In contrast, NH2OH itself did not activate cytosolic guanylate cyclase but it inhibited the basal activity of this enzyme in a concentration-dependent manner. The formation of cyclic GMP in the cytosolic fractions in response to NH2OH required the addition of catalase and H2O2. On the other hand, catalase and/or H2O2 lead to a decrease in L-arginine-induced cyclic GMP formation. Furthermore, NH2OH inhibited L-arginine- and sodium nitroprusside-induced cyclic GMP formation in the cytosol. The inhibition of L-arginine-induced cyclic GMP formation in the cytosol by NH2OH was not reversed by the addition of superoxide dismutase. These data strongly suggest that NH2OH is not a putative intermediate in the metabolism of L-arginine to an activator of guanylate cyclase. PMID:1671745

Functional interactions between arginine-133 and aspartate-88 in the human reduced folate carrier: evidence for a charge-pair association.

PubMed Central

Liu, X Y; Matherly, L H

2001-01-01

The human reduced folate carrier (hRFC) is an integral membrane protein that mediates cellular uptake of reduced folates and antifolates. hRFC contains several highly conserved charged residues predicted to lie in the transmembrane domains (TMDs). To explore the possible roles of the conserved arginine-133, located in TMD 4, in hRFC structure and function, this residue was systematically mutagenized to histidine, leucine, lysine and glutamate. When transfected into transport-impaired K562 cells, the mutant hRFC constructs were expressed at high levels; however, only lysine-133 hRFC was able to transport methotrexate and (6S)-5-formyl tetrahydrofolate. Substitution of aspartate-453 (in hRFC TMD 12) by valine largely preserved transport activity for both substrates. Although mutagenesis of aspartate-88 (in TMD 2) to leucine completely abolished transport activity in transfected cells, substitution with a glutamate preserved low levels ( approximately 12%) of transport. To assess the possibility that arginine-133 and aspartate-88 may form a charge-pair to stabilize hRFC tertiary structure, both charges were neutralized (by substituting leucine and valine, respectively) in the same construct. In contrast to the singly mutated hRFCs, the double mutant exhibited high levels of transport with both methotrexate and 5-formyl tetrahydrofolate. These results strongly suggest that arginine-133 and aspartate-88 form a charge-pair and that TMD 4 lies next to TMD 2 in the hRFC tertiary structure. PMID:11513752

Recent advances in targeting protein arginine methyltransferase enzymes in cancer therapy.

PubMed

Smith, Emily; Zhou, Wei; Shindiapina, Polina; Sif, Said; Li, Chenglong; Baiocchi, Robert A

2018-05-21

Exploration in the field of epigenetics has revealed the diverse roles of the protein arginine methyltransferase (PRMT) family of proteins in multiple disease states. These findings have led to the development of specific inhibitors and discovery of several new classes of drugs with potential to treat both benign and malignant conditions. Areas covered: We provide an overview on the role of PRMT enzymes in healthy and malignant cells, highlighting the role of arginine methylation in specific pathways relevant to cancer pathogenesis. Additionally, we describe structure and catalytic activity of PRMT and discuss the mechanisms of action of novel small molecule inhibitors of specific members of the arginine methyltransferase family. Expert opinion: As the field of PRMT biology advances, it's becoming clear that this class of enzymes is highly relevant to maintaining normal physiologic processes as well and disease pathogenesis. We discuss the potential impact of PRMT inhibitors as a broad class of drugs, including the pleiotropic effects, off target effects the need for more detailed PRMT-centric interactomes, and finally, the potential for targeting this class of enzymes in clinical development of experimental therapeutics for cancer.

Supplemental Citrulline Is More Efficient Than Arginine in Increasing Systemic Arginine Availability in Mice123

PubMed Central

Agarwal, Umang; Didelija, Inka C; Yuan, Yang; Wang, Xiaoying; Marini, Juan C

2017-01-01

Background: Arginine is considered to be an essential amino acid in various (patho)physiologic conditions of high demand. However, dietary arginine supplementation suffers from various drawbacks, including extensive first-pass extraction. Citrulline supplementation may be a better alternative than arginine, because its only fate in vivo is conversion into arginine. Objective: The goal of the present research was to determine the relative efficiency of arginine and citrulline supplementation to improve arginine availability. Methods: Six-week-old C57BL/6J male mice fitted with gastric catheters were adapted to 1 of 7 experimental diets for 2 wk. The basal diet contained 2.5 g l-arginine/kg, whereas the supplemented diets contained an additional 2.5, 7.5, and 12.5 g/kg diet of either l-arginine or l-citrulline. On the final day, after a 3-h food deprivation, mice were continuously infused intragastrically with an elemental diet similar to the dietary treatment, along with l-[13C6]arginine, to determine the splanchnic first-pass metabolism (FPM) of arginine. In addition, tracers were continuously infused intravenously to determine the fluxes and interconversions between citrulline and arginine. Linear regression slopes were compared to determine the relative efficiency of each supplement. Results: Whereas all the supplemented citrulline (105% ± 7% SEM) appeared in plasma and resulted in a marginal increase of 86% in arginine flux, supplemental arginine underwent an ∼70% FPM, indicating that only 30% of the supplemental arginine entered the peripheral circulation. However, supplemental arginine did not increase arginine flux. Both supplements linearly increased (P < 0.01) plasma arginine concentration from 109 μmol/L for the basal diet to 159 and 214 μmol/L for the highest arginine and citrulline supplementation levels, respectively. However, supplemental citrulline increased arginine concentrations to a greater extent (35%, P < 0.01). Conclusions: Citrulline

Roles of export genes cgmA and lysE for the production of L-arginine and L-citrulline by Corynebacterium glutamicum.

PubMed

Lubitz, Dorit; Jorge, João M P; Pérez-García, Fernando; Taniguchi, Hironori; Wendisch, Volker F

2016-10-01

L-arginine is a semi-essential amino acid with application in cosmetic, pharmaceutical, and food industries. Metabolic engineering strategies have been applied for overproduction of L-arginine by Corynebacterium glutamicum. LysE was the only known L-arginine exporter of this bacterium. However, an L-arginine-producing strain carrying a deletion of lysE still accumulated about 10 mM L-arginine in the growth medium. Overexpression of the putative putrescine and cadaverine export permease gene cgmA was shown to compensate for the lack of lysE with regard to L-arginine export. Moreover, plasmid-borne overexpression of cgmA rescued the toxic effect caused by feeding of the dipeptide Arg-Ala to lysE-deficient C. glutamicum and argO-deficient Escherichia coli strains. Deletion of the repressor gene cgmR improved L-arginine titers by 5 %. Production of L-lysine and L-citrulline was not affected by cgmA overexpression. Taken together, CgmA may function as an export system not only for the diamine putrescine and cadaverine but also for L-arginine. The major export system for L-lysine and L-arginine LysE may also play a role in L-citrulline export since production of L-citrulline was reduced when lysE was deleted and improved by 45 % when lysE was overproduced.

Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site.

PubMed

Cura, Vincent; Troffer-Charlier, Nathalie; Wurtz, Jean Marie; Bonnefond, Luc; Cavarelli, Jean

2014-09-01

Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 from Mus musculus refined at 1.7 Å resolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

Synthesis and cytotoxicity of azo nano-materials as new biosensors for L-Arginine determination.

PubMed

Shang, Xuefang; Luo, Leiming; Ren, Kui; Wei, Xiaofang; Feng, Yaqian; Li, Xin; Xu, Xiufang

2015-06-01

Inspired from biological counterparts, chemical modification of azo derivatives with function groups may provide a highly efficient method to detect amino acid. Herein, we have designed and prepared a series of azo nano-materials involving -NO2, -COOH, -SO3H and naphthyl group, which showed high response for Arginine (Arg) among normal twenty kinds of (Alanine, Valine, Leucine, Isoleucine, Methionine, Aspartic acid, Glutamic acid, Arginine, Glycine, Serine, Threonine, Asparagine, Phenylalanine, Histidine, Tryptophan, Proline, Lysine, Glutamine, Tyrosine and Cysteine). Furthermore, theoretical investigation further illustrated the possible binding mode in the host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. In addition, nano-material 3 exhibited high binding ability for Arg and low cytotoxicity to KYSE450 cells over a concentration range of 5-50μmol·L(-1) which may be used a biosensor for the Arg detection in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

Histone Arginine Methylation

PubMed Central

Lorenzo, Alessandra Di; Bedford, Mark T.

2012-01-01

Arginine methylation is a common posttranslational modification (PTM). This type of PTM occurs on both nuclear and cytoplasmic proteins, and is particularly abundant on shuttling proteins. In this review, we will focus on one aspect of this PTM: the diverse roles that arginine methylation of the core histone tails play in regulating chromatin function. A family of nine protein arginine methyltransferases (PRMTs) catalyze methylation reactions, and a subset target histones. Importantly, arginine methylation of histone tails can promote or prevent the docking of key transcriptional effector molecules, thus playing a central role in the orchestration of the histone code. PMID:21074527

Kinemage of action - Proposed reaction mechanism of glutamate-1-semialdehyde aminomutase at an atomic level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sorensen, John L., E-mail: John_Sorensen@umanitoba.ca; Stetefeld, Joerg, E-mail: stetefel@cc.umanitoba.ca

2011-10-07

Highlights: {yields} Inhibitors of tetrapyrrole cofactor biosynthesis may be useful antibiotics. {yields} Mechanism of critical enzyme, glutamate-1-semialdehyde aminomutase, is presented. {yields} Unique vitamin B6-dependant enzyme traps intermediate in active site. {yields} Molecular dynamics show that a re-orientation of the substrate is required. -- Abstract: Glutamate-1-semialdehyde aminomutase (GSAM), a key enzyme in tetrapyrrole cofactor biosynthesis, performs a unique transamination on a single substrate. The substrate, glutamate-1-semialdehyde (GSA), undergoes a reaction that exchanges the position of an amine and a carbonyl group to produce 5-aminolevulinic acid (ALA). This transamination reaction is unique in the fact that is does not require an externalmore » cofactor to act as a nitrogen donor or acceptor in this transamination reaction. One of the other remarkable features of the catalytic mechanism is the release free in the enzyme active site of the intermediate 4,5-diaminovaleric acid (DAVA). The action of a gating loop prevents the escape of DAVA from the active site. In a MD simulation approach, using snapshots provided by X-ray crystallography and protein crystal absorption spectrometry data, the individual catalytic steps in this unique intramolecular transamination have been elucidated.« less

A unique serpin P1' glutamate and a conserved β-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin).

PubMed

Ulbricht, David; Pippel, Jan; Schultz, Stephan; Meier, René; Sträter, Norbert; Heiker, John T

2015-09-15

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1') of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1' glutamate (Glu(379)) with an arginine residue (Arg(302)) of the β-sheet C. A P1' glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1' residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin-protease interaction. Arg(302) is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1' Glu(379), which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu(379) and Arg(302) influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin. © 2015 Authors; published by Portland Press Limited.

L-Arginine

MedlinePlus

... SAFE when taken by mouth appropriately for a short-term during pregnancy. Not enough is known about using L-arginine long-term in pregnancy or during breast-feeding. Stay on the safe side and avoid use. Children: L-arginine is POSSIBLY SAFE when used by ...

Effect of oral arginine supplementation on exhaled nitric oxide concentration in sickle cell anemia and acute chest syndrome.

PubMed

Sullivan, Kevin Joseph; Kissoon, Niranjan; Sandler, Eric; Gauger, Cynthia; Froyen, Melanie; Duckworth, Laurie; Brown, Martha; Murphy, Suzanne

2010-10-01

Decreased exhaled nitric oxide levels (FE(NO)) have been described in patients with sickle cell disease (SCD) and a history of acute chest syndrome (ACS) when compared with non-ACS controls. Oral arginine supplementation has been shown to increase FE(NO) in healthy participants, but its effect in SCD patients is not known. To determine the effect of oral arginine intake on FENO in sickle cell patients with and without history of ACS, and in healthy controls. No differences in the FE(NO) increase were seen in SCD patients with a history of ACS (ACS+) compared with healthy controls (HC) and SCD patients without history of ACS (ACS-). ACS+ (n=6), ACS- (n=9), and HC (n=7) patients were studied. At baseline, and after the administration of escalating doses of oral L-arginine (0.1, 0.2, and 0.4 g/kg), serial measurements were made of the following: FE(NO), plasma concentrations of arginine, ornithine, citrulline, aspartate, glutamate, arginine/ornithine ratio, nitrite, nitrate, heart rate (HR), respiratory rate (RR), blood pressure (BP), oxygen saturation (SpO2), forced expiratory volume in 1 second (FEV1), and forced vital capacity (FVC). At baseline, FE(NO) did not differ among the groups. ACS- and ACS+ groups were deficient in arginine, and had decreased FEV1, FVC, and SaO2 when compared with HC patients. After arginine supplementation, FE(NO), arginine, ornithine, citrulline, nitrite, and the arginine/ornithine ratio increased similarly in all groups. Changes from baseline for HR, BP, SpO2, RR, FEV1, and FVC were minimal and similar in all groups. In contrast to our earlier study, ACS+ patients had similar FE(NO) values when compared with ACS- and HC patients. All SCD patients were arginine deficient at baseline and showed impairment in respiratory physiology when compared with HC patients. After arginine supplementation, FE(NO) concentration increased in all groups to a similar degree, and lung function and physiologic parameters were minimally affected. The

The Effect of Carbohydrates and Arginine on Arginine Metabolism by Excised Bean Leaves in the Dark

PubMed Central

Stewart, Cecil R.

1975-01-01

The effect of carbohydrate on arginine utilization by excised bean (Phaseolus vulgaris L. var. Tendergreen) leaves in the dark was studied by adding arginine to leaves differing in carbohydrate levels, and measuring the arginine content of the leaves at intervals. In nonstarved leaves, the arginine content decreased steadily after vacuum infiltration of 10 mm arginine and was essentially completely utilized by 36 hours after infiltration. In starved leaves, the arginine content did not decrease except for a brief period of about 4 hours after infiltration. The distribution of 14C after adding 14C-arginine to starved and nonstarved leaves indicated that the presence of carbohydrates in the leaves stimulates the utilization of arginine for protein synthesis and conversion to other amino acids, organic acids, and CO2 (catabolism). Adding sucrose along with arginine to starved leaves stimulated this utilization of arginine for both protein synthesis and catabolism. This effect of sugar on catabolism is different than results of similar studies done previously with proline. Increasing the concentration of added arginine greatly increased arginine catabolism but had a relatively small effect on utilization of arginine for protein synthesis. This result is the same as similar results from adding different concentrations of proline to excised leaves. PMID:16659159

Ablation of Arginase II Spares Arginine and Abolishes the Arginine Requirement for Growth in Male Mice.

PubMed

Didelija, Inka C; Mohammad, Mahmoud A; Marini, Juan C

2017-08-01

Background: Arginine is considered a semiessential amino acid in many species, including humans, because under certain conditions its demand exceeds endogenous production. Arginine availability, however, is determined not only by its production but also by its disposal. Manipulation of disposal pathways has the potential to increase availability and thus abolish the requirement for arginine. Objective: The objective of the study was to test the hypothesis that arginase II ablation increases arginine availability for growth. Methods: In a completely randomized design with a factorial arrangement of treatments, postweaning growth was determined for 3 wk in male and female wild-type (WT) mice and arginase II knockout mice (ARGII) on a C57BL/6J background fed arginine-sufficient [Arg(+); 8 g arginine/kg] or arginine-free [Arg(-)] diets. Tracers were used to determine citrulline and arginine kinetics. Results: A sex dimorphism in arginine metabolism was detected; female mice had a greater citrulline flux (∼30%, P < 0.001), which translated to greater de novo synthesis of arginine (∼31%, P < 0.001). Female mice also had greater arginine fluxes ( P < 0.015) and plasma arginine concentrations ( P < 0.01), but a reduced arginine clearance rate ( P < 0.001). Ablation of arginase II increased plasma arginine concentrations in both sexes (∼27%, P < 0.01) but increased arginine flux only in males ( P < 0.01). The absence of arginine in the diet limited the growth of male WT mice ( P < 0.01), but had no effect on male ARGII mice ( P = 0.12). In contrast, WT females on the Arg(-) diet grew at the same rate and achieved final weight similar to that of female WT mice fed the Arg(+) diet ( P = 0.47). Conclusion: The ablation of arginase II in male mice spares arginine that can then be used for growth and to meet other metabolic functions, thus abolishing arginine requirements. © 2017 American Society for Nutrition.

The crystal structure of N-acetyl-L-glutamate synthase from Neisseria gonorrhoeae provides insights into mechanisms of catalysis and regulation.

PubMed

Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin; Yu, Xiaolin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M; Tuchman, Mendel

2008-03-14

The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.

Computational Study of Symmetric Methylation on Histone Arginine Catalyzed by Protein Arginine Methyltransferase PRMT5 through QM/MM MD and Free Energy Simulations

DOE PAGES

Yue, Yufei; CHu, Yuzhuo; Guo, Hong

2015-01-01

Protein arginine methyltransferases (PRMTs) catalyze the transfer of the methyl group from S-adenosyl-l-methionine (AdoMet) to arginine residues. There are three types of PRMTs (I, II and III) that produce different methylation products, including asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA). Since these different methylations can lead to different biological consequences, understanding the origin of product specificity of PRMTs is of considerable interest. In this article, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed to study SDMA catalyzed by the Type II PRMT5 on the basis of experimental observation that the dimethylated productmore » is generated through a distributive fashion. The simulations have identified some important interactions and proton transfers during the catalysis. Similar to the cases involving Type I PRMTs, a conserved Glu residue (Glu435) in PRMT5 is suggested to function as general base catalyst based on the result of the simulations. Moreover, our results show that PRMT5 has an energetic preference for the first methylation on N-1 followed by the second methylation on a different -guanidino nitrogen of arginine (N-2).The first and second methyl transfers are estimated to have free energy barriers of 19-20 and 18-19 kcal/mol respectively. The computer simulations suggest a distinctive catalytic mechanism of symmetric dimethylation that seems to be different from asymmetric dimethylation.« less

GLT-1: The elusive presynaptic glutamate transporter

PubMed Central

Rimmele, Theresa S.; Rosenberg, Paul A.

2016-01-01

Historically, glutamate uptake in the CNS was mainly attributed to glial cells for three reasons: 1) none of the glutamate transporters were found to be located in presynaptic terminals of excitatory synapses; 2) the putative glial transporters, GLT-1 and GLAST are expressed at high levels in astrocytes; 3) studies of the constitutive GLT-1 knockout as well as pharmacological studies demonstrated that >90% of glutamate uptake into forebrain synaptosomes is mediated by the operation of GLT-1. Here we summarize the history leading up to the recognition of GLT-1a as a presynaptic glutamate transporter. A major issue now is understanding the physiological and pathophysiologial significance of the expression of GLT-1 in presynaptic terminals. To elucidate the cell-type specific functions of GLT-1, a conditional knockout was generated with which to inactivate the GLT-1 gene in different cell types using Cre/lox technology. Astrocytic knockout led to an 80% reduction of GLT-1 expression, resulting in intractable seizures and early mortality as seen also in the constitutive knockout. Neuronal knockout was associated with no obvious phenotype. Surprisingly, synaptosomal uptake capacity (Vmax) was found to be significantly reduced, by 40%, in the neuronal knockout, indicating that the contribution of neuronal GLT-1 to synaptosomal uptake is disproportionate to its protein expression (5–10%). Conversely, the contribution of astrocytic GLT-1 to synaptosomal uptake was much lower than expected. In contrast, the loss of uptake into liposomes prepared from brain protein from astrocyte and neuronal knockouts was proportionate with the loss of GLT-1 protein, suggesting that a large portion of GLT-1 in astrocytic membranes in synaptosomal preparations is not functional, possibly because of a failure to reseal. These results suggest the need to reinterpret many previous studies using synaptosomal uptake to investigate glutamate transport itself as well as changes in glutamate

Repeated immobilization stress alters rat hippocampal and prefrontal cortical morphology in parallel with endogenous agmatine and arginine decarboxylase levels

PubMed Central

Zhu, Meng-Yang; Wang, Wei-Ping; Huang, Jingjing; Feng, Yang-Zheng; Regunathan, Soundar; Bissette, Garth

2008-01-01

Agmatine, an endogenous amine derived from decarboxylation of L-arginine catalyzed by arginine decarboxylase, has been proposed as a neurotransmitter or neuromodulator in the brain. In the present study we examined whether agmatine has neuroprotective effects against repeated immobilization-induced morphological changes in brain tissues and possible effects of immobilization stress on endogenous agmatine levels and arginine decarboxylase expression in rat brains. Sprague-Dawley rats were subjected to two hour immobilization stress daily for seven days. This paradigm significantly increased plasma corticosterone levels, and the glutamate efflux in the hippocampus as measured by in vivo microdialysis. Immunohistochemical staining with β-tubulin III showed that repeated immobilization caused marked morphological alterations in the hippocampus and medial prefrontal cortex that were prevented by simultaneous treatment with agmatine (50 mg/kg/day, i.p.). Likewise, endogenous agmatine levels measured by high performance liquid chromatography in the prefrontal cortex, hippocampus, striatum and hypothalamus were significantly increased by immobilization, as compared to controls. The increased endogenous agmatine levels, ranging from 92% to 265% of controls, were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. These results demonstrate that administration of exogenous agmatine protects the hippocampus and medial prefrontal cortex against neuronal insults caused by repeated immobilization. The parallel increase in endogenous brain agmatine and arginine decarboxylase protein levels triggered by repeated immobilization indicates that the endogenous agmatine system may play an important role in adaptation to stress as a potential neuronal self-protection mechanism. PMID:18832001

Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas

In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of delta(1)-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidativemore » stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.« less

Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas

In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidativemore » stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.« less

Cloning, expression, purification and preliminary X-ray crystallographic analysis of mouse protein arginine methyltransferase 7.

PubMed

Cura, Vincent; Troffer-Charlier, Nathalie; Lambert, Marie-Annick; Bonnefond, Luc; Cavarelli, Jean

2014-01-01

Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 97.4, c = 168.1 Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement.

The role of arginine and arginine-metabolizing enzymes during Giardia – host cell interactions in vitro

PubMed Central

2013-01-01

Background Arginine is a conditionally essential amino acid important in growing individuals and under non-homeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and arginine-metabolizing enzymes during intestinal protozoan infections. Results RNA expression analyses of major arginine-metabolizing enzymes revealed the arginine-utilizing pathways in human IECs (differentiated Caco-2 cells) grown in vitro. Most genes were constant or down-regulated (e.g. arginase 1 and 2) upon interaction with Giardia, whereas inducible NO synthase (iNOS) and ornithine decarboxylase (ODC) were up-regulated within 6 h of infection. Giardia was shown to suppress cytokine-induced iNOS expression, thus the parasite has both iNOS inducing and suppressive activities. Giardial arginine consumption suppresses NO production and the NO-degrading parasite protein flavohemoglobin is up-regulated in response to host NO. In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro. Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline. Conclusions Giardia affects the host’s arginine metabolism on many different levels. Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy. PMID:24228819

L-phenylalanyl-L-glutamate-stimulated, chloride-dependent glutamate binding represents glutamate sequestration mediated by an exchange system.

PubMed

Kessler, M; Petersen, G; Vu, H M; Baudry, M; Lynch, G

1987-04-01

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.

A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

PubMed Central

Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

2012-01-01

Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering. PMID:22792305

Supplemental citrulline is more efficient than arginine to increase systemic arginine availability in mice

USDA-ARS?s Scientific Manuscript database

Arginine is considered an essential amino acid in various (patho)physiological conditions of high demand. However, dietary arginine supplementation (ARG) suffers various drawbacks, including extensive first-pass extraction. Citrulline supplementation (CIT) may be a better alternative than arginine, ...

Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase

PubMed Central

Yu, Guang-Hui; Zou, Jie; Feng, Jing; Peng, Xiong-Bo; Wu, Ju-You; Wu, Ying-Liang; Palanivelu, Ravishankar; Sun, Meng-Xiang

2014-01-01

γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca2+-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca2+ increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca2+-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca2+-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes. PMID:24799560

Insights into the arginine paradox: evidence against the importance of subcellular location of arginase and eNOS

PubMed Central

Elms, Shawn; Chen, Feng; Wang, Yusi; Qian, Jin; Askari, Bardia; Yu, Yanfang; Pandey, Deepesh; Iddings, Jennifer; Caldwell, Ruth B.

2013-01-01

Reduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes overt cardiovascular disease. Increased expression of Arginase has been proposed as a mechanism to account for diminished NO production. Arginases consume l-arginine, the substrate for endothelial nitric oxide synthase (eNOS), and l-arginine depletion is thought to competitively reduce eNOS-derived NO. However, this simple relationship is complicated by the paradox that l-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. One mechanism proposed to explain this is compartmentalization of intracellular l-arginine into distinct, poorly interchangeable pools. In the current study, we investigated this concept by targeting eNOS and Arginase to different intracellular locations within COS-7 cells and also BAEC. We found that supplemental l-arginine and l-citrulline dose-dependently increased NO production in a manner independent of the intracellular location of eNOS. Cytosolic arginase I and mitochondrial arginase II reduced eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting arginase I to disparate regions of the cell did not differentially modify eNOS activity. Arginase-dependent suppression of eNOS activity was reversed by pharmacological inhibitors and absent in a catalytically inactive mutant. Arginase did not directly interact with eNOS, and the metabolic products of arginase or downstream enzymes did not contribute to eNOS inhibition. Cells expressing arginase had significantly lower levels of intracellular l-arginine and higher levels of ornithine. These results suggest that arginases inhibit eNOS activity by depletion of substrate and that the compartmentalization of l-arginine does not play a major role. PMID:23792682

Transient Kinetics Define a Complete Kinetic Model for Protein Arginine Methyltransferase 1*

PubMed Central

Hu, Hao; Luo, Cheng; Zheng, Y. George

2016-01-01

Protein arginine methyltransferases (PRMTs) are the enzymes responsible for posttranslational methylation of protein arginine residues in eukaryotic cells, particularly within the histone tails. A detailed mechanistic model of PRMT-catalyzed methylation is currently lacking, but it is essential for understanding the functions of PRMTs in various cellular pathways and for efficient design of PRMT inhibitors as potential treatments for a range of human diseases. In this work, we used stopped-flow fluorescence in combination with global kinetic simulation to dissect the transient kinetics of PRMT1, the predominant type I arginine methyltransferase. Several important mechanistic insights were revealed. The cofactor and the peptide substrate bound to PRMT1 in a random manner and then followed a kinetically preferred pathway to generate the catalytic enzyme-cofactor-substrate ternary complex. Product release proceeded in an ordered fashion, with peptide dissociation followed by release of the byproduct S-adenosylhomocysteine. Importantly, the dissociation rate of the monomethylated intermediate from the ternary complex was much faster than the methyl transfer. Such a result provided direct evidence for distributive arginine dimethylation, which means the monomethylated substrate has to be released to solution and rebind with PRMT1 before it undergoes further methylation. In addition, cofactor binding involved a conformational transition, likely an open-to-closed conversion of the active site pocket. Further, the histone H4 peptide bound to the two active sites of the PRMT1 homodimer with differential affinities, suggesting a negative cooperativity mechanism of substrate binding. These findings provide a new mechanistic understanding of how PRMTs interact with their substrates and transfer methyl groups. PMID:27834681

The Crystal Structure of N-Acetyl-L-glutamate Synthase from Neisseria gonorrhoeae Provides Insights into Mechanisms of Catalysis and Regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin

2010-01-07

The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomersmore » across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.« less

Effect of CDP-choline on age-dependent modifications of energy- and glutamate-linked enzyme activities in synaptic and non-synaptic mitochondria from rat cerebral cortex.

PubMed

Villa, Roberto Federico; Ferrari, Federica; Gorini, Antonella

2012-12-01

The effect of aging and CDP-choline treatment (20 mg kg⁻¹ body weight i.p. for 28 days) on the maximal rates (V(max)) of representative mitochondrial enzyme activities related to Krebs' cycle (citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase), glutamate and related amino acid metabolism (glutamate dehydrogenase, glutamate-oxaloacetate- and glutamate-pyruvate transaminases) were evaluated in non-synaptic and intra-synaptic "light" and "heavy" mitochondria from frontal cerebral cortex of male Wistar rats aged 4, 12, 18 and 24 months. During aging, enzyme activities vary in a complex way respect to the type of mitochondria, i.e. non-synaptic and intra-synaptic. This micro-heterogeneity is an important factor, because energy-related mitochondrial enzyme catalytic properties cause metabolic modifications of physiopathological significance in cerebral tissue in vivo, also discriminating pre- and post-synaptic sites of action for drugs and affecting tissue responsiveness to noxious stimuli. Results show that CDP-choline in vivo treatment enhances cerebral energy metabolism selectively at 18 months, specifically modifying enzyme catalytic activities in non-synaptic and intra-synaptic "light" mitochondrial sub-populations. This confirms that the observed changes in enzyme catalytic activities during aging reflect the bioenergetic state at each single age and the corresponding energy requirements, further proving that in vivo drug treatment is able to interfere with the neuronal energy metabolism. Copyright © 2012. Published by Elsevier Ltd.

Physiology of Growth and Sporulation in Bacillus cereus I. Effect of Glutamic and Other Amino Acids

PubMed Central

Buono, F.; Testa, R.; Lundgren, D. G.

1966-01-01

Buono, F. (Syracuse University, Syracuse, N.Y.), R. Testa, and D. G. Lundgren. Physiology of growth and sporulation in Bacillus cereus. I. Effect of glutamic and other amino acids. J. Bacteriol. 91:2291–2299. 1966.—Growth and sporulation were studied in Bacillus cereus by use of an active culture technique and a synthetic medium. A high level of glutamic acid (70 mm) was required for optimal growth and glucose oxidation followed by sporulation even though relatively little glutamic acid was consumed (14 mm). Optimal growth occurred with a combination of 14 mm glutamic acid and 56 mm (NH4)2SO4, aspartic acid, or alanine. Ornithine or arginine at 70 mm could replace glutamic acid in the synthetic medium without affecting the normal growth cycle. Glutamic acid was not replaced by any other amino acid, by (NH4)2SO4, or by a combination of either α-ketoglutarate or pyruvate plus (NH4)2SO4. Enzyme assays of cell-free extracts prepared from cells harvested at different times were used to study the metabolism of glutamic acid. Glutamic-oxaloacetic and glutamic-pyruvate transaminases were completely activated (or derepressed) during early stages of sporulation (period of 6 to 8 hr). Alanine dehydrogenase responded in a similar manner, but the levels of this enzyme were much higher throughout the culture cycle. Neither glutamic dehydrogenase nor α-ketoglutarate dehydrogenase was detected. Sporulation in a replacement salts medium was studied with cells harvested at different times from the synthetic medium. Cultures 2 to 6 hr old were unable to sporulate in the replacement salts medium unless glutamic acid (7.0 mm) was present. By the 6th hr, cells were in the early stages of sporulation, showing spore septa development. Cultures 8 hr old sporulated in the replacement salts medium. Other metabolic intermediates able to replace glutamic acid in the replacement salts medium were alanine, aspartic acid, and glutamine at equimolar concentrations. Also, ammonium ions in

Cloning, expression, purification and preliminary X-­ray crystallographic analysis of mouse protein arginine methyltransferase 7

PubMed Central

Cura, Vincent; Troffer-Charlier, Nathalie; Lambert, Marie-Annick; Bonnefond, Luc; Cavarelli, Jean

2014-01-01

Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 Å resolution. The crystal belonged to space group P43212, with unit-cell parameters a = b = 97.4, c = 168.1 Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement. PMID:24419624

Dual regulation of Ca2+-dependent glutamate release from astrocytes: vesicular glutamate transporters and cytosolic glutamate levels.

PubMed

Ni, Yingchun; Parpura, Vladimir

2009-09-01

Vesicular glutamate transporters (VGLUTs) are responsible for vesicular glutamate storage and exocytotic glutamate release in neurons and astrocytes. Here, we selectively and efficiently overexpressed individual VGLUT proteins (VGLUT1, 2, or 3) in solitary astrocytes and studied their effects on mechanical stimulation-induced Ca2+-dependent glutamate release. Neither VGLUT1 nor VGLUT2 overexpression changed the amount of glutamate release, whereas overexpression of VGLUT3 significantly enhanced Ca2+-dependent glutamate release from astrocytes. None of the VGLUT overexpression affected mechanically induced intracellular Ca2+ increase. Inhibition of glutamine synthetase activity by L-methionine sulfoximine in astrocytes, which leads to increased cytosolic glutamate concentration, greatly increased their mechanically induced Ca2+-dependent glutamate release, without affecting intracellular Ca2+ dynamics. Taken together, these data indicate that both VGLUT3 and the cytosolic concentration of glutamate are key limiting factors in regulating the Ca2+-dependent release of glutamate from astrocytes.

Safety and performance benefits of arginine supplements for military personnel: a systematic review.

PubMed

Brooks, James R; Oketch-Rabah, Hellen; Low Dog, Tieraona; Gorecki, Dennis K J; Barrett, Marilyn L; Cantilena, Louis; Chung, Mei; Costello, Rebecca B; Dwyer, Johanna; Hardy, Mary L; Jordan, Scott A; Maughan, Ronald J; Marles, Robin J; Osterberg, Robert E; Rodda, Bruce E; Wolfe, Robert R; Zuniga, Jorge M; Valerio, Luis G; Jones, Donnamaria; Deuster, Patricia; Giancaspro, Gabriel I; Sarma, Nandakumara D

2016-11-01

Dietary supplements are widely used by military personnel and civilians for promotion of health. The objective of this evidence-based review was to examine whether supplementation with l-arginine, in combination with caffeine and/or creatine, is safe and whether it enhances athletic performance or improves recovery from exhaustion for military personnel. Information from clinical trials and adverse event reports were collected from 17 databases and 5 adverse event report portals. Studies and reports were included if they evaluated the safety and the putative outcomes of enhanced performance or improved recovery from exhaustion associated with the intake of arginine alone or in combination with caffeine and/or creatine in healthy adults aged 19 to 50 years. Information related to population, intervention, comparator, and outcomes was abstracted. Of the 2687 articles screened, 62 articles meeting the inclusion criteria were analyzed. Strength of evidence was assessed in terms of risk of bias, consistency, directness, and precision. Most studies had few participants and suggested risk of bias that could negatively affect the results. l-Arginine supplementation provided little enhancement of athletic performance or improvements in recovery. Short-term supplementation with arginine may result in adverse gastrointestinal and cardiovascular effects. No information about the effects of arginine on the performance of military personnel was available. The available information does not support the use of l-arginine, either alone or in combination with caffeine, creatine, or both, to enhance athletic performance or improve recovery from exhaustion. Given the information gaps, an evidence-based review to assess the safety or effectiveness of multi-ingredient dietary supplements was not feasible, and therefore the development of a computational model-based approach to predict the safety of multi-ingredient dietary supplements is recommended. © The Author(s) 2016. Published by

Diminished Global Arginine Bioavailability and Increased Arginine Catabolism as Metabolic Profile of Increased Cardiovascular Risk

PubMed Central

Tang, W. H. Wilson; Wang, Zeneng; Cho, Leslie; Brennan, Danielle M.; Hazen, Stanley L.

2009-01-01

Objective We hypothesized that an integrated assessment of arginine with its catabolic products may better predict cardiovascular risks than arginine levels alone. Background Arginine is the sole nitrogen source for nitric oxide (NO) synthesis. The major catabolic products of arginine are ornithine and citrulline. Methods Plasma levels of free arginine, ornithine, citrulline and the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) were measured using LC/MS/MS. We examined the relationship of global arginine bioavailability ratio (GABR, defined as arginine/[ornithine+citrulline]) vs. arginine and its catabolic metabolites to prevalence of coronary artery disease (CAD) and incidence of major adverse cardiovascular events (MACE = death, myocardial infarction, stroke) over a 3-year follow-up in 1,010 subjects undergoing elective cardiac catheterization. Results Patients with CAD had significantly lower GABR [median(IQR); 1.06(0.75, 1.31) versus 1.27(0.96, 1.73), p<0.001] and arginine levels [mean: 68 ±20 μM versus 74 ±24 μM, p<0.001) than those without CAD. After adjusting for Framingham risk score, C-reactive protein, and renal function, lower GABR (but not arginine levels) and higher citrulline levels remained significantly associated with both prevalence of CAD [adjusted odds-ratio (OR) 3.93, p<0.001 and 5.98, p<0.001, respectively] and 3-year risk for incidence of MACE [adjusted Hazard ratio (HR) 1.98, p=0.025 and 2.40, p=0.01, respectively], and remained significant after adjusting for ADMA. Conclusions GABR may serve as a more comprehensive concept of reduced NO synthetic capacity compared to systemic arginine levels. Diminished GABR and high citrulline levels are associated with both development of atherosclerotic CAD and heightened long-term risk for major adverse cardiac events. PMID:19477356

Nicergoline enhances glutamate uptake via glutamate transporters in rat cortical synaptosomes.

PubMed

Nishida, Atsushi; Iwata, Hiroshi; Kudo, Yukitsuka; Kobayashi, Tsutomu; Matsuoka, Yuzo; Kanai, Yoshikatsu; Endou, Hitoshi

2004-06-01

To elucidate the mechanisms of neuroprotective action of nicergoline, we examined its effect on glutamate transport in rat cortical synaptosomes and cloned glutamate transporters. In synaptosomes, nicergoline enhanced the glutamate uptake at 1-10 microM in standard medium and suppressed the increase of extracellular glutamate by reversed transport in low Na(+) medium. Apparent increase of extracellular glutamate concentration by dihydrokinate, an inhibitor of glial glutamate transporter GLT-1, was antagonized by nicergoline. In Xenopus oocytes expressing mouse neuronal glutamate transporter (mEAAC1), the glutamate-induced inward current was enhanced by nicergoline. These results suggest that nicergoline reduces the extracellular glutamate concentration through its effect on glutamate transporters.

Glutamate Dysregulation and Glutamatergic Therapeutics for PTSD: Evidence from Human Studies

PubMed Central

Averill, Lynnette A.; Purohit, Prerana; Averill, Christopher L.; Boesl, Markus A.; Krystal, John H.; Abdallah, Chadi G.

2017-01-01

Posttraumatic stress disorder (PTSD) is a chronic and debilitating psychiatric disorder afflicting millions of individuals across the world. While the availability of robust pharmacologic interventions is quite lacking, our understanding of the putative neurobiological underpinnings of PTSD has significantly increased over the past two decades. Accumulating evidence demonstrates aberrant glutamatergic function in mood, anxiety, and trauma-related disorders and dysfunction in glutamate neurotransmission is increasingly considered a cardinal feature of stress-related psychiatric disorders including PTSD. As part of a PTSD Special Issue, this mini-review provides a concise discussion of (1) evidence of glutamatergic abnormalities in PTSD, with emphasis on human subjects data; (2) glutamate-modulating agents as potential alternative pharmacologic treatments for PTSD; and (3) selected gaps in the literature and related future directions. PMID:27916636

Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Torres, Rodrigo; Lan, Benson; Latif, Yama

2014-04-01

The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NOmore » levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully

The effects on plasma L-arginine levels of combined oral L-citrulline and L-arginine supplementation in healthy males.

PubMed

Suzuki, Takashi; Morita, Masahiko; Hayashi, Toshio; Kamimura, Ayako

2017-02-01

We investigated the effects of combining 1 g of l-citrulline and 1 g of l-arginine as oral supplementation on plasma l-arginine levels in healthy males. Oral l-citrulline plus l-arginine supplementation more efficiently increased plasma l-arginine levels than 2 g of l-citrulline or l-arginine, suggesting that oral l-citrulline and l-arginine increase plasma l-arginine levels more effectively in humans when combined.

Gene Cloning and Characterization of the Very Large NAD-Dependent l-Glutamate Dehydrogenase from the Psychrophile Janthinobacterium lividum, Isolated from Cold Soil▿

PubMed Central

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-01-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids. PMID:17526698

Gene cloning and characterization of the very large NAD-dependent l-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil.

PubMed

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-08-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.

pUL69 of Human Cytomegalovirus Recruits the Cellular Protein Arginine Methyltransferase 6 via a Domain That Is Crucial for mRNA Export and Efficient Viral Replication.

PubMed

Thomas, Marco; Sonntag, Eric; Müller, Regina; Schmidt, Stefanie; Zielke, Barbara; Fossen, Torgils; Stamminger, Thomas

2015-09-01

The regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor, facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a posttranslational modification of pUL69 via arginine methylation within the functionally important N terminus. First, we demonstrated a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of the catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6 and/or UAP56 recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69, we constructed various pUL69 derivatives harboring arginine-to-alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, nuclear magnetic resonance (NMR) analyses revealed the same α-helical structures for pUL69 sequences encoding either the wild type R1/R2 boxes or a UAP56/PRMT6 binding-deficient derivative, thereby excluding the possibility that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N terminus is methylated by PRMT6 and that this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus. The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a critical role for

Engagement of Arginine Finger to ATP Triggers Large Conformational Changes in NtrC1 AAA+ ATPase for Remodeling Bacterial RNA Polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat

The NtrC-like AAA+ ATPases control virulence and other important bacterial activities through delivering mechanical work to {sigma}54-RNA polymerase to activate transcription from {sigma}54-dependent genes. We report the first crystal structure for such an ATPase, NtrC1 of Aquifex aeolicus, in which the catalytic arginine engages the {gamma}-phosphate of ATP. Comparing the new structure with those previously known for apo and ADP-bound states supports a rigid-body displacement model that is consistent with large-scale conformational changes observed by low-resolution methods. First, the arginine finger induces rigid-body roll, extending surface loops above the plane of the ATPase ring to bind {sigma}54. Second, ATP hydrolysismore » permits Pi release and retraction of the arginine with a reversed roll, remodeling {sigma}54-RNAP. This model provides a fresh perspective on how ATPase subunits interact within the ring-ensemble to promote transcription, directing attention to structural changes on the arginine-finger side of an ATP-bound interface.« less

Arginine "Magic": Guanidinium Like-Charge Ion Pairing from Aqueous Salts to Cell Penetrating Peptides.

PubMed

Vazdar, Mario; Heyda, Jan; Mason, Philip E; Tesei, Giulio; Allolio, Christoph; Lund, Mikael; Jungwirth, Pavel

2018-06-19

It is a textbook knowledge that charges of the same polarity repel each other. For two monovalent ions in the gas phase at a close contact this repulsive interaction amounts to hundreds of kilojoules per mole. In aqueous solutions, however, this Coulomb repulsion is strongly attenuated by a factor equal to the dielectric constant of the medium. The residual repulsion, which now amounts only to units of kilojoules per mole, may be in principle offset by attractive interactions. Probably the smallest cationic pair, where a combination of dispersion and cavitation forces overwhelms the Coulomb repulsion, consists of two guanidinium ions in water. Indeed, by a combination of molecular dynamics with electronic structure calculations and electrophoretic, as well as spectroscopic, experiments, we have demonstrated that aqueous guanidinium cations form (weakly) thermodynamically stable like-charge ion pairs. The importance of pairing of guanidinium cations in aqueous solutions goes beyond a mere physical curiosity, since it has significant biochemical implications. Guanidinium chloride is known to be an efficient and flexible protein denaturant. This is due to the ability of the orientationally amphiphilic guanidinium cations to disrupt various secondary structural motifs of proteins by pairing promiscuously with both hydrophobic and hydrophilic groups, including guanidinium-containing side chains of arginines. The fact that the cationic guanidinium moiety forms the dominant part of the arginine side chain implies that the like-charge ion pairing may also play a role for interactions between peptides and proteins. Indeed, arginine-arginine pairing has been frequently found in structural protein databases. In particular, when strengthened by a presence of negatively charged glutamate, aspartate, or C-terminal carboxylic groups, this binding motif helps to stabilize peptide or protein dimers and is also found in or near active sites of several enzymes. The like

Polymerization on the rocks: beta-amino acids and arginine

NASA Technical Reports Server (NTRS)

Liu, R.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

1998-01-01

We have studied the accumulation of long oligomers of beta-amino acids on the surface of minerals using the 'polymerization on the rocks' protocol. We find that long oligopeptides of beta-glutamic acid which cannot be formed in homogeneous aqueous solution are accumulated efficiently on the surface of hydroxylapatite using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as condensing agent. The EDAC-induced oligomerization of aspartic acid on hydroxylapatite proceeds even more efficiently. Hydroxylapatite can also facilitate the ligation of the tripeptide (glu)3. The 'polymerization on the rocks' scenario is not restricted to negatively-charged amino acids. Oligoarginines are accumulated on the surface of illite using carbonyldiimidizole (CDI) as condensing agent. We find that FeS2 catalyzes the CDI-induced oligomerization of arginine, although it does not adsorb oligoarginines. These results are relevant to the formation of polypeptides on the primitive earth.

Diminished L-arginine bioavailability in hypertension.

PubMed

Moss, Monique B; Brunini, Tatiana M C; Soares De Moura, Roberto; Novaes Malagris, Lúcia E; Roberts, Norman B; Ellory, J Clive; Mann, Giovanni E; Mendes Ribeiro, Antônio C

2004-10-01

L-Arginine is the precursor of NO (nitric oxide), a key endogenous mediator involved in endothelium-dependent vascular relaxation and platelet function. Although the concentration of intracellular L-arginine is well above the Km for NO synthesis, in many cells and pathological conditions the transport of L-arginine is essential for NO production (L-arginine paradox). The present study was designed to investigate the modulation of L-arginine/NO pathway in systemic arterial hypertension. Transport of L-arginine into RBCs (red blood cells) and platelets, NOS (NO synthase) activity and amino acid profiles in plasma were analysed in hypertensive patients and in an animal model of hypertension. Influx of L-arginine into RBCs was mediated by the cationic amino acid transport systems y+ and y+L, whereas, in platelets, influx was mediated only via system y+L. Chromatographic analyses revealed higher plasma levels of L-arginine in hypertensive patients (175+/-19 micromol/l) compared with control subjects (137+/-8 micromol/l). L-Arginine transport via system y+L, but not y+, was significantly reduced in RBCs from hypertensive patients (60+/-7 micromol.l(-1).cells(-1).h(-1); n=16) compared with controls (90+/-17 micromol.l(-1).cells(-1).h(-1); n=18). In human platelets, the Vmax for L-arginine transport via system y+L was 86+/-17 pmol.10(9) cells(-1).min(-1) in controls compared with 36+/-9 pmol.10(9) cells(-1).min(-1) in hypertensive patients (n=10; P<0.05). Basal NOS activity was decreased in platelets from hypertensive patients (0.12+/-0.02 pmol/10(8) cells; n=8) compared with controls (0.22+/-0.01 pmol/10(8) cells; n=8; P<0.05). Studies with spontaneously hypertensive rats demonstrated that transport of L-arginine via system y+L was also inhibited in RBCs. Our findings provide the first evidence that hypertension is associated with an inhibition of L-arginine transport via system y+L in both humans and animals, with reduced availability of L-arginine limiting NO synthesis

The putative glutamate receptor 1.1 (AtGLR1.1) in Arabidopsis thaliana regulates abscisic acid biosynthesis and signaling to control development and water loss.

