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Sample records for arginine protein translocase

  1. TatE as a Regular Constituent of Bacterial Twin-arginine Protein Translocases.

    Science.gov (United States)

    Eimer, Ekaterina; Fröbel, Julia; Blümmel, Anne-Sophie; Müller, Matthias

    2015-12-01

    Twin-arginine translocation (Tat) systems mediate the transmembrane translocation of completely folded proteins that possess a conserved twin-arginine (RR) motif in their signal sequences. Many Tat systems consist of three essential membrane components named TatA, TatB, and TatC. It is not understood why some bacteria, in addition, constitutively express a functional paralog of TatA called TatE. Here we show, in live Escherichia coli cells, that, upon expression of a Tat substrate protein, fluorescently labeled TatE-GFP relocates from a rather uniform distribution in the plasma membrane into a number of discrete clusters. Clustering strictly required an intact RR signal peptide and the presence of the TatABC subunits, suggesting that TatE-GFP associates with functional Tat translocases. In support of this notion, site-specific photo cross-linking revealed interactions of TatE with TatA, TatB, and TatC. The same approach also disclosed a pronounced tendency of TatE and TatA to hetero-oligomerize. Under in vitro conditions, we found that TatE replaces TatA inefficiently. Our collective results are consistent with TatE being a regular constituent of the Tat translocase in E. coli.

  2. A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes

    NARCIS (Netherlands)

    Patel, Roshani; Vasilev, Cvetelin; Beck, Daniel; Monteferrante, Carmine G; van Dijl, Jan Maarten; Hunter, C Neil; Smith, Corinne; Robinson, Colin

    2014-01-01

    The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report

  3. Co-factor insertion and disulfide bond requirements for twin-arginine translocase-dependent export of the Bacillus subtilis Rieske protein QcrA

    NARCIS (Netherlands)

    Goosens, Vivianne J; Monteferrante, Carmine G; van Dijl, Jan Maarten

    2014-01-01

    Background: The Rieske protein QcrA was recently shown to be exported by twin-arginine translocation (Tat) in Bacillus subtilis. Results: QcrA has disulfide bond and co-factor requirements for effective Tat-dependent translocation. Conclusion: A hierarchy exists between disulfide bonding and co-fact

  4. Unique Photobleaching Phenomena of the Twin-Arginine Translocase Respiratory Enzyme Chaperone DmsD

    OpenAIRE

    Rivardo, Fabrizio; Leach, Thorin G.H.; Chan, Catherine S.; Winstone, Tara M.L.; Ladner, Carol L.; Sarfo, Kwabena J.; Turner, Raymond J.

    2014-01-01

    DmsD is a chaperone of the redox enzyme maturation protein family specifically required for biogenesis of DMSO reductase in Escherichia coli. It exists in multiple folding forms, all of which are capable of binding its known substrate, the twin-arginine leader sequence of the DmsA catalytic subunit. It is important for maturation of the reductase and targeting to the cytoplasmic membrane for translocation. Here, we demonstrate that DmsD exhibits an irreversible photobleaching phenomenon upon ...

  5. Unique Photobleaching Phenomena of the Twin-Arginine Translocase Respiratory Enzyme Chaperone DmsD

    Science.gov (United States)

    Rivardo, Fabrizio; Leach, Thorin G.H.; Chan, Catherine S.; Winstone, Tara M.L.; Ladner, Carol L.; Sarfo, Kwabena J.; Turner, Raymond J.

    2014-01-01

    DmsD is a chaperone of the redox enzyme maturation protein family specifically required for biogenesis of DMSO reductase in Escherichia coli. It exists in multiple folding forms, all of which are capable of binding its known substrate, the twin-arginine leader sequence of the DmsA catalytic subunit. It is important for maturation of the reductase and targeting to the cytoplasmic membrane for translocation. Here, we demonstrate that DmsD exhibits an irreversible photobleaching phenomenon upon 280 nm excitation irradiation. The phenomenon is due to quenching of the tryptophan residues in DmsD and is dependent on its folding and conformation. We also show that a tryptophan residue involved in DmsA signal peptide binding (W87) is important for photobleaching of DmsD. Mutation of W87, or binding of the DmsA twin-arginine signal peptide to DmsD in the pocket that includes W72, W80, and W91 significantly affects the degree of photobleaching. This study highlights the advantage of a photobleaching phenomenon to study protein folding and conformation changes within a protein that was once considered unusable in fluorescence spectroscopy. PMID:24497893

  6. Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins.

    Science.gov (United States)

    Stiller, Sebastian B; Höpker, Jan; Oeljeklaus, Silke; Schütze, Conny; Schrempp, Sandra G; Vent-Schmidt, Jens; Horvath, Susanne E; Frazier, Ann E; Gebert, Natalia; van der Laan, Martin; Bohnert, Maria; Warscheid, Bettina; Pfanner, Nikolaus; Wiedemann, Nils

    2016-05-10

    The mitochondrial inner membrane harbors three protein translocases. Presequence translocase and carrier translocase are essential for importing nuclear-encoded proteins. The oxidase assembly (OXA) translocase is required for exporting mitochondrial-encoded proteins; however, different views exist about its relevance for nuclear-encoded proteins. We report that OXA plays a dual role in the biogenesis of nuclear-encoded mitochondrial proteins. First, a systematic analysis of OXA-deficient mitochondria led to an unexpected expansion of the spectrum of OXA substrates imported via the presequence pathway. Second, biogenesis of numerous metabolite carriers depends on OXA, although they are not imported by the presequence pathway. We show that OXA is crucial for the biogenesis of the Tim18-Sdh3 module of the carrier translocase. The export translocase OXA is thus required for the import of metabolite carriers by promoting assembly of the carrier translocase. We conclude that OXA is of central importance for the biogenesis of the mitochondrial inner membrane.

  7. Mapping the twin-arginine protein translocation network of Bacillus subtilis

    NARCIS (Netherlands)

    Monteferrante, Carmine G.; MacKichan, Calum; Marchadier, Elodie; Prejean, Maria-Victoria; Carballido-Lopez, Rut; van Dijl, Jan Maarten

    2013-01-01

    Bacteria employ twin-arginine translocation (Tat) pathways for the transport of folded proteins to extracytoplasmic destinations. In recent years, most studies on bacterial Tat pathways addressed the membrane-bound TatA(B)C subunits of the Tat translocase, and the specific interactions between this

  8. A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid [Delta]pH/Tat translocase.

    Science.gov (United States)

    Mori, Hiroki; Cline, Kenneth

    2002-04-15

    The thylakoid DeltapH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane DeltapH as the sole energy source. Three known components of the transport machinery exist in two distinct subcomplexes. A cpTatC-Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition. Here we report that Tha4 assembles with cpTatC-Hcf106 during the translocation step. Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting. cpTatC and Hcf106 were consistently associated under all conditions tested. In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the DeltapH. Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly. The association of all three components was transient and dissipated upon the completion of protein translocation. Such an assembly-disassembly cycle could explain how the DeltapH/Tat system can assemble translocases to accommodate folded proteins of varied size. It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

  9. Structure of TatA paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that transports folded proteins of differing diameter.

    Science.gov (United States)

    Baglieri, Jacopo; Beck, Daniel; Vasisht, Nishi; Smith, Corinne J; Robinson, Colin

    2012-03-01

    The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ∼50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70-90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6-8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed.

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

    Science.gov (United States)

    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 (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. PMID:25775142

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

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    Juan C Marini

    Full Text Available 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.

  12. Evaluation of Porin Interaction with Adenine Nucleotide Translocase and Cyclophilin-D Proteins after Brain Ischemia and Reperfusion

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    Mohammad Ali Atlasi

    2011-01-01

    Full Text Available Objective (s Porin is a mitochondrial outer membrane channel, which usually functions as the pathway for the movement of various substances in and out of the mitochondria and is considered to be a component of the permeability transition (PT pore complex that plays a role in the PT. We addressed the hypothesis that porin interacts with other mitochondrial proteins after ischemic injury.Materials and MethodsFor this purpose, we used in vivo 4-vessel occlusion model of rat brain and porin purification method by hydroxyapatite column. After SDS gel electrophoresis and silver nitrate staining, Western blotting was done for porin, adenine nucleotide translocase and cyclophilin-D proteins.Results Porin was purified from mitochondrial mixture in ischemic brain and control groups. Investigation of interaction of adenine nucleotide transposes (ANT and cyclophilin-D with porin by Western blotting showed no proteins co-purified with porin from injured tissues.Conclusion The present study implies that there may not be interaction between porin, and ANT or cyclophilin-D, and if there is any, it is not maintained during the purification procedure.

  13. Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

    KAUST Repository

    Shah, Dhawal

    2011-09-21

    We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ XP, contrary to conventional thinking that positive Γ XP\\'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine\\'s preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine\\'s interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ XP, which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation. © 2011 American Institute of Chemical Engineers (AIChE).

  14. Theoretical insights into catalytic mechanism of protein arginine methyltransferase 1.

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    Ruihan Zhang

    Full Text Available Protein arginine methyltransferase 1 (PRMT1, the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD simulation and quantum mechanics/molecular mechanics (QM/MM calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical SN2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.

  15. Haloarchaeal Protein Translocation via the Twin Arginine Translocation Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Pohlschroder Mechthild

    2009-02-03

    Protein transport across hydrophobic membranes that partition cellular compartments is essential in all cells. The twin arginine translocation (Tat) pathway transports proteins across the prokaryotic cytoplasmic membranes. Distinct from the universally conserved Sec pathway, which secretes unfolded proteins, the Tat machinery is unique in that it secretes proteins in a folded conformation, making it an attractive pathway for the transport and secretion of heterologously expressed proteins that are Sec-incompatible. During the past 7 years, the DOE-supported project has focused on the characterization of the diversity of bacterial and archaeal Tat substrates as well as on the characterization of the Tat pathway of a model archaeon, Haloferax volcanii, a member of the haloarchaea. We have demonstrated that H. volcanii uses this pathway to transport most of its secretome.

  16. An allosteric inhibitor of protein arginine methyltransferase 3.

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    Siarheyeva, Alena; Senisterra, Guillermo; Allali-Hassani, Abdellah; Dong, Aiping; Dobrovetsky, Elena; Wasney, Gregory A; Chau, Irene; Marcellus, Richard; Hajian, Taraneh; Liu, Feng; Korboukh, Ilia; Smil, David; Bolshan, Yuri; Min, Jinrong; Wu, Hong; Zeng, Hong; Loppnau, Peter; Poda, Gennadiy; Griffin, Carly; Aman, Ahmed; Brown, Peter J; Jin, Jian; Al-Awar, Rima; Arrowsmith, Cheryl H; Schapira, Matthieu; Vedadi, Masoud

    2012-08-01

    PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.

  17. Arginine Inhibits Adsorption of Proteins on Polystyrene Surface

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    Shikiya, Yui; Tomita, Shunsuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2013-01-01

    Nonspecific adsorption of protein on solid surfaces causes a reduction of concentration as well as enzyme inactivation during purification and storage. However, there are no versatile inhibitors of the adsorption between proteins and solid surfaces at low concentrations. Therefore, we examined additives for the prevention of protein adsorption on polystyrene particles (PS particles) as a commonly-used material for vessels such as disposable test tubes and microtubes. A protein solution was mixed with PS particles, and then adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to PS particles through electrostatic, hydrophobic, and aromatic interactions, causing a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives, including arginine hydrochloride (Arg), lysine hydrochloride, guanidine hydrochloride, NaCl, glycine, and glucose, Arg was most effective in preventing the binding of proteins to PS particles as well as activity loss. Moreover, even after the mixing of protein and PS particles, the addition of Arg caused desorption of the bound protein from PS particles. This study demonstrated a new function of Arg, which expands the potential for application of Arg to proteins. PMID:23967100

  18. The Role of Protein Arginine Methyltransferases in Inflammatory Responses

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    Ji Hye Kim

    2016-01-01

    Full Text Available Protein arginine methyltransferases (PRMTs mediate the methylation of a number of protein substrates of arginine residues and serve critical functions in many cellular responses, including cancer development, progression, and aggressiveness, T-lymphocyte activation, and hepatic gluconeogenesis. There are nine members of the PRMT family, which are divided into 4 types (types I–IV. Although most PRMTs do not require posttranslational modification (PTM to be activated, fine-tuning modifications, such as interactions between cofactor proteins, subcellular compartmentalization, and regulation of RNA, via micro-RNAs, seem to be required. Inflammation is an essential defense reaction of the body to eliminate harmful stimuli, including damaged cells, irritants, or pathogens. However, chronic inflammation can eventually cause several types of diseases, including some cancers, atherosclerosis, rheumatoid arthritis, and periodontitis. Therefore, inflammation responses should be well modulated. In this review, we briefly discuss the role of PRMTs in the control of inflammation. More specifically, we review the roles of four PRMTs (CARM1, PRMT1, PRMT5, and PRMT6 in modulating inflammation responses, particularly in terms of modulating the transcriptional factors or cofactors related to inflammation. Based on the regulatory roles known so far, we propose that PRMTs should be considered one of the target molecule groups that modulate inflammatory responses.

  19. Mechanistic studies on transcriptional coactivator protein arginine methyltransferase 1.

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    Rust, Heather L; Zurita-Lopez, Cecilia I; Clarke, Steven; Thompson, Paul R

    2011-04-26

    Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to the guanidinium group of arginine residues in a number of important cell signaling proteins. PRMT1 is the founding member of this family, and its activity appears to be dysregulated in heart disease and cancer. To begin to characterize the catalytic mechanism of this isozyme, we assessed the effects of mutating a number of highly conserved active site residues (i.e., Y39, R54, E100, E144, E153, M155, and H293), which are believed to play key roles in SAM recognition, substrate binding, and catalysis. The results of these studies, as well as pH-rate studies, and the determination of solvent isotope effects (SIEs) indicate that M155 plays a critical role in both SAM binding and the processivity of the reaction but is not responsible for the regiospecific formation of asymmetrically dimethylated arginine (ADMA). Additionally, mutagenesis studies on H293, combined with pH studies and the lack of a normal SIE, do not support a role for this residue as a general base. Furthermore, the lack of a normal SIE with either the wild type or catalytically impaired mutants suggests that general acid/base catalysis is not important for promoting methyl transfer. This result, combined with the fact that the E144A/E153A double mutant retains considerably more activity then the single mutants alone, suggests that the PRMT1-catalyzed reaction is primarily driven by bringing the substrate guanidinium into the proximity of the S-methyl group of SAM and that the prior deprotonation of the substrate guanidinium is not required for methyl transfer.

  20. Evaluation of chemical labeling methods for identifying functional arginine residues of proteins by mass spectrometry.

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    Wanigasekara, Maheshika S K; Chowdhury, Saiful M

    2016-09-01

    Arginine residues undergo several kinds of post-translational modifications (PTMs). These PTMs are associated with several inflammatory diseases, such as rheumatoid arthritis, atherosclerosis, and diabetes. Mass spectrometric studies of arginine modified proteins and peptides are very important, not only to identify the reactive arginine residues but also to understand the tandem mass spectrometry behavior of these peptides for assigning the sequences unambiguously. Herein, we utilize tandem mass spectrometry to report the performance of two widely used arginine labeling reagents, 1,2-cyclohexanedione (CHD) and phenylglyoxal (PG) with several arginine containing peptides and proteins. Time course labeling studies were performed to demonstrate the selectivity of the reagents in proteins or protein digests. Structural studies on the proteins were also explored to better understand the reaction sites and position of arginine residues. We found CHD showed better labeling efficiencies compared to phenylglyoxal. Reactive arginine profiling on a purified albumin protein clearly pointed out the cellular glycation modification site for this protein with high confidence. We believe these detailed mass-spectrometric studies will provide significant input to profile reactive arginine residues in large-scale studies; therefore, targeted proteomics can be performed to the short listed reactive sites for cellular arginine modifications. PMID:27543028

  1. SecDF as part of the Sec-translocase facilitates efficient secretion of Bacillus cereus toxins and cell wall-associated proteins.

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    Vörös, Aniko; Simm, Roger; Slamti, Leyla; McKay, Matthew J; Hegna, Ida K; Nielsen-LeRoux, Christina; Hassan, Karl A; Paulsen, Ian T; Lereclus, Didier; Økstad, Ole Andreas; Molloy, Mark P; Kolstø, Anne-Brit

    2014-01-01

    The aim of this study was to explore the role of SecDF in protein secretion in Bacillus cereus ATCC 14579 by in-depth characterization of a markerless secDF knock out mutant. Deletion of secDF resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the ΔsecDF mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of secDF indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen B. cereus, and further supports the notion that B. cereus enterotoxins are secreted by the Sec-system.

  2. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM.

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    Daniel O Frank

    Full Text Available The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM. In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20 by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  3. Aflatoxin B1 induced upregulation of protein arginine methyltransferase 5 in human cell lines.

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    Ghufran, Md Sajid; Ghosh, Krishna; Kanade, Santosh R

    2016-09-01

    The exposure of naturally occurring mycotoxins affects human health and play a vital role in cancer initiation and progression. Aflatoxin B1 is a difuranocoumarin mycotoxin, classified as a group I carcinogen. The present study was conducted to assess the effect of aflatoxin B1 on epigenetic regulatory proteins. The protein arginine methyltransferase 5 expression was induced upon aflatoxin B1 treatment in a dose and time dependent manner. Further global arginine methylation was also increased in the same manner. This is the first report showing the induction of epigenetic regulatory protein, protein arginine methyltransferase 5 upon aflatoxin B1 treatment. Further study is required to establish the detailed pathway of PRMT5 induction. PMID:27242039

  4. Radiometric assay for determining the incorporation of L-canavanine or L-arginine into protein

    Energy Technology Data Exchange (ETDEWEB)

    Rosenthal, G.A.; Thomas, D.A.

    1985-06-01

    Procedures for a radiometric assay of L-(guanidinooxy-/sup 14/C)canavanine were developed which provide a convenient and accurate measure of the incorporation of (/sup 14/C)canavanine into de novo-synthesized proteins. These methods are also applicable to determining (/sup 14/C)arginine incorporation into protein. These procedures have been employed to study the synthesis of L-(guanidinooxy-/sup 14/C)canavanine- and L-(guanidino-/sup 14/C)arginine-containing proteins from the hemolymph of Manduca sexta and Heliothis virescens, two highly destructive insect pests.

  5. Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase.

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    Sinha, Devanjan; Srivastava, Shubhi; D'Silva, Patrick

    2016-08-12

    Mitochondrial J-proteins play a critical role in governing Hsp70 activity and, hence, are essential for organellar protein translocation and folding. In contrast to yeast, which has a single J-protein Pam18, humans involve two J-proteins, DnaJC15 and DnaJC19, associated with contrasting cellular phenotype, to transport proteins into the mitochondria. Mutation in DnaJC19 results in dilated cardiomyopathy and ataxia syndrome, whereas expression of DnaJC15 regulates the response of cancer cells to chemotherapy. In the present study we have comparatively assessed the biochemical properties of the J-protein paralogs in relation to their association with the import channel. Both DnaJC15 and DnaJC19 formed two distinct subcomplexes with Magmas at the import channel. Knockdown analysis suggested an essential role for Magmas and DnaJC19 in organellar protein translocation and mitochondria biogenesis, whereas DnaJC15 had dispensable supportive function. The J-proteins were found to have equal affinity for Magmas and could stimulate mitochondrial Hsp70 ATPase activity by equivalent levels. Interestingly, we observed that DnaJC15 exhibits bifunctional properties. At the translocation channel, it involves conserved interactions and mechanism to translocate the precursors into mitochondria. In addition to protein transport, DnaJC15 also showed a dual role in yeast where its expression elicited enhanced sensitivity of cells to cisplatin that required the presence of a functional J-domain. The amount of DnaJC15 expressed in the cell was directly proportional to the sensitivity of cells. Our analysis indicates that the differential cellular phenotype displayed by human mitochondrial J-proteins is independent of their activity and association with Magmas at the translocation channel.

  6. Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase.

    Science.gov (United States)

    Sinha, Devanjan; Srivastava, Shubhi; D'Silva, Patrick

    2016-08-12

    Mitochondrial J-proteins play a critical role in governing Hsp70 activity and, hence, are essential for organellar protein translocation and folding. In contrast to yeast, which has a single J-protein Pam18, humans involve two J-proteins, DnaJC15 and DnaJC19, associated with contrasting cellular phenotype, to transport proteins into the mitochondria. Mutation in DnaJC19 results in dilated cardiomyopathy and ataxia syndrome, whereas expression of DnaJC15 regulates the response of cancer cells to chemotherapy. In the present study we have comparatively assessed the biochemical properties of the J-protein paralogs in relation to their association with the import channel. Both DnaJC15 and DnaJC19 formed two distinct subcomplexes with Magmas at the import channel. Knockdown analysis suggested an essential role for Magmas and DnaJC19 in organellar protein translocation and mitochondria biogenesis, whereas DnaJC15 had dispensable supportive function. The J-proteins were found to have equal affinity for Magmas and could stimulate mitochondrial Hsp70 ATPase activity by equivalent levels. Interestingly, we observed that DnaJC15 exhibits bifunctional properties. At the translocation channel, it involves conserved interactions and mechanism to translocate the precursors into mitochondria. In addition to protein transport, DnaJC15 also showed a dual role in yeast where its expression elicited enhanced sensitivity of cells to cisplatin that required the presence of a functional J-domain. The amount of DnaJC15 expressed in the cell was directly proportional to the sensitivity of cells. Our analysis indicates that the differential cellular phenotype displayed by human mitochondrial J-proteins is independent of their activity and association with Magmas at the translocation channel. PMID:27330077

  7. Study of the five Rickettsia prowazekii proteins annotated as ATP/ADP translocases (Tlc): Only Tlc1 transports ATP/ADP, while Tlc4 and Tlc5 transport other ribonucleotides.

    Science.gov (United States)

    Audia, Jonathon P; Winkler, Herbert H

    2006-09-01

    The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tlc homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tlc genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a K(i) similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins. PMID:16923893

  8. Discovery of a Potent Class I Protein Arginine Methyltransferase Fragment Inhibitor.

    Science.gov (United States)

    Ferreira de Freitas, Renato; Eram, Mohammad S; Szewczyk, Magdalena M; Steuber, Holger; Smil, David; Wu, Hong; Li, Fengling; Senisterra, Guillermo; Dong, Aiping; Brown, Peter J; Hitchcock, Marion; Moosmayer, Dieter; Stegmann, Christian M; Egner, Ursula; Arrowsmith, Cheryl; Barsyte-Lovejoy, Dalia; Vedadi, Masoud; Schapira, Matthieu

    2016-02-11

    Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.

  9. Glucose Autoxidation Induces Functional Damage to Proteins via Modification of Critical Arginine Residues†

    Science.gov (United States)

    Chetyrkin, Sergei; Mathis, Missy; Pedchenko, Vadim; Sanchez, Otto A.; McDonald, W. Hayes; Hachey, David L.; Madu, Hartman; Stec, Donald; Hudson, Billy; Voziyan, Paul

    2011-01-01

    Non-enzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to αVβ3 integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while non-oxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation. PMID:21661747

  10. Glucose autoxidation induces functional damage to proteins via modification of critical arginine residues.

    Science.gov (United States)

    Chetyrkin, Sergei; Mathis, Missy; Pedchenko, Vadim; Sanchez, Otto A; McDonald, W Hayes; Hachey, David L; Madu, Hartman; Stec, Donald; Hudson, Billy; Voziyan, Paul

    2011-07-12

    Nonenzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to α(V)β(3) integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while nonoxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.

  11. Different methylation characteristics of protein arginine methyltransferase 1 and 3 toward the Ewing Sarcoma protein and a peptide.

    Science.gov (United States)

    Pahlich, Steffen; Bschir, Karim; Chiavi, Claudio; Belyanskaya, Larisa; Gehring, Heinz

    2005-10-01

    The multifunctional Ewing Sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, is extensively asymmetrically dimethylated at arginine residues within RGG consensus sequences. Using recombinant proteins we examined whether type I protein arginine methyltransferase (PRMT)1 or 3 is responsible for asymmetric dimethylations of the EWS protein. After in vitro methylation of the EWS protein by GST-PRMT1, we identified 27 dimethylated arginine residues out of 30 potential methylation sites by mass spectrometry-based techniques (MALDI-TOF MS and MS/MS). Thus, PRMT1 recognizes most if not all methylation sites of the EWS protein. With GST-PRMT3, however, only nine dimethylated arginines, located mainly in the C-terminal region of EWS protein, could be assigned, indicating that structural determinants prevent complete methylation. In contrary to previous reports this study also revealed that trypsin is able to cleave after methylated arginines. Pull-down experiments showed that endogenous EWS protein binds efficiently to GST-PRMT1 but less to GST-PRMT3, which is in accordance to the in vitro methylation results. Furthermore, methylation of a peptide containing different methylation sites revealed differences in the site selectivity as well as in the kinetic properties of GST-PRMT1 and GST-PRMT3. Kinetic differences due to an inhibition effect of the methylation inhibitor S-adenosyl-L-homocysteine could be excluded by determining the corresponding K(i) values of the two enzymes and the K(d) values for the methyl donor S-adenosyl-L-methionine. The study demonstrates the strength of MS-based methods for a qualitative and quantitative analysis of enzymic arginine methylation, a posttranslational modification that becomes more and more the object of investigations.

  12. Conformationally Constrained Peptidomimetics as Inhibitors of the Protein Arginine Methyl Transferases

    DEFF Research Database (Denmark)

    Knuhtsen, Astrid; Legrand, Baptiste; Van der Poorten, Olivier;

    2016-01-01

    Protein arginine N-methyl transferases (PRMTs) belong to a family of enzymes that modulate the epigenetic code through modifications of histones. In the present study, peptides emerging from a phage display screening were modified in the search for PRMT inhibitors through substitution with non-pr...

  13. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    Science.gov (United States)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-03-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  14. A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

    Science.gov (United States)

    Ansel-McKinney, P; Scott, S W; Swanson, M; Ge, X; Gehrke, L

    1996-01-01

    A defining feature of alfalfa mosaic virus (AMV) and ilarviruses [type virus: tobacco streak virus (TSV)] is that, in addition to genomic RNAs, viral coat protein is required to establish infection in plants. AMV and TSV coat proteins, which share little primary amino acid sequence identity, are functionally interchangeable in RNA binding and initiation of infection. The lysine-rich amino-terminal RNA binding domain of the AMV coat protein lacks previously identified RNA binding motifs. Here, the AMV coat protein RNA binding domain is shown to contain a single arginine whose specific side chain and position are crucial for RNA binding. In addition, the putative RNA binding domain of two ilarvirus coat proteins, TSV and citrus variegation virus, is identified and also shown to contain a crucial arginine. AMV and ilarvirus coat protein sequence alignment centering on the key arginine revealed a new RNA binding consensus sequence. This consensus may explain in part why heterologous viral RNA-coat protein mixtures are infectious. Images PMID:8890181

  15. Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis.

    Science.gov (United States)

    Ren, Jinqi; Wang, Yaqing; Liang, Yuheng; Zhang, Yongqing; Bao, Shilai; Xu, Zhiheng

    2010-04-23

    Modulation of ribosomal assembly is a fine tuning mechanism for cell number and organ size control. Many ribosomal proteins undergo post-translational modification, but their exact roles remain elusive. Here, we report that ribosomal protein s10 (RPS10) is a novel substrate of an oncoprotein, protein-arginine methyltransferase 5 (PRMT5). We show that PRMT5 interacts with RPS10 and catalyzes its methylation at the Arg(158) and Arg(160) residues. The methylation of RPS10 at Arg(158) and Arg(160) plays a role in the proper assembly of ribosomes, protein synthesis, and optimal cell proliferation. The RPS10-R158K/R160K mutant is not efficiently assembled into ribosomes and is unstable and prone to degradation by the proteasomal pathway. In nucleoli, RPS10 interacts with nucleophosmin/B23 and is predominantly concentrated in the granular component region, which is required for ribosome assembly. The RPS10 methylation mutant interacts weakly with nucleophosmin/B23 and fails to concentrate in the granular component region. Our results suggest that PRMT5 is likely to regulate cell proliferation through the methylation of ribosome proteins, and thus reveal a novel mechanism for PRMT5 in tumorigenesis.

  16. EWS is a substrate of type I protein arginine methyltransferase, PRMT8.

    Science.gov (United States)

    Kim, Jun-Dal; Kako, Koichiro; Kakiuchi, Misako; Park, Gwi Gun; Fukamizu, Akiyoshi

    2008-09-01

    EWS, a pro-oncoprotein which is encoded by the Ewing sarcoma (EWS) gene, contains arginine-glycine-glycine repeats (RGG box) in its COOH-terminus. We previously found that the RGG box of EWS is a target for dimethylation catalyzed by protein arginine methyltransferases (PRMTs). Although it has been observed that arginine residues in EWS are dimethylated in vivo, the endogenous enzyme(s) responsible for this reaction have not been identified to date. In the present study, we determined that EWS was physically associated with PRMT8, the novel eighth member of the PRMT family, through the COOH-terminal region of EWS including RGG3 with the NH2-terminal region of PRMT8 encompassing the S-adenosyl-L-methionine binding domain, and that arginine residues in EWS were asymmetrically dimethylated by PRMT8 using amino acid analysis with thin-layer chromatography. These results suggested that EWS is a substrate for PRMT8, as efficient as for PRMT1.

  17. Formation of functional Tat translocases from heterologous components

    OpenAIRE

    Widdick David A; Buchanan Grant; Guymer David; Hicks Matthew G; Caldelari Isabelle; Berks Ben C; Palmer Tracy

    2006-01-01

    Abstract Background The Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. In Eschericha coli, Tat transport requires the integral membrane proteins TatA, TatB and TatC. In this study we have tested the ability of tat genes from the eubacterial species Pseudomonas syringae, Streptomyces coelicolor and Aquifex aeolicus, to compensate for the absence of the cognate E. coli tat gene, and thus to form functional Tat translocase...

  18. Arginine appearance and nitric oxide synthesis in critically ill infants can be increased with a protein-energy-enriched enteral formula

    NARCIS (Netherlands)

    C.T. de Betue (Carlijn); K.F.M. Joosten (Koen); N.E.P. Deutz (Nicolaas); A.C.E. Vreugdenhil; D.A. van Waardenburg (Dick)

    2013-01-01

    textabstractBackground: Arginine is considered an essential amino acid during critical illness in children, and supplementation of arginine has been proposed to improve arginine availability to facilitate nitric oxide (NO) synthesis. Protein-energy-enriched enteral formulas (PE-formulas) can improve

  19. Periplasmic Binding Proteins in Thermophiles: Characterization and Potential Application of an Arginine-Binding Protein from Thermotoga maritima: A Brief Thermo-Story

    Directory of Open Access Journals (Sweden)

    Sabato D'Auria

    2013-02-01

    Full Text Available Arginine-binding protein from the extremophile Thermotoga maritima is a 27.7 kDa protein possessing the typical two-domain structure of the periplasmic binding proteins family. The protein is characterized by a very high specificity and affinity to bind to arginine, also at high temperatures. Due to its features, this protein could be taken into account as a potential candidate for the design of a biosensor for arginine. It is important to investigate the stability of proteins when they are used for biotechnological applications. In this article, we review the structural and functional features of an arginine-binding protein from the extremophile Thermotoga maritima with a particular eye on its potential biotechnological applications.

  20. 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.

    Science.gov (United States)

    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. PMID:27506270

  1. 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.

    Science.gov (United States)

    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.

  2. Structure of the TatC core of the twin-arginine protein transport system.

    Science.gov (United States)

    Rollauer, Sarah E; Tarry, Michael J; Graham, James E; Jääskeläinen, Mari; Jäger, Franziska; Johnson, Steven; Krehenbrink, Martin; Liu, Sai-Man; Lukey, Michael J; Marcoux, Julien; McDowell, Melanie A; Rodriguez, Fernanda; Roversi, Pietro; Stansfeld, Phillip J; Robinson, Carol V; Sansom, Mark S P; Palmer, Tracy; Högbom, Martin; Berks, Ben C; Lea, Susan M

    2012-12-13

    The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism. PMID:23201679

  3. Freeze-Drying of L-Arginine/Sucrose-Based Protein Formulations, Part 2: Optimization of Formulation Design and Freeze-Drying Process Conditions for an L-Arginine Chloride-Based Protein Formulation System.

    Science.gov (United States)

    Stärtzel, Peter; Gieseler, Henning; Gieseler, Margit; Abdul-Fattah, Ahmad M; Adler, Michael; Mahler, Hanns-Christian; Goldbach, Pierre

    2015-12-01

    We recently reported that the presence of chloride counter ions in freeze-dried l-arginine/sucrose formulations provided the greatest protein stability, but led to low collapse temperatures and glass transition temperatures of the freeze concentrates. The objectives of this study were to identify l-arginine chloride-based formulations and optimize freeze-drying process conditions to deliver a freeze-dried product with good physical quality attributes (including cake appearance, residual moisture, and reconstitution time). Additional properties were tested such as thermal properties, cake microstructure, and protein physical stability. Excipient concentrations were varied with and without a model protein (bovine serum albumin, BSA). Formulations were frozen with and without annealing or with and without controlled nucleation. Primary drying was conducted at high and low shelf temperature. Cakes with least defects and optimum physical attributes were achieved when protein to excipient ratios were high. Controlled nucleation led to elegant cakes for most systems at a low shelf temperature. Replacing BSA by a monoclonal antibody showed that protein (physical) stability was slightly improved under stress storage temperature (i.e., 40°C) in the presence of a low concentration of l-arginine in a sucrose-based formulation. At higher l-arginine concentrations, cake defects increased. Using optimized formulation design, addition of l-arginine chloride to a sucrose-based formulation provided elegant cakes and benefits for protein stability.

  4. Interaction of arginine with protein during refolding process probed by amide H/D exchange mass spectrometry and isothermal titration calorimetry.

    Science.gov (United States)

    Zhao, Dawei; Liu, Yongdong; Zhang, Guifeng; Zhang, Chun; Li, Xiunan; Wang, Qingqing; Shi, Hong; Su, Zhiguo

    2015-01-01

    Arginine has been widely used as low molecular weight additive to promote protein refolding by suppressing aggregate formation. However, methods to investigate the role of arginine in protein refolding are often limited on protein's global conformational properties. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used to study the effects of arginine on recombinant human granulocyte colony-stimulating factor (rhG-CSF) refolding at the scale of peptide mapping. It was found that deuteration levels of rhG-CSF refolded with arginine was higher than that without arginine during the whole refolding process, but they became almost the same when the refolding reached equilibrium. This phenomenon indicated that arginine could protect some amide deuterium atoms from being exchanged with hydrogen, but the protection diminished gradually along with refolding proceeding. Enzymatic digestion revealed six particular peptides of 16-47, 72-84, 84-93, 114-124, 145-153 and 154-162 were mainly responsible for the deuteration, and all of them dominantly located in protein's α-helix domain. Furthermore, thermodynamics analysis by isothermal titration calorimetry provided direct evidence that arginine could only react with denatured and partially refolded rhG-CSF. Taking all of the results together, we suggest that arginine suppresses protein aggregation by a reversible combination. At the initial refolding stage, arginine could combine with the denatured protein mainly through hydrogen bonding. Subsequently, arginine is gradually excluded from protein with protein's native conformation recovering.

  5. Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria

    Science.gov (United States)

    Jia, Qiong; Hu, Xinling; Shi, Dawei; Zhang, Yan; Sun, Meihao; Wang, Jianwei; Mi, Kaixia; Zhu, Guofeng

    2016-01-01

    The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smegmatis overexpressing M. tuberculosis universal stress protein Rv2624c exhibits increased survival in human monocyte THP-1 cells. Transcriptome analysis suggested that Rv2624c affects histidine metabolism, and arginine and proline metabolism. LC-MS/MS analysis showed that Rv2624c affects the abundance of arginine, a modulator of both mycobacteria and infected THP-1 cells. Biochemical analysis showed that Rv2624c is a nucleotide-binding universal stress protein, and an Rv2624c mutant incapable of binding ATP abrogated the growth advantage in THP-1 cells. Rv2624c may therefore modulate metabolic pathways in an ATP-dependent manner, changing the abundance of arginine and thus increasing survival in THP-1 cells. PMID:27762279

  6. Role of type II protein arginine methyltransferase 5 in the regulation of Circadian Per1 gene.

    Directory of Open Access Journals (Sweden)

    Jungtae Na

    Full Text Available Circadian clocks are the endogenous oscillators that regulate rhythmic physiological and behavioral changes to correspond to daily light-dark cycles. Molecular dissections have revealed that transcriptional feedback loops of the circadian clock genes drive the molecular oscillation, in which PER/CRY complexes inhibit the transcriptional activity of the CLOCK/BMAL1 heterodimer to constitute a negative feedback loop. In this study, we identified the type II protein arginine methyltransferase 5 (PRMT5 as an interacting molecule of CRY1. Although the Prmt5 gene was constitutively expressed, increased interaction of PRMT5 with CRY1 was observed when the Per1 gene was repressed both in synchronized mouse liver and NIH3T3 cells. Moreover, rhythmic recruitment of PRMT5 and CRY1 to the Per1 gene promoter was found to be associated with an increased level of histone H4R3 dimethylation and Per1 gene repression. Consistently, decreased histone H4R3 dimethylation and altered rhythmic Per1 gene expression were observed in Prmt5-depleted cells. Taken together, these findings provide an insight into the link between histone arginine methylation by PRMT5 and transcriptional regulation of the circadian Per1 gene.

  7. Protein arginine methyltransferase 6 enhances polyglutamine-expanded androgen receptor function and toxicity in spinal and bulbar muscular atrophy.

    Science.gov (United States)

    Scaramuzzino, Chiara; Casci, Ian; Parodi, Sara; Lievens, Patricia M J; Polanco, Maria J; Milioto, Carmelo; Chivet, Mathilde; Monaghan, John; Mishra, Ashutosh; Badders, Nisha; Aggarwal, Tanya; Grunseich, Christopher; Sambataro, Fabio; Basso, Manuela; Fackelmayer, Frank O; Taylor, J Paul; Pandey, Udai Bhan; Pennuto, Maria

    2015-01-01

    Polyglutamine expansion in androgen receptor (AR) is responsible for spinobulbar muscular atrophy (SBMA) that leads to selective loss of lower motor neurons. Using SBMA as a model, we explored the relationship between protein structure/function and neurodegeneration in polyglutamine diseases. We show here that protein arginine methyltransferase 6 (PRMT6) is a specific co-activator of normal and mutant AR and that the interaction of PRMT6 with AR is significantly enhanced in the AR mutant. AR and PRMT6 interaction occurs through the PRMT6 steroid receptor interaction motif, LXXLL, and the AR activating function 2 surface. AR transactivation requires PRMT6 catalytic activity and involves methylation of arginine residues at Akt consensus site motifs, which is mutually exclusive with serine phosphorylation by Akt. The enhanced interaction of PRMT6 and mutant AR leads to neurodegeneration in cell and fly models of SBMA. These findings demonstrate a direct role of arginine methylation in polyglutamine disease pathogenesis.

  8. Obese women on a low energy rice and bean diet: effects of leucine, arginine or glycine supplementation on protein turnover

    Directory of Open Access Journals (Sweden)

    J.S. Marchini

    2001-10-01

    Full Text Available This study examined if leucine, arginine or glycine supplementation in adult obese patients (body mass index of 33 ± 4 kg/m² consuming a Brazilian low energy and protein diet (4.2 MJ/day and 0.6 g protein/kg affects protein and amino acid metabolism. After four weeks adaptation to this diet, each subject received supplements of these amino acids (equivalent to 0.2 g protein kg-1 day-1 in random order. On the seventh day of each amino acid supplementation, a single-dose 15N-glycine study was carried out. There were no significant differences in protein flux, synthesis or breakdown. The protein flux (grams of nitrogen, gN/9 h was 55 ± 24 during the nonsupplemented diet intake and 39 ± 10, 44 ± 22 and 58 ± 35 during the leucine-, glycine- and arginine-supplemented diet intake, respectively; protein synthesis (gN/9 h was 57 ± 24, 36 ± 10, 41 ± 22 and 56 ± 36, respectively; protein breakdown (gN/9 h was 51 ± 24, 34 ± 10, 32 ± 28 and 53 ± 35, respectively; kinetic balance (gN/9 h was 3.2 ± 1.8, 4.1 ± 1.7, 3.4 ± 2.9 and 3.9 ± 1.6. There was no difference in amino acid profiles due to leucine, arginine or glycine supplementation. The present results suggest that 0.6 g/kg of dietary protein is enough to maintain protein turnover in obese women consuming a reduced energy diet and that leucine, arginine or glycine supplementation does not change kinetic balance or protein synthesis.

  9. Expression and Characterization of ArgR, An Arginine Regulatory Protein in Corynebacterium crenatum

    Institute of Scientific and Technical Information of China (English)

    CHEN Xue Lan; ZHANG Bin; TANG Li; JIAO Hai Tao; XU Heng Yi; XU Feng; XU Hong; WEI Hua; XIONG Yong Hua

    2014-01-01

    Objective Corynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain. Methods Quantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer. Results Arginine production assays showed a 69.9%reduction in arginine from 9.01±0.22 mg/mL in C. crenatum MT to 2.71±0.13 mg/mL (P Conclusion The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in arginine production.

  10. Molecular studies on bromovirus capsid protein. VII. Selective packaging on BMV RNA4 by specific N-terminal arginine residuals.

    Science.gov (United States)

    Choi, Y G; Rao, A L

    2000-09-15

    An arginine-rich RNA-binding motif (ARM) found at the N-proximal region of Brome mosaic virus (BMV) coat protein (CP) adopts alpha-helical conformation and shares homology with CPs of plant and insect RNA viruses, HIV-Rev and Tat proteins, bacterial antiterminators, and ribosomal splicing factors. The ARM of BMV CP, consisting of amino acids 9 through 21 with six arginine residues, is essential for RNA binding and subsequent packaging. In this study analysis of the alpha-helical contents of wild-type and mutant peptides by circular dichroism spectra identified protein determinants required for such conformation. Electrophoretic mobility-shift assays between viral RNA and BMV CP peptides with either proline or alanine substitutions revealed that the interaction is nonspecific. Expression in vivo of mature full-length BMV CP subunits, having the same substitutions for each arginine within the ARM, derived from biologically active clones was found to be competent to assemble into infectious virions and cause visible symptom phenotypes in whole plants. However, analysis of virion progeny RNA profiles of CP variants and subsequent in vitro reassembly assays between mutant CP and four BMV RNAs unveiled the ability of arginine residues at positions 10, 13, or 14 of the ARM to confer selective packaging of BMV RNA4. Thus, BMV CP contains determinants that specifically interact with RNA4 to ensure selective packaging.

  11. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Zhao, Bo; Kieff, Elliott [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States); Peng, Chih-Wen, E-mail: pengcw@mail.tcu.edu.tw [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)

    2013-01-18

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

  12. Response of maize serine/arginine-rich protein gene family in seedlings to drought stress.

    Science.gov (United States)

    Li, Jiao; Guo, Yuqi; Cui, Weiling; Xu, Aihua; Tian, Zengyuan

    2014-07-01

    Alternative splicing (AS) in eukaryotic organisms is closely related to the gene regulation in plant abiotic stress responses, in which serine/arginine-rich proteins (SR proteins) act as key regulators. The genome sequence of maize inbred line B73 was analyzed, showing that the promoter regions of SR genes possess about three to eight kinds of cis-acting regulatory elements. Twenty-seven SR genes encode alkaline proteins, and 23 of which are divided into five subgroups in terms of the first RNA recognition motif (RRM) at the amino terminal. The expression of SR genes showed tissue-specific and genotype-dependent features under drought stress in the hybrid Zhengdan-958 and its parents, Zheng-58 and Chang-7-2 via bidirectional hierarchical clustering. SR genes were down-regulated in roots while they were up-regulated in shoots under drought stress. However, SR genes were down-regulated in both roots and shoots in three different rehydration stages after severe drought stress. Additionally, a widespread alternative splicing exists in all SR genes although SR genes showed differential expression tendency under drought stress and/or during rehydration stages. Results above will deepen our understanding of the molecular mechanisms of plant response to abiotic stress from the perspective of AS-network.

  13. Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria

    NARCIS (Netherlands)

    Meissner, Daniel; Vollstedt, Angela; van Dijl, Jan Maarten; Freudl, Roland

    2007-01-01

    In contrast to the general protein secretion (Sec) system, the twin-arginine translocation (Tat) export pathway allows the translocation of proteins across the bacterial plasma membrane in a fully folded conformation. Due to this feature, the Tat pathway provides an attractive alternative to the sec

  14. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR).

    Science.gov (United States)

    Kanno, Yuichiro; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. PMID:25721668

  15. Identification and characterization of new molecular partners for the protein arginine methyltransferase 6 (PRMT6.

    Directory of Open Access Journals (Sweden)

    Alessandra Lo Sardo

    Full Text Available PRMT6 is a protein arginine methyltransferase that has been implicated in transcriptional regulation, DNA repair, and human immunodeficiency virus pathogenesis. Only few substrates of this enzyme are known and therefore its cellular role is not well understood. To identify in an unbiased manner substrates and potential regulators of PRMT6 we have used a yeast two-hybrid approach. We identified 36 new putative partners for PRMT6 and we validated the interaction in vivo for 7 of them. In addition, using invitro methylation assay we identified 4 new substrates for PRMT6, extending the involvement of this enzyme to other cellular processes beyond its well-established role in gene expression regulation. Holistic approaches create molecular connections that allow to test functional hypotheses. The assembly of PRMT6 protein network allowed us to formulate functional hypotheses which led to the discovery of new molecular partners for the architectural transcription factor HMGA1a, a known substrate for PRMT6, and to provide evidences for a modulatory role of HMGA1a on the methyltransferase activity of PRMT6.

  16. Characterization of an ATP translocase identified in the plant pathogen, Candidatus Liberibacter asiaticus

    Science.gov (United States)

    ATP/ADP translocases allow for the transport of ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate-intracellular bacteria. Of the bacterial ATP/ADP translo...

  17. Protein arginine methyltransferase 5 is associated with malignant phenotype and peritoneal metastasis in gastric cancer.

    Science.gov (United States)

    Kanda, Mitsuro; Shimizu, Dai; Fujii, Tsutomu; Tanaka, Haruyoshi; Shibata, Masahiro; Iwata, Naoki; Hayashi, Masamichi; Kobayashi, Daisuke; Tanaka, Chie; Yamada, Suguru; Nakayama, Goro; Sugimoto, Hiroyuki; Koike, Masahiko; Fujiwara, Michitaka; Kodera, Yasuhiro

    2016-09-01

    Identification of novel gastric cancer (GC)-related molecules is necessary to improve management of patients with GC in both diagnostic and therapeutic aspects. The aim of the present study was to determine whether protein arginine methyltransferase 5 (PRMT5) acts as an oncogene in the progression of GC and whether it serves as a novel diagnostic marker and therapeutic target. We conducted global expression profiling of GC cell lines and RNA interference experiments to evaluate the effect of PRMT5 expression on the phenotype of GC cells. We analysed tissues of 179 patients with GC to assess the association of PRMT5 mRNA levels with clinicopathological factors. Differential expression of PRMT5 mRNA by GC cell lines correlated positively with the levels of GEMIN2, STAT3 and TGFB3. PRMT5 knockdown reduced the proliferation, invasion and migration of a GC cell line. PRMT5 mRNA levels were significantly higher in GC tissues than the corresponding adjacent normal tissues and were independent of tumour depth, differentiation and lymph node metastasis. High PRMT5 expression was an independent risk factor of positive peritoneal lavage cytology (odds ratio 3.90, P=0.003) and decreased survival. PRMT5 enhances the malignant phenotype of GC cell lines and its expression in gastric tissues may serve as a biomarker for patient stratification and a potential target of therapy. PMID:27315569

  18. Novel Protein Arginine Methyltransferase 8 Isoform Is Essential for Cell Proliferation.

    Science.gov (United States)

    Hernandez, Sarah; Dominko, Tanja

    2016-09-01

    Identification of molecular mechanisms that regulate cellular replicative lifespan is needed to better understand the transition between a normal and a neoplastic cell phenotype. We have previously reported that low oxygen-mediated activity of FGF2 leads to an increase in cellular lifespan and acquisition of regeneration competence in human dermal fibroblasts (iRC cells). Though cells display a more plastic developmental phenotype, they remain non-tumorigenic when injected into SCID mice (Page et al. [2009] Cloning Stem Cells 11:417-426; Page et al. [2011] Eng Part A 17:2629-2640) allowing for investigation of mechanisms that regulate increased cellular lifespan in a non-tumorigenic system. Analysis of chromatin modification enzymes by qRT-PCR revealed a 13.3-fold upregulation of the arginine methyltransferase PRMT8 in iRC cells. Increased protein expression was confirmed in both iRC and human embryonic stem cells-the first demonstration of endogenous human PRMT8 expression outside the brain. Furthermore, iRC cells express a novel PRMT8 mRNA variant. Using siRNA-mediated knockdown we demonstrated that this novel variant was required for proliferation of human dermal fibroblasts (hDFs) and grade IV glioblastomas. PRMT8 upregulation in a non-tumorigenic system may offer a potential diagnostic biomarker and a therapeutic target for cells in pre-cancerous and cancerous states. J. Cell. Biochem. 117: 2056-2066, 2016. © 2016 Wiley Periodicals, Inc. PMID:26851891

  19. Facile synthesis of N-6 adenosine modified analogue toward S-adenosyl methionine derived probe for protein arginine methyltransferases

    Institute of Scientific and Technical Information of China (English)

    Wei Hong; James Dowden

    2011-01-01

    Chemically modified cellular co-factors that provide function, such as immobilization or incorporation of fluorescent dyes, are valuable probes of biological activity. A convenient route to obtain S-adenosyl methionine (AdoMet) analogues modified at N-6 adenosine to feature a linker terminating in azide functionality is described herein. Subsequent decoration of such AdoMet analogues with guanidinium terminated linkers leads to novel potential bisubstrate inhibitors for protein arginine methyltransferases, PRMTs.

  20. 精氨酸甲基转移酶与肺部疾病%Protein arginine methyltransferases and pulmonary diseases

    Institute of Scientific and Technical Information of China (English)

    梁彦超; 陈哲; 陈燕

    2014-01-01

    Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the transfer of a methyl group from S-adenosyl-L-methionine into a guanidino nitrogen of protein arginine.Protein arginine methylation is a novel posttranslational modification that plays a pivotal role in a variety of intracellular events,such as DNA repair,RNA processing,transcriptional regulation,and signal transduction.Recently,many studies reveal that PRMTs are significantly associated with pulmonary diseases,cardiovascular diseases,cancer,infections,glucose metabolic diseases,and autoimmune diseases.The paper introduces the recent progresses in the influence of dysregulation of PRMTs on pulmonary diseases.%蛋白质精氨酸甲基转移酶(protein arginine methyltransferases,PRMTs)是催化S-腺苷-甲硫氨酸的甲基转移至蛋白质精氨酸胍基氮原子上的酶.精氨酸甲基化是一种重要的翻译后修饰方式,参与许多重要的细胞过程,包括DNA修复、RNA加工、转录调控和信号转导.近年来大量研究证实,PRMTs与呼吸系统疾病、心血管系统疾病、肿瘤、病毒感染、糖代谢及其相关疾病、自身免疫性疾病密切相关.明确PRMTs的异常表达对肺部疾病的影响对疾病的治疗有重要作用.

  1. Formation of functional Tat translocases from heterologous components

    Directory of Open Access Journals (Sweden)

    Widdick David A

    2006-07-01

    Full Text Available Abstract Background The Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. In Eschericha coli, Tat transport requires the integral membrane proteins TatA, TatB and TatC. In this study we have tested the ability of tat genes from the eubacterial species Pseudomonas syringae, Streptomyces coelicolor and Aquifex aeolicus, to compensate for the absence of the cognate E. coli tat gene, and thus to form functional Tat translocases with E. coli Tat components. Results All three subunits of the Tat system from the Gram positive organism Streptomyces coelicolor were able to form heterologous translocases with substantive Tat transport activity. However, only the TatA and TatB proteins of Pseudomonas syringae were able to functionally interact with the E. coli Tat system even though the two organisms are closely related. Of the Tat components from the phylogenetically distant hyperthermophillic bacterium Aquifex aeolicus only the TatA proteins showed any detectable level of heterologous functionality. The heterologously expressed TatA proteins of S. coelicolor and A. aeolicus were found exclusively in the membrane fraction. Conclusion Our results show that of the three Tat proteins, TatA is most likely to show cross-species complementation. By contrast, TatB and TatC do not always show cross-complementation, probably because they must recognise heterologous signal peptides. Since heterologously-expressed S. coelicolor TatA protein was functional and found only in the membrane fraction, it suggests that soluble forms of Streptomyces TatA reported by others do not play a role in protein export.

  2. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    International Nuclear Information System (INIS)

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR

  3. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    Energy Technology Data Exchange (ETDEWEB)

    Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.

  4. Study of the Five Rickettsia prowazekii Proteins Annotated as ATP/ADP Translocases (Tlc): Only Tlc1 Transports ATP/ADP, While Tlc4 and Tlc5 Transport Other Ribonucleotides

    OpenAIRE

    Audia, Jonathon P.; Winkler, Herbert H.

    2006-01-01

    The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interes...

  5. The Ergogenic Potential of Arginine

    Directory of Open Access Journals (Sweden)

    La Bounty Paul M

    2004-12-01

    Full Text Available Abstract Arginine is a conditionally essential amino acid that is involved in protein synthesis, the detoxification of ammonia, and its conversion to glucose as well as being catabolized to produce energy. In addition to these physiological functions, arginine has been purported to have ergogenic potential. Athletes have taken arginine for three main reasons: 1 its role in the secretion of endogenous growth hormone; 2 its involvement in the synthesis of creatine; 3 its role in augmenting nitric oxide. These aspects of arginine supplementation will be discussed as well as a review of clinical investigations involving exercise performance and arginine ingestion.

  6. l-Arginine grafted alginate hydrogel beads: A novel pH-sensitive system for specific protein delivery

    Directory of Open Access Journals (Sweden)

    Mohamed S. Mohy Eldin

    2015-05-01

    Full Text Available Novel pH-sensitive hydrogels based on l-arginine grafted alginate (Arg-g-Alg hydrogel beads were synthesized and utilized as a new carrier for protein delivery (BSA in specific pH media. l-arginine was grafted onto the polysaccharide backbone of virgin alginate via amine functions. Evidences of grafting of alginate were extracted from FT-IR and thermal analysis, while the morphological structure of Arg-g-Alg hydrogel beads was investigated by SEM photographs. Factors affecting on the grafting process e.g. l-arginine concentration, reaction time, reaction temperature, reaction pH, and crosslinking conditions, have been studied. Whereas, grafting efficiency of each factor was evaluated. Grafting of alginate has improved both thermal and morphological properties of Arg-g-Alg hydrogel beads. The swelling behavior of Arg-g-Alg beads was determined as a function of pH and compared with virgin calcium alginate beads. The cumulative in vitro release profiles of BSA loaded beads were studied at different pHs for simulating the physiological environments of the gastrointestinal tract. The amount of BSA released from neat alginate beads at pH 2 was almost 15% after 5 h, while the Arg-g-Alg beads at the same conditions were clearly higher than 45%, then it increased to 90% at pH 7.2. Accordingly, grafting of alginate has improved its release profile behavior particularly in acidic media. The preliminary results clearly suggested that the Arg-g-Alg hydrogel may be a potential candidate for polymeric carrier for oral delivery of protein or drugs.

  7. Plant serine/arginine-rich proteins: roles in precursor messenger RNA splicing, plant development, and stress responses.

    Science.gov (United States)

    Reddy, Anireddy S N; Shad Ali, Gul

    2011-01-01

    Global analyses of splicing of precursor messenger RNAs (pre-mRNAs) have revealed that alternative splicing (AS) is highly pervasive in plants. Despite the widespread occurrence of AS in plants, the mechanisms that control splicing and the roles of splice variants generated from a gene are poorly understood. Studies on plant serine/arginine-rich (SR) proteins, a family of highly conserved proteins, suggest their role in both constitutive splicing and AS of pre-mRNAs. SR proteins have a characteristic domain structure consisting of one or two RNA recognition motifs at the N-terminus and a C-terminal RS domain rich in arginine/serine dipeptides. Plants have many more SR proteins compared to animals including several plant-specific subfamilies. Pre-mRNAs of plant SR proteins are extensively alternatively spliced to increase the transcript complexity by about six-fold. Some of this AS is controlled in a tissue- and development-specific manner. Furthermore, AS of SR pre-mRNAs is altered by various stresses, raising the possibility of rapid reprogramming of the whole transcriptome by external signals through regulation of the splicing of these master regulators of splicing. Most SR splice variants contain a premature termination codon and are degraded by up-frameshift 3 (UPF3)-mediated nonsense-mediated decay (NMD), suggesting a link between NMD and regulation of expression of the functional transcripts of SR proteins. Limited functional studies with plant SRs suggest key roles in growth and development and plant responses to the environment. Here, we discuss the current status of research on plant SRs and some promising approaches to address many unanswered questions about plant SRs.

  8. Human Papillomavirus Type 1 E1^E4 Protein Is a Potent Inhibitor of the Serine-Arginine (SR) Protein Kinase SRPK1 and Inhibits Phosphorylation of Host SR Proteins and of the Viral Transcription and Replication Regulator E2

    OpenAIRE

    Prescott, Emma L.; Brimacombe, Claire L.; Hartley, Margaret; Bell, Ian; Graham, Sheila; Roberts, Sally

    2014-01-01

    The serine-arginine-specific protein kinase SRPK1 is a common binding partner of the E1^E4 protein of diverse human papillomavirus types. We show here for the first time that the interaction between HPV1 E1^E4 and SRPK1 leads to potent inhibition of SRPK1 phosphorylation of host serine-arginine (SR) proteins that have critical roles in mRNA metabolism, including pre-mRNA processing, mRNA export, and translation. Furthermore, we show that SRPK1 phosphorylates serine residues of SR/RS dipeptide...

  9. Guipi decoction effects on arginine vasopressin protein and gene expression in the hippocampus, ventromedial hypothalamic nucleus, and prefrontal lobe in rats with spleen deficiency

    Institute of Scientific and Technical Information of China (English)

    Huinan Qian; Xueqin Hu; Libo Shen

    2008-01-01

    BACKGROUND: Arginine vasopressin has been shown to enhance learning in experimental animal models.OBJECTIVE: To determine whether Guipi decoction enhances memory and learning by increasing arginine vasopressin levels, and to verify the influence of Guipi decoction on arginine vasopressin protein and gene expression in the hippocampal CAI region, prefrontal lobe cortex, and ventral nucleus of hypothalamus in rats with spleen deficiency.DESIGN, TIME AND SETTING: The randomized, neuropharmacological, control study was performed in the College of Basic Medical Sciences, Beijing University of Chinese Medicine between March 2002 and March 2005.MATERIALS: Sixty, healthy, male, Wistar rats were used to establish spleen deficiency models according to the traditional Chinese medicine principle of bitter drugs for purgation, improper diet, and overstrain. Arginine vasopressin-I polyclonal anti-rabbit antibody immunohistocbemistry kit and arginine vasopressin in situ hybridization kit were provided by Department of Neuroanatomy in Shanghai Second Military Medical University of Chinese PLA.METHODS: Sixty rats were divided into five groups at random: normal control (n = 11), model (n = 13), Guipi decoction (n = 12), recipe control A (n = 12), and recipe control B groups (n = 12). Rats in the latter four groups received 7.5 g/kg of the drugs by intragastric administration each morning, which comprised Dahuang, Houpu, and Zhishi, prepared at a ratio of 2:1 : 1. The rats were lasted every other day, but were allowed free access to water at all times. The rats were forced to swim in 25℃ water until fatigued. Rats in the Guipi decoction and two recipe control groups were intragastrically administered 7.5 g/kg Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellets, respectively, each afternoon. Rats in the normal group were intragastrically administered the same amount of normal saline. All rats were treated for 6 weeks.MAIN OUTCOME MEASURES: At 6 weeks after drug

  10. Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins.