PubMed

Kang, Jiman; Mehta, Sohum; Turano, Frank J

2004-10-01

The involvement of the putative glutamate receptor 1.1 (AtGLR1.1) gene in the regulation of abscisic acid (ABA) biosynthesis and signaling was investigated in Arabidopsis. Seeds from AtGLR1.1-deficient (antiAtGLR1.1) lines had increased sensitivity to exogenous ABA with regard to the effect of the hormone on the inhibition of seed germination and root growth. Seed germination, which was inhibited by an animal ionotropic glutamate receptor antagonist, 6,7-dinitroquinoxaline-2,3-[1H,4H]-dione, was restored by co-incubation with an inhibitor of ABA biosynthesis, fluridone. These results confirm that germination in antiAtGLR1.1 lines was inhibited by increased ABA. When antiAtGLR1.1 and WT seeds were co-incubated in fluridone and exogenous ABA, the antiAtGLR1.1 seeds were more sensitive to ABA. In addition, the antiAtGLR1.1 lines exhibited altered expression of ABA biosynthetic (ABA) and signaling (ABI) genes, when compared with WT. Combining the physiological and molecular results suggest that ABA biosynthesis and signaling in antiAtGLR1.1 lines are altered. ABA levels in leaves of antiAtGLR1.1 lines are higher than those in WT. In addition, the antiAtGLR1.1 lines had reduced stomatal apertures, and exhibited enhanced drought tolerance due to deceased water loss compared with WT lines. The results from these experiments imply that ABA biosynthesis and signaling can be regulated through AtGLR1.1 to trigger pre- and post-germination arrest and changes in whole plant responses to water stress. Combined with our earlier results, these findings suggest that AtGLR1.1 integrates and regulates the different aspects of C, N and water balance that are required for normal plant growth and development.

Further studies of the effects of aging on arginine metabolites in the rat vestibular nucleus and cerebellum.

PubMed

Liu, P; Gupta, N; Jing, Y; Collie, N D; Zhang, H; Smith, P F

2017-04-21

Some studies have demonstrated that aging is associated with impaired vestibular reflexes, especially otolithic reflexes, resulting in postural instability. However, the neurochemical basis of these age-related changes is still poorly understood. The l-arginine metabolic system has been implicated in changes in the brain associated with aging. In the current study, we examined the levels of l-arginine and its metabolizing enzymes and downstream metabolites in the vestibular nucleus complex (VNC) and cerebellum (CE) of rats with and without behavioral testing which were young (4months old), middle-aged (12months old) or aged (24months old). We found that aging was associated with lower nitric oxide synthase activity in the CE of animals with testing and increased arginase in the VNC and CE of animals with testing. l-citrulline and l-ornithine were lower in the VNC of aged animals irrespective of testing, while l-arginine and l-citrulline were lower in the CE with and without testing, respectively. In the VNC and CE, aging was associated with lower levels of glutamate in the VNC, irrespective of testing. In the VNC it was associated with higher levels of agmatine and putrescine, irrespective of testing. In the CE, aging was associated with higher levels of putrescine in animals without testing and with higher levels of spermine in animals with testing, and spermidine, irrespective of testing. Multivariate analyses indicated significant predictive relationships between the different variables, and there were correlations between some of the neurochemical variables and behavioral measurements. Cluster analyses revealed that aging altered the relationships between l-arginine and its metabolites. The results of this study demonstrate that there are major changes occurring in l-arginine metabolism in the VNC and CE as a result of age, as well as behavioral activity. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Characterization and Expression of Glutamate Dehydrogenase in Response to Acute Salinity Stress in the Chinese Mitten Crab, Eriocheir sinensis

PubMed Central

Wang, Yueru; Li, Erchao; Yu, Na; Wang, Xiaodan; Cai, Chunfang; Tang, Boping; Chen, Liqiao; Van Wormhoudt, Alain

2012-01-01

Background Glutamate dehydrogenase (GDH) is a key enzyme for the synthesis and catabolism of glutamic acid, proline and alanine, which are important osmolytes in aquatic animals. However, the response of GDH gene expression to salinity alterations has not yet been determined in macro-crustacean species. Methodology/Principal Findings GDH cDNA was isolated from Eriocheir sinensis. Then, GDH gene expression was analyzed in different tissues from normal crabs and the muscle of crabs following transfer from freshwater (control) directly to water with salinities of 16‰ and 30‰, respectively. Full-length GDH cDNA is 2,349 bp, consisting of a 76 bp 5′- untranslated region, a 1,695 bp open reading frame encoding 564 amino acids and a 578 bp 3′- untranslated region. E. sinensis GDH showed 64–90% identity with protein sequences of mammalian and crustacean species. Muscle was the dominant expression source among all tissues tested. Compared with the control, GDH expression significantly increased at 6 h in crabs transferred to 16‰ and 30‰ salinity, and GDH expression peaked at 48 h and 12 h, respectively, with levels approximately 7.9 and 8.5 fold higher than the control. The free amino acid (FAA) changes in muscle, under acute salinity stress (16‰ and 30‰ salinities), correlated with GDH expression levels. Total FAA content in the muscle, which was based on specific changes in arginine, proline, glycine, alanine, taurine, serine and glutamic acid, tended to increase in crabs following transfer to salt water. Among these, arginine, proline and alanine increased significantly during salinity acclimation and accounted for the highest proportion of total FAA. Conclusions E. sinensis GDH is a conserved protein that serves important functions in controlling osmoregulation. We observed that higher GDH expression after ambient salinity increase led to higher FAA metabolism, especially the synthesis of glutamic acid, which increased the synthesis of proline and

Metabotropic glutamate receptors are required for the induction of long-term potentiation

NASA Technical Reports Server (NTRS)

Zheng, F.; Gallagher, J. P.

1992-01-01

Recent observations have led to the suggestion that the metabotropic glutamate receptor may play a role in the induction or maintenance of long-term potentiation (LTP). However, experimental evidence supporting a role for this receptor in the induction of LTP is still inconclusive and controversial. Here we report that, in rat dorsolateral septal nucleus (DLSN) neurons, which have the highest density of metabotropic receptors and show functional responses, the induction of LTP is not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate, but is blocked by two putative metabotropic glutamate receptor antagonists, L-2-amino-3-phosphonopropionic acid and L-2-amino-4-phosphonobutyrate. Furthermore, superfusion of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, a selective metabotropic glutamate agonist, resulted in a long-lasting potentiation of synaptic transmission similar to that induced by tetanic stimuli. Our results demonstrated that activation of postsynaptic metabotropic receptors is both necessary and sufficient for the induction of LTP in the DLSN, and we suggest that such a mechanism may be important at other CNS synapses.

Catalytic synthesis of biologically important metabolites from formaldehyde and ammonia as a putative way of their prebiotic emergence

NASA Astrophysics Data System (ADS)

Delidovich, Irina; Taran, Oxana; Simonov, Alexander; Parmon, Valentin

The modern `RNA-world' theory considering the primary life as a system of self-replicating informational and catalytic oligomers is the most widespread hypothesis of life's origin. At the same time an alternative major theory antithetic to the genetic one exists. It is based on the primary formation of important metabolites and building blocks of biopolymers and appearance of the primordial autocatalytic cycles in geochemical environment. [1]. Encouraged by this theory, we suggest the putative emergence pathways to biologically relevant metabolites from simple precursors: formaldehyde (FA) and ammonia, which are known to be present in the interstellar space [2]. Based on our earlier [3, 4] and novel findings as well as on the literature data [5, 6] the scheme of conjugate autocatalytic processes including photochemical condensation of FA yielding C-C bond and catalytic formation of pyruvaldehyde, pyruvic acid, sugars, aminoacids catalyzed by minerals (aluminum silicate, phoshates) and by aminoacids themselves is proposed. Acetaldehyde, glyoxal, glycol-and glyceraldehydes (GA and GCA) were identified in this work as products of photolysis of FA aqueous solution with summary selectivity up to 20% and can serve as substrates for the synthesis of more complex organic compounds. In turn, alanine and pyruvic acid are formed during UV-irradiation of aqueous solution of acetaldehyde and ammonium nitrate [6]. Pyruvaldehyde formation from GCA and FA in presence of amino acids was observed by A. Weber [5]. Finally, we revealed the catalytic activity of zeolite HZSM-5-17 in acid form towards formation of pyruvaldehyde from GA and GCA in presence of alanine with the yield about 5%. This observation indicates that the assumption of catalytic activity of silica-alumina towards the synthesis of metabolites could be realistic. Prebiotic synthesis of sugars from lower monosacharides, as we showed earlier, could take place over phosphate catalysts [4]. The substrates and the products

Synaptic Glutamate Spillover Due to Impaired Glutamate Uptake Mediates Heroin Relapse

PubMed Central

Scofield, Michael D.; Boger, Heather; Hensley, Megan; Kalivas, Peter W.

2014-01-01

Reducing the enduring vulnerability to relapse is a therapeutic goal in treating drug addiction. Studies with animal models of drug addiction show a marked increase in extrasynaptic glutamate in the core subcompartment of the nucleus accumbens (NAcore) during reinstated drug seeking. However, the synaptic mechanisms linking drug-induced changes in extrasynaptic glutamate to relapse are poorly understood. Here, we discovered impaired glutamate elimination in rats extinguished from heroin self-administration that leads to spillover of synaptically released glutamate into the nonsynaptic extracellular space in NAcore and investigated whether restoration of glutamate transport prevented reinstated heroin seeking. Through multiple functional assays of glutamate uptake and analyzing NMDA receptor-mediated currents, we show that heroin self-administration produced long-lasting downregulation of glutamate uptake and surface expression of the transporter GLT-1. This downregulation was associated with spillover of synaptic glutamate to extrasynaptic NMDA receptors within the NAcore. Ceftriaxone restored glutamate uptake and prevented synaptic glutamate spillover and cue-induced heroin seeking. Ceftriaxone-induced inhibition of reinstated heroin seeking was blocked by morpholino-antisense targeting GLT-1 synthesis. These data reveal that the synaptic glutamate spillover in the NAcore results from reduced glutamate transport and is a critical pathophysiological mechanism underling reinstated drug seeking in rats extinguished from heroin self-administration. PMID:24741055

Scavenging of blood glutamate for enhancing brain-to-blood glutamate efflux.

PubMed

Li, Yunhong; Hou, Xiaolin; Qi, Qi; Wang, Le; Luo, Lan; Yang, Shaoqi; Zhang, Yumei; Miao, Zhenhua; Zhang, Yanli; Wang, Fei; Wang, Hongyan; Huang, Weidong; Wang, Zhenhai; Shen, Ying; Wang, Yin

2014-01-01

The presence of excess glutamate in the brain interstitial fluid characterizes several acute pathological conditions of the brain, including traumatic brain injury and stroke. It has been demonstrated that it is possible to eliminate excess glutamate in the brain by decreasing blood glutamate levels and, accordingly, accelerating the brain-to-blood glutamate efflux. It is feasible to accomplish this process by activating blood resident enzymes in the presence of the respective glutamate cosubstrates. In the present study, several glutamate cosubstrates and cofactors were studied in an attempt to identify the optimal conditions to reduce blood glutamate levels. The administration of a mixture of 1 mM pyruvate and oxaloacetate (Pyr/Oxa) for 1 h decreased blood glutamate levels by ≤50%. The addition of lipoamide to this mixture resulted in a further reduction in blood glutamate levels of >80%. In addition, in vivo experiments showed that lipoamide together with Pyr/Oxa is able to decrease blood glutamate levels to a greater extent than Pyr/Oxa alone, and accordingly, this enhances the glutamate efflux from the brain to the blood. These results may outline a novel neuroprotective strategy with increased effectiveness for the removal of excess brain glutamate in various neurodegenerative conditions.

Kinetics of acrylodan-labelled cAMP-dependent protein kinase catalytic subunit denaturation.

PubMed

Kivi, Rait; Loog, Mart; Jemth, Per; Järv, Jaak

2013-10-01

Fluorescence spectroscopy was used to study denaturation of cAMP-dependent protein kinase catalytic subunit labeled with an acrylodan moiety. The dye was covalently bound to a cystein residue introduced into the enzyme by replacement of arginine in position 326 in the native sequence, located near the enzyme active center. This labeling had no effect on catalytic activity of the enzyme, but provided possibility to monitor changes in protein structure through measuring the fluorescence spectrum of the dye, which is sensitive to changes in its environment. This method was used to monitor denaturation of the protein kinase catalytic subunit and study the kinetics of this process as well as influence of specific ligands on stability of the protein. Stabilization of the enzyme structure was observed in the presence of adenosine triphosphate, peptide substrate RRYSV and inhibitor peptide PKI[5-24].

A comparison of DNA compaction by arginine and lysine peptides: A physical basis for arginine rich protamines

PubMed Central

DeRouchey, Jason; Hoover, Brandon

2013-01-01

Protamines are small, highly positively charged peptides used to package DNA to very high densities in sperm nuclei. Tight DNA packing is considered essential to minimize DNA damage by mutagens and reactive oxidizing species. A striking and general feature of protamines is the almost exclusive use of arginine over lysine for the positive charge to neutralize DNA. We have investigated whether this preference for arginine might arise from a difference in DNA condensation by arginine and lysine peptides. The forces underlying DNA compaction by arginine, lysine, and ornithine peptides are measured using the osmotic stress technique coupled with x-ray scattering. The equilibrium spacings between DNA helices condensed by lysine and ornithine peptides are significantly larger than the interhelical distances with comparable arginine peptides. The DNA surface-to-surface separation, for example, is some 50% larger with poly-lysine compared to poly-arginine. DNA packing by lysine rich peptides in sperm nuclei would allow much greater accessibility to small molecules that could damage DNA. The larger spacing with lysine peptides is due to both a weaker attraction and a stronger short ranged repulsion relative to the arginine peptides. A previously proposed model for poly-arginine and protamine binding to DNA provides a convenient framework for understanding the differences between the ability of lysine and arginine peptides to assemble DNA. PMID:23540557

Small-molecule inhibitors at the PSD-95/nNOS interface protect against glutamate-induced neuronal atrophy in primary cortical neurons.

PubMed

Doucet, M V; O'Toole, E; Connor, T; Harkin, A

2015-08-20

Glutamate and nitric oxide (NO) are important regulators of dendrite and axon development in the central nervous system. Excess glutamatergic stimulation is a feature of many pathological conditions and manifests in neuronal atrophy and shrinkage with eventual neurodegeneration and cell death. Here we demonstrate that treatment of cultured primary cortical rat neurons for 24h with glutamate (500μM) or N-methyl-d-aspartate (NMDA) (100-500μM) combined with glycine suppresses neurite outgrowth. A similar reduction of neurite outgrowth was observed with the NO precursor l-arginine and NO donor sodium nitroprusside (SNP) (100 and 300μM). The NMDA-receptor (NMDA-R) antagonists ketamine and MK-801 (10nM) counteracted the NMDA/glycine-induced reduction in neurite outgrowth and the neuronal NO synthase (nNOS) inhibitor 1-[2-(trifluoromethyl)phenyl] imidazole (TRIM) (100nM) counteracted both the NMDA/glycine and l-arginine-induced decreases in neurite outgrowth. Furthermore, targeting soluble guanylate cyclase (sGC), a downstream target of NO, with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10μM) also protected against l-arginine-induced decreases in neurite outgrowth. Since the NMDA-R is functionally coupled to nNOS via the postsynaptic protein 95kDa (PSD-95), inhibitors of the PSD-95/nNOS interaction were tested for their ability to protect against glutamate-induced suppression in neurite outgrowth. Treatment with the small-molecule inhibitors of the PSD-95/nNOS interface 2-((1H-benzo[d] [1,2,3]triazol-5-ylamino) methyl)-4,6-dichlorophenol (IC87201) (10 and 100nM) and 4-(3,5-dichloro-2-hydroxy-benzylamino)-2-hydroxybenzoic acid (ZL-006) (10 and 100nM) attenuated NMDA/glycine-induced decreases in neurite outgrowth. These data support the hypothesis that targeting the NMDA-R/PSD-95/nNOS interaction downstream of NMDA-R promotes neurotrophic effects by preventing neurite shrinkage in response to excess glutamatergic stimulation. The PSD-95/n

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

PubMed

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.

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

PubMed Central

Sarian, Fean D.; Janeček, Štefan; Pijning, Tjaard; Ihsanawati; Nurachman, Zeily; Radjasa, Ocky K.; Dijkhuizen, Lubbert; Natalia, Dessy; van der Maarel, Marc J. E. C.

2017-01-01

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13. PMID:28287181

l-Arginine administration attenuates airway inflammation by altering l-arginine metabolism in an NC/Nga mouse model of asthma.

PubMed

Zhang, Ran; Kubo, Masayuki; Murakami, Ikuo; Setiawan, Heri; Takemoto, Kei; Inoue, Kiyomi; Fujikura, Yoshihisa; Ogino, Keiki

2015-05-01

Changes in l-arginine metabolism, including increased arginase levels and decreased nitric oxide production, are involved in the pathophysiology of asthma. In this study, using an intranasal mite-induced NC/Nga mouse model of asthma, we examined whether administration of l-arginine ameliorated airway hyperresponsiveness and inflammation by altering l-arginine metabolism. Experimental asthma was induced in NC/Nga mice via intranasal administration of mite crude extract (50 µg/day) on 5 consecutive days (days 0-4, sensitization) and on day 11 (challenge). Oral administration of l-arginine (250 mg/kg) was performed twice daily on days 5-10 for prevention or on days 11-13 for therapy. On day 14, we evaluated the inflammatory airway response (airway hyperresponsiveness, the number of cells in the bronchoalveolar lavage fluid, and the changes in pathological inflammation of the lung), arginase expression and activity, l-arginine bioavailability, and the concentration of NOx, the end products of nitric oxide. Treatment with l-arginine ameliorated the mite-induced inflammatory airway response. Furthermore, l-arginine administration attenuated the increases in arginase expression and activity and elevated the NOx levels by enhancing l-arginine bioavailability. These findings indicate that l-arginine administration may contribute to the improvement of asthmatic symptoms by altering l-arginine metabolism.

Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice

USDA-ARS?s Scientific Manuscript database

Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depl...

Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria.

PubMed

Cui, Hongli; Wang, Yipeng; Wang, Yinchu; Qin, Song

2012-11-16

Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX) is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS). PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs) encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like) according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC) of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic mechanisms as eukaryotes. All cyanobacterial PRX proteins

Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria

PubMed Central

2012-01-01

Background Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX) is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS). PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Results Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs) encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like) according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC) of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. Conclusions The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic mechanisms as eukaryotes. All

Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice.

PubMed

Marini, Juan C; Didelija, Inka Cajo

2015-01-01

Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20) on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L), and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas) were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [14C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.

Advantages of a distant cellulase catalytic base.

PubMed

Burgin, Tucker; Ståhlberg, Jerry; Mayes, Heather B

2018-03-30

The inverting glycoside hydrolase Trichoderma reesei ( Hypocrea jecorina ) Cel6A is a promising candidate for protein engineering for more economical production of biofuels. Until recently, its catalytic mechanism had been uncertain: The best candidate residue to serve as a catalytic base, Asp-175, is farther from the glycosidic cleavage site than in other glycoside hydrolase enzymes. Recent unbiased transition path sampling simulations revealed the hydrolytic mechanism for this more distant base, employing a water wire; however, it is not clear why the enzyme employs a more distant catalytic base, a highly conserved feature among homologs across different kingdoms. In this work, we describe molecular dynamics simulations designed to uncover how a base with a longer side chain, as in a D175E mutant, affects procession and active site alignment in the Michaelis complex. We show that the hydrogen bond network is tuned to the shorter aspartate side chain, and that a longer glutamate side chain inhibits procession as well as being less likely to adopt a catalytically productive conformation. Furthermore, we draw comparisons between the active site in Trichoderma reesei Cel6A and another inverting, processive cellulase to deduce the contribution of the water wire to the overall enzyme function, revealing that the more distant catalytic base enhances product release. Our results can inform efforts in the study and design of enzymes by demonstrating how counterintuitive sacrifices in chemical reactivity can have worthwhile benefits for other steps in the catalytic cycle. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Ebselen: Mechanisms of Glutamate Dehydrogenase and Glutaminase Enzyme Inhibition.

PubMed

Yu, Yan; Jin, Yanhong; Zhou, Jie; Ruan, Haoqiang; Zhao, Han; Lu, Shiying; Zhang, Yue; Li, Di; Ji, Xiaoyun; Ruan, Benfang Helen

2017-12-15

Ebselen modulates target proteins through redox reactions with selenocysteine/cysteine residues, or through binding to the zinc finger domains. However, a recent contradiction in ebselen inhibition of kidney type glutaminase (KGA) stimulated our interest in investigating its inhibition mechanism with glutamate dehydrogenase (GDH), KGA, thioredoxin reductase (TrxR), and glutathione S-transferase. Fluorescein- or biotin-labeled ebselen derivatives were synthesized for mechanistic analyses. Biomolecular interaction analyses showed that only GDH, KGA, and TrxR proteins can bind to the ebselen derivative, and the binding to GDH and KGA could be competed off by glutamine or glutamate. From the gel shift assays, the fluorescein-labeled ebselen derivative could co-migrate with hexameric GDH and monomeric/dimeric TrxR in a dose-dependent manner; it also co-migrated with KGA but disrupted the tetrameric form of the KGA enzyme at a high compound concentration. Further proteomic analysis demonstrated that the ebselen derivative could cross-link with proteins through a specific cysteine at the active site of GDH and TrxR proteins, but for KGA protein, the binding site is at the N-terminal appendix domain outside of the catalytic domain, which might explain why ebselen is not a potent KGA enzyme inhibitor in functional assays. In conclusion, ebselen could inhibit enzyme activity by binding to the catalytic domain or disruption of the protein complex. In addition, ebselen is a relatively potent selective GDH inhibitor that might provide potential therapeutic opportunities for hyperinsulinism-hyperammonemia syndrome patients who have the mutational loss of GTP inhibition.

Arginine analogues incorporating carboxylate bioisosteric functions are micromolar inhibitors of human recombinant DDAH-1.

PubMed

Tommasi, Sara; Zanato, Chiara; Lewis, Benjamin C; Nair, Pramod C; Dall'Angelo, Sergio; Zanda, Matteo; Mangoni, Arduino A

2015-12-14

Dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme involved in the metabolism of asymmetric dimethylarginine (ADMA) and N-monomethyl arginine (NMMA), which are endogenous inhibitors of the nitric oxide synthase (NOS) family of enzymes. Two isoforms of DDAH have been identified in humans, DDAH-1 and DDAH-2. DDAH-1 inhibition represents a promising strategy to limit the overproduction of NO in pathological states without affecting the homeostatic role of this important messenger molecule. Here we describe the design and synthesis of 12 novel DDAH-1 inhibitors and report their derived kinetic parameters, IC50 and Ki. Arginine analogue 10a, characterized by an acylsulfonamide isosteric replacement of the carboxylate, showed a 13-fold greater inhibitory potential relative to the known DDAH-1 inhibitor, L-257. Compound 10a was utilized to study the putative binding interactions of human DDAH-1 inhibition using molecular dynamics simulations. The latter suggests that several stabilizing interactions occur in the DDAH-1 active-site, providing structural insights for the enhanced inhibitory potential demonstrated by in vitro inhibition studies.

CSmetaPred: a consensus method for prediction of catalytic residues.

PubMed

Choudhary, Preeti; Kumar, Shailesh; Bachhawat, Anand Kumar; Pandit, Shashi Bhushan

2017-12-22

Knowledge of catalytic residues can play an essential role in elucidating mechanistic details of an enzyme. However, experimental identification of catalytic residues is a tedious and time-consuming task, which can be expedited by computational predictions. Despite significant development in active-site prediction methods, one of the remaining issues is ranked positions of putative catalytic residues among all ranked residues. In order to improve ranking of catalytic residues and their prediction accuracy, we have developed a meta-approach based method CSmetaPred. In this approach, residues are ranked based on the mean of normalized residue scores derived from four well-known catalytic residue predictors. The mean residue score of CSmetaPred is combined with predicted pocket information to improve prediction performance in meta-predictor, CSmetaPred_poc. Both meta-predictors are evaluated on two comprehensive benchmark datasets and three legacy datasets using Receiver Operating Characteristic (ROC) and Precision Recall (PR) curves. The visual and quantitative analysis of ROC and PR curves shows that meta-predictors outperform their constituent methods and CSmetaPred_poc is the best of evaluated methods. For instance, on CSAMAC dataset CSmetaPred_poc (CSmetaPred) achieves highest Mean Average Specificity (MAS), a scalar measure for ROC curve, of 0.97 (0.96). Importantly, median predicted rank of catalytic residues is the lowest (best) for CSmetaPred_poc. Considering residues ranked ≤20 classified as true positive in binary classification, CSmetaPred_poc achieves prediction accuracy of 0.94 on CSAMAC dataset. Moreover, on the same dataset CSmetaPred_poc predicts all catalytic residues within top 20 ranks for ~73% of enzymes. Furthermore, benchmarking of prediction on comparative modelled structures showed that models result in better prediction than only sequence based predictions. These analyses suggest that CSmetaPred_poc is able to rank putative catalytic

Both neurons and astrocytes exhibited tetrodotoxin-resistant metabotropic glutamate receptor-dependent spontaneous slow Ca2+ oscillations in striatum.

PubMed

Tamura, Atsushi; Yamada, Naohiro; Yaguchi, Yuichi; Machida, Yoshio; Mori, Issei; Osanai, Makoto

2014-01-01

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson's disease. The group I mGluRs are known to modulate the intracellular Ca(2+) signaling. To characterize Ca(2+) signaling in striatal cells, spontaneous cytoplasmic Ca(2+) transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca(2+) transients (referred to as slow Ca(2+) oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca(2+) oscillation. Depletion of the intracellular Ca(2+) store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca(2+) oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca(2+) oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca(2+) oscillation in both putative-neurons and astrocytes. The slow Ca(2+) oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca(2+) oscillation may involve in the neuron-glia interaction in the striatum.

Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biterova, Ekaterina I.; Barycki, Joseph J.; UNL)

2009-12-01

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl{sub 2} (2.1 {angstrom};more » R = 18.2%, R{sub free} = 21.9%), and in complex with glutamate, MgCl{sub 2}, and ADP (2.7 {angstrom}; R = 19.0%, R{sub free} = 24.2%). Inspection of these structures reveals an unusual binding pocket for the {alpha}-carboxylate of the glutamate substrate and an ATP-independent Mg{sup 2+} coordination site, clarifying the Mg{sup 2+} dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.« less

Arginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors.

PubMed

Wickramasinghe, Susiji; Yatawara, Lalani; Nagataki, Mitsuru; Agatsuma, Takeshi

2016-10-01

To determine exon/intron organization of the Toxocara canis (T. canis) AK (TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1200 bp) of TCAK (wild type) was cloned into the BamH1/SalI site of pMAL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25 °C for the forward reaction (phosphagen synthesis). Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K m value of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K m value of the mutant (Serine to Glycine) increased to 0.19 mM. The K m value (0.19 mM) of the double mutant (Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type (0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica (A. indica), an aminoglycoside antibiotic (aminosidine), a citrus flavonoid glycoside (rutin) and a commercially available catechin mixture against TCAK. Green and black tea (1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK. Arginine kinase in T. canis has a seven-exon/six-intron gene

MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

Cancer.gov

The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

Protein arginine methylation/demethylation and cancer

PubMed Central

Poulard, Coralie; Corbo, Laura; Le Romancer, Muriel

2016-01-01

Protein arginine methylation is a common post-translational modification involved in numerous cellular processes including transcription, DNA repair, mRNA splicing and signal transduction. Currently, there are nine known members of the protein arginine methyltransferase (PRMT) family, but only one arginine demethylase has been identified, namely the Jumonji domain-containing 6 (JMJD6). Although its demethylase activity was initially challenged, its dual activity as an arginine demethylase and a lysine hydroxylase is now recognized. Interestingly, a growing number of substrates for arginine methylation and demethylation play key roles in tumorigenesis. Though alterations in the sequence of these enzymes have not been identified in cancer, their overexpression is associated with various cancers, suggesting that they could constitute targets for therapeutic strategies. In this review, we present the recent knowledge of the involvement of PRMTs and JMJD6 in tumorigenesis. PMID:27556302

Arginine and Citrulline and the Immune Response in Sepsis

PubMed Central

Wijnands, Karolina A.P.; Castermans, Tessy M.R.; Hommen, Merel P.J.; Meesters, Dennis M.; Poeze, Martijn

2015-01-01

Arginine, a semi-essential amino acid is an important initiator of the immune response. Arginine serves as a precursor in several metabolic pathways in different organs. In the immune response, arginine metabolism and availability is determined by the nitric oxide synthases and the arginase enzymes, which convert arginine into nitric oxide (NO) and ornithine, respectively. Limitations in arginine availability during inflammatory conditions regulate macrophages and T-lymfocyte activation. Furthermore, over the past years more evidence has been gathered which showed that arginine and citrulline deficiencies may underlie the detrimental outcome of inflammatory conditions, such as sepsis and endotoxemia. Not only does the immune response contribute to the arginine deficiency, also the impaired arginine de novo synthesis in the kidney has a key role in the eventual observed arginine deficiency. The complex interplay between the immune response and the arginine-NO metabolism is further underscored by recent data of our group. In this review we give an overview of physiological arginine and citrulline metabolism and we address the experimental and clinical studies in which the arginine-citrulline NO pathway plays an essential role in the immune response, as initiator and therapeutic target. PMID:25699985

Characterization and localization of arginine vasotocin receptors in the brain and kidney of an amphibian

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boyd, S.K.

1987-01-01

Because arginine vasotocin (AVT) activates male sexual behaviors in the rough-skinned newt (Taricha granulosa), quantitative autoradiography with radiolabeled arginine vasopressin (/sup 3/H-AVP) was used to localize and characterize putative AVT receptors in the brain of this amphibian. Binding of /sup 3/H-AVP to sites within the medial pallium was saturable, specific, reversible, of high affinity and low capacity. These binding sites appear to represent authentic central nervous system receptors for AVT. Furthermore, ligand specificity for the binding sites in this amphibian differs from that reported for AVP binding sites in rat brains. Dense concentrations of specific binding sites were located inmore » the olfactory nerve as it entered the olfactory bulb within the medial pallium, dorsal pallium, and amygdala pars lateralis of the telencephalon, and in the tegmental region of the medulla. Concentrations of binding sites differed significantly among various brain regions. A comparison of male and female newts collected during the breeding season revealed no sexual dimorphism. These areas may represent site(s) of action where AVT elicits sexual behaviors in male T. granulosa.« less

The difference in the effect of glutamate and NO synthase inhibitor on free calcium concentration and Na+, K+-ATPase activity in synaptosomes from various brain regions.

PubMed

Avrova, N F; Shestak, K I; Zakharova, I O; Sokolova, T V; Leont'ev, V G

1999-09-01

The significant increase of free calcium concentration ([Ca2+]i) was found in rat cerebral cortex synaptosomes and hippocampal crude synaptosomal fraction after their exposure to glutamate. But no change of [Ca2+]i was revealed in cerebellar synaptosomes, the slight increase of [Ca2+]i in striatal synaptosomes was not significant. The presence of Ng-nitro-L-arginine methyl ester (L-NAME) in the incubation medium practically prevented the increase of [Ca2+]i initiated by glutamate in cerebral cortex synaptosomes, but not in hippocampal ones. The significant diminution of [Ca2+]i in the presence of this inhibitor was shown in striatal synaptosomes exposed to glutamate. Na+,K+-ATPase activity is significantly lower in cerebral cortex, striatal and hippocampal synaptosomes exposed to glutamate. L-NAME prevented the inactivation of this enzyme by glutamate. In cerebellar synaptosomes the tendency to the decrease of enzymatic activity in the presence of L-NAME was on the contrary noticed. Thus, the data obtained provide evidence of the protective effect of NO synthase inhibitor in brain cortex and striatal synaptosomes, but not in cerebellar synaptosomes. Synaptosomes appear to be an adequate model to study the regional differences in the mechanism of toxic effect of excitatory amino acids.

An NMR-Based Metabolomic Approach to Investigate the Effects of Supplementation with Glutamic Acid in Piglets Challenged with Deoxynivalenol

PubMed Central

Ren, Wenkai; Yin, Jie; Hu, Jiayu; Duan, Jielin; Liu, Gang; Tan, Bie; Xiong, Xia; Oso, Abimbola Oladele; Adeola, Olayiwola; Yao, Kang; Yin, Yulong; Li, Tiejun

2014-01-01

Deoxynivalenol (DON) has various toxicological effects in humans and pigs that result from the ingestion of contaminated cereal products. This study was conducted to investigate the protective effects of dietary supplementation with glutamic acid on piglets challenged with DON. A total of 20 piglets weaned at 28 d of age were randomly assigned to receive 1 of 4 treatments (5 piglets/treatment): 1) basal diet, negative control (NC); 2) basal diet +4 mg/kg DON (DON); 3) basal diet +2% (g/g) glutamic acid (GLU); 4) basal diet +4 mg/kg DON +2% glutamic acid (DG). A 7-d adaptation period was followed by 30 days of treatment. A metabolite analysis using nuclear magnetic resonance spectroscopy (1H-NMR)-based metabolomic technology and the determination of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities for plasma, as well as the activity of Caspase-3 and the proliferation of epithelial cells were conducted. The results showed that contents of low-density lipoprotein, alanine, arginine, acetate, glycoprotein, trimethylamine-N-oxide (TMAO), glycine, lactate, and urea, as well as the glutamate/creatinine ratio were higher but high-density lipoprotein, proline, citrate, choline, unsaturated lipids and fumarate were lower in piglets of DON treatment than that of NC treatment (P<0.05). Compared with DON treatment, dietary supplementation with glutamic acid increased the plasma concentrations of proline, citrate, creatinine, unsaturated lipids, and fumarate, and decreased the concentrations of alanine, glycoprotein, TMAO, glycine, and lactate, as well as the glutamate/creatinine ratio (P<0.05). Addition glutamic acid to DON treatment increased the plasma activities of SOD and GSH-Px and the proliferating cell nuclear antigen (PCNA) labeling indexes for the jejunum and ileum (P<0.05). These novel findings indicate that glutamic acid has the potential to repair the injuries associated with oxidative stress as well as the disturbances of energy and amino

An NMR-based metabolomic approach to investigate the effects of supplementation with glutamic acid in piglets challenged with deoxynivalenol.

PubMed

Wu, Miaomiao; Xiao, Hao; Ren, Wenkai; Yin, Jie; Hu, Jiayu; Duan, Jielin; Liu, Gang; Tan, Bie; Xiong, Xia; Oso, Abimbola Oladele; Adeola, Olayiwola; Yao, Kang; Yin, Yulong; Li, Tiejun

2014-01-01

Deoxynivalenol (DON) has various toxicological effects in humans and pigs that result from the ingestion of contaminated cereal products. This study was conducted to investigate the protective effects of dietary supplementation with glutamic acid on piglets challenged with DON. A total of 20 piglets weaned at 28 d of age were randomly assigned to receive 1 of 4 treatments (5 piglets/treatment): 1) basal diet, negative control (NC); 2) basal diet +4 mg/kg DON (DON); 3) basal diet +2% (g/g) glutamic acid (GLU); 4) basal diet +4 mg/kg DON +2% glutamic acid (DG). A 7-d adaptation period was followed by 30 days of treatment. A metabolite analysis using nuclear magnetic resonance spectroscopy (1H-NMR)-based metabolomic technology and the determination of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities for plasma, as well as the activity of Caspase-3 and the proliferation of epithelial cells were conducted. The results showed that contents of low-density lipoprotein, alanine, arginine, acetate, glycoprotein, trimethylamine-N-oxide (TMAO), glycine, lactate, and urea, as well as the glutamate/creatinine ratio were higher but high-density lipoprotein, proline, citrate, choline, unsaturated lipids and fumarate were lower in piglets of DON treatment than that of NC treatment (P<0.05). Compared with DON treatment, dietary supplementation with glutamic acid increased the plasma concentrations of proline, citrate, creatinine, unsaturated lipids, and fumarate, and decreased the concentrations of alanine, glycoprotein, TMAO, glycine, and lactate, as well as the glutamate/creatinine ratio (P<0.05). Addition glutamic acid to DON treatment increased the plasma activities of SOD and GSH-Px and the proliferating cell nuclear antigen (PCNA) labeling indexes for the jejunum and ileum (P<0.05). These novel findings indicate that glutamic acid has the potential to repair the injuries associated with oxidative stress as well as the disturbances of energy and amino

Kinetics of the utilization of dietary arginine for nitric oxide and urea synthesis: insight into the arginine-nitric oxide metabolic system in humans.

PubMed

Mariotti, François; Petzke, Klaus J; Bonnet, Damien; Szezepanski, Isabelle; Bos, Cécile; Huneau, Jean-François; Fouillet, Hélène

2013-05-01

The systemic availability of oral/dietary arginine and its utilization for nitric oxide (NO) synthesis remains unknown and may be related to a competitive hydrolysis of arginine into urea in the splanchnic area and systemic circulation. We investigated the kinetics and dose-dependency of dietary arginine utilization for NO compared with urea synthesis and studied the characteristics of the arginine-NO metabolic system in healthy humans. We traced the metabolic fate and analyzed the utilization dynamics of dietary arginine after its ingestion at 2 nutritional amounts in healthy humans (n = 9) in a crossover design by using [(15)N-(15)N-(guanido)]-arginine, isotope ratio mass spectrometry techniques, and data analysis with a compartmental modeling approach. Whatever the amount of dietary arginine, 60 ± 3% (±SEM) was converted to urea, with kinetics indicative of a first-pass splanchnic phenomenon. Despite this dramatic extraction, intact dietary arginine made a major contribution to the postprandial increase in plasma arginine. However, the model identified that the plasma compartment was a very minor (~2%) precursor for the conversion of dietary arginine into NO, which, in any case, was small (<0.1% of the dose). The whole-body and plasma kinetics of arginine metabolism were consistent with the suggested competitive metabolism by the arginase and NO synthase pathways. The conversion of oral/dietary arginine into NO is not limited by the systemic availability of arginine but by a tight metabolic compartmentation at the systemic level. We propose an organization of the arginine metabolic system that explains the daily maintenance of NO homeostasis in healthy humans.

Influence of Volatile Anesthesia on the Release of Glutamate and other Amino Acids in the Nucleus Accumbens in a Rat Model of Alcohol Withdrawal: A Pilot Study

PubMed Central

Seidemann, Thomas; Spies, Claudia; Morgenstern, Rudolf; Wernecke, Klaus-Dieter; Netzhammer, Nicolai

2017-01-01

Background Alcohol withdrawal syndrome is a potentially life-threatening condition, which can occur when patients with alcohol use disorders undergo general anesthesia. Excitatory amino acids, such as glutamate, act as neurotransmitters and are known to play a key role in alcohol withdrawal syndrome. To understand this process better, we investigated the influence of isoflurane, sevoflurane, and desflurane anesthesia on the profile of excitatory and inhibitory amino acids in the nucleus accumbens (NAcc) of alcohol-withdrawn rats (AWR). Methods Eighty Wistar rats were randomized into two groups of 40, pair-fed with alcoholic or non-alcoholic nutrition. Nutrition was withdrawn and microdialysis was performed to measure the activity of amino acids in the NAcc. The onset time of the withdrawal syndrome was first determined in an experiment with 20 rats. Sixty rats then received isoflurane, sevoflurane, or desflurane anesthesia for three hours during the withdrawal period, followed by one hour of elimination. Amino acid concentrations were measured using chromatography and results were compared to baseline levels measured prior to induction of anesthesia. Results Glutamate release increased in the alcohol group at five hours after the last alcohol intake (p = 0.002). After 140 min, desflurane anesthesia led to a lower release of glutamate (p < 0.001) and aspartate (p = 0.0007) in AWR compared to controls. GABA release under and after desflurane anesthesia was also significantly lower in AWR than controls (p = 0.023). Over the course of isoflurane anesthesia, arginine release decreased in AWR compared to controls (p < 0.001), and aspartate release increased after induction relative to controls (p20min = 0.015 and p40min = 0.006). However, amino acid levels did not differ between the groups as a result of sevoflurane anesthesia. Conclusions Each of three volatile anesthetics we studied showed different effects on excitatory and inhibitory amino acid concentrations. Under

Influence of Volatile Anesthesia on the Release of Glutamate and other Amino Acids in the Nucleus Accumbens in a Rat Model of Alcohol Withdrawal: A Pilot Study.

PubMed

Seidemann, Thomas; Spies, Claudia; Morgenstern, Rudolf; Wernecke, Klaus-Dieter; Netzhammer, Nicolai

2017-01-01

Alcohol withdrawal syndrome is a potentially life-threatening condition, which can occur when patients with alcohol use disorders undergo general anesthesia. Excitatory amino acids, such as glutamate, act as neurotransmitters and are known to play a key role in alcohol withdrawal syndrome. To understand this process better, we investigated the influence of isoflurane, sevoflurane, and desflurane anesthesia on the profile of excitatory and inhibitory amino acids in the nucleus accumbens (NAcc) of alcohol-withdrawn rats (AWR). Eighty Wistar rats were randomized into two groups of 40, pair-fed with alcoholic or non-alcoholic nutrition. Nutrition was withdrawn and microdialysis was performed to measure the activity of amino acids in the NAcc. The onset time of the withdrawal syndrome was first determined in an experiment with 20 rats. Sixty rats then received isoflurane, sevoflurane, or desflurane anesthesia for three hours during the withdrawal period, followed by one hour of elimination. Amino acid concentrations were measured using chromatography and results were compared to baseline levels measured prior to induction of anesthesia. Glutamate release increased in the alcohol group at five hours after the last alcohol intake (p = 0.002). After 140 min, desflurane anesthesia led to a lower release of glutamate (p < 0.001) and aspartate (p = 0.0007) in AWR compared to controls. GABA release under and after desflurane anesthesia was also significantly lower in AWR than controls (p = 0.023). Over the course of isoflurane anesthesia, arginine release decreased in AWR compared to controls (p < 0.001), and aspartate release increased after induction relative to controls (p20min = 0.015 and p40min = 0.006). However, amino acid levels did not differ between the groups as a result of sevoflurane anesthesia. Each of three volatile anesthetics we studied showed different effects on excitatory and inhibitory amino acid concentrations. Under desflurane anesthesia, both glutamate

Estimation of dietary arginine requirements for Longyan laying ducks.

PubMed

Xia, Weiguang; Fouad, Ahmed Mohamed; Chen, Wei; Ruan, Dong; Wang, Shuang; Fan, Qiuli; Wang, Ying; Cui, Yiyan; Zheng, Chuntian

2017-01-01

This study aimed to establish the arginine requirements of Longyan ducks from 17 to 31 wk of age based on egg production, egg quality, plasma, and ovarian indices, as well as the expression of vitellogenesis-related genes. In total, 660 Longyan ducks with similar body weight at 15 wk of age were assigned randomly to 5 treatments, each with 6 replicates of 22 birds, and fed a corn-corn gluten meal basal diet (0.66% arginine) supplemented with either 0, 0.20%, 0.40%, 0.60%, or 0.80% arginine. Dietary arginine did not affect egg production by laying ducks, but it increased (linear, P < 0.01) the egg weight at 22 to 31 and 17 to 31 wk of age. Dietary arginine increased the yolk color score (linearly, P < 0.05) and the yolk percentage (quadratic, P < 0.05), where the maximum values were obtained with 1.26% arginine. Dietary arginine affected the total shell percentage and shell thickness, with the highest values using 1.46% arginine (P < 0.01). The weight and number of small yellow follicles (SYFs) increased (quadratic, P < 0.05) with the dietary arginine level and there was a quadratic response (P < 0.05) in terms of the SYFs weight/ovarian weight; the highest values were obtained in ducks fed 1.26% arginine. The plasma arginine concentration exhibited a quadratic (P < 0.05) response to dietary arginine. The plasma progesterone concentration decreased (linear, P < 0.05) as dietary arginine increased. The mRNA abundance of the very low density lipoprotein receptor-b increased in the second large yellow follicle membranes (quadratic, P < 0.05) with the dietary arginine level, where the highest value occurred with 1.26% arginine. According to the regression model, the dietary arginine requirements for Longyan laying ducks aged 17 to 31 wk are 1.06%, 1.13%, 1.22%, and 1.11% to obtain the maximum yolk percentage, SYFs number, SYFs weight, and SYFs weight/ovarian weight, respectively. © 2016 Poultry Science Association Inc.

A Type III Protein Arginine Methyltransferase from the Protozoan Parasite Trypanosoma brucei*

PubMed Central

Fisk, John C.; Sayegh, Joyce; Zurita-Lopez, Cecilia; Menon, Sarita; Presnyak, Vladimir; Clarke, Steven G.; Read, Laurie K.

2009-01-01

Arginine methylation is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). The ancient protozoan parasite, Trypanosoma brucei, possesses five putative PRMTs, a relatively large number for a single-celled eukaryote. Trypanosomatids lack gene regulation at the level of transcription, instead relying on post-transcriptional control mechanisms that act at the levels of RNA turnover, translation, and editing, all processes that likely involve multiple RNA-binding proteins, which are common targets of arginine methylation. Here, we report the characterization of a trypanosome PRMT, TbPRMT7, which is homologous to human PRMT7. Interestingly, trypanosomatids are the only single-celled eukaryotes known to harbor a PRMT7 homologue. TbPRMT7 differs dramatically from all known metazoan PRMT7 homologues in lacking the second AdoMet binding-like domain that is required for activity of the human enzyme. Nevertheless, bacterially expressed TbPRMT7 exhibits robust methyltransferase activity toward multiple targets in vitro. High resolution ion exchange chromatography analysis of methylated substrates reveals that TbPRMT7 is a type III PRMT, catalyzing the formation of only monomethylarginine, thereby representing the only exclusively type III PRMT identified to date. TbPRMT7 is expressed in both mammalian and insect stage T. brucei and is apparently dispensable for growth in both life cycle stages. The enzyme is cytoplasmically localized and is a component of several higher order complexes in vivo. Together, our studies indicate that TbPRMT7 is a Type III PRMT, and its robust activity and presence in numerous complexes suggest it plays multiple roles during the complex T. brucei life cycle. PMID:19254949

A type III protein arginine methyltransferase from the protozoan parasite Trypanosoma brucei.

PubMed

Fisk, John C; Sayegh, Joyce; Zurita-Lopez, Cecilia; Menon, Sarita; Presnyak, Vladimir; Clarke, Steven G; Read, Laurie K

2009-04-24

Arginine methylation is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). The ancient protozoan parasite, Trypanosoma brucei, possesses five putative PRMTs, a relatively large number for a single-celled eukaryote. Trypanosomatids lack gene regulation at the level of transcription, instead relying on post-transcriptional control mechanisms that act at the levels of RNA turnover, translation, and editing, all processes that likely involve multiple RNA-binding proteins, which are common targets of arginine methylation. Here, we report the characterization of a trypanosome PRMT, TbPRMT7, which is homologous to human PRMT7. Interestingly, trypanosomatids are the only single-celled eukaryotes known to harbor a PRMT7 homologue. TbPRMT7 differs dramatically from all known metazoan PRMT7 homologues in lacking the second AdoMet binding-like domain that is required for activity of the human enzyme. Nevertheless, bacterially expressed TbPRMT7 exhibits robust methyltransferase activity toward multiple targets in vitro. High resolution ion exchange chromatography analysis of methylated substrates reveals that TbPRMT7 is a type III PRMT, catalyzing the formation of only monomethylarginine, thereby representing the only exclusively type III PRMT identified to date. TbPRMT7 is expressed in both mammalian and insect stage T. brucei and is apparently dispensable for growth in both life cycle stages. The enzyme is cytoplasmically localized and is a component of several higher order complexes in vivo. Together, our studies indicate that TbPRMT7 is a Type III PRMT, and its robust activity and presence in numerous complexes suggest it plays multiple roles during the complex T. brucei life cycle.

Nonvesicular Release of Glutamate by Glial xCT Transporters Suppresses Glutamate Receptor Clustering In Vivo

PubMed Central

Augustin, Hrvoje; Grosjean, Yael; Chen, Kaiyun; Sheng, Qi; Featherstone, David E.

2008-01-01

We hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, we identified and functionally characterized a novel Drosophila xCT gene, which we subsequently named “genderblind” (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination of gb causes a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering, gb mutants show a large (200–300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additional in situ pharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, our results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamate in vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitized in vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses. PMID:17202478

Arginine production in the neonate

USDA-ARS?s Scientific Manuscript database

Endogenous arginine synthesis in adults is a complex multiorgan process, in which citrulline is synthesized in the gut, enters the general circulation, and is converted into arginine in the kidney, by what is known as the intestinal-renal axis. In neonates, the enzymes required to convert citrulline...

Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon.

PubMed

Takahara, Kentaro; Akashi, Kinya; Yokota, Akiho

2005-10-01

Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild watermelon glutamate N-acetyltransferase (CLGAT) that catalyses the transacetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutamate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 degrees C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought/strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought/strong-light stress.

The hydrophobic region of the DmsA twin-arginine leader peptide determines specificity with chaperone DmsD.

PubMed

Winstone, Tara M L; Tran, Vy A; Turner, Raymond J

2013-10-29

The system specific chaperone DmsD plays a role in the maturation of the catalytic subunit of dimethyl sulfoxide (DMSO) reductase, DmsA. Pre-DmsA contains a 45-amino acid twin-arginine leader peptide that is important for targeting and translocation of folded and cofactor-loaded DmsA by the twin-arginine translocase. DmsD has previously been shown to interact with the complete twin-arginine leader peptide of DmsA. In this study, isothermal titration calorimetry was used to investigate the thermodynamics of binding between synthetic peptides composed of different portions of the DmsA leader peptide and DmsD. Only those peptides that included the complete and contiguous hydrophobic region of the DmsA leader sequence were able to bind DmsD with a 1:1 stoichiometry. Each of the peptides that were able to bind DmsD also showed some α-helical structure as indicated by circular dichroism spectroscopy. Differential scanning calorimetry revealed that DmsD gained very little thermal stability upon binding any of the DmsA leader peptides tested. Together, these results suggest that a portion of the hydrophobic region of the DmsA leader peptide determines the specificity of binding and may produce helical properties upon binding to DmsD. Overall, this study demonstrates that the recognition of the DmsA twin-arginine leader sequence by the DmsD chaperone shows unexpected rules and confirms further that the biochemistry of the interaction of the chaperone with their leaders demonstrates differences in their molecular interactions.

Gas Phase Dissociation Behavior of Acyl-Arginine Peptides.

PubMed

McGee, William M; McLuckey, Scott A

2013-11-15

The gas phase dissociation behavior of peptides containing acyl-arginine residues is investigated. These acylations are generated via a combination of ion/ion reactions between arginine-containing peptides and N -hydroxysuccinimide (NHS) esters and subsequent tandem mass spectrometry (MS/MS). Three main dissociation pathways of acylated arginine, labeled Paths 1-3, have been identified and are dependent on the acyl groups. Path 1 involves the acyl-arginine undergoing deguanidination, resulting in the loss of the acyl group and dissociation of the guanidine to generate an ornithine residue. This pathway generates selective cleavage sites based on the recently discussed "ornithine effect". Path 2 involves the coordinated losses of H 2 O and NH 3 from the acyl-arginine side chain while maintaining the acylation. We propose that Path 2 is initiated via cyclization of the δ-nitrogen of arginine and the C-terminal carbonyl carbon, resulting in rapid rearrangement from the acyl-arginine side chain and the neutral losses. Path 3 occurs when the acyl group contains α-hydrogens and is observed as a rearrangement to regenerate unmodified arginine while the acylation is lost as a ketene.

A new specific ageusia: some humans cannot taste L-glutamate.

PubMed

Lugaz, O; Pillias, A-M; Faurion, A

2002-02-01

A new specific ageusia was found in human subjects for monosodium L-glutamate (MSG). Four tests were successively applied to discriminate non-tasters and hypotasters from tasters. (i) NaCl and MSG thresholds, and (ii) suprathreshold sensitivity were evaluated using the up-and-down procedure. Only 73% of 109 subjects common to both tests demonstrated a sensitivity for MSG significantly higher than their sensitivity to NaCl, and hence a specific sensitivity to L-glutamate. The remaining 27% who showed no significant difference in sensitivity to MSG and NaCl solutions were considered as putative hypotasters. (iii) Perception profiles (time-intensity) for MSG and NaCl were tested in 58 subjects and appeared significantly different in 47 tasters (81%). This technique helped in identifying among putative hypotasters of tests 1 and 2 a few tasters who perceived equal intensity for isoconcentration of NaCl and MSG but who could discriminate isomolar solutions on other cues. Thus, 19% of subjects, for whom no significant differences were found between MSG and NaCl time-intensity profiles, remained in the hypotaster group. (iv) A discrimination task including 24 triangular presentations per subject of NaCl and MSG 29 mM applied to the eight most severe hypotasters showed that two subjects at least (two of 58; 3.5%) could not discriminate between both stimuli. Moreover, these subjects probably perceived identical sensations for MSG and NaCl solutions. The six other hypotasters (10.3%) could discriminate both stimuli at the limit of significance. None of these eight subjects were able to identify the typical umami taste in 29 mM MSG.

Asymmetric arginine dimethylation of heterogeneous nuclear ribonucleoprotein K by protein-arginine methyltransferase 1 inhibits its interaction with c-Src.

PubMed

Ostareck-Lederer, Antje; Ostareck, Dirk H; Rucknagel, Karl P; Schierhorn, Angelika; Moritz, Bodo; Huttelmaier, Stefan; Flach, Nadine; Handoko, Lusy; Wahle, Elmar

2006-04-21

Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric N(G),N(G)-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.

Cooperativity in the two-domain arginine kinase from the sea anemone Anthopleura japonicus. II. Evidence from site-directed mutagenesis studies.

PubMed

Tada, Hiroshi; Suzuki, Tomohiko

2010-08-01

The arginine kinase (AK) from the sea anemone Anthopleura japonicus has an unusual two-domain structure (contiguous dimer; denoted by D1-D2). In a previous report, we suggested cooperativity in the contiguous dimer, which may be a result of domain-domain interactions, using MBP-fused enzymes. To further understand this observation, we inserted six-Lys residues into the linker region of the two-domain AK (D1-K6-D2 mutant) using His-tagged enzyme. The dissociation constants, K(a) and K(ia), of the mutant were similar to those of the wild-type enzyme but the catalytic constant, k(cat), was decreased to 28% that of the wild-type, indicating that some of the domain-domain interactions are lost due to the six-Lys insertion. Y68 plays a major role in arginine binding in the catalytic pocket in Limulus AK, and introduction of mutation at the Y68 position virtually abolishes catalytic activity. Thus, the constructed D1(Y68G)-D2 and D1-D2(Y68G) mutants mimic the D1(inactive)-D2(active) and D1(active)-D2(inactive) enzymes, respectively. The k(cat) values of both Y68 mutants were decreased to 13-18% that of the wild-type enzyme, which is much less than the 50% level of the two-domain enzyme. Thus, it is clear that substrate-binding to both domains is necessary for full expression of activity. In other words, substrate-binding appears to act as the trigger of the functional cooperativity in two-domain AK. Copyright 2010 Elsevier B.V. All rights reserved.

Different pools of glutamate receptors mediate sensitivity to ambient glutamate in the cochlear nucleus.

PubMed

Yang, Yang; Xu-Friedman, Matthew A

2015-06-01

Ambient glutamate plays an important role in pathological conditions, such as stroke, but its role during normal activity is not clear. In addition, it is not clear how ambient glutamate acts on glutamate receptors with varying affinities or subcellular localizations. To address this, we studied "endbulb of Held" synapses, which are formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. When ambient glutamate was increased by applying the glutamate reuptake inhibitor TFB-TBOA, BCs depolarized as a result of activation of N-methyl-D-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs). Application of antagonists against NMDARs (in 0 Mg(2+)) or mGluRs caused hyperpolarization, indicating that these receptors were bound by a tonic source of glutamate. AMPA receptors did not show these effects, consistent with their lower glutamate affinity. We also evaluated the subcellular localization of the receptors activated by ambient glutamate. The mGluRs were not activated by synaptic stimulation and thus appear to be exclusively extrasynaptic. By contrast, NMDARs in both synaptic and extrasynaptic compartments were activated by ambient glutamate, as shown using the use-dependent antagonist MK-801. Levels of ambient glutamate appeared to be regulated in a spike-independent manner, and glia likely play a major role. These low levels of ambient glutamate likely have functional consequences, as even low concentrations of TBOA caused significant increases in BC spiking following synaptic stimulation. These results indicate that normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate. Copyright © 2015 the American Physiological Society.

Different pools of glutamate receptors mediate sensitivity to ambient glutamate in the cochlear nucleus

PubMed Central

Yang, Yang

2015-01-01

Ambient glutamate plays an important role in pathological conditions, such as stroke, but its role during normal activity is not clear. In addition, it is not clear how ambient glutamate acts on glutamate receptors with varying affinities or subcellular localizations. To address this, we studied “endbulb of Held” synapses, which are formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. When ambient glutamate was increased by applying the glutamate reuptake inhibitor TFB-TBOA, BCs depolarized as a result of activation of N-methyl-d-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs). Application of antagonists against NMDARs (in 0 Mg2+) or mGluRs caused hyperpolarization, indicating that these receptors were bound by a tonic source of glutamate. AMPA receptors did not show these effects, consistent with their lower glutamate affinity. We also evaluated the subcellular localization of the receptors activated by ambient glutamate. The mGluRs were not activated by synaptic stimulation and thus appear to be exclusively extrasynaptic. By contrast, NMDARs in both synaptic and extrasynaptic compartments were activated by ambient glutamate, as shown using the use-dependent antagonist MK-801. Levels of ambient glutamate appeared to be regulated in a spike-independent manner, and glia likely play a major role. These low levels of ambient glutamate likely have functional consequences, as even low concentrations of TBOA caused significant increases in BC spiking following synaptic stimulation. These results indicate that normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate. PMID:25855696

Increased Erythrocytes By-Products of Arginine Catabolism Are Associated with Hyperglycemia and Could Be Involved in the Pathogenesis of Type 2 Diabetes Mellitus

PubMed Central

Ramírez-Zamora, Serafín; Méndez-Rodríguez, Miguel L.; Olguín-Martínez, Marisela; Sánchez-Sevilla, Lourdes; Quintana-Quintana, Miguel; García-García, Norberto; Hernández-Muñoz, Rolando

2013-01-01

Diabetes mellitus (DM) is a worldwide disease characterized by metabolic disturbances, frequently associated with high risk of atherosclerosis and renal and nervous system damage. Here, we assessed whether metabolites reflecting oxidative redox state, arginine and nitric oxide metabolism, are differentially distributed between serum and red blood cells (RBC), and whether significant metabolism of arginine exists in RBC. In 90 patients with type 2 DM without regular treatment for diabetes and 90 healthy controls, paired by age and gender, we measured serum and RBC levels of malondialdehyde (MDA), nitrites, ornithine, citrulline, and urea. In isolated RBC, metabolism of L-[14C]-arginine was also determined. In both groups, nitrites were equally distributed in serum and RBC; citrulline predominated in serum, whereas urea, arginine, and ornithine were found mainly in RBC. DM patients showed hyperglycemia and increased blood HbA1C, and increased levels of these metabolites, except for arginine, significantly correlating with blood glucose levels. RBC were observed to be capable of catabolizing arginine to ornithine, citrulline and urea, which was increased in RBC from DM patients, and correlated with an increased affinity for arginine in the activities of putative RBC arginase (Km = 0.23±0.06 vs. 0.50±0.13 mM, in controls) and nitric oxide synthase (Km = 0.28±0.06 vs. 0.43±0.09 mM, in controls). In conclusion, our results suggest that DM alters metabolite distribution between serum and RBC, demonstrating that RBC regulate serum levels of metabolites which affect nitrogen metabolism, not only by transporting them but also by metabolizing amino acids such as arginine. Moreover, we confirmed that urea can be produced also by human RBC besides hepatocytes, being much more evident in RBC from patients with type 2 DM. These events are probably involved in the specific physiopathology of this disease, i.e., endothelial damage and dysfunction. PMID:23826148

Ablation of arginase II spares arginine and abolishes the arginine requirement for growth in male mice

USDA-ARS?s Scientific Manuscript database

Arginine is considered a semi-essential amino acid in many species, including humans, because under certain conditions its demand exceeds endogenous production. Arginine availability, however, is not only determined by its production, but also by its disposal. Manipulation of disposal pathways has t...

Arginine de novo and nitric oxide production in disease states

PubMed Central

Luiking, Yvette C.; Ten Have, Gabriella A. M.; Wolfe, Robert R.

2012-01-01

Arginine is derived from dietary protein intake, body protein breakdown, or endogenous de novo arginine production. The latter may be linked to the availability of citrulline, which is the immediate precursor of arginine and limiting factor for de novo arginine production. Arginine metabolism is highly compartmentalized due to the expression of the enzymes involved in arginine metabolism in various organs. A small fraction of arginine enters the NO synthase (NOS) pathway. Tetrahydrobiopterin (BH4) is an essential and rate-limiting cofactor for the production of NO. Depletion of BH4 in oxidative-stressed endothelial cells can result in so-called NOS3 “uncoupling,” resulting in production of superoxide instead of NO. Moreover, distribution of arginine between intracellular transporters and arginine-converting enzymes, as well as between the arginine-converting and arginine-synthesizing enzymes, determines the metabolic fate of arginine. Alternatively, NO can be derived from conversion of nitrite. Reduced arginine availability stemming from reduced de novo production and elevated arginase activity have been reported in various conditions of acute and chronic stress, which are often characterized by increased NOS2 and reduced NOS3 activity. Cardiovascular and pulmonary disorders such as atherosclerosis, diabetes, hypercholesterolemia, ischemic heart disease, and hypertension are characterized by NOS3 uncoupling. Therapeutic applications to influence (de novo) arginine and NO metabolism aim at increasing substrate availability or at influencing the metabolic fate of specific pathways related to NO bioavailability and prevention of NOS3 uncoupling. These include supplementation of arginine or citrulline, provision of NO donors including inhaled NO and nitrite (sources), NOS3 modulating agents, or the targeting of endogenous NOS inhibitors like asymmetric dimethylarginine. PMID:23011059

Atorvastatin and Fluoxetine Prevent Oxidative Stress and Mitochondrial Dysfunction Evoked by Glutamate Toxicity in Hippocampal Slices.

PubMed

Ludka, Fabiana K; Dal-Cim, Tharine; Binder, Luisa Bandeira; Constantino, Leandra Celso; Massari, Caio; Tasca, Carla I

2017-07-01

Atorvastatin has been shown to exert a neuroprotective action by counteracting glutamatergic toxicity. Recently, we have shown atorvastatin also exerts an antidepressant-like effect that depends on both glutamatergic and serotonergic systems modulation. Excitotoxicity is involved in several brain disorders including depression; thus, it is suggested that antidepressants may target glutamatergic system as a final common pathway. In this study, a comparison of the mechanisms involved in the putative neuroprotective effect of a repetitive atorvastatin or fluoxetine treatment against glutamate toxicity in hippocampal slices was performed. Adult Swiss mice were treated with atorvastatin (10 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.), once a day during seven consecutive days. On the eighth day, animals were killed and hippocampal slices were obtained and subjected to an in vitro protocol of glutamate toxicity. An acute treatment of atorvastatin or fluoxetine was not neuroprotective; however, the repeated atorvastatin or fluoxetine treatment prevented the decrease in cellular viability induced by glutamate in hippocampal slices. The loss of cellular viability induced by glutamate was accompanied by increased D-aspartate release, increased reactive oxygen species (ROS) and nitric oxide (NO) production, and impaired mitochondrial membrane potential. Atorvastatin or fluoxetine repeated treatment also presented an antidepressant-like effect in the tail suspension test. Atorvastatin or fluoxetine treatment was effective in protecting mice hippocampal slices from glutamate toxicity by preventing the oxidative stress and mitochondrial dysfunction.

Polymorphism in glutamate cysteine ligase catalytic subunit (GCLC) is associated with sulfamethoxazole-induced hypersensitivity in HIV/AIDS patients

PubMed Central

2012-01-01

Background Sulfamethoxazole (SMX) is a commonly used antibiotic for prevention of infectious diseases associated with HIV/AIDS and immune-compromised states. SMX-induced hypersensitivity is an idiosyncratic cutaneous drug reaction with genetic components. Here, we tested association of candidate genes involved in SMX bioactivation and antioxidant defense with SMX-induced hypersensitivity. Results Seventy seven single nucleotide polymorphisms (SNPs) from 14 candidate genes were genotyped and assessed for association with SMX-induced hypersensitivity, in a cohort of 171 HIV/AIDS patients. SNP rs761142 T > G, in glutamate cysteine ligase catalytic subunit (GCLC), was significantly associated with SMX-induced hypersensitivity, with an adjusted p value of 0.045. This result was replicated in a second cohort of 249 patients (p = 0.025). In the combined cohort, heterozygous and homozygous carriers of the minor G allele were at increased risk of developing hypersensitivity (GT vs TT, odds ratio = 2.2, 95% CL 1.4-3.7, p = 0.0014; GG vs TT, odds ratio = 3.3, 95% CL 1.6 – 6.8, p = 0.0010). Each minor allele copy increased risk of developing hypersensitivity 1.9 fold (95% CL 1.4 – 2.6, p = 0.00012). Moreover, in 91 human livers and 84 B-lymphocytes samples, SNP rs761142 homozygous G allele carriers expressed significantly less GCLC mRNA than homozygous TT carriers (p < 0.05). Conclusions rs761142 in GCLC was found to be associated with reduced GCLC mRNA expression and with SMX-induced hypersensitivity in HIV/AIDS patients. Catalyzing a critical step in glutathione biosynthesis, GCLC may play a broad role in idiosyncratic drug reactions. PMID:22824134

Protein arginine deiminase 2 binds calcium in an ordered fashion: Implications for inhibitor design

DOE PAGES

Slade, Daniel J.; Fang, Pengfei; Dreyton, Christina J.; ...

2015-01-26

Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ionsmore » that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.« less

Protein Arginine Deiminase 2 Binds Calcium in an Ordered Fashion: Implications for Inhibitor Design

PubMed Central

2015-01-01

Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs. PMID:25621824

Arginine inactivates human herpesvirus 2 and inhibits genital herpesvirus infection.

PubMed

Ikeda, Keiko; Yamasaki, Hisashi; Minami, Sawako; Suzuki, Yukiko; Tsujimoto, Kazuko; Sekino, Yoshihisa; Irie, Hiroshi; Arakawa, Tsutomu; Koyama, A Hajime

2012-12-01

Arginine, among the amino acids, has demonstrated unique properties, including suppression of protein-protein interactions and virus inactivation. We investigated the effects of arginine on the infectivity of human herpesvirus 2 (HHV-2) and the potential application of arginine as a chemotherapeutic agent against genital herpes. Arginine directly inactivated HHV-2 and characterization of the inactivation demonstrated that 1 M arginine at pH 4.3 inactivated the virus more efficiently compared to 0.1 M citrate or 1 M sodium chloride, indicating that neither acidic pH nor ionic strength alone is sufficient for virus inactivation. The effect of arginine was rapid and concentration-dependent. Although virus inactivation was efficient at an acidic pH, arginine inactivated the virus even at a neutral pH, provided that a higher arginine concentration and prolonged incubation time were used. In addition, arginine suppressed the multiplication of HHV-2 under the conditions at which its effect on cell viability was insignificant. Pilot mouse model studies revealed a marked suppression of death by arginine when the mice were infected with HHV-2 through the vaginal route, followed by an intermittent application of acidic arginine by vaginal instillation.

Structure-based analysis reveals hydration changes induced by arginine hydrochloride.

PubMed

Nakakido, Makoto; Tanaka, Yoshikazu; Mitsuhori, Mariko; Kudou, Motonori; Ejima, Daisuke; Arakawa, Tsutomu; Tsumoto, Kouhei

2008-10-01

Arginine hydrochloride has been used to suppress protein aggregation during refolding and in various other applications. We investigated the structure of hen egg-white lysozyme (HEL) and solvent molecules in arginine hydrochloride solution by X-ray crystallography. Neither the backbone nor side-chain structure of HEL was altered by the presence of arginine hydrochloride. In addition, no stably bound arginine molecules were observed. The number of hydration water molecules, however, changed with the arginine hydrochloride concentration. We suggest that arginine hydrochloride suppresses protein aggregation by altering the hydration structure and the transient binding of arginine molecules that could not be observed.

Dependence of endotoxin-induced vascular hyporeactivity on extracellular L-arginine.

PubMed

Schott, C A; Gray, G A; Stoclet, J C

1993-01-01

1. The dependence on extracellular L-arginine of vascular hyporeactivity induced by bacterial lipopolysaccharide (LPS) was studied in vivo in rats infused with LPS and in vitro in endothelium-denuded rat thoracic aortic rings exposed to LPS. 2. Infusion of LPS during 50 min at a dose of 10 mg kg-1 h-1 produced a significant impairment of the pressor effect of noradrenaline, while in tissues collected 60 min after the start of LPS infusion, no significant alteration in either plasma arginine concentration or aortic arginine content was found compared to saline-infused controls (where plasma arginine was 78.5 +/- 7 microM and aortic arginine 394 +/- 124 nmol g-1 tissue). 3. Incubation of isolated, endothelium-denuded aortic rings with LPS (10 micrograms ml-1) in the absence of L-arginine for 4 h at 37 degrees C produced a 6 fold (P < 0.01) rightward shift in the noradrenaline concentration-effect curve compared to polymyxin B (1 micrograms ml-1, a LPS neutralizing agent) and reduced by 15% the maximum observed tension. 4. The presence of L-arginine (100 microM) during the incubation with LPS and throughout the following contraction experiments caused a 15 fold (P < 0.01) increase in the EC50 of noradrenaline and greater depression (45%) of the maximum observed tension compared to polymyxin B-treated controls. Responses in control, non LPS-treated rings were unaffected by the presence of L-arginine. 5. The addition of L-arginine to rings incubated with LPS in the absence of L-arginine and maximally precontracted with noradrenaline (10 microM) induced a dose-dependent relaxation. The EC50 of L-arginine was 8.0+/-0.3mu.6. The reactivity of LPS-treated rings to noradrenaline both in the absence and presence of L-arginine was restored to control levels by N0-nitro-L-arginine methyl ester (L-NAME, 300 mu), an inhibitor of NO production and by methylene blue (3 JAM), an inhibitor of guanylate cyclase.7. Incubation of isolated aortae in the absence of L-arginine did not

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

PubMed

Mathieu, Christoph; Macêdo, Juan P; Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander C; Suter Grotemeyer, Marianne; González-Salgado, Amaia; Schmidt, Remo S; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

2017-01-01

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei

PubMed Central

Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander C.; Suter Grotemeyer, Marianne; González-Salgado, Amaia; Schmidt, Remo S.; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

2017-01-01

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei. PMID:28045943

Influence of arginine-glycine-aspartic acid (RGD), integrins (alphaV and alpha5) and osteopontin on bovine sperm-egg binding, and fertilization in vitro.

PubMed

Gonçalves, R F; Wolinetz, C D; Killian, G J

2007-02-01

Osteopontin (OPN), a phosphoprotein containing an arginine-glycine-aspartic acid (RGD) sequence, has been identified in cow oviduct epithelium and fluid. To investigate the potential role OPN in fertilization, we evaluated the ability of RGD peptide (arginine-glycine-aspartic), RGE peptide (arginine-glycine-glutamic acid), integrins alphaV and alpha5 antibodies and OPN antibody to influence bovine in vitro sperm-egg binding and fertilization. Treatment of sperm or oocytes with the RGD peptide prior fertilization significantly decreased in vitro sperm-egg binding and fertilization compared to the non-treated controls or those treated with RGE peptide. Binding and fertilization were also significantly decreased when in vitro matured bovine oocytes or sperm were pre-incubated with integrins alphaV and alpha5 antibodies at concentration ranging from 5 to 20 microg/mL. Addition of a rabbit polyclonal IgG antibody against purified bovine milk OPN with sperm or/and oocytes decreased (P<0.05) fertilization compared to the in vitro-fertilized control. These data provided evidence that integrin ligands existed on bovine oocytes and spermatozoa that contained RGD recognition sequences, and that antibody to OPN, a protein that contains that RGD sequence, was capable of reducing sperm-egg binding and fertilization in vitro.

Ionotropic glutamate receptors activate cell signaling in response to glutamate in Schwann cells.

PubMed

Campana, Wendy M; Mantuano, Elisabetta; Azmoon, Pardis; Henry, Kenneth; Banki, Michael A; Kim, John H; Pizzo, Donald P; Gonias, Steven L

2017-04-01

In the peripheral nervous system, Schwann cells (SCs) demonstrate surveillance activity, detecting injury and undergoing trans -differentiation to support repair. SC receptors that detect peripheral nervous system injury remain incompletely understood. We used RT-PCR to profile ionotropic glutamate receptor expression in cultured SCs. We identified subunits required for assembly of N -methyl-d-aspartic acid (NMDA) receptors (NMDA-Rs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and kainate receptors. Treatment of SCs with 40-100 µM glutamate or with 0.5-1.0 µM NMDA robustly activated Akt and ERK1/2. The response was transient and bimodal; glutamate concentrations that exceeded 250 µM failed to activate cell signaling. Phosphoprotein profiling identified diverse phosphorylated proteins in glutamate-treated SCs in addition to ERK1/2 and Akt, including p70 S6-kinase, glycogen synthase kinase-3, ribosomal S6 kinase, c-Jun, and cAMP response element binding protein. Activation of SC signaling by glutamate was blocked by EGTA and dizocilpine and by silencing expression of the NMDA-R NR1 subunit. Phosphoinositide 3-kinase/PI3K functioned as an essential upstream activator of Akt and ERK1/2 in glutamate-treated SCs. When glutamate or NMDA was injected directly into crush-injured rat sciatic nerves, ERK1/2 phosphorylation was observed in myelinated and nonmyelinating SCs. Glutamate promoted SC migration by a pathway that required PI3K and ERK1/2. These results identified ionotropic glutamate receptors and NMDA-Rs, specifically, as potentially important cell signaling receptors in SCs.-Campana, W. M., Mantuano, E., Azmoon, P., Henry, K., Banki, M. A., Kim, J. H., Pizzo, D. P., Gonias, S. L. Ionotropic glutamate receptors activate cell signaling in response to glutamate in Schwann cells. © FASEB.

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1*

PubMed Central

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N.; Sif, Saïd

2012-01-01

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage. PMID:22761421

Protein arginine methyltransferase 7 regulates cellular response to DNA damage by methylating promoter histones H2A and H4 of the polymerase δ catalytic subunit gene, POLD1.

PubMed

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N; Sif, Saïd

2012-08-24

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.

Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes.

PubMed

Kandpal, M; Fouce, R B; Pal, A; Guru, P Y; Tekwani, B L

1995-05-01

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.

Ethylene production in relation to nitrogen metabolism in Saccharomyces cerevisiae.

PubMed

Johansson, Nina; Persson, Karl O; Quehl, Paul; Norbeck, Joakim; Larsson, Christer

2014-11-01

We have previously shown that ethylene production in Saccharomyces cerevisiae expressing the ethylene-forming enzyme (EFE) from Pseudomonas syringae is strongly influenced by variations in the mode of cultivation as well as the choice of nitrogen source. Here, we have studied the influence of nitrogen metabolism on the production of ethylene further. Using ammonium, glutamate, glutamate/arginine, and arginine as nitrogen sources, it was found that glutamate (with or without arginine) correlates with a high ethylene production, most likely linked to an observed increase in 2-oxoglutarate levels. Arginine as a sole nitrogen source caused a reduced ethylene production. A reduction of arginine levels, accomplished using an arginine auxotrophic ARG4-deletion strain in the presence of limiting amounts of arginine or through CAR1 overexpression, did however not correlate with an increased ethylene production. As expected, arginine was necessary for ethylene production as ethylene production in the ARG4-deletion strain ceased at the time when arginine was depleted. In conclusion, our data suggest that high levels of 2-oxoglutarate and a limited amount of arginine are required for successful ethylene production in yeast. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

[Pharmacology of glutamate sensitive synapses (I). Glutamate agonists (author's transl)].

PubMed

Shinozaki, H

1982-04-01

The actions of kainic acid, quisqualic acid, and ibotenic acid on the crayfish neuromuscular junction were described, and it was particularly interesting that the discrepancy between glutamate responses and EJPs was revealed by the use of kainic acid. On the other hand, there is increasing evidence showing that glutamate is an excitatory transmitter at the crayfish neuromuscular junction. At this stage, we are unable as yet to definitively support or reject glutamate's candidacy as the excitatory transmitter at the crayfish neuromuscular junction. The discrepancy revealed by the use of kainic acid may bring up some questions. Certainly, the differential action of kainic acid on the glutamate current and the excitatory synaptic current opens to doubt the transmitter role of glutamate. In the case of the study on a transmitter role for a substance of doubt status, the value of pharmacological studies seems to be greater in disproving than in asserting such the role. However, we have to consider the matter of the extra-junctional receptor postulated on the crayfish postsynaptic membrane as one of the major problems for pharmacological identification.

Arginine side chain interactions and the role of arginine as a gating charge carrier in voltage sensitive ion channels

PubMed Central

Armstrong, Craig T.; Mason, Philip E.; Anderson, J. L. Ross; Dempsey, Christopher E.

2016-01-01

Gating charges in voltage-sensing domains (VSD) of voltage-sensitive ion channels and enzymes are carried on arginine side chains rather than lysine. This arginine preference may result from the unique hydration properties of the side chain guanidinium group which facilitates its movement through a hydrophobic plug that seals the center of the VSD, as suggested by molecular dynamics simulations. To test for side chain interactions implicit in this model we inspected interactions of the side chains of arginine and lysine with each of the 19 non-glycine amino acids in proteins in the protein data bank. The arginine guanidinium interacts with non-polar aromatic and aliphatic side chains above and below the guanidinium plane while hydrogen bonding with polar side chains is restricted to in-plane positions. In contrast, non-polar side chains interact largely with the aliphatic part of the lysine side chain. The hydration properties of arginine and lysine are strongly reflected in their respective interactions with non-polar and polar side chains as observed in protein structures and in molecular dynamics simulations, and likely underlie the preference for arginine as a mobile charge carrier in VSD. PMID:26899474

Arginine side chain interactions and the role of arginine as a gating charge carrier in voltage sensitive ion channels

NASA Astrophysics Data System (ADS)

Armstrong, Craig T.; Mason, Philip E.; Anderson, J. L. Ross; Dempsey, Christopher E.

2016-02-01

Gating charges in voltage-sensing domains (VSD) of voltage-sensitive ion channels and enzymes are carried on arginine side chains rather than lysine. This arginine preference may result from the unique hydration properties of the side chain guanidinium group which facilitates its movement through a hydrophobic plug that seals the center of the VSD, as suggested by molecular dynamics simulations. To test for side chain interactions implicit in this model we inspected interactions of the side chains of arginine and lysine with each of the 19 non-glycine amino acids in proteins in the protein data bank. The arginine guanidinium interacts with non-polar aromatic and aliphatic side chains above and below the guanidinium plane while hydrogen bonding with polar side chains is restricted to in-plane positions. In contrast, non-polar side chains interact largely with the aliphatic part of the lysine side chain. The hydration properties of arginine and lysine are strongly reflected in their respective interactions with non-polar and polar side chains as observed in protein structures and in molecular dynamics simulations, and likely underlie the preference for arginine as a mobile charge carrier in VSD.

Parenteral or Enteral Arginine Supplementation Safety and Efficacy.

PubMed

Rosenthal, Martin D; Carrott, Phillip W; Patel, Jayshil; Kiraly, Laszlo; Martindale, Robert G

2016-12-01

Arginine supplementation has the potential to improve the health of patients. Its use in hospitalized patients has been a controversial topic in the nutrition literature, especially concerning supplementation of septic patients. In this article, we review the relevant literature both for and against the use of arginine in critically ill, surgical, and hospitalized patients. The effect of critical illness on arginine metabolism is reviewed, as is its use in septic and critically ill patients. Although mounting evidence supports immunonutrition, there are only a few studies that suggest that this is safe in patients with severe sepsis. The use of arginine has been shown to benefit a variety of critically ill patients. It should be considered for inclusion in combinations of immunonutrients or commercial formulations for groups in whom its benefit has been reported consistently, such as those who have suffered trauma and those in acute surgical settings. The aims of this review are to discuss the role of arginine in health, the controversy surrounding arginine supplementation of septic patients, and the use of arginine in critically ill patients. © 2016 American Society for Nutrition.

Agmatine: at the crossroads of the arginine pathways.

PubMed

Satriano, Joseph

2003-12-01

In acute inflammatory responses, such as wound healing and glomerulonephritis, arginine is the precursor for production of the cytostatic molecule nitric oxide (NO) and the pro-proliferative polyamines. NO is an early phase response whereas increased generation of polyamines is requisite for the later, repair phase response. The temporal switch of arginine as a substrate for the inducible nitric oxide synthase (iNOS)/NO axis to arginase/ornithine decarboxylase (ODC)/polyamine axis is subject to regulation by inflammatory cytokines as well as interregulation by the arginine metabolites themselves. Herein we describe the capacity of another arginine pathway, the metabolism of arginine to agmatine by arginine decarboxylase (ADC), to aid in this interregulation. Agmatine is an antiproliferative molecule due to its suppressive effects on intracellular polyamine levels, whereas the aldehyde metabolite of agmatine is a potent inhibitor of iNOS. We propose that the catabolism of agmatine to its aldehyde metabolite may act as a gating mechanism at the transition from the iNOS/NO axis to the arginase/ODC/polyamine axis. Thus, agmatine has the potential to serve in the coordination of the early and repair phase pathways of arginine in inflammation.

Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

PubMed Central

Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

2013-01-01

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions.

PubMed

Feng, You; Maity, Ranjan; Whitelegge, Julian P; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T; Bedford, Mark T; Masson, Jean-Yves; Clarke, Steven G

2013-12-27

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.

Enzymes of creatine biosynthesis, arginine and methionine metabolism in normal and malignant cells.

PubMed

Bera, Soumen; Wallimann, Theo; Ray, Subhankar; Ray, Manju

2008-12-01

The creatine/creatine kinase system decreases drastically in sarcoma. In the present study, an investigation of catalytic activities, western blot and mRNA expression unambiguously demonstrates the prominent expression of the creatine-synthesizing enzymes l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase in sarcoma, Ehrlich ascites carcinoma and Sarcoma 180 cells, whereas both enzymes were virtually undetectable in normal muscle. Compared to that of normal animals, these enzymes remained unaffected in the kidney or liver of sarcoma-bearing mice. High activity and expression of mitochondrial arginase II in sarcoma indicated increased ornithine formation. Slightly or moderately higher levels of ornithine, guanidinoacetate and creatinine were observed in sarcoma compared to muscle. Despite the intrinsically low level of creatine in Ehrlich ascites carcinoma and Sarcoma 180 cells, these cells could significantly take up and release creatine, suggesting a functional creatine transport, as verified by measuring mRNA levels of creatine transporter. Transcript levels of arginase II, ornithine-decarboxylase, S-adenosyl-homocysteine hydrolase and methionine-synthase were significantly upregulated in sarcoma and in Ehrlich ascites carcinoma and Sarcoma 180 cells. Overall, the enzymes related to creatine and arginine/methionine metabolism were found to be significantly upregulated in malignant cells. However, the low levels of creatine kinase in the same malignant cells do not appear to be sufficient for the building up of an effective creatine/phosphocreatine pool. Instead of supporting creatine biosynthesis, l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase appear to be geared to support cancer cell metabolism in the direction of polyamine and methionine synthesis because both these compounds are in high demand in proliferating cancer cells.

Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis.

PubMed

Gong, Jin-Song; Li, Wei; Zhang, Dan-Dan; Xie, Min-Feng; Yang, Biao; Zhang, Rong-Xian; Li, Heng; Lu, Zhen-Ming; Xu, Zheng-Hong; Shi, Jin-Song

2015-12-17

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-L-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba(2+). This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.

Ecological Effect of Arginine on Oral Microbiota.

PubMed

Zheng, Xin; He, Jinzhi; Wang, Lin; Zhou, Shuangshuang; Peng, Xian; Huang, Shi; Zheng, Liwei; Cheng, Lei; Hao, Yuqing; Li, Jiyao; Xu, Jian; Xu, Xin; Zhou, Xuedong

2017-08-03

Dental caries is closely associated with the microbial dybiosis between acidogenic/aciduric pathogens and alkali-generating commensal bacteria colonized in the oral cavity. Our recent studies have shown that arginine may represent a promising anti-caries agent by modulating microbial composition in an in vitro consortium. However, the effect of arginine on the oral microbiota has yet to be comprehensively delineated in either clinical cohort or in vitro biofilm models that better represent the microbial diversity of oral cavity. Here, by employing a clinical cohort and a saliva-derived biofilm model, we demonstrated that arginine treatment could favorably modulate the oral microbiota of caries-active individuals. Specifically, treatment with arginine-containing dentifrice normalized the oral microbiota of caries-active individuals similar to that of caries-free controls in terms of microbial structure, abundance of typical species, enzymatic activities of glycolysis and alkali-generation related enzymes and their corresponding transcripts. Moreover, we found that combinatory use of arginine with fluoride could better enrich alkali-generating Streptococcus sanguinis and suppress acidogenic/aciduric Streptococcus mutans, and thus significantly retard the demineralizing capability of saliva-derived oral biofilm. Hence, we propose that fluoride and arginine have a potential synergistic effect in maintaining an eco-friendly oral microbial equilibrium in favor of better caries management.

Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mayes, Heather B.; Knott, Brandon C.; Crowley, Michael F.

In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase ( TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleavingmore » the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2S O ring configuration as it reaches its binding site. Lastly, this work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.« less

Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases

DOE PAGES

Mayes, Heather B.; Knott, Brandon C.; Crowley, Michael F.; ...

2016-06-01

In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase ( TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleavingmore » the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2S O ring configuration as it reaches its binding site. Lastly, this work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.« less

Preservation of glutamic acid-iron chelate into montmorillonite to efficiently degrade Reactive Blue 19 in a Fenton system under sunlight irradiation at neutral pH

NASA Astrophysics Data System (ADS)

Huang, Zhujian; Wu, Pingxiao; Gong, Beini; Yang, Shanshan; Li, Hailing; Zhu, Ziao; Cui, Lihua

2016-05-01

To further enhance the visible light responsive property and the chemical stability of Fe/clay mineral catalysts, glutamic acid-iron chelate intercalated montmorillonite (G-Fe-Mt) was developed. The physiochemical properties of G-Fe-Mt were investigated by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), UV-vis diffuse reflectance spectroscopy (DRS), etc. The results showed that glutamic acid-iron chelates were successfully intercalated into the gallery of montmorillonite and the intercalated glutamic acid-iron chelate molecules were well preserved. The product G-Fe-Mt displayed excellent catalytic performance in heterogeneous photo-Fenton reaction under sunlight irradiation at acidic and neutral pH values. The chelation and the visible light responsiveness of glutamic acid produce a synergistic effect leading to greatly enhanced sunlight-Fenton reaction catalyzed by the heterogeneous G-Fe-Mt under neutral pH. G-Fe-Mt is a promising catalyst for advanced oxidation processes.

50 Hz hippocampal stimulation in refractory epilepsy: Higher level of basal glutamate predicts greater release of glutamate.

PubMed

Cavus, Idil; Widi, Gabriel A; Duckrow, Robert B; Zaveri, Hitten; Kennard, Jeremy T; Krystal, John; Spencer, Dennis D

2016-02-01

The effect of electrical stimulation on brain glutamate release in humans is unknown. Glutamate is elevated at baseline in the epileptogenic hippocampus of patients with refractory epilepsy, and increases during spontaneous seizures. We examined the effect of 50 Hz stimulation on glutamate release and its relationship to interictal levels in the hippocampus of patients with epilepsy. In addition, we measured basal and stimulated glutamate levels in a subset of these patients where stimulation elicited a seizure. Subjects (n = 10) were patients with medically refractory epilepsy who were undergoing intracranial electroencephalography (EEG) evaluation in an epilepsy monitoring unit. Electrical stimulation (50 Hz) was delivered through implanted hippocampal electrodes (n = 11), and microdialysate samples were collected every 2 min. Basal glutamate, changes in glutamate efflux with stimulation, and the relationships between peak stimulation-associated glutamate concentrations, basal zero-flow levels, and stimulated seizures were examined. Stimulation of epileptic hippocampi in patients with refractory epilepsy caused increases in glutamate efflux (p = 0.005, n = 10), and 4 of ten patients experienced brief stimulated seizures. Stimulation-induced increases in glutamate were not observed during the evoked seizures, but rather were related to the elevation in interictal basal glutamate (R(2) = 0.81, p = 0.001). The evoked-seizure group had lower basal glutamate levels than the no-seizure group (p = 0.04), with no stimulation-induced change in glutamate efflux (p = 0.47, n = 4). Conversely, increased glutamate was observed following stimulation in the no-seizure group (p = 0.005, n = 7). Subjects with an atrophic hippocampus had higher basal glutamate levels (p = 0.03, n = 7) and higher stimulation-induced glutamate efflux. Electrical stimulation of the epileptic hippocampus either increased extracellular glutamate efflux or induced seizures. The magnitude of stimulated

The growth of ZnO nanostructures using Arginine

NASA Astrophysics Data System (ADS)

Singh, Baljinder; Moudgil, Lovika; Singh, Gurinder; Kaura, Aman

2018-05-01

The growth mechanism of Zinc oxide (ZnO) nanomaterial with amino acid (Arginine) is explained at different shapes. The present study of ZnO nanostructures (NSs) in the presence of Arginine has enabled us to not only determine the growth mechanism of ZnO NSs but also to determine the effect of Arginine at different temperature of reactants. The synthesized samples are characterized using transmission electron microscopy (TEM) and X-ray diffraction (XRD). Results reveal that Arginine is responsible for formation of NSs. Based on these results, a plausible mechanism is explained.

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

PubMed

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

2006-11-01

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

Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

NASA Astrophysics Data System (ADS)

Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

2016-05-01

Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4-fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

PubMed

Jin, Yanhong; Li, Di; Lu, Shiying; Zhao, Han; Chen, Zhao; Hou, Wei; Ruan, Benfang Helen

Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

The role of arginine and the modified arginine deiminase enzyme ADI-PEG 20 in cancer therapy with special emphasis on Phase I/II clinical trials.

PubMed

Synakiewicz, Anna; Stachowicz-Stencel, Teresa; Adamkiewicz-Drozynska, Elzbieta

2014-11-01

The metabolic differences between normal, healthy cells and neoplastic cells have been exploited by anticancer therapies targeting metabolic pathways. Various studies of malignant processes have demonstrated disturbances in both arginine synthesis and metabolism that enhance or inhibit tumor cell growth. Consequently, there has been an increased interest in the arginine-depleting enzyme arginine deiminase (ADI) as a potential antineoplastic therapy. This review summarizes the literature on the potential anti-cancer therapeutics arginine and ADI, an arginine-catabolizing enzyme. The authors searched the MEDLINE database PubMed using the key words: 'arginine, 'ADI', 'arginine in cancer' and 'ADI and cancer'. The authors evaluate prospective randomized studies on cancer patients between 2004 and 2013 as well as ongoing research found through the US National Institutes of Health trial database. The results of current studies are promising but do not give unequivocal answers and so it is impossible to recommend arginine or its enzyme ADI as a therapeutic. In the opinion of the authors, further identification of arginine-dependent malignant tumors and their metabolism should be investigated. Furthermore, the use of these chemicals, in combination with other chemotherapeutics drugs, should be investigated and indeed may improve the success of arginine-depleting enzymes such as pegylated ADI (ADI-PEG20).

Glutamate dehydrogenase (RocG) in Bacillus licheniformis WX-02: Enzymatic properties and specific functions in glutamic acid synthesis for poly-γ-glutamic acid production.