    Science.gov (United States)

    Blobel, Jascha; Brath, Ulrika; Bernadó, Pau; Diehl, Carl; Ballester, Lidia; Sornosa, Alejandra; Akke, Mikael; Pons, Miquel

    2011-12-01

    Addition of a 50 mM mixture of L: -arginine and L: -glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE. PMID:21390527

  11. Evolutionary conservation of dual Sec translocases in the cyanelles of Cyanophora paradoxa

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    Löffelhardt Wolfgang

    2008-11-01

    Full Text Available Abstract Background Cyanelles, the peptidoglycan-armored plastids of glaucocystophytes, occupy a unique bridge position in between free-living cyanobacteria and chloroplasts. In some respects they side with cyanobacteria whereas other features are clearly shared with chloroplasts. The Sec translocase, an example for "conservative sorting" in the course of evolution, is found in the plasma membrane of all prokaryotes, in the thylakoid membrane of chloroplasts and in both these membrane types of cyanobacteria. Results In this paper we present evidence for a dual location of the Sec translocon in the thylakoid as well as inner envelope membranes of the cyanelles from Cyanophora paradoxa, i. e. conservative sorting sensu stricto. The prerequisite was the generation of specific antisera directed against cyanelle SecY that allowed immunodetection of the protein on SDS gels from both membrane types separated by sucrose density gradient floatation centrifugation. Immunoblotting of blue-native gels yielded positive but differential results for both the thylakoid and envelope Sec complexes, respectively. In addition, heterologous antisera directed against components of the Toc/Tic translocons and binding of a labeled precursor protein were used to discriminate between inner and outer envelope membranes. Conclusion The envelope translocase can be envisaged as a prokaryotic feature missing in higher plant chloroplasts but retained in cyanelles, likely for protein transport to the periplasm. Candidate passengers are cytochrome c6 and enzymes of peptidoglycan metabolism. The minimal set of subunits of the Toc/Tic translocase of a primitive plastid is proposed.

  12. Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum

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    Tian Shengli

    2009-08-01

    Full Text Available Abstract Background Serine/arginine (SR protein-specific kinases (SRPKs are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379 from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. Results We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS. Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328 was localized in the alkaline Ω-loop of a helix-loop-helix motif (HLHM of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. Conclusion This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Ω-loop structure could play a crucial role in the NLS function of PSRPK.

  13. Endoplasmic reticulum protein targeting of phospholamban: a common role for an N-terminal di-arginine motif in ER retention?

    Directory of Open Access Journals (Sweden)

    Parveen Sharma

    Full Text Available BACKGROUND: Phospholamban (PLN is an effective inhibitor of the sarco(endoplasmic reticulum Ca(2+-ATPase, which transports Ca(2+ into the SR lumen, leading to muscle relaxation. A mutation of PLN in which one of the di-arginine residues at positions 13 and 14 was deleted led to a severe, early onset dilated cardiomyopathy. Here we were interested in determining the cellular mechanisms involved in this disease-causing mutation. METHODOLOGY/PRINCIPAL FINDING: Mutations deleting codons for either or both Arg13 or Arg14 resulted in the mislocalization of PLN from the ER. Our data show that PLN is recycled via the retrograde Golgi to ER membrane traffic pathway involving COP-I vesicles, since co-immunoprecipitation assays determined that COP I interactions are dependent on an intact di-arginine motif as PLN RDelta14 did not co-precipitate with COP I containing vesicles. Bioinformatic analysis determined that the di-arginine motif is present in the first 25 residues in a large number of all ER/SR Gene Ontology (GO annotated proteins. Mutations in the di-arginine motif of the Sigma 1-type opioid receptor, the beta-subunit of the signal recognition particle receptor, and Sterol-O-acyltransferase, three proteins identified in our bioinformatic screen also caused mislocalization of these known ER-resident proteins. CONCLUSION: We conclude that PLN is enriched in the ER due to COP I-mediated transport that is dependent on its intact di-arginine motif and that the N-terminal di-arginine motif may act as a general ER retrieval sequence.

  14. Arginine transport in catabolic disease states.

    Science.gov (United States)

    Pan, Ming; Choudry, Haroon A; Epler, Mark J; Meng, Qinghe; Karinch, Anne; Lin, Chengmao; Souba, Wiley

    2004-10-01

    Arginine appears to be a semiessential amino acid in humans during critical illness. Catabolic disease states such as sepsis, injury, and cancer cause an increase in arginine utilization, which exceeds body production, leading to arginine depletion. This is aggravated by the reduced nutrient intake that is associated with critical illness. Arginine depletion may have negative consequences on tissue function under these circumstances. Nutritional regimens containing arginine have been shown to improve nitrogen balance and lymphocyte function, and stimulate arginine transport in the liver. We have studied the effects of stress mediators on arginine transport in vascular endothelium, liver, and gut epithelium. In vascular endothelium, endotoxin stimulates arginine uptake, an effect that is mediated by the cytokine tumor necrosis factor-alpha (TNF-alpha) and by the cyclo-oxygenase pathway. This TNF-alpha stimulation involves the activation of intracellular protein kinase C (PKC). A significant increase in hepatic arginine transport activity also occurs following burn injury and in rats with progressive malignant disease. Surgical removal of the growing tumor results in a normalization of the accelerated hepatic arginine transport within days. Chronic metabolic acidosis and sepsis individually augment intestinal arginine transport in rats and Caco-2 cell culture. PKC and mitogen-activated protein kinases are involved in mediating the sepsis/acidosis stimulation of arginine transport. Understanding the regulation of plasma membrane arginine transport will enhance our knowledge of nutrition and metabolism in seriously ill patients and may lead to the design of improved nutritional support formulas. PMID:15465794

  15. Effect of counter ions of arginine as an additive for the solubilization of protein and aromatic compounds.

    Science.gov (United States)

    Yoshizawa, Shunsuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2016-10-01

    Arginine is widely used in biotechnological application, but mostly with chloride counter ion. Here, we examined the effects of various anions on solubilization of aromatic compounds and reduced lysozyme and on refolding of the lysozyme. All arginine salts tested increased the solubility of propyl gallate with acetate much more effectively than chloride. The effects of arginine salts were compared with those of sodium or guanidine salts, indicating that the ability of anions to modulate the propyl gallate solubility is independent of the cation. Comparison of transfer free energy of propyl gallate between sodium and arginine salts indicates that the interaction of propyl gallate is more favorable with arginine than sodium. On the contrary, the solubility of aromatic amino acids is only slightly modulated by anions, implying that there is specific interaction between acetic acid and propyl gallate. Unlike their effects on the solubility of small aromatic compounds, the solubility of reduced lysozyme was much higher in arginine chloride than in arginine acetate or sulfate. Consistent with high solubility, refolding of reduced lysozyme was most effective in arginine chloride. These results suggest potential broader applications of arginine modulated by different anions. PMID:27234496

  16. Effect of counter ions of arginine as an additive for the solubilization of protein and aromatic compounds.

    Science.gov (United States)

    Yoshizawa, Shunsuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2016-10-01

    Arginine is widely used in biotechnological application, but mostly with chloride counter ion. Here, we examined the effects of various anions on solubilization of aromatic compounds and reduced lysozyme and on refolding of the lysozyme. All arginine salts tested increased the solubility of propyl gallate with acetate much more effectively than chloride. The effects of arginine salts were compared with those of sodium or guanidine salts, indicating that the ability of anions to modulate the propyl gallate solubility is independent of the cation. Comparison of transfer free energy of propyl gallate between sodium and arginine salts indicates that the interaction of propyl gallate is more favorable with arginine than sodium. On the contrary, the solubility of aromatic amino acids is only slightly modulated by anions, implying that there is specific interaction between acetic acid and propyl gallate. Unlike their effects on the solubility of small aromatic compounds, the solubility of reduced lysozyme was much higher in arginine chloride than in arginine acetate or sulfate. Consistent with high solubility, refolding of reduced lysozyme was most effective in arginine chloride. These results suggest potential broader applications of arginine modulated by different anions.

  17. Structural rationale for the recognition of arginine at P₃ in PEXEL motif containing proteins of Plasmodium falciparum by plasmepsin V.

    Science.gov (United States)

    Guruprasad, Lalitha; Tanneeru, Karunakar; Guruprasad, Kunchur

    2011-06-01

    The virulent form of malaria is caused by Plasmodium falciparum that infects red blood cells. In order to survive inside the host, the parasite remodels the infected erythrocytes by exporting more than 300 effector proteins outside the parasitophorous vacuole membrane into the cytosol. The main feature of all the export proteins is the presence of a pentapeptide sequence motif; RxLxE/Q/D. This sequence motif is hydrolysed between L-x and the proteins with the acetylated new N-terminus xE/Q/D are exported. The enzyme responsible for this hydrolysis is plasmepsin V which is one of the ten aspartic proteases in P. falciparum. In order to understand the structural rationale for the specificity of this protease towards cleavage of the above motif, we generated three-dimensional models of seven plasmepsins (I, V to X) for which experimental structures are not available and compared these along with the crystal structures of three P. falciparum plasmepsins (II to IV). The structure comparisons revealed the importance of Tyr13, Glu77 and Ala117 specific to plasmepsin V that facilitates the accommodation of arginine at P₃ in the RxLxE/Q/D motif. Our analysis correlates the structure-function relationship of plasmepsin V.

  18. Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa precore protein without C-terminal arginine-rich domain.

    Science.gov (United States)

    Kimura, Tatsuji; Ohno, Nobuhiko; Terada, Nobuo; Rokuhara, Akinori; Matsumoto, Akihiro; Yagi, Shintaro; Tanaka, Eiji; Kiyosawa, Kendo; Ohno, Shinichi; Maki, Noboru

    2005-06-10

    DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.

  19. 5′-Single-stranded/duplex DNA junctions are loading sites for E. coli UvrD translocase

    OpenAIRE

    Tomko, Eric J.; Jia, Haifeng; Park, Jeehae; Maluf, Nasib K.; Ha, Taekjip; Lohman, Timothy M.

    2010-01-01

    Single-molecule analyses of the dual DNA helicase/translocase enzyme UvrD reveal the particular substrate preferences of its monomeric, translocase form, with implications for understanding UvrD's translocase and anti-recombinase roles.

  20. Redox specificity of 2-hydroxyacid-coupled NAD(+/NADH dehydrogenases: a study exploiting "reactive" arginine as a reporter of protein electrostatics.

    Directory of Open Access Journals (Sweden)

    Pooja Gupta

    Full Text Available With "reactive" arginine as a kinetic reporter, 2-hydroxyacid dehydrogenases are assessed in basis of their specialization as NAD(+-reducing or NADH-oxidizing enzymes. Specifically, M4 and H4 lactate dehydrogenases (LDHs and cytoplasmic and mitochondrial malate dehydrogenases (MDHs are compared to assess if their coenzyme specificity may involve electrostatics of cationic or neutral nicotinamide structure as the basis. The enzymes from diverse eukaryote and prokaryote sources thus are assessed in "reactivity" of functionally-critical arginine as a function of salt concentration and pH. Electrostatic calculations were performed on "reactive" arginines and found good correspondence with experiment. The reductive and oxidative LDHs and MDHs are assessed in their count over ionizable residues and in placement details of the residues in their structures as proteins. The variants found to be high or low in ΔpKa of "reactive" arginine are found to be also strong or weak cations that preferentially oxidize NADH (neutral nicotinamide structure or reduce NAD(+ (cationic nicotinamide structure. The ionized groups of protein structure may thus be important to redox specificity of the enzyme on basis of electrostatic preference for the oxidized (cationic nicotinamide or reduced (neutral nicotinamide coenzyme. Detailed comparisons of isozymes establish that the residues contributing in their redox specificity are scrambled in structure of the reductive enzyme.

  1. Identification in Lupin Seed of a Serine-Endopeptidase Activity Cleaving between Twin Arginine Pairs and Causing Limited Proteolysis of Seed Storage Proteins

    Institute of Scientific and Technical Information of China (English)

    Chiara Magni; Fabio Sessa; Gabriella Tedeschi; Armando Negri; Alessio Scarafoni; Alessandro Consonni; Marcello Duranti

    2012-01-01

    The occurrence of twin-arginine motifs (-R-R-) in the amino acid sequences of animal pro-proteins frequently defines the cleavage site(s) for their structural/functional maturation.No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins.In this work,a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds.The endopeptidase was tested with a number of endogenous and exogenous protein substrates,which were selected according to the presence of one or more twin-arginine residue motifs in their amino acid sequences.The observed hydrolysis patterns were limited and highly specific.Partial proteolysis led to stable polypeptide fragments that were characterized by 1-and 2-D electrophoresis.Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry analyses.These approaches,supported by bioinformatic analysis of the available sequences,allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates.The endopeptidase activity was inhibited by 4-(2-AminoEthyl)Benzene-Sulphonyl Fluoride hydrochloride (AEBSF),leupeptin,and serine proteinase protein inhibitors,while it was not affected by pepstatin,transEpoxysuccinyI-L-leucylamido(4-guanidino)butane (E64),and ethylenediaminetetraacetic acid (EDTA),thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase.The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving-R-R-bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

  2. Uncovering the protein lysine and arginine methylation network in Arabidopsis chloroplasts.

    Directory of Open Access Journals (Sweden)

    Claude Alban

    Full Text Available Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division. Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.

  3. Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD.

    Directory of Open Access Journals (Sweden)

    Heng-Li Chen

    Full Text Available The rise of multidrug-resistant (MDR pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc contains an arginine-rich domain (ARD at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV. In this study, we demonstrated that the peptide containing the full-length ARD I-IV (HBc147-183 has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II-IV (HBc153-176 and ARD I-III (HBc147-167 were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS in several in vitro binding assays. Peptide ARD I-IV (HBc147-183 had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p. inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that

  4. Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat signal peptides in Escherichia coli.

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    Frank Lausberg

    Full Text Available The twin arginine translocation (Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria. Tat signal peptides contain a consensus motif (S/T-R-R-X-F-L-K that is thought to play a crucial role in substrate recognition by the Tat translocase. Replacement of the phenylalanine at the +2 consensus position in the signal peptide of a Tat-specific reporter protein (TorA-MalE by aspartate blocked export of the corresponding TorA(D(+2-MalE precursor, indicating that this mutation prevents a productive binding of the TorA(D(+2 signal peptide to the Tat translocase. Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D(+2-MalE when present in pairwise or triple combinations. The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine. Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment.

  5. A Proline/Arginine-Rich End Leucine-Rich Repeat Protein (PRELP) Variant Is Uniquely Expressed in Chronic Lymphocytic Leukemia Cells

    OpenAIRE

    Eva Mikaelsson; Anders Österborg; Mahmood Jeddi-Tehrani; Parviz Kokhaei; Mahyar Ostadkarampour; Reza Hadavi; Mehran Gholamin; Mehdi Akhondi; Fazel Shokri; Hodjattallah Rabbani; Håkan Mellstedt

    2013-01-01

    Proline/arginine-rich end leucine-rich repeat protein (PRELP) belongs to the small leucine-rich proteoglycan (SLRP) family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1). As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze...

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

    Science.gov (United States)

    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.

  7. Valine, isoleucine, arginine and glycine supplementation of low-protein diets for broiler chickens during the starter and grower phases.

    Science.gov (United States)

    Ospina-Rojas, I C; Murakami, A E; Duarte, C R A; Eyng, C; Oliveira, C A L; Janeiro, V

    2014-01-01

    1. Two experiments were performed to study the supplementation of valine, isoleucine, arginine and glycine (Val, Ile, Arg, Gly) in low-protein diets for broiler chickens in the starter (1-21 d; Exp. 1) and grower (22-42 d; Exp. 2) phases. 2. A low-crude protein (CP) diet was formulated to meet the requirements of all amino acids (AA) supplied by the control diet except for Val, Ile, Arg and Gly. The other experimental diets were obtained by the isolated or combined supplementation of the studied AA in the low-CP diet. 3. Growth, serum parameters and litter characteristics were taken in both of the experiments. Carcass measurements were taken in Experiment 2. 4. In the starter and grower phases, low-CP diets without supplementation resulted in birds with a poorer weight gain and feed conversion than those of the birds that received the control diet. 5. In the starter phase, individual supplementation with Val and Gly, but not Ile and Arg, restored the weight gain of the birds, while diets with the addition of Val + Gly, Val + Ile + Arg, Val + Ile + Gly and Val + Ile + Arg + Gly restored their feed conversion. 6. In the grower phase, weight gain was re-established at the same rate as the control diet for the diets supplemented with Val + Ile, Val + Ile + Arg, Val + Ile + Gly and Val + Ile + Arg + Gly. However, the feed conversion was restored only in birds that received the diet supplemented with all studied AA. 7. The supplementation of Val and Gly in low-CP diets was sufficient to avoid adverse effects in the performance and serum parameters of broilers in the starter phase. However, birds in the grower phase required the combined supplementation of Val, Ile, Arg and Gly, to prevent compromised performance.

  8. Protein arginine methyltransferase and the related diseases%蛋白质精氨酸甲基转移酶与相关疾病

    Institute of Scientific and Technical Information of China (English)

    罗举

    2011-01-01

    The family of protein arginine methyltransferases ( PRMTs) , which selectively methylates the arginine residues, is involved in many important cellular processes like DNA damage repair, RNA processing, transcription regulation, and signal transduction. PRMTs are also the major source for asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor. Many studies reveal that PRMTs are closely related to cardiovascular diseases, respiratory diseases, cancer, infections, and autoimmune diseases.%蛋白质精氨酸甲基转移酶家族(protein arginine methyltransferase,PRMTs)催化精氨酸残基甲基化,参与许多重要的细胞过程,包括DNA修复、RNA加工、转录调控和信号转导.此外,PRMTs还是内源性一氧化氮合酶抑制物非对称性二甲基精氨酸(asymmetric dimethylarginine,ADMA)的重要来源.大量研究证实,PRMTs与心血管疾病、呼吸系统疾病、肿瘤、病毒感染、自身免疫性疾病密切相关.

  9. Serine-arginine protein kinase 1 (SRPK1) inhibition as a potential novel targeted therapeutic strategy in prostate cancer.

    Science.gov (United States)

    Mavrou, A; Brakspear, K; Hamdollah-Zadeh, M; Damodaran, G; Babaei-Jadidi, R; Oxley, J; Gillatt, D A; Ladomery, M R; Harper, S J; Bates, D O; Oltean, S

    2015-08-13

    Angiogenesis is required for tumour growth and is induced principally by vascular endothelial growth factor A (VEGF-A). VEGF-A pre-mRNA is alternatively spliced at the terminal exon to produce two families of isoforms, pro- and anti-angiogenic, only the former of which is upregulated in prostate cancer (PCa). In renal epithelial cells and colon cancer cells, the choice of VEGF splice isoforms is controlled by the splicing factor SRSF1, phosphorylated by serine-arginine protein kinase 1 (SRPK1). Immunohistochemistry staining of human samples revealed a significant increase in SRPK1 expression both in prostate intra-epithelial neoplasia lesions as well as malignant adenocarcinoma compared with benign prostate tissue. We therefore tested the hypothesis that the selective upregulation of pro-angiogenic VEGF in PCa may be under the control of SRPK1 activity. A switch in the expression of VEGF165 towards the anti-angiogenic splice isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knockdown (KD). PC-3 SRPK1-KD cells resulted in tumours that grew more slowly in xenografts, with decreased microvessel density. No effect was seen as a result of SRPK1-KD on growth, proliferation, migration and invasion capabilities of PC-3 cells in vitro. Small-molecule inhibitors of SRPK1 switched splicing towards the anti-angiogenic isoform VEGF165b in PC-3 cells and decreased tumour growth when administered intraperitoneally in an orthotopic mouse model of PCa. Our study suggests that modulation of SRPK1 and subsequent inhibition of tumour angiogenesis by regulation of VEGF splicing can alter prostate tumour growth and supports further studies for the use of SRPK1 inhibition as a potential anti-angiogenic therapy in PCa. PMID:25381816

  10. Protein arginine methyltransferase Prmt5-Mep50 methylates histones H2A and H4 and the histone chaperone nucleoplasmin in Xenopus laevis eggs.

    Science.gov (United States)

    Wilczek, Carola; Chitta, Raghu; Woo, Eileen; Shabanowitz, Jeffrey; Chait, Brian T; Hunt, Donald F; Shechter, David

    2011-12-01

    Histone proteins carry information contained in post-translational modifications. Eukaryotic cells utilize this histone code to regulate the usage of the underlying DNA. In the maturing oocytes and eggs of the frog Xenopus laevis, histones are synthesized in bulk in preparation for deposition during the rapid early developmental cell cycles. During this key developmental time frame, embryonic pluripotent chromatin is established. In the egg, non-chromatin-bound histones are complexed with storage chaperone proteins, including nucleoplasmin. Here we describe the identification and characterization of a complex of the protein arginine methyltransferase 5 (Prmt5) and the methylosome protein 50 (Mep50) isolated from Xenopus eggs that specifically methylates predeposition histones H2A/H2A.X-F and H4 and the histone chaperone nucleoplasmin on a conserved motif (GRGXK). We demonstrate that nucleoplasmin (Npm), an exceedingly abundant maternally deposited protein, is a potent substrate for Prmt5-Mep50 and is monomethylated and symmetrically dimethylated at Arg-187. Furthermore, Npm modulates Prmt5-Mep50 activity directed toward histones, consistent with a regulatory role for Npm in vivo. We show that H2A and nucleoplasmin methylation appears late in oogenesis and is most abundant in the laid egg. We hypothesize that these very abundant arginine methylations are constrained to pre-mid blastula transition events in the embryo and therefore may be involved in the global transcriptional repression found in this developmental time frame.

  11. FlpS, the FNR-Like Protein of Streptococcus suis Is an Essential, Oxygen-Sensing Activator of the Arginine Deiminase System

    Directory of Open Access Journals (Sweden)

    Jörg Willenborg

    2016-07-01

    Full Text Available Streptococcus (S. suis is a zoonotic pathogen causing septicemia and meningitis in pigs and humans. During infection S. suis must metabolically adapt to extremely diverse environments of the host. CcpA and the FNR family of bacterial transcriptional regulators are important for metabolic gene regulation in various bacteria. The role of CcpA in S. suis is well defined, but the function of the FNR-like protein of S. suis, FlpS, is yet unknown. Transcriptome analyses of wild-type S. suis and a flpS mutant strain suggested that FlpS is involved in the regulation of the central carbon, arginine degradation and nucleotide metabolism. However, isotopologue profiling revealed no substantial changes in the core carbon and amino acid de novo biosynthesis. FlpS was essential for the induction of the arcABC operon of the arginine degrading pathway under aerobic and anaerobic conditions. The arcABC-inducing activity of FlpS could be associated with the level of free oxygen in the culture medium. FlpS was necessary for arcABC-dependent intracellular bacterial survival but redundant in a mice infection model. Based on these results, we propose that the core function of S. suis FlpS is the oxygen-dependent activation of the arginine deiminase system.

  12. FlpS, the FNR-Like Protein of Streptococcus suis Is an Essential, Oxygen-Sensing Activator of the Arginine Deiminase System.

    Science.gov (United States)

    Willenborg, Jörg; Koczula, Anna; Fulde, Marcus; de Greeff, Astrid; Beineke, Andreas; Eisenreich, Wolfgang; Huber, Claudia; Seitz, Maren; Valentin-Weigand, Peter; Goethe, Ralph

    2016-07-21

    Streptococcus (S.) suis is a zoonotic pathogen causing septicemia and meningitis in pigs and humans. During infection S. suis must metabolically adapt to extremely diverse environments of the host. CcpA and the FNR family of bacterial transcriptional regulators are important for metabolic gene regulation in various bacteria. The role of CcpA in S. suis is well defined, but the function of the FNR-like protein of S. suis, FlpS, is yet unknown. Transcriptome analyses of wild-type S. suis and a flpS mutant strain suggested that FlpS is involved in the regulation of the central carbon, arginine degradation and nucleotide metabolism. However, isotopologue profiling revealed no substantial changes in the core carbon and amino acid de novo biosynthesis. FlpS was essential for the induction of the arcABC operon of the arginine degrading pathway under aerobic and anaerobic conditions. The arcABC-inducing activity of FlpS could be associated with the level of free oxygen in the culture medium. FlpS was necessary for arcABC-dependent intracellular bacterial survival but redundant in a mice infection model. Based on these results, we propose that the core function of S. suis FlpS is the oxygen-dependent activation of the arginine deiminase system.

  13. The RNA-binding protein HOS5 and serine/arginine-rich proteins RS40 and RS41 participate in miRNA biogenesis in Arabidopsis

    KAUST Repository

    Chen, Tao

    2015-07-30

    MicroRNAs are a class of small regulatory RNAs that are generated from primary miRNA (pri-miRNA) transcripts with a stem-loop structure. Accuracy of the processing of pri-miRNA into mature miRNA in plants can be enhanced by SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1). HYL1 activity is regulated by the FIERY2 (FRY2)/RNA polymerase II C-terminal domain phosphatase-like 1 (CPL1). Here, we discover that HIGH OSMOTIC STRESS GENE EXPRESSION 5 (HOS5) and two serine/arginine-rich splicing factors RS40 and RS41, previously shown to be involved in pre-mRNA splicing, affect the biogenesis of a subset of miRNA. These proteins are required for correct miRNA strand selection and the maintenance of miRNA levels. FRY2 dephosphorylates HOS5 whose phosphorylation status affects its subnuclear localization. HOS5 and the RS proteins bind both intronless and intron-containing pri-miRNAs. Importantly, all of these splicing-related factors directly interact with both HYL1 and SE in nuclear splicing speckles. Our results indicate that these splicing factors are directly involved in the biogenesis of a group of miRNA.

  14. The RNA-binding protein HOS5 and serine/arginine-rich proteins RS40 and RS41 participate in miRNA biogenesis in Arabidopsis.

    Science.gov (United States)

    Chen, Tao; Cui, Peng; Xiong, Liming

    2015-09-30

    MicroRNAs are a class of small regulatory RNAs that are generated from primary miRNA (pri-miRNA) transcripts with a stem-loop structure. Accuracy of the processing of pri-miRNA into mature miRNA in plants can be enhanced by SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1). HYL1 activity is regulated by the FIERY2 (FRY2)/RNA polymerase II C-terminal domain phosphatase-like 1 (CPL1). Here, we discover that HIGH OSMOTIC STRESS GENE EXPRESSION 5 (HOS5) and two serine/arginine-rich splicing factors RS40 and RS41, previously shown to be involved in pre-mRNA splicing, affect the biogenesis of a subset of miRNA. These proteins are required for correct miRNA strand selection and the maintenance of miRNA levels. FRY2 dephosphorylates HOS5 whose phosphorylation status affects its subnuclear localization. HOS5 and the RS proteins bind both intronless and intron-containing pri-miRNAs. Importantly, all of these splicing-related factors directly interact with both HYL1 and SE in nuclear splicing speckles. Our results indicate that these splicing factors are directly involved in the biogenesis of a group of miRNA.

  15. Amino-acid selective experiments on uniformly 13C and 15N labeled proteins by MAS NMR: Filtering of lysines and arginines

    Science.gov (United States)

    Jehle, Stefan; Rehbein, Kristina; Diehl, Anne; van Rossum, Barth-Jan

    2006-12-01

    Amino-acid selective magic-angle spinning (MAS) NMR experiments can aid the assignment of ambiguous cross-peaks in crowded spectra of solid proteins. In particular for larger proteins, data analysis can be hindered by severe resonance overlap. In such cases, filtering techniques may provide a good alternative to site-specific spin-labeling to obtain unambiguous assignments that can serve as starting points in the assignment procedure. In this paper we present a simple pulse sequence that allows selective excitation of arginine and lysine residues. To achieve this, we make use of a combination of specific cross-polarization for selective excitation [M. Baldus, A.T. Petkova, J. Herzfeld, R.G. Griffin, Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems, Mol. Phys. 95 (1998) 1197-1207.] and spin diffusion for transfer along the amino-acid side-chain. The selectivity of the filter is demonstrated with the excitation of lysine and arginine side-chain resonances in a uniformly 13C and 15N labeled protein preparation of the α-spectrin SH3 domain. It is shown that the filter can be applied as a building block in a 13C- 13C lysine-only correlation experiment.

  16. A loose domain swapping organization confers a remarkable stability to the dimeric structure of the arginine binding protein from Thermotoga maritima.

    Directory of Open Access Journals (Sweden)

    Alessia Ruggiero

    Full Text Available The arginine binding protein from Thermatoga maritima (TmArgBP, a substrate binding protein (SBP involved in the ABC system of solute transport, presents a number of remarkable properties. These include an extraordinary stability to temperature and chemical denaturants and the tendency to form multimeric structures, an uncommon feature among SBPs involved in solute transport. Here we report a biophysical and structural characterization of the TmArgBP dimer. Our data indicate that the dimer of the protein is endowed with a remarkable stability since its full dissociation requires high temperature as well as SDS and urea at high concentrations. In order to elucidate the atomic level structural properties of this intriguing protein, we determined the crystallographic structures of the apo and the arginine-bound forms of TmArgBP using MAD and SAD methods, respectively. The comparison of the liganded and unliganded models demonstrates that TmArgBP tertiary structure undergoes a very large structural re-organization upon arginine binding. This transition follows the Venus Fly-trap mechanism, although the entity of the re-organization observed in TmArgBP is larger than that observed in homologous proteins. Intriguingly, TmArgBP dimerizes through the swapping of the C-terminal helix. This dimer is stabilized exclusively by the interactions established by the swapping helix. Therefore, the TmArgBP dimer combines a high level of stability and conformational freedom. The structure of the TmArgBP dimer represents an uncommon example of large tertiary structure variations amplified at quaternary structure level by domain swapping. Although the biological relevance of the dimer needs further assessments, molecular modelling suggests that the two TmArgBP subunits may simultaneously interact with two distinct ABC transporters. Moreover, the present protein structures provide some clues about the determinants of the extraordinary stability of the biomolecule

  17. Physiological implications of arginine metabolism in plants

    Directory of Open Access Journals (Sweden)

    Gudrun eWinter

    2015-07-01

    Full Text Available Nitrogen is a limiting resource for plant growth in most terrestrial habitats since large amounts of nitrogen are needed to synthesize nucleic acids and proteins. Among the 21 proteinogenic amino acids, arginine has the highest nitrogen to carbon ratio, which makes it especially suitable as a storage form of organic nitrogen. Synthesis in chloroplasts via ornithine is apparently the only operational pathway to provide arginine in plants, and the rate of arginine synthesis is tightly regulated by various feedback mechanisms in accordance with the overall nutritional status. While several steps of arginine biosynthesis still remain poorly characterized in plants, much wider attention has been paid to inter- and intracellular arginine transport as well as arginine-derived metabolites. A role of arginine as alternative source besides glutamate for proline biosynthesis is still discussed controversially and may be prevented by differential subcellular localization of enzymes. Apparently, arginine is a precursor for nitric oxide (NO, although the molecular mechanism of NO production from arginine remains unclear in higher plants. In contrast, conversion of arginine to polyamines is well documented, and in several plant species also ornithine can serve as a precursor for polyamines. Both NO and polyamines play crucial roles in regulating developmental processes as well as responses to biotic and abiotic stress. It is thus conceivable that arginine catabolism serves on the one hand to mobilize nitrogen storages, while on the other hand it may be used to fine-tune development and defense mechanisms against stress. This review summarizes the recent advances in our knowledge about arginine metabolism, with a special focus on the model plant Arabidopsis thaliana, and pinpoints still unresolved critical questions.

  18. Physiological implications of arginine metabolism in plants.

    Science.gov (United States)

    Winter, Gudrun; Todd, Christopher D; Trovato, Maurizio; Forlani, Giuseppe; Funck, Dietmar

    2015-01-01

    Nitrogen is a limiting resource for plant growth in most terrestrial habitats since large amounts of nitrogen are needed to synthesize nucleic acids and proteins. Among the 21 proteinogenic amino acids, arginine has the highest nitrogen to carbon ratio, which makes it especially suitable as a storage form of organic nitrogen. Synthesis in chloroplasts via ornithine is apparently the only operational pathway to provide arginine in plants, and the rate of arginine synthesis is tightly regulated by various feedback mechanisms in accordance with the overall nutritional status. While several steps of arginine biosynthesis still remain poorly characterized in plants, much wider attention has been paid to inter- and intracellular arginine transport as well as arginine-derived metabolites. A role of arginine as alternative source besides glutamate for proline biosynthesis is still discussed controversially and may be prevented by differential subcellular localization of enzymes. Apparently, arginine is a precursor for nitric oxide (NO), although the molecular mechanism of NO production from arginine remains unclear in higher plants. In contrast, conversion of arginine to polyamines is well documented, and in several plant species also ornithine can serve as a precursor for polyamines. Both NO and polyamines play crucial roles in regulating developmental processes as well as responses to biotic and abiotic stress. It is thus conceivable that arginine catabolism serves on the one hand to mobilize nitrogen storages, while on the other hand it may be used to fine-tune development and defense mechanisms against stress. This review summarizes the recent advances in our knowledge about arginine metabolism, with a special focus on the model plant Arabidopsis thaliana, and pinpoints still unresolved critical questions. PMID:26284079

  19. Genetics Home Reference: ornithine translocase deficiency

    Science.gov (United States)

    ... coma. Episodes of illness may coincide with the introduction of high-protein formulas or solid foods into ... Library of Medicine Lister Hill National Center for Biomedical Communications 8600 Rockville Pike, Bethesda, MD 20894, USA ...

  20. The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation.

    Science.gov (United States)

    Ramasamy, Sureshkumar; Abrol, Ravinder; Suloway, Christian J M; Clemons, William M

    2013-05-01

    In bacteria, two signal-sequence-dependent secretion pathways translocate proteins across the cytoplasmic membrane. Although the mechanism of the ubiquitous general secretory pathway is becoming well understood, that of the twin-arginine translocation pathway, responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0 Å and 6.8 Å resolution. The membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket, leading to structural changes that facilitate higher order assemblies. PMID:23583035

  1. Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2β1.

    Science.gov (United States)

    Jamros, Michael A; Aubol, Brandon E; Keshwani, Malik M; Zhang, Zhaiyi; Stamm, Stefan; Adams, Joseph A

    2015-07-10

    Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2β3), RS1 blocks phosphorylation of these serines in the full-length Tra2β1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2β1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2β1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis. PMID:26013829

  2. The role of arginine in infection and sepsis.

    Science.gov (United States)

    Luiking, Yvette C; Poeze, Martijn; Ramsay, Graham; Deutz, Nicolaas E P

    2005-01-01

    Sepsis is a systemic response to an infection, with high morbidity and mortality rates. Metabolic changes during infection and sepsis could be related to changes in metabolism of the amino acid L-arginine. In sepsis, protein breakdown is increased, which is a key process to maintain arginine delivery because both endogenous de novo arginine production from citrulline and food intake are reduced. Arginine catabolism, on the other hand, is markedly increased by enhanced use of arginine via the arginase and nitric oxide pathways. As a result, lowered plasma arginine levels are usually found. Arginine may therefore be considered as an essential amino acid in sepsis, and supplementation could be beneficial in sepsis by improving microcirculation and protein anabolism. L-Arginine supplementation in a hyperdynamic pig model of sepsis prohibits the increase in pulmonary arterial blood pressure, improves muscle and liver protein metabolism, and restores the intestinal motility pattern. Arguments raised against arginine supplementation are mainly pointed at stimulating nitric oxide (NO) production, with concerns about toxicity of increased NO and hemodynamic instability with refractory hypotension. NO synthase inhibition, however, increased mortality. Arginine supplementation in septic patients has transient effects on hemodynamics when supplied as a bolus but seems without hemodynamic side effects when supplied continuously. In conclusion, arginine could have an essential role in infection and sepsis.

  3. Poly-L-arginine-Induced internalization of tight junction proteins increases the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules.

    Science.gov (United States)

    Yamaki, Tsutomu; Ohtake, Kazuo; Ichikawa, Keiko; Uchida, Masaki; Uchida, Hiroyuki; Oshima, Shinji; Ohshima, Shinji; Juni, Kazuhiko; Kobayashi, Jun; Morimoto, Yasunori; Natsume, Hideshi

    2013-01-01

    We investigated whether poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 monolayer to hydrophilic macromolecules and clarified the disposition of tight junction (TJ) proteins. The transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran (FD-4) permeation were determined after treatment with PLA. TJ proteins were visualized using immunofluorescence microscopy after PLA exposure and depletion, and their expression levels were determined. The barrier function of TJs was also evaluated by measuring the alterations in the TEER and in the localization of TJ proteins. PLA induced an increase in hydrophilic macromolecule, FD-4, permeation through Caco-2 cell monolayers and a decrease in the TEER in a concentration-dependent manner, without any significant impact on the cell viability. This increased paracellular permeability induced by PLA was found to be internalized of claudin-4, ZO-1, tricellulin and mainly occludin from cell-cell junction to the subcellular space. ZO-1 appeared to play an important role in the reconstitution of TJ strand structures following PLA depletion. These results indicate that the PLA led to the internalization of TJ proteins to the subcellular space, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

  4. A proline/arginine-rich end leucine-rich repeat protein (PRELP) variant is uniquely expressed in chronic lymphocytic leukemia cells.

    Science.gov (United States)

    Mikaelsson, Eva; Österborg, Anders; Jeddi-Tehrani, Mahmood; Kokhaei, Parviz; Ostadkarampour, Mahyar; Hadavi, Reza; Gholamin, Mehran; Akhondi, Mehdi; Shokri, Fazel; Rabbani, Hodjattallah; Mellstedt, Håkan

    2013-01-01

    Proline/arginine-rich end leucine-rich repeat protein (PRELP) belongs to the small leucine-rich proteoglycan (SLRP) family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1). As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR) in all CLL patients (30/30), as well as in some patients with mantle cell lymphoma (3/5), but not in healthy donor leukocytes (0/20) or tumor samples from other hematological malignancies (0/35). PRELP was also detected in CLL cell-lines (4/4) but not in cell-lines from other hematological tumors (0/9). PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies. PMID:23826326

  5. A proline/arginine-rich end leucine-rich repeat protein (PRELP variant is uniquely expressed in chronic lymphocytic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Eva Mikaelsson

    Full Text Available Proline/arginine-rich end leucine-rich repeat protein (PRELP belongs to the small leucine-rich proteoglycan (SLRP family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD in patients with chronic lymphocytic leukemia (CLL. PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1. As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR in all CLL patients (30/30, as well as in some patients with mantle cell lymphoma (3/5, but not in healthy donor leukocytes (0/20 or tumor samples from other hematological malignancies (0/35. PRELP was also detected in CLL cell-lines (4/4 but not in cell-lines from other hematological tumors (0/9. PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies.

  6. Homology modeling of major intrinsic proteins in rice, maize and Arabidopsis: comparative analysis of transmembrane helix association and aromatic/arginine selectivity filters

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    Sankararamakrishnan Ramasubbu

    2007-04-01

    Full Text Available Abstract Background The major intrinsic proteins (MIPs facilitate the transport of water and neutral solutes across the lipid bilayers. Plant MIPs are believed to be important in cell division and expansion and in water transport properties in response to environmental conditions. More than 30 MIP sequences have been identified in Arabidopsis thaliana, maize and rice. Plasma membrane intrinsic proteins (PIPs, tonoplast intrinsic proteins (TIPs, Nod26-like intrinsic protein (NIPs and small and basic intrinsic proteins (SIPs are subfamilies of plant MIPs. Despite sequence diversity, all the experimentally determined structures belonging to the MIP superfamily have the same "hour-glass" fold. Results We have structurally characterized 39 rice and 31 maize MIPs and compared them with that of Arabidopsis. Homology models of 105 MIPs from all three plant species were built. Structure-based sequence alignments were generated and the residues in the helix-helix interfaces were analyzed. Small residues (Gly/Ala/Ser/Thr are found to be highly conserved as a group in the helix-helix interface of MIP structures. Individual families sometimes prefer one or another of the residues from this group. The narrow aromatic/arginine (ar/R selectivity filter in MIPs has been shown to provide an important constriction for solute permeability. Ar/R regions were analyzed and compared between the three plant species. Seventeen TIP, NIP and SIP members from rice and maize have ar/R signatures that are not found in Arabidopsis. A subgroup of rice and maize NIPs has small residues in three of the four positions in the ar/R tetrad, resulting in a wider constriction. These MIP members could transport larger solute molecules. Conclusion Small residues are group-conserved in the helix-helix interface of MIP structures and they seem to be important for close helix-helix interactions. Such conservation might help to preserve the hour-glass fold in MIP structures. Analysis and

  7. Severe Hypomyelination and Developmental Defects Are Caused in Mice Lacking Protein Arginine Methyltransferase 1 (PRMT1) in the Central Nervous System.

    Science.gov (United States)

    Hashimoto, Misuzu; Murata, Kazuya; Ishida, Junji; Kanou, Akihiko; Kasuya, Yoshitoshi; Fukamizu, Akiyoshi

    2016-01-29

    Protein arginine methyltransferase 1 (PRMT1) is involved in cell proliferation, DNA damage response, and transcriptional regulation. Although PRMT1 is extensively expressed in the CNS at embryonic and perinatal stages, the physiological role of PRMT1 has been poorly understood. Here, to investigate the primary function of PRMT1 in the CNS, we generated CNS-specific PRMT1 knock-out mice by the Cre-loxP system. These mice exhibited postnatal growth retardation with tremors, and most of them died within 2 weeks after birth. Brain histological analyses revealed prominent cell reduction in the white matter tracts of the mutant mice. Furthermore, ultrastructural analysis demonstrated that myelin sheath was almost completely ablated in the CNS of these animals. In agreement with hypomyelination, we also observed that most major myelin proteins including myelin basic protein (MBP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and myelin-associated glycoprotein (MAG) were dramatically decreased, although neuronal and astrocytic markers were preserved in the brain of CNS-specific PRMT1 knock-out mice. These animals had a reduced number of OLIG2(+) oligodendrocyte lineage cells in the white matter. We found that expressions of transcription factors essential for oligodendrocyte specification and further maturation were significantly suppressed in the brain of the mutant mice. Our findings provide evidence that PRMT1 is required for CNS development, especially for oligodendrocyte maturation processes.

  8. Feed intake and brain neuropeptide Y (NPY) and cholecystokinin (CCK) gene expression in juvenile cobia fed plant-based protein diets with different lysine to arginine ratios.

    Science.gov (United States)

    Nguyen, Minh Van; Jordal, Ann-Elise Olderbakk; Espe, Marit; Buttle, Louise; Lai, Hung Van; Rønnestad, Ivar

    2013-07-01

    Cobia (Rachycentron canadum, Actinopterygii, Perciformes;10.5±0.1g) were fed to satiation with three plant-based protein test diets with different lysine (L) to arginine (A) ratios (LL/A, 0.8; BL/A, 1.1; and HL/A, 1.8), using a commercial diet as control for six weeks. The test diets contained 730 g kg(-1) plant ingredients with 505-529 g protein, 90.2-93.9 g lipid kg(-1) dry matter; control diet contained 550 g protein and 95 g lipid kg(-1) dry matter. Periprandial expression of brain NPY and CCK (npy and cck) was measured twice (weeks 1 and 6). At week one, npy levels were higher in pre-feeding than postfeeding cobia for all diets, except LL/A. At week six, npy levels in pre-feeding were higher than in postfeeding cobia for all diets. cck in pre-feeding cobia did not differ from that in postfeeding for all diets, at either time point. Cobia fed LL/A had lower feed intake (FI) than cobia fed BL/A and control diet, but no clear correlations between dietary L/A ratio and FI, growth and expression of npy and cck were detected. The data suggest that NPY serves as an orexigenic factor, but further studies are necessary to describe links between dietary L/A and regulation of appetite and FI in cobia.

  9. Inhibition of activated protein C by recombinant alpha 1-antitrypsin variants with substitution of arginine or leucine for methionine358

    NARCIS (Netherlands)

    Heeb, M.J.; Bischoff, Rainer; Courtney, M.; Griffin, J.H.

    1990-01-01

    alpha 1-Antitrypsin (alpha 1-AT) was recently identified as a major physiologic plasma inhibitor of activated protein C. The reaction with activated protein C of recombinant alpha 1-AT containing amino acid substitutions at the reactive center was studied. The substitution of Arg358 for Met, as obse

  10. Protein arginine methyltransferase 1 may be involved in pregnane x receptor-activated overexpression of multidrug resistance 1 gene during acquired multidrug resistant

    Science.gov (United States)

    Li, Tingting; Kong, Ah-Ng Tony; Ma, Zhiqiang; Liu, Haiyan; Liu, Pinghua; Xiao, Yu; Jiang, Xuehua; Wang, Ling

    2016-01-01

    Purpose Pregnane x receptor (PXR) - activated overexpression of the multidrug resistance 1 (MDR1) gene is an important way for tumor cells to acquire drug resistance. However, the detailed mechanism still remains unclear. In the present study, we aimed to investigate whether protein arginine methyl transferase 1(PRMT1) is involved in PXR - activated overexpression of MDR1 during acquired multidrug resistant. Experimental Design Arginine methyltransferase inhibitor 1 (AMI-1) was used to pharmacologically block PRMT1 in resistant breast cancer cells (MCF7/adr). The mRNA and protein levels of MDR1 were detected by real-time PCR and western blotting analysis. Immunofluorescence microscopy and co-immunoprecipitation were used to investigate the physical interaction between PXR and PRMT1. Then, 136 candidate compounds were screened for PRMT1 inhibitors. Lastly, luciferase reporter gene and nude mice bearing resistant breast cancer xenografts were adopted to investigate the anti-tumor effect of PRMT1 inhibitors when combined with adriamycin. Results AMI-1 significantly suppressed the expression of MDR1 in MCF7/adr cells and increased cells sensitivity of MCF7/adr to adriamycin. Physical interaction between PRMT1 and PXR exists in MCF7/adr cells, which could be disrupted by AMI-1. Those results suggest that PRMT1 may be involved in PXR-activated overexpression of MDR1 in resistant breast cancer cells, and AMI-1 may suppress MDR1 by disrupting the interaction between PRMT1 and PXR. Then, five compounds including rutin, isoquercitrin, salvianolic acid A, naproxen, and felodipline were identified to be PRMT1 inhibitors. Finally, those PRMT1 inhibitors were observed to significantly decrease MDR1 promoter activity in vitro and enhance the antitumor effect of adriamycin in nude mice that bearing resistant breast cancer xenografts. Conclusions PRMT1 may be an important co-activator of PXR in activating MDR1 gene during acquired resistance, and PRMT1 inhibitor combined with

  11. L-arginine

    Science.gov (United States)

    ... that taking L-arginine, alone or together with antioxidants (Niteworks, Herbalife International, Inc), does not improve performance ... administered as a shot, or applied to the skin, short-term. It can cause some side effects ...

  12. Expression of Major Capsid Protein of Cainine Parvovirus by Yeast (Pichia pastoris and Efficient Purification using Arginine in Affinity Chromatography

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    Ying He

    2016-06-01

    Full Text Available An immunochromatographic (IC assay was developed for rapid detection of canine parvovirus using the monoclonal antibodies (McAbs against canine parvovirus (CPV-2. To prepare the McAbs, gene encoding the VP2 protein of CPV-2a was expressed in a Pichia pastoris expression vector pPICZ-A. The recombinant VP2 was similar antigenically function to the native capsid protein as demonstrated by Western blotting using CPV- 2 polyclonal antiserum. McAbs against CPV-2 were produced by fusing myeloma cell line SP2/0 with spleen cells from Balb/C mice immunized with purified recombinant VP2 protein. By ELISA it was shown that the McAbs specifically recognized VP2 epitopes of CPV-2 but not those of other canine viruses such as Canine distemper virus (CDV or canine adenovirus (CAV. An IC assay developed with the McAbs was suitable for rapid detection of canine parvovirus. Fecal samples (120 from dogs suspected of CPV-2 infection were analyzed by both haemaglutination (HA assay and the IC assay, and 52 and 53 samples were found positive for CPV-2, respectively. Comparison between the two different assays revealed that IC assay is as sensitive as HA; the sensitivity and specificity for the IC assay is 98.6% and 98.1%, respectively.

  13. Arginine methylation regulates the p53 response

    DEFF Research Database (Denmark)

    Jansson, Martin; Durant, Stephen T; Cho, Er-Chieh;

    2008-01-01

    on the p53 response. We show that the protein arginine methyltransferase (PRMT) 5, as a co-factor in a DNA damage responsive co-activator complex that interacts with p53, is responsible for methylating p53. Arginine methylation is regulated during the p53 response and affects the target gene specificity......Activation of the p53 tumour suppressor protein in response to DNA damage leads to apoptosis or cell-cycle arrest. Enzymatic modifications are widely believed to affect and regulate p53 activity. We describe here a level of post-translational control that has an important functional consequence...

  14. Arginine, citrulline and nitric oxide metabolism in sepsis

    Science.gov (United States)

    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...

  15. Nuclear factor-kB dependent protein arginine methyltransferase%核因子kB依赖的蛋白质精氨酸甲基转移酶1

    Institute of Scientific and Technical Information of China (English)

    王翠莲; 楚东岭; 金发光

    2008-01-01

    Arginine methylation involves in manifold cellular processes like signaling, transcription regulation,chromatin remolding, and apoptosis with extensive biology function. During recent years, new members of the family of protein arginine methyltransferases have been increasing, by now there are eleven protein arginine methyltransferases. Protein arginine methyltransferases are classified into two groups. Type I protein arginine methyltransferases catalyze the formation of monomethylarginine and asymmetric dimethylarginine,type Ⅱ enzymes form monomethylarginine and symmetric dimethylarginine. Coactivator- associated arginine methyltransferasel is a nuclear faetor-kB(NF-kB) dependent transcriptional coactivator and functions as a promoter-specific regulator of NF-kB recruitment to chromatin. Coactivator-associated arginine methyltransferasel may activate NF-kB signal transduction pathway by enhancing NF-kB recruitment to cognate sites and initate transcriptional activation of a variety of proinflammatory and immunoregulation genes, which plays critical role in transcription regulation of immune and inflammatory reaction genes. Now it is generally accepted that lung is the major source of NO,plays an important role in NO metabolism,and acts as the major source of NOS inhibitor asymmetric dimethylarginine. Asymmetric dimethylarginine serves as false substrates and competitively inhibits NOS activity, blocking the formation of endogenous NO. As a major source of not only NO but also the NOS inhibitor asymmetric dimethylarginine, the lung likely plays a critical role in the important and delicate balance of arginine-methylarginine-NO.%精氨酸甲基化参与信号转导、转录调控、染色质重塑和凋亡等多种细胞事件具有广泛的生物学功能.近年来蛋白质精氨酸甲基转移酶家族的新成员日益增多,目前已知有11个人类蛋白质精氨酸甲基转移酶.蛋白质精氨酸甲基转移酶可分成两型,Ⅰ型蛋白质精氨酸甲

  16. Active displacement of RecA filaments by UvrD translocase activity.

    Science.gov (United States)

    Petrova, Vessela; Chen, Stefanie H; Molzberger, Eileen T; Tomko, Eric; Chitteni-Pattu, Sindhu; Jia, Haifeng; Ordabayev, Yerdos; Lohman, Timothy M; Cox, Michael M

    2015-04-30

    The UvrD helicase has been implicated in the disassembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an active mechanism to remove RecA from the DNA. Efficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibility of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with enhanced DNA-binding properties. The ATPase rate of UvrD during RecA removal is much slower than the ATPase activity of UvrD when it is functioning either as a translocase or a helicase on DNA in the absence of RecA. Thus, in this context UvrD may operate in a specialized disassembly mode. PMID:25824953

  17. Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

    Directory of Open Access Journals (Sweden)

    Mario A. Moscarello

    2013-03-01

    Multiple sclerosis (MS is the most common CNS-demyelinating disease of humans, showing clinical and pathological heterogeneity and a general resistance to therapy. We first discovered that abnormal myelin hypercitrullination, even in normal-appearing white matter, by peptidylarginine deiminases (PADs correlates strongly with disease severity and might have an important role in MS progression. Hypercitrullination is known to promote focal demyelination through reduced myelin compaction. Here we report that 2-chloroacetamidine (2CA, a small-molecule, PAD active-site inhibitor, dramatically attenuates disease at any stage in independent neurodegenerative as well as autoimmune MS mouse models. 2CA reduced PAD activity and protein citrullination to pre-disease status. In the autoimmune models, disease induction uniformly induced spontaneous hypercitrullination with citrulline+ epitopes targeted frequently. 2CA rapidly suppressed T cell autoreactivity, clearing brain and spinal cord infiltrates, through selective removal of newly activated T cells. 2CA essentially prevented disease when administered before disease onset or before autoimmune induction, making hypercitrullination, and specifically PAD enzymes, a therapeutic target in MS models and thus possibly in MS.

  18. An evolutionarily conserved arginine is essential for Tre1 G protein-coupled receptor function during germ cell migration in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Angela R Kamps

    Full Text Available BACKGROUND: G protein-coupled receptors (GPCRs play central roles in mediating cellular responses to environmental signals leading to changes in cell physiology and behaviors, including cell migration. Numerous clinical pathologies including metastasis, an invasive form of cell migration, have been linked to abnormal GPCR signaling. While the structures of some GPCRs have been defined, the in vivo roles of conserved amino acid residues and their relationships to receptor function are not fully understood. Trapped in endoderm 1 (Tre1 is an orphan receptor of the rhodopsin class that is necessary for primordial germ cell migration in Drosophila melanogaster embryos. In this study, we employ molecular genetic approaches to identify residues in Tre1 that are critical to its functions in germ cell migration. METHODOLOGY/PRINCIPAL FINDINGS: First, we show that the previously reported scattershot mutation is an allele of tre1. The scattershot allele results in an in-frame deletion of 8 amino acids at the junction of the third transmembrane domain and the second intracellular loop of Tre1 that dramatically impairs the function of this GPCR in germ cell migration. To further refine the molecular basis for this phenotype, we assayed the effects of single amino acid substitutions in transgenic animals and determined that the arginine within the evolutionarily conserved E/N/DRY motif is critical for receptor function in mediating germ cell migration within an intact developing embryo. CONCLUSIONS/SIGNIFICANCE: These structure-function studies of GPCR signaling in native contexts will inform future studies into the basic biology of this large and clinically important family of receptors.

  19. The critical role of protein arginine methyltransferase prmt8 in zebrafish embryonic and neural development is non-redundant with its paralogue prmt1.

    Directory of Open Access Journals (Sweden)

    Yu-ling Lin

    Full Text Available Protein arginine methyltransferase (PRMT 1 is the most conserved and widely distributed PRMT in eukaryotes. PRMT8 is a vertebrate-restricted paralogue of PRMT1 with an extra N-terminal sequence and brain-specific expression. We use zebrafish (Danio rerio as a vertebrate model to study PRMT8 function and putative redundancy with PRMT1. The transcripts of zebrafish prmt8 were specifically expressed in adult zebrafish brain and ubiquitously expressed from zygotic to early segmentation stage before the neuronal development. Whole-mount in situ hybridization revealed ubiquitous prmt8 expression pattern during early embryonic stages, similar to that of prmt1. Knockdown of prmt8 with antisense morpholino oligonucleotide phenocopied prmt1-knockdown, with convergence/extension defects at gastrulation. Other abnormalities observed later include short body axis, curled tails, small and malformed brain and eyes. Catalytically inactive prmt8 failed to complement the morphants, indicating the importance of methyltransferase activity. Full-length prmt8 but not prmt1 cRNA can rescue the phenotypic changes. Nevertheless, cRNA encoding Prmt1 fused with the N-terminus of Prmt8 can rescue the prmt8 morphants. In contrast, N-terminus- deleted but not full-length prmt8 cRNA can rescue the prmt1 morphants as efficiently as prmt1 cRNA. Abnormal brain morphologies illustrated with brain markers and loss of fluorescent neurons in a transgenic fish upon prmt8 knockdown confirm the critical roles of prmt8 in neural development. In summery, our study is the first report showing the expression and function of prmt8 in early zebrafish embryogenesis. Our results indicate that prmt8 may play important roles non-overlapping with prmt1 in embryonic and neural development depending on its specific N-terminus.

  20. Histone H2A and H4 N-terminal tails are positioned by the MEP50 WD repeat protein for efficient methylation by the PRMT5 arginine methyltransferase.

    Science.gov (United States)

    Burgos, Emmanuel S; Wilczek, Carola; Onikubo, Takashi; Bonanno, Jeffrey B; Jansong, Janina; Reimer, Ulf; Shechter, David

    2015-04-10

    The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1-20) and H4(1-20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate Km, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.

  1. Two adenine nucleotide translocase paralogues involved in cell proliferation and spermatogenesis in the silkworm Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Ryohei Sugahara

    Full Text Available Mitochondrial adenine nucleotide translocase (ANT specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4 and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4 is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1 and the testis-specific paralogue (BmANTI2. The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1, but not those of other insect species (or PxANTI2, restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4.

  2. Octa-arginine mediated delivery of wild-type Lnk protein inhibits TPO-induced M-MOK megakaryoblastic leukemic cell growth by promoting apoptosis.

    Directory of Open Access Journals (Sweden)

    Chung Yeng Looi

    Full Text Available BACKGROUND: Lnk plays a non-redundant role by negatively regulating cytokine signaling of TPO, SCF or EPO. Retroviral expression of Lnk has been shown to suppress hematopoietic leukemic cell proliferation indicating its therapeutic value in cancer therapy. However, retroviral gene delivery carries risks of insertional mutagenesis. To circumvent this undesired consequence, we fused a cell permeable peptide octa-arginine to Lnk and evaluated the efficacy of inhibition of leukemic cell proliferation in vitro. METHODOLOGY/PRINCIPAL FINDINGS: In this study, proliferation assays, flow cytometry, Western Blot analyses were performed on wild-type (WT, mutant Lnk R8 or BSA treated M-MOK cells. We found that delivered WT, but not mutant Lnk R8 blocked TPO-induced M-MOK megakaryoblastic leukemic cell proliferation. In contrast, WT Lnk R8 showed no growth inhibitive effect on non-hematopoietic HELA or COS-7 cell. Moreover, we demonstrated that TPO-induced M-MOK cell growth inhibition by WT Lnk R8 was dose-dependent. Penetrated WT Lnk R8 induced cell cycle arrest and apoptosis. Immunoprecipitation and Western blots data indicated WT Lnk R8 interacted with endogeneous Jak2 and downregulated Jak-Stat and MAPK phosphorylation level in M-MOK cells after TPO stimulation. Treatment with specific inhibitors (TG101348 and PD98059 indicated Jak-Stat and MAPK pathways were crucial for TPO-induced proliferation of M-MOK cells. Further analyses using TF-1 and HEL leukemic cell-lines showed that WT Lnk R8 inhibited Jak2-dependent cell proliferation. Using cord blood-derived CD34+ stem cells, we found that delivered WT Lnk R8 blocked TPO-induced megakaryopoiesis in vitro. CONCLUSIONS/SIGNIFICANCE: Intracellular delivery of WT Lnk R8 fusion protein efficiently inhibited TPO-induced M-MOK leukemic cell growth by promoting apoptosis. WT Lnk R8 protein delivery may provide a safer and more practical approach to inhibit leukemic cell growth worthy of further development.