PubMed

Tian, Guangming; Wang, Qin; Wei, Xuetuan; Ma, Xin; Chen, Shouwen

2017-04-01

Poly-γ-glutamic acid (γ-PGA), a natural biopolymer, is widely used in cosmetics, medicine, food, water treatment, and agriculture owing to its features of moisture sequestration, cation chelation, non-toxicity and biodegradability. Intracellular glutamic acid, the substrate of γ-PGA, is a limiting factor for high yield in γ-PGA production. Bacillus subtilis and Bacillus licheniformis are both important γ-PGA producing strains, and B. subtilis synthesizes glutamic acid in vivo using the unique GOGAT/GS pathway. However, little is known about the glutamate synthesis pathway in B. licheniformis. The aim of this work was to characterize the glutamate dehydrogenase (RocG) in glutamic acid synthesis from B. licheniformis with both in vivo and in vitro experiments. By re-directing the carbon flux distribution, the rocG gene deletion mutant WX-02ΔrocG produced intracellular glutamic acid with a concentration of 90ng/log(CFU), which was only 23.7% that of the wild-type WX-02 (380ng/log(CFU)). Furthermore, the γ-PGA yield of mutant WX-02ΔrocG was 5.37g/L, a decrease of 45.3% compared to the wild type (9.82g/L). In vitro enzymatic assays of RocG showed that RocG has higher affinity for 2-oxoglutarate than glutamate, and the glutamate synthesis rate was far above degradation. This is probably the first study to reveal the glutamic acid synthesis pathway and the specific functions of RocG in B. licheniformis. The results indicate that γ-PGA production can be enhanced through improving intracellular glutamic acid synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Histone arginine methylations: their roles in chromatin dynamics and transcriptional regulation

PubMed Central

LITT, Michael; QIU, Yi; HUANG, Suming

2017-01-01

Synopsis PRMTs (protein arginine N-methyltransferases) specifically modify the arginine residues of key cellular and nuclear proteins as well as histone substrates. Like lysine methylation, transcriptional repression or activation is dependent upon the site and type of arginine methylation on histone tails. Recent discoveries imply that histone arginine methylation is an important modulator of dynamic chromatin regulation and transcriptional controls. However, under the shadow of lysine methylation, the roles of histone arginine methylation have been under-explored. The present review focuses on the roles of histone arginine methylation in the regulation of gene expression, and the interplays between histone arginine methylation, histone acetylation, lysine methylation and chromatin remodelling factors. In addition, we discuss the dynamic regulation of arginine methylation by arginine demethylases, and how dysregulation of PRMTs and their activities are linked to human diseases such as cancer. PMID:19220199

Arginine homopeptides for plasmid DNA purification using monolithic supports.

PubMed

Cardoso, Sara; Sousa, Ângela; Queiroz, João A; Azzoni, Adriano R; Sousa, Fani

2018-06-15

Purification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA. Additionally, some studies revealed the ability of arginine based cationic peptides to condense plasmid DNA, which increased lengthening can result in strongest interactions with higher binding capacities for chromatographic purposes of large molecules such as pDNA. In this work, arginine homopeptides were immobilized in monolithic supports and their performance was evaluated and compared with a single arginine monolithic column regarding supercoiled (sc) plasmid DNA purification. Specific interactions of arginine based peptides with several nucleic acids present in a clarified Escherichia coli lysate sample showed potential for the sc pDNA purification. Effectively, the immobilization of the arginine homopeptides became more functional compared with the single arginine amino acid, showing higher binding capacities, which was also reflected in the intensity of the interactions. The combination of structural versatilities of monoliths with the specificity of arginine peptides raised as a promising strategy for sc pDNA purification. Copyright © 2018 Elsevier B.V. All rights reserved.

Protein Arginine Methylation and Citrullination in Epigenetic Regulation

PubMed Central

2015-01-01

The post-translational modification of arginine residues represents a key mechanism for the epigenetic control of gene expression. Aberrant levels of histone arginine modifications have been linked to the development of several diseases including cancer. In recent years, great progress has been made in understanding the physiological role of individual arginine modifications and their effects on chromatin function. The present review aims to summarize the structural and functional aspects of histone arginine modifying enzymes and their impact on gene transcription. We will discuss the potential for targeting these proteins with small molecules in a variety of disease states. PMID:26686581

Cooperative Effects Between Arginine and Glutamic Acid in the Amino Acid-Catalyzed Aldol Reaction.

PubMed

Valero, Guillem; Moyano, Albert

2016-08-01

Catalysis of the aldol reaction between cyclohexanone and 4-nitrobenzaldehyde by mixtures of L-Arg and of L-Glu in wet dimethyl sulfoxide (DMSO) takes place with higher enantioselectivity (up to a 7-fold enhancement in the anti-aldol for the 1:1 mixture) than that observed when either L-Glu or L-Arg alone are used as the catalysts. These results can be explained by the formation of a catalytically active hydrogen-bonded complex between both amino acids, and demonstrate the possibility of positive cooperative effects in catalysis by two different α-amino acids. Chirality 28:599-605, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Glutamate and Neurodegenerative Disease

NASA Astrophysics Data System (ADS)

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.

Protein arginine methylation: Cellular functions and methods of analysis.

PubMed

Pahlich, Steffen; Zakaryan, Rouzanna P; Gehring, Heinz

2006-12-01

During the last few years, new members of the growing family of protein arginine methyltransferases (PRMTs) have been identified and the role of arginine methylation in manifold cellular processes like signaling, RNA processing, transcription, and subcellular transport has been extensively investigated. In this review, we describe recent methods and findings that have yielded new insights into the cellular functions of arginine-methylated proteins, and we evaluate the currently used procedures for the detection and analysis of arginine methylation.

Molecular dynamics simulations on the Tre1 G protein-coupled receptor: exploring the role of the arginine of the NRY motif in Tre1 structure

PubMed Central

2013-01-01

Background The arginine of the D/E/NRY motif in Rhodopsin family G protein-coupled receptors (GPCRs) is conserved in 96% of these proteins. In some GPCRs, this arginine in transmembrane 3 can form a salt bridge with an aspartic acid or glutamic acid in transmembrane 6. The Drosophila melanogaster GPCR Trapped in endoderm-1 (Tre1) is required for normal primordial germ cell migration. In a mutant form of the protein, Tre1sctt, eight amino acids RYILIACH are missing, resulting in a severe disruption of primordial germ cell development. The impact of the loss of these amino acids on Tre1 structure is unknown. Since the missing amino acids in Tre1sctt include the arginine that is part of the D/E/NRY motif in Tre1, molecular dynamics simulations were performed to explore the hypothesis that these amino acids are involved in salt bridge formation and help maintain Tre1 structure. Results Structural predictions of wild type Tre1 (Tre1+) and Tre1sctt were subjected to over 250 ns of molecular dynamics simulations. The ability of the model systems to form a salt bridge between the arginine of the D/E/NRY motif and an aspartic acid residue in transmembrane 6 was analyzed. The results indicate that a stable salt bridge can form in the Tre1+ systems and a weak salt bridge or no salt bridge, using an alternative arginine, is likely in the Tre1sctt systems. Conclusions The weak salt bridge or lack of a salt bridge in the Tre1sctt systems could be one possible explanation for the disrupted function of Tre1sctt in primordial germ cell migration. These results provide a framework for studying the importance of the arginine of the D/E/NRY motif in the structure and function of other GPCRs that are involved in cell migration, such as CXCR4 in the mouse, zebrafish, and chicken. PMID:24044607

Molecular dynamics simulations on the Tre1 G protein-coupled receptor: exploring the role of the arginine of the NRY motif in Tre1 structure.

PubMed

Pruitt, Margaret M; Lamm, Monica H; Coffman, Clark R

2013-09-18

The arginine of the D/E/NRY motif in Rhodopsin family G protein-coupled receptors (GPCRs) is conserved in 96% of these proteins. In some GPCRs, this arginine in transmembrane 3 can form a salt bridge with an aspartic acid or glutamic acid in transmembrane 6. The Drosophila melanogaster GPCR Trapped in endoderm-1 (Tre1) is required for normal primordial germ cell migration. In a mutant form of the protein, Tre1sctt, eight amino acids RYILIACH are missing, resulting in a severe disruption of primordial germ cell development. The impact of the loss of these amino acids on Tre1 structure is unknown. Since the missing amino acids in Tre1sctt include the arginine that is part of the D/E/NRY motif in Tre1, molecular dynamics simulations were performed to explore the hypothesis that these amino acids are involved in salt bridge formation and help maintain Tre1 structure. Structural predictions of wild type Tre1 (Tre1+) and Tre1sctt were subjected to over 250 ns of molecular dynamics simulations. The ability of the model systems to form a salt bridge between the arginine of the D/E/NRY motif and an aspartic acid residue in transmembrane 6 was analyzed. The results indicate that a stable salt bridge can form in the Tre1+ systems and a weak salt bridge or no salt bridge, using an alternative arginine, is likely in the Tre1sctt systems. The weak salt bridge or lack of a salt bridge in the Tre1sctt systems could be one possible explanation for the disrupted function of Tre1sctt in primordial germ cell migration. These results provide a framework for studying the importance of the arginine of the D/E/NRY motif in the structure and function of other GPCRs that are involved in cell migration, such as CXCR4 in the mouse, zebrafish, and chicken.

Arginine Catabolism by Sourdough Lactic Acid Bacteria: Purification and Characterization of the Arginine Deiminase Pathway Enzymes from Lactobacillus sanfranciscensis CB1

PubMed Central

De Angelis, Maria; Mariotti, Liberato; Rossi, Jone; Servili, Maurizio; Fox, Patrick F.; Rollán, Graciela; Gobbetti, Marco

2002-01-01

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37°C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1

PRMT7 is a member of the protein arginine methyltransferase family with a distinct substrate specificity.

PubMed

Miranda, Tina Branscombe; Miranda, Mark; Frankel, Adam; Clarke, Steven

2004-05-28

We have identified a mammalian arginine N-methyltransferase, PRMT7, that can catalyze the formation of omega-NG-monomethylarginine in peptides. This protein is encoded by a gene on human chromosome 16q22.1 (human locus AK001502). We expressed a full-length human cDNA construct in Escherichia coli as a glutathione S-transferase (GST) fusion protein. We found that GST-tagged PRMT7 catalyzes the S-adenosyl-[methyl-3H]-l-methionine-dependent methylation of the synthetic peptide GGPGGRGGPGG-NH2 (R1). The radiolabeled peptide was purified by high-pressure liquid chromatography and acid hydrolyzed to free amino acids. When the hydrolyzed products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species which co-migrated with an omega-NG-monomethylarginine standard. Surprisingly, GST-PRMT7 was not able to catalyze the in vitro methylation of a GST-fibrillarin (amino acids 1-148) fusion protein (GST-GAR), a methyl-accepting substrate for the previously characterized PRMT1, PRMT3, PRMT4, PRMT5, and PRMT6 enzymes. Nor was it able to methylate myelin basic protein or histone H2A, in vitro substrates of PRMT5. This specificity distinguishes PRMT7 from all of the other known arginine methyltransferases. An additional unique feature of PRMT7 is that it seems to have arisen from a gene duplication event and contains two putative AdoMet-binding motifs. To see if both motifs were necessary for activity, each putative domain was expressed as a GST-fusion and tested for activity with peptides R1 and R2 (acetyl-GGRGG-NH2). These truncated proteins were enzymatically inactive, suggesting that both domains are required for functionality.

L-arginine-induced vasodilation in healthy humans: pharmacokinetic–pharmacodynamic relationship

PubMed Central

Bode-Böger, Stefanie M; Böger, Rainer H; Galland, Andrea; Tsikas, Dimitrios; Frölich, Jürgen C

1998-01-01

Aims Administration of l-arginine by intravenous infusion or via oral absorption has been shown to induce peripheral vasodilation in humans, and to improve endothelium-dependent vasodilation. We investigated the pharmacokinetics and pharmacokinetic-pharmacodynamic relationship of l-arginine after a single intravenous infusion of 30 g or 6 g, or after a single oral application of 6 g, as compared with the respective placebo, in eight healthy male human subjects. Methods l-arginine levels were determined by h.p.l.c. The vasodilator effects of l-arginine were assessed non-invasively by blood pressure monitoring and impedance cardiography. Urinary nitrate and cyclic GMP excretion rates were measured as non-invasive indicators of endogenous NO production. Results Plasma l-arginine levels increased to (mean±s.e.mean) 6223±407 (range, 5100–7680) and 822±59 (527–955) μmol l−1 after intravenous infusion of 30 g and 6 g l-arginine, respectively, and to 310±152 (118–1219) μmol l−1 after oral ingestion of 6 g l-arginine. Oral bioavailability of l-arginine was 68±9 (51–87)%. Clearance was 544±24 (440–620), 894±164 (470–1190), and 1018±230 (710–2130) ml min−1, and elimination half-life was calculated as 41.6±2.3 (34–55), 59.6±9.1 (24–98), and 79.5±9.3 (50–121) min, respectively, for 30 g i.v., 6 g i.v., and 6 g p.o. of l-arginine. Blood pressure and total peripheral resistance were significantly decreased after intravenous infusion of 30 g l-arginine by 4.4±1.4% and 10.4±3.6%, respectively, but were not significantly changed after oral or intravenous administration of 6 g l-arginine. l-arginine (30 g) also significantly increased urinary nitrate and cyclic GMP excretion rates by 97±28 and 66±20%, respectively. After infusion of 6 g l-arginine, urinary nitrate excretion also significantly increased, (nitrate by 47±12% [P < 0.05], cyclic GMP by 67±47% [P = ns]), although to a lesser and more variable extent than after 30 g of l-arginine

Brain neuroprotection by scavenging blood glutamate.

PubMed

Zlotnik, Alexander; Gurevich, Boris; Tkachov, Sergei; Maoz, Ilana; Shapira, Yoram; Teichberg, Vivian I

2007-01-01

Excess glutamate in brain fluids characterizes acute brain insults such as traumatic brain injury and stroke. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As blood glutamate scavenging has been demonstrated to increase the efflux of excess glutamate from brain into blood, we tested the prediction that oxaloacetate-mediated blood glutamate scavenging causes neuroprotection in a pathological situation such as closed head injury (CHI), in which there is a well established deleterious increase of glutamate in brain fluids. We observed highly significant improvements of the neurological status of rats submitted to CHI following an intravenous treatment with 1 mmol oxaloacetate/100 g rat weight which decreases blood glutamate levels by 40%. No detectable therapeutic effect was obtained when rats were treated IV with 1 mmol oxaloacetate together with 1 mmol glutamate/100 g rat. The treatment with 0.005 mmol/100 g rat oxaloacetate was no more effective than saline but when it was combined with the intravenous administration of 0.14 nmol/100 g of recombinant glutamate-oxaloacetate transaminase, recovery was almost complete. Oxaloacetate provided neuroprotection when administered before CHI or at 60 min post CHI but not at 120 min post CHI. Since neurological recovery from CHI was highly correlated with the decrease of blood glutamate levels (r=0.89, P=0.001), we conclude that blood glutamate scavenging affords brain neuroprotection Blood glutamate scavenging may open now new therapeutic options.

Glutamic acid as anticancer agent: An overview

PubMed Central

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. 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. PMID:24227952

Glutamic acid as anticancer agent: An overview.

PubMed

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.

Combinatorial effects of arginine and fluoride on oral bacteria.

PubMed

Zheng, X; Cheng, X; Wang, L; Qiu, W; Wang, S; Zhou, Y; Li, M; Li, Y; Cheng, L; Li, J; Zhou, X; Xu, X

2015-02-01

Dental caries is closely associated with the microbial disequilibrium between acidogenic/aciduric pathogens and alkali-generating commensal residents within the dental plaque. Fluoride is a widely used anticaries agent, which promotes tooth hard-tissue remineralization and suppresses bacterial activities. Recent clinical trials have shown that oral hygiene products containing both fluoride and arginine possess a greater anticaries effect compared with those containing fluoride alone, indicating synergy between fluoride and arginine in caries management. Here, we hypothesize that arginine may augment the ecological benefit of fluoride by enriching alkali-generating bacteria in the plaque biofilm and thus synergizes with fluoride in controlling dental caries. Specifically, we assessed the combinatory effects of NaF/arginine on planktonic and biofilm cultures of Streptococcus mutans, Streptococcus sanguinis, and Porphyromonas gingivalis with checkerboard microdilution assays. The optimal NaF/arginine combinations were selected, and their combinatory effects on microbial composition were further examined in single-, dual-, and 3-species biofilm using bacterial species-specific fluorescence in situ hybridization and quantitative polymerase chain reaction. We found that arginine synergized with fluoride in suppressing acidogenic S. mutans in both planktonic and biofilm cultures. In addition, the NaF/arginine combination synergistically reduced S. mutans but enriched S. sanguinis within the multispecies biofilms. More importantly, the optimal combination of NaF/arginine maintained a "streptococcal pressure" against the potential growth of oral anaerobe P. gingivalis within the alkalized biofilm. Taken together, we conclude that the combinatory application of fluoride and arginine has a potential synergistic effect in maintaining a healthy oral microbial equilibrium and thus represents a promising ecological approach to caries management. © International & American

Combinatorial Effects of Arginine and Fluoride on Oral Bacteria

PubMed Central

Zheng, X.; Cheng, X.; Wang, L.; Qiu, W.; Wang, S.; Zhou, Y.; Li, M.; Li, Y.; Cheng, L.; Li, J.; Zhou, X.

2015-01-01

Dental caries is closely associated with the microbial disequilibrium between acidogenic/aciduric pathogens and alkali-generating commensal residents within the dental plaque. Fluoride is a widely used anticaries agent, which promotes tooth hard-tissue remineralization and suppresses bacterial activities. Recent clinical trials have shown that oral hygiene products containing both fluoride and arginine possess a greater anticaries effect compared with those containing fluoride alone, indicating synergy between fluoride and arginine in caries management. Here, we hypothesize that arginine may augment the ecological benefit of fluoride by enriching alkali-generating bacteria in the plaque biofilm and thus synergizes with fluoride in controlling dental caries. Specifically, we assessed the combinatory effects of NaF/arginine on planktonic and biofilm cultures of Streptococcus mutans, Streptococcus sanguinis, and Porphyromonas gingivalis with checkerboard microdilution assays. The optimal NaF/arginine combinations were selected, and their combinatory effects on microbial composition were further examined in single-, dual-, and 3-species biofilm using bacterial species–specific fluorescence in situ hybridization and quantitative polymerase chain reaction. We found that arginine synergized with fluoride in suppressing acidogenic S. mutans in both planktonic and biofilm cultures. In addition, the NaF/arginine combination synergistically reduced S. mutans but enriched S. sanguinis within the multispecies biofilms. More importantly, the optimal combination of NaF/arginine maintained a “streptococcal pressure” against the potential growth of oral anaerobe P. gingivalis within the alkalized biofilm. Taken together, we conclude that the combinatory application of fluoride and arginine has a potential synergistic effect in maintaining a healthy oral microbial equilibrium and thus represents a promising ecological approach to caries management. PMID:25477312

Glutamate cysteine ligase (GCL) in the freshwater bivalve Unio tumidus: impact of storage conditions and seasons on activity and identification of partial coding sequence of the catalytic subunit.

PubMed

Coffinet, Stéphanie; Cossu-Leguille, Carole; Rodius, François; Vasseur, Paule

2008-09-01

Glutamate cysteine ligase (GCL; EC 6.3.2.2) is the first enzyme involved in the synthesis of glutathione. A HPLC method with fluorimetric detection was used to measure GCL activity in the gills and the digestive gland of the freshwater bivalve, Unio tumidus. Storage conditions were optimized in order to prevent decrease of GCL activity and consisted in freezing the cytosolic fraction in the presence of protease (1 mM phenylmethylsulfonic fluoric acid) and gamma-glutamyltranspeptidase (1 mM L-serine borate mixture and 0.5 mM acivicin) inhibitors. Seasonal variations of activity in the digestive gland and to a lesser extent in the gills were found with activity increasing in spring compared to winter. No sex differences were revealed. The GCL coding sequence was identified using degenerated primers designed in the highly conserved regions of the catalytic subunit of GCL. The partial sequence identified encoded for 121 amino acids. The comparison of the identified partial coding sequence of U. tumidus with those available from vertebrates and invertebrates indicated that GCL sequence was highly conserved.

Lysine and glutamate transport in the erythrocytes of common brushtail possum, Tammar Wallaby and eastern grey, kangaroo.

PubMed

Ogawa, E; Kuchel, P W; Agar, N S

1998-04-01

It was recently coincidentally discovered, using 1H NMR spectroscopy, that the erythrocytes of two species of Australian marsupials, Tammar Wallaby (Macropus eugenii) and Bettong (Bettongia penicillata), contain relatively high concentrations of the essential amino acid lysine (Agar NS, Rae CD, Chapman BE, Kuchel PW. Comp Biochem Physiol 1991;99B:575-97). Hence, in the present work the rates of transport of lysine into the erythrocytes from the Common Brushtail Possum (Dactylopsilia trivirgata) and Eastern Grey Kangaroo (Macropus giganteus) (which both have low lysine concentrations), and Tammar Wallaby were studied, to explore the mechanistic basis of this finding. The concentration-dependence of the uptake was studied with lysine alone and in the presence of arginine, which may be a competitor of the transport in some species. In relation to GSH metabolism, glutamate uptake was determined in the presence and absence of Na+. The data was analysed to yield estimates of the maximal velocity (Vmax) and the Km in each of the species. Erythrocytes from Tammar Wallaby lacked saturable lysine transport in contrast to the other two species. The glutamate uptake was normal in all three animals for adequate GSH biosynthesis.

Regulation of accumulation of ammonium-inducible glutamate dehydrogenase catalytic activity and antigen during the cell cycle of fully induced, synchronous Chlorella sorokiniana cells.

PubMed

Yeung, A T; Bascomb, N F; Turner, K J; Schmidt, R R

1981-05-01

By use of a rocket immunoelectrophoresis-activity stain procedure, it was shown that catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) was accompanied by a coincident increase in enzyme antigen during the cell cycle of preinduced synchronous Chlorella sorokiniana cells growing in the continuous presence of ammonia. Between the fourth and fifth hours of the G-1 phase of the cell cycle, a three- to fourfold increase in linear accumulation of enzyme antigen was observed. Pulse-chase studies with [35S]sulfate, coupled with a specific indirect immunoadsorption procedure for enzyme antigen, showed that NADP-GDH antigen undergoes continuous degradation (i.e., a half-life of 88 to 110 min) during its linear pattern of accumulation during the cell cycle. The apparent half-life of the enzyme increased by approximately 23% of the 4.5-h positive rate change in antigen accumulation during the cell cycle. This increase in half-life is insufficient in itself to account for the large change in rate of NADP-GDH antigen accumulation. The data from immunoelectrophoresis, pulse-chase, and initial 35S incorporation rate experiments taken together support the inference that changes in the rate of NADP-GDH synthesis are primarily responsible for the accumulation patterns of NADP-GDH activity during the C. sorokiniana cell cycle.

Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant

PubMed Central

Hamasy, A; Wang, Q; Blomberg, K E M; Mohammad, D K; Yu, L; Vihinen, M; Berglöf, A; Smith, C I E

2017-01-01

Irreversible Bruton tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib have demonstrated remarkable clinical responses in multiple B-cell malignancies. Acquired resistance has been identified in a sub-population of patients in which mutations affecting BTK predominantly substitute cysteine 481 in the kinase domain for catalytically active serine, thereby ablating covalent binding of inhibitors. Activating substitutions in the BTK substrate phospholipase Cγ2 (PLCγ2) instead confers resistance independent of BTK. Herein, we generated all six possible amino acid substitutions due to single nucleotide alterations for the cysteine 481 codon, in addition to threonine, requiring two nucleotide substitutions, and performed functional analysis. Replacement by arginine, phenylalanine, tryptophan or tyrosine completely inactivated the catalytic activity, whereas substitution with glycine caused severe impairment. BTK with threonine replacement was catalytically active, similar to substitution with serine. We identify three potential ibrutinib resistance scenarios for cysteine 481 replacement: (1) Serine, being catalytically active and therefore predominating among patients. (2) Threonine, also being catalytically active, but predicted to be scarce, because two nucleotide changes are needed. (3) As BTK variants replaced with other residues are catalytically inactive, they presumably need compensatory mutations, therefore being very scarce. Glycine and tryptophan variants were not yet reported but likely also provide resistance. PMID:27282255

Arginine Improves pH Homeostasis via Metabolism and Microbiome Modulation.

PubMed

Agnello, M; Cen, L; Tran, N C; Shi, W; McLean, J S; He, X

2017-07-01

Dental caries can be described as a dysbiosis of the oral microbial community, in which acidogenic, aciduric, and acid-adapted bacterial species promote a pathogenic environment, leading to demineralization. Alkali generation by oral microbes, specifically via arginine catabolic pathways, is an essential factor in maintaining plaque pH homeostasis. There is evidence that the use of arginine in dentifrices helps protect against caries. The aim of the current study was to investigate the mechanistic and ecological effect of arginine treatment on the oral microbiome and its regulation of pH dynamics, using an in vitro multispecies oral biofilm model that was previously shown to be highly reflective of the in vivo oral microbiome. Pooled saliva from 6 healthy subjects was used to generate overnight biofilms, reflecting early stages of biofilm maturation. First, we investigated the uptake of arginine by the cells of the biofilm as well as the metabolites generated. We next explored the effect of arginine on pH dynamics by pretreating biofilms with 75 mM arginine, followed by the addition of sucrose (15 mM) after 0, 6, 20, or 48 h. pH was measured at each time point and biofilms were collected for 16S sequencing and targeted arginine quantification, and supernatants were prepared for metabolomic analysis. Treatment with only sucrose led to a sustained pH drop from 7 to 4.5, while biofilms treated with sucrose after 6, 20, or 48 h of preincubation with arginine exhibited a recovery to higher pH. Arginine was detected within the cells of the biofilms, indicating active uptake, and arginine catabolites citrulline, ornithine, and putrescine were detected in supernatants, indicating active metabolism. Sequencing analysis revealed a shift in the microbial community structure in arginine-treated biofilms as well as increased species diversity. Overall, we show that arginine improved pH homeostasis through a remodeling of the oral microbial community.

Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

PubMed Central

Krog, Anne; Heggeset, Tonje Marita Bjerkan; Ellingsen, Trond Erling

2013-01-01

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter−1 of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low Km value for ammonium (10 mM) and a high Km value for l-glutamate (250 mM), and the Vmax value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar Km values (1 to 1.4 mM) and Vmax values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism. PMID:23811508

Functional characterization of key enzymes involved in L-glutamate synthesis and degradation in the thermotolerant and methylotrophic bacterium Bacillus methanolicus.

PubMed

Krog, Anne; Heggeset, Tonje Marita Bjerkan; Ellingsen, Trond Erling; Brautaset, Trygve

2013-09-01

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter(-1) of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low Km value for ammonium (10 mM) and a high Km value for l-glutamate (250 mM), and the Vmax value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar Km values (1 to 1.4 mM) and Vmax values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.

The effect of arginine on oral biofilm communities.

PubMed

Nascimento, M M; Browngardt, C; Xiaohui, X; Klepac-Ceraj, V; Paster, B J; Burne, R A

2014-02-01

Alkali production by oral bacteria via the arginine deiminase system (ADS) increases the pH of oral biofilms and reduces the risk for development of carious lesions. This study tested the hypothesis that increased availability of arginine in the oral environment through an exogenous source enhances the ADS activity levels in saliva and dental plaque. Saliva and supra-gingival plaque samples were collected from 19 caries-free (CF) individuals (DMFT = 0) and 19 caries-active (CA) individuals (DMFT ≥ 2) before and after treatment, which comprised the use of a fluoride-free toothpaste containing 1.5% arginine, or a regular fluoride-containing toothpaste twice daily for 4 weeks. ADS activity was measured by quantification of ammonia produced from arginine by oral samples at baseline, after washout period, 4 weeks of treatment, and 2 weeks post-treatment. Higher ADS activity levels were observed in plaque samples from CF compared to those of CA individuals (P = 0.048) at baseline. The use of the arginine toothpaste significantly increased ADS activity in plaque of CA individuals (P = 0.026). The plaque microbial profiles of CA treated with the arginine toothpaste showed a shift in bacterial composition to a healthier community, more similar to that of CF individuals. Thus, an anti-caries effect may be expected from arginine-containing formulations due in large part to the enhancement of ADS activity levels and potential favorable modification to the composition of the oral microbiome. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Variability of arginine content and yield components in Valencia peanut germplasm.

PubMed

Aninbon, Chorkaew; Jogloy, Sanun; Vorasoot, Nimitr; Nuchadomrong, Suporn; Holbrook, C Corley; Kvien, Craig; Puppala, Naveen; Patanothai, Aran

2017-06-01

Peanut seeds are rich in arginine, an amino acid that has several positive effects on human health. Establishing the genetic variability of arginine content in peanut will be useful for breeding programs that have high arginine as one of their goals. The objective of this study was to evaluate the variation of arginine content, pods/plant, seeds/pod, seed weight, and yield in Valencia peanut germplasm. One hundred and thirty peanut genotypes were grown under field condition for two years. A randomized complete block design with three replications was used for this study. Arginine content was analyzed in peanut seeds at harvest using spectrophotometry. Yield and yield components were recorded for each genotype. Significant differences in arginine content and yield components were found in the tested Valencia peanut germplasm. Arginine content ranged from 8.68-23.35 μg/g seed. Kremena was the best overall genotype of high arginine content, number of pods/plant, 100 seed weight and pod yield.

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

PubMed

Liang, Bo; Zhang, Shu; Lang, Qiaolin; Song, Jianxia; Han, Lihui; Liu, Aihua

2015-07-16

A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP(+)-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(+) 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. Copyright © 2015 Elsevier B.V. All rights reserved.

Arginine, scurvy and Cartier's "tree of life"

PubMed Central

Durzan, Don J

2009-01-01

Several conifers have been considered as candidates for "Annedda", which was the source for a miraculous cure for scurvy in Jacques Cartier's critically ill crew in 1536. Vitamin C was responsible for the cure of scurvy and was obtained as an Iroquois decoction from the bark and leaves from this "tree of life", now commonly referred to as arborvitae. Based on seasonal and diurnal amino acid analyses of candidate "trees of life", high levels of arginine, proline, and guanidino compounds were also probably present in decoctions prepared in the severe winter. The semi-essential arginine, proline and all the essential amino acids, would have provided additional nutritional benefits for the rapid recovery from scurvy by vitamin C when food supply was limited. The value of arginine, especially in the recovery of the critically ill sailors, is postulated as a source of nitric oxide, and the arginine-derived guanidino compounds as controlling factors for the activities of different nitric oxide synthases. This review provides further insights into the use of the candidate "trees of life" by indigenous peoples in eastern Canada. It raises hypotheses on the nutritional and synergistic roles of arginine, its metabolites, and other biofactors complementing the role of vitamin C especially in treating Cartier's critically ill sailors. PMID:19187550

Safety of long-term dietary supplementation with L-arginine in pigs.

PubMed

Hu, Shengdi; Li, Xilong; Rezaei, Reza; Meininger, Cynthia J; McNeal, Catherine J; Wu, Guoyao

2015-05-01

This study was conducted with a swine model to determine the safety of long-term dietary supplementation with L-arginine-HCl or L-arginine free base. Beginning at 30 days of age, pigs were fed a corn- and soybean meal-based diet (31.5 g/kg body weight/day) supplemented with 0, 1.21, 1.81 or 2.42 % L-arginine-HCl (Experiment 1) or with 0, 1, 1.5 or 2 % L-arginine (Experiment 2). The supplemental doses of 0, 1, 1.5, and 2 % L-arginine provided pigs with 0, 315, 473, and 630 mg L-arginine/kg body weight/day, respectively, which were equivalent to 0, 286, 430, and 573 mg L-arginine/kg body weight/day, respectively, in humans. At 121 days of age (91 days after initiation of supplementation), blood samples were obtained from the jugular vein of pigs at 1 and 4 h after feeding for hematological and clinical chemistry tests. Dietary supplementation with L-arginine increased plasma concentrations of arginine, ornithine, proline, albumin and reticulocytes, while reducing plasma concentrations of ammonia, free fatty acids, triglyceride, cholesterol, and neutrophils. L-Arginine supplementation enhanced protein gain and reduced white-fat deposition in the body. Other variables in standard hematology and clinical chemistry tests, serum concentrations of insulin, growth hormone and insulin-like growth factor-I did not differ among all the groups of pigs. These results indicate that dietary supplementation with L-arginine (up to 630 mg/kg body weight/day) is safe in pigs for at least 91 days. Our findings help guide clinical studies to determine the safety of long-term oral administration of L-arginine to humans.

Inhibition of Protein Aggregation: Supramolecular Assemblies of Arginine Hold the Key

PubMed Central

Das, Utpal; Hariprasad, Gururao; Ethayathulla, Abdul S.; Manral, Pallavi; Das, Taposh K.; Pasha, Santosh; Mann, Anita; Ganguli, Munia; Verma, Amit K.; Bhat, Rajiv; Chandrayan, Sanjeev Kumar; Ahmed, Shubbir; Sharma, Sujata; Kaur, Punit; Singh, Tej P.; Srinivasan, Alagiri

2007-01-01

Background Aggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine. Methodology We have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimer's amyloid beta 1-42 (Aβ1-42) peptide is modified. Changes in the hydrodynamic volume of Aβ1-42 in the presence of arginine measured by size exclusion chromatography show that arginine binds to Aβ1-42. Arginine increases the solubility of Aβ1-42 peptide in aqueous medium. It decreases the aggregation of Aβ1-42 as observed by atomic force microscopy. Conclusions Based on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation. PMID:18000547

Glutamate and CO2 production from glutamine in incubated enterocytes of adult and very old rats.

PubMed

Meynial-Denis, Dominique; Bielicki, Guy; Beaufrère, Anne-Marie; Mignon, Michelle; Mirand, Philippe Patureau; Renou, Jean-Pierre

2013-04-01

Glutamine is the major fuel for enterocytes and promotes the growth of intestinal mucosa. Although oral glutamine exerts a positive effect on intestinal villus height in very old rats, how glutamine is used by enterocytes is unclear. Adult (8 months) and very old (27 months) female rats were exposed to intermittent glutamine supplementation for 50% of their age lifetime. Treated rats received glutamine added to their drinking water, and control rats received water alone. Jejunal epithelial cells (~300×10(6) cells) were incubated in oxygenated Krebs-Henseleit buffer for 30 min containing [1-(13)C] glutamine (~17 M) for analysis of glutamine metabolites by (13)C nuclear magnetic resonance ((13)C NMR). An aliquot fraction was incubated in the presence of [U-(14)C] glutamine to measure produced CO2. Glutamine pretreatment increased glutamate production and decreased CO2 production in very old rats. The ratio CO2/glutamate, which was very high in control very old rats, was similar at both ages after glutamine pretreatment, as if enterocytes from very old rats recovered the metabolic abilities of enterocytes from adult rats. Our results suggest that long-term treatment with glutamine started before advanced age (a) prevented the loss of rat body weight without limiting sarcopenia and (b) had a beneficial effect on enterocytes from very old rats probably by favoring the role of glutamate as a precursor for glutathione, arginine and proline biosynthesis, which was not detected in (13)C NMR spectra in our experimental conditions. Copyright © 2013 Elsevier Inc. All rights reserved.

Arginine dependence of tumor cells: targeting a chink in cancer’s armor

PubMed Central

Patil, MD; Bhaumik, J; Babykutty, S; Banerjee, UC; Fukumura, D

2017-01-01

Arginine, one among the 20 most common natural amino acids, has a pivotal role in cellular physiology as it is being involved in numerous cellular metabolic and signaling pathways. Dependence on arginine is diverse for both tumor and normal cells. Because of decreased expression of argininosuccinate synthetase and/or ornithine transcarbamoylase, several types of tumor are auxotrophic for arginine. Deprivation of arginine exploits a significant vulnerability of these tumor cells and leads to their rapid demise. Hence, enzyme-mediated arginine depletion is a potential strategy for the selective destruction of tumor cells. Arginase, arginine deiminase and arginine decarboxylase are potential enzymes that may be used for arginine deprivation therapy. These arginine catabolizing enzymes not only reduce tumor growth but also make them susceptible to concomitantly administered anti-cancer therapeutics. Most of these enzymes are currently under clinical investigations and if successful will potentially be advanced as anti-cancer modalities. PMID:27109103

Solubilization of aromatic and hydrophobic moieties by arginine in aqueous solutions

NASA Astrophysics Data System (ADS)

Li, Jianguo; Garg, Manju; Shah, Dhawal; Rajagopalan, Raj

2010-08-01

Experiments hold intriguing, circumstantial clues to the mechanisms behind arginine-mediated solubilization of small organic drugs and suppression of protein aggregation driven by hydrophobic or aromatic associations, but how exactly arginine's molecular structure and interactions contribute to its function remains unclear since attention has focused so far on the thermodynamics of the preferential exclusion or binding of arginine. Here, we examine, through molecular dynamics simulations, how arginine solubilizes nanoscale particles with hydrophobic surfaces or aromatic-ring-type surface interactions. We show that preferential, hydrophobic, and dispersion interactions of arginine's guanidinium group with the particles lead to a surfactant-like behavior of arginine around the particles and to a solvation layer with a protective polar mask creating a hydrophilic shell. Additionally, arginine-arginine association around the solvation layer further prevents aggregative contacts. The results shed some light on the mechanistic basis of arginine's function as a suppressant of protein aggregation, although the complex energy landscapes and kinetic pathways of aggregation are protein-dependent and pose formidable challenges to developing comprehensive mechanistic pictures. Our results suggest arginine's mode of interaction with hydrophobic patches and aromatic residues could reduce aggregation-prone intermediate states of proteins and shield protein-protein aggregative contacts. The approach used here offers a systematic way of exploring implications of other amino acid/excipient interactions by studying interactions of the excipient with particles grafted with amino acids.

The unique kinetic behavior of the very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum.

PubMed

Kawakami, Ryushi; Oyama, Masaki; Sakuraba, Haruhiko; Ohshima, Toshihisa

2010-01-01

The kinetics of a very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum showed positive cooperativity toward alpha-ketoglutarate and NADH, and the Michaelis-Menten type toward ammonium chloride in the absence of the catalytic activator, L-aspartate. An increase in the maximum activity accompanied the decrease in the S(0.5) values for alpha-ketoglutarate and NADH with the addition of L-aspartate, and the kinetic response for alpha-ketoglutarate changed completely to a typical Michaelis-Menten type in the presence of 10 mM L-aspartate.

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP.

PubMed

Yu, Qin; Hu, Liyan; Yao, Qing; Zhu, Yongqun; Dong, Na; Wang, Da-Cheng; Shao, Feng

2013-06-01

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

Plasma l-citrulline concentrations in l-arginine-supplemented healthy dogs.

PubMed

Flynn, K M; Kellihan, H B; Trepanier, L A

2017-08-01

To determine whether oral l-arginine increases plasma [l-citrulline] in dogs. Eleven healthy staff-owned dogs were used in this study. Dogs (n = 3) were given l-arginine (50mg/kg PO q8h) for 7 days, and plasma [l-arginine] and [l-citrulline] were analyzed by high performance liquid chromatography at baseline (BL), steady state trough, and 0.5, 1, 1.5, 2, 4, 6, and 8 h after final dosing on day 7. Eleven dogs were then treated with 100mg/kg l-arginine PO q8h for 7 days, and [l-arginine] and [l-citrulline] were measured at BL, steady state trough, and at peak 4 hrs after dosing (T4 hrs). - Plasma [l-arginine] and [l-citrulline] peaked at T4 hrs on the 50mg/kg dosage. Target outcome, modeled after human study results, of a doubling of [l-arginine] and a 25-30% increase in [l-citrulline] from BL were not reached. After the 100mg/kg dosage, plasma [l-arginine] increased from a BL median of 160.1 μM (range, 100.2-231.4 μM) to a peak of 417.4 μM (206.5-807.3 μM) at T4 hrs, and plasma [l-citrulline] increased from a BL median of 87.8 μM (59.1-117.1 μM) to peak of 102.2 μM (47.4-192.6 μM) at T4 hrs. Ten of eleven dogs showed a doubling of plasma [l-arginine] and 4/11 dogs achieved 25-30% or greater increases in plasma [l-citrulline]. No adverse effects on heart rate or blood pressure were noted. - Oral l-arginine dosage of 100mg/kg q8h doubles plasma [l-arginine] in healthy dogs, but conversion to l-citrulline is quite variable. Further evaluation of this dosage regimen in dogs with pulmonary hypertension is warranted. Copyright © 2017 Elsevier B.V. All rights reserved.

Intracellular L-arginine concentration does not determine NO production in endothelial cells: Implications on the 'L-arginine paradox'

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Soyoung; Mohan, Srinidi; Fung, Ho-Leung, E-mail: hlfung@buffalo.edu

2011-11-04

Highlights: Black-Right-Pointing-Pointer Our findings provide a possible solution to the 'L-arginine paradox'. Black-Right-Pointing-Pointer Extracellular L-arginine concentration is the major determinant of NO production. Black-Right-Pointing-Pointer Cellular L-arginine action is limited by cellular ARG transport, not the K{sub m} of NOS. Black-Right-Pointing-Pointer We explain how L-arginine supplementation can work to increase endothelial function. -- Abstract: We examined the relative contributory roles of extracellular vs. intracellular L-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of {sup 15}N{sub 4}-ARG, ARG, or L-arginine ethyl ester (ARG-EE) for 2 h.more » To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, {sup 15}N{sub 4}-ARG, dimethylarginines, and L-citrulline by an LC-MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by{sup 15}N-nitrite or estimated {sup 15}N{sub 3}-citrulline concentrations when {sup 15}N{sub 4}-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced {sup 15}N{sub 4}-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by {sup 15}N-nitrite, total nitrite and {sup 15}N{sub 3}-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside

Effects of three permeases on arginine utilization in Saccharomyces cerevisiae

PubMed Central

Zhang, Peng; Du, Guocheng; Zou, Huijun; Chen, Jian; Xie, Guangfa; Shi, Zhongping; Zhou, Jingwen

2016-01-01

Arginine plays an important role in cellular function and metabolism. Arginine uptake mainly occurs through three amino acid permeases, Alp1p, Gap1p and Can1p, which act as both transporters and receptors for amino acid utilization. In this study, seven mutants were constructed with different combinations of permease deficiencies that inhibit arginine utilization. Their effects on arginine metabolism were measured. The three amino acid permeases were also individually overexpressed in wild-type (WT), Δalp1Δgap1Δcan1 and Δnpr1 strains. The growth and arginine utilization of Δcan1, Δgap1Δcan1 and Δalp1Δgap1Δcan1 mutants were suppressed in YNB medium when arginine was the sole nitrogen source. Meanwhile, overexpression of Alp1p and Can1p enhanced growth and arginine utilization in WT, Δalp1Δgap1Δcan1 and Δnpr1. Besides, overexpression of Can1p caused a 26.7% increase in OD600 and 29.3% increase in arginine utilization compared to that of Alp1p in Δalp1Δgap1Δcan1. Transcription analysis showed that the effects of three amino acid permeases on the arginine utilization and the regulation of related genes, were tightly related to their individual characteristics. However, their overall effects were different for different combinations of mutants. The results presented here suggest some possible synergistic effects of different amino acid permeases on regulation of amino acid utilization and metabolism. PMID:26865023

In Vitro Functional Characterization of GET73 as Possible Negative Allosteric Modulator of Metabotropic Glutamate Receptor 5.

PubMed

Beggiato, Sarah; Borelli, Andrea C; Tomasini, Maria C; Castelli, M Paola; Pintori, Nicholas; Cacciaglia, Roberto; Loche, Antonella; Ferraro, Luca

2018-01-01

The present study was aimed to further characterize the pharmacological profile of N-[4-(trifluoromethyl) benzyl]-4-methoxybutyramide (GET73), a putative negative allosteric modulator (NAM) of metabotropic glutamate subtype 5 receptor (mGluR5) under development as a novel medication for the treatment of alcohol dependence. This aim has been accomplished by means of a series of in vitro functional assays. These assays include the measure of several down-stream signaling [intracellular Ca ++ levels, inositol phosphate (IP) formation and CREB phosphorylation (pCREB)] which are generally affected by mGluR5 ligands. In particular, GET73 (0.1 nM-10 μM) was explored for its ability to displace the concentration-response curve of some mGluR5 agonists/probes (glutamate, L-quisqualate, CHPG) in different native preparations. GET73 produced a rightward shift of concentration-response curves of glutamate- and CHPG-induced intracellular Ca ++ levels in primary cultures of rat cortical astrocytes. The compound also induced a rightward shift of concentration response curve of glutamate- and L-quisqualate-induced increase in IP turnover in rat hippocampus slices, along with a reduction of CHPG (10 mM)-induced increase in IP formation. Moreover, GET73 produced a rightward shift of concentration-response curve of glutamate-, CHPG- and L-quisqualate-induced pCREB levels in rat cerebral cortex neurons. Although the engagement of other targets cannot be definitively ruled out, these data support the view that GET73 acts as an mGluR5 NAM and support the significance of further investigating the possible mechanism of action of the compound.

Role of L-arginine in the pathogenesis and treatment of renal disease.

PubMed

Cherla, Gautam; Jaimes, Edgar A

2004-10-01

L-arginine is a semi essential amino acid and also a substrate for the synthesis of nitric oxide (NO), polyamines, and agmatine. These L-arginine metabolites may participate in the pathogenesis of renal disease and constitute the rationale for manipulating L-arginine metabolism as a strategy to ameliorate kidney disease. Modification of dietary L-arginine intake in experimental models of kidney diseases has been shown to have both beneficial as well as deleterious effects depending on the specific model studied. L-arginine supplementation in animal models of glomerulonephritis has been shown to be detrimental, probably by increasing the production of NO from increased local expression of inducible NO synthase (iNOS). L-arginine supplementation does not modify the course of renal disease in humans with chronic glomerular diseases. However, beneficial effects of L-arginine supplementation have been reported in several models of chronic kidney disease including renal ablation, ureteral obstruction, nephropathy secondary to diabetes, and salt-sensitive hypertension. L-arginine is reduced in preeclampsia and recent experimental studies indicate that L-arginine supplementation may be beneficial in attenuating the symptoms of preeclampsia. Administration of exogenous L-arginine has been shown to be protective in ischemic acute renal failure. In summary, the role of L-arginine in the pathogenesis and treatment of renal disease is not completely understood and remains to be established.

Arginine mimetic structures in biologically active antagonists and inhibitors.

PubMed

Masic, Lucija Peterlin

2006-01-01

Peptidomimetics have found wide application as bioavailable, biostable, and potent mimetics of naturally occurring biologically active peptides. L-Arginine is a guanidino group-containing basic amino acid, which is positively charged at neutral pH and is involved in many important physiological and pathophysiological processes. Many enzymes display a preference for the arginine residue that is found in many natural substrates and in synthetic inhibitors of many trypsin-like serine proteases, e.g. thrombin, factor Xa, factor VIIa, trypsin, and in integrin receptor antagonists, used to treat many blood-coagulation disorders. Nitric oxide (NO), which is produced by oxidation of L-arginine in an NADPH- and O(2)-dependent process catalyzed by isoforms of nitric oxide synthase (NOS), exhibits diverse roles in both normal and pathological physiologies and has been postulated to be a contributor to the etiology of various diseases. Development of NOS inhibitors as well as analogs and mimetics of the natural substrate L-arginine, is desirable for potential therapeutic use and for a better understanding of their conformation when bound in the arginine binding site. The guanidino residue of arginine in many substrates, inhibitors, and antagonists forms strong ionic interactions with the carboxylate of an aspartic acid moiety, which provides specificity for the basic amino acid residue in the active side. However, a highly basic guanidino moiety incorporated in enzyme inhibitors or receptor antagonists is often associated with low selectivity and poor bioavailability after peroral application. Thus, significant effort is focused on the design and preparation of arginine mimetics that can confer selective inhibition for specific trypsin-like serine proteases and NOS inhibitors as well as integrin receptor antagonists and possess reduced basicity for enhanced oral bioavailability. This review will describe the survey of arginine mimetics designed to mimic the function of the

The Structure and Function of a Microbial Allantoin Racemase Reveal the Origin and Conservation of a Catalytic Mechanism.

PubMed

Cendron, Laura; Ramazzina, Ileana; Puggioni, Vincenzo; Maccacaro, Eleonora; Liuzzi, Anastasia; Secchi, Andrea; Zanotti, Giuseppe; Percudani, Riccardo

2016-11-22

The S enantiomer of allantoin is an intermediate of purine degradation in several organisms and the final product of uricolysis in nonhominoid mammals. Bioinformatics indicated that proteins of the Asp/Glu racemase superfamily could be responsible for the allantoin racemase (AllR) activity originally described in Pseudomonas species. In these proteins, a cysteine of the catalytic dyad is substituted with glycine, yet the recombinant enzyme displayed racemization activity with a similar efficiency (k cat /K M ≈ 5 × 10 4 M -1 s -1 ) for the R and S enantiomers of allantoin. The protein crystal structure identified a glutamate residue located three residues downstream (E78) that can functionally replace the missing cysteine; the catalytic role of E78 was confirmed by site-directed mutagenesis. Allantoin can undergo racemization through formation of a bicyclic intermediate (faster) or proton exchange at the chiral center (slower). By monitoring the two alternative mechanisms by 13 C and 1 H nuclear magnetic resonance, we found that the velocity of the faster reaction is unaffected by the enzyme, whereas the velocity of the slower reaction is increased by 7 orders of magnitude. Protein phylogenies trace the origin of the racemization mechanism in enzymes acting on glutamate, a substrate for which proton exchange is the only viable reaction mechanism. This mechanism was inherited by allantoin racemase through divergent evolution and conserved in spite of the substitution of catalytic residues.

Arginine: Its pKa value revisited

PubMed Central

Fitch, Carolyn A; Platzer, Gerald; Okon, Mark; Garcia-Moreno E, Bertrand; McIntosh, Lawrence P

2015-01-01

Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pKa value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pKa value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pKa value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group. PMID:25808204

Arginine affects appetite via nitric oxide in ducks.

PubMed

Wang, C; Hou, S S; Huang, W; Xu, T S; Rong, G H; Xie, M

2014-08-01

The objective of the study was to investigate the mechanism by which arginine regulates feed intake in Pekin ducks. In experiment 1, one hundred forty-four 1-d-old male Pekin ducks were randomly allotted to 3 dietary treatments with 6 replicate pens of 8 birds per pen. Birds in each group were fed a corn-corn gluten meal diet containing 0.65, 0.95, and 1.45% arginine. Ducks fed the diet containing 0.65% arginine had lower feed intake and plasma nitric oxide level (P < 0.05) than the other 2 groups. In experiment 2, twenty 11-d-old ducks were allotted to 1 of 2 treatments. After 2 h fasting, birds in the 2 groups were intraperitoneally administrated saline and l-NG-nitro-arginine methyl ester HCl (L-NAME) for 3 d, respectively. Feed intake (P < 0.07) and plasma nitric oxide concentration (P < 0.05) 2 h postinjection in the L-NAME administered group were lower than those of the control group. In conclusion, the study implied that arginine modifies feeding behavior possibly through controlling endogenous synthesis of nitric oxide in Pekin ducks. © Poultry Science Association Inc.

Plasma membrane transporters for arginine.

PubMed

Closs, Ellen I; Simon, Alexandra; Vékony, Nicole; Rotmann, Alexander

2004-10-01

The supply of arginine may become rate limiting for enzymatic reactions that use this semiessential amino acid as a substrate (e.g., nitric oxide, agmatine, creatine, and urea synthesis), particularly under conditions of high demand such as growth, sepsis, or wound healing. In addition, arginine acts as a signaling molecule that regulates essential cellular functions such as protein synthesis, apoptosis, and growth. In the past decade, a number of carrier proteins for amino acids have been identified on the molecular level. They belong to different gene families, exhibit overlapping but distinctive substrate specificities, and can further be distinguished by their requirement for the cotransport or countertransport of inorganic ions. A number of these transporters function as exchangers rather than uniporters. Uptake of amino acids by these transporters therefore depends largely on the intracellular substrate composition. Hence, there is a complex crosstalk between transporters for cationic and neutral amino acids as well as for peptides. This article briefly reviews current knowledge regarding mammalian plasma membrane transporters that accept arginine as a substrate.

Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

PubMed Central

Gunawardana, Dilantha; Cheng, Heung-Chin; Gayler, Kenwyn R.

2008-01-01

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn2+- or Mg2+-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B. PMID:18025047

Glutamate synaptic inputs to ventral tegmental area neurons in the rat derive primarily from subcortical sources.

PubMed

Omelchenko, N; Sesack, S R

2007-05-25

Dopamine and GABA neurons in the ventral tegmental area project to the nucleus accumbens and prefrontal cortex and modulate locomotor and reward behaviors as well as cognitive and affective processes. Both midbrain cell types receive synapses from glutamate afferents that provide an essential control of behaviorally-linked activity patterns, although the sources of glutamate inputs have not yet been completely characterized. We used antibodies against the vesicular glutamate transporter subtypes 1 and 2 (VGlut1 and VGlut2) to investigate the morphology and synaptic organization of axons containing these proteins as putative markers of glutamate afferents from cortical versus subcortical sites, respectively, in rats. We also characterized the ventral tegmental area cell populations receiving VGlut1+ or VGlut2+ synapses according to their transmitter phenotype (dopamine or GABA) and major projection target (nucleus accumbens or prefrontal cortex). By light and electron microscopic examination, VGlut2+ as opposed to VGlut1+ axon terminals were more numerous, had a larger average size, synapsed more proximally, and were more likely to form convergent synapses onto the same target. Both axon types formed predominantly asymmetric synapses, although VGlut2+ terminals more often formed synapses with symmetric morphology. No absolute selectivity was observed for VGlut1+ or VGlut2+ axons to target any particular cell population. However, the synapses onto mesoaccumbens neurons more often involved VGlut2+ terminals, whereas mesoprefrontal neurons received relatively equal synaptic inputs from VGlut1+ and VGlut2+ profiles. The distinct morphological features of VGlut1 and VGlut2 positive axons suggest that glutamate inputs from presumed cortical and subcortical sources, respectively, differ in the nature and intensity of their physiological actions on midbrain neurons. More specifically, our findings imply that subcortical glutamate inputs to the ventral tegmental area

Dietary arginine and linear growth: the Copenhagen School Child Intervention Study.

PubMed

van Vught, Anneke J A H; Dagnelie, Pieter C; Arts, Ilja C W; Froberg, Karsten; Andersen, Lars B; El-Naaman, Bianca; Bugge, Anna; Nielsen, Birgit M; Heitman, Berit L

2013-03-28

The amino acid arginine is a well-known growth hormone (GH) stimulator and GH is an important modulator of linear growth. The aim of the present study was to investigate the effect of dietary arginine on growth velocity in children between 7 and 13 years of age. Data from the Copenhagen School Child Intervention Study during 2001-2 (baseline), and at 3-year and 7-year follow-up, were used. Arginine intake was estimated via a 7 d precoded food diary at baseline and 3-year follow-up. Data were analysed in a multilevel structure in which children were embedded within schools. Random intercept and slopes were defined to estimate the association between arginine intake and growth velocity, including the following covariates: sex; age; baseline height; energy intake; puberty stage at 7-year follow-up and intervention/control group. The association between arginine intake and growth velocity was significant for the third and fourth quintile of arginine intake (2.5-2.8 and 2.8-3.2 g/d, respectively) compared with the first quintile ( < 2.2 g/d) (P for trend = 0.04). Protein intake (excluding arginine) was significantly associated with growth velocity; however, the association was weaker than the association between arginine intake and growth velocity (P for trend = 0.14). The results of the present study suggest a dose-dependent physiological role of habitual protein intake, and specifically arginine intake, on linear growth in normally growing children. However, since the study was designed in healthy children, we cannot firmly conclude whether arginine supplementation represents a relevant clinical strategy. Further research is needed to investigate whether dietary arginine may represent a nutritional strategy potentially advantageous for the prevention and treatment of short stature.

Arg354 in the catalytic centre of bovine liver catalase is protected from methylglyoxal-mediated glycation.

PubMed

Scheckhuber, Christian Q

2015-12-30

In addition to controlled post-translational modifications proteins can be modified with highly reactive compounds. Usually this leads to a compromised functionality of the protein. Methylglyoxal is one of the most common agents that attack arginine residues. Methylglyoxal is also regarded as a pro-oxidant that affects cellular redox homeostasis by contributing to the formation of reactive oxygen species. Antioxidant enzymes like catalase are required to protect the cell from oxidative damage. These enzymes are also targets for methylglyoxal-mediated modification which could severely affect their catalytic activity in breaking down reactive oxygen species to less reactive or inert compounds. Here, bovine liver catalase was incubated with high levels of methylglyoxal to induce its glycation. This treatment did not lead to a pronounced reduction of enzymatic activity. Subsequently methylglyoxal-mediated arginine modifications (hydroimidazolone and dihydroxyimidazolidine) were quantitatively analysed by sensitive nano high performance liquid chromatography/electron spray ionisation/tandem mass spectrometry. Whereas several arginine residues displayed low to moderate levels of glycation (e.g., Arg93, Arg365, Arg444) Arg354 in the active centre of catalase was never found to be modified. Bovine liver catalase is able to tolerate very high levels of the modifying α-oxoaldehyde methylglyoxal so that its essential enzymatic function is not impaired.

Neurological effects of inorganic arsenic exposure: altered cysteine/glutamate transport, NMDA expression and spatial memory impairment.

PubMed

Ramos-Chávez, Lucio A; Rendón-López, Christian R R; Zepeda, Angélica; Silva-Adaya, Daniela; Del Razo, Luz M; Gonsebatt, María E

2015-01-01

Inorganic arsenic (iAs) is an important natural pollutant. Millions of individuals worldwide drink water with high levels of iAs. Chronic exposure to iAs has been associated with lower IQ and learning disabilities as well as memory impairment. iAs is methylated in tissues such as the brain generating mono and dimethylated species. iAs methylation requires cellular glutathione (GSH), which is the main antioxidant in the central nervous system (CNS). In humans, As species cross the placenta and are found in cord blood. A CD1 mouse model was used to investigate effects of gestational iAs exposure which can lead to oxidative damage, disrupted cysteine/glutamate transport and its putative impact in learning and memory. On postnatal days (PNDs) 1, 15 and 90, the expression of membrane transporters related to GSH synthesis and glutamate transport and toxicity, such as xCT, EAAC1, GLAST and GLT1, as well as LAT1, were analyzed. Also, the expression of the glutamate receptor N-methyl-D-aspartate (NMDAR) subunits NR2A and B as well as the presence of As species in cortex and hippocampus were investigated. On PND 90, an object location task was performed to associate exposure with memory impairment. Gestational exposure to iAs affected the expression of cysteine/glutamate transporters in cortex and hippocampus and induced a negative modulation of NMDAR NR2B subunit in the hippocampus. Behavioral tasks showed significant spatial memory impairment in males while the effect was marginal in females.

Neurological effects of inorganic arsenic exposure: altered cysteine/glutamate transport, NMDA expression and spatial memory impairment

PubMed Central

Ramos-Chávez, Lucio A.; Rendón-López, Christian R. R.; Zepeda, Angélica; Silva-Adaya, Daniela; Del Razo, Luz M.; Gonsebatt, María E.

2015-01-01

Inorganic arsenic (iAs) is an important natural pollutant. Millions of individuals worldwide drink water with high levels of iAs. Chronic exposure to iAs has been associated with lower IQ and learning disabilities as well as memory impairment. iAs is methylated in tissues such as the brain generating mono and dimethylated species. iAs methylation requires cellular glutathione (GSH), which is the main antioxidant in the central nervous system (CNS). In humans, As species cross the placenta and are found in cord blood. A CD1 mouse model was used to investigate effects of gestational iAs exposure which can lead to oxidative damage, disrupted cysteine/glutamate transport and its putative impact in learning and memory. On postnatal days (PNDs) 1, 15 and 90, the expression of membrane transporters related to GSH synthesis and glutamate transport and toxicity, such as xCT, EAAC1, GLAST and GLT1, as well as LAT1, were analyzed. Also, the expression of the glutamate receptor N-methyl-D-aspartate (NMDAR) subunits NR2A and B as well as the presence of As species in cortex and hippocampus were investigated. On PND 90, an object location task was performed to associate exposure with memory impairment. Gestational exposure to iAs affected the expression of cysteine/glutamate transporters in cortex and hippocampus and induced a negative modulation of NMDAR NR2B subunit in the hippocampus. Behavioral tasks showed significant spatial memory impairment in males while the effect was marginal in females. PMID:25709567

The influence of a novel pentadecapeptide, BPC 157, on N(G)-nitro-L-arginine methylester and L-arginine effects on stomach mucosa integrity and blood pressure.

PubMed

Sikirić, P; Seiwerth, S; Grabarević, Z; Rucman, R; Petek, M; Jagić, V; Turković, B; Rotkvić, I; Mise, S; Zoricić, I; Konjevoda, P; Perović, D; Jurina, L; Separović, J; Hanzevacki, M; Artuković, B; Bratulić, M; Tisljar, M; Gjurasin, M; Miklić, P; Stancić-Rokotov, D; Slobodnjak, Z; Jelovac, N; Marović, A

1997-07-30

The known effects of a novel stomach pentadecapeptide BPC157 (10 microg or 10 ng/kg), namely its salutary activity against ethanol (96%, i.g.)-induced gastric lesions (simultaneously applied i.p.) and in blood pressure maintenance (given i.v.), were investigated in rats challenged with a combination of N(G)-nitro-L-arginine methylester (L-NAME) (5 mg/kg i.v.), a competitive inhibitor of endothelium nitric oxide (NO)-generation and NO precursor, L-arginine (200 mg/kg i.v.) (D-arginine was ineffective). In the gastric lesions assay, NO agents were given 5 min before ethanol injury and BPC 157 medication. Given alone, BPC157 had an antiulcer effect, as did L-arginine, but L-NAME had no effect. L-NAME completely abolished the effect of L-arginine, whereas it only attenuated the effect of BPC 157. After application of the combination of L-NAME + L-arginine, the BPC157 effect was additionally impaired. In blood pressure studies, compared with L-arginine, pentadecapeptide BPC 157 (without effect on basal normal values) had both a mimicking effect (impaired L-NAME-blood pressure increase, when applied prophylactically and decreased already raised L-NAME values, given at the time of the maximal L-NAME-blood pressure increase (i.e., 10 min after L-NAME)) and preventive activity (L-arginine-induced moderate blood pressure decrease was prevented by BPC 157 pretreatment). When BPC 157 was given 10 min after L-NAME + L-arginine combination, which still led to a blood pressure increase, its previously clear effect (noted in L-NAME treated rats) disappeared. In vitro, in gastric mucosa from rat stomach tissue homogenates, BPC 157, given in the same dose (100 microM) as L-arginine, induced a comparable generation of NO. But, BPC 157 effect could not be inhibited by L-NAME, even when L-NAME was given in a tenfold (100 versus 1000 microM) higher dose than that needed for inhibition of the L-arginine effect. NO synthesis was blunted when the pentadecapeptide BPC 157 and L-arginine

Arginine depletion increases susceptibility to serious infections in preterm newborns

PubMed Central

Badurdeen, Shiraz; Mulongo, Musa; Berkley, James A.

2015-01-01

Preterm newborns are highly susceptible to bacterial infections. This susceptibility is regarded as being due to immaturity of multiple pathways of the immune system. However, it is unclear whether a mechanism that unifies these different, suppressed pathways exists. Here, we argue that the immune vulnerability of the preterm neonate is critically related to arginine depletion. Arginine, a “conditionally essential” amino acid, is depleted in acute catabolic states, including sepsis. Its metabolism is highly compartmentalized and regulated, including by arginase-mediated hydrolysis. Recent data suggest that arginase II-mediated arginine depletion is essential for the innate immune suppression that occurs in newborn models of bacterial challenge, impairing pathways critical for the immune response. Evidence that arginine depletion mediates protection from immune activation during first gut colonization suggests a regulatory role in controlling gut-derived pathogens. Clinical studies show that plasma arginine is depleted during sepsis. In keeping with animal studies, small clinical trials of L-arginine supplementation have shown benefit in reducing necrotizing enterocolitis in premature neonates. We propose a novel, broader hypothesis that arginine depletion during bacterial challenge is a key factor limiting the neonate's ability to mount an adequate immune response, contributing to the increased susceptibility to infections, particularly with respect to gut-derived sepsis. PMID:25360828

The Degradation of 14C-Glutamic Acid by L-Glutamic Acid Decarboxylase.

ERIC Educational Resources Information Center

Dougherty, Charles M; Dayan, Jean

1982-01-01

Describes procedures and semi-micro reaction apparatus (carbon dioxide trap) to demonstrate how a particular enzyme (L-Glutamic acid decarboxylase) may be used to determine the site or sites of labeling in its substrate (carbon-14 labeled glutamic acid). Includes calculations, solutions, and reagents used. (Author/SK)

Effect of starch ingestion on plasma glutamate concentrations in humans ingesting monosodium L-glutamate in soup.

PubMed

Stegink, L D; Filer, L J; Baker, G L

1985-02-01

Plasma glutamate concentrations in human subjects are markedly lower when monosodium L-glutamate is ingested in a water solution containing partially hydrolyzed starch than when ingested in water alone. This study was carried out to investigate whether starch ingested as crackers had a similar effect. Eight normal adult subjects (four male, four female) ingested three servings of a beef consommé providing 50 mg/kg body weight monosodium L-glutamate. One serving was consommé alone, the other two were accompanied by sufficient crackers to provide 0.25 or 0.5 g starch per kilogram body weight, respectively. Ingestion of consommé containing glutamate significantly increased the mean plasma glutamate concentration above baseline to a mean peak value 30 min later. The peak after consumption of 0.5 g starch per kilogram body weight, but not 0.25 g/kg body weight, was significantly lower than when consommé alone was ingested. These data indicate that simultaneous ingestion of metabolizable carbohydrate with glutamate has a marked effect on the plasma glutamate response and indicate that the threshold value for carbohydrate is greater than 0.25 g/kg body weight.

Arginine, citrulline and nitric oxide metabolism in sepsis

USDA-ARS?s Scientific Manuscript database

Arginine has vasodilatory effects, via its conversion by nitric oxide (NO) synthase into NO, and immunomodulatory actions that play important roles in sepsis. Protein breakdown affects arginine availability, and the release of asymmetric dimethylarginine, an inhibitor of NO synthase, may therefore a...

Ultrasonic assisted synthesis of adenosine triphosphate capped manganese-doped ZnS quantum dots for selective room temperature phosphorescence detection of arginine and methylated arginine in urine based on supramolecular Mg(2+)-adenosine triphosphate-arginine ternary system.

PubMed

Ren, Hu-Bo; Yan, Xiu-Ping

2012-08-15

An ultrasonic assisted approach was developed for rapid synthesis of highly water soluble phosphorescent adenosine triphosphate (ATP)-capped Mn-doped ZnS QDs. The prepared ATP-capped Mn-doped ZnS QDs allow selective phosphorescent detection of arginine and methylated arginine based on the specific recognition nature of supramolecular Mg(2+)-ATP-arginine ternary system in combination with the phosphorescence property of Mn-doped ZnS QDs. The developed QD based probe gives excellent selectivity and reproducibility (1.7% relative standard deviation for 11 replicate detections of 10 μM arginine) and low detection limit (3 s, 0.23 μM), and favors biological applications due to the effective elimination of interference from scattering light and autofluorescence. Copyright © 2012 Elsevier B.V. All rights reserved.

Metabotropic Glutamate Receptors in the Trafficking of Ionotropic Glutamate and GABAA Receptors at Central Synapses

PubMed Central

Xiao, Min-Yi; Gustafsson, Bengt; Niu, Yin-Ping

2006-01-01

The trafficking of ionotropic glutamate (AMPA, NMDA and kainate) and GABAA receptors in and out of, or laterally along, the postsynaptic membrane has recently emerged as an important mechanism in the regulation of synaptic function, both under physiological and pathological conditions, such as information processing, learning and memory formation, neuronal development, and neurodegenerative diseases. Non-ionotropic glutamate receptors, primarily group I metabotropic glutamate receptors (mGluRs), co-exist with the postsynaptic ionotropic glutamate and GABAA receptors. The ability of mGluRs to regulate postsynaptic phosphorylation and Ca2+ concentration, as well as their interactions with postsynaptic scaffolding/signaling proteins, makes them well suited to influence the trafficking of ionotropic glutamate and GABAA receptors. Recent studies have provided insights into how mGluRs may impose such an influence at central synapses, and thus how they may affect synaptic signaling and the maintenance of long-term synaptic plasticity. In this review we will discuss some of the recent progress in this area: i) long-term synaptic plasticity and the involvement of mGluRs; ii) ionotropic glutamate receptor trafficking and long-term synaptic plasticity; iii) the involvement of postsynaptic group I mGluRs in regulating ionotropic glutamate receptor trafficking; iv) involvement of postsynaptic group I mGluRs in regulating GABAA receptor trafficking; v) and the trafficking of postsynaptic group I mGluRs themselves. PMID:18615134

L-Arginine in the treatment of valproate overdose - five clinical cases.

PubMed

Schrettl, Verena; Felgenhauer, Norbert; Rabe, Christian; Fernando, Malkanthi; Eyer, Florian

2017-04-01

Valproic acid and its metabolites - particularly valproyl-CoA - are inhibitors of the enzyme N-acetylglutamate synthetase. The amino acid l-arginine can stimulate N-acetylglutamate synthetase activity and could be potentially used therapeutically to correct hyperammonemia caused by valproate therapy or overdose. Severely valproic-acid-poisoned patients are usually treated with l-carnitine or hemodialysis in order to decrease hyperammonemia. We herein report of five cases, in which l-arginine was administered. Observational study on five cases. Patients with hyperammonemia (i.e., ammonia 80 > μg/dL) and symptoms consistent with valproate overdose (i.e., drowsiness, coma) were selected for treatment with l-arginine. Data was collected retrospectively. l-Arginine decreased ammonia levels in a close temporal relation (case I ammonia in EDTA-plasma [μg/dL] decreased from 381 to 39; case II from 281 to 50; case III from 669 to 74; case IV from 447 to 56; case V from 202 to 60). In cases I and II, hemodialysis was performed and l-carnitine was given before the administration of l-arginine. In case III, hemodialysis was performed after the administration of l-arginine was already started. In cases IV and V, treatment with l-arginine was the sole measure to decrease ammonia levels in plasma. The results suggest that l-arginine may be beneficial in selected cases of valproate overdose complicated by hyperammonemia. l-Arginine could extend our conventional treatment options for valproic acid overdose.

Glutamate: Tastant and Neuromodulator in Taste Buds.

PubMed

Vandenbeuch, Aurelie; Kinnamon, Sue C

2016-07-01

In taste buds, glutamate plays a double role as a gustatory stimulus and neuromodulator. The detection of glutamate as a tastant involves several G protein-coupled receptors, including the heterodimer taste receptor type 1, member 1 and 3 as well as metabotropic glutamate receptors (mGluR1 and mGluR4). Both receptor types participate in the detection of glutamate as shown with knockout animals and selective antagonists. At the basal part of taste buds, ionotropic glutamate receptors [N-methyl-d-aspartate (NMDA) and non-NMDA] are expressed and participate in the modulation of the taste signal before its transmission to the brain. Evidence suggests that glutamate has an efferent function on taste cells and modulates the release of other neurotransmitters such as serotonin and ATP. This short article reviews the recent developments in the field with regard to glutamate receptors involved in both functions as well as the influence of glutamate on the taste signal. © 2016 American Society for Nutrition.

Receptor-mediated activation of nitric oxide synthesis by arginine in endothelial cells

PubMed Central

Joshi, Mahesh S.; Ferguson, T. Bruce; Johnson, Fruzsina K.; Johnson, Robert A.; Parthasarathy, Sampath; Lancaster, Jack R.

2007-01-01

Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and α-2 adrenoceptors (α-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and α-2 adrenoceptors, partly inhibited l-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of α-2 AR, at very low concentrations completely inhibited NO formation. Like l-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of α-2 AR was very potent in activating cellular NO, thus indicating a possible role for α-2 AR in l-arginine-mediated NO synthesis. d-arginine also activated NO production and could be inhibited by imidazoline and α-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated l-arginine-mediated NO synthesis, thus indicating mediation via G proteins. l-type Ca2+ channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the l-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of l-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development. PMID:17535904

Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum.

PubMed

Cao, Yan; Duan, Zuoying; Shi, Zhongping

2014-02-01

Biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic Corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with Tween 40 addition during fermentation. The transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (TP) of glutamate were investigated under the conditions of varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production.

Influence of L-arginine during bovine in vitro fertilization.

PubMed

Silva, Thiago Velasco Guimarães; da Silva, Bruno Baraúna; de Sá, André Luiz Alves; da Costa, Nathalia Nogueira; Sampaio, Rafael Vilar; Cordeiro, Marcela da Silva; Santana, Priscila Di Paula Bessa; Adona, Paulo Roberto; Santos, Simone do Socorro Damasceno; Miranda, Moysés dos Santos; Ohashi, Otávio Mitio

2014-12-01

The objective of this work was to evaluate the effect of using L-arginine during in vitro fertilization (IVF) on in vitro embryonic development using Bos taurus and Bos indicus semen. Effect of different concentrations (0, 1, 10 and 50 mM) of L-arginine, added to the IVF medium, was evaluated on the fertilization rate at 18 h post-fertilization (hpf), NO3(-)/NO2(-) production during IVF by the Griess colorimetric method (30 hpf), cleavage and blastocyst rates (on Day 2 and Day 7 of culture, respectively) and total blastocyst cell number (Day 7 of culture). The results reveal that the addition of 50 mM L-arginine to IVF medium, with either Bos taurus or Bos indicus spermatozoa, decreased the cleavage rate and blastocyst rate compared to the control group. Other concentrations did not affect embryo production. However, 1 mM L-arginine with Bos indicus semen increased the proportion of hatched blastocysts. These results indicate that high L-arginine concentrations may exhibit toxic effects on bovine gametes during in vitro fertilization.

Amine oxidation by d-arginine dehydrogenase in Pseudomonas aeruginosa.

PubMed

Ouedraogo, Daniel; Ball, Jacob; Iyer, Archana; Reis, Renata A G; Vodovoz, Maria; Gadda, Giovanni

2017-10-15

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) is a flavin-dependent oxidoreductase, which is part of a novel two-enzyme racemization system that functions to convert d-arginine to l-arginine. PaDADH contains a noncovalently linked FAD that shows the highest activity with d-arginine. The enzyme exhibits broad substrate specificity towards d-amino acids, particularly with cationic and hydrophobic d-amino acids. Biochemical studies have established the structure and the mechanistic properties of the enzyme. The enzyme is a true dehydrogenase because it displays no reactivity towards molecular oxygen. As established through solvent and multiple kinetic isotope studies, PaDADH catalyzes an asynchronous CH and NH bond cleavage via a hydride transfer mechanism. Steady-state kinetic studies with d-arginine and d-histidine are consistent with the enzyme following a ping-pong bi-bi mechanism. As shown by a combination of crystallography, kinetic and computational data, the shape and flexibility of loop L1 in the active site of PaDADH are important for substrate capture and broad substrate specificity. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of L-arginine on solubilization and purification of plant membrane proteins.

PubMed

Arakawa, Junji; Uegaki, Masamichi; Ishimizu, Takeshi

2011-11-01

Biochemical analysis of membrane proteins is problematic at the level of solubilization and/or purification because of their hydrophobic nature. Here, we developed methods for efficient solubilization and purification of membrane proteins using L-arginine. The addition of 100 mM of basic amino acids (L-arginine, L-lysine, and L-ornithine) to a detergent-containing solubilization buffer enhanced solubilization (by 2.6-4.3 fold) of a model membrane protein-polygalacturonic acid synthase. Of all the amino acids, arginine was the most effective additive for solubilization of this membrane protein. Arginine addition also resulted in the best solubilization of other plant membrane proteins. Next, we examined the effects of arginine on purification of a model membrane protein. In anion-exchange chromatography, the addition of arginine to the loading and elution buffers resulted in a greater recovery of a membrane protein. In ultrafiltration, the addition of arginine to a protein solution significantly improved the recovery of a membrane protein. These results were thought to be due to the properties of arginine that prevent aggregation of hydrophobic proteins. Taken together, the results of our study showed that arginine is useful for solubilization and purification of aggregate-prone membrane proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

l-Arginine modulates neonatal lymphocyte proliferation through an interleukin-2 independent pathway

PubMed Central

Yu, Hong-Ren; Kuo, Ho-Chang; Huang, Li-Tung; Chen, Chih-Cheng; Tain, You-Lin; Sheen, Jiunn-Ming; Tiao, Mao-Meng; Huang, Hsin-Chun; Yang, Kuender D; Ou, Chia-Yo; Hsu, Te-Yao

2014-01-01

In cases of arginine depletion, lymphocyte proliferation, cytokine production and CD3ζ chain expression are all diminished. In addition to myeloid suppressor cells, polymorphonuclear cells (PMN) also exert T-cell immune suppressive effects through arginase-induced l-arginine depletion, especially during pregnancy. In this study, we investigated how arginase/l-arginine modulates neonatal lymphocyte proliferation. Results showed that the neonatal plasma l-arginine level was lower than in adults (48·1 ± 11·3 versus 86·5 ± 14·6 μm; P = 0·003). Neonatal PMN had a greater abundance of arginase I protein than adult PMN. Both transcriptional regulation and post-transcriptional regulation were responsible for the higher arginase I expression of neonatal PMN. Exogenous l-arginine enhanced neonate lymphocyte proliferation but not that of adult cells. The RNA-binding protein HuR was important but was not the only modulation factor in l-arginine-regulated neonatal T-cell proliferation. l-Arginine-mediated neonatal lymphocyte proliferation could not be blocked by interleukin-2 receptor blocking antibodies. These results suggest that the altered arginase/l-arginine cascade may be one of the mechanisms that contribute to altered neonatal immune responses. Exogenous l-arginine could enhance neonate lymphocyte proliferation through an interleukin-2-independent pathway. PMID:24697328

Enzymatic production of α-ketoglutaric acid from l-glutamic acid via l-glutamate oxidase.

PubMed

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.

Comparative studies on glutamate decarboxylase and choline acetyltransferase activities in the vertebrate vestibule.

PubMed

López, I; Meza, G

1990-01-01

1. Vestibular putative neurotransmitters GABA and acetylcholine synthesizing enzymes were quantified in four vertebrate species to find a correlation between all-vertebrate vestibular hair cell II (HCII) and synaptic contacts and appearance of hair cell I (HCI) and related synapses in terrestrial species. 2. Glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT) values were: 3.76; 15.38; 21.68; 27.78 and 9.44; 450; 720; 970 n(pico)mol/mg protein/hr (min) in, respectively, frogs, guinea pigs, rats and chicks. 3. GAD and ChAT omnipresence may indicate constant GABAergic HCII and its cholinergic efferent synapses, their raised content, appearance of GABA-containing HCI and related cholinergic boutons in higher vertebrates.

Exploration of alternate catalytic mechanisms and optimization strategies for retroaldolase design.

PubMed

Bjelic, Sinisa; Kipnis, Yakov; Wang, Ling; Pianowski, Zbigniew; Vorobiev, Sergey; Su, Min; Seetharaman, Jayaraman; Xiao, Rong; Kornhaber, Gregory; Hunt, John F; Tong, Liang; Hilvert, Donald; Baker, David

2014-01-09

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches. © 2013.

Effects of arginine on heat-induced aggregation of concentrated protein solutions.

PubMed

Shah, Dhawal; Shaikh, Abdul Rajjak; Peng, Xinxia; Rajagopalan, Raj

2011-01-01

Arginine is one of the commonly used additives to enhance refolding yield of proteins, to suppress aggregation of proteins, and to increase solubility of proteins, and yet the molecular interactions that contribute to the role of arginine are unclear. Here, we present experiments, using bovine serum albumin (BSA), lysozyme (LYZ), and β-lactoglobulin (BLG) as model proteins, to show that arginine can enhance heat-induced aggregation of concentrated protein solutions, contrary to the conventional belief that arginine is a universal suppressor of aggregation. Results show that the enhancement in aggregation is caused only for BSA and BLG, but not for LYZ, indicating that arginine's preferential interactions with certain residues over others could determine the effect of the additive on aggregation. We use this previously unrecognized behavior of arginine, in combination with density functional theory calculations, to identify the molecular-level interactions of arginine with various residues that determine arginine's role as an enhancer or suppressor of aggregation of proteins. The experimental and computational results suggest that the guanidinium group of arginine promotes aggregation through the hydrogen-bond-based bridging interactions with the acidic residues of a protein, whereas the binding of the guanidinium group to aromatic residues (aggregation-prone) contributes to the stability and solubilization of the proteins. The approach, we describe here, can be used to select suitable additives to stabilize a protein solution at high concentrations based on an analysis of the amino acid content of the protein. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

Regenerating muscle with arginine methylation

PubMed Central

Blanc, Roméo S.; Richard, Stéphane

2017-01-01

ABSTRACT Protein arginine methyltransferase (PRMT) is a family of nine proteins catalyzing the methylation of arginine residues. They were recently shown to be essential for proper regeneration of skeletal muscles. However, the mechanisms triggering the methylation event, as well as how the methylated substrates regulate muscle stem cell function and fate decision remain to be determined. This point-of-view will discuss the recent findings on the specific role of PRMT1, CARM1/PRMT4, PRMT5, and PRMT7 in muscle stem cell fate guidance, and shed light on the future challenges which could help defining the therapeutic potential of PRMT inhibitors against muscular disorders and aging. PMID:28301308

Regenerating muscle with arginine methylation.

PubMed

Blanc, Roméo S; Richard, Stéphane

2017-05-27

Protein arginine methyltransferase (PRMT) is a family of nine proteins catalyzing the methylation of arginine residues. They were recently shown to be essential for proper regeneration of skeletal muscles. However, the mechanisms triggering the methylation event, as well as how the methylated substrates regulate muscle stem cell function and fate decision remain to be determined. This point-of-view will discuss the recent findings on the specific role of PRMT1, CARM1/PRMT4, PRMT5, and PRMT7 in muscle stem cell fate guidance, and shed light on the future challenges which could help defining the therapeutic potential of PRMT inhibitors against muscular disorders and aging.

Efficient reductive amination process for enantioselective synthesis of L-phosphinothricin applying engineered glutamate dehydrogenase.

PubMed

Yin, Xinjian; Wu, Jianping; Yang, Lirong

2018-05-01

The objective of this study was to identify and exploit a robust biocatalyst that can be applied in reductive amination for enantioselective synthesis of the competitive herbicide L-phosphinothricin. Applying a genome mining-based library construction strategy, eight NADPH-specific glutamate dehydrogenases (GluDHs) were identified for reductively aminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) to L-phosphinothricin. Among them, the glutamate dehydrogenase cloned from Pseudomonas putida (PpGluDH) exhibited relatively high catalytic activity and favorable soluble expression. This enzyme was purified to homogeneity for further characterization. The specific activity of PpGluDH was 296.1 U/g-protein, which is significantly higher than the reported value for a GluDH. To the best of our knowledge, there has not been any report on protein engineering of GluDH for PPO-oriented activity. Taking full advantage of the available information and the diverse characteristics of the enzymes in the enzyme library, PpGluDH was engineered by site-directed mutation based on multiple sequence alignment. The mutant I170M, which had 2.1-fold enhanced activity, was successfully produced. When the I170M mutant was applied in the batch production of L-phosphinothricin, it showed markedly improved catalytic efficiency compared with the wild type enzyme. The conversion reached 99% (0.1 M PPO) with an L-phosphinothricin productivity of 1.35 g/h·L, which far surpassed the previously reported level. These results show that PpGluDH I170M is a promising biocatalyst for highly enantioselective synthesis of L-phosphinothricin by reductive amination.

Ilex paraguariensis hydroalcoholic extract exerts antidepressant-like and neuroprotective effects: involvement of the NMDA receptor and the L-arginine-NO pathway.

PubMed

Ludka, Fabiana K; Tandler, Lori de Fátima; Kuminek, Gislaine; Olescowicz, Gislaine; Jacobsen, Jonatha; Molz, Simone

2016-06-01

Ilex paraguariensis St. Hilaire (Aquifoliaceae) is a typical plant from South America. Preclinical studies have reported the effect of I. paraguariensis-based preparations on different alterations in the brain. This study aimed to examine the antidepressant-like and neuroprotective effects of I. paraguariensis hydroalcoholic extract (IpHE). The role of the N-methyl-D-aspartate receptor and the L-arginine-nitric oxide pathway in the IpHE antidepressant-like effect was also evaluated. Using the tail suspension test, we showed that IpHE (0.1-10 mg/kg, orally) exerts an antidepressant-like effect similar to that of ketamine (1 mg/kg, intraperitoneally). The antidepressant-like effect depends on the N-methyl-D-aspartate receptor and L-arginine-nitric oxide pathway modulation as we observed a combinatory effect using subeffective doses of IpHE (0.01 mg/kg, orally) and ketamine (0.1 mg/kg, intraperitoneally) or MK-801 (0.001 mg/kg, intraperitoneally). Also, pretreatment of mice with L-arginine (750 mg/kg, intraperitoneally) abolished the antidepressant-like effect of IpHE. This effect coincides with the neuroprotective effect, given that glutamate toxicity (10 mmol/l) did not decrease cell viability in hippocampal or cortical slices from IpHE-treated mice. The chromatographic profile of IpHE showed the presence of the methylxanthines caffeine and theobromine. Administration of methylxanthines (2.7 µg/kg) in mice produced an antidepressant-like effect, but not neuroprotection. We suggest that methylxanthines are at least in part responsible for the antidepressant-like effect of IpHE; further studies are necessary to determine the biological compounds responsible for the neuroprotective effect.

Citrulline Supplementation Improves Organ Perfusion and Arginine Availability under Conditions with Enhanced Arginase Activity

PubMed Central

Wijnands, Karolina A.P.; Meesters, Dennis M.; van Barneveld, Kevin W.Y.; Visschers, Ruben G.J.; Briedé, Jacob J.; Vandendriessche, Benjamin; van Eijk, Hans M.H.; Bessems, Babs A.F.M.; van den Hoven, Nadine; von Wintersdorff, Christian J.H.; Brouckaert, Peter; Bouvy, Nicole D.; Lamers, Wouter H.; Cauwels, Anje; Poeze, Martijn

2015-01-01

Enhanced arginase-induced arginine consumption is believed to play a key role in the pathogenesis of sickle cell disease-induced end organ failure. Enhancement of arginine availability with l-arginine supplementation exhibited less consistent results; however, l-citrulline, the precursor of l-arginine, may be a promising alternative. In this study, we determined the effects of l-citrulline compared to l-arginine supplementation on arginine-nitric oxide (NO) metabolism, arginine availability and microcirculation in a murine model with acutely-enhanced arginase activity. The effects were measured in six groups of mice (n = 8 each) injected intraperitoneally with sterile saline or arginase (1000 IE/mouse) with or without being separately injected with l-citrulline or l-arginine 1 h prior to assessment of the microcirculation with side stream dark-field (SDF)-imaging or in vivo NO-production with electron spin resonance (ESR) spectroscopy. Arginase injection caused a decrease in plasma and tissue arginine concentrations. l-arginine and l-citrulline supplementation both enhanced plasma and tissue arginine concentrations in arginase-injected mice. However, only the citrulline supplementation increased NO production and improved microcirculatory flow in arginase-injected mice. In conclusion, the present study provides for the first time in vivo experimental evidence that l-citrulline, and not l-arginine supplementation, improves the end organ microcirculation during conditions with acute arginase-induced arginine deficiency by increasing the NO concentration in tissues. PMID:26132994

Acquired Amino Acid Deficiencies: A Focus on Arginine and Glutamine.

PubMed

Morris, Claudia R; Hamilton-Reeves, Jill; Martindale, Robert G; Sarav, Menaka; Ochoa Gautier, Juan B

2017-04-01

Nonessential amino acids are synthesized de novo and therefore not diet dependent. In contrast, essential amino acids must be obtained through nutrition since they cannot be synthesized internally. Several nonessential amino acids may become essential under conditions of stress and catabolic states when the capacity of endogenous amino acid synthesis is exceeded. Arginine and glutamine are 2 such conditionally essential amino acids and are the focus of this review. Low arginine bioavailability plays a pivotal role in the pathogenesis of a growing number of varied diseases, including sickle cell disease, thalassemia, malaria, acute asthma, cystic fibrosis, pulmonary hypertension, cardiovascular disease, certain cancers, and trauma, among others. Catabolism of arginine by arginase enzymes is the most common cause of an acquired arginine deficiency syndrome, frequently contributing to endothelial dysfunction and/or T-cell dysfunction, depending on the clinical scenario and disease state. Glutamine, an arginine precursor, is one of the most abundant amino acids in the body and, like arginine, becomes deficient in several conditions of stress, including critical illness, trauma, infection, cancer, and gastrointestinal disorders. At-risk populations are discussed together with therapeutic options that target these specific acquired amino acid deficiencies.

GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae.

PubMed Central

Avendaño, A; Deluna, A; Olivera, H; Valenzuela, L; Gonzalez, A

1997-01-01

It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP+-glutamate dehydrogenase (NADP+-GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP+-GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis. PMID:9287019

21 CFR 182.1045 - Glutamic acid.

Code of Federal Regulations, 2010 CFR

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...

21 CFR 182.1045 - Glutamic acid.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false 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...

Parenterally fed neonatal piglets have a low rate of endogenous arginine synthesis from circulating proline.

PubMed

Urschel, Kristine L; Evans, Amanda R; Wilkinson, Craig W; Pencharz, Paul B; Ball, Ronald O

2007-03-01

Parenterally fed neonatal piglets cannot synthesize sufficient arginine to maintain arginine status, presumably due to the intestinal atrophy that occurs with parenteral feeding. Parenteral feeding-induced atrophy can be reduced by the infusion of glucagon-like peptide 2 (GLP-2). GLP-2 infusion was hypothesized to increase the rate of endogenous arginine synthesis from proline, the major arginine precursor, in parenterally fed piglets receiving an arginine-deficient diet. Male piglets, fitted with jugular vein catheters for diet and isotope infusion, and femoral vein catheters for blood sampling (d 0), were allocated to a continuous infusion of either GLP-2 (n = 5; 10 nmol x kg(-1) x d(-1)) or saline (n = 5) for 7 d. Piglets received 2 d of a complete diet, followed by 5 d of an arginine-deficient [0.60 g x kg(-1) x d(-1)] diet. Piglets received primed, constant infusions of [guanido-(14)C]arginine to measure arginine flux (d 6) and [U-(14)C]proline (d 7) to measure proline conversion to arginine. Plasma arginine concentrations and arginine fluxes indicated a similar whole-body arginine status. Piglets receiving GLP-2 showed improvements in intestinal variables, including mucosal mass (P < 0.01) and villus height (P < 0.001), and a greater rate of arginine synthesis (micromol x kg(-1) x h(-1)) from proline (11.6 vs. 6.3) (P = 0.03). Mucosal mass (R(2) = 0.71; P = 0.002) and villus height were correlated (R(2) = 0.66; P = 0.004) with arginine synthesis. This study was the first to quantitate arginine synthesis in parenterally fed neonates and showed that although GLP-2 infusion increased arginine synthesis in a manner directly related to mucosal mass, this increased arginine synthesis was insufficient to improve whole-body arginine status in piglets receiving a low arginine diet.

Effect of ageing and ischemia on enzymatic activities linked to Krebs' cycle, electron transfer chain, glutamate and aminoacids metabolism of free and intrasynaptic mitochondria of cerebral cortex.

PubMed

Villa, Roberto Federico; Gorini, Antonella; Hoyer, Siegfried

2009-12-01

The effect of ageing and the relationships between the catalytic properties of enzymes linked to Krebs' cycle, electron transfer chain, glutamate and aminoacid metabolism of cerebral cortex, a functional area very sensitive to both age and ischemia, were studied on mitochondria of adult and aged rats, after complete ischemia of 15 minutes duration. The maximum rate (Vmax) of the following enzyme activities: citrate synthase, malate dehydrogenase, succinate dehydrogenase for Krebs' cycle; NADH-cytochrome c reductase as total (integrated activity of Complex I-III), rotenone sensitive (Complex I) and cytochrome oxidase (Complex IV) for electron transfer chain; glutamate dehydrogenase, glutamate-oxaloacetate-and glutamate-pyruvate transaminases for glutamate metabolism were assayed in non-synaptic, perikaryal mitochondria and in two populations of intra-synaptic mitochondria, i.e., the light and heavy mitochondrial fraction. The results indicate that in normal, steady-state cerebral cortex, the value of the same enzyme activity markedly differs according (a) to the different populations of mitochondria, i.e., non-synaptic or intra-synaptic light and heavy, (b) and respect to ageing. After 15 min of complete ischemia, the enzyme activities of mitochondria located near the nucleus (perikaryal mitochondria) and in synaptic structures (intra-synaptic mitochondria) of the cerebral tissue were substantially modified by ischemia. Non-synaptic mitochondria seem to be more affected by ischemia in adult and particularly in aged animals than the intra-synaptic light and heavy mitochondria. The observed modifications in enzyme activities reflect the metabolic state of the tissue at each specific experimental condition, as shown by comparative evaluation with respect to the content of energy-linked metabolites and substrates. The derangements in enzyme activities due to ischemia is greater in aged than in adult animals and especially the non-synaptic and the intra-synaptic light

Design and synthesis of inositolphosphoglycan putative insulin mediators.