  3. Arginine metabolism in wounds

    International Nuclear Information System (INIS)

    Arginine metabolism in wounds was investigated in the rat in 1) lambda-carrageenan-wounded skeletal muscle, 2) Schilling chambers, and 3) subcutaneous polyvinyl alcohol sponges. All showed decreased arginine and elevated ornithine contents and high arginase activity. Arginase could be brought to the wound by macrophages, which were found to contain arginase activity. However, arginase was expressed by macrophages only after cell lysis and no arginase was released by viable macrophages in vitro. Thus the extracellular arginase of wounds may derive from dead macrophages within the injured tissue. Wound and peritoneal macrophages exhibited arginase deiminase activity as demonstrated by the conversion of [guanido-14C]arginine to radiolabeled citrulline during culture, the inhibition of this reaction by formamidinium acetate, and the lack of prokaryotic contamination of the cultures. These findings and the known metabolic fates of the products of arginase and arginine deiminase in the cellular populations of the wound suggest the possibility of cooperativity among cells for the production of substrates for collagen synthesis

  4. Rotations of the 2B Sub-domain of E. coli UvrD Helicase/Translocase Coupled to Nucleotide and DNA Binding

    OpenAIRE

    Jia, Haifeng; Korolev, Sergey; Niedziela-Majka, Anita; Maluf, Nasib K.; Gauss, George H.; Myong, Sua; Ha, Taekjip; Waksman, Gabriel; Lohman, Timothy M.

    2011-01-01

    E. coli UvrD is a superfamily 1 (SF1) DNA helicase and single stranded (ss) DNA translocase that functions in DNA repair, plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA (dsDNA) and translocate along ssDNA with 3′ to 5′ directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Pre...

  5. Altered brain arginine metabolism in schizophrenia

    Science.gov (United States)

    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

  6. PGC-1β regulates mouse carnitine–acylcarnitine translocase through estrogen-related receptor α

    International Nuclear Information System (INIS)

    Highlights: ► The Cact gene is induced in mouse skeletal muscle after 24 h of fasting. ► The Cact gene contains a functional consensus sequence for ERR. ► This sequence binds ERRα both in vivo and in vitro. ► This ERRE is required for the activation of Cact expression by the PGC-1/ERR axis. ► Our results add Cact as a genuine gene target of these transcriptional regulators. -- Abstract: Carnitine/acylcarnitine translocase (CACT) is a mitochondrial-membrane carrier proteins that mediates the transport of acylcarnitines into the mitochondrial matrix for their oxidation by the mitochondrial fatty acid-oxidation pathway. CACT deficiency causes a variety of pathological conditions, such as hypoketotic hypoglycemia, cardiac arrest, hepatomegaly, hepatic dysfunction and muscle weakness, and it can be fatal in newborns and infants. Here we report that expression of the Cact gene is induced in mouse skeletal muscle after 24 h of fasting. To gain insight into the control of Cact gene expression, we examine the transcriptional regulation of the mouse Cact gene. We show that the 5′-flanking region of this gene is transcriptionally active and contains a consensus sequence for the estrogen-related receptor (ERR), a member of the nuclear receptor family of transcription factors. This sequence binds ERRαin vivo and in vitro and is required for the activation of Cact expression by the peroxisome proliferator-activated receptor gamma coactivator (PGC)-1/ERR axis. We also demonstrate that XTC790, the inverse agonist of ERRα, specifically blocks Cact activation by PGC-1β in C2C12 cells.

  7. PGC-1{beta} regulates mouse carnitine-acylcarnitine translocase through estrogen-related receptor {alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Gacias, Mar; Perez-Marti, Albert; Pujol-Vidal, Magdalena; Marrero, Pedro F. [Department of Biochemistry and Molecular Biology, School of Pharmacy and the Institute of Biomedicine of the University of Barcelona (IBUB) (Spain); Haro, Diego, E-mail: dharo@ub.edu [Department of Biochemistry and Molecular Biology, School of Pharmacy and the Institute of Biomedicine of the University of Barcelona (IBUB) (Spain); Relat, Joana [Department of Biochemistry and Molecular Biology, School of Pharmacy and the Institute of Biomedicine of the University of Barcelona (IBUB) (Spain)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer The Cact gene is induced in mouse skeletal muscle after 24 h of fasting. Black-Right-Pointing-Pointer The Cact gene contains a functional consensus sequence for ERR. Black-Right-Pointing-Pointer This sequence binds ERR{alpha} both in vivo and in vitro. Black-Right-Pointing-Pointer This ERRE is required for the activation of Cact expression by the PGC-1/ERR axis. Black-Right-Pointing-Pointer Our results add Cact as a genuine gene target of these transcriptional regulators. -- Abstract: Carnitine/acylcarnitine translocase (CACT) is a mitochondrial-membrane carrier proteins that mediates the transport of acylcarnitines into the mitochondrial matrix for their oxidation by the mitochondrial fatty acid-oxidation pathway. CACT deficiency causes a variety of pathological conditions, such as hypoketotic hypoglycemia, cardiac arrest, hepatomegaly, hepatic dysfunction and muscle weakness, and it can be fatal in newborns and infants. Here we report that expression of the Cact gene is induced in mouse skeletal muscle after 24 h of fasting. To gain insight into the control of Cact gene expression, we examine the transcriptional regulation of the mouse Cact gene. We show that the 5 Prime -flanking region of this gene is transcriptionally active and contains a consensus sequence for the estrogen-related receptor (ERR), a member of the nuclear receptor family of transcription factors. This sequence binds ERR{alpha}in vivo and in vitro and is required for the activation of Cact expression by the peroxisome proliferator-activated receptor gamma coactivator (PGC)-1/ERR axis. We also demonstrate that XTC790, the inverse agonist of ERR{alpha}, specifically blocks Cact activation by PGC-1{beta} in C2C12 cells.

  8. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.

    Science.gov (United States)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas; Lyon, David; Mullari, Meeli; Madsen, Maria V; Daniel, Jeremy A; Jensen, Lars J; Nielsen, Michael L

    2016-01-01

    The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation. PMID:27577262

  9. 5'-Single-stranded/duplex DNA junctions are loading sites for E. coli UvrD translocase.

    Science.gov (United States)

    Tomko, Eric J; Jia, Haifeng; Park, Jeehae; Maluf, Nasib K; Ha, Taekjip; Lohman, Timothy M

    2010-11-17

    Escherichia coli UvrD is a 3'-5' superfamily 1A helicase/translocase involved in a variety of DNA metabolic processes. UvrD can function either as a helicase or only as an single-stranded DNA (ssDNA) translocase. The switch between these activities is controlled in vitro by the UvrD oligomeric state; a monomer has ssDNA translocase activity, whereas at least a dimer is needed for helicase activity. Although a 3'-ssDNA partial duplex provides a high-affinity site for a UvrD monomer, here we show that a monomer also binds with specificity to DNA junctions possessing a 5'-ssDNA flanking region and can initiate translocation from this site. Thus, a 5'-ss-duplex DNA junction can serve as a high-affinity loading site for the monomeric UvrD translocase, whereas a 3'-ss-duplex DNA junction inhibits both translocase and helicase activity of the UvrD monomer. Furthermore, the 2B subdomain of UvrD is important for this junction specificity. This highlights a separation of helicase and translocase function for UvrD and suggests that a monomeric UvrD translocase can be loaded at a 5'-ssDNA junction when translocation activity alone is needed. PMID:20877334

  10. Inhibition of lytic infection of pseudorabies virus by arginine depletion

    International Nuclear Information System (INIS)

    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 catalyzing 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

  11. Plant PRMTs Broaden the Scope of Arginine Methylation

    Institute of Scientific and Technical Information of China (English)

    Ayaz Ahmad; Xiaofeng Cao

    2012-01-01

    Post-translational methylation at arginine residues is one of the most important covalent modifications of proteins,involved in a myriad of essential cellular processes in eukaryotes,such as transcriptional regulation,RNA processing,signal transduction,and DNA repair.Methylation at arginine residues is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs).PRMTs have been extensively studied in various taxa and there is a growing tendency to unveil their functional importance in plants.Recent studies in plants revealed that this evolutionarily conserved family of enzymes regulates essential traits including vegetative growth,flowering time,circadian cycle,and response to high medium salinity and ABA.In this review,we highlight recent advances in the field of posttranslational arginine methylation with special emphasis on the roles and future prospects of this modification in plants.

  12. Níveis de proteína e de arginina digestível na ração pré-inicial de frangos de corte Protein and digestible arginine levels in pre-starter broiler rations

    Directory of Open Access Journals (Sweden)

    Mônica Schaitl Thon

    2010-05-01

    Full Text Available O experimento foi conduzido para avaliar níveis de proteína bruta e arginina digestível na ração pré-inicial de frangos de corte e seus efeitos no desempenho das aves dos 7 aos 21 dias de idade. Foram utilizados 600 pintos da linhagem Cobb 500, distribuídos em delineamento de blocos casualizados em esquema fatorial 4 × 2, composto de quatro níveis de arginina digestível (1,363; 1,463; 1,563 e 1,663% e dois níveis de proteína bruta (20 e 22%, totalizando oito tratamentos, cada um com cinco repetições de 15 aves. Avaliaram-se o ganho de peso, o consumo de ração, o índice de conversão alimentar, a biometria dos órgãos do trato gastrintestinal e a digestibilidade e retenção de matéria seca e nitrogênio. O maior ganho de peso na fase de 1 a 14 dias de idade foi obtido com a ração com 22% de proteína bruta. Os níveis de arginina digestível tiveram efeito quadrático na conversão alimentar na fase de 1 a 10 dias de idade. O peso do esôfago e inglúvio foi maior nas aves alimentadas com a ração com 20% de proteína bruta, no entanto, houve efeito quadrático dos níveis de arginina digestível sobre o comprimento do intestino aos 10 dias de idade e sobre o peso do esôfago + inglúvio aos 3 dias de idade. Houve interação entre os níveis de proteína bruta e arginina digestível para o peso relativo do fígado aos 14 dias, que respondeu de forma quadrática ao nível de 20% de proteína bruta, e para o comprimento de intestino, cujo maior valor foi obtido com os níveis de 22% de proteína bruta e 1,603% de arginina digestível. O balanço e a retenção de nitrogênio foram maiores no nível de 22% de proteína bruta. O nível de 1,363% de arginina digestível atende às exigências nutricionais dos frangos de corte na fase pré-inicial.This experiment was carried out to evaluate levels of crude protein and digestible arginine in pre-starter broiler ration and their effects on the performance of the broilers from 7 to

  13. Arginine Supplementation Recovered the IFN-γ-Mediated Decrease in Milk Protein and Fat Synthesis by Inhibiting the GCN2/eIF2α Pathway, Which Induces Autophagy in Primary Bovine Mammary Epithelial Cells.

    Science.gov (United States)

    Xia, Xiaojing; Che, Yanyi; Gao, Yuanyuan; Zhao, Shuang; Ao, Changjin; Yang, Hongjian; Liu, Juxiong; Liu, Guowen; Han, Wenyu; Wang, Yuping; Lei, Liancheng

    2016-05-31

    During the lactation cycle of the bovine mammary gland, autophagy is induced in bovine mammary epithelial cells (BMECs) as a cellular homeostasis and survival mechanism. Interferon gamma (IFN-γ) is an important antiproliferative and apoptogenic factor that has been shown to induce autophagy in multiple cell lines in vitro. However, it remains unclear whether IFN-γ can induce autophagy and whether autophagy affects milk synthesis in BMECs. To understand whether IFN-γ affects milk synthesis, we isolated and purified primary BMECs and investigated the effect of IFN-γ on milk synthesis in primary BMECs in vitro. The results showed that IFN-γ significantly inhibits milk synthesis and that autophagy was clearly induced in primary BMECs in vitro within 24 h. Interestingly, autophagy was observed following IFN-γ treatment, and the inhibition of autophagy can improve milk protein and milk fat synthesis. Conversely, upregulation of autophagy decreased milk synthesis. Furthermore, mechanistic analysis confirmed that IFN-γ mediated autophagy by depleting arginine and inhibiting the general control nonderepressible-2 kinase (GCN2)/eukaryotic initiation factor 2α (eIF2α) signaling pathway in BMECs. Then, it was found that arginine supplementation could attenuate IFN-γ-induced autophagy and recover milk synthesis to some extent. These findings may not only provide a novel measure for preventing the IFN-γ-induced decrease in milk quality but also a useful therapeutic approach for IFN-γ-associated breast diseases in other animals and humans.

  14. Arginine starvation in colorectal carcinoma cells: Sensing, impact on translation control and cell cycle distribution.

    Science.gov (United States)

    Vynnytska-Myronovska, Bozhena O; Kurlishchuk, Yuliya; Chen, Oleh; Bobak, Yaroslav; Dittfeld, Claudia; Hüther, Melanie; Kunz-Schughart, Leoni A; Stasyk, Oleh V

    2016-02-01

    Tumor cells rely on a continued exogenous nutrient supply in order to maintain a high proliferative activity. Although a strong dependence of some tumor types on exogenous arginine sources has been reported, the mechanisms of arginine sensing by tumor cells and the impact of changes in arginine availability on translation and cell cycle regulation are not fully understood. The results presented herein state that human colorectal carcinoma cells rapidly exhaust the internal arginine sources in the absence of exogenous arginine and repress global translation by activation of the GCN2-mediated pathway and inhibition of mTOR signaling. Tumor suppressor protein p53 activation and G1/G0 cell cycle arrest support cell survival upon prolonged arginine starvation. Cells with the mutant or deleted TP53 fail to stop cell cycle progression at defined cell cycle checkpoints which appears to be associated with reduced recovery after durable metabolic stress triggered by arginine withdrawal.

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

    Science.gov (United States)

    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.

  16. Identification of oligonucleotide sequences that direct the movement of the Escherichia coli FtsK translocase

    OpenAIRE

    Levy, Oren; Ptacin, Jerod L.; Pease, Paul J.; Gore, Jeff; Eisen, Michael B.; Bustamante, Carlos; Cozzarelli, Nicholas R.

    2005-01-01

    FtsK from Escherichia coli is a fast and sequence-directed DNA translocase with roles in chromosome dimer resolution, segregation, and decatenation. From the movement of single FtsK particles on defined DNA substrates and an analysis of skewed DNA sequences in bacteria, we identify GNGNAGGG, its complement, or both as a sequence motif that controls translocation directionality. GNGNAGGG is skewed so that it is predominantly on the leading strand of chromosomal replication. Translocation acros...

  17. Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest

    DEFF Research Database (Denmark)

    Sylvestersen, Kathrine B; Horn, Heiko; Jungmichel, Stephanie;

    2014-01-01

    The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein......, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers...

  18. Glutamine, arginine, and leucine signaling in the intestine.

    Science.gov (United States)

    Marc Rhoads, J; Wu, Guoyao

    2009-05-01

    Glutamine and leucine are abundant constituents of plant and animal proteins, whereas the content of arginine in foods and physiological fluids varies greatly. Besides their role in protein synthesis, these three amino acids individually activate signaling pathway to promote protein synthesis and possibly inhibit autophagy-mediated protein degradation in intestinal epithelial cells. In addition, glutamine and arginine stimulate the mitogen-activated protein kinase and mammalian target of rapamycin (mTOR)/p70 (s6) kinase pathways, respectively, to enhance mucosal cell migration and restitution. Moreover, through the nitric oxide-dependent cGMP signaling cascade, arginine regulates multiple physiological events in the intestine that are beneficial for cell homeostasis and survival. Available evidence from both in vitro and in vivo animal studies shows that glutamine and arginine promote cell proliferation and exert differential cytoprotective effects in response to nutrient deprivation, oxidative injury, stress, and immunological challenge. Additionally, when nitric oxide is available, leucine increases the migration of intestinal cells. Therefore, through cellular signaling mechanisms, arginine, glutamine, and leucine play crucial roles in intestinal growth, integrity, and function.

  19. In search of lipid translocases and their biological functions

    NARCIS (Netherlands)

    Hoekstra, D; van Ijzendoorn, SCD

    2003-01-01

    In plasma membranes, lipids distribute asymmetrically across the bilayer, a process that requires proteins. Recent work identified novel lipid translocators in yeast, and their activity was functionally correlated to endocytosis, thus boosting investigations on identity, mechanism, and function of l

  20. Plastids contain a second sec translocase system with essential functions.

    Science.gov (United States)

    Skalitzky, Courtney A; Martin, Jonathan R; Harwood, Jessica H; Beirne, John J; Adamczyk, Benjamin J; Heck, Gregory R; Cline, Kenneth; Fernandez, Donna E

    2011-01-01

    Proteins that are synthesized on cytoplasmic ribosomes but function within plastids must be imported and then targeted to one of six plastid locations. Although multiple systems that target proteins to the thylakoid membranes or thylakoid lumen have been identified, a system that can direct the integration of inner envelope membrane proteins from the stroma has not been previously described. Genetics and localization studies were used to show that plastids contain two different Sec systems with distinct functions. Loss-of-function mutations in components of the previously described thylakoid-localized Sec system, designated as SCY1 (At2g18710), SECA1 (At4g01800), and SECE1 (At4g14870) in Arabidopsis (Arabidopsis thaliana), result in albino seedlings and sucrose-dependent heterotrophic growth. Loss-of-function mutations in components of the second Sec system, designated as SCY2 (At2g31530) and SECA2 (At1g21650) in Arabidopsis, result in arrest at the globular stage and embryo lethality. Promoter-swap experiments provided evidence that SCY1 and SCY2 are functionally nonredundant and perform different roles in the cell. Finally, chloroplast import and fractionation assays and immunogold localization of SCY2-green fluorescent protein fusion proteins in root tissues indicated that SCY2 is part of an envelope-localized Sec system. Our data suggest that SCY2 and SECA2 function in Sec-mediated integration and translocation processes at the inner envelope membrane. PMID:21051552

  1. Effects of L-Arginine on Physicochemical and Sensory Characteristics of Pork Sausage

    Directory of Open Access Journals (Sweden)

    Cunliu Zhou

    2014-05-01

    Full Text Available The objective of this study is to investigate the effects of L-arginine on physicochemical and sensory properties of pork sausage. CL decreased while pH increased with L-arginine levels (p<0.05. WHC increased at 0.8% L-arginine, but decreased at 0.2% L-arginine, compared with the control. L* decreased while a* increased at 0.4-0.8% L-arginine, compared with the control. Hardness, springiness and chewiness increased at 0.2-0.8% L-arginine (p<0.05, compared with the control. SEM illustrated that the addition of 0.6% L-arginine induced myofibrillar proteins to form a more smooth, compact and uniform gel matrix. DSC disclosed that the addition of 0.6% L-arginine increased the two thermal transition temperatures (Tp. The sample containing 0.6% L-arginine had higher sensory color, flavor, mouthfeel and slice traits than the control. Therefore, L-arginine showed a potential for improvement of yield, texture and sensory qualities of pork sausage.

  2. Conserved arginine residues in the carboxyl terminus of the equine arteritis virus E protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells.

    Science.gov (United States)

    Lu, Zhengchun; Sarkar, Sanjay; Zhang, Jianqiang; Balasuriya, Udeni B R

    2016-04-01

    Equine arteritis virus (EAV), the causative agent of equine viral arteritis, has relatively broad cell tropism in vitro. In horses, EAV primarily replicates in macrophages and endothelial cells of small blood vessels. Until now, neither the cellular receptor(s) nor the mechanism(s) of virus attachment and entry have been determined for this virus. In this study, we investigated the effect of heparin on EAV infection in equine endothelial cells (EECs). Heparin, but not other glycosaminoglycans, could reduce EAV infection up to 93 %. Sequence analysis of the EAV E minor envelope protein revealed a conserved amino acid sequence (52 RSLVARCSRGARYR 65) at the carboxy terminus of the E protein, which was predicted to be the heparin-binding domain. The basic arginine (R) amino acid residues were subsequently mutated to glycine by site-directed mutagenesis of ORF2a in an E protein expression vector and an infectious cDNA clone of EAV. Two single mutations in E (R52G and R57G) did not affect the heparin-binding capability, whereas the E double mutation (R52,60G) completely eliminated the interaction between the E protein and heparin. Although the mutant R52,60G EAV did not bind heparin, the mutations did not completely abolish infectivity, indicating that heparin is not the only critical factor for EAV infection. This also suggested that other viral envelope protein(s) might be involved in attachment through heparin or other cell-surface molecules, and this warrants further investigation.

  3. Homology modeling of major intrinsic proteins in rice, maize and Arabidopsis: comparative analysis of transmembrane helix association and aromatic/arginine selectivity filters

    OpenAIRE

    Sankararamakrishnan Ramasubbu; Bansal Anjali

    2007-01-01

    Abstract Background The major intrinsic proteins (MIPs) facilitate the transport of water and neutral solutes across the lipid bilayers. Plant MIPs are believed to be important in cell division and expansion and in water transport properties in response to environmental conditions. More than 30 MIP sequences have been identified in Arabidopsis thaliana, maize and rice. Plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), Nod26-like intrinsic protein (NIPs) and small...

  4. An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion.

    Directory of Open Access Journals (Sweden)

    Adele Goldman-Pinkovich

    2016-04-01

    Full Text Available 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.

  5. An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion.

    Science.gov (United States)

    Goldman-Pinkovich, Adele; Balno, Caitlin; Strasser, Rona; Zeituni-Molad, Michal; Bendelak, Keren; Rentsch, Doris; Ephros, Moshe; Wiese, Martin; Jardim, Armando; Myler, Peter J; Zilberstein, Dan

    2016-04-01

    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

  6. Characterization of arginine decarboxylase from Dianthus caryophyllus.

    Science.gov (United States)

    Ha, Byung Hak; Cho, Ki Joon; Choi, Yu Jin; Park, Ky Young; Kim, Kyung Hyun

    2004-04-01

    Arginine decarboxylase (ADC, EC 4.1.1.9) is a key enzyme in the biosynthesis of polyamines in higher plants, whereas ornithine decarboxylase represents the sole pathway of polyamine biosynthesis in animals. Previously, we characterized a genomic clone from Dianthus caryophyllus, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 78 kDa. In the present study, the ADC gene was subcloned into the pGEX4T1 expression vector in combination with glutathione S-transferase (GST). The fusion protein GST-ADC was water-soluble and thus was purified by sequential GSTrap-arginine affinity chromatography. A thrombin-mediated on-column cleavage reaction was employed to release free ADC from GST. Hiload superdex gel filtration FPLC was then used to obtain a highly purified ADC. The identity of the ADC was confirmed by immunoblot analysis, and its specific activity with respect to (14)C-arginine decarboxylation reaction was determined to be 0.9 CO(2) pkat mg(-1) protein. K(m) and V(max) of the reaction between ADC and the substrate were 0.077 +/- 0.001 mM and 6.0 +/- 0.6 pkat mg(-1) protein, respectively. ADC activity was reduced by 70% in the presence of 0.1 mM Cu(2+) or CO(2+), but was only marginally affected by Mg(2+), or Ca(2+) at the same concentration. Moreover, spermine at 1 mM significantly reduced its activity by 30%. PMID:15120115

  7. Characterization of arginine decarboxylase from Dianthus caryophyllus.

    Science.gov (United States)

    Ha, Byung Hak; Cho, Ki Joon; Choi, Yu Jin; Park, Ky Young; Kim, Kyung Hyun

    2004-04-01

    Arginine decarboxylase (ADC, EC 4.1.1.9) is a key enzyme in the biosynthesis of polyamines in higher plants, whereas ornithine decarboxylase represents the sole pathway of polyamine biosynthesis in animals. Previously, we characterized a genomic clone from Dianthus caryophyllus, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 78 kDa. In the present study, the ADC gene was subcloned into the pGEX4T1 expression vector in combination with glutathione S-transferase (GST). The fusion protein GST-ADC was water-soluble and thus was purified by sequential GSTrap-arginine affinity chromatography. A thrombin-mediated on-column cleavage reaction was employed to release free ADC from GST. Hiload superdex gel filtration FPLC was then used to obtain a highly purified ADC. The identity of the ADC was confirmed by immunoblot analysis, and its specific activity with respect to (14)C-arginine decarboxylation reaction was determined to be 0.9 CO(2) pkat mg(-1) protein. K(m) and V(max) of the reaction between ADC and the substrate were 0.077 +/- 0.001 mM and 6.0 +/- 0.6 pkat mg(-1) protein, respectively. ADC activity was reduced by 70% in the presence of 0.1 mM Cu(2+) or CO(2+), but was only marginally affected by Mg(2+), or Ca(2+) at the same concentration. Moreover, spermine at 1 mM significantly reduced its activity by 30%.

  8. Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae

    Directory of Open Access Journals (Sweden)

    Krehenbrink Martin

    2008-01-01

    Full Text Available Abstract Background Proteins secreted by bacteria play an important role in infection of eukaryotic hosts. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. Proteins secreted during the infection process by some rhizobial strains can influence infection and modify the plant defence signalling pathways. The aim of this study was to systematically analyse protein secretion in the recently sequenced strain Rhizobium leguminosarum bv. viciae 3841. Results Similarity searches using defined protein secretion systems from other Gram-negative bacteria as query sequences revealed that R. l. bv. viciae 3841 has ten putative protein secretion systems. These are the general export pathway (GEP, a twin-arginine translocase (TAT secretion system, four separate Type I systems, one putative Type IV system and three Type V autotransporters. Mutations in genes encoding each of these (except the GEP were generated, but only mutations affecting the PrsDE (Type I and TAT systems were observed to affect the growth phenotype and the profile of proteins in the culture supernatant. Bioinformatic analysis and mass fingerprinting of tryptic fragments of culture supernatant proteins identified 14 putative Type I substrates, 12 of which are secreted via the PrsDE, secretion system. The TAT mutant was defective for the symbiosis, forming nodules incapable of nitrogen fixation. Conclusion None of the R. l. bv. viciae 3841 protein secretion systems putatively involved in the secretion of proteins to the extracellular space (Type I, Type IV, Type V is required for establishing the symbiosis with legumes. The PrsDE (Type I system was shown to be the major route of protein secretion in non-symbiotic cells and to secrete proteins of widely varied size and predicted function. This is in contrast to many Type I systems from other bacteria, which typically secrete specific substrates encoded by genes often localised in close proximity to

  9. Co- and post-translational translocation through the protein-conducting channel : analogous mechanisms at work?

    NARCIS (Netherlands)

    Mitra, Kakoli; Frank, Joachim; Driessen, Arnold

    2006-01-01

    Many proteins are translocated across, or integrated into, membranes. Both functions are fulfilled by the 'translocon/translocase', which contains a membrane-embedded proteinconducting channel (PCC) and associated soluble factors that drive translocation and insertion reactions using nucleotide trip

  10. Inhibition of Nonsmall Cell Lung Cancer Cell Migration by Protein Arginine Methyltransferase 1-small Hairpin RNA Through Inhibiting Epithelial-mesenchymal Transition, Extracellular Matrix Degradation, and Src Phosphorylation In Vitro

    Directory of Open Access Journals (Sweden)

    Ting Zhang

    2015-01-01

    Full Text Available Background: Protein arginine methyltransferases 1 (PRMT1 is over-expressed in a variety of cancers, including lung cancer, and is correlated with a poor prognosis of tumor development. This study aimed to investigate the role of PRMT1 in nonsmall cell lung cancer (NSCLC migration in vitro. Methods: In this study, PRMT1 expression in the NSCLC cell line A549 was silenced using lentiviral vector-mediated short hairpin RNAs. Cell migration was measured using both scratch wound healing and transwell cell migration assays. The mRNA expression levels of matrix metalloproteinase 2 (MMP-2 and tissue inhibitor of metalloproteinase 1, 2 (TIMP1, 2 were measured using quantitative real-time reverse transcription-polymerase chain reaction. The expression levels of protein markers for epithelial-mesenchymal transition (EMT (E-cadherin, N-cadherin, focal adhesion kinase (FAK, Src, AKT, and their corresponding phosphorylated states were detected by Western blot. Results: Cell migration was significantly inhibited in the PRMT1 silenced group compared to the control group. The mRNA expression of MMP-2 decreased while TIMP1 and TIMP2 increased significantly. E-cadherin mRNA expression also increased while N-cadherin decreased. Only phosphorylated Src levels decreased in the silenced group while FAK or AKT remained unchanged. Conclusions: PRMT1-small hairpin RNA inhibits the migration abilities of NSCLC A549 cells by inhibiting EMT, extracellular matrix degradation, and Src phosphorylation in vitro.

  11. Identification and characterization of two temperature-induced surface-associated proteins of Streptococcus suis with high homologies to members of the arginine deiminase system of Streptococcus pyogenes

    NARCIS (Netherlands)

    Winterhoff, N.; Goethe, R.; Gruening, P.; Rohde, M.; Kalisz, H.; Smith, H.E.; Valentin-Weigand, P.

    2002-01-01

    The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42°C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42°C induc

  12. Dietary arginine and linear growth

    DEFF Research Database (Denmark)

    van Vught, Anneke J A H; Dagnelie, Pieter C; Arts, Ilja C W;

    2013-01-01

    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...

  13. An adenine nucleotide translocase (ANT) gene from Apostichopus japonicus; molecular cloning and expression analysis in response to lipopolysaccharide (LPS) challenge and thermal stress.

    Science.gov (United States)

    Liu, Qiu-Ning; Chai, Xin-Yue; Tu, Jie; Xin, Zhao-Zhe; Li, Chao-Feng; Jiang, Sen-Hao; Zhou, Chun-Lin; Tang, Bo-Ping

    2016-02-01

    The adenine nucleotide translocases (ANTs) play a vital role in energy metabolism via ADP/ATP exchange in eukaryotic cells. Apostichopus japonicus (Echinodermata: Holothuroidea) is an important economic species in China. Here, a cDNA representing an ANT gene of A. japonicus was isolated and characterized from respiratory tree and named AjANT. The full-length AjANT cDNA is 1924 bp, including a 5'-untranslated region (UTR) of 38 bp, 3'-UTR of 980 bp and an open reading frame (ORF) of 906 bp encoding a polypeptide of 301 amino acids. The protein contains three homologous repeat Mito_carr domains (Pfam00153). The deduced AjANT protein sequence has 49-81% in comparison to ANT proteins from other individuals. The predicted tertiary structure of AjANT protein is highly similar to animal ANT proteins. Phylogenetic analysis showed that the AjANT is closely related to Holothuroidea ANT genes. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that AjANT expression is higher in the respiratory tree than in other examined tissues. After thermal stress or LPS challenge, expression of AjANT was significantly fluctuant compared to the control. These results suggested that changes in the expression of ANT gene might be involved in immune defense and in protecting A. japonicus against thermal stress. PMID:26706223

  14. Inhibition of Nonsmall Cell Lung Cancer Cell Migration by Protein Arginine Methyltransferase 1-small Hairpin RNA Through Inhibiting Epithelial-mesenchymal Transition,Extracellular Matrix Degradation, and Src Phosphorylation In Vitro

    Institute of Scientific and Technical Information of China (English)

    Ting Zhang; Ge Cui; Yun-Liang Yao; Yue Guo; Qi-Chun Wang; Xi-Ning Li; Wen-Ming Feng

    2015-01-01

    Background:Protein arginine methyltransferases 1 (PRMT1) is over-expressed in a variety of cancers,including lung cancer,and is correlated with a poor prognosis of tumor development.This study aimed to investigate the role of PRMT1 in nonsmall cell lung cancer (NSCLC) migration in vitro.Methods:In this study,PRMT1 expression in the NSCLC cell line A549 was silenced using lentiviral vector-mediated short hairpin RNAs.Cell migration was measured using both scratch wound healing and transwell cell migration assays.The mRNA expression levels of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor ofmetalloproteinase 1,2 (TIMP l,2) were measured using quantitative real-time reverse transcription-polymerase chain reaction.The expression levels of protein markers for epithelial-mesenchymal transition (EMT) (E-cadherin,N-cadherin),focal adhesion kinase (FAK),Src,AKT,and their corresponding phosphorylated states were detected by Western blot.Results:Cell migration was significantly inhibited in the PRMT1 silenced group compared to the control group.The mRNA expression of MMP-2 decreased while TIMP 1 and TIMP2 increased significantly.E-cadherin mRNA expression also increased while N-cadherin decreased.Only phosphorylated Src levels decreased in the silenced group while FAK or AKT remained unchanged.Conclusions:PRMT1-small hairpin RNA inhibits the migration abilities of NSCLC A549 cells by inhibiting EMT,extracellular matrix degradation,and Src phosphorylation in vitro.

  15. Case-control studies show that a non-conservative amino-acid change from a glutamine to arginine in the P2RX7 purinergic receptor protein is associated with both bipolar- and unipolar-affective disorders.

    Science.gov (United States)

    McQuillin, A; Bass, N J; Choudhury, K; Puri, V; Kosmin, M; Lawrence, J; Curtis, D; Gurling, H M D

    2009-06-01

    Three linkage studies of bipolar disorder have implicated chromosome 12q24.3 with lod scores of over 3.0 and several other linkage studies have found lods between 2 and 3. Fine mapping within the original chromosomal linkage regions has identified several loci that show association with bipolar disorder. One of these is the P2RX7 gene encoding a central nervous system-expressed purinergic receptor. A non-synonymous single nucleotide polymorphism, rs2230912 (P2RX7-E13A, G allele) and a microsatellite marker NBG6 were both previously found to be associated with bipolar disorder (P=0.00071 and 0.008, respectively). rs2230912 has also been found to show association with unipolar depression. The effect of the polymorphism is non-conservative and results in a glutamine to arginine change (Gln460Arg), which is likely to affect P2RX7 dimerization and protein-protein interactions. We have confirmed the allelic associations between bipolar disorder and the markers rs2230912 (P2RX7-E13A, G allele, P=0.043) and NBG6 (P=0.010) in a London-based sample of 604 bipolar cases and 560 controls. When we combined these data with the published case-control studies of P2RX7 and mood disorder (3586 individuals) the association between rs2230912 (Gln460Arg) and affective disorders became more robust (P=0.002). The increase in Gln460Arg was confined to heterozygotes rather than homozygotes suggesting a dominant effect (odds ratio 1.302, CI=1.129-1.503). Although further research is needed to prove that the Gln460Arg change has an aetiological role, it is so far the most convincing mutation to have been found with a role for increasing susceptibility to bipolar and genetically related unipolar disorders.

  16. Structural organization of the rat gene for the arginine vasopressin-neurophysin precursor

    OpenAIRE

    Schmale, H.; Heinsohn, S; Richter, D

    1983-01-01

    The rat arginine vasopressin-neurophysin precursor gene has been isolated from a genomic library cloned in lambda phage Charon 4A. Restriction mapping and nucleotide sequence analysis demonstrated that the gene is 1.85 kilobase pairs long and contains two intervening sequences located in the protein coding region. Exon A encodes a putative signal peptide, the hormone arginine vasopressin and the variable N terminus of the carrier protein neurophysin, exon B encodes the highly conserved middle...

  17. Comparative analysis of serine/arginine-rich proteins across 27 eukaryotes: insights into sub-family classification and extent of alternative splicing.

    Directory of Open Access Journals (Sweden)

    Dale N Richardson

    Full Text Available Alternative splicing (AS of pre-mRNA is a fundamental molecular process that generates diversity in the transcriptome and proteome of eukaryotic organisms. SR proteins, a family of splicing regulators with one or two RNA recognition motifs (RRMs at the N-terminus and an arg/ser-rich domain at the C-terminus, function in both constitutive and alternative splicing. We identified SR proteins in 27 eukaryotic species, which include plants, animals, fungi and "basal" eukaryotes that lie outside of these lineages. Using RNA recognition motifs (RRMs as a phylogenetic marker, we classified 272 SR genes into robust sub-families. The SR gene family can be split into five major groupings, which can be further separated into 11 distinct sub-families. Most flowering plants have double or nearly double the number of SR genes found in vertebrates. The majority of plant SR genes are under purifying selection. Moreover, in all paralogous SR genes in Arabidopsis, rice, soybean and maize, one of the two paralogs is preferentially expressed throughout plant development. We also assessed the extent of AS in SR genes based on a splice graph approach (http://combi.cs.colostate.edu/as/gmap_SRgenes. AS of SR genes is a widespread phenomenon throughout multiple lineages, with alternative 3' or 5' splicing events being the most prominent type of event. However, plant-enriched sub-families have 57%-88% of their SR genes experiencing some type of AS compared to the 40%-54% seen in other sub-families. The SR gene family is pervasive throughout multiple eukaryotic lineages, conserved in sequence and domain organization, but differs in gene number across lineages with an abundance of SR genes in flowering plants. The higher number of alternatively spliced SR genes in plants emphasizes the importance of AS in generating splice variants in these organisms.

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

    Science.gov (United States)

    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.

  19. Proton transfer is rate-limiting for translocation of precursor proteins by the Escherichia coli translocase

    NARCIS (Netherlands)

    Driessen, Arnold J.M.; Wickner, William

    1991-01-01

    The protonmotive force stimulates translocation in vivo, in crude in vitro reactions, and in a purified, reconstituted reaction. Translocation activity is a function of the pH at the inner face of the membrane. Both the transmembrane pH gradient and the transmembrane electrical potential stimulate t

  20. Crystal structure of TDRD3 and methyl-arginine binding characterization of TDRD3, SMN and SPF30.

    Directory of Open Access Journals (Sweden)

    Ke Liu

    Full Text Available SMN (Survival motor neuron protein was characterized as a dimethyl-arginine binding protein over ten years ago. TDRD3 (Tudor domain-containing protein 3 and SPF30 (Splicing factor 30 kDa were found to bind to various methyl-arginine proteins including Sm proteins as well later on. Recently, TDRD3 was shown to be a transcriptional coactivator, and its transcriptional activity is dependent on its ability to bind arginine-methylated histone marks. In this study, we systematically characterized the binding specificity and affinity of the Tudor domains of these three proteins quantitatively. Our results show that TDRD3 preferentially recognizes asymmetrical dimethylated arginine mark, and SMN is a very promiscuous effector molecule, which recognizes different arginine containing sequence motifs and preferentially binds symmetrical dimethylated arginine. SPF30 is the weakest methyl-arginine binder, which only binds the GAR motif sequences in our library. In addition, we also reported high-resolution crystal structures of the Tudor domain of TDRD3 in complex with two small molecules, which occupy the aromatic cage of TDRD3.

  1. Geometry of guanidinium groups in arginines.

    Science.gov (United States)

    Malinska, Maura; Dauter, Miroslawa; Dauter, Zbigniew

    2016-09-01

    The restraints in common usage today have been obtained based on small molecule X-ray crystal structures available 25 years ago and recent reports have shown that the values of bond lengths and valence angles can be, in fact, significantly different from those stored in libraries, for example for the peptide bond or the histidine ring geometry. We showed that almost 50% of outliers found in protein validation reports released in the Protein Data Bank on 23 March 2016 come from geometry of guanidine groups in arginines. Therefore, structures of small molecules and atomic resolution protein crystal structures have been used to derive new target values for the geometry of this group. The most significant difference was found for NE-CZ-NH1 and NE-CZ-NH2 angles, showing that the guanidinium group is not symmetric. The NE-CZ-NH1 angle is larger, 121.5(10)˚, than NE-CZ-NH2, 119.2(10)˚, due to the repulsive interaction between NH1 and CD1 atom.

  2. Combined glutamine and arginine decrease proinflammatory cytokine production by biopsies from Crohn's patients in association with changes in nuclear factor-kappaB and p38 mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Lecleire, Stéphane; Hassan, Aktham; Marion-Letellier, Rachel; Antonietti, Michel; Savoye, Guillaume; Bôle-Feysot, Christine; Lerebours, Eric; Ducrotté, Philippe; Déchelotte, Pierre; Coëffier, Moïse

    2008-12-01

    Glutamine (Gln) and arginine (Arg) are conditionally essential amino acids with immunomodulatory properties. The aim of the study was to assess the effects of Gln and Arg alone or in combination on cytokine release by cultured colonic biopsies from patients with active Crohn's disease (CD). Ten consecutive patients [mean (range) age 26 (18-39) y] with active colonic CD (mean CD activity index: 383.7 +/- 129.8) were prospectively included in the study. Eight colonic biopsies were obtained via a colonoscopy and incubated during 18 h with low (physiological) or high (pharmacological) doses of Arg (0.1 or 2 mmol/L designated as Arg(low) or Arg(high), respectively) and Gln (0.6 or 10 mmol/L designated as Gln(low) or Gln(high), respectively). The concentrations of cytokines [interleukin (IL)-4, IL-10, IL-8, IL-6, tumor necrosis factor-alpha (TNFalpha), IL-1beta, interferon-gamma) were assessed by ELISA, and nitric oxide (NO) production was evaluated by Griess assay. Nuclear factor (NF)-kappaB p65 subunit, inhibitor of NFkappaB-alpha, and p38 mitogen-activated protein kinase (MAPK) were assessed by immunoblotting. Arg(high)/Gln(high) decreased the production of TNFalpha, IL-1beta, IL-8, and IL-6 (each P < 0.01). Arg(low)/Gln(high) decreased IL-6 and IL-8 production (both P < 0.01), whereas Arg(high)/Gln(low) did not affect cytokine and NO production. Arg(low)/Gln(high) and Arg(high)/Gln(high) decreased NF-kappaB p65 subunit expression, whereas p38 MAPK was decreased only by Arg(high)/Gln(high). Combined pharmacological doses of Arg and Gln decreased TNFalpha and the main proinflammatory cytokines release in active colonic CD biopsies via NF-kappaB and p38 MAPK pathways. These results could be the basis of prospective studies evaluating the effects of enteral supply of combined Arg and Gln during active CD.

  3. Influence of in ovo injection of L-arginine on productive and physiological performance of quail

    Directory of Open Access Journals (Sweden)

    W. K. Al–Hayani,

    2011-07-01

    Full Text Available This study evaluated the influence of inoculation of different levels of L–arginine into eggs of 0-day-old quail embryos. On 0 day of incubation, 480 eggs (120 for each treatment group were injected with 0% arginine (C group; 1% arginine (T1; 2% arginine (T2; or 3% arginine (T3. After hatching, 336 quail chicks (84 chicks produced from each in ovo injection treatment were placed in an experimental quail house and distributed into 4 treatment groups of 3 replicates each with 16 quail chicks for each replicate. Traits determined in this study were hatchability rate, initial body weight (7 days of age, final body weight (42 days old, feed intake, weight gain, feed conversion ratio, proportional weights of carcass, breast, legs, back bone, wings, neck, abdominal fat, liver, heart, and gizzard, blood serum glucose, protein, cholesterol, total lipids, triglycerides, calcium and phosphorus and Results revealed that in ovo injection with different levels of L–arginine on 0 day of incubation resulted in significant increase (P≤0.05 in hatchability rate, initial body weight, final body weight, feed conversion ratio and serum glucose, protein, total protein, calcium, phosphorus and proportional weights of carcass, breast, legs, liver, heart, and gizzard and significant decrease (P≤0.05 in serum cholesterol, total lipids, triglycerides and proportional weight of back bone, wings and abdominal fat. In conclusion, the inoculation of different levels of L–arginine into eggs of 0–day–old quail embryos especially at the levels of 2% and 3% resulted in significant improvement in productive and physiological performance of quail. Hence in ovo injection with L–arginine could be used as a beneficial tool for enhance productive performance of quail.

  4. Remodeling and Control of Homologous Recombination by DNA Helicases and Translocases that Target Recombinases and Synapsis.

    Science.gov (United States)

    Northall, Sarah J; Ivančić-Baće, Ivana; Soultanas, Panos; Bolt, Edward L

    2016-01-01

    Recombinase enzymes catalyse invasion of single-stranded DNA (ssDNA) into homologous duplex DNA forming "Displacement loops" (D-loops), a process called synapsis. This triggers homologous recombination (HR), which can follow several possible paths to underpin DNA repair and restart of blocked and collapsed DNA replication forks. Therefore, synapsis can be a checkpoint for controlling whether or not, how far, and by which pathway, HR proceeds to overcome an obstacle or break in a replication fork. Synapsis can be antagonized by limiting access of a recombinase to ssDNA and by dissociation of D-loops or heteroduplex formed by synapsis. Antagonists include DNA helicases and translocases that are identifiable in eukaryotes, bacteria and archaea, and which target synaptic and pre-synaptic DNA structures thereby controlling HR at early stages. Here we survey these events with emphasis on enabling DNA replication to be resumed from sites of blockage or collapse. We also note how knowledge of anti-recombination activities could be useful to improve efficiency of CRISPR-based genome editing. PMID:27548227

  5. Arginines Plasma Concentration and Oxidative Stress in Mild to Moderate COPD

    Science.gov (United States)

    Zinellu, Angelo; Fois, Alessandro Giuseppe; Sotgia, Salvatore; Sotgiu, Elisabetta; Zinellu, Elisabetta; Bifulco, Fabiana; Mangoni, Arduino A; Pirina, Pietro; Carru, Ciriaco

    2016-01-01

    Background Elevated plasma concentrations of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) have been observed in respiratory conditions such as asthma and cystic fibrosis. Since oxidative stress has been shown to increase the activity of arginine methylating enzymes, hence increased ADMA synthesis, and to reduce ADMA degrading enzymes, hence increased ADMA concentrations, we assessed methylated arginines concentrations in chronic obstructive pulmonary disease (COPD), a disease characterized by increased oxidative stress. Methods Plasma arginine, ADMA and symmetric dimethylarginine (SDMA), oxidative stress markers (thiobarbituric acid reactive substances, TBARS, and plasma proteins SH, PSH) and antioxidants (taurine and paraoxonase 1, PON1, activity) were measured in 43 COPD patients with mild (n = 29) or moderate (n = 14) disease and 43 age- and sex-matched controls. Results TBARS significantly increased with COPD presence and severity (median 2.93 vs 3.18 vs 3.64 μmol/L, respectively in controls, mild and moderate group, p<0.0001 by ANOVA) whereas PSH decreased (6.69±1.15 vs 6.04±0.85 vs 5.33±0.96 μmol/gr prot, p<0.0001 by ANOVA). Increased ADMA/arginine ratio, primarily due to reduced arginine concentrations, was also observed with COPD presence and severity (median 0.0067 vs 0.0075 vs 0.0100, p<0.0001 by ANOVA). In multiple logistic regression analysis, only TBARS (OR 0.44, 95% CI 0.25–0.77; p = 0.0045) and ADMA/Arginine ratio (OR 1.72, 95% CI 2.27–13.05; p = 0.02) were independently associated with COPD severity. Conclusion COPD presence and severity are associated with increased oxidative stress and alterations in arginine metabolism. The reduced arginine concentrations in COPD may offer a new target for therapeutic interventions increasing arginine availability. PMID:27479314

  6. A facile reporter system for the experimental identification of twin-arginine translocation (Tat) signal peptides from all kingdoms of life

    NARCIS (Netherlands)

    Widdick, David A.; Eijlander, Robyn T.; van Dijl, Jan Maarten; Kuipers, Oscar P.; Palmer, Tracy

    2008-01-01

    We have developed a reporter protein system for the experimental verification of twin-arginine signal peptides. This reporter system is based on the Streptomyces coelicolor agarase protein, which is secreted into the growth medium by the twin-arginine translocation (Tat) pathway and whose extracellu

  7. PRMT1-mediated arginine methylation controls ATXN2L localization

    Energy Technology Data Exchange (ETDEWEB)

    Kaehler, Christian; Guenther, Anika; Uhlich, Anja; Krobitsch, Sylvia, E-mail: krobitsc@molgen.mpg.de

    2015-05-15

    Arginine methylation is a posttranslational modification that is of importance in diverse cellular processes. Recent proteomic mass spectrometry studies reported arginine methylation of ataxin-2-like (ATXN2L), the paralog of ataxin-2, a protein that is implicated in the neurodegenerative disorder spinocerebellar ataxia type 2. Here, we investigated the methylation state of ATXN2L and its significance for ATXN2L localization. We first confirmed that ATXN2L is asymmetrically dimethylated in vivo, and observed that the nuclear localization of ATXN2L is altered under methylation inhibition. We further discovered that ATXN2L associates with the protein arginine-N-methyltransferase 1 (PRMT1). Finally, we showed that neither mutation of the arginine–glycine-rich motifs of ATXN2L nor methylation inhibition alters ATXN2L localization to stress granules, suggesting that methylation of ATXN2L is probably not mandatory. - Highlights: • ATXN2L is asymmetrically dimethylated in vivo. • ATXN2L interacts with PRMT1 under normal and stress conditions. • PRMT1-mediated dimethylation of ATXN2L controls its nuclear localization. • ATXN2L localization to stress granules appears independent of its methylation state.

  8. Characterization of the PRMT gene family in rice reveals conservation of arginine methylation.

    Directory of Open Access Journals (Sweden)

    Ayaz Ahmad

    Full Text Available Post-translational methylation of arginine residues profoundly affects the structure and functions of protein and, hence, implicated in a myriad of essential cellular processes such as signal transduction, mRNA splicing and transcriptional regulation. Protein arginine methyltransferases (PRMTs, the enzymes catalyzing arginine methylation have been extensively studied in animals, yeast and, to some extent, in model plant Arabidopsis thaliana. Eight genes coding for the PRMTs were identified in Oryza sativa, previously. Here, we report that these genes show distinct expression patterns in various parts of the plant. In vivo targeting experiment demonstrated that GFP-tagged OsPRMT1, OsPRMT5 and OsPRMT10 were localized to both the cytoplasm and nucleus, whereas OsPRMT6a and OsPRMT6b were predominantly localized to the nucleus. OsPRMT1, OsPRMT4, OsPRMT5, OsPRMT6a, OsPRMT6b and OsPRMT10 exhibited in vitro arginine methyltransferase activity against myelin basic protein, glycine-arginine-rich domain of fibrillarin and calf thymus core histones. Furthermore, they depicted specificities for the arginine residues in histones H3 and H4 and were classified into type I and Type II PRMTs, based on the formation of type of dimethylarginine in the substrate proteins. The two homologs of OsPRMT6 showed direct interaction in vitro and further titrating different amounts of these proteins in the methyltransferase assay revealed that OsPRMT6a inhibits the methyltransferase activity of OsPRMT6b, probably, by the formation of heterodimer. The identification and characterization of PRMTs in rice suggests the conservation of arginine methylation in monocots and hold promise for gaining further insight into regulation of plant development.

  9. Large-Scale Identification of the Arginine Methylome by Mass Spectrometry

    DEFF Research Database (Denmark)

    Sylvestersen, Kathrine B; Nielsen, Michael L

    2016-01-01

    The attachment of one or more methylation groups to the side chain of arginine residues is a regulatory mechanism for cellular proteins. Recent advances in mass spectrometry-based characterization allow comprehensive identification of arginine methylation sites by peptide-level enrichment...... strategies. Described in this unit is a 4-day protocol for enrichment of arginine-methylated peptides and subsequent identification of thousands of distinct sites by mass spectrometry. Specifically, the protocol explains step-by-step sample preparation, enrichment using commercially available antibodies......, prefractionation using strong cation exchange, and identification using liquid chromatography coupled to tandem mass spectrometry. A strategy for relative quantification is described using stable isotope labeling by amino acids in cell culture (SILAC). Approaches for analysis of arginine methylation site occupancy...

  10. Adenine Nucleotide Translocase 4 Is Expressed Within Embryonic Ovaries and Dispensable During Oogenesis

    Science.gov (United States)

    Lim, Chae Ho; Brower, Jeffrey V.; Resnick, James L.; Oh, S. Paul

    2015-01-01

    Adenine nucleotide translocase (Ant) facilitates the exchange of adenosine triphosphate across the mitochondrial inner membrane and plays a critical role for bioenergetics in eukaryotes. Mice have 3 Ant paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5), and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. We previously identified that Ant4 was expressed exclusively in testicular germ cells in adult mice and essential for spermatogenesis and subsequently male fertility. Further investigation into the process of spermatogenesis revealed that Ant4 was particularly highly expressed during meiotic prophase I and indispensable for normal progression of leptotene spermatocytes to the stages thereafter. In contrast, the expression and roles of Ant4 in female germ cells have not previously been elucidated. Here, we demonstrate that the Ant4 gene is expressed during embryonic ovarian development during which meiotic prophase I occurs. We confirmed embryonic ovary-specific Ant4 expression using a bacterial artificial chromosome transgene. In contrast to male, however, Ant4 null female mice were fertile although the litter size was slightly decreased. They showed apparently normal ovarian development which was morphologically indistinguishable from the control animals. These data indicate that Ant4 is a meiosis-specific gene expressed during both male and female gametogenesis however indispensable only during spermatogenesis and not oogenesis. The differential effects of Ant4 depletion within the processes of male and female gametogenesis may be explained by meiosis-specific inactivation of the X-linked Ant2 gene in male, a somatic paralog of the Ant4 gene. PMID:25031318

  11. Cationic amino acid transporters and Salmonella Typhimurium ArgT collectively regulate arginine availability towards intracellular Salmonella growth.

    Directory of Open Access Journals (Sweden)

    Priyanka Das

    Full Text Available Cationic amino acid transporters (mCAT1 and mCAT2B regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.

  12. Differential role of arginine mutations on the structure and functions of α-crystallin☆

    Science.gov (United States)

    Panda, Alok Kumar; Nandi, Sandip Kumar; Chakraborty, Ayon; Nagaraj, Ram H.; Biswas, Ashis

    2016-01-01

    Background α-Crystallin is a major protein of the eye lens in vertebrates. It is composed of two subunits, αA- and αB-crystallin. α-Crystallin is an oligomeric protein having these two subunits in 3:1 ratio. It belongs to small heat shock protein family and exhibits molecular chaperone function, which plays an important role in maintaining the lens transparency. Apart from chaperone function, both subunits also exhibit anti-apoptotic property. Comparison of their primary sequences reveals that αA- and αB-crystallin posses 13 and 14 arginine residues, respectively. Several of them undergo mutations which eventually lead to various eye diseases such as congenital cataract, juvenile cataract, and retinal degeneration. Interestingly, many arginine residues of these subunits are modified during glycation and even some are truncated during aging. All these facts indicate the importance of arginine residues in α-crystallin. Scope of review In this review, we will emphasize the recent in vitro and in vivo findings related to congenital cataract causing arginine mutations in α-crystallin. Major conclusions Congenital cataract causing arginine mutations alters the structure and decreases the chaperone function of α-crystallin. These mutations also affect the lens morphology and phenotypes. Interestingly, non-natural arginine mutations (generated for mimicking the glycation and truncation environment) improve the chaperone function of α-crystallin which may play an important role in maintaining the eye lens transparency during aging. General significance The neutralization of positive charge on the guanidino group of arginine residues is not always detrimental to the functionality of α-crystallin. PMID:26080000

  13. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Zheng Gong

    Full Text Available Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.

  14. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

    Science.gov (United States)

    Gong, Zheng; Tang, M Matt; Wu, Xueliang; Phillips, Nancy; Galkowski, Dariusz; Jarvis, Gary A; Fan, Huizhou

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.

  15. Relationship of arginine with lysine in diets for laying Japanese quails

    Directory of Open Access Journals (Sweden)

    Renata de Souza Reis

    2012-01-01

    Full Text Available To determine the relationship of arginine with lysine for Japanese quails during the period of production, an experiment was conducted using 360 subspecies of Japanese quails (Coturnix coturnix japonica with 162 days of age, distributed in a completely randomized design. Diets were formulated with corn, soybean meal, sorghum and wheat bran containing 20.0% crude protein and 2,800 kcal ME/kg. The basal diet contained suboptimal level of lysine equal to 1% and was supplemented with five levels of L-arginine 99% (0.032; 0.083; 0.134; 0.185 and 0.236% to replace the glutamic acid, corresponding to the relationship of arginine with digestible lysine of 1.16, 1.21, 1.26, 1.31 and 1.36. The parameters studied were: feed intake, egg production per hen/day, egg production per hen housed, commercial egg production, egg weight, egg mass, feed conversion by egg mass, feed conversion per dozen eggs, weight and percentage of components of the eggs (yolk, albumen and shell and specific gravity. There was no significant effect on the relationship of arginine with digestible lysine in the diet of Japanese quails for any of the parameters examined. The arginine/lysine ratio of 1.16, which corresponds to a daily intake of 288.84 mg of arginine, provides satisfactory performance and egg quality of Japanese quails.

  16. Arginine regulation of gramicidin S biosynthesis.

    OpenAIRE

    Poirier, A.; Demain, A L

    1981-01-01

    Several amino acids are known to affect the gramicidin S producer Bacillus brevis ATCC 9999 with respect ot growth, soluble gramicidin S synthetase formation, antibiotic production, or a combination of these. Our studies confirmed that arginine has paradoxical effects on the B. brevis fermentation; it markedly increased growth and antibiotic production, yet decreased the soluble heavy gramicidin S synthetase activity. We found that arginine did not repress heavy gramicidin S synthetase. The a...

  17. Three ways in, one way out: water dynamics in the trans-membrane domains of the inner membrane translocase AcrB.

    Science.gov (United States)

    Fischer, Nadine; Kandt, Christian

    2011-10-01

    Powered by proton-motive force, the inner membrane translocase AcrB is the engine of the AcrAB-TolC efflux pump in Escherichia coli. As proton conduction in proteins occurs along hydrogen-bonded networks of polar residues and water molecules, knowledge of the protein-internal water distribution and water-interacting residues allows drawing conclusions to possible pathways of proton conduction. Here, we report a series of 6× 50 ns independent molecular dynamics simulations of asymmetric AcrB embedded in a phospholipid/water environment. Simulating each monomer in its proposed protonation state, we calculated for each trans-membrane domain the average water distribution, identified residues interacting with these waters and quantified each residue's frequency of water hydrogen bond contact. Combining this information we find three possible routes of proton transfer connecting a continuously hydrated region of known key residues in the TMD interior to bulk water by one cytoplasmic and up to three periplasm water channels in monomer B and A. We find that water access of the trans-membrane domains is regulated by four groups of residues in a combination of side chain re-orientations and shifts of trans-membrane helices. Our findings support a proton release event via Arg971 during the C intermediate or in the transition to A, and proton uptake occurring in the A or B state or during a so far unknown intermediate in between B and C where cytoplasmic water access is still possible. Our simulations suggest experimentally testable hypotheses, which have not been investigated so far. PMID:21905112

  18. Arginine methylation of REF/ALY promotes efficient handover of mRNA to TAP/NXF1.

    Science.gov (United States)

    Hung, Ming-Lung; Hautbergue, Guillaume M; Snijders, Ambrosius P L; Dickman, Mark J; Wilson, Stuart A

    2010-06-01

    The REF/ALY mRNA export adaptor binds TAP/NXF1 via an arginine-rich region, which overlaps with its RNA-binding domain. When TAP binds a REF:RNA complex, it triggers transfer of the RNA from REF to TAP. Here, we have examined the effects of arginine methylation on the activities of the REF protein in mRNA export. We have mapped the arginine methylation sites of REF using mass spectrometry and find that several arginines within the TAP and RNA binding domains are methylated in vivo. However, arginine methylation has no effect on the REF:TAP interaction. Instead, arginine methylation reduces the RNA-binding activity of REF in vitro and in vivo. The reduced RNA-binding activity of REF in its methylated state is essential for efficient displacement of RNA from REF by TAP in vivo. Therefore, arginine methylation fine-tunes the RNA-binding activity of REF such that the RNA-protein interaction can be readily disrupted by export factors further down the pathway.

  19. Intravenous Selenium Modulates L-Arginine-Induced Experimental Acute Pancreatitis

    Directory of Open Access Journals (Sweden)

    Jonathan Hardman

    2005-09-01

    Full Text Available Context Oxidative stress is understood to have a critical role in the development of acinar injury in experimental acute pancreatitis. We have previously demonstrated that compound multiple antioxidant therapy ameliorates end-organ damage in the intra-peritoneal L-arginine rat model. As the principal co-factor for glutathione, selenium is a key constituent of multiple antioxidant preparations. Objective The intention of this study was to investigate the effect of selenium on pancreatic and remote organ injury in a wellvalidated experimental model of acute pancreatitis. Methods Male Sprague-Dawley rats were randomly allocated to one of 3 groups (n=5/group and sacrificed at 72 hours. Acute pancreatitis was induced by 250 mg per 100 g body weight of 20% L-arginine hydrochloride in 0.15 mol/L sodium chloride. Group allocations were: Group 1, control; Group 2, acute pancreatitis; Group 3, selenium. Main outcome measures Serum amylase, anti-oxidant levels, bronchoalveolar lavage protein, lung myeloperoxidase activity, and histological assessment of pancreatic injury. Results L-arginine induced acute pancreatitis characterised by oedema, neutrophil infiltration, acinar cell degranulation and elevated serum amylase. Selenium treatment was associated with reduced pancreatic oedema and inflammatory cell infiltration. Acinar degranulation and dilatation were completely absent. A reduction in bronchoalveolar lavage protein content was also demonstrated. Conclusion Intravenous selenium given 24 hours after induction of experimental acute pancreatitis was associated with a reduction in the histological stigmata of pancreatic injury and a dramatic reduction in broncho-alveolar lavage protein content. Serum selenium fell during the course of experimental acute pancreatitis and this effect was not reversed by exogenous selenium supplementation.

  20. Arginine and glutamine availability and macrophage functions in the obese insulin-resistant Zucker rat.

    Science.gov (United States)

    Blanc, Marie-Céline; Moinard, Christophe; Béziel, Aurélie; Darquy, Sylviane; Cynober, Luc; De Bandt, Jean-Pascal

    2005-01-01

    Increased susceptibility to infections in obese patients may be related to decreased availability of arginine and glutamine, which may affect immune cell functions. Our aim was to evaluate the in vitro effects of these amino acids on the function of macrophages from obese insulin-resistant Zucker rats. Macrophages, isolated from male Zucker obese or lean rats by peritoneal lavage, were incubated in Dulbecco's modified Eagle medium (DMEM) without arginine or glutamine. Arginine or glutamine was added to the medium at increasing final concentrations (0, 0.25, 0.5, 1 or 2 mM). After stimulation by lipopolysaccharide (LPS) from E. coli (40 microg/ml), productions of tumour necrosis factor alpha (TNFalpha) and of nitric oxide (NO) were measured after 3 or 48 h incubation, respectively. NO production, lower in macrophages from obese rats, decreased in macrophages from lean rats (0 mM: 2,423 +/- 1,174 vs. 2 mM: 198 +/- 31 microM/mg protein/24 h; P glutamine was added. TNFalpha production, lower in macrophages from obese rats, was inversely correlated with glutamine concentration. In the presence of arginine, NO production was constantly higher in macrophages from obese rats. It peaked at 0.5 mM arginine and decreased thereafter in both groups. TNFalpha production in macrophages from lean rats was unaffected by arginine, but decreased in macrophages from obese rats (0 mM: 1920 +/- 450 vs. 2 mM: 810 +/- 90 microM/mg protein/3 h; P arginine and glutamine metabolism in macrophages of obese rats, resulting in decreased TNFalpha production and increased NO release, may contribute to increased susceptibility to infection in insulin-resistant states.

  1. Detection of a novel arginine vasopression defect by dideoxy fingerprinting

    Energy Technology Data Exchange (ETDEWEB)

    Krishnamani, M.R.S.; Phillips, J.A. III; Copeland, K.C. (Vanderbilt Univ. School of Medicine, Nashville, TN (United States) Univ. of Vermont College of Medicine, Burlington, VT (United States))

    1993-09-01

    Autosomal dominant neurohypophyseal diabetes insipidus is a familial form of diabetes insipidus. This disorder is associated with variable levels of arginine vasopressin (AVP) and diabetes insipidus of varying severity, which responds to exogenous AVP. To determine the molecular basis of autosomal dominant neurohypophyseal diabetes insipidus, the AVP genes of members of a large kindred were analyzed. A new method, called dideoxy fingerprinting, was used to detect an AVP mutation that was characterized by DNA sequencing. The novel defect found changes the last codon of the AVP signal peptide from alanine to threonine, which should perturb cleavage of mature AVP from its precursor protein and inhibit its secretion or action. 18 refs., 3 figs.

  2. Rotations of the 2B Sub-domain of E. coli UvrD Helicase/Translocase Coupled to Nucleotide and DNA Binding

    Energy Technology Data Exchange (ETDEWEB)

    Jia, Haifeng; Korolev, Sergey; Niedziela-Majka, Anita; Maluf, Nasib K.; Gauss, George H.; Myong, Sua; Ha, Taekjip; Waksman, Gabriel; Lohman, Timothy M. (UIUC); (St. Louis-MED); (WU-MED); (UCL)

    2011-11-02

    Escherichia coli UvrD is a superfamily 1 DNA helicase and single-stranded DNA (ssDNA) translocase that functions in DNA repair and plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA and translocate along ssDNA with 3'-to-5' directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Previous crystal structures of UvrD bound to a ssDNA/duplex DNA junction show that its 2B sub-domain exists in a 'closed' state and interacts with the duplex DNA. Here, we report a crystal structure of an apo form of UvrD in which the 2B sub-domain is in an 'open' state that differs by an {approx} 160{sup o} rotation of the 2B sub-domain. To study the rotational conformational states of the 2B sub-domain in various ligation states, we constructed a series of double-cysteine UvrD mutants and labeled them with fluorophores such that rotation of the 2B sub-domain results in changes in fluorescence resonance energy transfer. These studies show that the open and closed forms can interconvert in solution, with low salt favoring the closed conformation and high salt favoring the open conformation in the absence of DNA. Binding of UvrD to DNA and ATP binding and hydrolysis also affect the rotational conformational state of the 2B sub-domain, suggesting that 2B sub-domain rotation is coupled to the function of this nucleic acid motor enzyme.

  3. Rotations of the 2B sub-domain of E. coli UvrD helicase/translocase coupled to nucleotide and DNA binding.

    Science.gov (United States)

    Jia, Haifeng; Korolev, Sergey; Niedziela-Majka, Anita; Maluf, Nasib K; Gauss, George H; Myong, Sua; Ha, Taekjip; Waksman, Gabriel; Lohman, Timothy M

    2011-08-19

    Escherichia coli UvrD is a superfamily 1 DNA helicase and single-stranded DNA (ssDNA) translocase that functions in DNA repair and plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA and translocate along ssDNA with 3'-to-5' directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Previous crystal structures of UvrD bound to a ssDNA/duplex DNA junction show that its 2B sub-domain exists in a "closed" state and interacts with the duplex DNA. Here, we report a crystal structure of an apo form of UvrD in which the 2B sub-domain is in an "open" state that differs by an ∼160° rotation of the 2B sub-domain. To study the rotational conformational states of the 2B sub-domain in various ligation states, we constructed a series of double-cysteine UvrD mutants and labeled them with fluorophores such that rotation of the 2B sub-domain results in changes in fluorescence resonance energy transfer. These studies show that the open and closed forms can interconvert in solution, with low salt favoring the closed conformation and high salt favoring the open conformation in the absence of DNA. Binding of UvrD to DNA and ATP binding and hydrolysis also affect the rotational conformational state of the 2B sub-domain, suggesting that 2B sub-domain rotation is coupled to the function of this nucleic acid motor enzyme. PMID:21704638

  4. Selection of Arginine-Rich Anti-Gold Antibodies Engineered for Plasmonic Colloid Self-Assembly

    CERN Document Server

    Jain, Purvi; Narayanan, S Shankara; Sharma, Jadab; Girard, Christian; Dujardin, Erik; Nizak, Clément

    2014-01-01

    Antibodies are affinity proteins with a wide spectrum of applications in analytical and therapeutic biology. Proteins showing specific recognition for a chosen molecular target can be isolated and their encoding sequence identified in vitro from a large and diverse library by phage display selection. In this work, we show that this standard biochemical technique rapidly yields a collection of antibody protein binders for an inorganic target of major technological importance: crystalline metallic gold surfaces. 21 distinct anti-gold antibody proteins emerged from a large random library of antibodies and were sequenced. The systematic statistical analysis of all the protein sequences reveals a strong occurrence of arginine in anti-gold antibodies, which corroborates recent molecular dynamics predictions on the crucial role of arginine in protein/gold interactions. Once tethered to small gold nanoparticles using histidine tag chemistry, the selected antibodies could drive the self-assembly of the colloids onto t...

  5. Arginine Adjunctive Therapy in Active Tuberculosis

    Directory of Open Access Journals (Sweden)

    Aliasghar Farazi

    2015-01-01

    Full Text Available Background. Dietary supplementation has been used as a mechanism to augment the immune system. Adjunctive therapy with L-arginine has the potential to improve outcomes in active tuberculosis. Methods. In a randomized clinical trial 63 participants with smear-positive pulmonary tuberculosis in Markazi Province of Iran were given arginine or placebo for 4 weeks in addition to conventional chemotherapy. The final treatment success, sputum conversion, weight gain, and clinical symptoms after one and two months were considered as primary outcomes and secondary outcomes were ESR, CRP, and Hg. Data were collected and analyzed with SPSS software (ver. 18. Results. Arginine supplementation reduced constitutional symptoms (P=0.032 in patients with smear-positive TB at the end of the first month of treatment. Arginine treated patients had significantly increased BMI at the end of the first and second months of treatment (P=0.032 and P=0.04 and a reduced CRP at the end of the first month of treatment (P=0.03 versus placebo group. Conclusion. Arginine is useful as an adjunctive therapy in patients with active tuberculosis, in which the effects are more likely mediated by the increased production of nitric oxide and improved constitutional symptoms and weight gain. This trial is registered with Clinical Trials Registry of Iran: IRCT201211179855N2.

  6. Arginase and Arginine Dysregulation in Asthma

    Directory of Open Access Journals (Sweden)

    Renée C. Benson

    2011-01-01

    Full Text Available In recent years, evidence has accumulated indicating that the enzyme arginase, which converts L-arginine into L-ornithine and urea, plays a key role in the pathogenesis of pulmonary disorders such as asthma through dysregulation of L-arginine metabolism and modulation of nitric oxide (NO homeostasis. Allergic asthma is characterized by airway hyperresponsiveness, inflammation, and remodeling. Through substrate competition, arginase decreases bioavailability of L-arginine for nitric oxide synthase (NOS, thereby limiting NO production with subsequent effects on airway tone and inflammation. By decreasing L-arginine bioavailability, arginase may also contribute to the uncoupling of NOS and the formation of the proinflammatory oxidant peroxynitrite in the airways. Finally, arginase may play a role in the development of chronic airway remodeling through formation of L-ornithine with downstream production of polyamines and L-proline, which are involved in processes of cellular proliferation and collagen deposition. Further research on modulation of arginase activity and L-arginine bioavailability may reveal promising novel therapeutic strategies for asthma.

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

    Directory of Open Access Journals (Sweden)

    Durzan Don J

    2009-02-01

    Full Text Available Abstract 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.

  8. [Antioxidant effects of L-arginine in the rat heart in experimental rhabdomyolysis].

    Science.gov (United States)

    Filimonenko, V P; Nikitchenko, I V; Kaliman, P A

    2009-01-01

    The glycerol administration in a dose of 1 ml of 50% water solution/100 g b. w. was found to cause considerable accumulation of the total heme in the rat blood serum that is accompanied by an increase of TBA-reactive products and protein carbonyl derivates contents and by changes of protein level. Heme entering in the heart tissue is observed in the first hours after glycerol injection. The breaches of heart antioxidant-prooxidant balance are noted in twenty-four hours: TBA-reactive products and protein carbonyl derivates accumulation, heme oxygenase and catalase activation, superoxide dismutase activity lowering and reduction of glutathione content elevation. Pretreatment by L-arginine (0.5 h before glycerol administration) almost did not affect the blood serum changes caused by glycerol injection. However in the rat heart L-arginine administration prevents from TBA-reactive products and protein carbonyl derivates accumulation and the breaches of superoxide dismutase and catalase activities. Besides L-arginine causes the ealier heme oxygenase induction. Possible mechanisms of L-arginine protective action in the rat heart under experimental rhabdomyolysis are discussed. PMID:19877424

  9. Rosiglitazone reduces fatty acid translocase and increases AMPK in skeletal muscle in aged rats: a possible mechanism to prevent high-fat-induced insulin resistance

    Institute of Scientific and Technical Information of China (English)

    SONG Guang-yao; GAO Yu; WANG Chao; HU Shu-guo; WANG Jing; QU Dong-ming; MA Hui-juan

    2010-01-01

    Background As an agonist of peroxisome proliferator-activated receptor-gamma (PPARy), rosiglitazone can prevent acute fatty acid-induced insulin resistance in rats, however, the precise mechanisms by which rosiglitazone alleviates insulin resistance induced by high-fat diet need to be further investigated.Methods Wistar rats aged 23-24 weeks were divided into three groups: (1) aged control group (OC), (2) high-fat diet (HF) group and (3) high-fat diet plus rosiglitazone maleate tablets (HF+Rosi) treatment group (n=20 in each group). Insulin sensitivity was evaluated by conscious hyperinsulinemic-euglycemic clamp technique. mRNA levels of fatty acid translocase (FAT/CD36), AMP-activated protein kinase α1 (AMPKα1), AMPKα2 and acetyl CoA carboxylase (ACC) of rat skeletal muscle were determined using real-time PCR, while muscle camitine palmitoyltransferase-1 (CPT-1β) was determined using semi-quantitative PCR. Protein expression levels of FAT/CD36, AMPK phosphorylation (reflecting AMPK activity), P-ACC (inversely related with ACC activity) and muscle CPT-1M in rat skeletal muscles were measured using Western blotting.Results Aged rats fed by diet rich in fat for more than 8 weeks led to significant increases of plasma lipids, skeletal muscle intramuscular triglyceride and long-chain fatty acyl-CoA (LCACoA) compared to aged rats fed by normal chow diet (OC) (P <0.05), which might correlate with the lower (reduced by 42.4%) whole body insulin sensitivity in HF rats. FAT/CD36 protein concentrations and mRNA levels increased in untreated HF aged rats (P <0.01) and high-fat diet induced a significant decrease in P-AMPK, P-ACC, CPT-1M protein concentrations and AMPKα2 and CPT-1β mRNA levels in rat skeletal muscles (P <0.05). No change in AMPKα1 mRNA levels was observed in the HF group.Conclusion High-fat diet in aged rats results in a lipid accumulation and subsequent insulin resistance, while rosiglitazone can alleviate the insulin resistance by reducing fatty

  10. Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods

    Directory of Open Access Journals (Sweden)

    Cabrera-Luque Juan

    2008-09-01

    Full Text Available Abstract Background The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG, an essential allosteric activator of carbamylphosphate synthetase I (CPSI in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS, which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS. Results Information from mutagenesis studies of E. coli and P. aeruginosa NAGS was combined with structural information from the related bacterial N-acetylglutamate kinases to identify four residues in mammalian NAGS that interact with arginine. Substitutions of these four residues were engineered in mouse NAGS and into the vertebrate-like N-acetylglutamate synthase-kinase (NAGS-K of Xanthomonas campestris, which is inhibited by arginine. All mutations resulted in arginine losing the ability to activate mouse NAGS, and inhibit X. campestris NAGS-K. To examine at what point in evolution inversion of arginine effect on NAGS occur, we cloned NAGS from fish and frogs and examined the arginine response of their corresponding proteins. Fish NAGS were partially inhibited by arginine and frog NAGS were activated by arginine. Conclusion Difference in arginine effect on bacterial and mammalian NAGS most likely stems from the difference in the type of conformational change triggered by arginine binding to these proteins. The change from arginine

  11. Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

    Energy Technology Data Exchange (ETDEWEB)

    Min, Li; Jin, Zhongmin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang (GUW); (Maryland); (GWU); (Georgia)

    2010-01-07

    N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in L-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by L-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with L-arginine bound and in the active R-state complexed with CoA and L-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of L-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by {approx}10 {angstrom} and decreases its height by {approx}20{angstrom}. AAK dimers move 5{angstrom} outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by {approx}4{sup o}. The NAT domains rotate {approx}109{sup o} relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the L-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.

  12. L-Arginine Intake Effect on Adenine Nucleotide Metabolism in Rat Parenchymal and Reproductive Tissues

    Directory of Open Access Journals (Sweden)

    G. Kocic

    2012-01-01

    Full Text Available L-arginine is conditionally essetcial amino acid, required for normal cell growth, protein synthesis, ammonia detoxification, tissue growth and general performance, proposed in the treatment of men sterility and prevention of male impotence. The aim of the present paper was to estimate the activity of the enzymes of adenine nucleotide metabolism: 5′-nucleotidase (5′-NU, adenosine deaminase (ADA, AMP deaminase, and xanthine oxidase (XO, during dietary intake of L-arginine for a period of four weeks of male Wistar rats. Adenosine concentration in tissues is maintained by the relative activities of the adenosine-producing enzyme, 5′-NU and the adenosine-degrading enzyme-ADA adenosine deaminase. Dietary L-arginine intake directed adenine nucleotide metabolism in liver, kidney, and testis tissue toward the activation of adenosine production, by increased 5′-NU activity and decreased ADA activity. Stimulation of adenosine accumulation could be of importance in mediating arginine antiatherosclerotic, vasoactive, immunomodulatory, and antioxidant effects. Assuming that the XO activity reflects the rate of purine catabolism in the cell, while the activity of AMP deaminase is of importance in ATP regeneration, reduced activity of XO, together with the increased AMP-deaminase activity, may suggest that adenine nucleotides are presumably directed to the ATP regenerating process during dietary L-arginine intake.

  13. Modulators of arginine metabolism support cancer immunosurveillance

    Directory of Open Access Journals (Sweden)

    Freschi Massimo

    2009-01-01

    Full Text Available Abstract Background Tumor-associated accrual of myeloid derived suppressor cells (MDSC in the blood, lymphoid organs and tumor tissues may lead to perturbation of the arginine metabolism and impairment of the endogenous antitumor immunity. The objective of this study was to evaluate whether accumulation of MDSC occurred in Th2 prone BALB/c and Th1 biased C57BL/6 mice bearing the C26GM colon carcinoma and RMA T lymphoma, respectively, and to investigate whether N(G nitro-L-arginine methyl ester (L-NAME and sildenafil, both modulators of the arginine metabolism, restored antitumor immunity. Results We report here that MDSC accumulate in the spleen and blood of mice irrespective of the mouse and tumor model used. Treatment of tumor-bearing mice with either the phosphodiesterase-5 inhibitor sildenafil or the nitric-oxide synthase (NOS inhibitor L-NAME significantly restrained tumor growth and expanded the tumor-specific immune response. Conclusion Our data emphasize the role of MDSC in modulating the endogenous tumor-specific immune response and underline the anti-neoplastic therapeutic potential of arginine metabolism modulators.

  14. Determination of arginine catabolism by salivary pellet

    NARCIS (Netherlands)

    M.A. Hoogenkamp; J.M. ten Cate

    2014-01-01

    To determine the formation of ammonium from arginine by oral bacteria residing in saliva and dental plaque, an arginolytic activity assay based on the work described by Nascimento et al. [2] was developed. Following the original methodology, insufficient ammonium production could be determined. To i

  15. Low plasma arginine:asymmetric dimethyl arginine ratios predict mortality after intracranial aneurysm rupture

    DEFF Research Database (Denmark)

    Staalsø, Jonatan Myrup; Bergström, Anita; Edsen, Troels;

    2013-01-01

    Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases, predicts mortality in cardiovascular disease and has been linked to cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). In this prospective study, we assessed whether circulating ADMA, arginine...

  16. Mass spectrometry-based identification and characterisation of lysine and arginine methylation in the human proteome.

    Science.gov (United States)

    Bremang, Michael; Cuomo, Alessandro; Agresta, Anna Maria; Stugiewicz, Magdalena; Spadotto, Valeria; Bonaldi, Tiziana

    2013-09-01

    Protein methylation is a post-translational modification (PTM) by which a variable number of methyl groups are transferred to lysine and arginine residues within proteins. Despite increased interest in this modification due to its reversible nature and its emerging role in a diverse set of biological pathways beyond chromatin, global identification of protein methylation has remained an unachieved goal. To characterise sites of lysine and arginine methylation beyond histones, we employed an approach that combines heavy methyl stable isotope labelling by amino acids in cell culture (hmSILAC) with high-resolution mass spectrometry-based proteomics. Through a broad evaluation of immuno-affinity enrichment and the application of two classical protein separation techniques prior to mass spectrometry, to nucleosolic and cytosolic fractions separately, we identified a total of 501 different methylation types, on 397 distinct lysine and arginine sites, present on 139 unique proteins. Our results considerably extend the number of known in vivo methylation sites and indicate their significant presence on several protein complexes involved at all stages of gene expression, from chromatin remodelling and transcription to splicing and translation. In addition, we describe the potential of the hmSILAC approach for accurate relative quantification of methylation levels between distinct functional states. PMID:23748837

  17. Noncanonical Myo9b-RhoGAP Accelerates RhoA GTP Hydrolysis by a Dual-Arginine-Finger Mechanism.

    Science.gov (United States)

    Yi, Fengshuang; Kong, Ruirui; Ren, Jinqi; Zhu, Li; Lou, Jizhong; Wu, Jane Y; Feng, Wei

    2016-07-31

    The GTP hydrolysis activities of Rho GTPases are stimulated by GTPase-activating proteins (GAPs), which contain a RhoGAP domain equipped with a characteristic arginine finger and an auxiliary asparagine for catalysis. However, the auxiliary asparagine is missing in the RhoGAP domain of Myo9b (Myo9b-RhoGAP), a unique motorized RhoGAP that specifically targets RhoA for controlling cell motility. Here, we determined the structure of Myo9b-RhoGAP in complex with GDP-bound RhoA and magnesium fluoride. Unexpectedly, Myo9b-RhoGAP contains two arginine fingers at its catalytic site. The first arginine finger resembles the one within the canonical RhoGAP domains and inserts into the nucleotide-binding pocket of RhoA, whereas the second arginine finger anchors the Switch I loop of RhoA and interacts with the nucleotide, stabilizing the transition state of GTP hydrolysis and compensating for the lack of the asparagine. Mutating either of the two arginine fingers impaired the catalytic activity of Myo9b-RhoGAP and affected the Myo9b-mediated cell migration. Our data indicate that Myo9b-RhoGAP accelerates RhoA GTP hydrolysis by a previously unknown dual-arginine-finger mechanism, which may be shared by other noncanonical RhoGAP domains lacking the auxiliary asparagine. PMID:27363609

  18. Biosynthetic arginine decarboxylase in phytopathogenic fungi.

    Science.gov (United States)

    Khan, A J; Minocha, S C

    1989-01-01

    It has been reported that while bacteria and higher plants possess two different pathways for the biosynthesis of putrescine, via ornithine decarboxylase (ODC) and arginine decarboxylase (ADC); the fungi, like animals, only use the former pathway. We found that contrary to the earlier reports, two of the phytopathogenic fungi (Ceratocystis minor and Verticillium dahliae) contain significant levels of ADC activity with very little ODC. The ADC in these fungi has high pH optimum (8.4) and low Km (0.237 mM for C. minor, 0.103 mM for V. dahliae), and is strongly inhibited by alpha-difluoromethylarginine (DFMA), putrescine and spermidine, further showing that this enzyme is probably involved in the biosynthesis of polyamines and not in the catabolism of arginine as in Escherichia coli. The growth of these fungi is strongly inhibited by DFMA while alpha-difluoromethylornithine (DFMO) has little effect.

  19. Differential expression of protein kinase C beta I (PKC beta I) but not PKC alpha and PKC beta II in the suprachiasmatic nucleus of selected house mouse lines, and the relationship to arginine-vasopressin

    NARCIS (Netherlands)

    Kobylk, ME; Van der Zee, EA; Bult, Abel

    2001-01-01

    The functional significance of the suprachiasmatic nucleus (SCN) in circadian rhythm control of mammals has been well documented. The role of protein phosphorylation mediated by protein kinase C (PKC), however, is not well known. We report the immunocytochemical localization of three Ca2+-dependent

  20. Differential expression of protein kinase C βI (PKCβI) but not PKCα and PKCβII in the suprachiasmatic nucleus of selected house mouse lines, and the relationship to arginine-vasopressin

    NARCIS (Netherlands)

    Bult, Abel; Kobylk, Micki E.; Zee, Eddy A. van der

    2001-01-01

    The functional significance of the suprachiasmatic nucleus (SCN) in circadian rhythm control of mammals has been well documented. The role of protein phosphorylation mediated by protein kinase C (PKC), however, is not well known. We report the immunocytochemical localization of three Ca2+-dependent

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

    OpenAIRE

    Durzan Don J

    2009-01-01

    Abstract 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 compoun...

  2. Protective Effects of Arginine on Saccharomyces cerevisiae Against Ethanol Stress

    Science.gov (United States)

    Cheng, Yanfei; Du, Zhaoli; Zhu, Hui; Guo, Xuena; He, Xiuping

    2016-01-01

    Yeast cells are challenged by various environmental stresses in the process of industrial fermentation. As the currently main organism for bio-ethanol production, Saccharomyces cerevisiae suffers from ethanol stress. Some amino acids have been reported to be related to yeast tolerance to stresses. Here the relationship between arginine and yeast response to ethanol stress was investigated. Marked inhibitions of ethanol on cell growth, expression of genes involved in arginine biosynthesis and intracellular accumulation of arginine were observed. Furthermore, extracellular addition of arginine can abate the ethanol damage largely. To further confirm the protective effects of arginine on yeast cells, yeast strains with different levels of arginine content were constructed by overexpression of ARG4 involved in arginine biosynthesis or CAR1 encoding arginase. Intracellular arginine was increased by 18.9% or 13.1% respectively by overexpression of ARG4 or disruption of CAR1, which enhanced yeast tolerance to ethanol stress. Moreover, a 41.1% decrease of intracellular arginine was observed in CAR1 overexpressing strain, which made yeast cells keenly sensitive to ethanol. Further investigations indicated that arginine protected yeast cells from ethanol damage by maintaining the integrity of cell wall and cytoplasma membrane, stabilizing the morphology and function of organellae due to low ROS generation. PMID:27507154

  3. Reconstruction and modeling protein translocation and compartmentalization in Escherichia coli at the genome-scale

    DEFF Research Database (Denmark)

    Liu, Joanne K.; O’Brien, Edward J.; Lerman, Joshua A.;

    2014-01-01

    translocation pathways, (2) assignment of all cellular proteins to one of four compartments (cytoplasm, inner membrane, periplasm, and outer membrane) and a translocation pathway, (3) experimentally determined translocase catalytic and porin diffusion rates, and (4) a novel membrane constraint that reflects...

  4. A direct screen for c-di-GMP modulators reveals a Salmonella Typhimurium periplasmic ʟ-arginine-sensing pathway.

    Science.gov (United States)

    Mills, Erez; Petersen, Erik; Kulasekara, Bridget R; Miller, Samuel I

    2015-06-01

    Cyclic-di-GMP (c-di-GMP) is a bacterial second messenger that transduces internal and external signals and regulates bacterial motility and biofilm formation. Some organisms encode more than 100 c-di-GMP-modulating enzymes, but only for a few has a signal been defined that modulates their activity. We developed and applied a high-throughput, real-time flow cytometry method that uses a fluorescence resonance energy transfer (FRET)-based biosensor of free c-di-GMP to screen for signals that modulate its concentration within Salmonella Typhimurium. We identified multiple compounds, including glucose, N-acetyl-d-glucosamine, salicylic acid, and ʟ-arginine, that modulated the FRET signal and therefore the free c-di-GMP concentration. By screening a library of mutants, we identified proteins required for the c-di-GMP response to each compound. Furthermore, low micromolar concentrations of ʟ-arginine induced a rapid translation-independent increase in c-di-GMP concentrations and c-di-GMP-dependent cellulose synthesis, responses that required the regulatory periplasmic domain of the diguanylate cyclase STM1987. ʟ-Arginine signaling also required the periplasmic putative ʟ-arginine-binding protein ArtI, implying that ʟ-arginine sensing occurred in the periplasm. Among the 20 commonly used amino acids, S. Typhimurium specifically responded to ʟ-arginine with an increase in c-di-GMP, suggesting that ʟ-arginine may serve as a signal during S. Typhimurium infection. Our results demonstrate that a second-messenger biosensor can be used to identify environmental signals and define pathways that alter microbial behavior.

  5. [Effect of L-arginine on pro- and antioxidant status of the rat vessels and lungs in experimental rhabdomyolysis].

    Science.gov (United States)

    Fylymonenko, V P; Nikitchenko, I V; Kaliman, P A

    2009-01-01

    The glycerol administration was found to cause accumulation of the total heme in rat blood serum, vessels and lungs that are accompanied by increase of TBA-reactive products and protein carbonyl derivates contents. A decrease of superoxide dismutase activity and an increase of reduced glutathione in lung were observed. Heme entering the vessels and lungs is accompanied by elevation in heme oxygenase activity. Pretreatment by L-arginine (0.5 h before glycerol administration) didn't affect blood serum and vessels changes caused by glycerol injection. However, in lungs, L-arginine prevents TBA-reactive products and protein carbonyl derivates accumulation, the decrease ofsuperoxide dismutase activity and causes the ealier heme oxygenase induction. Prooxidant effects of heme in tissues studied and possible mechanisms of L-arginine protective action in lung under experimental rhabdomyolysis are discussed. PMID:20095386

  6. Over-expression of Adenine Nucleotide Translocase 1 (ANT1) Induces Apoptosis and Tumor Regression in vivo

    International Nuclear Information System (INIS)

    Adenine nucleotide translocase (ANT) is located in the inner mitochondrial membrane and catalyzes the exchange of mitochondrial ATP for cytosolic ADP. ANT has been known to be a major component of the permeability transition pore complex of mitochondria and contributes to mitochondria-mediated apoptosis. Human ANT has four isoforms (ANT1, ANT2, ANT3, and ANT4), and the expression of the ANT isoforms is variable depending on the tissue and cell type, developmental stage, and proliferation status. Among the isoforms, ANT1 is highly expressed in terminally-differentiated tissues, but expressed in low levels in proliferating cells, such as cancer cells. In particular, over-expression of ANT1 induces apoptosis in cultured tumor cells. We applied an ANT1 gene transfer approach to induce apoptosis and to evaluate the anti-tumor effect of ANT1 in a nude mouse model. We demonstrated that ANT1 transfection induced apoptosis of MDA-MB-231 cells, inactivated NF-κB activity, and increased Bax expression. ANT1-inducing apoptosis was accompanied by the disruption of mitochondrial membrane potential, cytochrome c release and the activation of caspases-9 and -3. Moreover, ANT1 transfection significantly suppressed tumor growth in vivo. Our results suggest that ANT1 transfection may be a useful therapeutic modality for the treatment of cancer

  7. Plastids Contain a Second Sec Translocase System with Essential Functions1[W][OA

    Science.gov (United States)

    Skalitzky, Courtney A.; Martin, Jonathan R.; Harwood, Jessica H.; Beirne, John J.; Adamczyk, Benjamin J.; Heck, Gregory R.; Cline, Kenneth; Fernandez, Donna E.

    2011-01-01

    Proteins that are synthesized on cytoplasmic ribosomes but function within plastids must be imported and then targeted to one of six plastid locations. Although multiple systems that target proteins to the thylakoid membranes or thylakoid lumen have been identified, a system that can direct the integration of inner envelope membrane proteins from the stroma has not been previously described. Genetics and localization studies were used to show that plastids contain two different Sec systems with distinct functions. Loss-of-function mutations in components of the previously described thylakoid-localized Sec system, designated as SCY1 (At2g18710), SECA1 (At4g01800), and SECE1 (At4g14870) in Arabidopsis (Arabidopsis thaliana), result in albino seedlings and sucrose-dependent heterotrophic growth. Loss-of-function mutations in components of the second Sec system, designated as SCY2 (At2g31530) and SECA2 (At1g21650) in Arabidopsis, result in arrest at the globular stage and embryo lethality. Promoter-swap experiments provided evidence that SCY1 and SCY2 are functionally nonredundant and perform different roles in the cell. Finally, chloroplast import and fractionation assays and immunogold localization of SCY2-green fluorescent protein fusion proteins in root tissues indicated that SCY2 is part of an envelope-localized Sec system. Our data suggest that SCY2 and SECA2 function in Sec-mediated integration and translocation processes at the inner envelope membrane. PMID:21051552

  8. Spectrophotometric Determination of Arginine in Grape Juice Using 8-Hydroquinoline

    Institute of Scientific and Technical Information of China (English)

    WANG Hua; LIANG Xin-hong; ZHAO Rui-xiang; FENG Li-dan; LI Hua

    2008-01-01

    Arginine in grape juice can be metabolized by wine yeasts and malolactic bacteria to precursors of ethyl carbamate, known as carcinogen. The aim of this study was to develop a simple, fast, and accurate method for determining arginine in grape juice with Sakaguchi reaction by separating arginine with strong cation-exchange resins. Parameters were optimized including the concentrations of 8-hydroquinoline and sodium hydrobromite. The color stability lasted for 4 min, which is sufficient to finish the measurement. The method is simple, reproducible and accurate, and can be applied for quick measurement of arginine in grape juice to take necessary measures for controlling the level of ethyl carbamate.

  9. A mutation in the human phospholamban gene, deleting arginine 14, results in lethal, hereditary cardiomyopathy

    OpenAIRE

    Haghighi, Kobra; Kolokathis, Fotis; Gramolini, Anthony O.; Waggoner, Jason R.; Pater, Luke; Lynch, Roy A.; Fan, Guo-Chang; Tsiapras, Dimitris; Parekh, Rohan R.; Dorn, Gerald W., II; MacLennan, David H.; Kremastinos, Dimitrios Th; Kranias, Evangelia G.

    2006-01-01

    The sarcoplasmic reticulum Ca2+-cycling proteins are key regulators of cardiac contractility, and alterations in sarcoplasmic reticulum Ca2+-cycling properties have been shown to be causal of familial cardiomyopathies. Through genetic screening of dilated cardiomyopathy patients, we identified a previously uncharacterized deletion of arginine 14 (PLN-R14Del) in the coding region of the phospholamban (PLN) gene in a large family with hereditary heart failure. No homozygous individuals were ide...

  10. Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex

    OpenAIRE

    Tsurumura, Toshiharu; Tsumori, Yayoi; Qiu, Hao; Oda, Masataka; Sakurai, Jun; Nagahama, Masahiro; Tsuge, Hideaki

    2012-01-01

    Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD+ analog βTAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD+...

  11. Catabolism and safety of supplemental L-arginine in animals.

    Science.gov (United States)

    Wu, Zhenlong; Hou, Yongqing; Hu, Shengdi; Bazer, Fuller W; Meininger, Cynthia J; McNeal, Catherine J; Wu, Guoyao

    2016-07-01

    L-arginine (Arg) is utilized via multiple pathways to synthesize protein and low-molecular-weight bioactive substances (e.g., nitric oxide, creatine, and polyamines) with enormous physiological importance. Furthermore, Arg regulates cell signaling pathways and gene expression to improve cardiovascular function, augment insulin sensitivity, enhance lean tissue mass, and reduce obesity in humans. Despite its versatile roles, the use of Arg as a dietary supplement is limited due to the lack of data to address concerns over its safety in humans. Data from animal studies are reviewed to assess arginine catabolism and the safety of long-term Arg supplementation. The arginase pathway was responsible for catabolism of 76-85 and 81-96 % Arg in extraintestinal tissues of pigs and rats, respectively. Dietary supplementation with Arg-HCl or the Arg base [315- and 630-mg Arg/(kg BW d) for 91 d] had no adverse effects on male or female pigs. Similarly, no safety issues were observed for male or female rats receiving supplementation with 1.8- and 3.6-g Arg/(kg BW d) for at least 91 d. Intravenous administration of Arg-HCl to gestating sheep at 81 and 180 mg Arg/(kg BW d) is safe for at least 82 and 40 d, respectively. Animals fed conventional diets can well tolerate large amounts of supplemental Arg [up to 630-mg Arg/(kg BW d) in pigs or 3.6-g Arg/(kg BW d) in rats] for 91 d, which are equivalent to 573-mg Arg/(kg BW d) for humans. Collectively, these results can help guide studies to determine the safety of long-term oral administration of Arg in humans. PMID:27156062

  12. Converting the yeast arginine can1 permease to a lysine permease.

    Science.gov (United States)

    Ghaddar, Kassem; Krammer, Eva-Maria; Mihajlovic, Natalija; Brohée, Sylvain; André, Bruno; Prévost, Martine

    2014-03-01

    Amino acid uptake in yeast cells is mediated by about 16 plasma membrane permeases, most of which belong to the amino acid-polyamine-organocation (APC) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino acid permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Although Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H(+)-driven arginine-specific permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a three-dimensional Can1 structural model based on the crystal structure of the bacterial APC family arginine/agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q and E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H(+) coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport.

  13. Supplemental arginine above the requirement during suckling causes obesity and insulin resistance in rats.

    Science.gov (United States)

    Otani, Lila; Mori, Tomomi; Koyama, Ayaka; Takahashi, Shin-Ichiro; Kato, Hisanori

    2016-06-01

    Nutrition in early life is important in determining susceptibility to adult obesity, and arginine may promote growth acceleration in infants. We hypothesized that maternal arginine supplementation may promote growth in their pups and contribute to obesity and alteration of the metabolic system in later life. Dams and pups of Wistar rats were given a normal diet (15% protein) as a control (CN) or a normal diet with 2% arginine (ARG). Altered profiles of free amino acids in breast milk were observed in that the concentrations of threonine and glycine were lower in the ARG dams compared with the CN dams. The offspring of the CN and ARG dams were further subdivided into normal-diet (CN-CN and ARG-CN) groups and a high fat-diet groups (CN-HF and ARG-HF). In response to the high fat-diet feeding, the visceral fat deposits were significantly increased in the ARG-HF group (although not compared with the CN-HF group); no difference was observed between the CN-CN and ARG-CN groups. The blood glucose and insulin levels after glucose loading were significantly higher in the ARG-HF group compared with the CN-HF group. The results suggest that the offspring of dams supplemented with arginine during lactation acquired increased susceptibility to a high-fat diet, resulting in visceral obesity and insulin resistance. The lower supply of threonine and glycine to pups may be one of the contributing causes to the programming of lifelong obesity risk in offspring. Our findings also indicated that maternal arginine supplementation during suckling causes obesity and insulin resistance in rats. PMID:27188903

  14. ARCD, THE 1ST GENE OF THE ARC OPERON FOR ANAEROBIC ARGININE CATABOLISM IN PSEUDOMONAS-AERUGINOSA, ENCODES AN ARGININE-ORNITHINE EXCHANGER

    NARCIS (Netherlands)

    VERHOOGT, HJC; SMIT, H; ABEE, T; GAMPER, M; DRIESSEN, AJM; KONINGS, WN

    1992-01-01

    In the absence of oxygen and nitrate, Pseudomonas aeruginosa metabolizes arginine via the arginine deiminase pathway, which allows slow growth on rich media. The conversion of arginine to ornithine, CO2, and NH3 is coupled to the production of ATP from ADP. The enzymes of the arginine deiminase path

  15. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    Energy Technology Data Exchange (ETDEWEB)

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong

    2011-12-31

    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  16. Arginine specific aminopeptidase from Lactobacillus brevis

    Directory of Open Access Journals (Sweden)

    Arya Nandan

    2010-12-01

    Full Text Available The proteolytic system of lactic acid bacteria contribute to the development of flavor during the ripening of cheese through the generation of short peptides and free amino acids, which directly or indirectly act as flavor precursors. Newly isolated lactic acid bacteria (LAB as well as those procured from culture collection centers were screened for the production of various substrate specific aminopeptidases. Among all the strains screened, L. brevis (NRRL B-1836 was found to produce quantifiable amount of intracellular arginine specific aminopeptidase (EC 3.4.11.6. The productivity of arginine aminopeptidase in 5 L fermentor was 36 IU/L/h. The Luedeking and Piret model was tested for intracellular production of aminopeptidase and the data seemed to fit well, as the correlation coefficient was 0.9964 for MRS. The αAP and βAP was 0.4865 and 0.0046, respectively in MRS medium indicating that the yield was predominantly depended on growth. The culture produced lactic acid and also tolerated pH 2.0-3.0 and 0.3-0.5% bile salts, the most important probiotic features.

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

    Directory of Open Access Journals (Sweden)

    Ruijter Jan M

    2008-11-01

    Full Text Available Abstract 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.

  18. Active displacement of RecA filaments by UvrD translocase activity

    OpenAIRE

    Petrova, Vessela; Chen, Stefanie H.; Molzberger, Eileen T.; Tomko, Eric; Chitteni-Pattu, Sindhu; Jia, Haifeng; Ordabayev, Yerdos; Lohman, Timothy M.; Cox, Michael M.

    2015-01-01

    The UvrD helicase has been implicated in the disassembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an active mechanism to remove RecA from the DNA. Efficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibility of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with ...

  19. Arginine Deiminase Resistance in Melanoma Cells Is Associated with Metabolic Reprogramming, Glucose Dependence and Glutamine Addiction

    OpenAIRE

    Long, Yan; Tsai, Wen-Bin; Wangpaichitr, Medhi; Tsukamoto, Takashi; Savaraj, Niramol; Feun, Lynn G.; Kuo, Macus Tien

    2013-01-01

    Many malignant human tumors, including melanomas are auxotrophic for arginine due to reduced expression of argininosuccinate synthetase1 (ASS1), the rate-limiting enzyme for arginine biosynthesis. Pegylated arginine deiminase (ADI-PEG20), which degrades extracellular arginine resulting in arginine deprivation, has shown favorable results in clinical trials for treating arginine-auxotrophic tumors. Drug resistance is the major obstacle for effective ADI-PEG20 usage. To elucidate mechanisms of ...

  20. BASIC AMINO ACID CARRIER 2 gene expression modulates arginine and urea content and stress recovery in Arabidopsis leaves.

    Directory of Open Access Journals (Sweden)

    Séverine ePlanchais

    2014-07-01

    Full Text Available In plants, basic amino acids are important for the synthesis of proteins and signaling molecules and for nitrogen recycling. The Arabidopsis nuclear gene BASIC AMINO ACID CARRIER 2 (BAC2 encodes a mitochondria-located carrier that transports basic amino acids in vitro. We present here an analysis of the physiological and genetic function of BAC2 in planta. When BAC2 is overexpressed in vivo, it triggers catabolism of arginine, a basic amino acid, leading to arginine depletion and urea accumulation in leaves. BAC2 expression was known to be strongly induced by stress. We found that compared to wild type plants, bac2 null mutants (bac2-1 recover poorly from hyperosmotic stress when restarting leaf expansion. The bac2-1 transcriptome differs from the wild-type transcriptome in control conditions and under hyperosmotic stress. The expression of genes encoding stress-related transcription factors, arginine metabolism enzymes, and transporters is particularly disturbed in bac2-1, and in control conditions, the bac2-1 transcriptome has some hallmarks of a wild-type stress transcriptome. The BAC2 carrier is therefore involved in controlling the balance of arginine and arginine-derived metabolites and its associated amino acid metabolism is physiologically important in equipping plants to respond to and recover from stress.

  1. Anti-aging effects of l-arginine

    Directory of Open Access Journals (Sweden)

    Mohamed Z. Gad

    2010-07-01

    Full Text Available l-Arginine is one of the most metabolically versatile amino acids. In addition to its role in the synthesis of nitric oxide, l-arginine serves as a precursor for the synthesis of polyamines, proline, glutamate, creatine, agmatine and urea. Several human and experimental animal studies have indicated that exogenous l-arginine intake has multiple beneficial pharmacological effects when taken in doses larger than normal dietary consumption. Such effects include reduction in the risk of vascular and heart diseases, reduction in erectile dysfunction, improvement in immune response and inhibition of gastric hyperacidity. This review summarises several positive studies and personal experiences of l-arginine. The demonstrated anti-aging benefits of l-arginine show greater potential than any pharmaceutical or nutraceutical agent ever previously discovered.

  2. L-Arginine Pathway in COPD Patients with Acute Exacerbation

    DEFF Research Database (Denmark)

    Ruzsics, Istvan; Nagy, Lajos; Keki, Sandor;

    2016-01-01

    (ADMA, SDMA) is related to hypoxia. In COPD, a rise in ADMA results in a shift of L-arginine breakdown, contributing to airway obstruction. We aimed to compare serum levels of ADMA, SDMA and L-arginine in patients with and without AECOPD. METHODS: L-arginine metabolites quantified by high......BACKGROUND: Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) remains a major cause of mortality. Clinical criteria of AECOPD are subjective. Biomarkers for AECOPD may aid in the initiation of early treatment. Increased production of asymmetric and symmetric dimethylarginine......-arginine, ADMA and SDMA serum levels. In patients with AECOPD, production of ADMA and SDMA are more pronounced presumably due to more severe hypoxic insult. Methylated arginine derivatives in the sera may help early recognition of AECOPD....

  3. l-Arginine metabolism in cardiovascular and renal tissue from hyper- and hypothyroid rats

    Science.gov (United States)

    Moliz, Juan N; Quesada, Andrés; Montoro-Molina, Sebastian; Vargas-Tendero, Pablo; Osuna, Antonio; Wangensteen, Rosemary; Vargas, Félix

    2015-01-01

    This study assessed the effects of thyroid hormones on the enzymes involved in l-arginine metabolism and the metabolites generated by the different metabolic pathways. Compounds of l-arginine metabolism were measured in the kidney, heart, aorta, and liver of euthyroid, hyperthyroid, and hypothyroid rats after 6 weeks of treatment. Enzymes studied were NOS isoforms (neuronal [nNOS], inducible [iNOS], and endothelial [eNOS]), arginases I and II, ornithine decarboxylase (ODC), ornithine aminotransferase (OAT), and l-arginine decarboxylase (ADC). Metabolites studied were l-arginine, l-citrulline, spermidine, spermine, and l-proline. Kidney heart and aorta levels of eNOS and iNOS were augmented and reduced (P < 0.05, for each tissue and enzyme) in hyper- and hypothyroid rats, respectively. Arginase I abundance in aorta, heart, and kidney was increased (P < 0.05, for each tissue) in hyperthyroid rats and was decreased in kidney and aorta of hypothyroid rats (P < 0.05, for each tissue). Arginase II was augmented in aorta and kidney (P < 0.05, for each tissue) of hyperthyroid rats and remained unchanged in all organs of hypothyroid rats. The substrate for these enzymes, l-arginine, was reduced (P < 0.05, for all tissues) in hyperthyroid rats. Levels of ODC and spermidine, its product, were increased and decreased (P < 0.05) in hyper- and hypothyroid rats, respectively, in all organs studied. OAT and proline levels were positively modulated by thyroid hormones in liver but not in the other tissues. ADC protein levels were positively modulated by thyroid hormones in all tissues. According to these findings, thyroid hormone treatment positively modulates different l-arginine metabolic pathways. The changes recorded in the abundance of eNOS, arginases I and II, and ADC protein in renal and cardiovascular tissues may play a role in the hemodynamic and renal manifestations observed in thyroid disorders. Furthermore, the changes in ODC and spermidine might

  4. Influence of a Hydrophobic Environment on the Structure of Arginine-Carboxylate Salt Bridge

    Institute of Scientific and Technical Information of China (English)

    FENG,Yong(封勇); LIU,Lei(刘磊); MU,Ting-Wei(穆廷巍); GUO,Qing-Xiang(郭庆祥)

    2002-01-01

    The exact structure of an arginine-carboxylate salt bridge in different chemical environments remains a controversial problem. In the present work, the zwitterionic and neutral forms of arginine-carboxylate salt bridge were studied by the B3LYP/6-311G(d,p)//PM3 method. It turns out that the neutral forms are more stable than the zwitterionic counterparis in gas phase.However, when bound by c-cyclodextrin, the zwitterionic forms become more stable than the corresponding neutral ones.It is suggested that the hydrophobic environment provided by the cyclodextrin cavity leads to such behavior. Tnerefore, the salt bridge still could be in a zwitterionic form in the hydrophobic interior of the real proteins.

  5. The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond.

    Science.gov (United States)

    Stopa, Nicole; Krebs, Jocelyn E; Shechter, David

    2015-06-01

    Post-translational arginine methylation is responsible for regulation of many biological processes. The protein arginine methyltransferase 5 (PRMT5, also known as Hsl7, Jbp1, Skb1, Capsuleen, or Dart5) is the major enzyme responsible for mono- and symmetric dimethylation of arginine. An expanding literature demonstrates its critical biological function in a wide range of cellular processes. Histone and other protein methylation by PRMT5 regulate genome organization, transcription, stem cells, primordial germ cells, differentiation, the cell cycle, and spliceosome assembly. Metazoan PRMT5 is found in complex with the WD-repeat protein MEP50 (also known as Wdr77, androgen receptor coactivator p44, or Valois). PRMT5 also directly associates with a range of other protein factors, including pICln, Menin, CoPR5 and RioK1 that may alter its subcellular localization and protein substrate selection. Protein substrate and PRMT5-MEP50 post-translation modifications induce crosstalk to regulate PRMT5 activity. Crystal structures of C. elegans PRMT5 and human and frog PRMT5-MEP50 complexes provide substantial insight into the mechanisms of substrate recognition and procession to dimethylation. Enzymological studies of PRMT5 have uncovered compelling insights essential for future development of specific PRMT5 inhibitors. In addition, newly accumulating evidence implicates PRMT5 and MEP50 expression levels and their methyltransferase activity in cancer tumorigenesis, and, significantly, as markers of poor clinical outcome, marking them as potential oncogenes. Here, we review the substantial new literature on PRMT5 and its partners to highlight the significance of understanding this essential enzyme in health and disease.

  6. Asymmetric Dimethyl Arginine in Hypothyroid Patients

    International Nuclear Information System (INIS)

    Thyroid diseases may lead to endothelial dysfunction, however, the mechanism underlying the endothelial dysfunction in thyroid disease is still not clear. Asymmetric dimethyl arginine (ADMA), a novel inhibitor of endothelial nitric oxide synthetase (eNOS), was reported to inhibit nitric oxide (NO) synthesis from L-arginine. The present study was carried out to investigate ADMA levels together with effects of dislipidemia in sub-clinical and overt hypothyroid females. There were significant increase in the levels of total cholesterol, low density lipoprotein-cholesterol (LDL-c), high density lipoprotein-cholesterol (HDL-c), thyroid stimulating hormone (TSH) and ADMA in hypothyroid females as compared to controls while the levels of NO and free T4 were significantly decreased than controls. Sub-clinical hypothyroid females had significant high TSH, LDL-c and non-significantly high ADMA levels and total cholesterol as compared to controls while they had significant decrease in NO, HDL-c and non-significant decrease in free T4 as compared to controls. There were significant negative correlations between NO and both ADMA (r2 = 0.84) and free T4 (r2 = 0.95) in overt hypothyroid group while significant positive correlation (r2 = 0.85) was detected between TSH and HDL-c in the same group. These results are highly suggestive that the decrease of nitric oxide secondary to accumulation of ADMA represent an important pathogenic factor together with dyslipidemia in endothelial dysfunction and increased cardiovascular risk especially in hypothyroid females

  7. Arginine and proline applied as food additives stimulate high freeze tolerance in larvae of Drosophila melanogaster.

    Science.gov (United States)

    Koštál, Vladimír; Korbelová, Jaroslava; Poupardin, Rodolphe; Moos, Martin; Šimek, Petr

    2016-08-01

    The fruit fly Drosophila melanogaster is an insect of tropical origin. Its larval stage is evolutionarily adapted for rapid growth and development under warm conditions and shows high sensitivity to cold. In this study, we further developed an optimal acclimation and freezing protocol that significantly improves larval freeze tolerance (an ability to survive at -5°C when most of the freezable fraction of water is converted to ice). Using the optimal protocol, freeze survival to adult stage increased from 0.7% to 12.6% in the larvae fed standard diet (agar, sugar, yeast, cornmeal). Next, we fed the larvae diets augmented with 31 different amino compounds, administered in different concentrations, and observed their effects on larval metabolomic composition, viability, rate of development and freeze tolerance. While some diet additives were toxic, others showed positive effects on freeze tolerance. Statistical correlation revealed tight association between high freeze tolerance and high levels of amino compounds involved in arginine and proline metabolism. Proline- and arginine-augmented diets showed the highest potential, improving freeze survival to 42.1% and 50.6%, respectively. Two plausible mechanisms by which high concentrations of proline and arginine might stimulate high freeze tolerance are discussed: (i) proline, probably in combination with trehalose, could reduce partial unfolding of proteins and prevent membrane fusions in the larvae exposed to thermal stress (prior to freezing) or during freeze dehydration; (ii) both arginine and proline are exceptional among amino compounds in their ability to form supramolecular aggregates which probably bind partially unfolded proteins and inhibit their aggregation under increasing freeze dehydration. PMID:27489218

  8. Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation.

    Science.gov (United States)

    Fox, Barbara A; Gigley, Jason P; Bzik, David J

    2004-03-01

    Two separate carbamoyl phosphate synthetase activities are required for the de novo synthesis of pyrimidines and arginine in most eukaryotes. Toxoplasma gondii is novel in possessing a single carbamoyl phosphate synthetase II gene that corresponds to a glutamine-dependent form required for pyrimidine biosynthesis. We therefore examined arginine acquisition in T. gondii to determine whether the single carbamoyl phosphate synthetase II activity could provide both pyrimidine and arginine biosynthesis. We found that arginine deprivation efficiently blocks the replication of intracellular T. gondii, yet has little effect on long-term parasite viability. Addition of citrulline, but not ornithine, rescues the growth defect observed in the absence of exogenous arginine. This rescue with citrulline is ablated when parasites are cultured in a human citrullinemia fibroblast cell line that is deficient in argininosuccinate synthetase activity. These results reveal the absence of genes and activities of the arginine biosynthetic pathway and demonstrate that T. gondii is an arginine auxotroph. Arginine starvation was also found to efficiently trigger differentiation of replicative tachyzoites into bradyzoites contained within stable cyst-like structures. These same parasites expressing bradyzoite antigens can be efficiently switched back to rapidly proliferating tachyzoites several weeks after arginine starvation. We hypothesise that the absence of gene activities that are essential for the biosynthesis of arginine from carbamoyl phosphate confers a selective advantage by increasing bradyzoite switching during the host response to T. gondii infection. These findings are consistent with a model of host-parasite evolution that allowed host control of bradyzoite induction by trading off virulence for increased transmission. PMID:15003493

  9. Caries-free subjects have high levels of urease and arginine deiminase activity

    Directory of Open Access Journals (Sweden)

    Evelyn REYES

    2014-06-01

    Full Text Available Objectives: This study investigated the relationship between urease and arginine deiminase system (ADS activities and dental caries through a cross-sectional study. Material and Methods: Urease and ADS activities were measured in saliva and plaque samples from 10 caries-free subjects and 13 caries-active. Urease activity was obtained from the ammonia produced by incubation of plaque and saliva samples in urea. ADS activity was obtained from the ammonia generated by the arginine-HCl and Tris-maleate buffer. Specific activity was defined as micromoles of ammonia per minute per milligram of protein. Shapiro-Wilk statistical test was used to analyze the distribution of the data, and Mann-Whitney test was used to determine the significance of the data. Results: The specific urease activity in saliva and plaque was significantly higher in individuals with low DMFT scores. ADS activity in saliva (6.050 vs 1.350, p=0.0154 and plaque (8.830 vs 1.210, p=0.025 was also higher in individuals with low DMFT scores. Conclusions: Caries-free subjects had a higher ammonia generation activity by urease and arginine deiminase system for both saliva and plaque samples than low caries-active subjects. High levels of alkali production in oral environment were related to caries-free subjects.

  10. Measurement of arginine metabolites: regulators of nitric oxide metabolism.

    Science.gov (United States)

    Augustine, Molly S; Rogers, Lynette K

    2013-01-01

    Arginine is the substrate for nitric oxide synthases (NOS), and arginine availability regulates the production of nitric oxide. Through the activity of methyltransferases, arginine can be methylated to form monomethylarginine (NMMA), asymmetrical dimethylarginine (ADMA), and symmetrical dimethylarginine (SDMA). NMMA and ADMA directly inhibit NOS, whereas SDMA inhibits the cellular import of arginine through the cationic amino acid transporter. Increased levels of methylarginine compounds have been associated with many diseases including atherosclerosis, renal failure, pulmonary hypertension, and preeclampsia. Previous HPLC methods to measure these molecules rely on derivatization with ortho-phthalaldehyde, which is unstable and requires immediate pre- or post-column reactions. We have identified a new fluorometric agent that is stable for at least 1 week and provides chromatographic properties that facilitate separation of these chemically similar compounds by reverse phase chromatography. PMID:24510541

  11. Cellular Mechanisms of L-arginine Induced Experimental Acute Pancreatitis

    OpenAIRE

    Masood, Omar

    2013-01-01

    AbstractThe University Of ManchesterOmar MasoodMD Thesis 2013Cellular Mechanisms of L-arginine Induced Experimental Acute Pancreatitis. IntroductionImpairment of cytosolic calcium ([Ca2+]i) signaling and in particular calcium overload has emerged as a possible unifying mechanism for precipitating acute pancreatitis (AP.)In the L-arginine (L-arg) experimental model of AP, nitric oxide (NO) has been implicated however the disease progression is largely unaffected by nitric oxide synthase (NOS) ...