PubMed

López-Prados, Javier; Cuevas, Félix; Reichardt, Niels-Christian; de Paz, José-Luis; Morales, Ezequiel Q; Martín-Lomas, Manuel

2005-03-07

The binding modes of a series of molecules, containing the glucosamine (1-->6) myo-inositol structural motif, into the ATP binding site of the catalytic subunit of cAMP-dependent protein kinase (PKA) have been analysed using molecular docking. These calculations predict that the presence of a phosphate group at the non-reducing end in pseudodisaccharide and pseudotrisaccharide structures properly orientate the molecule into the binding site and that pseudotrisaccharide structures present the best shape complementarity. Therefore, pseudodisaccharides and pseudotrisaccharides have been synthesised from common intermediates using effective synthetic strategies. On the basis of this synthetic chemistry, the feasibility of constructing small pseudotrisaccharide libraries on solid-phase using the same intermediates has been explored. The results from the biological evaluation of these molecules provide additional support to an insulin-mediated signalling system which involves the intermediacy of inositolphosphoglycans as putative insulin mediators.

Long-term enteral arginine supplementation in rats with intestinal ischemia and reperfusion.

PubMed

Lee, Chien-Hsing; Hsiao, Chien-Chou; Hung, Ching-Yi; Chang, Yu-Jun; Lo, Hui-Chen

2012-06-01

The effects of short-term enteral arginine supplementation on intestinal ischemia-reperfusion (IR) injury have been widely studied, especially the ischemic preconditioning supplementation. The aim of this study was to investigate the effects of long-term intra-duodenal supplementation of arginine on intestinal morphology, arginine-associated amino acid metabolism, and inflammatory responses in rats with intestinal IR. Male Wistar rats with or without three hours of ileal ischemia underwent duodenal cannulation for continuous infusion of formula with 2% arginine or commercial protein powder for 7 d. The serological examinations, plasma amino acid and cytokine profiles, and intestinal morphology were assessed. Intestinal IR injury had significant impacts on the decreases in circulating red blood cells, hemoglobin, ileum mass, and villus height and crypt depth of the distal jejunum. In addition, arginine supplementation decreased serum cholesterol and increased plasma arginine concentrations. In rats with intestinal IR injury, arginine supplementation significantly decreased serum nitric oxide, plasma citrulline and ornithine, and the mucosal protein content of the ileum. These results suggest that long-term intra-duodenal arginine administration may not have observable benefits on intestinal morphology or inflammatory response in rats with intestinal ischemia and reperfusion injury. Therefore, the necessity of long-term arginine supplementation for patients with intestinal ischemia and reperfusion injury remains questionable and requires further investigation. Copyright © 2012 Elsevier Inc. All rights reserved.

Restoration of impaired nitric oxide production in MELAS syndrome with citrulline and arginine supplementation

PubMed Central

El-Hattab, Ayman W.; Hsu, Jean W.; Emrick, Lisa T.; Wong, Lee-Jun C.; Craigen, William J.; Jahoor, Farook; Scaglia, Fernando

2014-01-01

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most common mitochondrial disorders. Although the pathogenesis of stroke-like episodes remains unclear, it has been suggested that mitochondrial proliferation may result in endothelial dysfunction and decreased nitric oxide (NO) availability leading to cerebral ischemic events. This study aimed to assess NO production in subjects with MELAS syndrome and the effect of the NO precursors arginine and citrulline. Using stable isotope infusion techniques, we assessed arginine, citrulline, and NO metabolism in control subjects and subjects with MELAS syndrome before and after arginine or citrulline supplementation. The results showed that subjects with MELAS had lower NO synthesis rate associated with reduced citrulline flux, de novo arginine synthesis rate, and plasma arginine and citrulline concentrations, and higher plasma asymmetric dimethylarginine (ADMA) concentration and arginine clearance. We conclude that the observed impaired NO production is due to multiple factors including elevated ADMA, higher arginine clearance, and, most importantly, decreased de novo arginine synthesis secondary to decreased citrulline availability. Arginine and, to a greater extent, citrulline supplementation increased the de novo arginine synthesis rate, the plasma concentrations and flux of arginine and citrulline, and NO production. De novo arginine synthesis increased markedly with citrulline supplementation, explaining the superior efficacy of citrulline in increasing NO production. The improvement in NO production with arginine or citrulline supplementation supports their use in MELAS and suggests that citrulline may have a better therapeutic effect than arginine. These findings can have a broader relevance for other disorders marked by perturbations in NO metabolism. PMID:22325939

Dietary arginine depletion reduces depressive-like responses in male, but not female, mice.

PubMed

Workman, Joanna L; Weber, Michael D; Nelson, Randy J

2011-09-30

Previous behavioral studies have manipulated nitric oxide (NO) production either by pharmacological inhibition of its synthetic enzyme, nitric oxide synthase (NOS), or by deletion of the genes that code for NOS. However manipulation of dietary intake of the NO precursor, L-arginine, has been understudied in regard to behavioral regulation. L-Arginine is a common amino acid present in many mammalian diets and is essential during development. In the brain L-arginine is converted into NO and citrulline by the enzyme, neuronal NOS (nNOS). In Experiment 1, paired mice were fed a diet comprised either of an L-arginine-depleted, L-arginine-supplemented, or standard level of L-arginine during pregnancy. Offspring were continuously fed the same diets and were tested in adulthood in elevated plus maze, forced swim, and resident-intruder aggression tests. L-Arginine depletion reduced depressive-like responses in male, but not female, mice and failed to significantly alter anxiety-like or aggressive behaviors. Arginine depletion throughout life reduced body mass overall and eliminated the sex difference in body mass. Additionally, arginine depletion significantly increased corticosterone concentrations, which negatively correlated with time spent floating. In Experiment 2, adult mice were fed arginine-defined diets two weeks prior to and during behavioral testing, and again tested in the aforementioned tests. Arginine depletion reduced depressive-like responses in the forced swim test, but did not alter behavior in the elevated plus maze or the resident intruder aggression test. Corticosterone concentrations were not altered by arginine diet manipulation in adulthood. These results indicate that arginine depletion throughout development, as well as during a discrete period during adulthood ameliorates depressive-like responses. These results may yield new insights into the etiology and sex differences of depression. Copyright © 2011 Elsevier B.V. All rights reserved.

Arginine Metabolism in Bacterial Pathogenesis and Cancer Therapy

PubMed Central

Xiong, Lifeng; Teng, Jade L. L.; Botelho, Michael G.; Lo, Regina C.; Lau, Susanna K. P.; Woo, Patrick C. Y.

2016-01-01

Antibacterial resistance to infectious diseases is a significant global concern for health care organizations; along with aging populations and increasing cancer rates, it represents a great burden for government healthcare systems. Therefore, the development of therapies against bacterial infection and cancer is an important strategy for healthcare research. Pathogenic bacteria and cancer have developed a broad range of sophisticated strategies to survive or propagate inside a host and cause infection or spread disease. Bacteria can employ their own metabolism pathways to obtain nutrients from the host cells in order to survive. Similarly, cancer cells can dysregulate normal human cell metabolic pathways so that they can grow and spread. One common feature of the adaption and disruption of metabolic pathways observed in bacterial and cancer cell growth is amino acid pathways; these have recently been targeted as a novel approach to manage bacterial infections and cancer therapy. In particular, arginine metabolism has been illustrated to be important not only for bacterial pathogenesis but also for cancer therapy. Therefore, greater insights into arginine metabolism of pathogenic bacteria and cancer cells would provide possible targets for controlling of bacterial infection and cancer treatment. This review will summarize the recent progress on the relationship of arginine metabolism with bacterial pathogenesis and cancer therapy, with a particular focus on arginase and arginine deiminase pathways of arginine catabolism. PMID:26978353

The utility of ionotropic glutamate receptor antagonists in the treatment of nociception induced by epidural glutamate infusion in rats.

PubMed

Osgood, Doreen B; Harrington, William F; Kenney, Elizabeth V; Harrington, J Frederick

2013-01-01

The authors have previously demonstrated that human herniated disc material contains high concentrations of free glutamate. In an experimental model, elevated epidural glutamate concentrations in the lumbar spine can cause a focal hyperesthetic state. Rats underwent epidural glutamate infusion in the lumbar spine by a miniosmotic pump over a 72-hour period. Some rats underwent coinfusion with glutamate and ionotropic glutamate antagonists. Nociception was assessed by von Frey fibers and by assessment of glutamate receptor expression in the corresponding dorsal horn of the spinal cord. The kainic acid antagonist, UBP 301, decreased epidural glutamate-based hyperesthesia in a dose dependent manner. Concordant with these findings, there was significant decrease in kainate receptor expression in the dorsal horn. The N-Methyl-4-isoxazoleproionic acid (NMDA) antagonist Norketamine also significantly diminished hyperesthesia and decreased receptor expression in the dorsal horn. Both UBP 301, the kainic acid receptor antagonist and Norketamine, an NMDA receptor antagonist, dampened epidural glutamate-based nociception. Focal epidural injections of Kainate or NMDA receptor antagonists could be effective treatments for disc herniation-based lumbar radiculopathy.

Metabotropic glutamate receptors in the trafficking of ionotropic glutamate and GABA(A) receptors at central synapses.

PubMed

Xiao, Min-Yi; Gustafsson, Bengt; Niu, Yin-Ping

2006-01-01

The trafficking of ionotropic glutamate (AMPA, NMDA and kainate) and GABA(A) receptors in and out of, or laterally along, the postsynaptic membrane has recently emerged as an important mechanism in the regulation of synaptic function, both under physiological and pathological conditions, such as information processing, learning and memory formation, neuronal development, and neurodegenerative diseases. Non-ionotropic glutamate receptors, primarily group I metabotropic glutamate receptors (mGluRs), co-exist with the postsynaptic ionotropic glutamate and GABA(A) receptors. The ability of mGluRs to regulate postsynaptic phosphorylation and Ca(2+) concentration, as well as their interactions with postsynaptic scaffolding/signaling proteins, makes them well suited to influence the trafficking of ionotropic glutamate and GABA(A) receptors. Recent studies have provided insights into how mGluRs may impose such an influence at central synapses, and thus how they may affect synaptic signaling and the maintenance of long-term synaptic plasticity. In this review we will discuss some of the recent progress in this area: i) long-term synaptic plasticity and the involvement of mGluRs; ii) ionotropic glutamate receptor trafficking and long-term synaptic plasticity; iii) the involvement of postsynaptic group I mGluRs in regulating ionotropic glutamate receptor trafficking; iv) involvement of postsynaptic group I mGluRs in regulating GABA(A) receptor trafficking; v) and the trafficking of postsynaptic group I mGluRs themselves.

Consequences of Phosphate-Arginine Complexes in Voltage-Gated Ion Channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Green, Michael E.

2008-11-01

There are two reasons for suspecting that phosphate complexes of arginine make it very difficult to derive gating charge in voltage gated potassium (and presumably sodium) channels from the motion of charged arginines. For one thing, the arginines should be complexed with phosphate, thereby neutralizing the charge, at least partially. Second, Li et al.(1) have shown that there is a large energy penalty for putting a charged arginine into a membrane. on channel gating current is generally attributed to S4 motion, in that the S4 segment of the voltage sensing domain (VSD) of these channels contains arginines, some of whichmore » are not (or at least not obviously) salt bridged, or otherwise charge compensated. There is, however, good reason to expect that there should be a complex of these arginines with phosphate, very probably from lipid headgroups. This has consequences for gating current; the complexed arginines, if they moved, would carry too much of the membrane along. This leads to the suggestion that an alternative to S4 physical motion, H+ transport, should be considered as a possible resolution of the apparent paradox. The consequences for a gating model that was proposed in our earlier work are discussed; there is one major difference in the model in the present form (a conformational change), but the proton cascade as gating current and the role of water in the closed state are reinforced.« less

Astroglial Glutamate Signaling and Uptake in the Hippocampus

PubMed Central

Rose, Christine R.; Felix, Lisa; Zeug, Andre; Dietrich, Dirk; Reiner, Andreas; Henneberger, Christian

2018-01-01

Astrocytes have long been regarded as essentially unexcitable cells that do not contribute to active signaling and information processing in the brain. Contrary to this classical view, it is now firmly established that astrocytes can specifically respond to glutamate released from neurons. Astrocyte glutamate signaling is initiated upon binding of glutamate to ionotropic and/or metabotropic receptors, which can result in calcium signaling, a major form of glial excitability. Release of so-called gliotransmitters like glutamate, ATP and D-serine from astrocytes in response to activation of glutamate receptors has been demonstrated to modulate various aspects of neuronal function in the hippocampus. In addition to receptors, glutamate binds to high-affinity, sodium-dependent transporters, which results in rapid buffering of synaptically-released glutamate, followed by its removal from the synaptic cleft through uptake into astrocytes. The degree to which astrocytes modulate and control extracellular glutamate levels through glutamate transporters depends on their expression levels and on the ionic driving forces that decrease with ongoing activity. Another major determinant of astrocytic control of glutamate levels could be the precise morphological arrangement of fine perisynaptic processes close to synapses, defining the diffusional distance for glutamate, and the spatial proximity of transporters in relation to the synaptic cleft. In this review, we will present an overview of the mechanisms and physiological role of glutamate-induced ion signaling in astrocytes in the hippocampus as mediated by receptors and transporters. Moreover, we will discuss the relevance of astroglial glutamate uptake for extracellular glutamate homeostasis, focusing on how activity-induced dynamic changes of perisynaptic processes could shape synaptic transmission at glutamatergic synapses. PMID:29386994

Plasma growth hormone (GH), insulin and amino acid responses to arginine with or without aspartic acid in pigs. Effect of the dose.

PubMed

Cochard, A; Guilhermet, R; Bonneau, M

1998-01-01

The aim of the present study was to examine, for the first time in pigs, the dose-dependent effect of arginine (ARG) on growth hormone (GH) and insulin release and the effect of the combined ARG and aspartic acid (ASP) treatment on GH and insulin release. ARG (0.5 or 1 g/kg body weight) with or without an equimolar supplement of ASP (0.38 or 0.76 g/kg, respectively) was administered in piglets via the duodenum. ARG increased plasma arginine, ornithine, urea, proline and branched chain amino acid concentrations. ASP increased specifically plasma aspartic acid, glutamic acid, alanine and citrulline concentrations. Plasma insulin increased with no apparent difference between treatments. Maximum GH level and the area under the GH curve (AUC) were increased in a dose-dependent manner in response to ARG treatment. GH response to the combined ARG and ASP treatment (ARGASP) was delayed compared to ARG alone and was not dose-dependent. AUC for GH after ARGASP treatments were intermediate between those observed after the two ARG doses. Our data suggest that high ASP doses transiently inhibit and delay ARG-induced GH release in pigs and that an equimolar supplement of ASP stimulates or inhibits ARG-induced GH release depending on the dose used.

Microbial production of poly-γ-glutamic acid.

PubMed

Sirisansaneeyakul, Sarote; Cao, Mingfeng; Kongklom, Nuttawut; Chuensangjun, Chaniga; Shi, Zhongping; Chisti, Yusuf

2017-09-05

Poly-γ-glutamic acid (γ-PGA) is a natural, biodegradable and water-soluble biopolymer of glutamic acid. This review is focused on nonrecombinant microbial production of γ-PGA via fermentation processes. In view of its commercial importance, the emphasis is on L-glutamic acid independent producers (i.e. microorganisms that do not require feeding with the relatively expensive amino acid L-glutamic acid to produce γ-PGA), but glutamic acid dependent production is discussed for comparison. Strategies for improving production, reducing costs and using renewable feedstocks are discussed.

Effect of methylation on the side-chain pKa value of arginine.

PubMed

Evich, Marina; Stroeva, Ekaterina; Zheng, Yujun George; Germann, Markus W

2016-02-01

Arginine methylation is important in biological systems. Recent studies link the deregulation of protein arginine methyltransferases with certain cancers. To assess the impact of methylation on interaction with other biomolecules, the pKa values of methylated arginine variants were determined using NMR data. The pKa values of monomethylated, symmetrically dimethylated, and asymmetrically dimethylated arginine are similar to the unmodified arginine (14.2 ± 0.4). Although the pKa value has not been significantly affected by methylation, consequences of methylation include changes in charge distribution and steric effects, suggesting alternative mechanisms for recognition. © 2015 The Protein Society.

The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

PubMed

Prokop, Pavol

2015-08-01

A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching.

Variability in arginine content and yield components in Valencia peanut germplasm

USDA-ARS?s Scientific Manuscript database

Peanut seeds are rich in arginine, an amino acid that has several positive effects on human health. Establishing the genetic variability of arginine content in peanut will be useful for breeding programs that have high arginine as one of their goals. The objective of this study was to evaluate the...

Regulation of Skeletal Muscle Plasticity by Protein Arginine Methyltransferases and Their Potential Roles in Neuromuscular Disorders

PubMed Central

Stouth, Derek W.; vanLieshout, Tiffany L.; Shen, Nicole Y.; Ljubicic, Vladimir

2017-01-01

Protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the methylation of arginine residues on target proteins, thereby mediating a diverse set of intracellular functions that are indispensable for survival. Indeed, full-body knockouts of specific PRMTs are lethal and PRMT dysregulation has been implicated in the most prevalent chronic disorders, such as cancers and cardiovascular disease (CVD). PRMTs are now emerging as important mediators of skeletal muscle phenotype and plasticity. Since their first description in muscle in 2002, a number of studies employing wide varieties of experimental models support the hypothesis that PRMTs regulate multiple aspects of skeletal muscle biology, including development and regeneration, glucose metabolism, as well as oxidative metabolism. Furthermore, investigations in non-muscle cell types strongly suggest that proteins, such as peroxisome proliferator-activated receptor-γ coactivator-1α, E2F transcription factor 1, receptor interacting protein 140, and the tumor suppressor protein p53, are putative downstream targets of PRMTs that regulate muscle phenotype determination and remodeling. Recent studies demonstrating that PRMT function is dysregulated in Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS) suggests that altering PRMT expression and/or activity may have therapeutic value for neuromuscular disorders (NMDs). This review summarizes our understanding of PRMT biology in skeletal muscle, and identifies uncharted areas that warrant further investigation in this rapidly expanding field of research. PMID:29163212

Guanidinium Group Remains Protonated in a Strongly Basic Arginine Solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Bo; Jacobs, Michael I.; Kostko, Oleg

Knowledge of the acid dissociation constant of an amino acid has very important ramifications in the biochemistry of proteins and lipid bilayers in aqueous environments because charge and proton transfer depend on its value. The acid dissociation constant for the guanidinium group in arginine has historically been posited as 12.5, but there is substantial variation in published values over the years. Recent experiments suggest that the dissociation constant for arginine is much higher than 12.5, which explains why the arginine guanidinium group retains its positive charge under all physiological conditions. Here, we use X-ray photoelectron spectroscopy to study unsupported, aqueousmore » arginine nanoparticles. By varying the pH of the constituent solution, we provide evidence that the guanidinium group is protonated even in a very basic solution. By analyzing the energy shifts in the C and N X-ray photoelectron spectra, we establish a molecular level picture of how charge and proton transport in aqueous solutions of arginine occur.« less

Guanidinium Group Remains Protonated in a Strongly Basic Arginine Solution

DOE PAGES

Xu, Bo; Jacobs, Michael I.; Kostko, Oleg; ...

2017-05-16

Knowledge of the acid dissociation constant of an amino acid has very important ramifications in the biochemistry of proteins and lipid bilayers in aqueous environments because charge and proton transfer depend on its value. The acid dissociation constant for the guanidinium group in arginine has historically been posited as 12.5, but there is substantial variation in published values over the years. Recent experiments suggest that the dissociation constant for arginine is much higher than 12.5, which explains why the arginine guanidinium group retains its positive charge under all physiological conditions. Here, we use X-ray photoelectron spectroscopy to study unsupported, aqueousmore » arginine nanoparticles. By varying the pH of the constituent solution, we provide evidence that the guanidinium group is protonated even in a very basic solution. By analyzing the energy shifts in the C and N X-ray photoelectron spectra, we establish a molecular level picture of how charge and proton transport in aqueous solutions of arginine occur.« less

Arginine kinase in Phytomonas, a trypanosomatid parasite of plants.

PubMed

Canepa, Gaspar E; Carrillo, Carolina; Miranda, Mariana R; Sayé, Melisa; Pereira, Claudio A

2011-09-01

Phytomonas are trypanosomatid plant parasites closely related to parasites that cause several human diseases. Little is known about the biology of these organisms including aspects of their metabolism. Arginine kinase (E.C. 2.7.3.3) is a phosphotransferase which catalyzes the interconversion between the phosphagen phosphoarginine and ATP. This enzyme is present in some invertebrates and is a homolog of another widely distributed phosphosphagen kinase, creatine kinase. In this work, a single canonical arginine kinase isoform was detected in Phytomonas Jma by enzymatic activity assays, PCR, and Western Blot. This arginine kinase is very similar to the canonical isoforms found in T. cruzi and T. brucei, presenting about 70% of amino acid sequence identity and a very similar molecular weight (40kDa). The Phytomonas phosphagen system seems to be very similar to T. cruzi, which has only one isoform, or T. brucei (three isoforms); establishing a difference with other trypanosomatids, such as Leishmania, which completely lacks phosphagen kinases, probably by the presence of the arginine-consuming enzyme, arginase. Finally, phylogenetic analysis suggests that Kinetoplastids' arginine kinase was acquired, during evolution, from the arthropod vectors by horizontal gene transfer. Copyright © 2011 Elsevier Inc. All rights reserved.

Demonstration of vesicular glutamate transporter-1 in corticotroph cells in the anterior pituitary of the rat.

PubMed

Kocsis, Zsuzsa S; Molnár, Csilla S; Watanabe, Masahiko; Daneels, Guy; Moechars, Dieder; Liposits, Zsolt; Hrabovszky, Erik

2010-02-01

Recent immunohistochemical studies of the rat adenohypophysis identified type-2 vesicular glutamate transporter (VGLUT2), a marker for glutamatergic neuronal phenotype, in high percentages of adenohypophysial gonadotrophs and thyrotrophs. The presence and molecular identity of amino acid neurotransmitters in the remaining hormone producing cell types are unknown. In the present study we addressed the putative synthesis of another glutamatergic marker, VGLUT1 by adenohypophysial cells. Immunohistochemical studies revealed VGLUT1 immunoreactivity in a small subset of polygonal medium-sized cells in the anterior lobe. Western blot analysis revealed a single major 60 kDa protein band in the adenohypophysis. Furthermore, the expression of VGLUT1 mRNA was confirmed by reverse transcription-polymerase chain reaction followed by sequence analysis of the amplicon. In contrast with rats which only showed VGLUT1 signal in the anterior lobe of the pituitary, mice contained high levels of VGLUT1 immunoreactivity in the intermediate, in addition to the anterior lobe. No signal was present in VGLUT1-knockout mice, providing evidence for specificity. In rats, results of colocalization studies with dual-immunofluorescent labeling provided evidence for VGLUT1 immunoreactivity in 45.9% of corticotrophs and 7.7% of luteinizing hormone beta-immunopositive gonadotrophs. Cells of the other peptide hormone phenotypes were devoid of VGLUT1 signal. A few cells in the adenohypophysis expressed both VGLUT1 and VGLUT2 immunoreactivities. The presence of the glutamate markers VGLUT1 and VGLUT2 in distinct populations of peptide hormone-secreting hypophysial cells highly indicates the involvement of endogenous glutamate release in autocrine/paracrine regulatory mechanisms. The biological function of adenohypophysial glutamate will require clarification. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

The arginine-ornithine antiporter ArcD contributes to biological fitness of Streptococcus suis.

PubMed

Fulde, Marcus; Willenborg, Joerg; Huber, Claudia; Hitzmann, Angela; Willms, Daniela; Seitz, Maren; Eisenreich, Wolfgang; Valentin-Weigand, Peter; Goethe, Ralph

2014-01-01

The arginine-ornithine antiporter (ArcD) is part of the Arginine Deiminase System (ADS), a catabolic, energy-providing pathway found in a variety of different bacterial species, including the porcine zoonotic pathogen Streptococcus suis. The ADS has recently been shown to play a role in the pathogenicity of S. suis, in particular in its survival in host cells. The contribution of arginine and arginine transport mediated by ArcD, however, has yet to be clarified. In the present study, we showed by experiments using [U-(13)C6]arginine as a tracer molecule that S. suis is auxotrophic for arginine and that bacterial growth depends on the uptake of extracellular arginine. To further study the role of ArcD in arginine metabolism, we generated an arcD-specific mutant strain and characterized its growth compared to the wild-type (WT) strain, a virulent serotype 2 strain. The mutant strain showed a markedly reduced growth in chemically defined media supplemented with arginine when compared to the WT strain, suggesting that ArcD promotes arginine uptake. To further evaluate the in vivo relevance of ArcD, we studied the intracellular bacterial survival of the arcD mutant strain in an epithelial cell culture infection model. The mutant strain was substantially attenuated, and its reduced intracellular survival rate correlated with a lower ability to neutralize the acidified environment. Based on these results, we propose that ArcD, by its function as an arginine-ornithine antiporter, is important for supplying arginine as substrate of the ADS and, thereby, contributes to biological fitness and virulence of S. suis in the host.

The arginine-ornithine antiporter ArcD contributes to biological fitness of Streptococcus suis

PubMed Central

Fulde, Marcus; Willenborg, Joerg; Huber, Claudia; Hitzmann, Angela; Willms, Daniela; Seitz, Maren; Eisenreich, Wolfgang; Valentin-Weigand, Peter; Goethe, Ralph

2014-01-01

The arginine-ornithine antiporter (ArcD) is part of the Arginine Deiminase System (ADS), a catabolic, energy-providing pathway found in a variety of different bacterial species, including the porcine zoonotic pathogen Streptococcus suis. The ADS has recently been shown to play a role in the pathogenicity of S. suis, in particular in its survival in host cells. The contribution of arginine and arginine transport mediated by ArcD, however, has yet to be clarified. In the present study, we showed by experiments using [U-13C6]arginine as a tracer molecule that S. suis is auxotrophic for arginine and that bacterial growth depends on the uptake of extracellular arginine. To further study the role of ArcD in arginine metabolism, we generated an arcD-specific mutant strain and characterized its growth compared to the wild-type (WT) strain, a virulent serotype 2 strain. The mutant strain showed a markedly reduced growth in chemically defined media supplemented with arginine when compared to the WT strain, suggesting that ArcD promotes arginine uptake. To further evaluate the in vivo relevance of ArcD, we studied the intracellular bacterial survival of the arcD mutant strain in an epithelial cell culture infection model. The mutant strain was substantially attenuated, and its reduced intracellular survival rate correlated with a lower ability to neutralize the acidified environment. Based on these results, we propose that ArcD, by its function as an arginine-ornithine antiporter, is important for supplying arginine as substrate of the ADS and, thereby, contributes to biological fitness and virulence of S. suis in the host. PMID:25161959

New Insights into the Methodology of L-Arginine-Induced Acute Pancreatitis

PubMed Central

Kui, Balázs; Balla, Zsolt; Vasas, Béla; Végh, Eszter T.; Pallagi, Petra; Kormányos, Eszter S.; Venglovecz, Viktória; Iványi, Béla; Takács, Tamás; Hegyi, Péter; Rakonczay, Zoltán

2015-01-01

Animal models are ideal to study the pathomechanism and therapy of acute pancreatitis (AP). The use of L-arginine-induced AP model is nowadays becoming increasingly popular in mice. However, carefully looking through the literature, marked differences in disease severity could be observed. In fact, while setting up the L-arginine (2×4 g/kg i.p.)-induced AP model in BALB/c mice, we found a relatively low rate (around 15%) of pancreatic necrosis, whereas others have detected much higher rates (up to 55%). We suspected that this may be due to differences between mouse strains. We administered various concentrations (5–30%, pH = 7.4) and doses (2×4, 3×3, or 4×2.5 g/kg) of L-arginine-HCl in BALB/c, FVB/n and C57BL/6 mice. The potential gender-specific effect of L-arginine was investigated in C57BL/6 mice. The fate of mice in response to the i.p. injections of L arginine followed one of three courses. Some mice (1) developed severe AP or (2) remained AP-free by 72 h, whereas others (3) had to be euthanized (to avoid their death, which was caused by the high dose of L-arginine and not AP) within 12 h., In FVB/n and C57BL/6 mice, the pancreatic necrosis rate (about 50%) was significantly higher than that observed in BALB/c mice using 2×4 g/kg 10% L–arginine, but euthanasia was necessary in a large proportion of animals, The i.p. injection of lower L-arginine concentrations (e.g. 5–8%) in case of the 2×4 g/kg dose, or other L-arginine doses (3×3 or 4×2.5 g/kg, 10%) were better for inducing AP. We could not detect any significant differences between the AP severity of male and female mice. Taken together, when setting up the L-arginine-induced AP model, there are several important factors that are worth consideration such as the dose and concentration of the administered L arginine-HCl solution and also the strain of mice. PMID:25688985

Arginine-Dependent Acid Resistance in Salmonella enterica Serovar Typhimurium

PubMed Central

Kieboom, Jasper; Abee, Tjakko

2006-01-01

Salmonella enterica serovar Typhimurium does not survive a pH 2.5 acid challenge under conditions similar to those used for Escherichia coli (J. W. Foster, Nat. Rev. Microbiol. 2:898-907, 2004). Here, we provide evidence that S. enterica serovar Typhimurium can display arginine-dependent acid resistance (AR) provided the cells are grown under anoxic conditions and not under the microaerobic conditions used for assessment of AR in E. coli. The role of the arginine decarboxylase pathway in Salmonella AR was shown by the loss of AR in mutants lacking adiA, which encodes arginine decarboxylase; adiC, which encodes the arginine-agmatine antiporter; or adiY, which encodes an AraC-like regulator. Transcription of adiA and adiC was found to be dependent on AdiY, anaerobiosis, and acidic pH. PMID:16855258

Stimulated Nitric Oxide Production and Arginine Deficiency in Cystic Fibrosis Children with Nutritional Failure

PubMed Central

Engelen, Mariëlle PKJ; Com, Gulnur; Luiking, Yvette C; Deutz, Nicolaas EP

2013-01-01

Objective Reduced nitric oxide (NO) concentrations are found in the airways of many patients with cystic fibrosis (CF) and are associated with increased airflow obstruction. We determined whether upregulated whole body de novo arginine synthesis and protein breakdown are present as a compensatory mechanism to meet the increased demand for arginine and nitric oxide production in pediatric patients with CF and nutritional failure. Study design In 16 children with CF, studied at the end of antibiotic treatment for a pulmonary exacerbation, and 17 healthy controls, whole body arginine, citrulline, and protein turnover were assessed by stable isotope methodology and de novo arginine synthesis, arginine clearance, NO synthesis, protein synthesis and breakdown, and net protein balance were calculated. The plasma isotopic enrichments and amino acid concentrations were measured by LC-MS/MS. Results Increased arginine clearance was found in patients with CF (p<0.001) whereas whole body NO production rate and plasma arginine levels were not different. Whole body arginine production (P<0.001), de novo arginine synthesis, and protein breakdown and synthesis (P<0.05) were increased in patients with CF, but net protein balance was comparable. Patients with CF with nutritional failure (n=7) had significantly higher NO production (P<0.05), de novo arginine synthesis, citrulline production (P<0.001), and plasma citrulline concentration (P<0.05) and lower plasma arginine concentration (P<0.05) than those without nutritional failure (n=9). Conclusions Nutritional failure in CF is associated with increased NO production. However, upregulation of de novo arginine synthesis and citrulline production was not sufficient to meet the increased arginine needs leading to arginine deficiency. PMID:23419590

Structure of the C-terminal domain of the arginine repressor protein from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cherney, Leonid T.; Cherney, Maia M.; Garen, Craig R.

2008-09-01

The structure of the core domain of the arginine repressor protein from M. tuberculosis has been determined with (1.85 Å resolution) and without (2.15 Å resolution) the arginine corepressor bound. Three additional arginine molecules have been found to bind to the core domain hexamer at high (0.2 M) arginine concentration. The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the l-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of argininemore » repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 Å resolution with bound arginine and at 2.15 Å resolution in the unliganded form. These structures show that six molecules of MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11° upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.« less

Restoration of impaired nitric oxide production in MELAS syndrome with citrulline and arginine supplementation.

PubMed

El-Hattab, Ayman W; Hsu, Jean W; Emrick, Lisa T; Wong, Lee-Jun C; Craigen, William J; Jahoor, Farook; Scaglia, Fernando

2012-04-01

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most common mitochondrial disorders. Although the pathogenesis of stroke-like episodes remains unclear, it has been suggested that mitochondrial proliferation may result in endothelial dysfunction and decreased nitric oxide (NO) availability leading to cerebral ischemic events. This study aimed to assess NO production in subjects with MELAS syndrome and the effect of the NO precursors arginine and citrulline. Using stable isotope infusion techniques, we assessed arginine, citrulline, and NO metabolism in control subjects and subjects with MELAS syndrome before and after arginine or citrulline supplementation. The results showed that subjects with MELAS had lower NO synthesis rate associated with reduced citrulline flux, de novo arginine synthesis rate, and plasma arginine and citrulline concentrations, and higher plasma asymmetric dimethylarginine (ADMA) concentration and arginine clearance. We conclude that the observed impaired NO production is due to multiple factors including elevated ADMA, higher arginine clearance, and, most importantly, decreased de novo arginine synthesis secondary to decreased citrulline availability. Arginine and, to a greater extent, citrulline supplementation increased the de novo arginine synthesis rate, the plasma concentrations and flux of arginine and citrulline, and NO production. De novo arginine synthesis increased markedly with citrulline supplementation, explaining the superior efficacy of citrulline in increasing NO production. The improvement in NO production with arginine or citrulline supplementation supports their use in MELAS and suggests that citrulline may have a better therapeutic effect than arginine. These findings can have a broader relevance for other disorders marked by perturbations in NO metabolism. Copyright © 2012 Elsevier Inc. All rights reserved.

Arginine Vasopressin selectively enhances recognition of sexual cues in male humans.

PubMed

Guastella, Adam J; Kenyon, Amanda R; Unkelbach, Christian; Alvares, Gail A; Hickie, Ian B

2011-02-01

Arginine Vasopressin modulates complex social and sexual behavior by enhancing social recognition, pair bonding, and aggression in non-human mammals. The influence of Arginine Vasopressin in human social and sexual behavior is, however, yet to be fully understood. We evaluated whether Arginine Vasopressin nasal spray facilitated recognition of positive and negative social and sexual stimuli over non-social stimuli. We used a recognition task that has already been shown to be sensitive to the influence of Oxytocin nasal spray (Unkelbach et al., 2008). In a double-blind, randomized, placebo-controlled, between-subjects design, 41 healthy male volunteers were administered Arginine Vasopressin (20 IU) or a placebo nasal spray after a 45 min wait period and then completed the recognition task. Results showed that the participants administered Arginine Vasopressin nasal spray were faster to detect sexual words over other types of words. This effect appeared for both positively and negatively valenced words. Results demonstrate for the first time that Arginine Vasopressin selectively enhances human cognition for sexual stimuli, regardless of valence. They further extend animal and human genetic studies linking Arginine Vasopressin to sexual behavior in males. Findings suggest an important cognitive mechanism that could enhance sexual behaviors in humans. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

Synthesis, characterization and properties of L-arginine-passivated silver nanocolloids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunatkari, A. L., E-mail: ashok.sunatkari@rediffmail.com; Talwatkar, S. S.; Tamgadge, Y. S.

2016-05-06

We investigate the effect of L-arginine-surface passivation on localised surface plasmon resonance (LSPR), size and stability of colloidal Silver Nanoparticles (AgNPs) synthesized by chemical reduction method. The surface Plasmon resonance absorption peak of AgNPs shows blue shift with the increase in L-arginine concentration. Transmission electron microscopy (TEM) analysis confirmed that the average size of AgNPs reduces from 10 nm to 6 nm as the concentration of L-Arginine increased from 1 to 5 mM. The X-ray diffraction study (XRD) confirmed the formation face-centred cubic (fcc) structured AgNPs. FT-IR studies revealed strong bonding between L-arginine functional groups and AgNPs.

The Nitric Oxide Synthase Inhibitor NG-Nitro-L-Arginine Methyl Ester Diminishes the Immunomodulatory Effects of Parental Arginine in Rats with Subacute Peritonitis

PubMed Central

Lo, Hui-Chen; Hung, Ching-Yi; Huang, Fu-Huan; Su, Tzu-Cheng; Lee, Chien-Hsing

2016-01-01

The combined treatment of parenteral arginine and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) have been shown to improve liver function and systemic inflammation in subacute peritonitic rats. Here, we investigated the effects of single and combined parenteral arginine and L-NAME treatments on leukocyte and splenocyte immunity. Male Wistar rats were subjected to cecal punctures and were intravenously given total parenteral nutrition solutions with or without arginine and/or L-NAME supplementations for 7 days. Non-surgical and sham-operated rats with no cecal puncture were given a chow diet and parenteral nutrition, respectively. Parenteral feeding elevated the white blood cell numbers and subacute peritonitis augmented the parenteral nutrition-induced alterations in the loss of body weight gain, splenomegaly, and splenocyte decreases. Parenteral arginine significantly increased the B-leukocyte level, decreased the natural killer T (NKT)-leukocyte and splenocyte levels, alleviated the loss in body weight gain and total and cytotoxic T-splenocyte levels, and attenuated the increases in plasma nitrate/nitrite and interferon-gamma production by T-splenocytes. L-NAME infusion significantly decreased NKT-leukocyte level, tumor-necrosis factor (TNF)-alpha production by T-splenocytes and macrophages, and interferon-gamma production by T-leukocytes, monocytes, and T-splenocytes, as well as increased interleukin-6 production by T-leukocytes and monocytes and nitrate/nitrite production by T-leukocytes. Combined treatment significantly decreased plasma nitrate/nitrite, the NKT-leukocyte level, and TNF-alpha production by T-splenocytes. Parenteral arginine may attenuate immune impairment and L-NAME infusion may augment leukocyte proinflammatory response, eliminate splenocyte proinflammatory and T-helper 1 responses, and diminish arginine-induced immunomodulation in combined treatment in subacute peritonitic rats. PMID:27007815

The influence of gastric pentadecapeptide BPC 157 on acute and chronic ethanol administration in mice. The effect of N(G)-nitro-L-arginine methyl ester and L-arginine.

PubMed

Boban-Blagaic, Alenka; Blagaic, Vladimir; Romic, Zeljko; Jelovac, Nikola; Dodig, Goran; Rucman, Rudolf; Petek, Marijan; Turkovic, Branko; Seiwerth, Sven; Sikiric, Predrag

2006-01-01

Alcohol disturbances, NO stimulation (by the NO-precursor L-arginine), and/or NO-synthesis blockade (by N(G)-nitro-L-arginine methyl ester, i.e. L-NAME) were challenged with stable gastric pentadecapeptide BPC 157, which inhibits both acute alcohol intoxication and alcohol withdrawal symptoms. Mice received intraperitoneally (i.p.) BPC 157 (10 microg/kg), L-NAME (10 mg/kg), and L-arginine (400 mg/kg), alone or in combination, 5 minutes before or after acute ethanol (4 g/kg i.p.) intoxication or after 0, 3, or 7 hours of withdrawal after drinking 20% alcohol for 13 days. BPC 157 rapidly opposes the strongest disturbance presentations in acute intoxication (sustained ethanol anesthesia, complete loss of righting reflex, no reaction to external stimuli, hypothermia, 25% mortality) and withdrawal (prominent seizures). NO-agents: Aggravation of acute alcohol intoxication and opposition to withdrawal are common, but the later intervals affected by L-arginine and the action throughout the experiment by L-NAME are distinctive. Given together, L-arginine and L-NAME counteract each other, while either the "L-NAME presentation" (acute intoxication) or the "L-arginine presentation" (withdrawal) predominates. BPC157+NO-agent: In acute intoxication (L-NAME predominating in NO-system functioning to aggravate intoxication), both BPC157+L-NAME and BPC157+L-arginine follow the presentation of L-NAME, but without worsened mortality. In withdrawal (L-arginine predominating in NO-system functioning to oppose disturbance symptoms), BPC157+L-NAME follows the presentation of L-NAME, while BPC 157+L-arginine imitates that of L-arginine. The relationships among pentadecapeptide BPC 157, the NO-system, acute alcohol intoxication, and opposed withdrawal may be important, presenting pentadecapeptide BPC 157 as a suitable alcohol antagonist.

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

PubMed

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

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

PubMed Central

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

2017-01-01

Objective 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. Methods 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. Results 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. Conclusion 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

Hepatic Adaptation Compensates Inactivation of Intestinal Arginine Biosynthesis in Suckling Mice

PubMed Central

Marion, Vincent; Sankaranarayanan, Selvakumari; de Theije, Chiel; van Dijk, Paul; Hakvoort, Theo B. M.; Lamers, Wouter H.; Köhler, Eleonore S.

2013-01-01

Suckling mammals, including mice, differ from adults in the abundant expression of enzymes that synthesize arginine from citrulline in their enterocytes. To investigate the importance of the small-intestinal arginine synthesis for whole-body arginine production in suckling mice, we floxed exon 13 of the argininosuccinate synthetase (Ass) gene, which codes for a key enzyme in arginine biosynthesis, and specifically and completely ablated Ass in enterocytes by crossing Ass fl and Villin-Cre mice. Unexpectedly, Ass fl/fl /VilCre tg/- mice showed no developmental impairments. Amino-acid fluxes across the intestine, liver, and kidneys were calculated after determining the blood flow in the portal vein, and hepatic and renal arteries (86%, 14%, and 33%, respectively, of the transhepatic blood flow in 14-day-old mice). Relative to control mice, citrulline production in the splanchnic region of Ass fl/fl /VilCre tg/- mice doubled, while arginine production was abolished. Furthermore, the net production of arginine and most other amino acids in the liver of suckling control mice declined to naught or even changed to consumption in Ass fl/fl /VilCre tg/- mice, and had, thus, become remarkably similar to that of post-weaning wild-type mice, which no longer express arginine-biosynthesizing enzymes in their small intestine. The adaptive changes in liver function were accompanied by an increased expression of genes involved in arginine metabolism (Asl, Got1, Gpt2, Glud1, Arg1, and Arg2) and transport (Slc25a13, Slc25a15, and Slc3a2), whereas no such changes were found in the intestine. Our findings suggest that the genetic premature deletion of arginine synthesis in enterocytes causes a premature induction of the post-weaning pattern of amino-acid metabolism in the liver. PMID:23785515

Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency

PubMed Central

Burrage, Lindsay C.; Sun, Qin; Elsea, Sarah H.; Jiang, Ming-Ming; Nagamani, Sandesh C.S.; Frankel, Arthur E.; Stone, Everett; Alters, Susan E.; Johnson, Dale E.; Rowlinson, Scott W.; Georgiou, George; Lee, Brendan H.

2015-01-01

Arginase deficiency is caused by deficiency of arginase 1 (ARG1), a urea cycle enzyme that converts arginine to ornithine. Clinical features of arginase deficiency include elevated plasma arginine levels, spastic diplegia, intellectual disability, seizures and growth deficiency. Unlike other urea cycle disorders, recurrent hyperammonemia is typically less severe in this disorder. Normalization of plasma arginine levels is the consensus treatment goal, because elevations of arginine and its metabolites are suspected to contribute to the neurologic features. Using data from patients enrolled in a natural history study conducted by the Urea Cycle Disorders Consortium, we found that 97% of plasma arginine levels in subjects with arginase deficiency were above the normal range despite conventional treatment. Recently, arginine-degrading enzymes have been used to deplete arginine as a therapeutic strategy in cancer. We tested whether one of these enzymes, a pegylated human recombinant arginase 1 (AEB1102), reduces plasma arginine in murine models of arginase deficiency. In neonatal and adult mice with arginase deficiency, AEB1102 reduced the plasma arginine after single and repeated doses. However, survival did not improve likely, because this pegylated enzyme does not enter hepatocytes and does not improve hyperammonemia that accounts for lethality. Although murine models required dosing every 48 h, studies in cynomolgus monkeys indicate that less frequent dosing may be possible in patients. Given that elevated plasma arginine rather than hyperammonemia is the major treatment challenge, we propose that AEB1102 may have therapeutic potential as an arginine-reducing agent in patients with arginase deficiency. PMID:26358771

Plasma L-arginine levels distinguish pulmonary arterial hypertension from left ventricular systolic dysfunction.

PubMed

Sandqvist, Anna; Schneede, Jörn; Kylhammar, David; Henrohn, Dan; Lundgren, Jakob; Hedeland, Mikael; Bondesson, Ulf; Rådegran, Göran; Wikström, Gerhard

2018-03-01

Pulmonary arterial hypertension (PAH) is a life-threatening condition, characterized by an imbalance of vasoactive substances and remodeling of pulmonary vasculature. Nitric oxide, formed from L-arginine, is essential for homeostasis and smooth muscle cell relaxation in PAH. Our aim was to compare plasma concentrations of L-arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) in PAH compared to left ventricular systolic dysfunction (LVSD) and healthy subjects. This was an observational, multicenter study comparing 21 patients with PAH to 14 patients with LVSD and 27 healthy subjects. Physical examinations were obtained and blood samples were collected. Plasma levels of ADMA, SDMA, L-arginine, L-ornithine, and L-citrulline were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma levels of ADMA and SDMA were higher, whereas L-arginine and L-arginine/ADMA ratio were lower in PAH patients compared to healthy subjects (p < 0.001). Patients with PAH also had lower levels of L-arginine than patients with LVSD (p < 0.05). L-Arginine correlated to 6 min walking distance (6MWD) (r s  = 0.58, p = 0.006) and L-arginine/ADMA correlated to WHO functional class (r s  = -0.46, p = 0.043) in PAH. In conclusion, L-arginine levels were significantly lower in treatment naïve PAH patients compared to patients with LVSD. Furthermore, L-arginine correlated with 6MWD in PAH. L-arginine may provide useful information in differentiating PAH from LVSD.