  12. Local Administration of L-Arginine Accelerates Wound Closure

    Directory of Open Access Journals (Sweden)

    Masoumeh Varedi

    2009-09-01

    Full Text Available Objective(sThe process of wound healing involves tightly integrated events including inflammation, granulation tissue formation and remodeling. Systemic administration of L arginine promotes wound healing but its global side effects are undesirable. To confine the action of L-arginine at the site of injury, we tested the effects of local administration of L arginine on the healing of excisional wound in the rat.Materials and MethodsFull thickness excisional wounds were generated on the dorsum of adult male rats. The test wounds received 200 µm or 400 µm of L-arginine on day 3 and 5 post-wounding. Normal saline was injected into the sham wounds which were otherwise treated as the test wounds. Control wounds remained unmanipulated. The wound size was monitored daily by imaging. To determine the rate of wound closure, wound images were scanned and the rate of size reduction was analyzed and quantified by ScnImage software. The repaired tissues were harvested on day 12 post-wounding. The tissue sections were prepared and stained for microscopic examination. ResultsWounds treated with L-arginine showed a significant increase in the rate of wound closure. The morphology of basal keratinocytes was altered, and the thickness of neoepidermis was markedly reduced in the wounds treated with L-arginine. Both tested dose of L-arginine were equally effective. ConclusionLocal administration of L-arginine accelerates wound closure and has profound effects on keratinocytes performance during the process of healing. Therefore, it can be potentially used for treatment of skin disorders, in particular, those characterized by hyperkeratosis.

  13. Acellular matrix of bovine pericardium bound with L-arginine

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyo Joo [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Bae, Jin Woo [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Chun Ho [Laboratory of Tissue Engineering, Korea Cancer Center Hospital, Seoul 139-240 (Korea, Republic of); Lee, Jin Woo [Department of Orthopaedic Surgery, College of Medicine, Yonsei University, Seoul 120-749 (Korea, Republic of); Shin, Jung Woog [Department of Biomedical Engineering, Inje University, Gimhae 621-749 (Korea, Republic of); Park, Ki Dong [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

    2007-09-15

    Surface immobilization of bioactive molecules onto natural tissues has been interestingly studied for the development of new functional matrices for the replacement of lost or malfunctioning tissues. In this study, an acellular matrix of bovine pericardium (ABP) was chemically modified by the direct coupling of L-arginine after glutaraldehyde (GA) cross-linking. The effects of L-arginine coupling on durability and calcification were investigated and the biocompatibility was evaluated in vitro and in vivo. A four-step detergent and enzymatic extraction process has been utilized to remove cellular components from fresh bovine pericardium (BP). Microscopic observation confirmed that nearly all cellular constituents are removed. Thermal and mechanical properties showed that the durability of L-arginine-treated matrices increased as compared with control ABP and GA-treated ABP. Resistance to collagenase digestion revealed that modified matrices have greater resistance to enzyme digestion than control ABP and GA-treated ABP. The in vivo calcification study demonstrated much less calcium deposition on L-arginine-treated ABP than GA-treated one. In vitro cell viability results showed that ABP modified with L-arginine leads to a significant increase in attachment of human dermal fibroblasts. The obtained results attest to the usefulness of L-arginine-treated ABP matrices for cardiovascular bioprostheses.

  14. L-Arginine ameliorates cardiac left ventricular oxidative stress by upregulating eNOS and Nrf2 target genes in alloxan-induced hyperglycemic rats

    Energy Technology Data Exchange (ETDEWEB)

    Ramprasath, Tharmarajan; Hamenth Kumar, Palani; Syed Mohamed Puhari, Shanavas; Senthil Murugan, Ponniah; Vasudevan, Varadaraj [Molecular Cardiology Unit, Department of Biochemistry, Center for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, Tamilnadu (India); Selvam, Govindan Sadasivam, E-mail: drselvamgsbiochem@rediffmail.com [Molecular Cardiology Unit, Department of Biochemistry, Center for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, Tamilnadu (India)

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer L-Arginine treatment reduced the metabolic disturbances in diabetic animals. Black-Right-Pointing-Pointer Antioxidant marker proteins were found high in myocardium by L-arginine treatment. Black-Right-Pointing-Pointer Elevated antioxidant status, mediates the reduced TBA-reactivity in left ventricle. Black-Right-Pointing-Pointer L-Arginine treatment enhanced the Nrf2 and eNOS signaling in left ventricle. Black-Right-Pointing-Pointer Improved cell survival signaling by arginine, offers a novel tactic for targeting. -- Abstract: Hyperglycemia is independently related with excessive morbidity and mortality in cardiovascular disorders. L-Arginine-nitric oxide (NO) pathway and the involvement of NO in modulating nuclear factor-E2-related factor-2 (Nrf2) signaling were well established. In the present study we investigated, whether L-arginine supplementation would improve the myocardial antioxidant defense under hyperglycemia through activation of Nrf2 signaling. Diabetes was induced by alloxan monohydrate (90 mg kg{sup -1} body weight) in rats. Both non-diabetic and diabetic group of rats were divided into three subgroups and they were administered either with L-arginine (2.25%) or L-NAME (0.01%) in drinking water for 12 days. Results showed that L-arginine treatment reduced the metabolic disturbances in diabetic rats. Antioxidant enzymes and glutathione levels were found to be increased in heart left ventricles, thereby reduction of lipid peroxidation by L-arginine treatment. Heart histopathological analysis further validates the reversal of typical diabetic characteristics consisting of alterations in myofibers and myofibrillary degeneration. qRT-PCR studies revealed that L-arginine treatment upregulated the transcription of Akt and downregulated NF-{kappa}B. Notably, transcription of eNOS and Nrf2 target genes was also upregulated, which were accompanied by enhanced expression of Nrf2 in left ventricular tissue from diabetic

  15. Plasma arginine and ornithine are the main citrulline precursors in mice infused with arginine-free diets.

    Science.gov (United States)

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

    2010-08-01

    Dietary arginine is the main dietary precursor for citrulline synthesis, but it is not known if other precursors can compensate when arginine is absent in the diet. To address this question, the contributions of plasma and dietary precursors were determined by using multitracer protocols in conscious mice infused i.g. either an arginine-sufficient diet [Arg(+)] or an arginine-free diet [Arg(-)]. The plasma entry rate of citrulline and arginine did not differ between the 2 diet groups (156 +/- 6 and 564 +/- 30 micromol kg(-1) h(-1), respectively); however, the entry rate of ornithine was greater in the mice fed the Arg(+) than the Arg(-) diet (332 +/- 33 vs. 180 +/- 16 micromol kg(-1) h(-1)). There was a greater utilization of plasma ornithine for the synthesis of citrulline (49 +/- 4 vs. 36 +/- 3 micromol kg(-1) h(-1), 30 +/- 3% vs. 24 +/- 2% of citrulline entry rate) in the mice fed the Arg(-) diet than the Arg(+) diet. The utilization of plasma arginine did not differ between the 2 diet groups for citrulline synthesis, either through plasma ornithine (approximately 29 +/- 3 micromol kg(-1) h(-1)) or at the site of citrulline synthesis (approximately 12 +/- 3 micromol kg(-1) h(-1)). The contribution of dietary proline to the synthesis of citrulline was mainly at the site of citrulline production (17 +/- 1 micromol kg(-1) h(-1)), rather than through plasma ornithine (5 +/- 0.4 micromol kg(-1) h(-1)). Dietary glutamine was utilized only at the site of citrulline synthesis (4 +/- 0.2 micromol kg(-1) h(-1)). Dietary glutamine and proline made a greater contribution to the synthesis of citrulline in mice fed the Arg(-) diet but remained minor sources for citrulline production. Plasma arginine and ornithine are able to support citrulline synthesis during arginine-free feeding.

  16. NCBI nr-aa BLAST: CBRC-TTRU-01-1371 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available ndent protein export [Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)] gb|ACQ68339.1| twin-arginin...e translocase subunit, sec-independent protein export [Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)] YP_002924487.1 0.89 24% ...

  17. Differential induction of PPAR-gamma by luminal glutamine and iNOS by luminal arginine in the rodent postischemic small bowel.

    Science.gov (United States)

    Sato, N; Moore, F A; Kone, B C; Zou, L; Smith, M A; Childs, M A; Moore-Olufemi, S; Schultz, S G; Kozar, R A

    2006-04-01

    Using a rodent model of gut ischemia-reperfusion (I/R), we have previously shown that the induction of inducible nitric oxide synthase (iNOS) is harmful, whereas the induction of heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is protective. In the present study, we hypothesized that the luminal nutrients arginine and glutamine differentially modulate these molecular events in the postischemic gut. Jejunal sacs were created in rats at laparotomy, filled with either 60 mM glutamine, arginine, or magnesium sulfate (osmotic control) followed by 60 min of superior mesenteric artery occlusion and 6 h of reperfusion, and compared with shams. The jejunum was harvested for histology or myeloperoxidase (MPO) activity (inflammation). Heat shock proteins and iNOS were quantitated by Western blot analysis and PPAR-gamma by DNA binding activity. In some experiments, rats were pretreated with the PPAR-gamma inhibitor G9662 or with the iNOS inhibitor N-[3(aminomethyl)benzyl]acetamidine (1400W). iNOS was significantly increased by arginine but not by glutamine following gut I/R and was associated with increased MPO activity and mucosal injury. On the other hand, PPAR-gamma was significantly increased by glutamine but decreased by arginine, whereas heat shock proteins were similarly increased in all experimental groups. The PPAR-gamma inhibitor G9662 abrogated the protective effects of glutamine, whereas the iNOS inhibitor 1400W attenuated the injurious effects of arginine. We concluded that luminal arginine and glutamine differentially modulate the molecular events that regulate injurious I/R-mediated gut inflammation and injury. The induction of PPAR-gamma by luminal glutamine is a novel protective mechanism, whereas luminal arginine appears harmful to the postischemic gut due to enhanced expression of iNOS.

  18. Research advances in arginine nutrition of fish%鱼类精氨酸营养研究进展

    Institute of Scientific and Technical Information of China (English)

    王连生; 徐奇友

    2014-01-01

    Arginine has been shown to be an essential amino acid for fish, it is involved in protein, nitric oxide, polyamine and creatine synthesis and plays important roles in the growth and immune function. This review summarized the effects of arginine on growth, body composition and immune function of fish and the balance of arginine and lysine, in order to provide reference for the application of arginine in fish.%精氨酸(Arg)作为鱼类必需氨基酸,参与机体蛋白质合成,一氧化氮、多胺及肌酸的生成,在生长和免疫方面发挥重要作用。文章综述近年来精氨酸对鱼类生长、体成分及免疫功能影响及Arg和赖氨酸(Lys)平衡,为精氨酸在鱼类生产中的应用提供参考。

  19. The L-arginine Pathway in Acute Ischemic Stroke and Severe Carotid Stenosis

    DEFF Research Database (Denmark)

    Molnar, Tihamer; Pusch, Gabriella; Papp, Viktoria;

    2014-01-01

    BACKGROUND: Endothelial dysfunction is associated with increased levels of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) resulting in a decreased production of nitric oxide, which regulates the vascular tone. METHODS: Patients with acute ischemic stroke (AIS, n = 55......) and asymptomatic significant carotid stenosis (AsCS, n = 44) were prospectively investigated. L-arginine, ADMA, SDMA, S100 B, and high-sensitivity C-reactive protein (hsCRP) were serially measured within 6 hours after the onset of stroke, at 24 and 72 poststroke hours. All markers were compared with healthy...

  20. L-Arginine Supplementation and Metabolism in Asthma

    Directory of Open Access Journals (Sweden)

    Angela Linderholm

    2011-01-01

    Full Text Available L-Arginine, the amino acid substrate for nitric oxide synthase, has been tested as a therapeutic intervention in a variety of chronic diseases and is commonly used as a nutritional supplement. In this study, we hypothesized that a subset of moderate to severe persistent asthma patients would benefit from supplementation with L-arginine by transiently increasing nitric oxide levels, resulting in bronchodilation and a reduction in inflammation. The pilot study consisted of a 3 month randomized, double-blind, placebo-controlled trial of L-arginine (0.05 g/kg twice daily in patients with moderate to severe asthma. We measured spirometry, exhaled breath nitric oxide, serum arginine metabolites, questionnaire scores, daily medication use and PEFR with the primary endpoint being the number of minor exacerbations at three months. Interim analysis of the 20 subjects showed no difference in the number of exacerbations, exhaled nitric oxide levels or lung function between groups, though participants in the L-arginine group had higher serum L-arginine at day 60 (2.0 ± 0.6 × 10−3 vs. 1.1 ± 0.2 × 10−3 µmol/L, p < 0.05, ornithine at day 30 (2.4 ± 0.9 vs. 1.2 ± 0.3 µmol/L serum, p < 0.05 and ADMA at day 30 (6.0 ± 1.5 × 10−1 vs. 2.6 ± 0.6 × 10−1 µmol/L serum, p < 0.05 on average compared to the placebo group. The study was terminated prematurely. Supplementing asthma subjects with L-arginine increases plasma levels; whether subgroups might benefit from such supplementation requires further study.

  1. The effect of arginine on oral biofilm communities.

    Science.gov (United States)

    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.

  2. Data supporting the involvement of the adenine nucleotide translocase conformation in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

    Science.gov (United States)

    Korotkov, Sergey M

    2016-06-01

    There we made available information about the effects of the adenine nucleotide translocase (ANT) 'c' conformation fixers (phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside) as well as thiol reagent (4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS)) on isolated rat liver mitochondria. We observed a decrease in A540 (mitochondrial swelling) and respiratory control rates (RCRADP [state 3/state 4] and RCRDNP [2,4-dinitrophenol-uncoupled state/basal state or state 4]), as well as an increase in Ca(2+)-induced safranin fluorescence (F485/590, arbitrary units), showed a dissipation in the inner membrane potential (ΔΨmito), in experiments with energized rat liver mitochondria, injected into the buffer containing 25-75 mM TlNO3, 125 mM KNO3, and 100 µM Ca(2+). The fixers and DIDS, in comparison to Ca(2+) alone, greatly increased A540 decline and the rate of Ca(2+)-induced ΔΨmito dissipation. These reagents also markedly decreased RCRADP and RCRDNP. The MPTP inhibitors (ADP, cyclosporin A, bongkrekic acid, and N-ethylmaleimide) fixing the ANT in 'm' conformation significantly hindered the above-mentioned effects of the fixers and DIDS. A more complete scientific analysis of these findings may be obtained from the manuscript "To involvement the conformation of the adenine nucleotide translocase in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria" (Korotkov et al., 2016 [1]). PMID:27054168

  3. Prevention of muscle fibers atrophy during gravitational unloading: The effect of L-arginine administration

    Science.gov (United States)

    Kartashkina, N.; Lomonosova, Y.; Shevchenko, T. F.; Bugrova, A. E.; Turtikova, O. V.; Kalamkarov, G. R.; Nemirovskaya, T. L.

    2011-05-01

    Gravitational unloading results in pronounced atrophy of m.soleus. Probably, the output of NO is controlled by the muscle activity. We hypothesized that NO may be involved in the protein metabolism and increase of its concentration in muscle can prevent atrophic changes induced by gravitational unloading. In order to test the hypothesis we applied NO donor L-arginine during gravitational unloading. 2.5-month-old male Wistar rats weighing 220-230g were divided into sedentary control group (CTR, n=7), 14-day hindlimb suspension (HS, n=7), 14 days of hindlimb suspension+ L-arginine (HSL, n=7) (with a daily supplementation of 500 mg/kg wt L-arginine) and 14 days of hindlimb suspension+ L-NAME (HSN, n=7) (90 mg/kg wt during 14 days). Cross sectional area (CSA) of slow twitch (ST) and fast twitch (FT) soleus muscle fibers decreased by 45% and 28% in the HS group ( pHSL group in comparison with the HS group ( pHSL group was completely prevented since FT fiber CSA had no significant differences from the CTR group. In HS group, the percentage of fibers revealing either gaps/disruption of the dystrophin layer of the myofiber surface membrane increased by 27% and 17%, respectively, as compared to the controls (CTR group, pHSL group. NO concentration decreased by 60% in the HS group (as well as HSN group) and at the same time no changes were detectable in the HSL group. This fact indicates the compensation of NO content in the unloaded muscle under L-arginine administration. The levels of atrogin-1 mRNA were considerably altered in suspended animals (HS group: plus 27%, HSL group: minus 13%) as compared to the control level. Conclusion: L-arginine administration allows maintaining NO concentration in m.soleus at the level of cage control group, prevents from dystrophin layer destruction, decreases the atrogin mRNA concentration in the muscle and atrophy level under gravitational unloading.

  4. Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs

    Science.gov (United States)

    Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.

    2016-01-01

    Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

  5. The formation of argpyrimidine, a methylglyoxal-arginine adduct, in the nucleus of neural cells

    International Nuclear Information System (INIS)

    Methylglyoxal (MG) is an endogenous metabolite in glycolysis and forms stable adducts primarily with arginine residues of intracellular proteins. The biological role of this modification in cell function is not known. In the present study, we found that a MG-detoxification enzyme glyoxalase I (GLO1) is mainly expressed in the ventricular zone (VZ) at embryonic day 16 which neural stem and progenitor cells localize. Moreover, immunohistochemical analysis revealed that argpyrimidine, a major MG-arginine adduct, is predominantly produced in cortical plate neurons not VZ during cerebral cortex development and is exclusively located in the nucleus. Immunoblotting experiment showed that the formation of argpyrimidine occurs on some nuclear proteins of cortical neurons. To our knowledge, this is first report of the argpyrimidine formation in the nucleus of neuron. These findings suggest that GLO1, which is dominantly expressed in the embryonic VZ, reduces the intracellular level of MG and suppresses the formation of argpyrimidine in neural stem and progenitor cells. Argpyrimidine may contribute to the neural differentiation and/or the maintenance of the differentiated state via the modification of nuclear proteins.

  6. Influence of L-arginine during bovine in vitro fertilization.

    Science.gov (United States)

    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. PMID:25651608

  7. Identification of arginine- and lysine-methylation in the proteome of Saccharomyces cerevisiae and its functional implications

    Directory of Open Access Journals (Sweden)

    Gasteiger Elisabeth

    2010-02-01

    Full Text Available Abstract Background The methylation of eukaryotic proteins has been proposed to be widespread, but this has not been conclusively shown to date. In this study, we examined 36,854 previously generated peptide mass spectra from 2,607 Saccharomyces cerevisiae proteins for the presence of arginine and lysine methylation. This was done using the FindMod tool and 5 filters that took advantage of the high number of replicate analysis per protein and the presence of overlapping peptides. Results A total of 83 high-confidence lysine and arginine methylation sites were found in 66 proteins. Motif analysis revealed many methylated sites were associated with MK, RGG/RXG/RGX or WXXXR motifs. Functionally, methylated proteins were significantly enriched for protein translation, ribosomal biogenesis and assembly and organellar organisation and were predominantly found in the cytoplasm and ribosome. Intriguingly, methylated proteins were seen to have significantly longer half-life than proteins for which no methylation was found. Some 43% of methylated lysine sites were predicted to be amenable to ubiquitination, suggesting methyl-lysine might block the action of ubiquitin ligase. Conclusions This study suggests protein methylation to be quite widespread, albeit associated with specific functions. Large-scale tandem mass spectroscopy analyses will help to further confirm the modifications reported here.

  8. Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA.

    Science.gov (United States)

    Snijders, Ambrosius P; Hautbergue, Guillaume M; Bloom, Alex; Williamson, James C; Minshull, Thomas C; Phillips, Helen L; Mihaylov, Simeon R; Gjerde, Douglas T; Hornby, David P; Wilson, Stuart A; Hurd, Paul J; Dickman, Mark J

    2015-03-01

    Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.

  9. Transsulfuration pathway thiols and methylated arginines: the Hunter Community Study.

    Directory of Open Access Journals (Sweden)

    Arduino A Mangoni

    Full Text Available BACKGROUND: Serum homocysteine, when studied singly, has been reported to be positively associated both with the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine [ADMA, via inhibition of dimethylarginine dimethylaminohydrolase (DDAH activity] and with symmetric dimethylarginine (SDMA. We investigated combined associations between transsulfuration pathway thiols, including homocysteine, and serum ADMA and SDMA concentrations at population level. METHODS: Data on clinical and demographic characteristics, medication exposure, C-reactive protein, serum ADMA and SDMA (LC-MS/MS, and thiols (homocysteine, cysteine, taurine, glutamylcysteine, total glutathione, and cysteinylglycine; capillary electrophoresis were collected from a sample of the Hunter Community Study on human ageing [n = 498, median age (IQR = 64 (60-70 years]. RESULTS: REGRESSION ANALYSIS SHOWED THAT: a age (P = 0.001, gender (P = 0.03, lower estimated glomerular filtration rate (eGFR, P = 0.08, body mass index (P = 0.008, treatment with beta-blockers (P = 0.03, homocysteine (P = 0.02, and glutamylcysteine (P = 0.003 were independently associated with higher ADMA concentrations; and b age (P = 0.001, absence of diabetes (P = 0.001, lower body mass index (P = 0.01, lower eGFR (P<0.001, cysteine (P = 0.007, and glutamylcysteine (P < 0.001 were independently associated with higher SDMA concentrations. No significant associations were observed between methylated arginines and either glutathione or taurine concentrations. CONCLUSIONS: After adjusting for clinical, demographic, biochemical, and pharmacological confounders the combined assessment of transsulfuration pathway thiols shows that glutamylcysteine has the strongest and positive independent associations with ADMA and SDMA. Whether this reflects a direct effect of glutamylcysteine on DDAH activity (for ADMA and/or cationic amino acid transport requires further investigations.

  10. Comparison of the effect of topical versus systemic L-arginine on wound healing in acute incisional diabetic rat model

    Directory of Open Access Journals (Sweden)

    Alireza Zandifar

    2015-01-01

    Full Text Available Background: Diabetes is associated with endothelial dysfunction and impaired wound healing. The amino acid L-arginine is the only substrate for nitric oxide (NO synthesis. The purpose of this study was to compare the topical versus systemic L-arginine treatment on total nitrite (NO x and vascular endothelial growth factor (VEGF concentrations in wound fluid and rate of wound healing in an acute incisional diabetic wound model. Materials and Methods: A total of 56 Sprague-Dawley rats were used of which 32 were rendered diabetic. Animals underwent a dorsal skin incision. Dm-sys-arg group (N = 8, diabetic and Norm-sys-arg group (N = 8, normoglycemic were gavaged with L-arginine. Dm-sys-control group (N = 8, diabetic and Norm-sys-control group (N = 8, normoglycemic were gavaged with water. Dm-top-arg group (N = 8, diabetic and norm-top-arg group (N = 8, normoglycemic received topical L-arginine gel. Dm-top-control group (N = 8, diabetic received gel vehicle. On the day 5 the amount of NO x in wound fluid was measured by Griess reaction. VEGF/total protein in wound fluids was also measured on day 5 using enzyme-linked immunosorbent assay. All wound tissue specimens were fixed and stained to be evaluated for rate of healing. Data were analyzed using SPSS software (version 18.0, Chicago, IL, USA through One-way analysis of variance test and Tukey′s post-hoc. Results: In dm-sys-arg group, the level of NO x on day 5 was significantly more than dm-top-arg group (P < 0.05. VEGF content in L-arginine treated groups were significantly more than controls (P < 0.05. Rate of diabetic wound healing in dm-sys-arg group was significantly more than dm-top-arg group. Conclusion: Systemic L-arginine is more efficient than topical L-arginine in wound healing. This process is mediated at least in part, by increasing VEGF and NO in the wound fluid.

  11. Structure and function of ubiquitin: evidence for differential interactions of arginine-74 with the activating enzyme and the proteases of ATP-dependent proteolysis

    International Nuclear Information System (INIS)

    Ubiquitin was modified with the anionic, arginine-specific reagent 4-(oxoacetyl)phenoxyacetic acid in order to study the relationship between structure and function of the molecule. Four different derivatives (A, B, C, and D) were purified from the reaction mixture by anion-exchange high-performance liquid chromatography and subjected to tryptic peptide mapping to determine the location of the modification(s). These derivatives were stable throughout the procedures required for purification, tryptic hydrolysis, and peptide mapping. Derivative A was modified at arginine-42, derivative B at arginine-72, derivative C at arginines-42 and -72, and derivative D at arginine-74. Modification of ubiquitin with 14C-labeled 4-(oxoacetyl)phenoxyacetic acid indicated that the reagent formed a stable, 1:1 complex with arginine residues of the protein. Native ubiquitin and each of the four derivatives were tested for their ability to stimulate 32P exchange between ATP and pyrophosphate, a reaction catalyzed by enzyme 1 of the ubiquitin-dependent proteolytic pathway. A and C were capable of promoting this exchange at a rate only 15% that of native ubiquitin, B stimulated the exchange to 25%, and D stimulated exchange to 60% of the native level. None of the derivative was capable of promoting a significant level of ubiquitin-dependent proteolysis. These results indicate that in this system, the integrity or arginines-42, -72, and -74 is essential for full function of ubiquitin and suggest that the ubiquitin activating enzyme (E1) and the protease(s) of the system recognize different regions or conformations of ubiquitin

  12. Local Structures and Chemical Properties of Deprotonated Arginine

    Institute of Scientific and Technical Information of China (English)

    Hong-bao Li; Zi-jing Lin; Yi Luo

    2012-01-01

    The potential energy surface of gaseous deprotonated arginine has been systematically investigated by first principles calculations.At the B3LYP/6-31G(d) level,apart from the identification of several stable local structures,a new global minimum is located which is about 6.56 kJ/mol more stable than what has been reported.The deprotonated arginine molecule has two distinct forms with the deprotonation at the carboxylate group (COO-).These two forms are bridged by a very high energy barrier and possess very different IR spectral profiles.Our calculated proton dissociation energy and gas-phase acidity of arginine molecule are found to be in good agreement with the corresponding experimental results.The predicted geometries,dipole moments,rotational constants,vertical ionization energies and IR spectra of low energy conformers will be useful for future experimental measurements.

  13. Mitochondria: role of citrulline and arginine supplementation in MELAS syndrome.

    Science.gov (United States)

    El-Hattab, Ayman W; Emrick, Lisa T; Chanprasert, Sirisak; Craigen, William J; Scaglia, Fernando

    2014-03-01

    Mitochondria are found in all nucleated human cells and generate most of the cellular energy. Mitochondrial disorders result from dysfunctional mitochondria that are unable to generate sufficient ATP to meet the energy needs of various organs. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a frequent maternally inherited mitochondrial disorder. There is growing evidence that nitric oxide (NO) deficiency occurs in MELAS syndrome and results in impaired blood perfusion that contributes significantly to several complications including stroke-like episodes, myopathy, and lactic acidosis. Both arginine and citrulline act as NO precursors and their administration results in increased NO production and hence can potentially have therapeutic utility in MELAS syndrome. Citrulline raises NO production to a greater extent than arginine, therefore, citrulline may have a better therapeutic effect. Controlled studies assessing the effects of arginine or citrulline supplementation on different clinical aspects of MELAS syndrome are needed.

  14. Reduced arginine availability and nitric oxide synthesis in cancer is related to impaired endogenous arginine synthesis.

    Science.gov (United States)

    Engelen, Mariëlle P K J; Safar, Ahmed M; Bartter, Thaddeus; Koeman, Fari; Deutz, Nicolaas E P

    2016-07-01

    Reduced plasma arginine (ARG) concentrations are found in various types of cancer. ARG and its product nitric oxide (NO) are important mediators in the immune function and the defense against tumour cells. It remains unclear whether the diminished systemic ARG availability in cancer is related to insufficient endogenous ARG synthesis, negatively affecting NO synthesis, and whether a dietary amino acid mixture is able to restore this. In 13 patients with advanced non-small cell lung cancer (NSCLC) and 11 healthy controls, whole body ARG and CIT (citrulline) rates of appearance were measured by stable isotope methodology before and after intake of a mixture of amino acids as present in whey protein. The conversions of CIT to ARG (indicator of de novo ARG synthesis) and ARG to CIT (marker of NO synthesis), and ARG clearance (reflecting ARG disposal capacity) were calculated. Plasma isotopic enrichments and amino acid concentrations were measured by LC-MS/MS. Conversions of CIT to ARG and ARG to CIT (P<0.05), and CIT rate of appearance (P=0.07) were lower in NSCLC. ARG rate of appearance and clearance were comparable suggesting no enhanced systemic ARG production and disposal capacity in NSCLC. After intake of the mixture, ARG rate of appearance and concentration increased (P<0.001), and ARG to CIT conversion was restored in NSCLC. In conclusion, an impaired endogenous ARG synthesis plays a role in the reduced systemic ARG availability and NO synthesis in advanced NSCLC. Nutritional approaches may restore systemic ARG availability and NO synthesis in cancer, but the clinical implication remains unclear. PMID:27129191

  15. Arginine protection against ammonia toxicity in exhausted rat.

    Science.gov (United States)

    Krishna Mohan, P; Indira, K; Rajendra, W

    1987-01-01

    Arginine administration (5 m moles/kg/day) to albino rats for 7 days, revealed that this vital basic amino acid possesses latent potentiality for the accentuation of urea cycle or at least for arginase activity. The mitigation of ammonia toxicity was observed to be more effective in the case of gastrocnemius and red vastus as compared to white vastus. Further, ammonia and lactate levels were also decreased by arginine in blood and thereby delaying the onset of fatigue by preventing ammonotoxemia and lactic acidemia. PMID:3666875

  16. Requirement of dietary arginine for juvenile orange-spotted grouper, Epinephelus coioides%斜带石斑鱼幼鱼的饲料精氨酸需求量

    Institute of Scientific and Technical Information of China (English)

    韩凤禄; 张琴; 黄国强; 迟淑艳; 谭北平; 董晓慧; 杨奇慧; 刘泓宇; 章双

    2016-01-01

    通过配制7种含不同水平精氨酸(2.13%、2.42%、2.71%、2.95%、3.20%、3.48%、3.74%干物质)的等氮、等脂的实验饲料,研究精氨酸对斜带石斑鱼(Epinephelus coioides)幼鱼生长、饲料利用和血清一氧化氮合酶等的影响。每组设3个重复,每个重复30尾鱼(7.52±0.02g),饲喂8周。结果显示,饲料精氨酸水平对各组存活率无显著影响(P>0.05);精氨酸水平为2.95%的斜带石斑鱼获得最大增重率和特定生长率,显著高于其他各组(P0.05). However, the weight gain rate and specific growth rate were significantly higher in the 2.95% arginine group than in the other groups (P<0.05). The feed coefficient of the fish fed 2.95% arginine was significantly lower than that of the fish fed 2.13%, 2.42%, or 2.71% arginine (P<0.05). The whole-body protein of the fish fed 3.20% arginine was signifi-cantly higher than that of the fish fed 2.13% arginine (P<0.05). Serum glucose tended to decrease as dietary ar-ginine increased, and was significantly higher in the fish fed 2.13% or 2.42% arginine than in those of the other groups (P<0.05). Total protein and total nitric oxide synthase were significantly higher in the sera of fish fed 2.95% arginine than those in the sera of fish in the other groups (P<0.05). These results indicate that dietary ar-ginine not only promotes growth and body protein synthesis, but also improves the immunity of the grouper. A quadratic regression analysis of the specific growth rate against dietary arginine indicated that the optimum argin-ine level for the maximum growth of the juvenile grouper is 3.06%, corresponding to 6.07% of dietary protein on a dry weight basis.

  17. Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex.

    Science.gov (United States)

    You, Zhiying; Ode, Koji L; Shindo, Mayumi; Takisawa, Haruhiko; Masai, Hisao

    2016-05-01

    All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2∼7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity. PMID:26940553

  18. Salmonella Type III Secretion-Associated Protein InvE Controls Translocation of Effector Proteins into Host Cells

    OpenAIRE

    Kubori, Tomoko; Galán, Jorge E.

    2002-01-01

    Salmonella enterica encodes a type III secretion system (TTSS) within a pathogenicity island located at centisome 63 (SPI-1), which is essential for its pathogenicity. This system mediates the transfer of a battery of bacterial proteins into the host cell with the capacity to modulate cellular functions. The transfer process is dependent on the function of protein translocases SipB, SipC, and SipD. We report here that Salmonella protein InvE, which is also encoded within SPI-1, is essential f...

  19. Molecular and immunological characterization of arginine kinase from the Indianmeal moth, Plodia interpunctella, a novel cross-reactive invertebrate pan-allergen.

    Science.gov (United States)

    Binder, M; Mahler, V; Hayek, B; Sperr, W R; Schöller, M; Prozell, S; Wiedermann, G; Valent, P; Valenta, R; Duchêne, M

    2001-11-01

    IgE recognition of indoor allergens represents a major cause of allergic asthma in atopic individuals. We found that 52 of 102 patients suffering from allergic symptoms indoors contained IgE Abs against allergens from the Indianmeal moth (Plodia interpunctella), a ubiquitous food pest. Using serum IgE from a moth-sensitized patient we screened an expression cDNA library constructed from P. interpunctella larvae. cDNAs coding for arginine kinase (EC 2.7.3.3), a 40-kDa enzyme commonly occurring in invertebrates that is involved in the storage of such high-energy phosphate bonds as phosphoarginine, were isolated. Recombinant moth arginine kinase, designated Plo i 1, was expressed in Escherichia coli as a histidine-tagged protein with enzymatic activity, and purified to homogeneity by nickel chelate affinity chromatography. Purified recombinant arginine kinase induced specific basophil histamine release and immediate as well as late-phase skin reactions. It reacted with serum IgE from 13 of the 52 (25%) moth-allergic patients and inhibited the binding of allergic patients' IgE to an immunologically related 40-kDa allergen present in house dust mite, cockroach, king prawn, lobster, and mussel. Our results indicate that arginine kinases represent a new class of cross-reactive invertebrate pan-allergens. Recombinant arginine kinase may be used to identify a group of polysensitized indoor allergic patients and for immunotherapy of these individuals.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Minaeva, Ekaterina; Forchhammer, Karl; Ermilova, Elena

    2015-11-01

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

  2. Contents of corticotropin-releasing hormone and arginine vasopressin immunoreativity in the spleen and thymus during a chronic inflammatory stress

    DEFF Research Database (Denmark)

    Chowdrey, H.S.; Lightman, S.L.; Harbuz, M.S.;

    1994-01-01

    Corticotropin-releasing hormone, spleen, thymus, immune system, stress, arthritis, arginine vasopressin......Corticotropin-releasing hormone, spleen, thymus, immune system, stress, arthritis, arginine vasopressin...

  3. O-Crystallin, arginine kinase and ferritin from the octopus lens.

    Science.gov (United States)

    Zinovieva, R D; Piatigorsky, J; Tomarev, S I

    1999-05-18

    Three proteins have been identified in the eye lens of the octopus, Octopus dofleini. A 22 kDa protein comprising 3-5% of the soluble protein of the lens is 35-43% identical to a family of phosphatidylethanolamine-binding proteins of vertebrates. Other members of this family include the immunodominant antigen of the filarial parasite, Onchocerca volvulus, putative odorant-binding proteins of Drosophila and a protein with unknown function of Caenorhabditis elegans. We have called this protein O-crystallin on the basis of its abundance in the transparent lens. O-Crystallin mRNA was detected only in the lens. Two tryptic peptides of another octopus lens protein, less abundant than O-crystallin, showed 80% identity to arginine kinase of invertebrates, a relative of creatine kinase of vertebrates. Finally, ferritin cDNA was isolated as an abundant cDNA from the octopus lens library. Northern blots showed that ferritin mRNA is not lens-specific. PMID:10350626

  4. Effect of oral L-arginine administration on exhaled nitric oxide (no) concentration in healthy volunteers

    OpenAIRE

    Ogata, Hiroshi; Yatabe, Midori; Misaka, Shingen; Shikama, Yayoi; Sato, Suguru; Munakata, Mitsuru; Kimura, Junko

    2013-01-01

    We previously reported a case of pulmonary hypertension, where the symptoms were improved by oral L-arginine (arginine) administration. Arginine may increase nitric oxide (NO) production in the pulmonary artery. Exhaled NO may reflect pulmonary artery NO production. It has been demonstrated that exhaled NO concentration is higher in patients with allergic diseases, but whether oral arginine administration alters exhaled NO is unknown. Therefore, in this study, we investigated whether oral arg...

  5. Arginine Functionally Improves Clinically Relevant Human Galactose-1-Phosphate Uridylyltransferase (GALT) Variants Expressed in a Prokaryotic Model.

    Science.gov (United States)

    Coelho, Ana I; Trabuco, Matilde; Silva, Maria João; de Almeida, Isabel Tavares; Leandro, Paula; Rivera, Isabel; Vicente, João B

    2015-01-01

    Classic galactosemia is a rare genetic disease of the galactose metabolism, resulting from deficient activity of galactose-1-phosphate uridylyltransferase (GALT). The current standard of care is lifelong dietary restriction of galactose, which however fails to prevent the development of long-term complications. Structural-functional studies demonstrated that the most prevalent GALT mutations give rise to proteins with increased propensity to aggregate in solution. Arginine is a known stabilizer of aggregation-prone proteins, having already shown a beneficial effect in other inherited metabolic disorders.Herein we developed a prokaryotic model of galactose sensitivity that allows evaluating in a cellular context the mutations' impact on GALT function, as well as the potential effect of arginine in functionally rescuing clinically relevant variants.This study revealed that some hGALT variants, previously described to exhibit no detectable activity in vitro, actually present residual activity when determined in vivo. Furthermore, it revealed that arginine presents a mutation-specific beneficial effect, particularly on the prevalent p.Q188R and p.K285N variants, which led us to hypothesize that it might constitute a promising therapeutic agent in classic galactosemia. PMID:25814382

  6. Interplay among coactivator-associated arginine methyltransferase 1, CBP, and CIITA in IFN-gamma-inducible MHC-II gene expression.

    Science.gov (United States)

    Zika, Eleni; Fauquier, Lucas; Vandel, Laurence; Ting, Jenny P-Y

    2005-11-01

    Class II major histocompatibility (MHC-II) genes are prototype targets of IFN-gamma. IFN-gamma activates the expression of the non-DNA-binding master regulator of MHC-II, class II transactivator (CIITA), which is crucial for enhanceosome formation and gene activation. This report shows the importance of the histone methyltransferase, coactivator-associated arginine methyltransferase (CARM1/PRMT4), during IFN-gamma-induced MHC-II gene activation. It also demonstrates the coordinated regulation of CIITA, CARM1, and the acetyltransferase cyclic-AMP response element binding (CREB)-binding protein (CBP) during this process. CARM1 synergizes with CIITA in activating MHC-II transcription and synergy is abrogated when an arginine methyltransferase-defective CARM1 mutant is used. Protein-arginine methyltransferase 1 has much less effect on MHC-II transcription. Specific RNA interference reduced CARM1 expression as well as MHC-II expression. The recruitment of CARM1 to the promoter requires endogenous CIITA and results in methylation of histone H3-R17; hence, CIITA is an upstream regulator of histone methylation. Previous work has shown that CARM1 can methylate CBP at three arginine residues. Using wild-type CBP and a mutant of CBP lacking the CARM1-targeted arginine residues (R3A), we show that arginine methylation of CBP is required for IFN-gamma induction of MHC-II. A kinetic analysis shows that CIITA, CARM1, and H3-R17 methylation all precede CBP loading on the MHC-II promoter during IFN-gamma treatment. These results suggest functional and temporal relationships among CIITA, CARM1, and CBP for IFN-gamma induction of MHC-II.

  7. In vivo arginine production and intravascular nitric oxide synthesis in hypotensive sepsis

    Science.gov (United States)

    Arginine is important in the response to infections and is a precursor for the synthesis of the vasodilator nitric oxide (NO). Low plasma arginine is correlated with a worse prognosis in patients with sepsis, and increased NO has been implicated in the hypotension of sepsis. Data on in vivo arginine...

  8. High plasma arginine concentrations in critically ill patients suffering from hepatic failure

    NARCIS (Netherlands)

    R. Nijveldt (Robin); M.P.C. Siroen; B. van der Hoven (Ben); T. Teerlink (Tom); H.A. Prins (Hubert); A.R.J. Girbes (Armand); P.A.M. van Leeuwen

    2004-01-01

    textabstractObjective: In physiological conditions, the liver plays an important role in the regulation of plasma arginine concentrations by taking up large amounts of arginine from the hepatic circulation. When hepatic failure is present, arginine metabolism may be disturbed. Therefore, we hypothes

  9. Arginine dimethylation products in pediatric patients with chronic kidney disease

    Directory of Open Access Journals (Sweden)

    Akram E. El-Sadek

    2016-08-01

    Conclusion: Disturbed serum levels of arginine and its dimethyl derivatives may underlie development and/or progression of CKD. Elevated serum SDMA level is strongly correlated with impaired kidney functions and could be considered as a predictor for kidney functions deterioration and CKD progression.

  10. L-Arginine improves multiple physiological parameters in mice exposed to diet-induced metabolic disturbances

    DEFF Research Database (Denmark)

    Clemmensen, Christoffer; Madsen, Andreas Nygaard; Smajilovic, Sanela;

    2012-01-01

    L: -Arginine (L: -Arg) is a conditionally essential amino acid and a natural constituent of dietary proteins. Studies in obese rats and type 2 diabetic humans have indicated that dietary supplementation with L: -Arg can diminish gain in white adipose tissue (WAT) and improve insulin sensitivity...... on locomotor activity, substrate metabolism or expression of uncoupling proteins (UCP1 and UCP2) in adipose tissues was displayed. In conclusion, dietary L: -Arg supplementation substantially affects an array of metabolic-associated parameters including a reduction in WAT, hyperphagia, improved insulin...... groups. Glucose homeostasis experiments revealed a major effect of L: -Arg supplementation on glucose tolerance and insulin sensitivity, interestingly, independent of a parallel regulation in whole-body adiposity. Increased L: -Arg ingestion also raised energy expenditure; however, no concurrent effect...

  11. Safety, efficacy and physiological actions of a lysine-free, arginine-rich formula to treat glutaryl-CoA dehydrogenase deficiency: focus on cerebral amino acid influx.

    Science.gov (United States)

    Strauss, Kevin A; Brumbaugh, Joan; Duffy, Alana; Wardley, Bridget; Robinson, Donna; Hendrickson, Christine; Tortorelli, Silvia; Moser, Ann B; Puffenberger, Erik G; Rider, Nicholas L; Morton, D Holmes

    2011-01-01

    Striatal degeneration from glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type 1, GA1) is associated with cerebral formation and entrapment of glutaryl-CoA and its derivatives that depend on cerebral lysine influx. In 2006 we designed a lysine-free study formula enriched with arginine to selectively block lysine transport across cerebral endothelia and thereby limit glutaryl-CoA production by brain. Between 2006 and present, we treated twelve consecutive children with study formula (LYSx group) while holding all other treatment practices constant. Clinical and biochemical outcomes were compared to 25 GA1 patients (PROx group) treated between 1995 and 2005 with natural protein restriction (dietary lysine/arginine ratio of 1.7±0.3 mg:mg). We used published kinetic parameters of the y+and LAT1 blood-brain barrier transporters to model the influx of amino acids into the brain. Arginine fortification to achieve a mean dietary lysine/arginine ratio of 0.7±0.2 mg:mg was neuroprotective. All 12 LYSx patients are physically and neurologically healthy after 28 aggregate patient-years of follow up (current ages 28±21 months) and there were no adverse events related to formula use. This represents a 36% reduction of neurological risk (95% confidence interval 14-52%, p=0.018) that we can directly attribute to altered amino acid intake. During the first year of life, 20% lower lysine intake and two-fold higher arginine intake by LYSx patients were associated with 50% lower plasma lysine, 3-fold lower plasma lysine/arginine concentration ratio, 42% lower mean calculated cerebral lysine influx, 54% higher calculated cerebral arginine influx, 15-26% higher calculated cerebral influx of several anaplerotic precursors (isoleucine, threonine, methionine, and leucine), 50% less 3-hydroxyglutarate excretion, and a 3-fold lower hospitalization rate (0.8 versus 2.3 hospitalizations per patient per year). The relationship between arginine fortification and plasma lysine

  12. Circadian variation of plasma arginine vasopressin concentration, or arginine vasopressin in enuresis.

    Science.gov (United States)

    Aikawa, T; Kasahara, T; Uchiyama, M

    1999-01-01

    The objective of these studies was to determine a relationship between primary nocturnal enuresis and arginine vasopressin (AVP) secretion. The first study compared 24-h AVP secretion profiles of enuretic (n = 9) and non-enuretic children (n = 8). Blood samples were collected at 1-h intervals for 24 h. In the second study, nocturnal AVP secretion in group A (n = 40)--with low urinary osmotic pressure (UOP) and large nocturnal urine output (NUO)--was compared with that in group D (n = 11) with normal UOP and small NUO. Plasma AVP levels were measured at 30-min intervals, immediately after falling asleep until 06.00 the following morning. The results of the first study showed that the plasma AVP level was significantly lower (p < 0.05-0.001) in the enuretic group between 23.00 and 04.00. The second study showed that group A had significantly lower AVP levels (p < 0.05-0.001) than group D throughout the night. The mean AVP level during night sleep was 0.64 +/- 0.23 pg/ml in group A and 1.43 +/- 0.66 pg/ml in group D. The results of the first study suggest that decreased nocturnal AVP secretion is a cause of bedwetting. However, the results of the second study suggest that nocturnal enuresis cannot be explained by a decrease in nocturnal AVP secretion alone.

  13. Escherichia coli exhibits a membrane-related response to a small arginine- and tryptophan-rich antimicrobial peptide.

    Science.gov (United States)

    Fränzel, Benjamin; Penkova, Maya; Frese, Christian; Metzler-Nolte, Nils; Andreas Wolters, Dirk

    2012-08-01

    Since multiresistant bacterial strains are more widespread and the victim numbers steadily increase, it is very important to possess a broad bandwidth of antimicrobial substances. Antibiotics often feature membrane-associated effect mechanisms. So, we present a membrane proteomic approach to shed light on the cellular response of Escherichia coli as model organism to the hexapeptide MP196, which is arginine and tryptophan rich. Analyzing integral membrane proteins are still challenging, although various detection strategies have been developed in the past. In particular, membrane proteomics in bacteria have been conducted very little due to the special physical properties of these membrane proteins. To obtain more information on the cellular response of the new compound group of small peptides, the tryptophan- and arginine-rich hexapeptide MP196 was subject to a comprehensive quantitative membrane proteomic study on E. coli by means of metabolic labeling in combination with membrane lipid analyses. This study provides in total 767 protein identifications including 185 integral membrane proteins, from which 624 could be quantified. Among these proteins, 134 were differentially expressed. Thereby, functional groups such as amino acid and membrane biosynthesis were affected, stress response could be observed, and the lipid composition of the membrane was significantly altered. Especially, the strong upregulation of the envelope stress induced protein. Spy indicates membrane damage, as well as the downregulation of the mechano-sensitive channel MscL beside others. Finally, the exceptional downregulation of transport systems strengthens these findings.

  14. Partial purification and characterization of arginine decarboxylase from avocado fruit, a thermostable enzyme.

    Science.gov (United States)

    Winer, L; Vinkler, C; Apelbaum, A

    1984-09-01

    A partially purified preparation of arginine decarboxylase (EC 4.1.1.19), a key enzyme in polyamine metabolism in plants, was isolated from avocado (Persea americana Mill. cv Fuerte) fruit. The preparation obtained from the crude extract after ammonium sulfate precipitation, dialysis, and heat treatment, had maximal activity between pH 8.0 and 9.0 at 60 degrees C, in the presence of 1.2 millimolar MnCl(2), 2 millimolar dithiothreitol, and 0.06 millimolar pyridoxal phosphate. The K(m), of arginine for the decarboxylation reaction was determined for enzymes prepared from the seed coat of both 4-week-old avocado fruitlet and fully developed fruit, and was found to have a value of 1.85 and 2.84 millimolar, respectively. The value of V(app) (max) of these enzymes was 1613 and 68 nanomoles of CO(2) produced per milligram of protein per hour for the fruitlet and the fully developed fruit, respectively. Spermine, an end product of polyamine metabolism, caused less than 5% inhibition of the enzyme from fully developed fruit and 65% inhibition of the enzyme from the seed coat of 4-week-old fruitlets at 1 millimolar under similar conditions. The effect of different inhibitors on the enzyme and the change in the nature of the enzyme during fruit development are discussed.

  15. Analysis of an Alanine/Arginine Mixture by Using TLC/FTIR Technique

    Directory of Open Access Journals (Sweden)

    Jun Liu

    2014-01-01

    Full Text Available We applied TLC/FTIR coupled with mapping technique to analyze an alanine/arginine mixture. Narrow band TLC plates prepared by using AgI as a stationary phase were used to separate alanine and arginine. The distribution of alanine and arginine spots was manifested by a 3D chromatogram. Alanine and arginine can be successfully separated by the narrow band TLC plate. In addition, the FTIR spectra of the separated alanine and arginine spots on the narrow band TLC plate are roughly the same as the corresponding reference IR spectra.

  16. Expression of arg genes of Escherichia coli during arginine limitation dependent upon stringent control of translation.

    OpenAIRE

    Williams, M.G.; Rogers, P

    1987-01-01

    The transcription and translation of operons for arginine biosynthetic enzymes after arginine removal (arginine down shift) were studied in relA and relA+ strains of Escherichia coli. After arginine down shift, derepression of synthesis of the arginine biosynthetic enzymes ornithine carbamoyltransferase (argF) and argininosuccinate lyase (argH) began at about 15 min in relA+ cells but was delayed in relA cells for more than 2 h. However, both relA+ and relA cells accumulated high levels of ar...

  17. A triple-arginine motif in the amino-terminal domain and oligomerization are required for HIV-1 inhibition by human MX2.

    Science.gov (United States)

    Goujon, Caroline; Greenbury, Rebecca A; Papaioannou, Stelios; Doyle, Tomas; Malim, Michael H

    2015-04-01

    We have employed molecular genetic approaches to understand the domain organization of the HIV-1 resistance factor myxovirus resistance 2 (MX2). First, we describe an essential triple-arginine motif in the amino-terminal domain. Second, we demonstrate that this 91-residue domain mediates antiviral activity when appended to heterologous proteins, and we provide genetic evidence that protein oligomerization is required for MX2 function. These insights will facilitate future work aiming to elucidate MX2's mechanism of action.

  18. Differential effects of arginine, glutamate and phosphoarginine on Ca(2+)-activation properties of muscle fibres from crayfish and rat.

    Science.gov (United States)

    Jame, David W; West, Jan M; Dooley, Philip C; Stephenson, D George

    2004-01-01

    The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca2+-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log10[Ca2+]) that either contained the test solute ("test") or not ("control"). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l(-1)) significantly shifted the force-pCa curve by 0.08-0.13 pCa units in the direction of increased sensitivity to Ca2+-activated contraction in all fibre types; (ii) phosphoarginine (9-10 mmol l(-1)) induced a significant shift of the force-pCa curve by 0.18-0.24 pCa units in the direction of increased sensitivity to Ca2+ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force-pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36-40 mmol l(-1)), like arginine affected the force-pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca2+-activated contraction by 0.08-0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca2+-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca2+ and the regulatory and other myofibrillar proteins. PMID:15711880

  19. Temporal Expression of the Bacillus subtilis secA Gene, Encoding a Central Component of the Preprotein Translocase

    OpenAIRE

    Herbort, Markus; Klein, Michael; Manting, Erik H.; Driessen, Arnold J. M.; Freudl, Roland

    1999-01-01

    In Bacillus subtilis, the secretion of extracellular proteins strongly increases upon transition from exponential growth to the stationary growth phase. It is not known whether the amounts of some or all components of the protein translocation apparatus are concomitantly increased in relation to the increased export activity. In this study, we analyzed the transcriptional organization and temporal expression of the secA gene, encoding a central component of the B. subtilis preprotein transloc...

  20. Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

    Science.gov (United States)

    Takechi-Haraya, Yuki; Nadai, Ryo; Kimura, Hitoshi; Nishitsuji, Kazuchika; Uchimura, Kenji; Sakai-Kato, Kumiko; Kawakami, Kohsaku; Shigenaga, Akira; Kawakami, Toru; Otaka, Akira; Hojo, Hironobu; Sakashita, Naomi; Saito, Hiroyuki

    2016-06-01

    The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

  1. To involvement the conformation of the adenine nucleotide translocase in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

    Science.gov (United States)

    Korotkov, Sergey M; Konovalova, Svetlana A; Brailovskaya, Irina V; Saris, Nils-Erik L

    2016-04-01

    The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl(+)-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria. Using the Tl(+)-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds. PMID:26835787

  2. Identification of novel inhibitors of phospho-MurNAc-pentapeptide translocase MraY from library screening: Isoquinoline alkaloid michellamine B and xanthene dye phloxine B.

    Science.gov (United States)

    Mihalyi, Agnes; Jamshidi, Shirin; Slikas, Justinas; Bugg, Timothy D H

    2014-09-01

    The National Cancer Institute (NCI) Diversity Set was screened for potential inhibitors of phospho-MurNAc-pentapeptide translocase MraY from Escherichia coli using a primary fluorescence enhancement assay, followed by a secondary radiochemical assay. One new MraY inhibitor was identified from this screen, a naphthylisoquinoline alkaloid michellamine B, which inhibited E. coli MraY (IC50 456μM) and Bacillus subtilis MraY (IC50 386μM), and which showed antimicrobial activity against B. subtilis (MIC 16μg/mL). Following an earlier report of halogenated fluoresceins identified from a combined MraY/MurG screen, three halogenated fluoresceins were tested as inhibitors of E. coli MraY and E. coli MurG, and phloxine B was identified as an inhibitor of E. coli MraY (IC50 32μM). Molecular docking of inhibitor structures against the structure of Aquifex aeolicus MraY indicates that phloxine B appears to bind to the Mg(2+) cofactor in the enzyme active site, while michellamine B binds to a hydrophobic groove formed between transmembrane helices 5 and 9. PMID:25127465

  3. Top-dressing 1% arginine supplementation in the lactation diet of sows does not affect the litter performance and milk composition

    Directory of Open Access Journals (Sweden)

    Djane Dallanora

    2016-08-01

    Full Text Available ABSTRACT: The study aimed to evaluate the effects of arginine supplementation in the lactation diet of sows on their milk composition, litter performance and piglet survival. Sixty-four lactating Landrace x Large White sows, parity 1 to 7, were randomly assigned to two treatments: 1 Control - a corn/soybean meal based diet with 1.10% standardized ileal digestible (SID lysine and 3,475kcal of metabolizable energy (ME kg-1, and 2 arginine - the control diet top-dressed daily with arginine at 1% of feed allowance. The daily feed allowance per sow was 5.0 and 7.5kg from day (D0 to D7 and D8 to D21, respectively. The average litter size was 12.8 piglets after cross-fostering. Litters were weighed on D1, D10, and D21 of lactation and pre-weaning mortality was recorded. Samples of milk (60mL were collected from all functional teats at D10 and D20 of lactation. There were no effects (P>0.05 of arginine supplementation on piglet weight, litter weight, and average daily gain of piglets at D10 and D21 of lactation. The interaction between weight day and treatment was not significant (P>0.05 for any of these response variables. The percentages of piglets that survived until D10 and D21 were 90.3% and 88.3%, respectively, with no difference (P>0.05 between treatments. There were no effects (P>0.05 of the lactation day (D10 or D20, treatment or the interaction between them on crude protein and amino acid content in milk. Top-dressing arginine at 1% of feed allowance of the lactation diet of sows does not affect litter performance and survival and does not influence the amino acid content or arginine: lysine ratio of milk.

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

    Science.gov (United States)

    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.

  5. Protective effect of quercetin and/or l-arginine against nano-zinc oxide-induced cardiotoxicity in rats

    Energy Technology Data Exchange (ETDEWEB)

    Faddah, L. M.; Baky, Nayira A. Abdel [King Saud University, Pharmacology Department, Faculty of Pharmacy (Saudi Arabia); Mohamed, Azza M., E-mail: azzamohamed99@yahoo.com [King Abdulaziz University, Biochemistry Department, Faculty of Science for Girls (Saudi Arabia); Al-Rasheed, Nouf M.; Al-Rasheed, Nawal M. [King Saud University, Pharmacology Department, Faculty of Pharmacy (Saudi Arabia)

    2013-04-15

    The aim of this study was to investigate the protective role of quercetin and/or l-arginine against the cardiotoxic potency of zinc oxide nanoparticle (ZnO-NP)-induced cardiac infarction. ZnO-NPs (50 nm) were administered orally at either 600 mg or 1 g/kg body weight for 5 consecutive days. The results revealed that co-administration of quercetin and/or l-arginine (each 200 mg/kg body weight) daily for 3 weeks to rats intoxicated by either of the two doses markedly ameliorated increases in serum markers of cardiac infarction, including troponin T, creatine kinase-MB, and myoglobin, as well as increases in proinflammatory biomarkers, including tumor necrosis factor-{alpha}, interleukin-6, and C-reactive protein, compared with intoxicated, untreated rats. Each agent alone or in combination also successfully modulated the alterations in serum vascular endothelial growth factor, cardiac calcium concentration, and oxidative DNA damage as well as the increase in the apoptosis marker caspase 3 of cardiac tissue in response to ZnO-NP toxicity. In conclusion, early treatment with quercetin and l-arginine may protect cardiac tissue from infarction induced by the toxic effects of ZnO-NPs.

  6. Graphene Functionalized with Arginine Decreases the Development of Glioblastoma Multiforme Tumor in a Gene-Dependent Manner.

    Science.gov (United States)

    Sawosz, Ewa; Jaworski, Sławomir; Kutwin, Marta; Vadalasetty, Krishna Prasad; Grodzik, Marta; Wierzbicki, Mateusz; Kurantowicz, Natalia; Strojny, Barbara; Hotowy, Anna; Lipińska, Ludwika; Jagiełło, Joanna; Chwalibog, André

    2015-10-23

    Our previous studies revealed that graphene had anticancer properties in experiments in vitro with glioblastoma multiforme (GBM) cells and in tumors cultured in vivo. We hypothesized that the addition of arginine or proline to graphene solutions might counteract graphene agglomeration and increase the activity of graphene. Experiments were performed in vitro with GBM U87 cells and in vivo with GBM tumors cultured on chicken embryo chorioallantoic membranes. The measurements included cell morphology, mortality, viability, tumor morphology, histology, and gene expression. The cells and tumors were treated with reduced graphene oxide (rGO) and rGO functionalized with arginine (rGO + Arg) or proline (rGO + Pro). The results confirmed the anticancer effect of graphene on GBM cells and tumor tissue. After functionalization with amino acids, nanoparticles were distributed more specifically, and the flakes of graphene were less agglomerated. The molecule of rGO + Arg did not increase the expression of TP53 in comparison to rGO, but did not increase the expression of MDM2 or the MDM2/TP53 ratio in the tumor, suggesting that arginine may block MDM2 expression. The expression of NQO1, known to be a strong protector of p53 protein in tumor tissue, was greatly increased. The results indicate that the complex of rGO + Arg has potential in GBM therapy.