Enhancing poly-γ-glutamic acid production in Bacillus amyloliquefaciens by introducing the glutamate synthesis features from Corynebacterium glutamicum.

PubMed

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 �

Guanosine-5'-monophosphate induces cell death in rat hippocampal slices via ionotropic glutamate receptors activation and glutamate uptake inhibition.

PubMed

Molz, Simone; Dal-Cim, Tharine; Tasca, Carla I

2009-12-01

Guanine derivatives modulate the glutamatergic system through displacement of binding of glutamate to its receptors acting as antagonist of glutamate receptors in moderate to high micromolar concentrations. Guanosine-5'-monophosphate (GMP) is shown to be neuroprotective against glutamate- or oxygen/glucose deprivation-induced neurotoxicity and also against NMDA-induced apoptosis in hippocampal slices. However, in this study we are showing that high extracellular GMP concentrations (5mM) reduced cell viability in hippocampal brain slices. The toxic effect of GMP was not blocked by dipyridamole, a nucleoside transport inhibitor, nor mimicked by guanosine, suggesting an extracellular mode of action to GMP which does not involve its hydrolysis to guanosine. GMP-dependent cell damage was not blocked by P1 purinergic receptor antagonists, neither altered by adenosine A(1) or A(2A) receptor agonists. The blockage of the ionotropic glutamate receptors AMPA or NMDA, but not KA or metabotropic glutamate receptors, reversed the toxicity induced by GMP. GMP (5mM) induced a decrease in glutamate uptake into hippocampal slices, which was reversed by dl-TBOA. Therefore, GMP-induced hippocampal cell damage involves activation of ionotropic glutamate receptors and inhibition of glutamate transporters activity.

Glial glutamate transporters expression, glutamate uptake, and oxidative stress in an experimental rat model of intracerebral hemorrhage.

PubMed

Neves, J D; Vizuete, A F; Nicola, F; Da Ré, C; Rodrigues, A F; Schmitz, F; Mestriner, R G; Aristimunha, D; Wyse, A T S; Netto, C A

2018-06-01

Glial glutamate transporters (EAAT1 and EAAT2), glutamate uptake, and oxidative stress are important players in the pathogenesis of ischemic brain injury. However, the changes in EAAT1 and EAAT2 expression, glutamate uptake and the oxidative profile during intracerebral hemorrhage (ICH) development have not been described. The present study sought to investigate the changes of the above-mentioned variables, as well as the Na + /K + -ATP ase and glutamine synthetase activities (as important contributors of glutamate homeostasis) and the percentage of neuronal cells after 6 h, 24 h, 72 h and 7 days of ICH. An injection of 0.2U of bacterial collagenase in the ipsilateral striatum was used to induce ICH in male Wistar rats; naïve animals were used as controls. EAAT1 and EAAT2 expression and glutamate uptake in the ipsilateral striatum were assessed. Additionally, the percentage of MAP2+ cells, Na + /K + -ATP ase and GS activities, as well as the oxidative profile were analyzed. It is shown a decrease of EAAT1 expression and glutamate uptake 6 h post-ICH, whereas EAAT2 decreased 72 h after the event; conversely EAAT2 and glutamate uptake were increased after 7 days. The oxidative stress and endogenous defense system exhibited a remarkable response at 72 h of injury. ICH also increased Na + /K + -ATP ase activity and selectively decreased GS activity, variables known to be important contributors of glial glutamate transporters activities. Altogether, present findings indicate that ICH induces different temporal EAAT1 and EAAT2 responses, culminating with an imbalance of glutamate uptake capacity, increased oxidative stress and sustained neuronal loss. Copyright © 2018 Elsevier Ltd. All rights reserved.

Glutamate Dehydrogenase from Apodachlya (Oomycetes) 1

PubMed Central

Price, Jeffrey S.; Gleason, Frank H.

1972-01-01

A glutamate dehydrogenase specific for nicotinamide-adenine-dinucleotide has been purified 50-fold from Apodachlya brachynema (Leptomitales). Certain physical, chemical, and kinetic properties of this enzyme have been studied, particularly specificity for coenzymes and substrates. With glucose as the sole carbon source, the synthesis of glutamate dehydrogenase was repressed, whereas glutamate, proline, alanine, or ornithine plus aspartate as sole carbon sources induced synthesis of the enzyme. These data indicate that the function of this enzyme is primarily degradative, although there is no evidence for a nicotinamide-adenine-dinucleotide-phosphate-specific biosynthetic glutamate dehydrogenase in Apodachlya. PMID:16657902

Arginine Consumption by the Intestinal Parasite Giardia intestinalis Reduces Proliferation of Intestinal Epithelial Cells

PubMed Central

Stadelmann, Britta; Merino, María C.; Persson, Lo; Svärd, Staffan G.

2012-01-01

In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the host´s production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that

Biocatalytic synthesis, antimicrobial properties and toxicity studies of arginine derivative surfactants.

PubMed

Fait, M Elisa; Garrote, Graciela L; Clapés, Pere; Tanco, Sebastian; Lorenzo, Julia; Morcelle, Susana R

2015-07-01

Two novel arginine-based cationic surfactants were synthesized using as biocatalyst papain, an endopeptidase from Carica papaya latex, adsorbed onto polyamide. The classical substrate N (α)-benzoyl-arginine ethyl ester hydrochloride for the determination of cysteine and serine proteases activity was used as the arginine donor, whereas decyl- and dodecylamine were used as nucleophiles for the condensation reaction. Yields higher than 90 and 80 % were achieved for the synthesis of N (α)-benzoyl-arginine decyl amide (Bz-Arg-NHC10) and N (α)-benzoyl-arginine dodecyl amide (Bz-Arg-NHC12), respectively. The purification process was developed in order to make it more sustainable, by using water and ethanol as the main separation solvents in a single cationic exchange chromatographic separation step. Bz-Arg-NHC10 and Bz-Arg-NHC12 proved antimicrobial activity against both Gram-positive and Gram-negative bacteria, revealing their potential use as effective disinfectants as they reduced 99 % the initial bacterial population after only 1 h of contact. The cytotoxic effect towards different cell types of both arginine derivatives was also measured. Bz-Arg-NHCn demonstrated lower haemolytic activity and were less eye-irritating than the commercial cationic surfactant cetrimide. A similar trend could also be observed when cytotoxicity was tested on hepatocytes and fibroblast cell lines: both arginine derivatives were less toxic than cetrimide. All these properties would make the two novel arginine compounds a promising alternative to commercial cationic surfactants, especially for their use as additives in topical formulations.

NO-producing compounds transform neuron responses to glutamate.

PubMed

D'yakonova, T L

2000-01-01

We have previously shown that NO increases the excitatory effects of glutamate and blocks the desensitization of neurons to glutamate in the brain of the common snail. The aim of the present work was to identify the possible effect of NO on inhibitory responses to glutamate in the neurons of this mollusk. Electrophysiological investigations were performed on three identified neurons. The results showed that glutamate (0.05-0.1 mM) initially induced hyperpolarization and blocked the spike activity of these neurons. Simultaneous exposure to glutamate and the NO donor nitroprusside or preincubation with an NO donor had the effect that cells again responded to glutamate with depolarization and excitation. The transformed excitatory response lasted several minutes and could be reproduced even after 24 h of washing. The NO synthase blocker monomethylarginine blocked the excitatory response to glutamate. Another agonist of glutamate receptors, N-methyl-D-aspartate (NMDA, 0.1-1 mM), initially had excitatory effects on these neurons; this effect was significantly enhanced after transformation of the response to glutamate by NO donors. The results obtained here show that NO is involved in transforming the inhibitory responses to glutamate to excitatory responses, and that this effect may be mediated by NMDA-type receptors.

L-arginine: a new opportunity in the management of clinical derangements in dialysis patients.

PubMed

Bellinghieri, Guido; Santoro, Domenico; Mallamace, Agostino; Di Giorgio, Rosa Maria; De Luca, Grazia; Savica, Vincenzo

2006-07-01

L-Arginine is an essential amino acid for infants and growing children, as well as for pregnant women. This amino acid is a substrate for at least 5 enzymes identified in mammals, including arginase, arginine-glycine transaminase, kyotorphine synthase, nitric oxide synthase, and arginine decarboxylase. L-arginine is essential for the synthesis of creatine, urea, polyamines, nitric oxide, and agmatine. Arginine may be considered an essential amino acid in sepsis, and its supplementation could be beneficial in this clinical setting by improving microcirculation and protein anabolism. Rats receiving arginine-supplemented parenteral nutrition showed an increased ability to synthesize acute phase proteins when challenged with sepsis. Finally, L-arginine exerts antihypertensive and antiproliferative effects on vascular smooth muscles. It has been shown to reduce systemic blood pressure in some forms of experimental hypertension. Endothelial dysfunction and reduced nitric oxide bioactivity are associated with increased incidence of cardiovascular diseases. A beneficial effect of acute and chronic L-arginine supplementation on endothelial derived nitric oxide production and endothelial function has been shown. In end-stage renal disease patients, the rate of de novo arginine synthesis seemed to be preserved. Our preliminary data on a group of dialysis patients showed that predialysis arginine levels were stable in a normal range during the dialysis session and that hypertensive patients had lower arginine-citrulline ratio than normotensive patients.

L-arginine as dietary supplement for improving microvascular function.

PubMed

Melik, Ziva; Zaletel, Polona; Virtic, Tina; Cankar, Ksenija

2017-01-01

Reduced availability of nitric oxide leads to dysfunction of endothelium which plays an important role in the development of cardiovascular diseases. The aim of the present study was to determine whether the dietary supplement L-arginine improves the endothelial function of microvessels by increasing nitric oxide production. We undertook experiments on 51 healthy male volunteers, divided into 4 groups based on their age and physical activity since regular physical activity itself increases endothelium-dependent vasodilation. The skin laser Doppler flux was measured in the microvessels before and after the ingestion of L-arginine (0.9 g). The endothelium-dependent vasodilation was assessed by acetylcholine iontophoresis and the endothelium-independent vasodilation by sodium nitroprusside iontophoresis. In addition, we measured endothelium-dependent and endothelium-independent vasodilation in 81 healthy subjects divided into four age groups. After the ingestion of L-arginine, the endothelium-dependent vasodilation in the young trained subjects increased (paired t-test, p < 0.05), while in the other groups it remained the same. There were no differences in the endothelium-independent vasodilation after ingestion of L-arginine. With aging endothelium-independent vasodilation decreased while endothelium-dependent vasodilation remained mainly unchanged. Obtained results demonstrated that a single dose of L-arginine influences endothelium-dependent vasodilation predominantly in young, trained individuals.

Arginine Methylation: The Coming of Age.

PubMed

Blanc, Roméo S; Richard, Stéphane

2017-01-05

Arginine methylation is a common post-translational modification functioning as an epigenetic regulator of transcription and playing key roles in pre-mRNA splicing, DNA damage signaling, mRNA translation, cell signaling, and cell fate decision. Recently, a wealth of studies using transgenic mouse models and selective PRMT inhibitors helped define physiological roles for protein arginine methyltransferases (PRMTs) linking them to diseases such as cancer and metabolic, neurodegenerative, and muscular disorders. This review describes the recent molecular advances that have been uncovered in normal and diseased mammalian cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Does L-arginine induce intestinal adaptation by epithelial growth factor?

PubMed

Camli, Alparslan; Barlas, Meral; Yagmurlu, Aydin

2005-01-01

To evaluate whether L-Arginine has an effect on endogenous epidermal growth factor secretion and intestinal adaptation in massive small bowel resection an experimental study was performed. Fourteen albino Wistar rats weighing 250-300 g were used for the study. After performing 50% small bowel resection and anastomosis the rats were randomly divided into two groups. The first group received 500 mg/kg/day of L-Arginine intraperitoneally for 14 days just after the surgical procedure. The control group received isotonic saline instead. Body weight measurement was preformed daily. At the end of the second postoperative week all rats underwent relaparotomy. Small bowel was resected for histopathological examination. Levels of epidermal growth factor were measured by enzyme-linked immunosorbent assay in serum, saliva, and urine at the end of second postoperative week in both groups. The weight gain was higher in the L-Arginine treated group (P < 0.05). Serum, saliva and urinary epidermal growth factor levels were significantly higher at the end of the second week compared to the control group (P < 0.05). The villus height was higher on histopathological examination in L-Arginine treated group compared to the control group (P < 0.05). L-Arginine resulted in a better intestinal adaptation after massive bowel resection. The high levels of epidermal growth factor in body fluids of L-Arginine treated rats could be the explanation for this effect.

Direct stimulation of pituitary prolactin release by glutamate.

PubMed

Login, I S

1990-01-01

The ability of glutamate and other excitatory amino acids to stimulate prolactin secretion when administered to adult animals is hypothesized to depend on a central site of action in the brain, but there are no data to support this position. An alternative hypothesis was tested that glutamate would stimulate prolactin release when applied directly to primary cultures of dispersed adult female rat anterior pituitary cells studied in a perifusion protocol. Glutamate increased the rate of prolactin release within two minutes in a self-limited manner. Glutamate-stimulated prolactin release was augmented about 4-fold by elimination of magnesium from the perfusate and was associated with stimulation of pituitary calcium flux. Ketamine and MK-801 both reduced the basal rate of prolactin release and abolished the effects of glutamate. Pituitary cells of 10-day-old rats responded similarly to glutamate. Exposure to glutamate did not influence subsequent responses to physiological hypothalamic secretagogues, thus the likelihood of toxicity was minimized. These results suggest that the N-methyl-D-aspartate (NMDA) subclass of the glutamate receptor complex is involved. Prolactin secretion may be regulated physiologically through a functional glutamate receptor on pituitary cells.

Stimulated nitric oxide production and arginine deficiency in children with cystic fibrosis with nutritional failure.

PubMed

Engelen, Mariëlle P K J; Com, Gulnur; Luiking, Yvette C; Deutz, Nicolaas E P

2013-08-01

To determine whether upregulated whole body de novo arginine synthesis and protein breakdown are present as a compensatory mechanism to meet the increased demand for arginine and nitric oxide (NO) production in pediatric patients with cystic fibrosis (CF) and nutritional failure. In 16 children with CF, studied at the end of antibiotic treatment for a pulmonary exacerbation, and 17 healthy controls, whole body arginine, citrulline (Cit), and protein turnover were assessed by stable isotope methodology and de novo arginine synthesis, arginine clearance, NO synthesis, protein synthesis and breakdown, and net protein balance were calculated. The plasma isotopic enrichments and amino acid concentrations were measured by liquid chromatography-tandem mass spectrometry. Increased arginine clearance was found in patients with CF (P < .001), whereas whole body NO production rate and plasma arginine levels were not different. Whole body arginine production (P < .001), de novo arginine synthesis, and protein breakdown and synthesis (P < .05) were increased in patients with CF, but net protein balance was comparable. Patients with CF with nutritional failure (n = 7) had significantly higher NO production (P < .05), de novo arginine synthesis, Cit production (P < .001), and plasma Cit concentration (P < .05) and lower plasma arginine concentration (P < .05) than those without nutritional failure (n = 9). Nutritional failure in CF is associated with increased NO production. However, up-regulation of de novo arginine synthesis and Cit production was not sufficient to meet the increased arginine needs leading to arginine deficiency. Copyright © 2013 Mosby, Inc. All rights reserved.

Association of plasma arginine with breast cancer molecular subtypes in women of Liaoning province.

PubMed

Hu, Lu; Gao, Yu; Cao, Yunfeng; Zhang, Yinxu; Xu, Minghao; Wang, Yuanyuan; Jing, Yu; Guo, Shengnan; Jing, Fangyu; Hu, Xiaodan; Zhu, Zhitu

2016-12-01

Arginine is one of the human nonessential amino acids critical for the growth of human cancers. The aim of this study is to investigate the variation of arginine between breast cancer (BC) patients and benign mammary gland disease (control) patients to determine its value in predicting the risk of BC. We also explore the associations between arginine levels and breast cancer subtypes. Preoperative blood samples were obtained from 267 patients (102 BC and 165 controls) in 2015. Plasma arginine values were determined for all preoperative blood samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyse differences in arginine levels between BC patients and control patients and the correlations between arginine and clinicopathologic parameters in BC. The arginine levels of BC patients were significantly lower than those of control patients (5.96 [3.76-12.47] vs. 12.54 [7.14-24.94], P = 0.000). The area under the curve (AUC) for arginine was 0.721 (95% CI, 0.660-0.782, P < 0.0001). The concentration of arginine was significantly different among different molecular BC subtypes (P = 0.030). Our results suggested that plasma arginine was associated with breast cancer molecular subtypes. © 2016 IUBMB Life, 68(12):980-984, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

Modafinil attenuates reinstatement of cocaine seeking: role for cystine-glutamate exchange and metabotropic glutamate receptors.

PubMed

Mahler, Stephen V; Hensley-Simon, Megan; Tahsili-Fahadan, Pouya; LaLumiere, Ryan T; Thomas, Charles; Fallon, Rebecca V; Kalivas, Peter W; Aston-Jones, Gary

2014-01-01

Modafinil may be useful for treating stimulant abuse, but the mechanisms by which it acts to do so are unknown. Indeed, a primary effect of modafinil is to inhibit dopamine transport, which typically promotes rather than inhibits motivated behavior. Therefore, we examined the role of nucleus accumbens extracellular glutamate and the group II metabotropic glutamate receptor (mGluR2/3) in modafinil effects. One group of rats was trained to self-administer cocaine for 10 days and extinguished, then given priming injections of cocaine to elicit reinstatement. Modafinil (300 mg/kg, intraperitoneal) inhibited reinstated cocaine seeking (but did not alter extinction responding by itself), and this effect was prevented by pre-treatment with bilateral microinjections of the mGluR2/3 antagonist LY-341495 (LY) into nucleus accumbens core. No reversal of modafinil effects was seen after unilateral accumbens core LY, or bilateral LY in the rostral pole of accumbens. Next, we sought to explore effects of modafinil on extracellular glutamate levels in accumbens after chronic cocaine. Separate rats were administered non-contingent cocaine, and after 3 weeks of withdrawal underwent accumbens microdialysis. Modafinil increased extracellular accumbens glutamate in chronic cocaine, but not chronic saline-pre-treated animals. This increase was prevented by reverse dialysis of cystine-glutamate exchange or voltage-dependent calcium channel antagonists. Voltage-dependent sodium channel blockade partly attenuated the increase in glutamate, but mGluR1 blockade did not. We conclude that modafinil increases extracellular glutamate in nucleus accumbens from glial and neuronal sources in cocaine-exposed rats, which may be important for its mGluR2/3-mediated antirelapse properties. © 2012 The Authors, Addiction Biology © 2012 Society for the Study of Addiction.

Mannitol/l-Arginine-Based Formulation Systems for Freeze Drying of Protein Pharmaceuticals: Effect of the l-Arginine Counter Ion and Formulation Composition on the Formulation Properties and the Physical State of Mannitol.

PubMed

Stärtzel, Peter; Gieseler, Henning; Gieseler, Margit; Abdul-Fattah, Ahmad M; Adler, Michael; Mahler, Hanns-Christian; Goldbach, Pierre

2016-10-01

Previous studies have shown that protein storage stability in freeze-dried l-arginine-based systems improved in the presence of chloride ions. However, chloride ions reduced the glass transition temperature of the freeze concentrate (Tg') and made freeze drying more challenging. In this study, l-arginine was freeze dried with mannitol to obtain partially crystalline solids that can be freeze dried in a fast process and result in elegant cakes. We characterized the effect of different l-arginine counter ions on physicochemical properties of mannitol compared with mannitol/sucrose systems. Thermal properties of formulations with different compositions were correlated to thermal history during freeze drying and to physicochemical properties (cake appearance, residual moisture, reconstitution time, crystallinity). Partially crystalline solids were obtained even at the highest l-arginine level (mannitol:l-arginine of 2:1) used in this study. All l-arginine-containing formulations yielded elegant cakes. Only cakes containing l-arginine chloride and succinate showed a surface "crust" formed by phase separation. X-ray powder diffraction showed that inhibition of mannitol crystallization was stronger for l-arginine compared with sucrose and varied with the type of l-arginine counter ion. The counter ion affected mannitol polymorphism and higher levels of mannitol hemi-hydrate were obtained at high levels of l-arginine chloride. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Arginine and citrulline for the treatment of MELAS syndrome

PubMed Central

El-Hattab, Ayman W.; Almannai, Mohammed; Scaglia, Fernando

2017-01-01

MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome is a maternally inherited mitochondrial disease with a broad spectrum of manifestations. In addition to impaired energy production, nitric oxide (NO) deficiency occurs in MELAS syndrome and leads to impaired blood perfusion in microvasculature that can contribute to several complications including stroke-like episodes, myopathy, and lactic acidosis. The supplementation of NO precursors, L-arginine and L-citrulline, increases NO production and hence can potentially have therapeutic utility in MELAS syndrome. L-citrulline raises NO production to a greater extent than L-arginine; therefore, L-citrulline may have a better therapeutic effect. The clinical effect of L-citrulline has not yet been studied and clinical studies on L-arginine, which are limited, only evaluated the stroke-like episodes aspect of the disease. Controlled studies are still needed to assess the clinical effects of L-arginine and L-citrulline on different aspects of MELAS syndrome. PMID:28736735

Arginine and citrulline for the treatment of MELAS syndrome.

PubMed

El-Hattab, Ayman W; Almannai, Mohammed; Scaglia, Fernando

2017-01-01

MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome is a maternally inherited mitochondrial disease with a broad spectrum of manifestations. In addition to impaired energy production, nitric oxide (NO) deficiency occurs in MELAS syndrome and leads to impaired blood perfusion in microvasculature that can contribute to several complications including stroke-like episodes, myopathy, and lactic acidosis. The supplementation of NO precursors, L-arginine and L-citrulline, increases NO production and hence can potentially have therapeutic utility in MELAS syndrome. L-citrulline raises NO production to a greater extent than L-arginine; therefore, L-citrulline may have a better therapeutic effect. The clinical effect of L-citrulline has not yet been studied and clinical studies on L-arginine, which are limited, only evaluated the stroke-like episodes aspect of the disease. Controlled studies are still needed to assess the clinical effects of L-arginine and L-citrulline on different aspects of MELAS syndrome.

Lactic acid bacteria isolated from apples are able to catabolise arginine.

PubMed

Savino, María J; Sánchez, Leandro A; Saguir, Fabiana M; de Nadra, María C Manca

2012-03-01

We investigated the potentiality of lactic acid bacteria (LAB) isolated from two apples variety to utilize arginine at different initial pH values. Apples surface contained average levels of bacteria ranging from log 2.49 ± 0.53 to log 3.73 ± 0.48 cfu/ml for Red Delicious and Golden Delicious varieties, respectively. Thirty-one strains able to develop in presence of arginine at low pH were phenotypically and genotipically identified as belonging to Lactobacillus, Pediococcus and Leuconostoc genera. In general, they did not produce ammonia from arginine when cultivated in basal medium with arginine (BMA) at pH 4.5 or 5.2. When this metabolite was quantified only six strains belonging to Leuconostoc dextranicum, Lactobacillus brevis and Lactobacillus plantarum species formed higher ammonia amounts in BMA as compared to control. This was correlated with arginine utilization and it was more pronounced at pH 4.5 than 5.2. Analysis of citrulline production confirmed the arginine utilization in these bacteria by the arginine deiminase (ADI) pathway. Maxima citrulline production was observed for Lactobacillus brevis M15 at the two pH values. In this strain ammonia was formed at higher rate than citrulline, which was detected in concentration lower than 1 mM. Thus, main LAB species found on apple surfaces with abilities to degrade arginine by the ADI pathway under different conditions were reported here at the first time. The results suggested that the ADI pathway in apples LAB might not be mainly relevant for their survival in the acid natural environmental, despite leading to the ammonia formation, which may contribute to the increase in pH, coping the acid stress.

Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimura, Shuhei; Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574; Sawatsubashi, Shun

2008-07-11

Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressedmore » EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly.« less

An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion

PubMed Central

Strasser, Rona; Zeituni-Molad, Michal; Bendelak, Keren; Rentsch, Doris; Ephros, Moshe; Wiese, Martin; Jardim, Armando; Myler, Peter J.; Zilberstein, Dan

2016-01-01

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade. PMID:27043018

Discovery and mechanistic study of a class of protein arginine methylation inhibitors.

PubMed

Feng, You; Li, Mingyong; Wang, Binghe; Zheng, Yujun George

2010-08-26

Protein arginine methylation regulates multiple biological processes such as chromatin remodeling and RNA splicing. Malfunction of protein arginine methyltransferases (PRMTs) is correlated with many human diseases. Thus, small molecule inhibitors of protein arginine methylation are of great potential for therapeutic development. Herein, we report a type of compound that blocks PRMT1-mediated arginine methylation at micromolar potency through a unique mechanism. Most of the discovered compounds bear naphthalene and sulfonate groups and are structurally different from typical PRMT substrates, for example, histone H4 and glycine- and arginine-rich sequences. To elucidate the molecular basis of inhibition, we conducted a variety of kinetic and biophysical assays. The combined data reveal that this type of naphthyl-sulfo (NS) molecule directly targets the substrates but not PRMTs for the observed inhibition. We also found that suramin effectively inhibited PRMT1 activity. These findings about novel PRMT inhibitors and their unique inhibition mechanism provide a new way for chemical regulation of protein arginine methylation.

Structural and Functional insights into the catalytic mechanism of the Type II NADH:quinone oxidoreductase family

PubMed Central

Marreiros, Bruno C.; Sena, Filipa V.; Sousa, Filipe M.; Oliveira, A. Sofia F.; Soares, Cláudio M.; Batista, Ana P.; Pereira, Manuela M.

2017-01-01

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains. These proteins contribute indirectly to the establishment of the transmembrane difference of electrochemical potential by catalyzing the reduction of quinone by oxidation of NAD(P)H. NDH-2s are widespread enzymes being present in the three domains of life. In this work, we explored the catalytic mechanism of NDH-2 by investigating the common elements of all NDH-2s, based on the rationale that conservation of such elements reflects their structural/functional importance. We observed conserved sequence motifs and structural elements among 1762 NDH-2s. We identified two proton pathways possibly involved in the protonation of the quinone. Our results led us to propose the first catalytic mechanism for NDH-2 family, in which a conserved glutamate residue, E172 (in NDH-2 from Staphylococcus aureus) plays a key role in proton transfer to the quinone pocket. This catalytic mechanism may also be extended to the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, such as sulfide:quinone oxidoreductases. PMID:28181562

Integration between Glycolysis and Glutamate-Glutamine Cycle Flux May Explain Preferential Glycolytic Increase during Brain Activation, Requiring Glutamate

PubMed Central

Hertz, Leif; Chen, Ye

2017-01-01

The 1988 observation by Fox et al. (1988) that brief intense brain activation increases glycolysis (pyruvate formation from glucose) much more than oxidative metabolism has been abundantly confirmed. Specifically glycolytic increase was unexpected because the amount of ATP it generates is much smaller than that formed by subsequent oxidative metabolism of pyruvate. The present article shows that preferential glycolysis can be explained by metabolic processes associated with activation of the glutamate-glutamine cycle. The flux in this cycle, which is essential for production of transmitter glutamate and GABA, equals 75% of brain glucose utilization and each turn is associated with utilization of ~1 glucose molecule. About one half of the association between cycle flux and glucose metabolism occurs during neuronal conversion of glutamine to glutamate in a process similar to the malate-aspartate shuttle (MAS) except that glutamate is supplied from glutamine, not formed from α-ketoglutarate (αKG) as during operation of conventional MAS. Regular MAS function is triggered by one oxidative process in the cytosol during glycolysis causing NAD+ reduction to NADH. Since NADH cannot cross the mitochondrial membrane (MEM) for oxidation NAD+ is re-generated by conversion of cytosolic oxaloacetate (OAA) to malate, which enters the mitochondria for oxidation and in a cyclic process regenerates cytosolic OAA. Therefore MAS as well as the “pseudo-MAS” necessary for neuronal glutamate formation can only operate together with cytosolic reduction of NAD+ to NADH. The major process causing NAD+ reduction is glycolysis which therefore also must occur during neuronal conversion of glutamine to glutamate and may energize vesicular glutamate uptake which preferentially uses glycolytically derived energy. Another major contributor to the association between glutamate-glutamine cycle and glucose utilization is the need for astrocytic pyruvate to generate glutamate. Although some

Importance of Loop L1 Dynamics for Substrate Capture and Catalysis in Pseudomonas aeruginosa d-Arginine Dehydrogenase.

PubMed

Ouedraogo, Daniel; Souffrant, Michael; Vasquez, Sheena; Hamelberg, Donald; Gadda, Giovanni

2017-05-16

Mobile loops located at the active site entrance in enzymes often participate in conformational changes required to shield the reaction from bulk solvent, to control the access of the substrate to the active site, and to position residues for substrate binding and catalysis. In d-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH), previous crystallographic data suggested that residues 45-47 in the FAD-binding domain and residues 50-56 in the substrate-binding domain in loop L1 could adopt two distinct conformations. In this study, we have used molecular dynamics, kinetics, and fluorescence spectroscopy on the S45A and A46G enzyme variants of PaDADH to investigate the impact of mutations in loop L1 on the catalytic function of the enzyme. Molecular dynamics showed that the mutant enzymes have probabilities of being in open conformations that are higher than that of wild-type PaDADH of loop L1, yielding an increased level of solvent exposure of the active site. In agreement, the flavin fluorescence intensity was ∼2-fold higher in the S45A and A46G enzymes than in wild-type PaDADH, with a 9 nm bathochromic shift of the emission band. In the variant enzymes, the k cat /K m values with d-arginine were ∼13-fold lower than in wild-type PaDADH. Moreover, the pH profiles for the k cat value with d-arginine showed a hollow, consistent with restricted proton movements in catalysis, and no saturation was achieved with the alternate substrate d-leucine in the reductive half-reaction of the variant enzymes. Taken together, the computational and experimental data are consistent with the dynamics of loop L1 being important for substrate capture and catalysis in PaDADH.

Effects of a chronic l-arginine supplementation on the arginase pathway in aged rats.

PubMed

Moretto, Johnny; Guglielmetti, Anne-Sophie; Tournier-Nappey, Maude; Martin, Hélène; Prigent-Tessier, Anne; Marie, Christine; Demougeot, Céline

2017-04-01

While ageing is frequently associated with l-arginine deficiency, clinical and experimental studies provided controversial data on the interest of a chronic l-arginine supplementation with beneficial, no or even deleterious effects. It was hypothesized that these discrepancies might relate to a deviation of l-arginine metabolism towards production of l-ornithine rather than nitric oxide as a result of age-induced increase in arginase activity. This study investigated the effect of ageing on arginase activity/expression in target tissues and determined whether l-arginine supplementation modulated the effect of ageing on arginase activity. Arginase activity and expression were measured in the heart, vessel, brain, lung, kidney and liver in young rats (3-months old) and aged Wistar rats (22-24-months-old) with or without l-arginine supplementation (2.25% in drinking water for 6weeks). Plasma levels of l-arginine and l-ornithine were quantified in order to calculate the plasma l-arginine/l-ornithine ratio, considered as a reflection of arginase activity. Cardiovascular parameters (blood pressure, heart rate) and aortic vascular reactivity were also studied. Ageing dramatically reduced plasma l-arginine and l-arginine/l-ornithine ratio, decreased liver and kidney arginase activities but did not change activities in other tissues. l-Arginine supplementation normalized plasma l-arginine and l-arginine/l-ornithine ratio, improved endothelial function and decreased systolic blood pressure. These effects were associated with decreased arginase activity in aorta along with no change in the other tissues except in the lung in which activity was increased. A strong mismatch was therefore observed between arginase activity and expression in analyzed tissues. The present study reveals that ageing selectively changes arginase activity in clearance tissues, but does not support a role of the arginase pathway in the potential deleterious effect of the l-arginine supplementation in

Glutamate and Brain Glutaminases in Drug Addiction.

PubMed

Márquez, Javier; Campos-Sandoval, José A; Peñalver, Ana; Matés, José M; Segura, Juan A; Blanco, Eduardo; Alonso, Francisco J; de Fonseca, Fernando Rodríguez

2017-03-01

Glutamate is the principal excitatory neurotransmitter in the central nervous system and its actions are related to the behavioral effects of psychostimulant drugs. In the last two decades, basic neuroscience research and preclinical studies with animal models are suggesting a critical role for glutamate transmission in drug reward, reinforcement, and relapse. Although most of the interest has been centered in post-synaptic glutamate receptors, the presynaptic synthesis of glutamate through brain glutaminases may also contribute to imbalances in glutamate homeostasis, a key feature of the glutamatergic hypothesis of addiction. Glutaminases are the main glutamate-producing enzymes in brain and dysregulation of their function have been associated with neurodegenerative diseases and neurological disorders; however, the possible implication of these enzymes in drug addiction remains largely unknown. This mini-review focuses on brain glutaminase isozymes and their alterations by in vivo exposure to drugs of abuse, which are discussed in the context of the glutamate homeostasis theory of addiction. Recent findings from mouse models have shown that drugs induce changes in the expression profiles of key glutamatergic transmission genes, although the molecular mechanisms that regulate drug-induced neuronal sensitization and behavioral plasticity are not clear.

Asymmetric dimethylarginine, related arginine derivatives, and incident atrial fibrillation.

PubMed

Schnabel, Renate B; Maas, Renke; Wang, Na; Yin, Xiaoyan; Larson, Martin G; Levy, Daniel; Ellinor, Patrick T; Lubitz, Steven A; McManus, David D; Magnani, Jared W; Atzler, Dorothee; Böger, Rainer H; Schwedhelm, Edzard; Vasan, Ramachandran S; Benjamin, Emelia J

2016-06-01

Oxidative stress plays an important role in the development of atrial fibrillation (AF). Arginine derivatives including asymmetric dimethylarginine (ADMA) are central to nitric oxide metabolism and nitrosative stress. Whether blood concentrations of arginine derivatives are related to incidence of AF is uncertain. In 3,310 individuals (mean age 58 ± 10 years, 54% women) from the community-based Framingham Study, we prospectively examined the relations of circulating levels of ADMA, l-arginine, symmetric dimethylarginine (SDMA), and the ratio of l-arginine/ADMA to incidence of AF using proportional hazards regression models. Over a median follow-up time of 10 years, 247 AF cases occurred. Using age- and sex-adjusted regression models, ADMA was associated with a hazard ratio of 1.15 per 1-SD increase in loge-biomarker concentration (95% CI 1.02-1.29, P = .02) for AF, which was no longer significant after further risk factor adjustment (hazard ratio 1.09, 95% CI 0.97-1.23, P = .15). Neither l-arginine nor SDMA was related to new-onset AF. A clinical model comprising clinical risk factors for AF (for age, sex, height, weight, systolic blood pressure, diastolic blood pressure, current smoking, diabetes, hypertension treatment, myocardial infarction, and heart failure; c statistic = 0.781; 95% CI 0.753-0.808) was not improved by the addition of ADMA (0.782; 95% CI 0.755-0.809). Asymmetric dimethylarginine and related arginine derivatives were not associated with incident AF in the community after accounting for other clinical risk factors and confounders. Its role in the pathogenesis of AF needs further refinement. Copyright © 2016 Elsevier Inc. All rights reserved.

Potential of single cationic amino acid molecule "Arginine" for stimulating oral absorption of insulin.

PubMed

Kamei, Noriyasu; Khafagy, El-Sayed; Hirose, Jun; Takeda-Morishita, Mariko

2017-04-15

We have reported that cell-penetrating peptides, such as oligoarginine, act as powerful absorption enhancers for the development of oral insulin delivery systems. However, the minimal essential sequence of oligoarginine that stimulates intestinal insulin absorption remains unclear. Therefore, the present study was conducted to clarify this minimum sequence of oligoarginine and to examine the effect of single cationic amino acid arginine on the intestinal and oral absorption of insulin. The results demonstrated that a remarkable enhancement of intestinal insulin absorption was observed after coadministration of insulin with l-arginine. The efficacy of d-forms of oligoarginine/arginine tended to decrease with a decreasing number of amino acid residues, whereas the effect of l-arginine was the strongest of any of the l-forms of oligoarginine/arginine. Interestingly, the effect of l-arginine was stronger than that of d-arginine at various concentrations, and the effect of other cationic amino acids such as lysine and histidine was relatively lower than that of arginine. In addition, no leakage of lactate dehydrogenase from the intestinal epithelium and no change in the transepithelial electrical resistance of a Caco-2 cell monolayer were detected after administration of l-arginine as the single amino acid, which suggests that there were no undesirable effects of arginine on the integrity of cell membranes and paracellular tight junctions. Oral administration study in mice demonstrated that the stronger hypoglycemic effects were observed after coadministration of insulin with l-arginine. In this study, we found that arginine is a key cationic amino acid for delivering insulin across intestinal epithelial barriers and hopefully accelerating the clinical development of oral insulin delivery systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanism of arginine sensing by CASTOR1 upstream of mTORC1

PubMed Central

Saxton, Robert A.; Chantranupong, Lynne; Knockenhauer, Kevin E.; Schwartz, Thomas U.; Sabatini, David M.

2016-01-01

Summary The mechanistic Target of Rapamycin Complex 1 (mTORC1) is a major regulator of eukaryotic growth that coordinates anabolic and catabolic cellular processes with inputs such as growth factors and nutrients, including amino acids1–3. In mammals, arginine is particularly important and promotes diverse physiological effects including immune cell activation, insulin secretion, and muscle growth, largely through activation of mTORC14–7. Arginine activates mTORC1 upstream of the Rag GTPases8, through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting CASTOR1 (Cellular Arginine Sensor for mTORC1)9–12. However, the mechanism by which the mTORC1 pathway detects and transmits the arginine signal has been elusive. Here, we present the 1.8 Å crystal structure of arginine-bound CASTOR1. Homodimeric CASTOR1 binds arginine at the interface of two ACT domains, enabling allosteric control of the adjacent GATOR2-binding site to trigger dissociation from GATOR2 and the downstream activation of mTORC1. Our data reveal that CASTOR1 shares substantial structural homology with the lysine-binding regulatory domain of prokaryotic aspartate kinases, suggesting that the mTORC1 pathway exploited an ancient amino-acid-dependent allosteric mechanism to acquire arginine sensitivity. Together, these results establish a structural basis for arginine sensing by the mTORC1 pathway and provide insights into the evolution of a mammalian nutrient sensor. PMID:27487210

Protein Arginine Methylation in Mammals: Who, What, and Why

PubMed Central

Bedford, Mark T.; Clarke, Steven G.

2012-01-01

The covalent marking of proteins by methyl group addition to arginine residues can promote their recognition by binding partners or can modulate their biological activity. A small family of gene products that catalyze such methylation reactions in eukaryotes (PRMTs) work in conjunction with a changing cast of associated subunits to recognize distinct cellular substrates. These reactions display many of the attributes of reversible covalent modifications such as protein phosphorylation or protein lysine methylation; however, it is unclear to what extent protein arginine demethylation occurs. Physiological roles for protein arginine methylation have been established in signal transduction, mRNA splicing, transcriptional control, DNA repair, and protein translocation. PMID:19150423

Glutamate mediates platelet activation through the AMPA receptor

PubMed Central

Morrell, Craig N.; Sun, Henry; Ikeda, Masahiro; Beique, Jean-Claude; Swaim, Anne Marie; Mason, Emily; Martin, Tanika V.; Thompson, Laura E.; Gozen, Oguz; Ampagoomian, David; Sprengel, Rolf; Rothstein, Jeffrey; Faraday, Nauder; Huganir, Richard; Lowenstein, Charles J.

2008-01-01

Glutamate is an excitatory neurotransmitter that binds to the kainate receptor, the N-methyl-D-aspartate (NMDA) receptor, and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each receptor was first characterized and cloned in the central nervous system (CNS). Glutamate is also present in the periphery, and glutamate receptors have been identified in nonneuronal tissues, including bone, heart, kidney, pancreas, and platelets. Platelets play a central role in normal thrombosis and hemostasis, as well as contributing greatly to diseases such as stroke and myocardial infarction. Despite the presence of glutamate in platelet granules, the role of glutamate during hemostasis is unknown. We now show that activated platelets release glutamate, that platelets express AMPAR subunits, and that glutamate increases agonist-induced platelet activation. Furthermore, we demonstrate that glutamate binding to the AMPAR increases intracellular sodium concentration and depolarizes platelets, which are important steps in platelet activation. In contrast, platelets treated with the AMPAR antagonist CNQX or platelets derived from GluR1 knockout mice are resistant to AMPA effects. Importantly, mice lacking GluR1 have a prolonged time to thrombosis in vivo. Our data identify glutamate as a regulator of platelet activation, and suggest that the AMPA receptor is a novel antithrombotic target. PMID:18283118

Exercise training reverses the negative effects of chronic L-arginine supplementation on insulin sensitivity.

PubMed

Salgueiro, Rafael Barrera; Gerlinger-Romero, Frederico; Guimarães-Ferreira, Lucas; de Castro Barbosa, Thais; Nunes, Maria Tereza

2017-12-15

L-Arginine has emerged as an important supplement for athletes and non-athletes in order to improve performance. Arginine has been extensively used as substrate for nitric oxide synthesis, leading to increased vasodilatation and hormonal secretion. However, the chronic consumption of arginine has been shown to impair insulin sensitivity. In the present study, we aimed to evaluate whether chronic arginine supplementation associated with exercise training would have a beneficial impact on insulin sensitivity. We, therefore, treated Wistar rats for 4weeks with arginine, associated or not with exercise training (treadmill). We assessed the somatotropic activation, by evaluating growth hormone (GH) gene expression and protein content in the pituitary, as well is GH concentration in the serum. Additionally, we evaluate whole-body insulin sensitivity, by performing an insulin tolerance test. Skeletal muscle morpho-physiological parameters were also assessed. Insulin sensitivity was impaired in the arginine-treated rats. However, exercise training reversed the negative effects of arginine. Arginine and exercise training increased somatotropic axis function, muscle mass and body weight gain. The combination arginine and exercise training further decreased total fat mass. Our results confirm that chronic arginine supplementation leads to insulin resistance, which can be reversed in the association with exercise training. We provide further evidence that exercise training is an important tool to improve whole-body metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

Intravenous glutamine supplementation enhances renal de novo arginine synthesis in humans: a stable isotope study.

PubMed

Buijs, Nikki; Brinkmann, Saskia J H; Oosterink, J Efraim; Luttikhold, Joanna; Schierbeek, Henk; Wisselink, Willem; Beishuizen, Albertus; van Goudoever, Johannes B; Houdijk, Alexander P J; van Leeuwen, Paul A M; Vermeulen, Mechteld A R

2014-11-01

Arginine plays a role in many different pathways in multiple cell types. Consequently, a shortage of arginine, caused by pathologic conditions such as cancer or injury, has the potential to disturb many cellular and organ functions. Glutamine is the ultimate source for de novo synthesis of arginine in humans via the intestinal-renal axis. Therefore, we hypothesized that parenteral glutamine supplementation may stimulate the interorgan pathway of arginine production. The objectives were to quantify arginine production from its precursor glutamine and to establish the contribution of the kidneys to de novo synthesis of arginine in patients receiving intravenous supplementation of glutamine dipeptide during major abdominal surgery. Whole-body and renal metabolism of glutamine, citrulline, and arginine was assessed by stable isotope techniques in 7 patients receiving a perioperative supplement of intravenous alanyl-glutamine (0.5 g · kg(-1) · d(-1)). Plasma glutamine, citrulline, and arginine concentrations increased significantly in patients receiving intravenous glutamine dipeptide. At whole-body level, 91% of total citrulline turnover was derived from glutamine, whereas 49% of whole-body citrulline turnover was used for de novo synthesis of arginine. The kidneys were responsible for 75% of whole-body arginine production from citrulline. Glutamine and citrulline are important sources for de novo arginine synthesis. The kidneys are the main production site for endogenous arginine. After comparison of these results with previous similar studies, our data suggest that an intravenous glutamine supplement doubles renal arginine production from citrulline. This trial was registered at www.trialregister.nl as NTR2914. © 2014 American Society for Nutrition.

21 CFR 182.1047 - Glutamic acid hydrochloride.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Glutamic acid hydrochloride. 182.1047 Section 182.1047 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Food Substances § 182.1047 Glutamic acid hydrochloride. (a) Product. Glutamic acid hydrochloride. (b...

21 CFR 182.1047 - Glutamic acid hydrochloride.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Glutamic acid hydrochloride. 182.1047 Section 182.1047 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Food Substances § 182.1047 Glutamic acid hydrochloride. (a) Product. Glutamic acid hydrochloride. (b...

Efficiency of cardioplegic solutions containing L-arginine and L-aspartic acid.

PubMed

Pisarenko, O I; Shul'zhenko, V S; Studneva, I M

2006-04-01

In experiments on rats we studied the effects of cardioplegic solutions with L-aspartic acid or L-arginine on functional recovery and metabolism of isolated working heart after 40-min normothermal global ischemia and 30-min reperfusion. After reperfusion of the hearts preventively protected with cardioplegic solution containing L-aspartic acid or L-arginine, coronary flow decreased in comparison with the initial values. As a component of cardioplegic solution, L-arginine was less efficient in recovery of contractility and cardiac output of the hearts in comparison with L-aspartic acid. In hearts protected with L-aspartic acid, the postischemic levels of ATP and phosphocreatine were significantly higher, and the level of lactate was significantly lower than in hearts protected with L-arginine. In comparison with L-arginine, L-aspartic acid is a more efficient component of cardioplegic solution in protection of the heart from metabolic and functional damages caused by global ischemia and reperfusion.

Inhibition of lytic infection of pseudorabies virus by arginine depletion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, H.-C.; Kao, Y.-C.; Chang, T-J.