  7. Effects of nutritional supplementation with l-arginine on repair of injuries due to muscle strain: experimental study on rats

    Directory of Open Access Journals (Sweden)

    Lauren Izabel Medeiros Couto

    2015-08-01

    Full Text Available ABSTRACTOBJECTIVE: To evaluate the influence of oral supplementation with arginine on regeneration of injuries due to straining of the anterior tibial muscle of rats. METHODS: Twenty-four Wistar rats of weight 492.5 ± 50.45 g were used. Injuries were induced through straining the anterior tibial muscles. The rats were separated into three groups of eight rats each. In the untreated group (UTG, after induction of injuries, the rats were observed for 24 h. In the simulation group (SG and the arginine group (AG respectively, the rats received isotonic saline solution and arginine solution via direct gavage, over a seven-day period. At the end of the period, blood samples were collected for serum evaluations of creatine kinase (CK, lactic dehydrogenase (LDH, aspartate aminotransferase (AST and C-reactive protein (CRP. The right and left anterior tibial muscles were resected for histopathological evaluations on the muscle injuries, investigating edema, hemorrhage and disorganization or morphometric alteration of the muscle fibers. The tissue repair was investigated in terms of proliferation of adipose tissue, angiogenesis and collagen fibers. The ANOVA and Student's tmethods were used and p≤ 0.05 was taken to be statistically significant. RESULTS: In the serum evaluations, the AG showed lower CK assay values and higher AST values. In the histopathological evaluation, the UTG presented edema and hemorrhage compatible with injuries due to strain; the SG presented edema and hemorrhage with proliferation of adipose tissue and collagen fibers; and the AG presented not only the findings of the SG but also, especially, intense angiogenesis. CONCLUSION: Oral supplementation with arginine did not cause any significant metabolic alterations that would contraindicate its use and it induced angiogenesis during the repair of muscles injured due to strain.

  8. Mutation of the Erwinia amylovora argD gene causes arginine auxotrophy, nonpathogenicity in apples, and reduced virulence in pears.

    Science.gov (United States)

    Ramos, Laura S; Lehman, Brian L; Peter, Kari A; McNellis, Timothy W

    2014-11-01

    Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.

  9. Effects of non-surgical periodontal treatment on the L-arginine-nitric oxide pathway and oxidative status in platelets.

    Science.gov (United States)

    Siqueira, Mariana Alves de Sá; Fischer, Ricardo Guimarães; Pereira, Natália Rodrigues; Martins, Marcela Anjos; Moss, Monique Bandeira; Mendes-Ribeiro, Antônio Cláudio; Figueredo, Carlos Marcelo da Silva; Brunini, Tatiana Marlowe Cunha

    2013-06-01

    Several studies have suggested an increase of cardiovascular disease (CVD) risk on periodontitis patients. An enhancement has been demonstrated on both platelet activation and oxidative stress on periodontitis patients, which may contribute for this association. Therefore, the aim of this study was to evaluate the effects of non-surgical periodontal treatment on the l-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway and oxidative status in platelets. A total of eight periodontitis patients and eight controls were included in this study. Clinical, laboratory and experimental evaluations were performed on baseline and 90 days after periodontal treatment (except for western blot analysis). The clinical periodontal evaluation included measurements of probing pocket depth (PPD), clinical attachment loss (CAL), % of sites with plaque and % of sites with bleeding on probing. We evaluated: l-[(3)H]arginine influx; nitric oxide synthase (NOS) and arginase enzymes activity and expression; expression of guanylate cyclase and phosphodiesterase-5 enzymes; cGMP levels; platelet aggregation; oxidative status through superoxide dismutase (SOD) and catalase activities, and measurement of reactive oxygen species (ROS) levels and C-reactive protein (CRP) levels. The initial results showed an activation of both l-arginine influx and via system y (+ )L associated with reduced intraplatelet cGMP levels in periodontitis patients and increased systemic levels of CRP. After periodontal treatment, there was a significant reduction of the % of sites with PPD 4-5mm, % of sites with CAL 4-5 mm, and an enhancement in cGMP levels and SOD activity. Moreover, CRP levels were reduced after treatment. Therefore, alterations in the intraplatelet l-arginine-NO-cGMP pathway and oxidant-antioxidant balance associated with a systemic inflammatory response may lead to platelet dysfunction, which may contribute to a higher risk of CVD in periodontitis. PMID:23918883

  10. Gliclazide directly inhibits arginine-induced glucagon release

    DEFF Research Database (Denmark)

    Cejvan, Kenan; Coy, David H; Holst, Jens Juul;

    2002-01-01

    Arginine-stimulated insulin and somatostatin release is enhanced by the sulfonylurea gliclazide. In contrast, gliclazide inhibits the glucagon response. The aim of the present study was to investigate whether this inhibition of glucagon release was mediated by a direct suppressive effect of...... specific antagonist of type 2 somatostatin receptor, DC-41-33 (2 micro mol/l), which fully antagonizes the suppressive somatostatin effect on rat A cells. Gliclazide (30 micro mol/l) inhibited glucagon release by 54% in the perfusion experiments, whereas the somatostatin response was nearly abolished. In...... islet perifusions with DC-41-33, arginine-induced glucagon release was inhibited by 66%. We therefore concluded that gliclazide inhibits glucagon release by a direct action on the pancreatic A cell....

  11. Arginine vasopressin in septic shock: supplement or substitute for norepinephrine?

    OpenAIRE

    Rehberg, Sebastian; Enkhbaatar, Perenlei; Traber, Daniel L

    2009-01-01

    In the current issue of Critical Care, Simon and coworkers investigated the effects of first-line arginine vasopressin (AVP) on organ function and systemic metabolism compared with norepinephrine in a pig model of fecal peritonitis. AVP was titrated according to the mean arterial pressure suggesting a vasopressor rather than a hormone replacement therapy. The study provides some evidence for the safety of this therapeutic approach. It needs to be determined whether AVP is most beneficial as a...

  12. Structural basis for signal-sequence recognition by the translocase motor SecA determined by NMR

    NARCIS (Netherlands)

    Gelis, I.; Bonvin, A.M.J.J.; Keramisanou, D.; Koukaki, M.; Gouridis, G.; Karamanou, S.; Economou, A.; Kalodimos, C.G.

    2007-01-01

    Recognition of signal sequences by cognate receptors controls the entry of virtually all proteins to export pathways. Despite its importance, this process remains poorly understood. Here, we present the solution structure of a signal peptide bound to SecA, the 204 kDa ATPase motor of the Sec translo

  13. The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues.

    Science.gov (United States)

    Scrima, Andrea; Thomas, Christoph; Deaconescu, Delia; Wittinghofer, Alfred

    2008-04-01

    The GTP-binding protein Rap1 regulates integrin-mediated and other cell adhesion processes. Unlike most other Ras-related proteins, it contains a threonine in switch II instead of a glutamine (Gln61 in Ras), a residue crucial for the GTPase reaction of most G proteins. Furthermore, unlike most other GTPase-activating proteins (GAPs) for small G proteins, which supply a catalytically important Arg-finger, no arginine residue of RapGAP makes a significant contribution to the GTPase reaction of Rap1. For a detailed understanding of the reaction mechanism, we have solved the structure of Rap1 in complex with Rap1GAP. It shows that the Thr61 of Rap is away from the active site and that an invariant asparagine of RapGAPs, the Asn-thumb, takes over the role of the cis-glutamine of Ras, Rho or Ran. The structure and biochemical data allow to further explain the mechanism and to define the important role of a conserved tyrosine. The structure and biochemical data furthermore show that the RapGAP homologous region of the tumour suppressor Tuberin is sufficient for catalysis on Rheb.

  14. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production.

    Science.gov (United States)

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

    2016-01-01

    L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L(-1) L-arginine with yield up to 0.431 g L-arginine g(-1) glucose in fed-batch fermentation. PMID:27338253

  15. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production

    Science.gov (United States)

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

    2016-01-01

    L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L−1 L-arginine with yield up to 0.431 g L-arginine g−1 glucose in fed-batch fermentation. PMID:27338253

  16. Principles of protein targeting to the nucleolus.

    Science.gov (United States)

    Martin, Robert M; Ter-Avetisyan, Gohar; Herce, Henry D; Ludwig, Anne K; Lättig-Tünnemann, Gisela; Cardoso, M Cristina

    2015-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution.

  17. Structural Basis for Recognizing Phosphoarginine and Evolving Residue-Specific Protein Phosphatases in Gram-Positive Bacteria

    Directory of Open Access Journals (Sweden)

    Jakob Fuhrmann

    2013-06-01

    Full Text Available Many cellular pathways are regulated by the competing activity of protein kinases and phosphatases. The recent identification of arginine phosphorylation as a protein modification in bacteria prompted us to analyze the molecular basis of targeting phospho-arginine. In this work, we characterize an annotated tyrosine phosphatase, YwlE, that counteracts the protein arginine kinase McsB. Strikingly, structural studies of YwlE reaction intermediates provide a direct view on a captured arginine residue. Together with biochemical data, the crystal structures depict the evolution of a highly specific phospho-arginine phosphatase, with the use of a size-and-polarity filter for distinguishing phosphorylated arginine from other phosphorylated side chains. To confirm the proposed mechanism, we performed bioinformatic searches for phosphatases, employing a similar selectivity filter, and identified a protein in Drosophila melanogaster exhibiting robust arginine phosphatase activity. In sum, our findings uncover the molecular framework for specific targeting of phospho-arginine and suggest that protein arginine (dephosphorylation may be relevant in eukaryotes.

  18. L-arginine and L-glutamine as immunonutrients and modulating agents for oxidative stress and toxicity induced by sodium nitrite in rats.

    Science.gov (United States)

    El-Sheikh, Nora M; Khalil, Fatma A

    2011-04-01

    Sodium nitrite (NaNO(2)) is a flavoring, coloring and preservative agent in meat and fish products. The study aimed to evaluate the efficacy of L-arginine and L-glutamine supplementation as a potentially novel and useful strategy for the modulation of oxidative stress and toxicity induced by NaNO(2) in male rats. Rats were divided into six groups each of 10 rats and treated for 6 weeks: group 1 as normal control; group 2 fed standard diet containing 0.2% NaNO(2); group 3 and 4 fed the previous diet supplemented with 1% and 2% arginine, respectively; group 5 and 6 fed NaNO(2) diet supplemented with 1% and 2% glutamine, respectively. NaNO(2) treatment induced a significant increase in serum malondialdehyde, nitric oxide, arginase, glutathione-S-transferase activities, urea and creatinine as well as differential leucocytes%. However, a significant decrease was recorded in reduced glutathione, catalase activity, total protein, albumin and some hematological parameters as well as immunoglobulin G. On the other hand, arginine or glutamine showed a remarkable modulation of these abnormalities as indicated by reduction of malondialdehyde and improvement of the investigated antioxidant and hematological parameters. It can be concluded that arginine or glutamine supplementation may reduce oxidative stress and improve the hazard effects of NaNO(2).

  19. The arginine methyltransferase Rmt2 is enriched in the nucleus and co-purifies with the nuclear porins Nup49, Nup57 and Nup100

    International Nuclear Information System (INIS)

    Arginine methylation is a post-translational modification of proteins implicated in RNA processing, protein compartmentalization, signal transduction, transcriptional regulation and DNA repair. In a screen for proteins associated with the nuclear envelope in the yeast Saccharomyces cerevisiae, we have identified the arginine methyltransferase Rmt2, previously shown to methylate the ribosomal protein L12. By indirect immunofluorescence and subcellular fractionations we demonstrate here that Rmt2 has nuclear and cytoplasmic localizations. Biochemical analysis of a fraction enriched in nuclei reveals that nuclear Rmt2 is resistant to extractions with salt and detergent, indicating an association with structural components. This was supported by affinity purification experiments with TAP-tagged Rmt2. Rmt2 was found to co-purify with the nucleoporins Nup49, Nup57 and Nup100, revealing a novel link between arginine methyltransferases and the nuclear pore complex. In addition, a genome-wide transcription study of the rmt2Δ mutant shows significant downregulation of the transcription of MYO1, encoding the Type II myosin heavy chain required for cytokinesis and cell separation

  20. Oral L-Arginine Stimulates GLP-1 Secretion to Improve Glucose Tolerance in Male Mice

    DEFF Research Database (Denmark)

    Clemmensen, Christoffer; Smajilovic, Sanela; Smith, Eric P;

    2013-01-01

    -induced obesity, may provide an alternative therapeutic approach. Importantly, recent evidence suggests the amino acid l-arginine, a well-known insulin secretagogue, can also stimulate release of GLP-1 from isolated rat intestine. Here we tested the hypothesis that oral l-arginine acts as a GLP-1 secretagogue...... in vivo, to augment postprandial insulin secretion and improve glucose tolerance. To test this, we administered l-arginine or vehicle by oral gavage, immediately prior to an oral glucose tolerance test in lean and diet-induced obese mice. In both lean and obese mice oral l-arginine increased plasma GLP-1...... and insulin and substantially improved glucose clearance. To directly assess the contribution of GLP-1 receptor (GLP-1R)-signaling to these improvements, l-arginine was given to Glp1r knockout mice and their wild-type littermates. In this experiment oral l-arginine significantly augmented insulin secretion...

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

    Directory of Open Access Journals (Sweden)

    Marcus eFulde

    2014-08-01

    Full Text Available 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 rate in chemically defined media supplemented with arginine when compared to the WT strain, indicating 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.

  2. Remission of diabetes mellitus in cats cannot be predicted by the arginine stimulation test

    OpenAIRE

    Tschuor, F

    2011-01-01

    Background: Responsiveness of β-cells to arginine persists the longest during diabetes progression, making the intravenous arginine stimulation test (IVAST) a useful tool to assess residual insulin and glucagon secretion. Hypothesis: Diabetic cats with and without remission will have different arginine-induced insulin or glucagon response. Animals: 17 cats with diabetes, 7 healthy cats. Methods: Response to IVAST was assessed by calculating insulin and glucagon area under the c...

  3. Arginine synthesis from enteral glutamine in healthy adults in the fed state.

    Science.gov (United States)

    Tomlinson, Chris; Rafii, Mahroukh; Ball, Ronald O; Pencharz, Paul

    2011-08-01

    Recent studies have documented transfer of labeled nitrogen from [2-(15)N]glutamine to citrulline and arginine in fasting human adults. Conversely, in neonates and piglets we have shown no synthesis of arginine from [2-(15)N]glutamate, and others have shown in mice that glutamine is a nitrogen, but not a carbon donor, for arginine synthesis. Therefore, we performed a multitracer study to determine whether glutamine is a nitrogen and/or carbon donor for arginine in healthy adult men. Two glutamine tracers, 2-(15)N and 1-(13)C, were given enterally to five healthy men fed a standardized milkshake diet. There was no difference in plasma enrichments between the two glutamine tracers. 1-(13)C isotopomers of citrulline and arginine were synthesized from [1-(13)C]glutamine. Three isotopomers each of citrulline and arginine were synthesized from the [2-(15)N]glutamine tracer: 2-(15)N, 5-(15)N, and 2,5-(15)N(2). Significantly greater enrichment was found of both [5-(15)N]arginine (0.75%) and citrulline (3.98%) compared with [2-(15)N]arginine (0.44%) and [2-(15)N]citrulline (2.62%), indicating the amino NH(2) from glutamine is mostly transferred to arginine and citrulline by transamination. Similarly, the enrichment of the 1-(13)C isotopomers was significantly less than the 2-(15)N isotopomers, suggesting rapid formation of α-ketoglutarate and recycling of the nitrogen label. Our results show that the carbon for 50% of newly synthesized arginine comes from dietary glutamine but that glutamine acts primarily as a nitrogen donor for arginine synthesis. Hence, studies using [2-(15)N]glutamine will overestimate arginine synthesis rates.

  4. Hyponatraemia in the first week of life in preterm infants. Part I. Arginine vasopressin secretion.

    OpenAIRE

    Rees, L; Brook, C G; Shaw, J C; Forsling, M L

    1984-01-01

    Continuous sequential urinary arginine vasopressin measurements in 14 preterm, ventilated infants suggest that both osmoreceptor and volume receptor systems are able to stimulate the prolonged secretion of arginine vasopressin from 26 weeks' gestation. The kidney is able to respond to arginine vasopressin stimulation from the first day of life and from 26 weeks' gestation. A maximum urine osmolality not exceeding 550 mOsm/kg was reached which varied with hydration of the infant. Excretion of ...

  5. Vasodilator effects of L-arginine are stereospecific and augmented by insulin in humans.

    Science.gov (United States)

    Dallinger, Susanne; Sieder, Anna; Strametz, Jeanette; Bayerle-Eder, Michaela; Wolzt, Michael; Schmetterer, Leopold

    2003-06-01

    The amino acid l-arginine, the precursor of nitric oxide (NO) synthesis, induces vasodilation in vivo, but the mechanism behind this effect is unclear. There is, however, some evidence to assume that the l-arginine membrane transport capacity is dependent on insulin plasma levels. We hypothesized that vasodilator effects of l-arginine may be dependent on insulin plasma levels. Accordingly, we performed two randomized, double-blind crossover studies in healthy male subjects. In protocol 1 (n = 15), subjects received an infusion of insulin (6 mU x kg(-1) x min(-1) for 120 min) or placebo and, during the last 30 min, l-arginine or d-arginine (1 g/min for 30 min) x In protocol 2 (n = 8), subjects received l-arginine in stepwise increasing doses in the presence (1.5 mU x kg(-1) x min(-1)) or absence of insulin. Renal plasma flow and glomerular filtration rate were assessed by the para-aminohippurate and inulin plasma clearance methods, respectively. Pulsatile choroidal blood flow was assessed with laser interferometric measurement of fundus pulsation, and mean flow velocity in the ophthalmic artery was measured with Doppler sonography. l-arginine, but not d-arginine, significantly increased renal and ocular hemodynamic parameters. Coinfusion of l-arginine with insulin caused a dose-dependent leftward shift of the vasodilator effect of l-arginine. This stereospecific renal and ocular vasodilator potency of l-arginine is enhanced by insulin, which may result from facilitated l-arginine membrane transport, enhanced intracellular NO formation, or increased NO bioavailability.

  6. Protective Effect of Arginine on Oxidative Stress in Transgenic Sickle Mouse Models

    OpenAIRE

    Dasgupta, Trisha; Hebbel, Robert P.; Kaul, Dhananjay K.

    2006-01-01

    Sickle cell disease (SCD) is characterized by reperfusion injury and chronic oxidative stress. Oxidative stress and hemolysis in SCD result in inactivation of nitric oxide (NO) and depleted arginine levels. We hypothesized that augmenting NO production by arginine supplementation will reduce oxidative stress in SCD. To this end, we measured the effect of arginine (5% in mouse chow) on NO metabolites (NOx), lipid peroxidation (LPO) and selected antioxidants in transgenic sickle mouse models. U...

  7. The functional relationship between the Cdc50p-Drs2p putative aminophospholipid translocase and the Arf GAP Gcs1p in vesicle formation in the retrieval pathway from yeast early endosomes to the TGN.

    OpenAIRE

    Sakane, Hiroshi; Yamamoto, Takaharu; Tanaka, Kazuma

    2006-01-01

    Drs2p, the catalytic subunit of the Cdc50p-Drs2p putative aminophospholipid translocase, has been implicated in conjunction with the Arf1 signaling pathway in the formation of clathrin-coated vesicles (CCVs) from the TGN. Herein, we searched for Arf regulator genes whose mutations were synthetically lethal with cdc50Δ, and identified the Arf GAP gene GCS1. Most of the examined transport pathways in the Cdc50p-depleted gcs1Δ mutant were nearly normal, including endocytic transport to vacuoles,...

  8. Purification and characterization of an arginine ester hydrolase from the venom of Trimeresurus mucrosqumatus in Hunan province of China

    Institute of Scientific and Technical Information of China (English)

    YU Xiao-dong; LI Bo; YU Zheng-ping

    2005-01-01

    Objective: To study the physical and chemical properties of an arginine ester hydrolase from the venom of Trimeresurus mucrosqumatus in Hunan province of China. Methods :The arginine ester hydrolase (AEH) was isolated from the venom of Chinese Trimeresurus mucrosqumatus by a combination of ionexchange chromatography on DEAE-Sephadex A-50, CM-Sepharose Cl-6B and gel filtration on Sephadex G-100. Results: The purified protein named TM-AEH,a glycoprotein with carbohydrate content of 0.5 % neutral hexose and 0. 75 % sialic acid,a relative molecular mass of 29.0 kDa,and an isoelectric point (pI) of 5. 2. It shares with an extinction coefficient (E0.1%/cm) of 1.332 at 280 nm,consisted of 225 amino acid residues ,and migrated as a band under reduced or non-reduced condition in basic PAGE. TM-AEH was a highly thermostable protein and was stable to pH changes between 5 and 9. The optimum temperature and optimum pH were 55℃ and 8. 4 for its catalytic activity respectively,which was inhibited by Fe3+ and Cu2+. Conclusion:This protein can exhibit higher BAEE-hydrolysing activity and fibrinogenolytic activity as compared to that of whole venom.

  9. Bioinformatic evaluation of L-arginine catabolic pathways in 24 cyanobacteria and transcriptional analysis of genes encoding enzymes of L-arginine catabolism in the cyanobacterium Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Pistorius Elfriede K

    2007-11-01

    Full Text Available Abstract Background So far very limited knowledge exists on L-arginine catabolism in cyanobacteria, although six major L-arginine-degrading pathways have been described for prokaryotes. Thus, we have performed a bioinformatic analysis of possible L-arginine-degrading pathways in cyanobacteria. Further, we chose Synechocystis sp. PCC 6803 for a more detailed bioinformatic analysis and for validation of the bioinformatic predictions on L-arginine catabolism with a transcript analysis. Results We have evaluated 24 cyanobacterial genomes of freshwater or marine strains for the presence of putative L-arginine-degrading enzymes. We identified an L-arginine decarboxylase pathway in all 24 strains. In addition, cyanobacteria have one or two further pathways representing either an arginase pathway or L-arginine deiminase pathway or an L-arginine oxidase/dehydrogenase pathway. An L-arginine amidinotransferase pathway as a major L-arginine-degrading pathway is not likely but can not be entirely excluded. A rather unusual finding was that the cyanobacterial L-arginine deiminases are substantially larger than the enzymes in non-photosynthetic bacteria and that they are membrane-bound. A more detailed bioinformatic analysis of Synechocystis sp. PCC 6803 revealed that three different L-arginine-degrading pathways may in principle be functional in this cyanobacterium. These are (i an L-arginine decarboxylase pathway, (ii an L-arginine deiminase pathway, and (iii an L-arginine oxidase/dehydrogenase pathway. A transcript analysis of cells grown either with nitrate or L-arginine as sole N-source and with an illumination of 50 μmol photons m-2 s-1 showed that the transcripts for the first enzyme(s of all three pathways were present, but that the transcript levels for the L-arginine deiminase and the L-arginine oxidase/dehydrogenase were substantially higher than that of the three isoenzymes of L-arginine decarboxylase. Conclusion The evaluation of 24

  10. Different roles of cell surface and exogenous glycosaminoglycans in controlling gene delivery by arginine-rich peptides with varied distribution of arginines.

    Science.gov (United States)

    Naik, Rangeetha J; Chatterjee, Anindo; Ganguli, Munia

    2013-06-01

    The role of cell surface and exogenous glycosaminoglycans (GAGs) in DNA delivery by cationic peptides is controlled to a large extent by the peptide chemistry and the nature of its complex with DNA. We have previously shown that complexes formed by arginine homopeptides with DNA adopt a GAG-independent cellular internalization mechanism and show enhanced gene delivery in presence of exogenous GAGs. In contrast, lysine complexes gain cellular entry primarily by a GAG-dependent pathway and are destabilized by exogenous GAGs. The aim of the current study was to elucidate the factors governing the role of cell surface and soluble glycosaminoglycans in DNA delivery by sequences of arginine-rich peptides with altered arginine distributions (compared to homopeptide). Using peptides with clustered arginines which constitute known heparin-binding motifs and a control peptide with arginines alternating with alanines, we show that complexes formed by these peptides do not require cell surface GAGs for cellular uptake and DNA delivery. However, the charge distribution and the spacing of arginine residues affects DNA delivery efficiency of these peptides in presence of soluble GAGs, since these peptides show only a marginal increase in transfection in presence of exogenous GAGs unlike that observed with arginine homopeptides. Our results indicate that presence of arginine by itself drives these peptides to a cell surface GAG-independent route of entry to efficiently deliver functional DNA into cells in vitro. However, the inherent stability of the complexes differ when the distribution of arginines in the peptides is altered, thereby modulating its interaction with exogenous GAGs.

  11. Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1

    International Nuclear Information System (INIS)

    Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering. Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM128–507 and two structural states of CARM1140–480 were expressed, purified and crystallized. Crystals of CARM128–507 belong to space group P6222, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM128–507 was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1140–480 belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1140–480 in complex with S-adenosyl-l-homocysteine belong to space P21212, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1140–480 were solved by molecular-replacement techniques from the structure of CARM128–507

  12. Risk assessment for the amino acids taurine, L-glutamine and L-arginine.

    Science.gov (United States)

    Shao, Andrew; Hathcock, John N

    2008-04-01

    Taurine, glutamine and arginine are examples of amino acids which have become increasingly popular as ingredients in dietary supplements and functional foods and beverages. Animal and human clinical research suggests that oral supplementation of these amino acids provides additional health and/or performance benefits beyond those observed from normal intake of dietary protein. The increased consumer awareness and use of these amino acids as ingredients in dietary supplements and functional foods warrant a comprehensive review of their safety through quantitative risk assessment, and identification of a potential safe upper level of intake. The absence of a systematic pattern of adverse effects in humans in response to orally administered taurine (Tau), l-glutamine (Gln) and l-arginine (Arg) precluded the selection of a no observed adverse effect level (NOAEL) or lowest observed adverse effect level (LOAEL). Therefore, by definition, the usual approach to risk assessment for identification of a tolerable upper level of intake (UL) could not be used. Instead, the newer method described as the Observed Safe Level (OSL) or Highest Observed Intake (HOI) was utilized. The OSL risk assessments indicate that based on the available published human clinical trial data, the evidence for the absence of adverse effects is strong for Tau at supplemental intakes up to 3 g/d, Gln at intakes up to 14 g/d and Arg at intakes up to 20 g/d, and these levels are identified as the respective OSLs for normal healthy adults. Although much higher levels of each of these amino acids have been tested without adverse effects and may be safe, the data for intakes above these levels are not sufficient for a confident conclusion of long-term safety, and therefore these values are not selected as the OSLs.

  13. Type 1 diabetes: can exercise impair the autoimmune event? The L-arginine/glutamine coupling hypothesis.

    Science.gov (United States)

    Krause, Maurício da Silva; de Bittencourt, Paulo Ivo Homem

    2008-06-01

    Prevention of type 1 diabetes mellitus (T1DM) requires early intervention in the autoimmune process directed against beta-cells of the pancreatic islets of Langerhans, which is believed to result from a disorder of immunoregulation. According to this concept, a T-helper lymphocyte of type 1 (Th1) subset of T-lymphocytes and their cytokine products, the type 1 cytokines [e.g. interleukin 2 (IL-2), interferon gamma (IFN-gamma) and tumour necrosis factor beta (TNF-beta)] prevail over immunoregulatory (anti-inflammatory) Th2 subset and its cytokine products, i.e. type 2 cytokines (e.g. IL-4, IL-6 and IL-10). This allows type 1 cytokines to initiate a cascade of immune/inflammatory processes in the islet (insulitis), culminating in beta-cell destruction. Activation of sympathetic-corticotropin-releasing hormone (CRH) axis by psychological stress induces specifically Th1 cell overactivity that determines enhanced glutamine utilization and consequent poor L-arginine supply for nitric oxide (NO)-assisted insulin secretion. This determines the shift of intraislet glutamate metabolism from the synthesis of glutathione (GSH) to that of L-arginine, leading to a redox imbalance that activates nuclear factor kappaB exacerbating inflammation and NO-mediated cytotoxicity. Physical exercise is capable of inducing changes in the pattern of cytokine production and release towards type 2 class and to normalize the glutamine supply to the circulation, which reduces the need for glutamate, whose metabolic fate may be restored in the direction of GSH synthesis and antioxidant defence. Also, the 70-kDa heat shock protein (hsp70), which is immunoregulatory, may modulate exercise-induced anti-inflammation. In this work, we envisage how exercise can intervene in the mechanisms involved in the autoimmune process against beta-cells and how novel therapeutic approaches may be inferred from these observations.

  14. Type 1 diabetes: can exercise impair the autoimmune event? The L-arginine/glutamine coupling hypothesis.

    Science.gov (United States)

    Krause, Maurício da Silva; de Bittencourt, Paulo Ivo Homem

    2008-06-01

    Prevention of type 1 diabetes mellitus (T1DM) requires early intervention in the autoimmune process directed against beta-cells of the pancreatic islets of Langerhans, which is believed to result from a disorder of immunoregulation. According to this concept, a T-helper lymphocyte of type 1 (Th1) subset of T-lymphocytes and their cytokine products, the type 1 cytokines [e.g. interleukin 2 (IL-2), interferon gamma (IFN-gamma) and tumour necrosis factor beta (TNF-beta)] prevail over immunoregulatory (anti-inflammatory) Th2 subset and its cytokine products, i.e. type 2 cytokines (e.g. IL-4, IL-6 and IL-10). This allows type 1 cytokines to initiate a cascade of immune/inflammatory processes in the islet (insulitis), culminating in beta-cell destruction. Activation of sympathetic-corticotropin-releasing hormone (CRH) axis by psychological stress induces specifically Th1 cell overactivity that determines enhanced glutamine utilization and consequent poor L-arginine supply for nitric oxide (NO)-assisted insulin secretion. This determines the shift of intraislet glutamate metabolism from the synthesis of glutathione (GSH) to that of L-arginine, leading to a redox imbalance that activates nuclear factor kappaB exacerbating inflammation and NO-mediated cytotoxicity. Physical exercise is capable of inducing changes in the pattern of cytokine production and release towards type 2 class and to normalize the glutamine supply to the circulation, which reduces the need for glutamate, whose metabolic fate may be restored in the direction of GSH synthesis and antioxidant defence. Also, the 70-kDa heat shock protein (hsp70), which is immunoregulatory, may modulate exercise-induced anti-inflammation. In this work, we envisage how exercise can intervene in the mechanisms involved in the autoimmune process against beta-cells and how novel therapeutic approaches may be inferred from these observations. PMID:18383559

  15. Arginine vasopressin stimulates proliferation of adult rat cardiac fibroblasts via protein kinase C-extracellular signal-regulated kinase 1/2 pathway%蛋白激酶C-细胞外信号调节激酶1/2通路介导精氨酸升压素对成年大鼠心肌成纤维细胞的促增殖作用

    Institute of Scientific and Technical Information of China (English)

    何燕萍; 赵连友; 郑强荪; 刘少伟; 赵晓燕; 陆晓龙; 牛晓琳

    2008-01-01

    精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用.我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖.本研究旨在进一步观察AVP是否对成年大鼠CFs具有促增殖作用,并探计其机制.采用组织块法培养成年大鼠CFs,用[3H]-TdR掺入法和流式细胞仪方法观察AVP作用下CFs的DNA合成和细胞周期分布.根据特异性底物髓磷脂基质蛋白(myelin basic protein, MBP)的磷酸化水平测定细胞外信号调节激酶1/2 (extracellular signal-regulated kinase 1/2, ERK1/2)的活性.用Western blot检测ERK1/2的磷酸化和p27Kip1、细胞周期蛋白D1、 A、 E的表达.结果显示,AVP(0.1μmol/L)可促进成年大鼠CFs的DNA合成,该作用可被V1受体拮抗剂d(CH2)5[Tyr2(Me),Arg8]-vasopressin (0.1μmol/L)阻断,而不受V2受体拮抗剂desglycinamide [d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin (0.1μmol/L)的影响.AVP可激活ERK1/2,用蛋白激酶C(protein kinase C, PKC)激动剂佛波酯(phorbol 12-myristate 13-acetate, PMA, 30nmol/L, 5min)急性刺激可模拟该作用,而PMA持续慢性作用(2.5μmol/L,24h)耗竭PKC后则抑制AVP对ERK1/2的激活.AVP可抑制p27Kip1的蛋白表达,升高细胞周期蛋白D1、 A和E的表达,同时促进细胞周期由G0/G1期进入S期.ERK1/2抑制剂PD98059 (30μmol/L)阻断AVP对DNA合成、p27Kip1、细胞周期蛋白D1、A和E蛋白表达的作用,并抑制细胞周期进程.以上结果表明,AVP可促进成年大鼠CFs增殖,该作用由V1受体和PKC-ERK1/2通路介导.AVP可通过ERK1/2调控p27Kip1、细胞周期蛋白D1、A和E的表达,从而促进成年大鼠CFs的细胞周期进程.%Arginine vasopressin (AVP), a neurohormone and hemodynamic factor implicated in the pathophysiology of hypertension and congestive heart failure, can also act as a growth-stimulating factor. Our previous work

  16. Dietary L-arginine supplementation reduces Methotrexate-induced intestinal mucosal injury in rat

    Directory of Open Access Journals (Sweden)

    Koppelmann Tal

    2012-04-01

    Full Text Available Abstract Background Arginine (ARG and nitric oxide maintain the mucosal integrity of the intestine in various intestinal disorders. In the present study, we evaluated the effects of oral ARG supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX-induced intestinal damage in a rat. Methods Male rats were divided into four experimental groups: Control rats, CONTR-ARG rats, were treated with oral ARG given in drinking water 72 hours before and 72 hours following vehicle injection, MTX rats were treated with a single dose of methotrexate, and MTX-ARG rats were treated with oral ARG following injection of MTX. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 hours following MTX injection. RT-PCR was used to determine bax and bcl-2 mRNA expression. Results MTX-ARG rats demonstrated greater jejunal and ileal bowel weight, greater ileal mucosal weight, greater ileal mucosal DNA and protein levels, greater villus height in jejunum and ileum and crypt depth in ileum, compared to MTX animals. A significant decrease in enterocyte apoptosis in the ileum of MTX-ARG rats (vs MTX was accompanied by decreased bax mRNA and protein expression and increased bcl-2 protein levels. Conclusions Treatment with oral ARG prevents mucosal injury and improves intestinal recovery following MTX- injury in the rat.

  17. Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol–xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum

    Science.gov (United States)

    Scheele, Sandra; Oertel, Dan; Bongaerts, Johannes; Evers, Stefan; Hellmuth, Hendrik; Maurer, Karl-Heinz; Bott, Michael; Freudl, Roland

    2013-01-01

    Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor-containing sorbitol–xylitol oxidase from Streptomyces coelicolor was achieved by using the twin-arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor-containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies. PMID:23163932

  18. Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol-xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum.

    Science.gov (United States)

    Scheele, Sandra; Oertel, Dan; Bongaerts, Johannes; Evers, Stefan; Hellmuth, Hendrik; Maurer, Karl-Heinz; Bott, Michael; Freudl, Roland

    2013-03-01

    Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor-containing sorbitol-xylitol oxidase from Streptomyces coelicolor was achieved by using the twin-arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor-containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies.

  19. The subcellular compartmentalization of arginine metabolizing enzymes and their role in endothelial dysfunction

    Directory of Open Access Journals (Sweden)

    Feng eChen

    2013-07-01

    Full Text Available The endothelial production of nitric oxide (NO mediates endothelium-dependent vasorelaxation and restrains vascular inflammation, smooth muscle proliferation and platelet aggregation. Impaired production of NO is a hallmark of endothelial dysfunction and promotes the development of cardiovascular disease. In endothelial cells, NO is generated by endothelial nitric oxide synthase (eNOS through the conversion of its substrate, L-arginine to L-citrulline. Reduced access to L-arginine has been proposed as a major mechanism underlying reduced eNOS activity and NO production in cardiovascular disease. The arginases (Arg1 and Arg2 metabolize L-arginine to generate L-ornithine and urea and increased expression of arginase has been proposed as a mechanism of reduced eNOS activity secondary to the depletion of L-arginine. Indeed, supplemental L-arginine and suppression of arginase activity has been shown to improve endothelium-dependent relaxation and ameliorate cardiovascular disease. However, L-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis suggesting additional mechanisms. The compartmentalization of intracellular L-arginine into poorly interchangeable pools has been proposed to allow for the local depletion of L-arginine. Indeed the subcellular location of L-arginine metabolizing enzymes plays important functional roles. In endothelial cells, eNOS is found in discrete intracellular locations and the capacity to generate NO is heavily influenced by its localtion. Arg1 and Arg2 also reside in different subcellular environments and are thought to differentially influence endothelial function. The plasma membrane solute transporter, CAT-1 and the arginine recycling enzyme, ASL, co-localize with eNOS and facilitate NO release. This review highlights the importance of the subcellular location of eNOS and arginine transporting and metabolizing enzymes to NO release and cardiovascular disease.

  20. Oral L-arginine supplementation impacts several reproductive parameters during the postpartum period in mares.

    Science.gov (United States)

    Kelley, Dale E; Warren, Lori K; Mortensen, Christopher J

    2013-05-01

    L-arginine is an amino acid which can alter pituitary function and increase blood flow to the reproductive tract. The objective was to determine the effect of supplementing 100g of L-arginine on plasma arginine concentrations, follicular dynamics and ovarian and uterine artery blood flow during the estrus that occurs subsequent to foaling. In Experiment 1, mares were fed 100g L-arginine for 1 day during the last 3 weeks of pregnancy and plasma samples taken for every hour for the first 4h and every other hour until 12h.L-arginine supplementation elevated plasma arginine concentrations from 1 to 8h post feeding; arginine peaked at 6h (arginine: 515±33μmol/L; control: 80±33μmol/L). In Experiment 2, mares received either 100g L-arginine or control diets beginning 21 d before the expected foaling date and continued for 30 d postpartum. The reproductive tract was evaluated by transrectal Doppler ultrasonography from Day 1 postpartum through Day 30. There were no differences in ovarian follicular dynamics, ovarian or uterine resistance indices between groups. Vascular perfusion of the F1 follicular wall was greater in L-arginine supplemented mares (37.3±2.6%) than controls (25.4±2.7%; Pmares had a smaller uterine body and horns and accumulated less uterine fluid than controls (Pfollicular development, raises the possible use of L-arginine supplementation as a breeding management tool during the postpartum period to increase reproductive success. PMID:23523236

  1. Adverse effects associated with arginine alpha-ketoglutarate containing supplements.

    Science.gov (United States)

    Prosser, J M; Majlesi, N; Chan, G M; Olsen, D; Hoffman, R S; Nelson, L S

    2009-05-01

    The athletic performance supplement industry is a multibillion-dollar business and one popular category claims to increase nitric oxide (NO) production. We report three patients presenting to the emergency department with adverse effects. A 33-year-old man presented with palpitations, dizziness, vomiting, and syncope, after the use of NO(2) platinum. His examination and electrocardiogram (ECG) were normal. The dizziness persisted, requiring admission overnight. A 21-year-old man with palpitations and near syncope had used a "nitric oxide" supplement. He was tachycardic to 115 bpm with otherwise normal examination. Laboratory values including methemoglobin, and ECG were unremarkable. He was treated with 1 L of saline with no change in heart rate. He was admitted for observation. A 24-year-old man presented after taking NO-Xplode with palpitations and a headache. His examination, laboratory values, and ECG were normal. He was discharged. The purported active ingredient in these products is arginine alpha-ketoglutarate (AAKG), which is claimed to increase NO production by supplying the precursor L-arginine. The symptoms could be due to vasodilation from increased levels of NO, though other etiologies cannot be excluded. AAKG containing supplements may be associated with adverse effects requiring hospital admission. PMID:19755457

  2. Supplementation with l-arginine stabilizes plasma arginine and nitric oxide metabolites, suppresses elevated liver enzymes and peroxidation in sickle cell anaemia.

    Science.gov (United States)

    Jaja, S I; Ogungbemi, S O; Kehinde, M O; Anigbogu, C N

    2016-06-01

    The effect of l-arginine on liver function in SCD has received little or no attention. The effect of a chronic, oral, low-dose supplementation with l-arginine (1gm/day for 6 weeks) on some liver enzymes, lipid peroxidation and nitric oxide metabolites was studied in 20 normal (non-sickle cell anaemia; NSCA) subjects and 20 sickle cell anaemia (SCA) subjects. Ten milliliters of blood was withdrawn from an ante-cubital vein for the estimation of plasma arginine concentration ([R]), alanine aminotransaminase (ALT), aspartate aminotransaminase (AST) and alkaline phosphatase (ALP), plasma total bilirubin concentration [TB], malondialdehyde concentration [MDA] and nitric oxide metabolites concentration [NOx]. Before supplementation, ALT, AST, ALP (pconcentration and nitric oxide metabolites levels in NSCA and SCA subjects. Responses in SCA subjects to l-arginine were more sensitive than in NSCA subjects.

  3. Short- and Long-Term Soy Diet Versus Casein Protects Liver Steatosis Independent of the Arginine Content.

    Science.gov (United States)

    Hakkak, Reza; Zeng, Huawei; Dhakal, Ishwori B; Korourian, Soheila

    2015-11-01

    Nonalcoholic fatty liver disease, a major cause of abnormal liver function, is often associated with obesity. Arginine (ARG) plays a role in modulating body weight/fat, but limited data exist as to the role of ARG in soy protein's ability to protect from liver steatosis. We investigated the role of native ARG in the soy protein isolate (SPI) in reducing liver steatosis in male obese Zucker rats. Rats (N=48; 6 weeks old) were randomly assigned to one of three diets for 8 or 16 weeks: the casein (CAS) diet as control (0.6% ARG), CAS diet supplemented to contain 1.3% ARG, or an SPI diet containing isoflavones (1.3% ARG). SPI and ARG rats gained significantly more weight (Pdiet had no effect on steatosis or ALT and AST levels. We found that the SPI diet reduced (Pdiets after 8 and 16 weeks. The SPI diet significantly reduced (Pdiet at 8 weeks, but there was no significant difference at 16 weeks. Based on the findings of our study, the protective effect of SPI in reducing liver steatosis is not modulated by its native arginine content.

  4. Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine.

    Science.gov (United States)

    Dutta, Asim K; Ramnath; Dkhar, Barilin; Tandon, Veena; Das, Bidyadhar

    2016-09-01

    Phosphoenolpyruvate carboxykinase is an essential regulatory enzyme of glycolysis in the cestode parasite, Raillietina echinobothrida, and is considered a potential target for anthelmintic action because of its differential activity from that of its avian host. However, due to the unavailability of its structure, the mechanism of regulation of PEPCK from R. echinobothrida (rePEPCK) and its interaction with possible modulators remain unclear. Hence, in this study, the rePEPCK gene was cloned into pGEX-4T-3 and overexpressed for its characterization. On being induced by IPTG, the recombinant rePEPCK was expressed as inclusion bodies (IBs); hence, various agents, like different inducer concentrations, temperature, time, host cell types, culture media, pH, and additives, were used to bring the protein to soluble form. Finally, a significant amount (∼46%) of rePEPCK was solubilized from IBs by adding 2M l-arginine. Near-UV circular dichroism spectra analysis indicated that l-arginine (2M) had no effect on the conformation of the protein. In this study, we have reported a yield of ∼73mg of purified rePEPCK per 1L of culture. The purified rePEPCK retained its biological activity, and Km of the enzyme for its substrate was determined and discussed. The availability of recombinant rePEPCK may help in biochemical- and biophysical-studies to explore its molecular mechanisms and regulations.

  5. Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine.

    Science.gov (United States)

    Dutta, Asim K; Ramnath; Dkhar, Barilin; Tandon, Veena; Das, Bidyadhar

    2016-09-01

    Phosphoenolpyruvate carboxykinase is an essential regulatory enzyme of glycolysis in the cestode parasite, Raillietina echinobothrida, and is considered a potential target for anthelmintic action because of its differential activity from that of its avian host. However, due to the unavailability of its structure, the mechanism of regulation of PEPCK from R. echinobothrida (rePEPCK) and its interaction with possible modulators remain unclear. Hence, in this study, the rePEPCK gene was cloned into pGEX-4T-3 and overexpressed for its characterization. On being induced by IPTG, the recombinant rePEPCK was expressed as inclusion bodies (IBs); hence, various agents, like different inducer concentrations, temperature, time, host cell types, culture media, pH, and additives, were used to bring the protein to soluble form. Finally, a significant amount (∼46%) of rePEPCK was solubilized from IBs by adding 2M l-arginine. Near-UV circular dichroism spectra analysis indicated that l-arginine (2M) had no effect on the conformation of the protein. In this study, we have reported a yield of ∼73mg of purified rePEPCK per 1L of culture. The purified rePEPCK retained its biological activity, and Km of the enzyme for its substrate was determined and discussed. The availability of recombinant rePEPCK may help in biochemical- and biophysical-studies to explore its molecular mechanisms and regulations. PMID:26363119

  6. Serum amino acids profile and the beneficial effects of L-arginine or L-glutamine supplementation in dextran sulfate sodium colitis.

    Directory of Open Access Journals (Sweden)

    Wenkai Ren

    Full Text Available This study was conducted to investigate serum amino acids profile in dextran sulfate sodium (DSS-induced colitis, and impacts of graded dose of arginine or glutamine supplementation on the colitis. Using DSS-induced colitis model, which is similar to human ulcerative colitis, we determined serum profile of amino acids at day 3, 7, 10 and 12 (5 days post DSS treatment. Meanwhile, effects of graded dose of arginine (0.4%, 0.8%, and 1.5% or glutamine (0.5%, 1.0% and 2.0% supplementation on clinical parameters, serum amino acids, colonic tight junction proteins, colonic anti-oxidative indicators [catalase, total superoxide dismutase (T-SOD, glutathione peroxidase (GSH-Px], colonic pro-inflammatory cytokines [interleukin-1 beta (IL-1β, IL-6, IL-17 and tumor necrosis factor alpha (TNF-α] in DSS-induced colitis were fully analyzed at day 7 and 12. Additionally, the activation of signal transduction pathways, including nuclear factor kappa B (NF-κB, mitogen-activated protein kinases (MAPK, phosphoinositide-3-kinases (PI3K/PI3K-protein kinase B (Akt, and myosin light chain kinase (MLCK-myosin light chain (MLC20, were analyzed using immunoblotting. Serum amino acids analysis showed that DSS treatment changed the serum contents of amino acids, such as Trp, Glu, and Gln (P<0.05. Dietary arginine or glutamine supplementation had significant (P<0.05 influence on the clinical and biochemical parameters (T-SOD, IL-17 and TNF-α in colitis model. These results were associated with colonic NF-κB, PI3K-Akt and MLCK signaling pathways. In conclusion, arginine or glutamine could be a potential therapy for intestinal inflammatory diseases.

  7. Giardia duodenalis arginine deiminase modulates the phenotype and cytokine secretion of human dendritic cells by depletion of arginine and formation of ammonia.

    Science.gov (United States)

    Banik, Stefanie; Renner Viveros, Pablo; Seeber, Frank; Klotz, Christian; Ignatius, Ralf; Aebischer, Toni

    2013-07-01

    Depletion of arginine is a recognized strategy that pathogens use to evade immune effector mechanisms. Depletion depends on microbial enzymes such as arginases, which are considered virulence factors. The effect is mostly interpreted as being a consequence of successful competition with host enzymes for the substrate. However, both arginases and arginine deiminases (ADI) have been associated with pathogen virulence. Both deplete arginine, but their reaction products differ. An ADI has been implicated in the virulence of Giardia duodenalis, an intestinal parasite that infects humans and animals, causing significant morbidity. Dendritic cells (DC) play a critical role in host defense and also in a murine G. duodenalis infection model. The functional properties of these innate immune cells depend on the milieu in which they are activated. Here, the dependence of the response of these cells on arginine was studied by using Giardia ADI and lipopolysaccharide-stimulated human monocyte-derived DC. Arginine depletion by ADI significantly increased tumor necrosis factor alpha and decreased interleukin-10 (IL-10) and IL-12p40 secretion. It also reduced the upregulation of surface CD83 and CD86 molecules, which are involved in cell-cell interactions. Arginine depletion also reduced the phosphorylation of S6 kinase in DC, suggesting the involvement of the mammalian target of rapamycin signaling pathway. The changes were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Comparison of NH(4)(+) and urea revealed distinct immunomodulatory activities of these products of deiminases and arginases, respectively. The data suggest that a better understanding of the role of arginine-depleting pathogen enzymes for immune evasion will have to take enzyme class and reaction products into consideration.

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

    OpenAIRE

    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 al...

  9. Effect of L-arginine, dimercaptosuccinic acid (DMSA and the association of L-arginine and DMSA on tissue lead mobilization and blood pressure level in plumbism

    Directory of Open Access Journals (Sweden)

    Malvezzi C.K.

    2001-01-01

    Full Text Available Lead (Pb-induced hypertension is characterized by an increase in reactive oxygen species (ROS and a decrease in nitric oxide (NO. In the present study we evaluated the effect of L-arginine (NO precursor, dimercaptosuccinic acid (DMSA, a chelating agent and ROS scavenger, and the association of L-arginine/DMSA on tissue Pb mobilization and blood pressure levels in plumbism. Tissue Pb levels and blood pressure evolution were evaluated in rats exposed to: 1 Pb (750 ppm, in drinking water, for 70 days, 2 Pb plus water for 30 more days, 3 Pb plus DMSA (50 mg kg-1 day-1, po, L-arginine (0.6%, in drinking water, and the combination of L-arginine/DMSA for 30 more days, and 4 their respective matching controls. Pb exposure increased Pb levels in the blood, liver, femur, kidney and aorta. Pb levels in tissues decreased after cessation of Pb administration, except in the aorta. These levels did not reach those observed in nonintoxicated rats. All treatments mobilized Pb from the kidney, femur and liver. Pb mobilization from the aorta was only effective with the L-arginine/DMSA treatment. Blood Pb concentrations in Pb-treated groups were not different from those of the Pb/water group. Pb increased blood pressure starting from the 5th week. L-arginine and DMSA treatments (4th week and the combination of L-arginine/DMSA (3rd and 4th weeks decreased blood pressure levels of intoxicated rats. These levels did not reach those of nonintoxicated rats. Treatment with L-arginine/DMSA was more effective than the isolated treatments in mobilizing Pb from tissues and in reducing the blood pressure of intoxicated rats.

  10. Giardia duodenalis Arginine Deiminase Modulates the Phenotype and Cytokine Secretion of Human Dendritic Cells by Depletion of Arginine and Formation of Ammonia

    Science.gov (United States)

    Banik, Stefanie; Renner Viveros, Pablo; Seeber, Frank; Klotz, Christian; Ignatius, Ralf

    2013-01-01

    Depletion of arginine is a recognized strategy that pathogens use to evade immune effector mechanisms. Depletion depends on microbial enzymes such as arginases, which are considered virulence factors. The effect is mostly interpreted as being a consequence of successful competition with host enzymes for the substrate. However, both arginases and arginine deiminases (ADI) have been associated with pathogen virulence. Both deplete arginine, but their reaction products differ. An ADI has been implicated in the virulence of Giardia duodenalis, an intestinal parasite that infects humans and animals, causing significant morbidity. Dendritic cells (DC) play a critical role in host defense and also in a murine G. duodenalis infection model. The functional properties of these innate immune cells depend on the milieu in which they are activated. Here, the dependence of the response of these cells on arginine was studied by using Giardia ADI and lipopolysaccharide-stimulated human monocyte-derived DC. Arginine depletion by ADI significantly increased tumor necrosis factor alpha and decreased interleukin-10 (IL-10) and IL-12p40 secretion. It also reduced the upregulation of surface CD83 and CD86 molecules, which are involved in cell-cell interactions. Arginine depletion also reduced the phosphorylation of S6 kinase in DC, suggesting the involvement of the mammalian target of rapamycin signaling pathway. The changes were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Comparison of NH4+ and urea revealed distinct immunomodulatory activities of these products of deiminases and arginases, respectively. The data suggest that a better understanding of the role of arginine-depleting pathogen enzymes for immune evasion will have to take enzyme class and reaction products into consideration. PMID:23589577

  11. Enteral L-Arginine and Glutamine Supplementation for Prevention of NEC in Preterm Neonates.

    Science.gov (United States)

    El-Shimi, M S; Awad, H A; Abdelwahed, M A; Mohamed, M H; Khafagy, S M; Saleh, G

    2015-01-01

    Objective. Evaluating the efficacy and safety of arginine and glutamine supplementation in decreasing the incidence of NEC among preterm neonates. Methods. Prospective case-control study done on 75 preterm neonates ≤34 weeks, divided equally into L-arginine group receiving enteral L-arginine, glutamine group receiving enteral glutamine, and control group. Serum L-arginine and glutamine levels were measured at time of enrollment (sample 1), after 14 days of enrollment (sample 2), and at time of diagnosis of NEC (sample 3). Results. The incidence of NEC was 9.3%. There was no difference in the frequency of NEC between L-arginine and control groups (P > 0.05). NEC was not detected in glutamine group; L-arginine concentrations were significantly lower in arginine group than control group in both samples while glutamine concentrations were comparable in glutamine and control groups in both samples. No significant difference was found between groups as regards number of septic episodes, duration to reach full oral intake, or duration of hospital stay. Conclusion. Enteral L-arginine supplementation did not seem to reduce the incidence of NEC. Enteral glutamine may have a preventive role against NEC if supplied early to preterm neonates. However, larger studies are needed to confirm these findings. This work is registered in ClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT01263041).

  12. Enteral L-Arginine and Glutamine Supplementation for Prevention of NEC in Preterm Neonates

    Directory of Open Access Journals (Sweden)

    M. S. El-Shimi

    2015-01-01

    Full Text Available Objective. Evaluating the efficacy and safety of arginine and glutamine supplementation in decreasing the incidence of NEC among preterm neonates. Methods. Prospective case-control study done on 75 preterm neonates ≤34 weeks, divided equally into L-arginine group receiving enteral L-arginine, glutamine group receiving enteral glutamine, and control group. Serum L-arginine and glutamine levels were measured at time of enrollment (sample 1, after 14 days of enrollment (sample 2, and at time of diagnosis of NEC (sample 3. Results. The incidence of NEC was 9.3%. There was no difference in the frequency of NEC between L-arginine and control groups (P>0.05. NEC was not detected in glutamine group; L-arginine concentrations were significantly lower in arginine group than control group in both samples while glutamine concentrations were comparable in glutamine and control groups in both samples. No significant difference was found between groups as regards number of septic episodes, duration to reach full oral intake, or duration of hospital stay. Conclusion. Enteral L-arginine supplementation did not seem to reduce the incidence of NEC. Enteral glutamine may have a preventive role against NEC if supplied early to preterm neonates. However, larger studies are needed to confirm these findings. This work is registered in ClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT01263041.

  13. Synthesis, characterization and behaviour of trans-bis (argininate) copper (II) to gamma radiation

    International Nuclear Information System (INIS)

    The synthesis, the characterization and the behaviour to gamma radiation of trans-bis (argininate) copper (II) are presented. The synthesis is made from copper sulfate, sodium hydroxide and hydrochloride of L (+) arginine, in aqueous medium, and the characterization by infrared spectroscopy, visible and ultraviolet spectroscopy and elementary analysis. (C.G.C.)

  14. Antibacterial action of a novel functionalized chitosan-arginine against Gram-negative bacteria.

    Science.gov (United States)

    Tang, Hong; Zhang, Peng; Kieft, Thomas L; Ryan, Shannon J; Baker, Shenda M; Wiesmann, William P; Rogelj, Snezna

    2010-07-01

    The antimicrobial activity of chitosan and chitosan derivatives has been well established. However, although several mechanisms have been proposed, the exact mode of action is still unclear. Here we report on the investigation of antibacterial activity and the antibacterial mode of action of a novel water-soluble chitosan derivative, arginine-functionalized chitosan, on the Gram-negative bacteria Pseudomonas fluorescens and Escherichia coli. Two different arginine-functionalized chitosans (6% arginine-substituted and 30% arginine-substituted) each strongly inhibited P. fluorescens and E. coli growth. Time-dependent killing efficacy experiments showed that 5000 mg l(-1) of 6%- and 30%-substituted chitosan-arginine killed 2.7 logs and 4.5 logs of P. fluorescens, and 4.8 logs and 4.6 logs of E. coli in 4h, respectively. At low concentrations, the 6%-substituted chitosan-arginine was more effective in inhibiting cell growth even though the 30%-substituted chitosan-arginine appeared to be more effective in permeabilizing the cell membranes of both P. fluorescens and E. coli. Studies using fluorescent probes, 1-N-phenyl-naphthylamine (NPN), nile red (NR) and propidium iodide (PI), and field emission scanning electron microscopy (FESEM) suggest that chitosan-arginine's antibacterial activity is, at least in part, due to its interaction with the cell membrane, in which it increases membrane permeability. PMID:20060936

  15. Facilitation of peptide fibre formation by arginine-phosphate/carboxylate interactions

    Indian Academy of Sciences (India)

    K Krishna Prasad; Sandeep Verma

    2008-01-01

    This study describes peptide fibre formation in a hexapeptide, derived from the V3 loop of HIV-1, mediated by the interactions between arginine residues and phosphate/carboxylate anions. This charge neutralization approach was further confirmed when the deletion of arginine residue from the hexapeptide sequence resulted in fibre formation, which was studied by a combination of microscopic techniques.

  16. Enzymatic Synthesis of Agmatine by Immobilized Escherichia coli Cells with Arginine Decarboxylase Activity

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei-guo; ZHAO Gen-hai; LIU Jun-zhong; LIU Qian; JIAO Qing-cai

    2011-01-01

    A new method for the enzymatic synthesis of agmatine by immobilized Escherichia coli cells with arginine decarboxylase(ADC)activity was established and a series of optimal reaction conditions was set down.The arginine decarboxylase showed the maximum activity when the pyridoxal phosphate(PLP)concentration was 50 mmol/L,pH=7 and 45 ℃.The arginine decarboxylase exhibited the maximum production efficiency when the substrate concentration was 100 mmol/L and the reaction time was 15 h.It was also observed that the appropriate concentration of Mg2+,especially at 0.5 mmol/L promoted the arginine decarboxylase activity; Mn2+ had little effect on the arginine decarboxylase activity.The inhibition of Cu2+ and Zn2+ to the arginine decarboxylase activity was significant.The immobilized cells were continuously used 6 times and the average conversion rate during the six-time usage was 55.6%.The immobilized cells exhibited favourable operational stability.After optimization,the maximally cumulative amount of agmatine could be up to 20 g/L.In addition,this method can also catalyze D,L-arginine to agmatine,leaving the pure optically D-arginine simultaneously.The method has a very important guiding significance to the enzymatic preparation of agmatine.

  17. Acute hypothalamic administration of L-arginine increases feed intake in rats

    OpenAIRE

    Carlos Ricardo Maneck Malfatti; Luiz Augusto da Silva; Ricardo Aparecido Pereira; Renan Garcia Michel; André Luiz Snak; Fabio Seidel dos Santos

    2015-01-01

    Objective: This study investigated the chronic (oral) and acute (hypothalamic infusion) effects of L-arginine supplementation on feed intake, body composition, and behavioral changes in rats. Methods: Twenty rats were divided into two groups treated orally for 60 days; one group received L-arginine (1 g/kg body weight) and one group received saline (1 mL/NaCl ...