2005-08-26

Pseudorabies virus (PRV) is a member of Alphahepesviruses; it is an enveloped virus with a double-stranded DNA genome. Polyamines (such as spermine and spermidine) are ubiquitous in animal cells and participate in cellular proliferation and differentiation. Previous results of our laboratory showed that the PRV can accomplish lytic infection either in the presence of exogenous spermine (or spermidine) or depletion of cellular polyamines. The amino acid arginine is a precursor of polyamine biosynthesis. In this work, we investigated the role of arginine in PRV infection. It was found that the plaque formation of PRV was inhibited by arginase (enzyme catalyzingmore » the conversion of arginine into ornithine and urea) treatment whereas this inhibition can be reversed by exogenous arginine, suggesting that arginine is essential for PRV proliferation. Western blotting was conducted to study the effect of arginine depletion on the levels of structural proteins of PRV in virus-infected cells. Four PRV structural proteins (gB, gE, UL47, and UL48) were chosen for examination, and results revealed that the levels of viral proteins were obviously reduced in long time arginase treatment. However, the overall protein synthesis machinery was apparently not influenced by arginase treatment either in mock or PRV-infected cells. Analyzing with native gel, we found that arginase treatment affected the mobility of PRV structural proteins, suggesting the conformational change of viral proteins by arginine depletion. Heat shock proteins, acting as molecular chaperons, participate in protein folding and translocation. Our results demonstrated that long time arginase treatment could reduce the expression of cellular heat shock proteins 70 (hsc70 and hsp70), and transcriptional suppression of heat shock protein 70 gene promoter was one of the mechanisms involved in this reduced expression.« less

Introduction to the Glutamate-Glutamine Cycle.

PubMed

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.

The role of glutamate and its receptors in autism and the use of glutamate receptor antagonists in treatment

PubMed Central

Rojas, Donald C.

2014-01-01

Glutamate is the major excitatory neurotransmitter in the brain and may be a key neurotransmitter involved in autism. Literature pertaining to glutamate and autism or related disorders (e.g., Fragile X syndrome) is reviewed in this article. Interest in glutamatergic dysfunction in autism is high due to increasing convergent evidence implicating the system in the disorder from peripheral biomarkers, neuroimaging, protein expression, genetics and animal models. Currently, there are no pharmaceutical interventions approved for autism that address glutamate deficits in the disorder. New treatments related to glutamatergic neurotransmission, however, are emerging. In addition, older glutamate-modulating medications with approved indications for use in other disorders are being investigated for re-tasking as treatments for autism. This review presents evidence in support of glutamate abnormalities in autism and the potential for translation into new treatments for the disorder. PMID:24752754

The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif

PubMed Central

Barillà, Daniela; Carmelo, Emma; Hayes, Finbarr

2007-01-01

The ParF protein of plasmid TP228 belongs to the ubiquitous superfamily of ParA ATPases that drive DNA segregation in bacteria. ATP-bound ParF polymerizes into multistranded filaments. The partner protein ParG is dimeric, consisting of C-termini that interweave into a ribbon–helix–helix domain contacting the centromeric DNA and unstructured N-termini. ParG stimulates ATP hydrolysis by ParF ≈30-fold. Here, we establish that the mobile tails of ParG are crucial for this enhancement and that arginine R19 within the tail is absolutely required for activation of ParF nucleotide hydrolysis. R19 is part of an arginine finger-like loop in ParG that is predicted to intercalate into the ParF nucleotide-binding pocket thereby promoting ATP hydrolysis. Significantly, mutations of R19 abrogated DNA segregation in vivo, proving that intracellular stimulation of ATP hydrolysis by ParG is a key regulatory process for partitioning. Furthermore, ParG bundles ParF-ATP filaments as well as promoting nucleotide-independent polymerization. The N-terminal flexible tail is required for both activities, because N-terminal ΔParG polypeptides are defective in both functions. Strikingly, the critical arginine finger-like residue R19 is dispensable for ParG-mediated remodeling of ParF polymers, revealing that the ParG N-terminal tail possesses two separable activities in the interplay with ParF: a catalytic function during ATP hydrolysis and a mechanical role in modulation of polymerization. We speculate that activation of nucleotide hydrolysis via an arginine finger loop may be a conserved, regulatory mechanism of ParA family members and their partner proteins, including ParA-ParB and Soj-Spo0J that mediate DNA segregation and MinD-MinE that determine septum localization. PMID:17261809

The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif.

PubMed

Barillà, Daniela; Carmelo, Emma; Hayes, Finbarr

2007-02-06

The ParF protein of plasmid TP228 belongs to the ubiquitous superfamily of ParA ATPases that drive DNA segregation in bacteria. ATP-bound ParF polymerizes into multistranded filaments. The partner protein ParG is dimeric, consisting of C-termini that interweave into a ribbon-helix-helix domain contacting the centromeric DNA and unstructured N-termini. ParG stimulates ATP hydrolysis by ParF approximately 30-fold. Here, we establish that the mobile tails of ParG are crucial for this enhancement and that arginine R19 within the tail is absolutely required for activation of ParF nucleotide hydrolysis. R19 is part of an arginine finger-like loop in ParG that is predicted to intercalate into the ParF nucleotide-binding pocket thereby promoting ATP hydrolysis. Significantly, mutations of R19 abrogated DNA segregation in vivo, proving that intracellular stimulation of ATP hydrolysis by ParG is a key regulatory process for partitioning. Furthermore, ParG bundles ParF-ATP filaments as well as promoting nucleotide-independent polymerization. The N-terminal flexible tail is required for both activities, because N-terminal DeltaParG polypeptides are defective in both functions. Strikingly, the critical arginine finger-like residue R19 is dispensable for ParG-mediated remodeling of ParF polymers, revealing that the ParG N-terminal tail possesses two separable activities in the interplay with ParF: a catalytic function during ATP hydrolysis and a mechanical role in modulation of polymerization. We speculate that activation of nucleotide hydrolysis via an arginine finger loop may be a conserved, regulatory mechanism of ParA family members and their partner proteins, including ParA-ParB and Soj-Spo0J that mediate DNA segregation and MinD-MinE that determine septum localization.

Molecular dynamics simulation of the last step of a catalytic cycle: product release from the active site of the enzyme chorismate mutase from Mycobacterium tuberculosis.

PubMed

Choutko, Alexandra; van Gunsteren, Wilfred F

2012-11-01

The protein chorismate mutase MtCM from Mycobacterium tuberculosis catalyzes one of the few pericyclic reactions known in biology: the transformation of chorismate to prephenate. Chorismate mutases have been widely studied experimentally and computationally to elucidate the transition state of the enzyme catalyzed reaction and the origin of the high catalytic rate. However, studies about substrate entry and product exit to and from the highly occluded active site of the enzyme have to our knowledge not been performed on this enzyme. Crystallographic data suggest a possible substrate entry gate, that involves a slight opening of the enzyme for the substrate to access the active site. Using multiple molecular dynamics simulations, we investigate the natural dynamic process of the product exiting from the binding pocket of MtCM. We identify a dominant exit pathway, which is in agreement with the gate proposed from the available crystallographic data. Helices H2 and H4 move apart from each other which enables the product to exit from the active site. Interestingly, in almost all exit trajectories, two residues arginine 72 and arginine 134, which participate in the burying of the active site, are accompanying the product on its exit journey from the catalytic site. Copyright © 2012 The Protein Society.

Role of L-arginine in the biological effects of blue light

NASA Astrophysics Data System (ADS)

Makela, Anu M.

2005-11-01

Arginine, a semi-essential amino acid, and metabolites of arginine exert multiple biological effects. It has been known that arginine causes the release of various hormones such as insulin, glucagon, growth hormone, prolactin, and adrenal catecholamines. Arginine infusion also produces vasodilation, and in the kidney increased plasma flow accompanied by increases in glomerular filtration rate (GFR). Recent studies have showed that blue and red light irradiation in vitro and in vivo can increase production of nitric oxide (NO), superoxide anion, and related reactive oxygen species (ROS). These then can modulate the production and secretion of several cytokines and other mediators and play an important role as regulatory mediators in signaling processes which can then modulate the production, mobilization and homing of stem cells. It is proposed that some of the therapeutic effects of light can be considered to be due to the changes in the metabolism of L-arginine. The regulation of L-arginine turnover by the use of light at blue wavelengths between 400nm and 510nm can be the explanation for some of the observed effects of blue light: lowering of blood pressure, pain killing effect, regulating insulin production, anti-inflammatory action, and possible effects on the release and homing of stem cells.

Constitutive production of catalytic antibodies to a Staphylococcus aureus virulence factor and effect of infection.

PubMed

Brown, Eric L; Nishiyama, Yasuhiro; Dunkle, Jesse W; Aggarwal, Shreya; Planque, Stephanie; Watanabe, Kenji; Csencsits-Smith, Keri; Bowden, M Gabriela; Kaplan, Sheldon L; Paul, Sudhir

2012-03-23

Antibodies that recognize microbial B lymphocyte superantigenic epitopes are produced constitutively with no requirement for adaptive immune maturation. We report cleavage of the Staphylococcus aureus virulence factor extracellular fibrinogen-binding protein (Efb) by catalytic antibodies produced with no exposure to the bacterium and reduction of the catalytic antibody activity following infection. IgG catalytic antibodies that specifically hydrolyzed Efb via a nucleophilic catalytic mechanism were found in the blood of healthy humans and aseptic mice free of S. aureus infection. IgG hydrolyzed peptide bonds on the C-terminal side of basic amino acids, including a bond located within the C3b-binding domain of Efb. Efb digested with the IgG lost its ability to bind C3b and inhibit complement-dependent antibody-mediated red blood cell lysis. In addition to catalysis, the IgG expressed saturable Efb binding activity. IgG from S. aureus-infected mice displayed reduced Efb cleaving activity and increased Efb binding activity compared with uninfected controls, suggesting differing effects of the infection on the antibody subsets responsible for the two activities. IgG from children hospitalized for S. aureus infection also displayed reduced Efb cleavage compared with healthy children. These data suggest a potential defense function for constitutively produced catalytic antibodies to a putative superantigenic site of Efb, but an adaptive catalytic response appears to be proscribed.

Glutamate and Its Receptors as Therapeutic Targets for Migraine.

PubMed

Hoffmann, Jan; Charles, Andrew

2018-04-01

There is substantial evidence indicating a role for glutamate in migraine. Levels of glutamate are higher in the brain and possibly also in the peripheral circulation in migraine patients, particularly during attacks. Altered blood levels of kynurenines, endogenous modulators of glutamate receptors, have been reported in migraine patients. Population genetic studies implicate genes that are involved with glutamate signaling in migraine, and gene mutations responsible for familial hemiplegic migraine and other familial migraine syndromes may influence glutamate signaling. Animal studies indicate that glutamate plays a key role in pain transmission, central sensitization, and cortical spreading depression. Multiple therapies that target glutamate receptors including magnesium, topiramate, memantine, and ketamine have been reported to have efficacy in the treatment of migraine, although with the exception of topiramate, the evidence for the efficacy of these therapies is not strong. Also, because all of these therapies have other mechanisms of action, it is not possible to conclude that the efficacy of these drugs is entirely due to their effects on glutamate receptors. Further studies are needed to more clearly delineate the possible roles of glutamate and its specific receptor subtypes in migraine and to identify new ways of targeting glutamate for migraine therapy.

Using glutamate homeostasis as a target for treating addictive disorders

PubMed Central

Reissner, Kathryn J.; Kalivas, Peter W.

2010-01-01

Well-developed cellular mechanisms exist to preserve glutamate homeostasis and regulate extrasynaptic glutamate levels. Accumulating evidence indicates that disruptions in glutamate homeostasis are associated with addictive disorders. The disruptions in glutamate concentrations observed following prolonged exposure to drugs of abuse are associated with changes in the function and activity of several key components within the homeostatic control mechanism, including the cystine/glutamate exchanger xc− and the glial glutamate transporter EAAT2/GLT-1. Changes in the balance between synaptic and extrasynaptic glutamate levels in turn influence signaling through pre- and postsynaptic glutamate receptors, and thus affect synaptic plasticity and circuit-level activity. In this review we describe the evidence for impaired glutamate homestasis as a critical mediator of long-term drug-seeking behaviors, how chronic neuroadaptations in xc− and GLT-1 mediate a disruption in glutamate homeostasis, and how targeting these components restores glutamate levels and inhibits drug-seeking behaviors. PMID:20634691

Through the central V2, not V1 receptors influencing the endogenous opiate peptide system, arginine vasopressin, not oxytocin in the hypothalamic paraventricular nucleus involves in the antinociception in the rat.

PubMed

Yang, Jun; Chen, Jian-min; Song, Cao-You; Liu, Wen-Yan; Wang, Gen; Wang, Cheng-hai; Lin, Bao-Chen

2006-01-19

Our previous study has proven that hypothalamic paraventricular nucleus (PVN) played a role in the antinociception. The central bioactive substances involving in the PVN regulating antinociception were investigated in the rat. The results showed that electrical stimulation of the PVN increased the pain threshold, and L-glutamate sodium injection into the PVN elevated the pain threshold, but the PVN cauterization decreased the pain threshold; pain stimulation raised the arginine vasopressin (AVP), not oxytocin (OXT), leucine-enkephalin (L-Ek), beta-endorphin (beta-Ep) and DynorphinA1-13 (DynA1-13) concentrations in the PVN tissue using micropunch method, heightened AVP, L-Ek, beta-Ep and DynA1-13, not OXT concentrations in the PVN perfuse liquid, and reduced the number of AVP-, not OXT, L-Ek, beta-Ep and DynA1-13-immunoreactive neurons in the PVN especially in the posterior magnocellular part of the PVN using immunocytochemistry. There was a negative relationship between the PVN AVP concentration and the pain threshold; pain stimulation enhanced the AVP, not OXT mRNA expression in the PVN using in situ hybridization and RT-PCR; intraventricular injection of anti-AVP serum completely reversed L-glutamate sodium injection into the PVN-induced antinociception, and administration of naloxone - the opiate peptide antagonist, partly blocked this L-glutamate sodium effect, but anti-OXT serum pretreatment did not influence this L-glutamate sodium effect; L-glutamate sodium injection into the PVN-induced analgesia was inhibited by V2 receptor antagonist - d(CH2)5[D-Ile2, Ile4, Ala-NH2(9)]AVP, not V1 receptor antagonist - d(CH2)5Tyr(Me)AVP. The data suggested that the PVN was limited to the central AVP, not OXT, which was through V2, not V1 receptors influencing the endogenous opiate peptide system, to regulate antinociception.

Patch-clamp, ion-sensing, and glutamate-sensing techniques to study glutamate transport in isolated retinal glial cells.

PubMed

Billups, B; Szatkowski, M; Rossi, D; Attwell, D

1998-01-01

We have described how a combination of electrical, ion-sensing, and glutamate-sensing techniques has advanced our understanding of glutamate uptake into isolated salamander retinal glial cells. The next steps in understanding glutamate transport will inevitably depend strongly on molecular biological methods, as described elsewhere in this book, but will also require more detailed study of transporters in their normal environment, perhaps by using patch-clamping or imaging techniques to study cells in situ.

Differential effects of repetitive oral administration of monosodium glutamate on interstitial glutamate concentration and muscle pain sensitivity.

PubMed

Shimada, Akiko; Baad-Hansen, Lene; Castrillon, Eduardo; Ghafouri, Bijar; Stensson, Niclas; Gerdle, Björn; Ernberg, Malin; Cairns, Brian; Svensson, Peter; Svensson Odont, Peter

2015-02-01

The aim of this study was to determine the relationship of high daily monosodium glutamate (MSG) consumption with glutamate concentrations in jaw muscle, saliva, and serum, and muscle pain sensitivity in healthy participants. A randomized, double-blinded, placebo-controlled study was conducted to investigate the effect of repetitive consumption of high-dose MSG on glutamate concentration in the masseter muscles measured by microdialysis and muscle pain sensitivity. In five contiguous experimental daily sessions, 32 healthy participants drank MSG (150 mg/kg) or NaCl (24 mg/kg) diluted with a 400 mL soda. The concentrations of glutamate before and after the ingestion were assessed in dialysate and plasma samples on the first and last days. Saliva glutamate concentration was assessed every day. Pressure pain threshold, pressure pain tolerance, autonomic parameters (heart rate, systolic and diastolic blood pressures) and reported side effects also were assessed. No significant change was noted in the baseline concentration of glutamate in the masseter muscle, blood, or saliva, but the peak concentration in the masseter muscle increased significantly between day 1 and 5. A statistically significant increase in systolic and diastolic blood pressures after MSG administration was observed, as well as a significantly higher frequency of reports of nausea and headache in the MSG group. No robust effect of MSG on muscle sensitivity was found. Interstitial glutamate concentration in the masseter muscle is not highly disturbed by excessive repetitive intake of MSG in healthy man. Copyright © 2015 Elsevier Inc. All rights reserved.

Human Protein Arginine Methyltransferase 7 (PRMT7) Is a Type III Enzyme Forming ω-NG-Monomethylated Arginine Residues*

PubMed Central

Zurita-Lopez, Cecilia I.; Sandberg, Troy; Kelly, Ryan; Clarke, Steven G.

2012-01-01

Full-length human protein arginine methyltransferase 7 (PRMT7) expressed as a fusion protein in Escherichia coli was initially found to generate only ω-NG-monomethylated arginine residues in small peptides, suggesting that it is a type III enzyme. A later study, however, characterized fusion proteins of PRMT7 expressed in bacterial and mammalian cells as a type II/type I enzyme, capable of producing symmetrically dimethylated arginine (type II activity) as well as small amounts of asymmetric dimethylarginine (type I activity). We have sought to clarify the enzymatic activity of human PRMT7. We analyzed the in vitro methylation products of a glutathione S-transferase (GST)-PRMT7 fusion protein with robust activity using a variety of arginine-containing synthetic peptides and protein substrates, including a GST fusion with the N-terminal domain of fibrillarin (GST-GAR), myelin basic protein, and recombinant human histones H2A, H2B, H3, and H4. Regardless of the methylation reaction conditions (incubation time, reaction volume, and substrate concentration), we found that PRMT7 only produces ω-NG-monomethylarginine with these substrates. In control experiments, we showed that mammalian GST-PRMT1 and Myc-PRMT5 were, unlike PRMT7, able to dimethylate both peptide P-SmD3 and SmB/D3 to give the expected asymmetric and symmetric products, respectively. These experiments show that PRMT7 is indeed a type III human methyltransferase capable of forming only ω-NG-monomethylarginine, not asymmetric ω-NG,NG-dimethylarginine or symmetric ω-NG,NG′-dimethylarginine, under the conditions tested. PMID:22241471

l-Arginine is a Radioprotector for Hematopoietic Progenitor Cells

PubMed Central

Pearce, Linda L.; Zheng, Xichen; Martinez-Bosch, Sandra; Kerr, Patrick P.; Khlangwiset, Pornsri; Epperly, Michael W.; Fink, Mitchell P.; Greenberger, Joel S.; Peterson, Jim

2012-01-01

l-Arginine is shown to protect hematopoietic progenitor (32D cl 3) cells from death due to exposure to γ radiation (137Cs). Some of the other intermediates in the urea cycle, namely ornithine and citrulline, plus urea itself, were not found to have any significant impact on cell survival after irradiation. Intriguingly, supplementation of irradiated cells with l-arginine results in decreased production of peroxynitrite, suggesting that suppression of superoxide generation by nitric oxide synthase in one or more microenvironments is an important factor in the observed radioprotection. The absence of any radioprotective effect of l-arginine in cells at 3% oxygen also confirms the involvement of one or more oxygen-derived species. Knockdown experiments with nitric oxide synthase (NOS) siRNAs in cells and NOS knockout animals confirm that the observed radioprotection is associated with nNOS (NOS-1). l-Arginine also ameliorates the transient inhibition of the electron-transport chain complex I that occurs within 30 min of completing the dose (10 Gy) and that appears to be a functional marker for postirradiation mitochondrial oxidant production. PMID:22175298

Dishevelled3 is a novel arginine methyl transferase substrate.

PubMed

Bikkavilli, Rama Kamesh; Avasarala, Sreedevi; Vanscoyk, Michelle; Sechler, Marybeth; Kelley, Nicole; Malbon, Craig C; Winn, Robert A

2012-01-01

Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation. Interestingly, Wnt3a stimulation of F9 teratocarcinoma cells results in reduced Dishevelled methylation. Similarly, the methylation-deficient mutant of Dishevelled, R271K, displayed spontaneous membrane localization and robust activation of Wnt signaling; suggesting that differential methylation of Dishevelled plays an important role in Wnt signaling. Thus arginine methylation is shown to be an important switch in regulation of Dishevelled function and Wnt signaling.

Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation.

PubMed

Koopman, Jessica E; Röling, Wilfred F M; Buijs, Mark J; Sissons, Christopher H; ten Cate, Jacob M; Keijser, Bart J F; Crielaard, Wim; Zaura, Egija

2015-02-01

Dysbiosis induced by low pH in the oral ecosystem can lead to caries, a prevalent bacterial disease in humans. The amino acid arginine is one of the pH-elevating agents in the oral cavity. To obtain insights into the effect of arginine on oral microbial ecology, a multi-plaque "artificial mouth" (MAM) biofilm model was inoculated with saliva from a healthy volunteer and microcosms were grown for 4 weeks with 1.6 % (w/v) arginine supplement (Arginine) or without (Control), samples were taken at several time-points. A cariogenic environment was mimicked by sucrose pulsing. The bacterial composition was determined by 16S rRNA gene amplicon sequencing, the presence and amount of Candida and arginine deiminase system genes arcA and sagP by qPCR. Additionally, ammonium and short-chain fatty acid concentrations were determined. The Arginine microcosms were dominated by Streptococcus, Veillonella, and Neisseria and remained stable in time, while the composition of the Control microcosms diverged significantly in time, partially due to the presence of Megasphaera. The percentage of Candida increased 100-fold in the Control microcosms compared to the Arginine microcosms. The pH-raising effect of arginine was confirmed by the pH and ammonium results. The abundances of sagP and arcA were highest in the Arginine microcosms, while the concentration of butyrate was higher in the Control microcosms. We demonstrate that supplementation with arginine serves a health-promoting function; it enhances microcosm resilience toward acidification and suppresses outgrowth of the opportunistic pathogen Candida. Arginine facilitates stability of oral microbial communities and prevents them from becoming cariogenic.

L-arginine prevents xanthoma development and inhibits atherosclerosis in LDL receptor knockout mice.

PubMed

Aji, W; Ravalli, S; Szabolcs, M; Jiang, X C; Sciacca, R R; Michler, R E; Cannon, P J

1997-01-21

The potential antiatherosclerotic actions of NO were investigated in four groups of mice (n = 10 per group) lacking functional LDL receptor genes, an animal model of familial hypercholesterolemia. Group 1 was fed a regular chow diet. Groups 2 through 4 were fed a 1.25% high-cholesterol diet. In addition, group 3 received supplemental L-arginine and group 4 received L-arginine and N omega-nitro-L-arginine (L-NA), an inhibitor of NO synthase (NOS). Animals were killed at 6 months; aortas were stained with oil red O for planimetry and with antibodies against constitutive and inducible NOSs. Plasma cholesterol was markedly increased in the animals receiving the high-cholesterol diet. Xanthomas appeared in all mice fed the high-cholesterol diet alone but not in those receiving L-arginine. Aortic atherosclerosis was present in all mice on the high-cholesterol diet. The mean atherosclerotic lesion area was reduced significantly (P < .01) in the cholesterol-fed mice given L-arginine compared with those receiving the high-cholesterol diet alone. The mean atherosclerotic lesion area was significantly larger (P < .01) in cholesterol-fed mice receiving L-arginine + L-NA than in those on the high-cholesterol diet alone. Within the atherosclerotic plaques, endothelial cells immunoreacted for endothelial cell NOS; macrophages, foam cells, and smooth muscle cells immunostained strongly for inducible NOS and nitrotyrosine residues. The data indicate that L-arginine prevents xanthoma formation and reduces atherosclerosis in LDL receptor knockout mice fed a high-cholesterol diet. The abrogation of the beneficial effects of L-arginine by L-NA suggests that the antiatherosclerotic actions of L-arginine are mediated by NOS. The data suggest that L-arginine may be beneficial in familial hypercholesterolemia.

Nanoenzyme-Augmented Cancer Sonodynamic Therapy by Catalytic Tumor Oxygenation.

PubMed

Zhu, Piao; Chen, Yu; Shi, Jianlin

2018-04-24

Ultrasound (US)-triggered sonodynamic therapy (SDT) can solve the critical issue of low tissue-penetrating depth of traditional phototriggered therapies, but the SDT efficacy is still not satisfactorily high in combating cancer at the current stage. Here we report on augmenting the SDT efficacy based on catalytic nanomedicine, which takes the efficient catalytic features of nanoenzymes to modulate the tumor microenvironment (TME). The multifunctional nanosonosensitizers have been successfully constructed by the integration of a MnO x component with biocompatible/biodegradable hollow mesoporous organosilica nanoparticles, followed by conjugation with protoporphyrin (as the sonosensitizer) and cyclic arginine-glycine-aspartic pentapeptide (as the targeting peptide). The MnO x component in the composite nanosonosensitizer acts as an inorganic nanoenzyme for converting the tumor-overexpressed hydrogen peroxide (H 2 O 2 ) molecules into oxygen and enhancing the tumor oxygen level subsequently, which has been demonstrated to facilitate SDT-induced reactive oxygen species production and enhance SDT efficacy subsequently. The targeted accumulation of these composite nanosonosensitizers efficiently suppressed the growth of U87 tumor xenograft on nude mice after US-triggered SDT treatment. The high in vivo biocompatibility and easy excretion of these multifunctional nanosonosensitizers from the body have also been evaluated and demonstrated to guarantee their future clinical translation, and their TME-responsive T 1 -weighted magnetic resonance imaging capability provides the potential for therapeutic guidance and monitoring during SDT.

Nitric oxide donor augments antineoplastic effects of arginine deprivation in human melanoma cells.

PubMed

Mayevska, Oksana; Chen, Oleh; Karatsai, Olena; Bobak, Yaroslav; Barska, Maryna; Lyniv, Liliana; Pavlyk, Iuliia; Rzhepetskyy, Yuriy; Igumentseva, Natalia; Redowicz, Maria Jolanta; Stasyk, Oleh

2017-06-15

Anticancer therapy based on recombinant arginine-degrading enzymes has been proposed for the treatment of several types of malignant cells deficient in arginine biosynthesis. One of the predicted side effects of such therapy is restricted bioavailability of nitric oxide as arginine catabolic product. Prolonged NO limitation may lead to unwanted disturbances in NO-dependent vasodilation, cardiovascular and immune systems. This problem can be overcome by co-supplementation with exogenous NO donor. However, NO may potentially counteract anticancer effects of therapy based on arginine deprivation. In this study, we evaluate for the first time the effects of an exogenous NO donor, sodium nitroprusside, on viability and metastatic properties of two human melanoma cell lines SK-MEL-28 and WM793 under arginine-deprived conditions. It was revealed that NO did not rescue melanoma cells from specific effects evoked by arginine deprivation, namely decreased viability and induction of apoptosis, dramatically reduced motility, invasiveness and clonogenic potential. Moreover, sodium nitroprusside co-treatment augmented several of these antineoplastic effects. We report that a combination of NO-donor and arginine deprivation strongly and specifically impaired metastatic behavior of melanoma cells. Thus, sodium nitroprusside can be considered as an adjuvant for the more efficient treatment of malignant melanoma and possibly other tumors with arginine-degrading enzymes. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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.

Arginine for gestating sows and foetal development: A systematic review.

PubMed

Palencia, J Y P; Lemes, M A G; Garbossa, C A P; Abreu, M L T; Pereira, L J; Zangeronimo, M G

2018-02-01

The use of functional amino acids during pregnancy has been linked to improved reproduction in mammals. In this context, arginine is a precursor in the synthesis of numerous molecules, such as nitric oxide and polyamines, which play an important role during reproduction. However, contradictory studies are found in the literature, particularly regarding the amount of supplementation and the period of pregnancy in which it is used. The objective of this study was to evaluate the effects of dietary arginine supplementation for pregnant sows on foetal development via a systematic review. The search for papers was performed during the month of December 2015, in the databases ISI Web of Science, Science Direct, Scopus, and SciELO. From a total of 5675 returned studies, only 13 papers were selected after applying selection criteria. Most (47%) of the studies that evaluated the effects of dietary arginine supplementation on foetal development in pigs used 1% arginine. Supplementation was initiated in the first third of pregnancy in 47% of tests, including in both primiparous and multiparous sows. These studies showed positive results for embryo survival and foetal development, evidenced by the increase in placental weight and the number and weight of piglets born alive. Of all evaluated studies, 53% showed benefits on foetal development. It is concluded that supplementing dietary arginine in gestating sows can benefit embryo survival and foetal development. However, to establish a supplementation plan with this amino acid, aspects related to the period of pregnancy, supplementation levels, and source of arginine must be well defined. © 2017 Blackwell Verlag GmbH.

Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells.

PubMed

Gou, Qing; He, ShuJiao; Zhou, ZeJian

2017-02-01

Hepatocellular carcinoma is the most common subtype of liver cancer. Protein arginine N-methyltransferase 1 was shown to be upregulated in various cancers. However, the role of protein arginine N-methyltransferase 1 in hepatocellular carcinoma progression remains incompletely understood. We investigated the clinical and functional significance of protein arginine N-methyltransferase 1 in a series of clinical hepatocellular carcinoma samples and a panel of hepatocellular carcinoma cell lines. We performed suppression analysis of protein arginine N-methyltransferase 1 using small interfering RNA to determine the biological roles of protein arginine N-methyltransferase 1 in hepatocellular carcinoma. In addition, the expression of epithelial-mesenchymal transition indicators was verified by western blotting in hepatocellular carcinoma cell lines after small interfering RNA treatment. Protein arginine N-methyltransferase 1 expression was found to be significantly upregulated in hepatocellular carcinoma cell lines and clinical tissues. Moreover, downregulation of protein arginine N-methyltransferase 1 in hepatocellular carcinoma cells by small interfering RNA could inhibit cell proliferation, migration, and invasion in vitro. These results indicate that protein arginine N-methyltransferase 1 may contribute to hepatocellular carcinoma progression and serves as a promising target for the treatment of hepatocellular carcinoma patients.

An optimized fluorescent probe for visualizing glutamate neurotransmission.

PubMed

Marvin, Jonathan S; Borghuis, Bart G; Tian, Lin; Cichon, Joseph; Harnett, Mark T; Akerboom, Jasper; Gordus, Andrew; Renninger, Sabine L; Chen, Tsai-Wen; Bargmann, Cornelia I; Orger, Michael B; Schreiter, Eric R; Demb, Jonathan B; Gan, Wen-Biao; Hires, S Andrew; Looger, Loren L

2013-02-01

We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

Moclobemide attenuates anoxia and glutamate-induced neuronal damage in vitro independently of interaction with glutamate receptor subtypes.

PubMed

Verleye, Marc; Steinschneider, Remy; Bernard, François Xavier; Gillardin, Jean-Marie

2007-03-23

Recent data suggested the existence of a bidirectional relation between depression and neurodegenerative diseases resulting from cerebral ischemia injury. Glutamate, a major excitatory neurotransmitter, has long been recognised to play a key role in the pathophysiology of anoxia or ischemia, due to its excessive accumulation in the extracellular space and the subsequent activation of its receptors. A characteristic response to glutamate is the increase in cytosolic Na(+) and Ca(2+) levels which is due mainly to influx from the extracellular space, with a consequent cell swelling and oxidative metabolism dysfunction. The present study examined the in vitro effects of the antidepressant and type-A monoamine oxidase inhibitor, moclobemide, in neuronal-astroglial cultures from rat cerebral cortex exposed to anoxia (for 5 and 7 h) or to glutamate (2 mM for 6 h), two in vitro models of brain ischemia. In addition, the affinity of moclobemide for the different glutamate receptor subtypes and an interaction with the cell influx of Na(+) and of Ca(2+) enhanced by veratridine and K(+) excess, respectively, were evaluated. Moclobemide (10-100 microM) included in the culture medium during anoxia or with glutamate significantly increased in a concentration-dependent manner the amount of surviving neurons compared to controls. Moclobemide displayed no binding affinity for the different glutamate receptor subtypes (IC(50)>100 microM) and did not block up to 300 microM the entry of Na(+) and of Ca(2+) activated by veratridine and K(+), respectively. These results suggest that the neuroprotective properties of moclobemide imply neither the glutamate neurotransmission nor the Na(+) and Ca(2+) channels.

High asymmetric dimethylarginine, symmetric dimethylarginine and L-arginine levels in migraine patients.

PubMed

Reyhani, Aylin; Celik, Yahya; Karadag, Hakan; Gunduz, Ozgur; Asil, Talip; Sut, Necdet

2017-07-01

Experimental and clinical data strongly suggests that nitric oxide (NO) plays a pivotal role in migraine. This is also supported by studies of migraine induced by substances that release NO. NO is synthesized from L-arginine by endothelial NO synthase (NOS). Asymmetric dimethylarginine (ADMA) is the major endogenous competitive inhibitor of NOS. Symmetric dimethylarginine (SDMA) is an inactive stereoisomer of ADMA. It may reduce NO production by competing with arginine for cellular uptake. The aim of this study was to measure the levels of ADMA, SDMA and L-arginine in migraine patients during the interictal period. One hundred migraine patients and 100 healthy volunteers were recruited. The patients were in the interictal period and classified into two groups as having migraine with aura and migraine without aura. Their serum ADMA, SDMA and L-arginine levels were measured by high-performance liquid chromotography (HPLC) method. ADMA, SDMA and L-arginine levels were significantly higher in migraine patients compared to the control group. But there was no difference between the patients with and without aura. These results suggest that NOS inhibitors and L-arginine/NO pathway plays an important role in migraine pathopysiology.

Intestinal absorption of an arginine-containing peptide in cystinuria

PubMed Central

Asatoor, A. M.; Harrison, B. D. W.; Milne, M. D.; Prosser, D. I.

1972-01-01

Separate tolerance tests involving oral intake of the dipeptide, L-arginyl-L-aspartate, and of a corresponding free amino acid mixture, were carried out in a single type 2 cystinuric patient. Absorption of aspartate was within normal limits, whilst that of arginine was normal after the peptide but considerably reduced after the amino acid mixture. The results are compared with the increments of serum arginine found in eight normal subjects after the oral intake of the free amino acid mixture. Analyses of urinary pyrrolidine and of tetramethylenediamine in urine samples obtained after the two tolerance tests in the patient support the view that arginine absorption was subnormal after the amino acid mixture but within normal limits after the dipeptide. PMID:5045711

An examination of the roles of glutamate and sex in latent inhibition: Relevance to the glutamate hypothesis of schizophrenia?

PubMed

Huang, Andrew Chih Wei; Bo-Han He, Alan; Chen, Chih-Chung

2017-10-01

The present study examined the effects of the glutamate receptor antagonist MK-801, the glutamate receptor agonist N-methyl-D-aspartate (NMDA), and sexual dimorphism on latent inhibition to elucidate the glutamate hypothesis of schizophrenia. During the pre-exposure phase, 56 male and 65 female Wistar rats were intracerebroventricularly administered normal saline, MK-801 or NMDA, in the left ventricle and then exposed to a passive avoidance box (or a different context) in three trials over 3 days. Then, all of the rats were placed in the light compartment of the passive avoidance box and were allowed to enter the dark compartment, where they each received a footshock (1mA, 2s) in five trials over 5 days. Injections of the glutamate drugs NMDA and MK-801 did not affect latent inhibition. Sexual dimorphism did not occur in latent inhibition. The present data on the male rats indicated that the glutamate system did not affect latent inhibition, indicating that the glutamate system was not like the dopamine system in terms of mediating the positive symptoms of schizophrenia. The glutamate system might be involved in the negative and cognitive symptoms of schizophrenia. The results may provide information for novel treatments of the negative and cognitive symptoms of schizophrenia. Copyright © 2017 Elsevier B.V. All rights reserved.

Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming ω-NG-monomethylated arginine residues.

PubMed

Zurita-Lopez, Cecilia I; Sandberg, Troy; Kelly, Ryan; Clarke, Steven G

2012-03-09

Full-length human protein arginine methyltransferase 7 (PRMT7) expressed as a fusion protein in Escherichia coli was initially found to generate only ω-N(G)-monomethylated arginine residues in small peptides, suggesting that it is a type III enzyme. A later study, however, characterized fusion proteins of PRMT7 expressed in bacterial and mammalian cells as a type II/type I enzyme, capable of producing symmetrically dimethylated arginine (type II activity) as well as small amounts of asymmetric dimethylarginine (type I activity). We have sought to clarify the enzymatic activity of human PRMT7. We analyzed the in vitro methylation products of a glutathione S-transferase (GST)-PRMT7 fusion protein with robust activity using a variety of arginine-containing synthetic peptides and protein substrates, including a GST fusion with the N-terminal domain of fibrillarin (GST-GAR), myelin basic protein, and recombinant human histones H2A, H2B, H3, and H4. Regardless of the methylation reaction conditions (incubation time, reaction volume, and substrate concentration), we found that PRMT7 only produces ω-N(G)-monomethylarginine with these substrates. In control experiments, we showed that mammalian GST-PRMT1 and Myc-PRMT5 were, unlike PRMT7, able to dimethylate both peptide P-SmD3 and SmB/D3 to give the expected asymmetric and symmetric products, respectively. These experiments show that PRMT7 is indeed a type III human methyltransferase capable of forming only ω-N(G)-monomethylarginine, not asymmetric ω-N(G),N(G)-dimethylarginine or symmetric ω-N(G),N(G')-dimethylarginine, under the conditions tested.

Prefrontal glutamate correlates of methamphetamine sensitization and preference

PubMed Central

Lominac, Kevin D.; Quadir, Sema G.; Barrett, Hannah M.; McKenna, Courtney L.; Schwartz, Lisa M.; Ruiz, Paige N.; Wroten, Melissa G.; Campbell, Rianne R.; Miller, Bailey W.; Holloway, John J.; Travis, Katherine O.; Rajasekar, Ganesh; Maliniak, Dan; Thompson, Andrew B.; Urman, Lawrence E.; Kippin, Tod E.; Phillips, Tamara J.; Szumlinski, Karen K.

2016-01-01

Methamphetamine (MA) is a widely abused, highly addictive, psychostimulant that elicits pronounced deficits in neurocognitive function related to hypo-functioning of the prefrontal cortex (PFC). Our understanding of how repeated methamphetamine impacts excitatory glutamatergic transmission within the PFC is limited, as is information about the relation between PFC glutamate and addiction vulnerability/resiliency. In vivo microdialysis and immunoblotting studies characterized the effects of methamphetamine (10 injections of 2 mg/kg, IP) upon extracellular glutamate in C57BL/6J mice and upon glutamate receptor and transporter expression, within the medial PFC. Glutamatergic correlates of both genetic and idiopathic variance in MA preference/intake were determined through studies of high versus low MA-drinking selectively bred mouse lines (MAHDR versus MALDR, respectively) and inbred C57BL/6J mice exhibiting spontaneously divergent place-conditioning phenotypes. Repeated methamphetamine sensitized drug-induced glutamate release and lowered indices of NMDA receptor expression in C57BL/6J mice, but did not alter basal extracellular glutamate content or total protein expression of Homer proteins, or metabotropic or AMPA glutamate receptors. Elevated basal glutamate, blunted methamphetamine-induced glutamate release and ERK activation, as well as reduced protein expression of mGlu2/3 and Homer2a/b were all correlated biochemical traits of selection for high versus low methamphetamine drinking, and Homer2a/b levels were inversely correlated with the motivational valence of methamphetamine in C57BL/6J mice. These data provide novel evidence that repeated, low-dose, methamphetamine is sufficient to perturb pre- and post-synaptic aspects of glutamate transmission within the medial PFC and that glutamate anomalies within this region may contribute to both genetic and idiopathic variance in methamphetamine addiction vulnerability/resiliency. PMID:26742098

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.

PubMed

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.

Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

PubMed

Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

2014-02-01

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.

L-arginine and Arginase Products Potentiate Dexmedetomidine-induced Contractions in the Rat Aorta.

PubMed

Wong, Emily S W; Man, Ricky Y K; Ng, Kwok F J; Leung, Susan W S; Vanhoutte, Paul M

2018-03-01

The α2-adrenergic sedative/anesthetic agent dexmedetomidine exerts biphasic effects on isolated arteries, causing endothelium-dependent relaxations at concentrations at or below 30 nM, followed by contractions at higher concentrations. L-arginine is a common substrate of endothelial nitric oxide synthase and arginases. This study was designed to investigate the role of L-arginine in modulating the overall vascular response to dexmedetomidine. Isometric tension was measured in isolated aortic rings of Sprague Dawley rats. Cumulative concentrations of dexmedetomidine (10 nM to 10 μM) were added to quiescent rings (with and without endothelium) after previous incubation with vehicle, N-nitro-L-arginine methyl ester hydrochloride (L-NAME; nitric oxide synthase inhibitor), prazosin (α1-adrenergic antagonist), rauwolscine (α2-adrenergic antagonist), L-arginine, (S)-(2-boronethyl)-L-cysteine hydrochloride (arginase inhibitor), N-hydroxy-L-arginine (arginase inhibitor), urea and/or ornithine. In some preparations, immunofluorescent staining, immunoblotting, or measurement of urea content were performed. Dexmedetomidine did not contract control rings with endothelium but evoked concentration-dependent increases in tension in such rings treated with L-NAME (Emax 50 ± 4%) or after endothelium-removal (Emax 74 ± 5%; N = 7 to 12). Exogenous L-arginine augmented the dexmedetomidine-induced contractions in the presence of L-NAME (Emax 75 ± 3%). This potentiation was abolished by (S)-(2-boronethyl)-L-cysteine hydrochloride (Emax 16 ± 4%) and N-hydroxy-L-arginine (Emax 18 ± 4%). Either urea or ornithine, the downstream arginase products, had a similar potentiating effect as L-arginine. Immunoassay measurements demonstrated an upregulation of arginase I by L-arginine treatment in the presence of L-NAME (N = 4). These results suggest that when vascular nitric oxide homeostasis is impaired, the potentiation of the vasoconstrictor effect of

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

PubMed

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.

Grewia tiliaefolia and its active compound vitexin regulate the expression of glutamate transporters and protect Neuro2a cells from glutamate toxicity.

PubMed

Malar, Dicson Sheeja; Prasanth, Mani Iyer; Shafreen, Rajamohamed Beema; Balamurugan, Krishnaswamy; Devi, Kasi Pandima

2018-04-25

Glutamate is a major neurotransmitter involved in several brain functions and glutamate excitotoxicity is involved in Alzheimer's disease (AD). In the current study, the neuroprotective effect of the Indian medicinal plant Grewia tiliaefolia (GT) and its active component vitexin was evaluated in Neuro-2a cells against glutamate toxicity. Neuro-2a cells were exposed to glutamate to cause excitotoxicity and the neuroprotective effect of GT and vitexin were evaluated using biochemical studies (estimation of reactive oxygen species, reactive nitrogen species, protein carbonyl content, lipid peroxidation level, mitochondrial membrane potential and caspase-3 activity), molecular docking studies, gene expression and western blot analysis. Glutamate exposure to Neuro-2a cells induced oxidative stress, loss of membrane potential, suppressed the expression of antioxidant response genes (Nrf-2, HO-1, NQO-1), glutamate transporters (GLAST-1, GLT-1) and induced the expression of NMDAR, Calpain. However, pre-treatment of cells with GT/vitexin inhibited oxidative stress mediated damage by augmenting the expression of Nrf-2/HO-1 pathway, inducing the expression of glutamate transporters and downregulating Calpain, NMDAR. Molecular docking showed that vitexin effectively binds to NMDAR and GSK-3β and thereby can inhibit their activation. GT/vitexin also inhibited glutamate induced Bax expression. Methanol extract of G. tiliaefolia and its active component vitexin can act in an antioxidant dependent mechanism as well as by regulating glutamate in mitigating the toxicity exerted by glutamate in Neuro-2a cells. Our results conclude that GT/vitexin can act as potential drug leads for the therapeutic intervention of AD. Copyright © 2017. Published by Elsevier Inc.

Increased pain and muscle glutamate concentration after single ingestion of monosodium glutamate by myofascial temporomandibular disorders patients.

PubMed

Shimada, A; Castrillon, E E; Baad-Hansen, L; Ghafouri, B; Gerdle, B; Wåhlén, K; Ernberg, M; Cairns, B E; Svensson, P

2016-10-01

A randomized, double-blinded, placebo-controlled study was conducted to investigate if single monosodium glutamate (MSG) administration would elevate muscle/serum glutamate concentrations and affect muscle pain sensitivity in myofascial temporomandibular disorders (TMD) patients more than in healthy individuals. Twelve myofascial TMD patients and 12 sex- and age-matched healthy controls participated in two sessions. Participants drank MSG (150 mg/kg) or NaCl (24 mg/kg; control) diluted in 400 mL of soda. The concentration of glutamate in the masseter muscle, blood plasma and saliva was determined before and after the ingestion of MSG or control. At baseline and every 15 min after the ingestion, pain intensity was scored on a 0-10 numeric rating scale. Pressure pain threshold, pressure pain tolerance (PPTol) and autonomic parameters were measured. All participants were asked to report adverse effects after the ingestion. In TMD, interstitial glutamate concentration was significantly greater after the MSG ingestion when compared with healthy controls. TMD reported a mean pain intensity of 2.8/10 at baseline, which significantly increased by 40% 30 min post MSG ingestion. At baseline, TMD showed lower PPTols in the masseter and trapezius, and higher diastolic blood pressure and heart rate than healthy controls. The MSG ingestion resulted in reports of headache by half of the TMD and healthy controls, respectively. These findings suggest that myofascial TMD patients may be particularly sensitive to the effects of ingested MSG. WHAT DOES THIS STUDY ADD?': Elevation of interstitial glutamate concentration in the masseter muscle caused by monosodium glutamate (MSG) ingestion was significantly greater in myofascial myofascial temporomandibular disorders (TMD) patients than healthy individuals. This elevation of interstitial glutamate concentration in the masseter muscle significantly increased the intensity of spontaneous pain in myofascial TMD patients. © 2016

The second case of a young man with L-arginine-induced acute pancreatitis.

PubMed

Binet, Quentin; Dufour, Inès; Agneessens, Emmanuel; Debongnie, Jean-Claude; Aouattah, Tarik; Covas, Angélique; Coche, Jean-Charles; De Koninck, Xavier

2018-04-21

Dietary supplementation of arginine has been used by numerous world-class athletes and professional bodybuilders over the past 30 years. L-Arginine indeed enhances muscular power and general performance via maintaining ATP level. However, L-arginine is also known to induce acute pancreatitis in murine models. We report the case of young man presenting with upper abdominal pain and increased serum lipase levels. Contrast-enhanced computed tomography confirms a mild acute pancreatitis. Common etiologies have been ruled out and toxicological anamnestic screening reveals the intake of protein powder. This is, to the best of our knowledge, the second case in human of arginine-induced acute pancreatitis. This case report suggests that every patient presenting with acute pancreatitis without obvious etiology should be evaluated for the intake of toxics other than alcohol, including L-arginine.