  18. Solid-state properties and dissolution behaviour of tablets containing co-amorphous indomethacin-arginine

    DEFF Research Database (Denmark)

    Lenz, Elisabeth; Jensen, Katrine Birgitte Tarp; Blaabjerg, Lasse Ingerslev;

    2015-01-01

    arginine in a larger production scale. In this work, a tablet formulation was developed for a co-amorphous salt, namely spray dried indomethacin–arginine (SD IND–ARG). The effects of compaction pressure on tablet properties, physical stability and dissolution profiles under non-sink conditions were examined...

  19. Supplementation with l-arginine stabilizes plasma arginine and nitric oxide metabolites, suppresses elevated liver enzymes and peroxidation in sickle cell anaemia.

    Science.gov (United States)

    Jaja, S I; Ogungbemi, S O; Kehinde, M O; Anigbogu, C N

    2016-06-01

    The effect of l-arginine on liver function in SCD has received little or no attention. The effect of a chronic, oral, low-dose supplementation with l-arginine (1gm/day for 6 weeks) on some liver enzymes, lipid peroxidation and nitric oxide metabolites was studied in 20 normal (non-sickle cell anaemia; NSCA) subjects and 20 sickle cell anaemia (SCA) subjects. Ten milliliters of blood was withdrawn from an ante-cubital vein for the estimation of plasma arginine concentration ([R]), alanine aminotransaminase (ALT), aspartate aminotransaminase (AST) and alkaline phosphatase (ALP), plasma total bilirubin concentration [TB], malondialdehyde concentration [MDA] and nitric oxide metabolites concentration [NOx]. Before supplementation, ALT, AST, ALP (pNOx] were higher in NSCA subjects (pNOX] in SCA than in NSCA subjects (plow-dose supplementation with l-arginine improved liver function, oxidative stress, plasma arginine concentration and nitric oxide metabolites levels in NSCA and SCA subjects. Responses in SCA subjects to l-arginine were more sensitive than in NSCA subjects. PMID:27156372

  20. Differential effects of cranial radiation on growth hormone response to arginine and insulin infusion

    International Nuclear Information System (INIS)

    The growth hormone responses to arginine infusion and to insulin-induced hypoglycemia were studied in 13 patients with neoplastic disease after treatment with radiation and chemotherapy. Patients who received intensive cranial radiation (greater than 2,400 rads) had no response to either arginine or insulin; those who received moderate cranial radiation (greater than or equal to 2,400 rads) had GH response to arginine but not to insulin; patients receiving no cranial radiation responded to both arginine and insulin. These data support the hypothesis that GH secretion in response to arginine infusion has a different mechanism in contrast to the response to insulin-induced hypoglycemia and that the latter is more vulnerable to cranial radiation

  1. Arginine does not exacerbate markers of inflammation in cocultures of human enterocytes and leukocytes

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Negrier, I.; Neveux, N.;

    2007-01-01

    with arginine did not affect epithelial integrity, production of any of the cytokines investigated, or the amount of nitric oxide. The amino acid used primarily by nonstimulated intestinal epithelial cells cocultured with leukocytes was glutamine. Activation of IEC with bacteria significantly enhanced...... the catabolism of serine, asparagine, and lysine, and reduced glutamine catabolism. Addition of arginine increased ornithine formation and moderately reduced transepithelial transport of methionine and other amino acids. Hence, arginine supplementation does not interfere with inflammation-associated cross......Enteral arginine supplementation in the critically ill has become a matter of controversy. In this study, we investigated effects of the addition of 0.4 and 1.2 mmol/L arginine in a coculture model on markers of inflammation, enterocyte layer integrity, and amino acid transport. In this model...

  2. Life on arginine for Mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas.

    Directory of Open Access Journals (Sweden)

    Sabine Pereyre

    2009-10-01

    Full Text Available Mycoplasma hominis is an opportunistic human mycoplasma. Two other pathogenic human species, M. genitalium and Ureaplasma parvum, reside within the same natural niche as M. hominis: the urogenital tract. These three species have overlapping, but distinct, pathogenic roles. They have minimal genomes and, thus, reduced metabolic capabilities characterized by distinct energy-generating pathways. Analysis of the M. hominis PG21 genome sequence revealed that it is the second smallest genome among self-replicating free living organisms (665,445 bp, 537 coding sequences (CDSs. Five clusters of genes were predicted to have undergone horizontal gene transfer (HGT between M. hominis and the phylogenetically distant U. parvum species. We reconstructed M. hominis metabolic pathways from the predicted genes, with particular emphasis on energy-generating pathways. The Embden-Meyerhoff-Parnas pathway was incomplete, with a single enzyme absent. We identified the three proteins constituting the arginine dihydrolase pathway. This pathway was found essential to promote growth in vivo. The predicted presence of dimethylarginine dimethylaminohydrolase suggested that arginine catabolism is more complex than initially described. This enzyme may have been acquired by HGT from non-mollicute bacteria. Comparison of the three minimal mollicute genomes showed that 247 CDSs were common to all three genomes, whereas 220 CDSs were specific to M. hominis, 172 CDSs were specific to M. genitalium, and 280 CDSs were specific to U. parvum. Within these species-specific genes, two major sets of genes could be identified: one including genes involved in various energy-generating pathways, depending on the energy source used (glucose, urea, or arginine and another involved in cytadherence and virulence. Therefore, a minimal mycoplasma cell, not including cytadherence and virulence-related genes, could be envisaged containing a core genome (247 genes, plus a set of genes required for

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

    Directory of Open Access Journals (Sweden)

    Jin-Song Gong

    2015-12-01

    Full Text Available 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 Ba2+. 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.

  4. Evidence that L-Arginine inhibits glycation of human serum albumin (HSA) in vitro

    International Nuclear Information System (INIS)

    Previous work by Brownlee has shown that glycation of bovine serum albumin can be reduced in the presence of aminoguanidine (AG). Presumably, the guanidinium group on AG interferes with further rearrangement of amadori products to advanced glycosylated end products (AGE). Since L-arginine (ARG) also contains a guanidinium group, its ability to inhibit the formation of AGE products was investigated. HSA was incubated at 37 degrees C in the presence or absence of glucose; with glucose and fructose; or with sugars in the presence or absence of ARG or AG. A tracer amount of U-14C-glucose was added to each tube containing sugars. Protein bound glucose was separated from unreacted glucose by gel filtration. Radioactivity, total protein, fluorescence, and glucose concentration were measured. Preliminary data show enhanced binding of 14C-glucose to HSA with fructose at all time points. A 30-40% decrease in 14C-glucose incorporation was observed when ARG or AG as present. ARG and AG were equally effective in inhibiting incorporation of 14C-glucose. FPLC analysis is in progress to determine the type and degree of HSA crosslinking during the 2 week incubation period

  5. Effects of "Bioactive" amino acids leucine, glutamate, arginine and tryptophan on feed intake and mRNA expression of relative neuropeptides in broiler chicks

    Directory of Open Access Journals (Sweden)

    Wang Songbo

    2012-08-01

    Full Text Available Abstract Feed intake control is vital to ensuring optimal nutrition and achieving full potential for growth and development in poultry. The aim of the present study was to investigate the effects of L-leucine, L-glutamate, L-tryptophan and L-arginine on feed intake and the mRNA expression levels of hypothalamic Neuropeptide involved in feed intake regulation in broiler chicks. Leucine, glutamate, tryptophan or arginine was intra-cerebroventricularly (ICV administrated to 4d-old broiler chicks respectively and the feed intake were recorded at various time points. Quantitative PCR was performed to determine the hypothalamic mRNA expression levels of Neuropeptide Y (NPY, agouti related protein (AgRP, pro-opiomelanocortin (POMC, melanocortin receptor 4 (MC4R and corticotrophin releasing factor (CRF. Our results showed that ICV administration of L-leucine (0.15 or 1.5  μmol significantly (P P 

  6. Altered Nitrogen Balance and Decreased Urea Excretion in Male Rats Fed Cafeteria Diet Are Related to Arginine Availability

    Directory of Open Access Journals (Sweden)

    David Sabater

    2014-01-01

    rats, but low arginine levels point to a block in the urea cycle between ornithine and arginine, thereby preventing the elimination of excess nitrogen as urea. The ultimate consequence of this paradoxical block in the urea cycle seems to be the limitation of arginine production and/or availability.

  7. Microwave heating of arginine yields highly fluorescent nanoparticles

    International Nuclear Information System (INIS)

    Brightly fluorescent nanoparticles were produced via a single-step, single-precursor procedure based on microwave heating of an aqueous solution of the amino acid arginine. Key structural and optical properties of the resulting Arg nanoparticles, Arg-dots, are reported and discussed with emphasis on the pH dependence of their fluorescence emission. The surface of the Arg-dots was functionalised through coupling to folic acid, opening up ways for connecting fluorescent nanoparticles to cancer cells. The generality and versatility of the microwave heating procedure was further demonstrated by the synthesis of different types of carbon nanoparticles, such as CE-dots, that were produced by use of citric acid and ethanolamine as precursors and compared to the Arg-dots.

  8. Microwave heating of arginine yields highly fluorescent nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Philippidis, Aggelos [Foundation for Research and Technology-Hellas, Institute of Electronic Structure and Laser (Greece); Stefanakis, Dimitrios [University of Crete, Department of Chemistry (Greece); Anglos, Demetrios, E-mail: anglos@iesl.forth.gr [Foundation for Research and Technology-Hellas, Institute of Electronic Structure and Laser (Greece); Ghanotakis, Demetrios, E-mail: ghanotakis@chemistry.uoc.gr [University of Crete, Department of Chemistry (Greece)

    2013-01-15

    Brightly fluorescent nanoparticles were produced via a single-step, single-precursor procedure based on microwave heating of an aqueous solution of the amino acid arginine. Key structural and optical properties of the resulting Arg nanoparticles, Arg-dots, are reported and discussed with emphasis on the pH dependence of their fluorescence emission. The surface of the Arg-dots was functionalised through coupling to folic acid, opening up ways for connecting fluorescent nanoparticles to cancer cells. The generality and versatility of the microwave heating procedure was further demonstrated by the synthesis of different types of carbon nanoparticles, such as CE-dots, that were produced by use of citric acid and ethanolamine as precursors and compared to the Arg-dots.

  9. Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome.

    Science.gov (United States)

    Wu, Tsu-Fang; Yao, Ya-Li; Lai, I-Lu; Lai, Chien-Chen; Lin, Pei-Lun; Yang, Wen-Ming

    2015-08-14

    PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome. PMID:26149688

  10. Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome*

    Science.gov (United States)

    Wu, Tsu-Fang; Yao, Ya-Li; Lai, I-Lu; Lai, Chien-Chen; Lin, Pei-Lun; Yang, Wen-Ming

    2015-01-01

    PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome. PMID:26149688

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

    Science.gov (United States)

    Vogelsgesang, Martin; Aktories, Klaus

    2006-01-24

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

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

    Science.gov (United States)

    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.

  13. The Nitric Oxide Synthase Inhibitor NG-Nitro-L-Arginine Methyl Ester Diminishes the Immunomodulatory Effects of Parental Arginine in Rats with Subacute Peritonitis.

    Science.gov (United States)

    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.

  14. Enteral Glutamine Administration in Critically Ill Nonseptic Patients Does Not Trigger Arginine Synthesis

    Directory of Open Access Journals (Sweden)

    Mechteld A. R. Vermeulen

    2016-01-01

    Full Text Available Glutamine supplementation in specific groups of critically ill patients results in favourable clinical outcome. Enhancement of citrulline and arginine synthesis by glutamine could serve as a potential mechanism. However, while receiving optimal enteral nutrition, uptake and enteral metabolism of glutamine in critically ill patients remain unknown. Therefore we investigated the effect of a therapeutically relevant dose of L-glutamine on synthesis of L-citrulline and subsequent L-arginine in this group. Ten versus ten critically ill patients receiving full enteral nutrition, or isocaloric isonitrogenous enteral nutrition including 0.5 g/kg L-alanyl-L-glutamine, were studied using stable isotopes. A cross-over design using intravenous and enteral tracers enabled splanchnic extraction (SE calculations. Endogenous rate of appearance and SE of glutamine citrulline and arginine was not different (SE controls versus alanyl-glutamine: glutamine 48 and 48%, citrulline 33 versus 45%, and arginine 45 versus 42%. Turnover from glutamine to citrulline and arginine was not higher in glutamine-administered patients. In critically ill nonseptic patients receiving adequate nutrition and a relevant dose of glutamine there was no extra citrulline or arginine synthesis and glutamine SE was not increased. This suggests that for arginine synthesis enhancement there is no need for an additional dose of glutamine when this population is adequately fed. This trial is registered with NTR2285.

  15. Enteral Glutamine Administration in Critically Ill Nonseptic Patients Does Not Trigger Arginine Synthesis.

    Science.gov (United States)

    Vermeulen, Mechteld A R; Brinkmann, Saskia J H; Buijs, Nikki; Beishuizen, Albertus; Bet, Pierre M; Houdijk, Alexander P J; van Goudoever, Johannes B; van Leeuwen, Paul A M

    2016-01-01

    Glutamine supplementation in specific groups of critically ill patients results in favourable clinical outcome. Enhancement of citrulline and arginine synthesis by glutamine could serve as a potential mechanism. However, while receiving optimal enteral nutrition, uptake and enteral metabolism of glutamine in critically ill patients remain unknown. Therefore we investigated the effect of a therapeutically relevant dose of L-glutamine on synthesis of L-citrulline and subsequent L-arginine in this group. Ten versus ten critically ill patients receiving full enteral nutrition, or isocaloric isonitrogenous enteral nutrition including 0.5 g/kg L-alanyl-L-glutamine, were studied using stable isotopes. A cross-over design using intravenous and enteral tracers enabled splanchnic extraction (SE) calculations. Endogenous rate of appearance and SE of glutamine citrulline and arginine was not different (SE controls versus alanyl-glutamine: glutamine 48 and 48%, citrulline 33 versus 45%, and arginine 45 versus 42%). Turnover from glutamine to citrulline and arginine was not higher in glutamine-administered patients. In critically ill nonseptic patients receiving adequate nutrition and a relevant dose of glutamine there was no extra citrulline or arginine synthesis and glutamine SE was not increased. This suggests that for arginine synthesis enhancement there is no need for an additional dose of glutamine when this population is adequately fed. This trial is registered with NTR2285.

  16. L-arginine, the substrate of nitric oxide synthase,inhibits fertility of male rats

    Institute of Scientific and Technical Information of China (English)

    W. D. Ramasooriya; M. G. Dharmasiri

    2001-01-01

    Aim: To examine the effect of L-arginine, the substrate of nitric oxide (NO) synthase, on reproductive function of male rots. Methods: Male rats were gavaged with either L-arginine (100 or 200 mg@ kg- 1@ d-1), D-arginine (200 mg@ kg- 1@ d-1 ) or vehicle (0.9% NaCl) for seven consecutive days. Their sexual behaviour and fertility were evaluat ed using receptive females. Results: L-arginine (200 mg/kg) had no significant effect on sexual competence (in terms of sexual arousal, libido, sexual vigour and sexual performance). In mating experiments, the higher dose of L arginine effectively and reversibly inhibited fertility, whilst the lower dose and the inactive stereoisomer D-arginine had no significant effect. The antifertility effect caused by L-arginine was due to a profound elevation in the preimplantation loss mediated possibly by impairment in epididymal sperm maturation, hyperactivated sperm motility and sperm capaci ration. Conclusion: Elevated NO production may be detrimental to male fertility.

  17. Enteral Glutamine Administration in Critically Ill Nonseptic Patients Does Not Trigger Arginine Synthesis

    Science.gov (United States)

    Vermeulen, Mechteld A. R.; Brinkmann, Saskia J. H.; Buijs, Nikki; Beishuizen, Albertus; Bet, Pierre M.; Houdijk, Alexander P. J.; van Goudoever, Johannes B.; van Leeuwen, Paul A. M.

    2016-01-01

    Glutamine supplementation in specific groups of critically ill patients results in favourable clinical outcome. Enhancement of citrulline and arginine synthesis by glutamine could serve as a potential mechanism. However, while receiving optimal enteral nutrition, uptake and enteral metabolism of glutamine in critically ill patients remain unknown. Therefore we investigated the effect of a therapeutically relevant dose of L-glutamine on synthesis of L-citrulline and subsequent L-arginine in this group. Ten versus ten critically ill patients receiving full enteral nutrition, or isocaloric isonitrogenous enteral nutrition including 0.5 g/kg L-alanyl-L-glutamine, were studied using stable isotopes. A cross-over design using intravenous and enteral tracers enabled splanchnic extraction (SE) calculations. Endogenous rate of appearance and SE of glutamine citrulline and arginine was not different (SE controls versus alanyl-glutamine: glutamine 48 and 48%, citrulline 33 versus 45%, and arginine 45 versus 42%). Turnover from glutamine to citrulline and arginine was not higher in glutamine-administered patients. In critically ill nonseptic patients receiving adequate nutrition and a relevant dose of glutamine there was no extra citrulline or arginine synthesis and glutamine SE was not increased. This suggests that for arginine synthesis enhancement there is no need for an additional dose of glutamine when this population is adequately fed. This trial is registered with NTR2285. PMID:27200186

  18. One-pot green synthesis of biocompatible arginine-stabilized magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Wang Zhongjun; Zhu Hui; Wang Xiaolei; Yang Fan; Yang Xiurong, E-mail: xryang@ciac.jl.c [State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022 (China)

    2009-11-18

    A green one-step approach has been developed for the synthesis of amino-functionalized magnetite nanoparticles. The synthesis was accomplished by simply mixing FeCl{sub 2} with arginine under ambient conditions. It was found that the Fe{sup 2+}/arginine molar ratio, reaction duration and temperature greatly influence the size, morphology and composition of magnetic nanoparticles. The arginine-stabilized magnetic nanoparticles were characterized by transmission electron microscopy, x-ray diffraction, x-ray photoelectron spectroscopy, thermogravimetric analysis, and Fourier transform infrared spectroscopy techniques. The results show that the prepared nanoparticles are spherically shaped with a nearly uniform size distribution and pure magnetite phase. The presence of arginine on the magnetic nanoparticle surface has been confirmed and the amount of surface arginine varies with the Fe{sup 2+}/arginine molar ratio. The surface amine densities are calculated to be 5.60 and 7.84 {mu}mol mg{sup -1} for magnetic nanoparticles prepared at 1:1 and 1:2 Fe{sup 2+}/arginine molar ratio, respectively. The as-synthesized nanoparticles show superparamagnetic behavior at room temperature and good solubility in water. In addition, using a similar synthesis procedure, we have been able to synthesize superparamagnetic manganese and cobalt ferrite nanoparticles.

  19. One-pot green synthesis of biocompatible arginine-stabilized magnetic nanoparticles

    International Nuclear Information System (INIS)

    A green one-step approach has been developed for the synthesis of amino-functionalized magnetite nanoparticles. The synthesis was accomplished by simply mixing FeCl2 with arginine under ambient conditions. It was found that the Fe2+/arginine molar ratio, reaction duration and temperature greatly influence the size, morphology and composition of magnetic nanoparticles. The arginine-stabilized magnetic nanoparticles were characterized by transmission electron microscopy, x-ray diffraction, x-ray photoelectron spectroscopy, thermogravimetric analysis, and Fourier transform infrared spectroscopy techniques. The results show that the prepared nanoparticles are spherically shaped with a nearly uniform size distribution and pure magnetite phase. The presence of arginine on the magnetic nanoparticle surface has been confirmed and the amount of surface arginine varies with the Fe2+/arginine molar ratio. The surface amine densities are calculated to be 5.60 and 7.84 μmol mg-1 for magnetic nanoparticles prepared at 1:1 and 1:2 Fe2+/arginine molar ratio, respectively. The as-synthesized nanoparticles show superparamagnetic behavior at room temperature and good solubility in water. In addition, using a similar synthesis procedure, we have been able to synthesize superparamagnetic manganese and cobalt ferrite nanoparticles.

  20. Investigation on the remineralization effect of arginine toothpaste for early enamel caries: nanotribological and nanomechanical properties

    Science.gov (United States)

    Yu, Ping; Arola, Dwayne D.; Min, Jie; Yu, Dandan; Xu, Zhou; Li, Zhi; Gao, Shanshan

    2016-11-01

    Remineralization is confirmed as a feasible method to restore early enamel caries. While there is evidence that the 8% arginine toothpaste has a good remineralization effect by increasing surface microhardness, the repair effect on wear-resistance and nanomechanical properties still remains unclear. Therefore, this research was conducted to reveal the nanotribological and nanomechanical properties changes of early caries enamel after remineralized with arginine toothpaste. Early enamel caries were created in bovine enamel blocks, and divided into three groups according to the treatment solutions: distilled and deionized water (DDW group), arginine toothpaste slurry (arginine group) and fluoride toothpaste slurry (fluoride group). All of the samples were subjected to pH cycling for 12 d. The nanotribological and nanomechanical properties were evaluated via the nanoscratch and nanoindentation tests. The wear depth and scratch morphology were observed respectively by scanning probe microscopic (SPM) and scanning electron microscopy (SEM). Finally, x-ray photoelectron spectroscopy (XPS) was used for element analysis of remineralized surfaces. Results showed that the wear depth of early caries enamel decreased after remineralization treatment and both the nanohardness and elastic modulus increased. Compared with the fluoride group, the arginine group exhibited higher nanohardness and elastic modulus with higher levels of calcium, fluoride, nitrogen and phosphorus; this group also underwent less wear and related damage. Overall, the synergistic effect of arginine and fluoride in arginine toothpaste achieves better nanotribological and nanomechanical properties than the single fluoride toothpaste, which could have significant impact on fight against early enamel caries.

  1. Asymmetric arginine dimethylation of RelA provides a repressive mark to modulate TNFα/NF-κB response.

    Science.gov (United States)

    Reintjes, Anja; Fuchs, Julian E; Kremser, Leopold; Lindner, Herbert H; Liedl, Klaus R; Huber, Lukas A; Valovka, Taras

    2016-04-19

    Nuclear factor kappa B (NF-κB) is an inducible transcription factor that plays critical roles in immune and stress responses and is often implicated in pathologies, including chronic inflammation and cancer. Although much has been learned about NF-κB-activating pathways, the specific repression of NF-κB is far less well understood. Here we identified the type I protein arginine methyltransferase 1 (PRMT1) as a restrictive factor controlling TNFα-induced activation of NF-κB. PRMT1 forms a cellular complex with NF-κB through direct interaction with the Rel homology domain of RelA. We demonstrate that PRMT1 methylates RelA at evolutionary conserved R30, located in the DNA-binding L1 loop, which is a critical residue required for DNA binding. Asymmetric R30 dimethylation inhibits the binding of RelA to DNA and represses NF-κB target genes in response to TNFα. Molecular dynamics simulations of the DNA-bound RelA:p50 predicted structural changes in RelA caused by R30 methylation or a mutation that interferes with the stability of the DNA-NF-κB complex. Our findings provide evidence for the asymmetric arginine dimethylation of RelA and unveil a unique mechanism controlling TNFα/NF-κB signaling. PMID:27051065

  2. Atropine and ODQ antagonize tetanic fade induced by L-arginine in cats

    Directory of Open Access Journals (Sweden)

    J.M. Cruciol-Souza

    1999-10-01

    Full Text Available Although it has been demonstrated that nitric oxide (NO released from sodium nitrite induces tetanic fade in the cat neuromuscular preparations, the effect of L-arginine on tetanic fade and its origin induced by NO have not been studied in these preparations. Furthermore, atropine reduces tetanic fade induced by several cholinergic and anticholinergic drugs in these preparations, whose mechanism is suggested to be mediated by the interaction of acetylcholine with inhibitory presynaptic muscarinic receptors. The present study was conducted in cats to determine the effects of L-arginine alone or after pretreatment with atropine or 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ on neuromuscular preparations indirectly stimulated at high frequency. Drugs were injected into the middle genicular artery. L-arginine (2 mg/kg and S-nitroso-N-acetylpenicillamine (SNAP; 16 µg/kg induced tetanic fade. The Nw-nitro-L-arginine (L-NOARG; 2 mg/kg alone did not produce any effect, but reduced the tetanic fade induced by L-arginine. D-arginine (2 mg/kg did not induce changes in tetanic fade. The tetanic fade induced by L-arginine or SNAP was reduced by previous injection of atropine (1.0 µg/kg or ODQ (15 µg/kg. ODQ alone did not change tetanic fade. The data suggest that the NO-synthase-GC pathway participates in the L-arginine-induced tetanic fade in cat neuromuscular preparations. The tetanic fade induced by L-arginine probably depends on the action of NO at the presynaptic level. NO may stimulate guanylate cyclase increasing acetylcholine release and thereby stimulating presynaptic muscarinic receptors.

  3. Hepatic adaptation compensates inactivation of intestinal arginine biosynthesis in suckling mice.

    Directory of Open Access Journals (Sweden)

    Vincent Marion

    Full Text Available 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.

  4. The effect of L-arginine on guinea-pig and rabbit airway smooth muscle function in vitro

    OpenAIRE

    Perez A.C.; Paul W.; Harrison S.; Page C.P.; Spina D.

    1998-01-01

    We have investigated the effects of L-arginine, D-arginine and L-lysine on airway smooth muscle responsiveness to spasmogens in vitro. Both L-arginine and D-arginine (100 mM) significantly reduced the contractile potency and maximal contractile response to histamine but not to methacholine or potassium chloride in guinea-pig epithelium-denuded isolated trachea. Similarly, the contractile response to histamine was significantly reduced by L-arginine (100 mM) in rabbit epithelium-denuded isolat...

  5. High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors

    Science.gov (United States)

    Bageshwar, Umesh K.; VerPlank, Lynn; Baker, Dwight; Dong, Wen; Hamsanathan, Shruthi; Whitaker, Neal; Sacchettini, James C.; Musser, Siegfried M.

    2016-01-01

    The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors. PMID:26901445

  6. 斑节对虾腺苷酸转移酶(PmANT)基因的cDNA克隆与表达分析%Molecular cloning and expression analysis of adenine nucleotide translocase (PmANT) in Penaeus monodon

    Institute of Scientific and Technical Information of China (English)

    孙文文; 周发林; 黄建华; 邱丽华; 杨其彬; 江世贵

    2013-01-01

    利用RACE技术获得了斑节对虾(Penaeus monodon)ANT基因(PmANT)的cDNA序列.该序列全长1 388 bp,开放阅读框(ORF)为930 bp,3’非编码区(UTR)为393 bp,5 '非编码区(UTR)为65 bp.ORF可编码309个氨基酸,分子量大约为33.622 ku.与所有ANT家族成员一样,PmANT蛋白具有3个重复同源的线粒体跨膜结构域,但不含信号肽和糖基化位点.相似性、同源性及系统进化树分析显示,斑节对虾的ANT基因与凡纳滨对虾的同源性和相似性最高,并与其聚为一支.采用荧光定量的方法研究了ANT基因在雌雄个体不同组织、卵巢不同发育阶段及未成熟和成熟精巢的差异表达情况.结果表明:PmANT的mRNA在各组织中都有表达,其中,在雄性个体的肌肉中表达量最高,其次为雌性肌肉,在精巢的表达量最低,且未成熟精巢低于成熟精巢.PmANT的mRNA在卵巢的表达量高于精巢,且在Ⅲ期卵巢表达量最高,Ⅳ期最低.为今后进一步研究该基因在斑节对虾性腺发育中的作用提供基础材料.%The adenine nucleotide translocase (ANT) is the most abundant mitochondrial inner membrane protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. In the present study, the full sequence of P. Monodon ANT gene was cloned and named PmANT. The full length cDNA of PmANT contained a 5' untranslated region (UTR) of 65 bp, a 3' UTR of 393 bp and an ORF of 930 bp encoding a polypeptide of 309 amino acids with an estimated molecular mass of 33. 622 ku. Like other animal ANTs, the structure of the PmANT protein consists of three homologous repeated domains. But there are no signal peptide and glycosylation sites in PmANT protein. Sequence alignment analysis showed that the PmANT with the ANT of Litopenaeus vannamei shared a similarity of 98. 7% and the homology of 97. 4%. Analysis of the tissue expression pattern of the PmANT showed that the PmANT m

  7. Structure-based molecular design for thermostabilization of N-acetyltransferase Mpr1 involved in a novel pathway of L-arginine synthesis in yeast.

    Science.gov (United States)

    Nasuno, Ryo; Hirase, Saeka; Norifune, Saki; Watanabe, Daisuke; Takagi, Hiroshi

    2016-02-01

    Previously, N-Acetyltransferase Mpr1 was suggested to be involved in a novel pathway of L-arginine biosynthesis in yeast. Our recent crystallographic analysis demonstrated that the overall structure of Mpr1 is a typical folding among proteins in the Gcn5-related N-acetyltransferase superfamily, and also provided clues to the design of mutations for improvement of the enzymatic functions. Here, we constructed new stable variants, Asn203Lys- and Asn203Arg-Mpr1, which exhibited 2.4-fold and 2.2-fold longer activity half-lives than wild-type Mpr1, respectively, by structure-based molecular design. The replacement of Asn203 with a basic amino acid was suggested to stabilize α-helix 2, which is important for the Mpr1 structure, probably by neutralizing its dipole. In addition, the combination of two amino acid substitutions at positions 65 and 203 in Mpr1, Phe65Leu, which was previously isolated by the screening from PCR random mutagenesis library of MPR1, and Asn203Lys or Asn203Arg, led to further stabilization of Mpr1. Our growth assay suggests that overexpression of the stable Mpr1 variants increase L-arginine synthesis in yeast cells. Our finding is the first report on the rational engineering of Mpr1 for thermostabilization and could be useful in the construction of new yeast strains with higher L-arginine synthetic activity and also improved fermentation ability.

  8. Mutation of Residue Arginine18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    It has been known that arginine is used as the basic amino acid in the ?subunit of cytochrome b559 (Cyt b559) except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem (PS) Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSII complexes. In the present study,two arginine18 (R18) mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid (E) and glycine (G). The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type (WT) levels and that, after irradiation at high light intensity, oxygen evolution for the PSII of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSII (Fv/Fm) of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the ?subunit of Cyt b559 and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt b559 maintain the structure of the PSII complex and its activity,although it is not directly bound to the heme group.

  9. Purification, cloning, and immunological characterization of arginine kinase, a novel allergen of Octopus fangsiao.

    Science.gov (United States)

    Shen, Hai-Wang; Cao, Min-Jie; Cai, Qiu-Feng; Ruan, Mi-Mi; Mao, Hai-Yan; Su, Wen-Jin; Liu, Guang-Ming

    2012-03-01

    Arginine kinase (AK) is an important enzyme participating in energy metabolism in invertebrates, but, to date, there have been no reports that AK from octopus is an allergen. In this study, octopus AK was purified, and its molecular biological, immunological, and physicochemical characterizations were analyzed. The results showed that octopus AK was purified and confirmed by mass spectrometry for the first time, and its molecular mass was 38 kDa. The full-length gene sequence of octopus AK encompassed 1209 bp and was predicted to encode a protein with 348 amino acid residues. The homology of octopus AK and crustacean AK was about 54%, but the similarity between their three-dimensional structures was high. Octopus AK could react with mouse anti-shrimp AK and rabbit anti-crab AK polyclonal antibody singly. Octopus AK could also react with specific IgE of the sera from octopus-allergic patients effectively, whereas crab AK could inhibit the reaction between them. Finally, the IgE-binding activity of octopus AK could be reduced in the processes of thermal or acid-alkali treatment. In summary, AK was identified as a novel allergen in octopus, which had a sensitizing ability similar to that of crustacean AK. This is significant in allergy diagnosis and the treatment of octopus-allergic disorders.

  10. Effect of poly-L-arginine on intestinal absorption of hydrophilic macromolecules in rats.

    Science.gov (United States)

    Yamaki, Tsutomu; Uchida, Masaki; Kuwahara, Yusuke; Shimazaki, Yohei; Ohtake, Kazuo; Kimura, Mitsutoshi; Uchida, Hiroyuki; Kobayashi, Jun; Ogihara, Masahiko; Morimoto, Yasunori; Natsume, Hideshi

    2013-01-01

    We have already reported that poly-L-arginine (PLA) remarkably enhanced the in vivo nasal absorption of hydrophilic macromolecules without producing any significant epithelial damage in rats. In the present study, we examined whether PLA could enhance the absorption of a model hydrophilic macromolecule, fluorescein isothiocyanate-dextran (FD-4), across the intestinal mucosa, as well as the nasal mucosa, by an in situ closed-loop method using the rat intestine. PLA was found to enhance the intestinal absorption of FD-4 in a concentration-dependent manner within the concentrations investigated in this study, but segment-specific differences were found to be associated with this effect (ileum>jejunum>duodenum≧colon). The factors responsible for the segment-specific differences were also investigated by intestinal absorption studies using aprotinin, a trypsin inhibitor, and an analysis of the expression of occludin, a tight junction protein. In the small intestine, the differences in the effect of PLA on the absorption of FD-4 may be related to the enzymatic degradation of PLA. In the colon, the reduced effect of PLA on the absorption of FD-4 may be related to the smaller surface area for absorption and the higher expression of occludin compared with other segments.

  11. Transgenic approach to express the channelrhodopsin 2 gene in arginine vasopressin neurons of rats.

    Science.gov (United States)

    Ishii, Masahiro; Hashimoto, Hirofumi; Ohkubo, Jun-Ichi; Ohbuchi, Toyoaki; Saito, Takeshi; Maruyama, Takashi; Yoshimura, Mitsuhiro; Yamamoto, Yukiyo; Kusuhara, Koichi; Ueta, Yoichi

    2016-09-01

    Optogenetics provides a powerful tool to regulate neuronal activity by light-sensitive ion channels such as channelrhodopsin 2 (ChR2). Arginine vasopressin (AVP; also known as the anti-diuretic hormone) is a multifunctional hormone which is synthesized in the magnocellular neurosecretory cells (MNCs) of the hypothalamus. Here, we have generated a transgenic rat that expresses an AVP-ChR2-enhanced green fluorescent protein (eGFP) fusion gene in the MNCs of the hypothalamus. The eGFP fluorescence that indicates the expression of ChR2-eGFP was observed in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN) that is known to contain AVP-secreting neurons. The eGFP fluorescence intensities in those nuclei and posterior pituitary were markedly increased after chronic salt loading (2% NaCl in drinking water for 5days). ChR2-eGFP was localized mainly in the membrane of AVP-positive MNCs. Whole-cell patch-clamp recordings were performed from single MNCs isolated from the SON of the transgenic rats, and blue light evoked repetitive action potentials. Our work provides for the first time an optogenetic approach to selectively activate AVP neurons in the rat. PMID:27493075

  12. The transporter and permeability interactions of asymmetric dimethylarginine (ADMA) and L-arginine with the human blood-brain barrier in vitro.

    Science.gov (United States)

    Watson, Christopher P; Pazarentzos, Evangelos; Fidanboylu, Mehmet; Padilla, Beatriz; Brown, Rachel; Thomas, Sarah A

    2016-10-01

    The blood-brain barrier (BBB) is a biological firewall that carefully regulates the cerebral microenvironment by acting as a physical, metabolic and transport barrier. This selectively permeable interface was modelled using the immortalised human cerebral microvascular endothelial cell line (hCMEC/D3) to investigate interactions with the cationic amino acid (CAA) L-arginine, the precursor for nitric oxide (NO), and with asymmetric dimethylarginine (ADMA), an endogenously derived analogue of L-arginine that potently inhibits NO production. The transport mechanisms utilised by L-arginine are known but they are not fully understood for ADMA, particularly at the BBB. This is of clinical significance giving the emerging role of ADMA in many brain and cerebrovascular diseases and its potential as a therapeutic target. We discovered that high concentrations of ADMA could induce endothelial dysfunction in the hCMEC/D3s BBB permeability model, leading to an increase in paracellular permeability to the paracellular marker FITC-dextran (40kDa). We also investigated interactions of ADMA with a variety of transport mechanisms, comparing the data with L-arginine interactions. Both molecules are able to utilise the CAA transport system y(+). Furthermore, the expression of CAT-1, the best known protein from this group, was confirmed in the hCMEC/D3s. It is likely that influx systems, such as y(+)L and b(0,+), have an important physiological role in ADMA transport at the BBB. These data are not only important with regards to the brain, but apply to other microvascular endothelia where ADMA is a major area of investigation. PMID:27431938

  13. Resveratrol inhibits Trypanosoma cruzi arginine kinase and exerts a trypanocidal activity.

    Science.gov (United States)

    Valera Vera, Edward A; Sayé, Melisa; Reigada, Chantal; Damasceno, Flávia S; Silber, Ariel M; Miranda, Mariana R; Pereira, Claudio A

    2016-06-01

    Arginine kinase catalyzes the reversible transphosphorylation between ADP and phosphoarginine which plays a critical role in the maintenance of cellular energy homeostasis. Arginine kinase from the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, meets the requirements to be considered as a potential therapeutic target for rational drug design including being absent in its mammalian hosts. In this study a group of polyphenolic compounds was evaluated as potential inhibitors of arginine kinase using molecular docking techniques. Among the analyzed compounds with the lowest free binding energy to the arginine kinase active site (market price; and (3) has as a well-defined target enzyme which is absent in the mammalian host, it is a promising compound as a trypanocidal drug for Chagas disease. PMID:26976067

  14. Corrosion Inhibition Effect of Carbon Steel in Sea Water by L-Arginine-Zn2+ System

    Directory of Open Access Journals (Sweden)

    S. Gowri

    2014-01-01

    Full Text Available The inhibition efficiency of L-Arginine-Zn2+ system in controlling corrosion of carbon steel in sea water has been evaluated by the weight-loss method. The formulation consisting of 250 ppm of L-Arginine and 25 ppm of Zn2+ has 91% IE. A synergistic effect exists between L-Arginine and Zn2+. Polarization study reveals that the L-Arginine-Zn2+ system functions as an anodic inhibitor and the formulation controls the anodic reaction predominantly. AC impedance spectra reveal that protective film is formed on the metal surface. Cyclic voltammetry study reveals that the protective film is more compact and stable even in a 3.5% NaCl environment. The nature of the protective film on a metal surface has been analyzed by FTIR, SEM, and AFM analysis.

  15. Endothelial arginine resynthesis contributes to the maintenance of vasomotor function in male diabetic mice

    DEFF Research Database (Denmark)

    Chennupati, Ramesh; Meens, Merlijn J P M T; Marion, Vincent;

    2014-01-01

    AIM: Argininosuccinate synthetase (ASS) is essential for recycling L-citrulline, the by-product of NO synthase (NOS), to the NOS substrate L-arginine. Here, we assessed whether disturbed arginine resynthesis modulates endothelium-dependent vasodilatation in normal and diabetic male mice. METHODS...... AND RESULTS: Endothelium-selective Ass-deficient mice (Assfl/fl/Tie2Cretg/- = Ass-KOTie2) were generated by crossing Assfl/fl mice ( = control) with Tie2Cre mice. Gene ablation in endothelial cells was confirmed by immunohistochemistry. Blood pressure (MAP) was recorded in 34-week-old male mice. Vasomotor...... of endothelial citrulline recycling to arginine did not affect blood pressure and systemic arterial vasomotor responses in healthy mice. EDNO-mediated vasodilatation was significantly more impaired in diabetic Ass-KOTie2 than in control mice demonstrating that endothelial arginine recycling becomes a limiting...

  16. Metabolomic analysis of plasma and liver from surplus arginine fed Atlantic salmon

    Science.gov (United States)

    Andersen, Synne M.; Assaad, Houssein I.; Lin, Gang; Wang, Junjun; Aksnes, Anders; Wu, Guoyao; Espe, Marit

    2016-01-01

    The aim of this study was to determine the metabolic effect of surplus arginine (36.1 g/kg dry matter) compared to a control diet with required arginine (21.1 g/kg dry matter) in adult Atlantic salmon (Salmo salar L.). Although the feeding trial had no significant effect on growth, there were significant differences in the metabolite profile in both plasma and liver in experimental group as compared to the control group. There was increased concentrations of biliverdin, PGF-2 alpha, oxidized glutathione, selenocysteine, two monoacylglycerols and a tripeptide in the liver as well as decreased concentrations of valine and a vitamin D3 metabolite in plasma of arginine supplemented fish. These results indicate that while surplus arginine does not affect growth or body weight, it induces metabolic changes in Atlantic salmon. PMID:25553364

  17. Utilization of ornithine and arginine as specific precursors of clavulanic acid.

    Science.gov (United States)

    Romero, J; Liras, P; Martín, J F

    1986-01-01

    Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine. PMID:2877616

  18. Increased arginine vasopressin mRNA expression in the human hypothalamus in depression: A preliminary report

    NARCIS (Netherlands)

    G. Meynen; U.A. Unmehopa; J.J. van Heerikhuize; M.A. Hofman; D.F. Swaab; W.J.G. Hoogendijk

    2006-01-01

    Background: Elevated arginine vasopressin (AVP) plasma levels have been observed in major depression, particularly in relation to the melancholic subtype. Two hypothalamic structures produce plasma vasopressin: the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). The aim of this study

  19. The effect of citrulline and arginine supplementation on lactic acidemia in MELAS syndrome.

    Science.gov (United States)

    El-Hattab, Ayman W; Emrick, Lisa T; Williamson, Kaitlin C; Craigen, William J; Scaglia, Fernando

    2013-12-01

    Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a mitochondrial disorder in which nitric oxide (NO) deficiency may play a role in the pathogenesis of several complications including stroke-like episodes and lactic acidosis. Supplementing the NO precursors arginine and citrulline restores NO production in MELAS syndrome. In this study we evaluated the effect of arginine or citrulline on lactic acidemia in adults with MELAS syndrome. Plasma lactate decreased significantly after citrulline supplementation, whereas the effect of arginine supplementation did not reach statistical significance. These results support the potential therapeutic utility of arginine and citrulline in MELAS syndrome and suggest that citrulline supplementation may be more efficacious. However, therapeutic efficacy of these compounds should be further evaluated in clinical trials.

  20. Streptococcus pneumoniae arginine synthesis genes promote growth and virulence in pneumococcal meningitis

    NARCIS (Netherlands)

    J.R. Piet; M. Geldhoff; B.D.C. van Schaik; M.C. Brouwer; M. Valls Seron; M.E. Jakobs; K. Schipper; Y. Pannekoek; A.H. Zwinderman; T. van der Poll; A.H.C. van Kampen; F. Baas; A van der Ende; D. van de Beek

    2014-01-01

    Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing pneumonia, sepsis and bacterial meningitis. Using a clinical phenotype based approach with bacterial whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with

  1. Thermal, FT–IR and SHG efficiency studies of L-arginine doped KDP crystals

    Indian Academy of Sciences (India)

    K D Parikh; D J Dave; B B Parekh; M J Joshi

    2007-04-01

    Potassium dihydrogen phosphate (KDP) is a well known nonlinear optical (NLO) material with different applications. Since most of the amino acids exhibit NLO property, it is of interest to dope them in KDP. In the present study, amino acid L-arginine was doped in KDP. The doping of L-arginine was confirmed by FT–IR and paper chromatography. Thermogravimetry suggested that as the amount of doping increases the thermal stability decreases as well as the value of thermodynamic and kinetic parameters decreases. The second harmonic generation (SHG) efficiency of L-arginine doped KDP crystals was found to be increasing with doping concentration of L-arginine. The results are discussed here.

  2. Arginine metabolism by macrophages promotes cardiac and muscle fibrosis in mdx muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Michelle Wehling-Henricks

    Full Text Available BACKGROUND: Duchenne muscular dystrophy (DMD is the most common, lethal disease of childhood. One of 3500 new-born males suffers from this universally-lethal disease. Other than the use of corticosteroids, little is available to affect the relentless progress of the disease, leading many families to use dietary supplements in hopes of reducing the progression or severity of muscle wasting. Arginine is commonly used as a dietary supplement and its use has been reported to have beneficial effects following short-term administration to mdx mice, a genetic model of DMD. However, the long-term effects of arginine supplementation are unknown. This lack of knowledge about the long-term effects of increased arginine metabolism is important because elevated arginine metabolism can increase tissue fibrosis, and increased fibrosis of skeletal muscles and the heart is an important and potentially life-threatening feature of DMD. METHODOLOGY: We use both genetic and nutritional manipulations to test whether changes in arginase metabolism promote fibrosis and increase pathology in mdx mice. Our findings show that fibrotic lesions in mdx muscle are enriched with arginase-2-expressing macrophages and that muscle macrophages stimulated with cytokines that activate the M2 phenotype show elevated arginase activity and expression. We generated a line of arginase-2-null mutant mdx mice and found that the mutation reduced fibrosis in muscles of 18-month-old mdx mice, and reduced kyphosis that is attributable to muscle fibrosis. We also observed that dietary supplementation with arginine for 17-months increased mdx muscle fibrosis. In contrast, arginine-2 mutation did not reduce cardiac fibrosis or affect cardiac function assessed by echocardiography, although 17-months of dietary supplementation with arginine increased cardiac fibrosis. Long-term arginine treatments did not decrease matrix metalloproteinase-2 or -9 or increase the expression of utrophin, which have

  3. Nonspecific blockade of vascular free radical signals by methylated arginine analogues

    Directory of Open Access Journals (Sweden)

    Pedro M.A.

    1998-01-01

    Full Text Available Methylated arginine analogues are often used as probes of the effect of nitric oxide; however, their specificity is unclear and seems to be frequently overestimated. This study analyzed the effects of NG-methyl-L-arginine (L-NMMA on the endothelium-dependent release of vascular superoxide radicals triggered by increased flow. Plasma ascorbyl radical signals measured by direct electron paramagnetic resonance spectroscopy in 25 rabbits increased by 3.8 ± 0.7 nmol/l vs baseline (28.7 ± 1.4 nmol/l, P<0.001 in response to papaverine-induced flow increases of 121 ± 12%. In contrast, after similar papaverine-induced flow increases simultaneously with L-NMMA infusions, ascorbyl levels were not significantly changed compared to baseline. Similar results were obtained in isolated rabbit aortas perfused ex vivo with the spin trap a-phenyl-N-tert-butylnitrone (N = 22. However, in both preparations, this complete blockade was not reversed by co-infusion of excess L-arginine and was also obtained by N-methyl-D-arginine, thus indicating that it is not related to nitric oxide synthase. L-arginine alone was ineffective, as previously demonstrated for NG-methyl-L-arginine ester (L-NAME. In vitro, neither L-arginine nor its analogues scavenged superoxide radicals. This nonspecific activity of methylated arginine analogues underscores the need for careful controls in order to assess nitric oxide effects, particularly those related to interactions with active oxygen species.

  4. Effect of L-arginine supplement on liver regeneration after partial hepatectomy in rats

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    Kurokawa Tsuyoshi

    2012-05-01

    Full Text Available Abstract Background Nitric oxide (NO has been reported to be a key mediator in hepatocyte proliferation during liver regeneration. NO is the oxidative metabolite of L-arginine, and is produced by a family of enzymes, collective termed nitric oxide synthase (NOS. Thus, administration of L-arginine might enhance liver regeneration after a hepatectomy. Another amino acid, L-glutamine, which plays an important role in catabolic states and is a crucial factor in various cellular and organ functions, is widely known to enhance liver regeneration experimentally. Thus, the present study was undertaken to evaluate the effects of an L-arginine supplement on liver regeneration, and to compared this with supplementation with L-glutamine and L-alanine (the latter as a negative control, using a rat partial hepatectomy model. Methods Before and after a 70% hepatectomy, rats received one of three amino acid solutions (L-arginine, L-glutamine, or L-alanine. The effects on liver regeneration of the administered solutions were examined by assessment of restituted liver mass, staining for proliferating cell nuclear antigen (PCNA, and total RNA and DNA content 24 and 72 hours after the operation. Results At 72 hours after the hepatectomy, the restituted liver mass, the PCNA labeling index and the DNA quantity were all significantly higher in the L-arginine and L-glutamine groups than in the control. There were no significant differences in those parameters between the L-arginine and L-glutamine groups, nor were any significant differences found between the L-alanine group and the control. Conclusion Oral supplements of L-arginine and L-glutamine enhanced liver regeneration after hepatectomy in rats, suggesting that an oral arginine supplement can clinically improve recovery after a major liver resection.

  5. L-Arginine but not L-glutamine likely increases exogenous carbohydrate oxidation during endurance exercise.

    Science.gov (United States)

    Rowlands, David S; Clarke, Jim; Green, Jackson G; Shi, Xiaocai

    2012-07-01

    The addition of L-arginine or L-glutamine to glucose-electrolyte solutions can increase intestinal water, glucose, and sodium absorption in rats and humans. We evaluated the utility of L-arginine and L-glutamine in energy-rehydration beverages through assessment of exogenous glucose oxidation and perceptions of exertion and gastrointestinal distress during endurance exercise. Eight cyclists rode 150 min at 50% of peak power on four occasions while ingesting solutions at a rate of 150 mL 15 min(-1) that contained (13)C-enriched glucose (266 mmol L(-1)) and sodium citrate ([Na(+)] 60 mmol L(-1)), and either: 4.25 mmol L(-1) L-arginine or 45 mmol L(-1) L-glutamine, and as controls glucose only or no glucose. Relative to glucose only, L-arginine invoked a likely 12% increase in exogenous glucose oxidation (90% confidence limits: ± 8%); however, the effect of L-glutamine was possibly trivial (4.5 ± 7.3%). L-Arginine also led to very likely small reductions in endogenous fat oxidation rate relative to glucose (12 ± 4%) and L-glutamine (14 ± 4%), and relative to no glucose, likely reductions in exercise oxygen consumption (2.6 ± 1.5%) and plasma lactate concentration (0.20 ± 0.16 mmol L(-1)). Effects on endogenous and total carbohydrate oxidation were inconsequential. Compared with glucose only, L-arginine and L-glutamine caused likely small-moderate effect size increases in perceptions of stomach fullness, abdominal cramp, exertion, and muscle tiredness during exercise. Addition of L-arginine to a glucose and electrolyte solution increases the oxidation of exogenous glucose and decreases the oxygen cost of exercise, although the mechanisms responsible and impact on endurance performance require further investigation. However, L-arginine also increases subjective feelings of gastrointestinal distress, which may attenuate its other benefits.

  6. Effects of inhaled L-arginine administration in a murine model of acute asthma.

    Directory of Open Access Journals (Sweden)

    Zeynep Arikan-Ayyildiz

    2014-10-01

    Full Text Available Increased arginase activity in the airways decreases L-arginine and causes deficiency of bronchodilating and anti-inflammatory nitric oxide (NO in asthma. As, it is suggested that L-arginine may have therapeutic potential in asthma treatment, we aimed to investigate the effects of inhaled L-arginine on oxygen saturation (SaO₂ and airway histology in a murine model of acute asthma. Twenty eight BALB/c mice were divided into four groups; I, II, III and IV (control. All groups except the control were sensitized and challenged with ovalbumin. After establishement of acute asthma attack by metacholine administration, the mice were treated with inhaled L-arginine (Group I, saline (Group II and budesonide (Group III, respectively. SaO₂was measured by pulse oximeter just before and 5 min after methacholine. A third measurement of SaO₂was also obtained 15 min after drug administration in these study groups. Inflammation in the lung tissues of the sacrificed animals were scored to determine the effects of the study drugs. The number of eosinophils in bronchoalveolar lavage (BAL was determined. The results indicated that inflammatory scores significantly improved in groups receiving study drugs when compared with placebo and L-arginine was similar in decreasing scores when compared with budesonide. SaO₂had a tendency to increase after L-arginine administration after acute asthma attack and this increase was statistically significant (p=0.043. Eosinophilia in BAL significantly reduced in group receiving L-arginine when compared with placebo (p<0.05. Thus in this study we demonstrated that L-arginine improved SaO₂and inflammatory scores in an acute model of asthma.

  7. Chiral pharmacokinetics and inversion of NG-nitro-arginine in the rat

    Institute of Scientific and Technical Information of China (English)

    Yan-feiXIN; RuiTONG; YangFANG; Xiang-junZHOU; Yong-xiangWANG

    2004-01-01

    AIM: To explore pharmacokinetics of NG-nitro-D-arginine (D-NNA) and NG-nitro-L-arginine (L-NNA) in conscious rats.METHODS: The plasma concentration of D-NNA and L-NNA were determined by chiral ligand exchange method with capillary electrochromatography (CEC). Pharmacokinetic parameters were estimated using non-compartment model and were fitted using a computer program DAS. Chiral inversion rate of D-NNA to L-

  8. L-Arginine but not L-glutamine likely increases exogenous carbohydrate oxidation during endurance exercise.

    Science.gov (United States)

    Rowlands, David S; Clarke, Jim; Green, Jackson G; Shi, Xiaocai

    2012-07-01

    The addition of L-arginine or L-glutamine to glucose-electrolyte solutions can increase intestinal water, glucose, and sodium absorption in rats and humans. We evaluated the utility of L-arginine and L-glutamine in energy-rehydration beverages through assessment of exogenous glucose oxidation and perceptions of exertion and gastrointestinal distress during endurance exercise. Eight cyclists rode 150 min at 50% of peak power on four occasions while ingesting solutions at a rate of 150 mL 15 min(-1) that contained (13)C-enriched glucose (266 mmol L(-1)) and sodium citrate ([Na(+)] 60 mmol L(-1)), and either: 4.25 mmol L(-1) L-arginine or 45 mmol L(-1) L-glutamine, and as controls glucose only or no glucose. Relative to glucose only, L-arginine invoked a likely 12% increase in exogenous glucose oxidation (90% confidence limits: ± 8%); however, the effect of L-glutamine was possibly trivial (4.5 ± 7.3%). L-Arginine also led to very likely small reductions in endogenous fat oxidation rate relative to glucose (12 ± 4%) and L-glutamine (14 ± 4%), and relative to no glucose, likely reductions in exercise oxygen consumption (2.6 ± 1.5%) and plasma lactate concentration (0.20 ± 0.16 mmol L(-1)). Effects on endogenous and total carbohydrate oxidation were inconsequential. Compared with glucose only, L-arginine and L-glutamine caused likely small-moderate effect size increases in perceptions of stomach fullness, abdominal cramp, exertion, and muscle tiredness during exercise. Addition of L-arginine to a glucose and electrolyte solution increases the oxidation of exogenous glucose and decreases the oxygen cost of exercise, although the mechanisms responsible and impact on endurance performance require further investigation. However, L-arginine also increases subjective feelings of gastrointestinal distress, which may attenuate its other benefits. PMID:22048324

  9. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1.

    Science.gov (United States)

    Saxton, Robert A; Chantranupong, Lynne; Knockenhauer, Kevin E; Schwartz, Thomas U; Sabatini, David M

    2016-08-11

    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 acids. In mammals arginine is particularly important, promoting diverse physiological effects such as immune cell activation, insulin secretion, and muscle growth, largely mediated through activation of mTORC1 (refs 4, 5, 6, 7). Arginine activates mTORC1 upstream of the Rag family of GTPases, through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting Cellular Arginine Sensor for mTORC1 (CASTOR1). However, the mechanism by which the mTORC1 pathway detects and transmits this 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 Aspartate kinase, Chorismate mutase, TyrA (ACT) domains, enabling allosteric control of the adjacent GATOR2-binding site to trigger dissociation from GATOR2 and 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

  10. Abnormal mitochondrial L-arginine transport contributes to the pathogenesis of heart failure and rexoygenation injury.

    Directory of Open Access Journals (Sweden)

    David Williams

    Full Text Available BACKGROUND: Impaired mitochondrial function is fundamental feature of heart failure (HF and myocardial ischemia. In addition to the effects of heightened oxidative stress, altered nitric oxide (NO metabolism, generated by a mitochondrial NO synthase, has also been proposed to impact upon mitochondrial function. However, the mechanism responsible for arginine transport into mitochondria and the effect of HF on such a process is unknown. We therefore aimed to characterize mitochondrial L-arginine transport and to investigate the hypothesis that impaired mitochondrial L-arginine transport plays a key role in the pathogenesis of heart failure and myocardial injury. METHODS AND RESULTS: In mitochondria isolated from failing hearts (sheep rapid pacing model and mouse Mst1 transgenic model we demonstrated a marked reduction in L-arginine uptake (p<0.05 and p<0.01 respectively and expression of the principal L-arginine transporter, CAT-1 (p<0.001, p<0.01 compared to controls. This was accompanied by significantly lower NO production and higher 3-nitrotyrosine levels (both p<0.05. The role of mitochondrial L-arginine transport in modulating cardiac stress responses was examined in cardiomyocytes with mitochondrial specific overexpression of CAT-1 (mtCAT1 exposed to hypoxia-reoxygenation stress. mtCAT1 cardiomyocytes had significantly improved mitochondrial membrane potential, respiration and ATP turnover together with significantly decreased reactive oxygen species production and cell death following mitochondrial stress. CONCLUSION: These data provide new insights into the role of L-arginine transport in mitochondrial biology and cardiovascular disease. Augmentation of mitochondrial L-arginine availability may be a novel therapeutic strategy for myocardial disorders involving mitochondrial stress such as heart failure and reperfusion injury.

  11. Abnormal Mitochondrial L-Arginine Transport Contributes to the Pathogenesis of Heart Failure and Rexoygenation Injury

    Science.gov (United States)

    Byrne, Melissa; Joshi, Mandar; Horlock, Duncan; Lam, Nicholas T.; Gregorevic, Paul; McGee, Sean L.; Kaye, David M.

    2014-01-01

    Background Impaired mitochondrial function is fundamental feature of heart failure (HF) and myocardial ischemia. In addition to the effects of heightened oxidative stress, altered nitric oxide (NO) metabolism, generated by a mitochondrial NO synthase, has also been proposed to impact upon mitochondrial function. However, the mechanism responsible for arginine transport into mitochondria and the effect of HF on such a process is unknown. We therefore aimed to characterize mitochondrial L-arginine transport and to investigate the hypothesis that impaired mitochondrial L-arginine transport plays a key role in the pathogenesis of heart failure and myocardial injury. Methods and Results In mitochondria isolated from failing hearts (sheep rapid pacing model and mouse Mst1 transgenic model) we demonstrated a marked reduction in L-arginine uptake (p<0.05 and p<0.01 respectively) and expression of the principal L-arginine transporter, CAT-1 (p<0.001, p<0.01) compared to controls. This was accompanied by significantly lower NO production and higher 3-nitrotyrosine levels (both p<0.05). The role of mitochondrial L-arginine transport in modulating cardiac stress responses was examined in cardiomyocytes with mitochondrial specific overexpression of CAT-1 (mtCAT1) exposed to hypoxia-reoxygenation stress. mtCAT1 cardiomyocytes had significantly improved mitochondrial membrane potential, respiration and ATP turnover together with significantly decreased reactive oxygen species production and cell death following mitochondrial stress. Conclusion These data provide new insights into the role of L-arginine transport in mitochondrial biology and cardiovascular disease. Augmentation of mitochondrial L-arginine availability may be a novel therapeutic strategy for myocardial disorders involving mitochondrial stress such as heart failure and reperfusion injury. PMID:25111602

  12. L-canavanine and protein synthesis in the tobacco hornworm Manduca sexta

    Energy Technology Data Exchange (ETDEWEB)

    Rosenthal, G.A.; Dahlman, D.L.

    1986-01-01

    L-Canavanine, a nonprotein amino acid of certain leguminous plants, manifests potent insecticidal properties in a canavanine-sensitive insect such as the tobacco hornworm Manduca sexta (L.) (Sphingidae). This arginine analog is activated and aminoacylated by arginyl-tRNA synthetase and incorporated into nascent polypeptide chains to create structurally aberrant, canavanine-containing proteins. Analysis of incorporation of (TH)leucine into protein in M. sexta larvae that had been injected with canavanine revealed that this arginine analog stimulates protein synthesis. During the first 3 hr after injection of canavanine, canavanine-mediated net stimulation of protein formation was readily discerned. Thereafter, the stimulation of protein synthesis appeared to be offset by the preferential of degradation of anomalous proteins. Double-label protein-turnover experiments with larvae injected with ( UC)canavanine- and (TH)arginine-containing hemolymph proteins showed that canavanine-containing proteins were degraded preferentially.

  13. L-canavanine and protein synthesis in the tobacco hornworm Manduca sexta

    International Nuclear Information System (INIS)

    L-Canavanine, a nonprotein amino acid of certain leguminous plants, manifests potent insecticidal properties in a canavanine-sensitive insect such as the tobacco hornworm Manduca sexta (L.) (Sphingidae). This arginine analog is activated and aminoacylated by arginyl-tRNA synthetase and incorporated into nascent polypeptide chains to create structurally aberrant, canavanine-containing proteins. Analysis of incorporation of [3H]leucine into protein in M. sexta larvae that had been injected with canavanine revealed that this arginine analog stimulates protein synthesis. During the first 3 hr after injection of canavanine, canavanine-mediated net stimulation of protein formation was readily discerned. Thereafter, the stimulation of protein synthesis appeared to be offset by the preferential of degradation of anomalous proteins. Double-label protein-turnover experiments with larvae injected with [14C]canavanine- and [3H]arginine-containing hemolymph proteins showed that canavanine-containing proteins were degraded preferentially

  14. Adaptations of Arginine's Intestinal-Renal Axis in Cachectic Tumor-Bearing Rats.

    Science.gov (United States)

    Buijs, Nikki; Vermeulen, Mechteld A R; Weeda, Viola B; Bading, James R; Houdijk, Alexander P J; van Leeuwen, Paul A M

    2015-01-01

    Malignancies induce disposal of arginine, an important substrate for the immune system. To sustain immune function, the tumor-bearing host accelerates arginine's intestinal-renal axis by glutamine mobilization from skeletal muscle and this may promote cachexia. Glutamine supplementation stimulates argi-nine production in healthy subjects. Arginine's intestinal-renal axis and the effect of glutamine supplementation in cancer cach-exia have not been investigated. This study evaluated the long-term adaptations of the interorgan pathway for arginine production following the onset of cachexia and the metabolic effect of glutamine supplementation in the cachectic state. Fischer-344 rats were randomly divided into a tumor-bearing group (n = 12), control group (n = 7) and tumor-bearing group receiving a glutamine-enriched diet (n = 9). Amino acid fluxes and net fractional extractions across intestine, kidneys, and liver were studied. Compared to controls, the portal-drained viscera of tumor-bearing rats took up significantly more glutamine and released significantly less citrulline. Renal metabolism was unchanged in the cachectic tumor-bearing rats compared with controls. Glutamine supplementation had no effects on intestinal and renal adaptations. In conclusion, in the cachectic state, an increase in intestinal glutamine uptake is not accompanied by an increase in renal arginine production. The adaptations found in the cachectic, tumor-bearing rat do not depend on glutamine availability.

  15. N-terminal arginines modulate plasma-membrane localization of Kv7.1/KCNE1 channel complexes.

    Directory of Open Access Journals (Sweden)

    Zenawit Girmatsion

    Full Text Available BACKGROUND AND OBJECTIVE: The slow delayed rectifier current (I(Ks is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I(Ks and atrial fibrillation (a human arrhythmia. Structure-function relationship of the KCNE1 N-terminus for I(Ks modulation is poorly understood and was subject of this study. METHODS: We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy. RESULTS: All KCNE1 constructs physically interacted with Kv7.1. I(Ks resulting from co-expression of Kv7.1 with non-atrial fibrillation '38S' was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions ('38G-3xA' were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct ('Δ1-38'. Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct. CONCLUSIONS: The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I(Ks. Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex.

  16. Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences.

    Science.gov (United States)

    Suzuki, T; Inoue, N; Higashi, T; Mizobuchi, R; Sugimura, N; Yokouchi, K; Furukohri, T

    2000-12-01

    Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a Km value of 0.6 mM for arginine and a Vmax value of 13 micromole Pi min(-1) mg protein(-1) for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1,608 and 1,239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66-73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63-71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55-57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered. PMID:11281267

  17. Protective effect of Mimosa pudica L. in an L-arginine model of acute necrotising pancreatitis in rats.

    Science.gov (United States)

    Kaur, Jagdeep; Sidhu, Shabir; Chopra, Kanwaljit; Khan, M U

    2016-07-01

    Mimosa pudica is used in traditional medicine for treating various disorders such as inflammatory conditions, diarrhoea, insomnia, alopecia, urogenital infections and wounds. The present study investigated the effect of M. pudica extract (MPE) on L-arginine-induced acute necrotising pancreatitis in rats. The ethanolic extract of M. pudica leaves was studied for the presence of quercetin and gallic acid using high-performance liquid chromatography. Four groups were employed-normal control rats, L-arginine control rats (two intraperitoneal [i.p.] injections of 2 g/kg at an interval of 1 h), MPE-treated rats (400 mg/kg orally) and melatonin-treated rats (positive control 10 mg/kg i.p.), which were further divided into subgroups according to time points (24 h, 3 days and 14 days). Serum amylase, lipase, tumour necrosis factor-α (TNF-α), pancreatic amylase, nucleic acid content, protein, transforming growth factor-β1 (TGF-β1), thiobarbituric reactive substances, glutathione, nitrite/nitrate, collagen content and histopathological examination were carried out. MPE significantly improved acute necrotising pancreatitis by modulating diagnostic markers of pancreatitis such as serum lipase and pancreatic amylase, inflammation (TNF-α), and oxidative and nitrosative stress. Moreover, MPE administration induced regenerative changes in the pancreas evidenced by increased levels of pancreatic proteins, nucleic acid content and histopathology report. In addition, MPE improved TGF-β1 and collagen levels thereby preventing fibrosis. The current investigation indicates the novel role of MPE in reducing the severity of acute necrotising pancreatitis by plausible mechanisms such as anti-inflammatory and anti-fibrotic activity and by promoting repair and regeneration of the pancreas. PMID:27164910

  18. Effects of Arginine Vasopressin on musical short-term memory

    Directory of Open Access Journals (Sweden)

    Roni Y. Granot

    2013-10-01

    Full Text Available Previous genetic studies showed an association between variations in the gene coding for the 1a receptor of the neuro-hormone arginine vasopressin (AVP and musical working memory (WM. The current study set out to test the influence of intranasal administration (INA of AVP on musical as compared to verbal WM using a double blind crossover (AVP – placebo design. Two groups of 25 males were exposed to 20 IU of AVP in one session, and 20 IU of saline water (placebo in a second session, one week apart. In each session subjects completed the tonal subtest from Gordon's Musical Aptitude Profile, the interval subtest from the Montreal Battery for Evaluation of Amusias (MBEA, and the forward and backward digit span tests. Scores in the digit span tests were not influenced by AVP. In contrast, in the music tests there was an AVP effect. In the MBEA test, scores for the group receiving placebo in the first session (PV were higher than for the group receiving vasopressin in the first session (VP (p < .05 with no main Session effect nor Group * Session interaction. In the Gordon test there was a main Session effect (p < .05 with scores higher in the second as compared to the first session, a marginal main Group effect (p = .093 and a marginal Group X Session interaction (p = 0.88. In addition we found that the group that received AVP in the first session scored higher on scales indicative of happiness, and alertness on the Positive and Negative Affect Scale, (PANAS. Only in this group and only in the music test these scores were significantly correlated with memory scores. Together the results reflect a complex interaction between AVP, musical memory, arousal, and contextual effects such as session, and base levels of memory. The results are interpreted in light of music's universal use as a means to modulate arousal on the one hand, and AVP's influence on mood, arousal, and social interactions on the other.

  19. Spatiotemporal profiles of arginine vasopressin transcription in cultured suprachiasmatic nucleus.

    Science.gov (United States)

    Yoshikawa, Tomoko; Nakajima, Yoshihiro; Yamada, Yoshiko; Enoki, Ryosuke; Watanabe, Kazuto; Yamazaki, Maya; Sakimura, Kenji; Honma, Sato; Honma, Ken-ichi

    2015-11-01

    Arginine vasopressin (AVP), a major neuropeptide in the suprachiasmatic nucleus (SCN), is postulated to mediate the output of the circadian oscillation. Mice carrying a reporter gene of AVP transcription (AVP(ELuc)) were produced by knocking-in a cDNA of Emerald-luciferase (ELuc) in the translational initiation site. Homozygous mice did not survive beyond postnatal day 7. Using the heterozygous (AVP(ELuc/+)) mice, a bioluminescence reporter system was developed that enabled to monitor AVP transcription through AVP-ELuc measurement in real time for more than 10 cycles in the cultured brain slice. AVP(ELuc/+) mice showed circadian behaviour rhythms and light responsiveness indistinguishable from those of the wild-type. Robust circadian rhythms in AVP-ELuc were detected in the cultured SCN slice at a single cell as well as tissue levels. The circadian rhythm of the whole SCN slice was stable, with the peak at the mid-light phase of a light-dark cycle, while that of a single cell was more variable. By comparison, rhythmicity in the paraventricular nucleus and supraoptic nucleus in the hypothalamus was unstable and damped rapidly. Spatiotemporal profiles of AVP expression at the pixel level revealed significant circadian rhythms in the entire area of AVP-positive cells in the SCN, and at least two clusters that showed different circadian oscillations. Contour analysis of bioluminescence intensity in a cell-like region demonstrated the radiation area was almost identical to the cell size. This newly developed reporter system for AVP gene expression is a useful tool for the study of circadian rhythms. PMID:26342201

  20. Effects of arginine vasopressin on musical working memory.

    Science.gov (United States)

    Granot, Roni Y; Uzefovsky, Florina; Bogopolsky, Helena; Ebstein, Richard P

    2013-01-01

    Previous genetic studies showed an association between variations in the gene coding for the 1a receptor of the neuro-hormone arginine vasopressin (AVP) and musical working memory (WM). The current study set out to test the influence of intranasal administration (INA) of AVP on musical as compared to verbal WM using a double blind crossover (AVP-placebo) design. Two groups of 25 males were exposed to 20 IU of AVP in one session, and 20 IU of saline water (placebo) in a second session, 1 week apart. In each session subjects completed the tonal subtest from Gordon's "Musical Aptitude Profile," the interval subtest from the "Montreal Battery for Evaluation of Amusias (MBEA)," and the forward and backward digit span tests. Scores in the digit span tests were not influenced by AVP. In contrast, in the music tests there was an AVP effect. In the MBEA test, scores for the group receiving placebo in the first session (PV) were higher than for the group receiving vasopressin in the first session (VP) (p effect nor Group × Session interaction. In the Gordon test there was a main Session effect (p effect (p = 0.093) and a marginal Group × Session interaction (p = 0.88). In addition we found that the group that received AVP in the first session scored higher on scales indicative of happiness, and alertness on the positive and negative affect scale, (PANAS). Only in this group and only in the music test these scores were significantly correlated with memory scores. Together the results reflect a complex interaction between AVP, musical memory, arousal, and contextual effects such as session, and base levels of memory. The results are interpreted in light of music's universal use as a means to modulate arousal on the one hand, and AVP's influence on mood, arousal, and social interactions on the other. PMID:24151474

  1. Copper-induced tight junction mRNA expression changes, apoptosis and antioxidant responses via NF-κB, TOR and Nrf2 signaling molecules in the gills of fish: Preventive role of arginine

    International Nuclear Information System (INIS)

    Highlights: • Cu exposure induced oxidative stress via disruption of antioxidant system. • Cu exposure disrupted TJ mRNA expression through regulation of cytokines in fish. • Cu induced gill apoptosis partly via intrinsic pathway but not extrinsic pathway. • Cu exposure can regulate Nrf2, NF-κB and TOR signaling molecules in fish. • Arginine can effectively prevent Cu-induced fish gill damage. - Abstract: This study explored the possible preventive effects of dietary arginine on copper (Cu)-induced tight junction mRNA expression changes, apoptosis and antioxidant responses in the gills of young grass carp (Ctenopharyngodon idella). The results indicated that exposure to 0.7 mg/L (11.01 μmol/L) Cu for 96 h induced the production of reactive oxygen species (ROS), thereby increasing protein oxidation, lipid peroxidation and DNA damage in the gills of fish. However, these oxidative effects were prevented by arginine supplementation. Arginine also prevented the toxic effects of Cu on the activities of copper/zinc superoxide dismutase (SOD1), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) and the glutathione (GSH) content (P < 0.05). However, Cu induced an adaptive increase in the activity of catalase (CAT), and arginine supplementation further increased CAT activity (P < 0.05). Moreover, Cu induced increases in the relative mRNA expressions of SOD1, CAT, GPx, GST, caspase-3, caspase-9, NF-E2-related factor 2 (Nrf2), Kelch-like-ECH-associated protein 1a (Keap1a), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-8 (IL-8), transforming growth factor-β (TGF-β) and nuclear transcription factor-κB p65 (NF-κB p65) in the gills of grass carp (P < 0.05). In contrast, the relative mRNA expression levels of occludin, zonula occludens-1 (ZO-1), claudin b, claudin 3, claudin 12, target of rapamycin (TOR) and inhibitor factor κBα (IκBα) in the gills were decreased by Cu (P < 0.05). However, pre

  2. Copper-induced tight junction mRNA expression changes, apoptosis and antioxidant responses via NF-κB, TOR and Nrf2 signaling molecules in the gills of fish: Preventive role of arginine

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Biao [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Feng, Lin; Jiang, Wei-Dan [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Wu, Pei [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Kuang, Sheng-Yao [Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu, 610066, Sichuan (China); Jiang, Jun [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Tang, Ling; Tang, Wu-Neng [Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu, 610066, Sichuan (China); Zhang, Yong-An [Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Liu, Yang, E-mail: kyckgk@hotmail.com [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); and others

    2015-01-15

    Highlights: • Cu exposure induced oxidative stress via disruption of antioxidant system. • Cu exposure disrupted TJ mRNA expression through regulation of cytokines in fish. • Cu induced gill apoptosis partly via intrinsic pathway but not extrinsic pathway. • Cu exposure can regulate Nrf2, NF-κB and TOR signaling molecules in fish. • Arginine can effectively prevent Cu-induced fish gill damage. - Abstract: This study explored the possible preventive effects of dietary arginine on copper (Cu)-induced tight junction mRNA expression changes, apoptosis and antioxidant responses in the gills of young grass carp (Ctenopharyngodon idella). The results indicated that exposure to 0.7 mg/L (11.01 μmol/L) Cu for 96 h induced the production of reactive oxygen species (ROS), thereby increasing protein oxidation, lipid peroxidation and DNA damage in the gills of fish. However, these oxidative effects were prevented by arginine supplementation. Arginine also prevented the toxic effects of Cu on the activities of copper/zinc superoxide dismutase (SOD1), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) and the glutathione (GSH) content (P < 0.05). However, Cu induced an adaptive increase in the activity of catalase (CAT), and arginine supplementation further increased CAT activity (P < 0.05). Moreover, Cu induced increases in the relative mRNA expressions of SOD1, CAT, GPx, GST, caspase-3, caspase-9, NF-E2-related factor 2 (Nrf2), Kelch-like-ECH-associated protein 1a (Keap1a), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-8 (IL-8), transforming growth factor-β (TGF-β) and nuclear transcription factor-κB p65 (NF-κB p65) in the gills of grass carp (P < 0.05). In contrast, the relative mRNA expression levels of occludin, zonula occludens-1 (ZO-1), claudin b, claudin 3, claudin 12, target of rapamycin (TOR) and inhibitor factor κBα (IκBα) in the gills were decreased by Cu (P < 0.05). However, pre

  3. The role of nitric oxide in L-arginine-stimulated growth hormone secretion%一氧化氮在L-精氨酸促生长激素分泌中的作用

    Institute of Scientific and Technical Information of China (English)

    蒋明玉; 蔡德培

    2011-01-01

    Amino acids have been pointed out as potent stimulators of growth hormone (GH) secretion. Among them, L-arginine is considered one of the most effective GH releasers. L-arginine increases GH release in pituitary when given orallyor intravenously. L-arginine-stimulated GH secretion can be mediated by several signaling systems, but nitric oxide (NO) dependent soluble guanylyl cyclase (sGC) -cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway plays a key role in the process of L-arginine-stimulated GH secretion.%氨基酸是促进垂体生长激素(GH)分泌的潜在刺激因子.在所有氨基酸中,左旋精氨酸(L-精氨酸)促GH分泌作用最为明显.口服或静脉滴注精氨酸均可刺激垂体GH的分泌.L-精氨酸促GH的分泌可能涉及多种机制,但一氧化氮(NO)依赖的可溶性鸟苷酸环化酶-环鸟苷酸-蛋白激酶G(sGC-cGMP-PKG)信号转导通路在此过程中起着重要作用.

  4. A novel mutation affecting the arginine-137 residue of AVPR2 in dizygous twins leads to nephrogenic diabetes insipidus and attenuated urine exosome aquaporin-2

    DEFF Research Database (Denmark)

    Hinrichs, Gitte R; Hansen, Louise H; Nielsen, Maria R;

    2016-01-01

    Mutations in the vasopressin V2 receptor gene AVPR2 may cause X-linked nephrogenic diabetes insipidus by defective apical insertion of aquaporin-2 in the renal collecting duct principal cell. Substitution mutations with exchange of arginine at codon 137 can cause nephrogenic syndrome of inappropr...... administration. While a similar urine exosome release rate was shown between probands and controls by western blotting for the marker ALIX, there was a selective decrease in exosome aquaporin-2 versus aquaporin-1 protein in probands compared to controls....

  5. The hydrophobic core of twin-arginine signal sequences orchestrates specific binding to Tat-pathway related chaperones.

    Directory of Open Access Journals (Sweden)

    Anitha Shanmugham

    Full Text Available Redox enzyme maturation proteins (REMPs bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA and TorD (specific for trimethylamine N-oxide reductase, TorA. Green fluorescent protein (GFP was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function.

  6. Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.

    Science.gov (United States)

    Sinzel, Monika; Tan, Tao; Wendling, Philipp; Kalbacher, Hubert; Özbalci, Cagakan; Chelius, Xenia; Westermann, Benedikt; Brügger, Britta; Rapaport, Doron; Dimmer, Kai Stefan

    2016-07-01

    Mitochondria are separated from the remainder of the eukaryotic cell by the mitochondrial outer membrane (MOM). The MOM plays an important role in different transport processes like lipid trafficking and protein import. In yeast, the ER-mitochondria encounter structure (ERMES) has a central, but poorly defined role in both activities. To understand the functions of the ERMES, we searched for suppressors of the deficiency of one of its components, Mdm10, and identified a novel mitochondrial protein that we named Mdm10 complementing protein 3 (Mcp3). Mcp3 partially rescues a variety of ERMES-related phenotypes. We further demonstrate that Mcp3 is an integral protein of the MOM that follows a unique import pathway. It is recognized initially by the import receptor Tom70 and then crosses the MOM via the translocase of the outer membrane. Mcp3 is next relayed to the TIM23 translocase at the inner membrane, gets processed by the inner membrane peptidase (IMP) and finally integrates into the MOM. Hence, Mcp3 follows a novel biogenesis route where a MOM protein is processed by a peptidase of the inner membrane. PMID:27226123

  7. Arginine deiminase resistance in melanoma cells is associated with metabolic reprogramming, glucose dependence, and glutamine addiction.

    Science.gov (United States)

    Long, Yan; Tsai, Wen-Bin; Wangpaichitr, Medhi; Tsukamoto, Takashi; Savaraj, Niramol; Feun, Lynn G; Kuo, Macus Tien

    2013-11-01

    Many malignant human tumors, including melanomas, are auxotrophic for arginine due to reduced expression of argininosuccinate synthetase-1 (ASS1), the rate-limiting enzyme for arginine biosynthesis. Pegylated arginine deiminase (ADI-PEG20), which degrades extracellular arginine, resulting in arginine deprivation, has shown favorable results in clinical trials for treating arginine-auxotrophic tumors. Drug resistance is the major obstacle for effective ADI-PEG20 usage. To elucidate mechanisms of resistance, we established several ADI-PEG20-resistant (ADI(R)) variants from A2058 and SK-Mel-2 melanoma cells. Compared with the parental lines, these ADI(R) variants showed the following characteristics: (i) all ADI(R) cell lines showed elevated ASS1 expression, resulting from the constitutive binding of the transcription factor c-Myc on the ASS1 promoter, suggesting that elevated ASS1 is the major mechanism of resistance; (ii) the ADI(R) cell lines exhibited enhanced AKT signaling and were preferentially sensitive to PI3K/AKT inhibitors, but reduced mTOR signaling, and were preferentially resistant to mTOR inhibitor; (iii) these variants showed enhanced expression of glucose transporter-1 and lactate dehydrogenase-A, reduced expression of pyruvate dehydrogenase, and elevated sensitivity to the glycolytic inhibitors 2-deoxy-glucose and 3-bromopyruvate, consistent with the enhanced glycolytic pathway (the Warburg effect); (iv) the resistant cells showed higher glutamine dehydrogenase and glutaminase expression and were preferentially vulnerable to glutamine inhibitors. We showed that c-Myc, not elevated ASS1 expression, is involved in upregulation of many of these enzymes because knockdown of c-Myc reduced their expression, whereas overexpressed ASS1 by transfection reduced their expression. This study identified multiple targets for overcoming ADI-PEG resistance in cancer chemotherapy using recombinant arginine-degrading enzymes.

  8. Arginine deiminase resistance in melanoma cells is associated with metabolic reprogramming, glucose dependence, and glutamine addiction.

    Science.gov (United States)

    Long, Yan; Tsai, Wen-Bin; Wangpaichitr, Medhi; Tsukamoto, Takashi; Savaraj, Niramol; Feun, Lynn G; Kuo, Macus Tien

    2013-11-01

    Many malignant human tumors, including melanomas, are auxotrophic for arginine due to reduced expression of argininosuccinate synthetase-1 (ASS1), the rate-limiting enzyme for arginine biosynthesis. Pegylated arginine deiminase (ADI-PEG20), which degrades extracellular arginine, resulting in arginine deprivation, has shown favorable results in clinical trials for treating arginine-auxotrophic tumors. Drug resistance is the major obstacle for effective ADI-PEG20 usage. To elucidate mechanisms of resistance, we established several ADI-PEG20-resistant (ADI(R)) variants from A2058 and SK-Mel-2 melanoma cells. Compared with the parental lines, these ADI(R) variants showed the following characteristics: (i) all ADI(R) cell lines showed elevated ASS1 expression, resulting from the constitutive binding of the transcription factor c-Myc on the ASS1 promoter, suggesting that elevated ASS1 is the major mechanism of resistance; (ii) the ADI(R) cell lines exhibited enhanced AKT signaling and were preferentially sensitive to PI3K/AKT inhibitors, but reduced mTOR signaling, and were preferentially resistant to mTOR inhibitor; (iii) these variants showed enhanced expression of glucose transporter-1 and lactate dehydrogenase-A, reduced expression of pyruvate dehydrogenase, and elevated sensitivity to the glycolytic inhibitors 2-deoxy-glucose and 3-bromopyruvate, consistent with the enhanced glycolytic pathway (the Warburg effect); (iv) the resistant cells showed higher glutamine dehydrogenase and glutaminase expression and were preferentially vulnerable to glutamine inhibitors. We showed that c-Myc, not elevated ASS1 expression, is involved in upregulation of many of these enzymes because knockdown of c-Myc reduced their expression, whereas overexpressed ASS1 by transfection reduced their expression. This study identified multiple targets for overcoming ADI-PEG resistance in cancer chemotherapy using recombinant arginine-degrading enzymes. PMID:23979920

  9. Metabolism via arginase or nitric oxide synthase: two competing arginine pathways in macrophages

    Directory of Open Access Journals (Sweden)

    Meera eRath

    2014-10-01

    Full Text Available Macrophages play a major role in the immune system, both as antimicrobial effector cells and as immunoregulatory cells, which induce, suppress or modulate adaptive immune responses. These key aspects of macrophage biology are fundamentally driven by the phenotype of macrophage arginine metabolism that is prevalent in an evolving or ongoing immune response. M1 macrophages express the enzyme nitric oxide synthase (NOS, which metabolizes arginine to nitric oxide (NO and citrulline. NO can be metabolized to further downstream reactive nitrogen species, while citrulline might be reused for efficient NO synthesis via the citrulline-NO cycle. M2 macrophages are characterized by expression of the enzyme arginase, which hydrolyzes arginine to ornithine and urea. The arginase pathway limits arginine availability for NO synthesis and ornithine itself can further feed into the important downstream pathways of polyamine and proline syntheses, which are important for cellular proliferation and tissue repair. M1 versus M2 polarization leads to opposing outcomes of inflammatory reactions, but depending on the context, M1 and M2 macrophages can be both pro- and antiinflammatory. Notably, M1/M2 macrophage polarization can be driven by microbial infection or innate danger signals without any influence of adaptive immune cells, secondarily driving the T helper (Th1/Th2 polarization of the evolving adaptive immune response. Since both arginine metabolic pathways cross-inhibit each other on the level of the respective arginine break-down products and Th1 and Th2 lymphocytes can drive or amplify macrophage M1/M2 dichotomy via cytokine activation, this forms the basis of a self-sustaining M1/M2 polarization of the whole immune response. Understanding the arginine metabolism of M1/M2 macrophage phenotypes is therefore central to find new possibilities to manipulate immune responses in infection, autoimmune diseases, chronic inflammatory conditions and cancer.

  10. Expression pattern of a nuclear encoded mitochondrial arginine-ornithine translocator gene from Arabidopsis

    Directory of Open Access Journals (Sweden)

    Schneider Anja

    2003-01-01

    Full Text Available Abstract Background Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine. Results High-level expression of a nuclear encoded Arabidopsis thaliana homolog (AtmBAC2 of Ort1p/Arg11p was able to suppress the growth deficiency of arg11. RT-PCR analysis demonstrated expression of AtmBAC2 in all tissues with highest levels in flowers. Promoter-GUS fusions showed preferential expression in flowers, i.e. pollen, in the vasculature of siliques and in aborted seeds. Variable expression was observed in leaf vasculature. Induction of the promoter was not observed during the first two weeks in seedlings grown on media containing NH4NO3, arginine or ornithine as sole nitrogen sources. Conclusion AtmBAC2 was isolated as a mitochondrial transporter for arginine in Arabidopsis. The absence of expression in developing seeds and in cotyledons of seedlings indicates that other transporters are responsible for storage and mobilization of arginine in seeds.

  11. Serum amino acids profile and the beneficial effects of L-arginine or L-glutamine supplementation in dextran sulfate sodium colitis.

    Science.gov (United States)

    Ren, Wenkai; Yin, Jie; Wu, Miaomiao; Liu, Gang; Yang, Guan; Xion, Yan; Su, Dingding; Wu, Li; Li, Tiejun; Chen, Shuai; Duan, Jielin; Yin, Yulong; Wu, Guoyao

    2014-01-01

    This study was conducted to investigate serum amino acids profile in dextran sulfate sodium (DSS)-induced colitis, and impacts of graded dose of arginine or glutamine supplementation on the colitis. Using DSS-induced colitis model, which is similar to human ulcerative colitis, we determined serum profile of amino acids at day 3, 7, 10 and 12 (5 days post DSS treatment). Meanwhile, effects of graded dose of arginine (0.4%, 0.8%, and 1.5%) or glutamine (0.5%, 1.0% and 2.0%) supplementation on clinical parameters, serum amino acids, colonic tight junction proteins, colonic anti-oxidative indicators [catalase, total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px)], colonic pro-inflammatory cytokines [interleukin-1 beta (IL-1β), IL-6, IL-17 and tumor necrosis factor alpha (TNF-α)] in DSS-induced colitis were fully analyzed at day 7 and 12. Additionally, the activation of signal transduction pathways, including nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt), and myosin light chain kinase (MLCK)-myosin light chain (MLC20), were analyzed using immunoblotting. Serum amino acids analysis showed that DSS treatment changed the serum contents of amino acids, such as Trp, Glu, and Gln (Parginine or glutamine supplementation had significant (Parginine or glutamine could be a potential therapy for intestinal inflammatory diseases.

  12. Absence of Btn1p in the yeast model for juvenile Batten disease may cause arginine to become toxic to yeast cells.

    Science.gov (United States)

    Vitiello, Seasson Phillips; Wolfe, Devin M; Pearce, David A

    2007-05-01

    Lymphoblast cell lines established from individuals with juvenile Batten disease (JNCL) bearing mutations in CLN3 and yeast strains lacking Btn1p (btn1-Delta), the homolog to CLN3, have decreased intracellular levels of arginine and defective lysosomal/vacuolar transport of arginine. It is important to establish the basis for this decrease in arginine levels and whether restoration of arginine levels would be of therapeutic value for Batten disease. Previous studies have suggested that synthesis and degradation of arginine are unaltered in btn1-Delta. Using the yeast model for the Batten disease, we have determined that although btn1-Delta results in decreased intracellular arginine levels, it does not result from altered arginine uptake, arginine efflux or differences in arginine incorporation into peptides. However, expression of BTN1 is dependent on arginine and Gcn4p, the master regulator of amino acid biosynthesis. Moreover, deletion of GCN4 (gcn4-Delta), in combination with btn1-Delta, results in a very specific growth requirement for arginine. In addition, increasing the intracellular levels of arginine through overexpression of Can1p, the plasma membrane basic amino acid permease, results in increased cell volume and a severe growth defect specific to basic amino acid availability for btn1-Delta, but not wild-type cells. Therefore, elevation of intracellular levels of arginine in btn1-Delta cells is detrimental and is suggestive that btn1-Delta and perhaps mutation of CLN3 predispose cells to keep arginine levels lower than normal.

  13. Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

    Science.gov (United States)

    Mandal, Abhishek; Das, Sushmita; Roy, Saptarshi; Ghosh, Ayan Kumar; Sardar, Abul Hasan; Verma, Sudha; Saini, Savita; Singh, Ruby; Abhishek, Kumar; Kumar, Ajay; Mandal, Chitra; Das, Pradeep

    2016-01-01

    The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis. PMID:26808657

  14. Arginine: A Potent Prey Attractant to Predatory Newts in Mountain Streams

    Science.gov (United States)

    Ferrer, R. P.; Zimmer, R. K.

    2005-05-01

    Chemoreception of aquatic organisms has been well-studied in the laboratory, but rarely in the field. The California newt, Taricha torosa, in natural stream habitats is an excellent animal for exploring behavioral responses to prey odors. Here, we selected 13 amino acids for field bioassays based on their concentrations in prey tissue extracts. Bioassays were calibrated for stimulus dilution by means of fluorescent dye releases and flow-through spectrofluorometry. Moreover, hydrodynamic properties of stream flows were determined using an electromagnetic current meter. Of all amino acids tested, only arginine, alanine and glycine were significantly attractive (relative to stream water controls). These three substances caused free-ranging newts to turn upstream and swim towards the odor sources. Additional experiments showed that arginine was the most effective attractant, evoking plume-tracking behavior at concentrations as low as 10 nM. In subsequent trials, nine arginine analogs were tested, but each compound failed to elicit a significant response. Even subtle changes to arginine, such as the addition of a single carbon to the side chain, destroyed all bioactivity. Within its natural habitat, the California newt thus exhibits keen sensitivity and narrow tuning to the free amino acid, arginine, a chemical signal of its prey.

  15. The development of poly-L-arginine-coated liposomes for gene delivery

    Directory of Open Access Journals (Sweden)

    Opanasopit P

    2011-10-01

    Full Text Available Praneet Opanasopit1, Jintana Tragulpakseerojn1, Auayporn Apirakaramwong1, Tanasait Ngawhirunpat1, Theerasak Rojanarata1, Uracha Ruktanonchai21Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand; 2National Nanotechnology Center, Thailand Science Park, Pathumthani, Thailand Abstract: In this study, liposomes coated with cationic polymers, poly-L-arginine (PLA, were assessed as a promising gene transfer system in human cervical carcinoma (HeLa cells and human hepatoma cell line (Huh7 cells. The liposomes were prepared using egg yolk phosphatidylcholine and sodium oleate in the molar ratio of 10:2 with an ultrasonic generator and then coated with PLA. The PLA-coated liposomes (PCLs formed complexes with plasmid DNA encoding green fluorescent protein. The complexes were characterized by agarose gel electrophoresis and investigated for their transfection efficiency in HeLa and Huh7 cells. The data were compared with PLA/DNA complexes and the positive control Lipofectamine 2000TM. The results showed that complete PCL/DNA complexes were formed at weight ratios of more than 0.05. Efficient gene transfer by PCLs was dependent on the cell type. The transfection efficiency of PCLs was about two times higher than that of PLA/DNA complexes in both HeLa cells and Huh7 cells. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay and showed that 80%-100% of both of the cells were viable after treating PCL/DNA complexes. The present results demonstrate that PCLs are a promising, nonviral gene carrier with low toxicity.Keywords: PLA-coated liposomes, PLA, gene delivery, transfection efficiency 

  16. Mapping and characterization of antigenic epitopes of arginine kinase of Scylla paramamosain.

    Science.gov (United States)

    Yang, Yang; Cao, Min-Jie; Alcocer, Marcos; Liu, Qing-Mei; Fei, Dan-Xia; Mao, Hai-Yan; Liu, Guang-Ming

    2015-06-01

    Arginine kinase (AK) is a panallergen present in crustaceans, which can induce an immunoglobulin (Ig) E-mediated immune response in humans. The aim of this work was to map and characterize the antigenic epitopes of Scylla paramamosain AK. Specific-protein-A-enriched IgG raised in rabbits against purified S. paramamosain AK was used to screen a phage display random peptide library. Five AK mimotope clones were identified among 20 random clones after biopanning. Four conformational epitopes D3A4K43M1A5T49T44I7, L31K33V35T32E11E18F14S34D37, V177G172M173D176Q178T174L181K175L187, and R202L170Y203E190P205W204L187T206Y145 were identified with the program LocaPep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively. On the basis of biopanning, six IgE-specific peptides were mapped with synthetic overlapping peptides using the sera from crab-allergic patients, and four seropositive peptides (amino acids 113-127, 127-141, 141-155, and 204-218) were confirmed as linear epitopes in a degranulation assay in RBL-2H3 cells. Stability experiments showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability. PMID:25728640

  17. Deletion of Genes Encoding Arginase Improves Use of "Heavy" Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast.

    Directory of Open Access Journals (Sweden)

    Weronika E Borek

    Full Text Available The use of "heavy" isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC mass spectrometry in the fission yeast Schizosaccharomyces pombe is hindered by the fact that under normal conditions, arginine is extensively catabolized in vivo, resulting in the appearance of "heavy"-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This "arginine conversion problem" significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism. Here we show that although this method is indeed successful when (13C6-arginine (Arg-6 is used for labeling, it is less successful when (13C6(15N4-arginine (Arg-10, a theoretically preferable label, is used. In particular, we find that with this method, "heavy"-isotope label derived from Arg-10 is observed in amino acids other than arginine, indicating metabolic conversion of Arg-10. Arg-10 conversion, which severely complicates both MS and MS/MS analysis, is further confirmed by the presence of (13C5(15N2-arginine (Arg-7 in arginine-containing peptides from Arg-10-labeled cells. We describe how all of the problems associated with the use of Arg-10 can be overcome by a simple modification of our original method. We show that simultaneous deletion of the fission yeast arginase genes car1+ and aru1+ prevents virtually all of the arginine conversion that would otherwise result from the use of Arg-10. This solution should enable a wider use of heavy isotope-labeled amino acids in fission yeast SILAC.

  18. Regulatory role for L-arginine in the utilization of amino acids by pig small-intestinal bacteria.

    Science.gov (United States)

    Dai, Zhao-Lai; Li, Xi-Long; Xi, Peng-Bin; Zhang, Jing; Wu, Guoyao; Zhu, Wei-Yun

    2012-07-01

    We recently reported that bacteria from the pig small intestine rapidly utilize and metabolize amino acids (AA). This study investigated the effect of L-arginine on the utilization of AA by pure bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the pig small intestine. Bacteria were incubated at 37°C for 3 h in anaerobic AA media containing 0-5 mmol/L of arginine to determine the effect of arginine on the bacterial utilization of AA. Amino acids in the medium plus cell extracts were analyzed by high-performance liquid chromatography. Results indicated concentration-dependent increases in the bacterial utilization of arginine and altered fluxes of arginine into ornithine and citrulline in the bacteria. Net glutamine utilization increased in pure bacterial strains with increased concentrations of arginine. With the addition of arginine, net utilization of threonine, glycine, phenylalanine and branched-chain AA increased (P<0.05) in Streptococcus sp. and Klebsiella sp., but decreased in E. coli. Net utilization of lysine, threonine, isoleucine, leucine, glycine and alanine by jejunal or ileal mixed bacteria decreased (P<0.05) with the addition of arginine. Complete utilization of asparagine, aspartate and serine were observed in pig small-intestinal bacteria after 3 h of incubation. Overall, the addition of arginine affected the metabolism of the arginine-family of AA and the serine- and aspartate-family of AA in small-intestinal bacteria and reduced the utilization of most AA in ileal mixed bacteria. These novel findings indicate that arginine exerts its beneficial effects on swine nutrition partially by regulating AA utilization and metabolism in the small-intestinal microbiota.

  19. Signifiance of Arginine 20 in the 2A protease for swine vesicular disease virus pathogenicity

    DEFF Research Database (Denmark)

    Inoue, Toru; Zhang, Zhidong; Wang, Leyuan;

    2007-01-01

    of the 2A protease is particularly significant. Inoculation of pigs with mutant viruses containing single amino acid substitutions at this residue leads to the appearance of revertants, often containing an arginine at this position encoded by an AGA codon, one of six codons for this residue. The properties...... in pigs of two chimeric viruses, each with an arginine residue at this position but encoded by different codons, have been investigated in parallel with the parental pathogenic and attenuated strains. Presence of the arginine residue, but not of the AGA codon, is essential for induction of high viraemia......Pathogenic and attenuated strains of swine vesicular disease virus (SVDV), an enterovirus, have been characterized previously and, by using chimeric infectious cDNA clones, the key determinants of pathogenicity in pigs have been mapped to the coding region for 1D–2A. Within this region, residue 20...

  20. Oral arginine metabolism may decrease the risk for dental caries in children.

    Science.gov (United States)

    Nascimento, M M; Liu, Y; Kalra, R; Perry, S; Adewumi, A; Xu, X; Primosch, R E; Burne, R A

    2013-07-01

    Arginine metabolism by oral bacteria via the arginine deiminase system (ADS) increases the local pH, which can neutralize the effects of acidification from sugar metabolism and reduce the cariogenicity of oral biofilms. To explore the relationship between oral arginine metabolism and dental caries experience in children, we measured ADS activity in oral samples from 100 children and correlated it with their caries status and type of dentition. Supragingival dental plaque was collected from tooth surfaces that were caries-lesion-free (PF) and from dentinal (PD) and enamel (PE) caries lesions. Regardless of children's caries status or type of dentition, PF (378.6) had significantly higher ADS activity compared with PD (208.4; p caries status. Mixed-model analysis showed that plaque caries status is significantly associated with ADS activity despite children's age, caries status, and dentition (p caries.

  1. Yeast Interacting Proteins Database: YML064C, YPL111W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available both induction by arginine and nitrogen catabolite repression; disruption enhanc...esponsible for arginine degradation, expression responds to both induction by arginine and nitrogen catab

  2. GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate.

    Science.gov (United States)

    Laroute, Valérie; Yasaro, Chonthicha; Narin, Waranya; Mazzoli, Roberto; Pessione, Enrica; Cocaign-Bousquet, Muriel; Loubière, Pascal

    2016-01-01

    Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes. PMID:27458444

  3. Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

    Directory of Open Access Journals (Sweden)

    Markus Munder

    Full Text Available Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i CD8(+ T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

  4. Potential protective effect of arginine against 4-nitrophenol-induced ovarian damage in rats.

    Science.gov (United States)

    Xu, Wei-Feng; Li, Yan-Sen; Dai, Peng-Yuan; Li, Chun-Mei

    2016-01-01

    4-nitrophenol (PNP) is generally regarded as a diesel exhaust particle (DEP). Arginine plays an important role as a new feed additive, possessing highly efficient antioxidant activities. Here we investigated the effects of dietary supplementation with arginine against ovarian damage induced by PNP in rats. A total of thirty-two female rats postnatal day 28 (PND 28) were randomly divided into four groups. Two groups were fed with basal diet or 13 g/kg arginine in diet for 4 weeks, respectively; the other two groups were given PNP (100 mg/kg b.w.) daily by subcutaneous injection for 2 weeks following pretreatment with either basal diet or arginine diet for 2 weeks. The values of body weight gain (BWG), average daily gain (ADG) and percentage weight gain (PWG) upon PNP treatment were significantly reduced than those in other groups. The relative liver weight in the PNP group was significantly decreased compared with the control group. Treatment with PNP significant reduced the number of corpora lutea, although serum 17β-estradiol (E2) and progesterone (P4) concentrations were unchanged. The morphology of the ovaries in PNP-treated rats displayed necrosis, follicular deformation and granulosa cells irregular arrangement. Moreover, exposure to PNP enhanced production of malondialdehyde (MDA) and hydrogen peroxide (H2O2), and decreased the activities of total superoxide dismutase (T-SOD) and catalase (CAT), and the co-administration of arginine can attenuate the oxidative stress caused by PNP. These results suggest that arginine may have a protective effect against ovarian damage induced by PNP owing to its antioxidant capacity effect. PMID:27193729

  5. Linear short histidine and cysteine modified arginine peptides constitute a potential class of DNA delivery agents.

    Science.gov (United States)

    Mann, Anita; Shukla, Vasundhara; Khanduri, Richa; Dabral, Spoorti; Singh, Harpal; Ganguli, Munia

    2014-03-01

    The success of gene therapy relies on the development of safe and efficient multifunctional carriers of nucleic acids that can overcome extra- and intracellular barriers, protect the nucleic acid and mediate its release at the desired site allowing gene expression. Peptides bear unique properties that are indispensable for any carrier, e.g., they can mediate DNA condensation, cellular targeting, membrane translocation, endosomal escape and nuclear localization. In an effort to design a multifunctional peptide, we have modified an arginine homopeptide R16 by replacement of seven arginines with histidines and addition of one cysteine at each end respectively to impart endosomal escape property while maintaining the DNA condensation and release balance. Addition of histidines imparts endosomal escape property to arginine homopeptide, but their arrangement with respect to arginines is more critical in controlling DNA condensation, release and transfection efficiency. Intriguingly, R5H7R4 peptide where charge/arginine is distributed in blocks is preferred for strong condensation while more efficient transfection is seen in the variants R9H7 and H4R9H3, which exhibit weak condensation and strong release. Addition of cysteine to each of these peptides further fine-tuned the condensation-release balance without application of any oxidative procedure unlike other similar systems reported in the literature. This resulted in a large increase in the transfection efficiency in all of the histidine modified peptides irrespective of the arginine and histidine positions. This series of multifunctional peptides shows comparable transfection efficiency to commercially available transfection reagent Lipofectamine 2000 at low charge ratios, with simple preparative procedure and exhibits much less toxicity. PMID:24476132

  6. Effects of chronic oral L-arginine administration on the L-arginine/NO pathway in patients with peripheral arterial occlusive disease or coronary artery disease: L-Arginine prevents renal loss of nitrite, the major NO reservoir.

    Science.gov (United States)

    Schneider, Jessica Y; Rothmann, Sabine; Schröder, Frank; Langen, Jennifer; Lücke, Thomas; Mariotti, François; Huneau, Jean François; Frölich, Jürgen C; Tsikas, Dimitrios

    2015-09-01

    Despite saturation of nitric oxide (NO) synthase (NOS) by its substrate L-arginine (Arg), oral and intravenous supplementation of Arg may enhance NO synthesis, a phenomenon known as "The L-arginine paradox". Yet, Arg is not only a source of NO, but is also a source for guanidine-methylated (N (G)) arginine derivatives which are all inhibitors of NOS activity. Therefore, Arg supplementation may not always result in enhanced NO synthesis. Concomitant synthesis of N (G)-monomethyl arginine (MMA), N (G),N (G)-dimethylarginine (asymmetric dimethylarginine, ADMA) and N (G),N (G´)-dimethylarginine (symmetric dimethylarginine, SDMA) from supplemented Arg may outweigh and even outbalance the positive effects of Arg on NO. Another possible, yet little investigated effect of Arg supplementation may be alteration of renal function, notably the influence on the excretion of nitrite in the urine. Nitrite is the autoxidation product of NO and the major reservoir of NO in the circulation. Nitrite and Arg are reabsorbed in the proximal tubule of the nephron and this reabsorption is coupled, at least in part, to the renal carbonic anhydrase (CA) activity. In the present placebo-controlled studies, we investigated the effect of chronic oral Arg supplementation of 10 g/day for 3 or 6 months in patients suffering from peripheral arterial occlusive disease (PAOD) or coronary artery disease (CAD) on the urinary excretion of nitrite relative to nitrate. We determined the urinary nitrate-to-nitrite molar ratio (UNOxR), which is a measure of nitrite-dependent renal CA activity before and after oral intake of Arg or placebo by the patients. The UNOxR was also determined in 6 children who underwent the Arg test, i.e., intravenous infusion of Arg (0.5 g Arg/kg bodyweight) for 30 min. Arg was well tolerated by the patients of the three studies. Oral Arg supplementation increased Arg (plasma and urine) and ADMA (urine) concentrations. No appreciable changes were seen in NO (in PAOD and CAD) and

  7. Nonspecific blockade of vascular free radical signals by methylated arginine analogues

    OpenAIRE

    Pedro M.A.; Augusto O.; Barbeiro H.V.; Carvalho M.H.C.; da-Luz P.L.; Laurindo F.R.M.

    1998-01-01

    Methylated arginine analogues are often used as probes of the effect of nitric oxide; however, their specificity is unclear and seems to be frequently overestimated. This study analyzed the effects of NG-methyl-L-arginine (L-NMMA) on the endothelium-dependent release of vascular superoxide radicals triggered by increased flow. Plasma ascorbyl radical signals measured by direct electron paramagnetic resonance spectroscopy in 25 rabbits increased by 3.8 ± 0.7 nmol/l vs baseline (28.7 ± 1.4 nmol...

  8. The Role of Arginin and Uric Acid on Portulaca Grandiflora Growth under Saline Conditions.

    OpenAIRE

    Mahmoud Yagi

    2014-01-01

    Seeds of Portulaca grandiflora were soaked in distilled water, NaCl or NaCl with uric acid or arginine or in one of the amino acids. Treatment of seeds of Portulaca grandiflora with arginine and uric acid under saline conditions increased the percent of seed germination from 55% to 80%. Incorporation of these amino acids in the nutrient medium also significantly enhanced the dry weights as well as the contents of chlorophyll and ascorbic acid in the seedlings. Levels of both total amino acids...

  9. Efficacy L-Arginine In Patients With Nonalcoholic Steatohepatitis Associated With Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Oleksandr Fediv

    2015-01-01

    Full Text Available Abstract Background and Purpose Recent research in the field of hematology indicate that among the many pathogenic mechanisms of development and progression of nonalcoholic steatohepatitis NASH which occurs on the background of the metabolic syndrome an important role is played by endothelial dysfunction and violations of haemocoagulation. The aim of this research was to study the effectiveness of L-arginine as it corrects endothelial dysfunction and disorders of homeostasis haemocoagulation link in patients with NASH associated with the metabolic syndrome. Subjects and Methods 128 patients with nonalcoholic steatohepatitis associated with metabolic syndrome were examined. Some patients 63 persons received standard treatment according to national guidelines. To another group 65 patients on the background of basic therapy L-arginine hydrochloride followed by transition to oral form of L-arginine aspartate was administered. Blood levels of stable nitrogen monoxide metabolites nitrites nitrates endothelin-1 and plasma recalcification time prothrombin time thrombin time activated partial thromboplastin time fibrinogen plasma level activity of antithrombin III and coagulation factor XIII potential activity of plasminogen plasma fibrinolytic blood activity were studied. Results Originally significantly increased levels of endothelin-1 decreased after the therapy in all studied groups but more noticeable changes in the group with L-arginine appointment were observed p0.05. In the studied groups normalization of stable nitrogen monoxide metabolites after treatment was also noticed. Significant p0.05 increase in all haemocoagulation time characteristics and activities of antithrombin-III and factor XIII was found. The positive effect of L-arginine on blood fibrinolytic activity was noted. Discussion and Conclusion Combined therapy of nonalcoholic steatohepatitis associated with metabolic syndrome with a differentiated degreeal L-arginine assignment by

  10. Effect of L-arginine on neuromuscular transmission of the chick biventer cervicis muscle

    Directory of Open Access Journals (Sweden)

    B. Esfandiar

    2008-01-01

    Full Text Available biventer cervicis muscleD. Effect of L-arginine on neuromuscular transmission of the chick EsfandiarAbstractBackground and Purpose: NO is a short-lived gas molecule generated by degradation of L-arg to citrulline and by the activation of enzyme NOS Ca2+/calmodulin-dependent. There are multiple NOS isoforms that strongly are expressed in skeletal muscle, suggesting the crucial role of NO in regulating muscular metabolism and function. In this study, the effect of L-arginine was examined at the neuromuscular junction of the chick biventer cervicis muscle.Materials and Methods: Biventer cervicis muscle preparations from chick’s age of 3 weeks were set up in the organ bath. The organ bath had a vessel with volume of about 70 ml; it contained Tyrode solution aerated with oxygen and was kept at 37º C. NO levels was also measured in the chick biventer cervicis muscle homogenates, using spectrophotometer method for the direct detection of NO, nitrite and nitrate. Total nitrite (nitrite+nitrate was measured by a spectrophotometer at 540 nm after the conversion of nitrate to nitrite by copperized cadmium granules.Results: L-Arginine at 500 µg/ml, decreased twitch response to electrical stimulation, and produced rightward shift of the dose-response curve for acetylcholine or carbachol. L-arginine at 1000 µg/ml produced a strong shift to the right of the dose-response curve for acetylcholine or carbachol with a reduction in efficacy. The inhibitory effect of L-arginine on the twitch response was blocked by caffeine (200 µg/ml. NO levels were found to be significantly increased in concentrations 500 and 1000 µg/ml of L-arginine in comparison with the control group (p < 0.001.Conclusion: These findings indicate a possible role of increased NO levels in the suppressive action of L-arginie on the twitch response. In addition, the results indicate that the post-junctional antagonistic action of L-arginine is probably the result of impaired sarcoplasmic

  11. Arginine vasopressin stimulates phosphoinositide turnover in an enriched rat Leydig cell preparation

    DEFF Research Database (Denmark)

    Nielsen, J.R.; Hansen, Harald S.; Jensen, B.

    1989-01-01

    An enriched rat Leydig cell preparation was preincubated with [C]arachidonic acid. Stimulation of the cells with arginine vasopressin (AVP) (1 µM) for 2 min caused a significant increase in labelled phosphatidic acid and a significant fall in radioactivity in phosphatidylinositol and phosphatidyl......An enriched rat Leydig cell preparation was preincubated with [C]arachidonic acid. Stimulation of the cells with arginine vasopressin (AVP) (1 µM) for 2 min caused a significant increase in labelled phosphatidic acid and a significant fall in radioactivity in phosphatidylinositol and...

  12. Dietary l-Arginine Supplementation Protects Weanling Pigs from Deoxynivalenol-Induced Toxicity

    OpenAIRE

    Li Wu; Peng Liao; Liuqin He; Zemeng Feng; Wenkai Ren; Jie Yin; Jielin Duan; Tiejun Li; Yulong Yin

    2015-01-01

    This study was conducted to determine the positive effects of dietary supplementation with l-arginine (Arg) on piglets fed a deoxynivalenol (DON)-contaminated diet. A total of eighteen, 28-day-old healthy weanling pigs were randomly assigned into one of three groups: uncontaminated basal diet (control group), 6 mg/kg DON-contaminated diet (DON group) and 6 mg/kg DON + 1% l-arginine (DON + ARG group). After 21 days of Arg supplementation, piglets in the DON and DON + ARG groups were challenged...

  13. DNA strand break dependence on Tris and arginine scavenger concentrations under ultra-soft X-ray irradiation: the contribution of secondary arginine radicals.

    Science.gov (United States)

    Souici, Mounir; Khalil, Talat Tariq; Boulanouar, Omar; Belafrites, Abdelfettah; Mavon, Christophe; Fromm, Michel

    2016-05-01

    In this study, we used a bench-top cold-cathode ultra-soft X-ray (USX) generator to expose aqueous DNA plasmid solutions to low-LET radiation under various scavenging conditions. Single- and double-strand breaks were assessed using classic gel electrophoresis quantification of linear, circular and supercoiled plasmid DNA topologies. With their very low penetration range in water, USX can only interact with matter up to short distances, of the order of 50 μm. We validated a stirring procedure which makes it possible to expose 100 µL of aqueous samples (2 mm thick). The scavenging of OH radicals by Tris buffer was studied at ambient temperature under aerobic conditions and compared to data gathered in the literature. A very good agreement was found with the rare data dealing with DNA plasmid exposed to Al Kα photons at low temperature (T ≤ 277 K), which therefore validated the experimental procedure. The yields for DNA single-strand breaks determined during this study enabled the ratio of indirect to direct effects to be determined at 96.2%, in good agreement with the value of 97.7% stemming from a study based on γ-ray irradiation of frozen solutions of plasmid DNA. Then, arginine was used both to create a "biological-like" chemical environment around the DNA plasmids and as an OH radical scavenger, in vitro. Although arginine has a greater scavenging (protecting) power than Tris, surprisingly, it led to higher rates of strand breakage. Based on the specific binding modes of arginine to DNA, we suggest that the side effects observed are due to the presence of arginine near to, but also inside, the DNA double helix. PMID:26994994

  14. Functional variation in the arginine vasopressin 2 receptor as a modifier of human plasma von Willebrand factor levels

    DEFF Research Database (Denmark)

    Nossent, Anne Yaël; Robben, J H; Deen, P M T;

    2010-01-01

    SUMMARY OBJECTIVES: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that gain-of-function variations in the V2R gene (AVPR2) would lead to higher plasma levels of V...

  15. An alternative, arginase-independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme

    NARCIS (Netherlands)

    Romagnoli, G.; Verhoeven, M.D.; Mans, R.; Fleury Rey, Y.; Bel-Rhlid, R.; Van den Broek, M.; Maleki Seifar, R.; Ten Pierick, A.; Thompson, M.; Müller, V.; Wahl, S.A.; Pronk, J.T.; Daran, J.M.

    2014-01-01

    Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were clon

  16. The microbiome, intestinal function, and arginine metabolism of healthy Indian women are different from those of American and Jamaican women

    Science.gov (United States)

    Indian women have slower arginine flux during pregnancy compared with American and Jamaican women. Arginine is a semi-essential amino acid that becomes essential during periods of rapid lean tissue deposition. It is synthesized only from citrulline, a nondietary amino acid produced mainly in the gut...

  17. Molecular Cloning and Sequence Analysis of Adenine Nucleotide Translocase Gene in Antheraea pernyi%柞蚕腺苷酸转移酶基因的克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    李玉萍; 王欢; 武松; 夏润玺; 刘彦群; 秦利; 李喜升; 姜德富

    2009-01-01

    腺苷酸转移酶(adenine nucleotide translocase,ANT)是线粒体内膜上的转运蛋白家族成员.从柞蚕(An-theraea pernyi)蛹全长cDNA文库中获得了柞蚕腺苷酸转移酶基因(ApANT)的cDNA序列(GenBank登录号FJ788509).对该基因进行生物信息学分析表明:ApANT cDNA全长1 282 bp,含有1个903 bp的开放阅读框(ORF)序列,编码300个氨基酸;ApANT与烟草天蛾(Manduca sexta)等鳞翅目昆虫的ANT基因在核苷酸和氨基酸序列水平分别具有80%和90%以上的同源性,说明ANT蛋白在这些昆虫中是高度保守的;与其它已知鳞翅目昆虫的ANT、蛋白一样,ApANT蛋白含有3个线粒体穿膜结构域,并且这3个保守结构域之间也显示出较高的相似性.

  18. Biochemical enrichment and biophysical characterization of a taste receptor for L-arginine from the catfish, Ictalurus puntatus

    Directory of Open Access Journals (Sweden)

    Spielman Andrew I

    2004-07-01

    Full Text Available Abstract Background The channel catfish, Ictalurus punctatus, is invested with a high density of cutaneous taste receptors, particularly on the barbel appendages. Many of these receptors are sensitive to selected amino acids, one of these being a receptor for L-arginine (L-Arg. Previous neurophysiological and biophysical studies suggested that this taste receptor is coupled directly to a cation channel and behaves as a ligand-gated ion channel receptor (LGICR. Earlier studies demonstrated that two lectins, Ricinus communis agglutinin I (RCA-I and Phaseolus vulgaris Erythroagglutinin (PHA-E, inhibited the binding of L-Arg to its presumed receptor sites, and that PHA-E inhibited the L-Arg-stimulated ion conductance of barbel membranes reconstituted into lipid bilayers. Results Both PHA-E and RCA-I almost exclusively labeled an 82–84 kDa protein band of an SDS-PAGE of solubilized barbel taste epithelial membranes. Further, both rhodamine-conjugated RCA-I and polyclonal antibodies raised to the 82–84 kDa electroeluted peptides labeled the apical region of catfish taste buds. Because of the specificity shown by RCA-I, lectin affinity was chosen as the first of a three-step procedure designed to enrich the presumed LGICR for L-Arg. Purified and CHAPS-solubilized taste epithelial membrane proteins were subjected successively to (1, lectin (RCA-I affinity; (2, gel filtration (Sephacryl S-300HR; and (3, ion exchange chromatography. All fractions from each chromatography step were evaluated for L-Arg-induced ion channel activity by reconstituting each fraction into a lipid bilayer. Active fractions demonstrated L-Arg-induced channel activity that was inhibited by D-arginine (D-Arg with kinetics nearly identical to those reported earlier for L-Arg-stimulated ion channels of native barbel membranes reconstituted into lipid bilayers. After the final enrichment step, SDS-PAGE of the active ion channel protein fraction revealed a single band at 82–84 k

  19. Application of Response Surface Methodology for Optimizing Arginine Deiminase Production Medium for Enterococcus faecium sp. GR7

    Directory of Open Access Journals (Sweden)

    Baljinder Kaur

    2013-01-01

    Full Text Available Arginine metabolism in Enterococcus faecium sp. GR7 was enhanced via arginine deiminase pathway. Process parameters including fermentation media and environmental conditions were optimized using independent experiments and response surface methodology (central composite design. Fermentation media (EAPM were optimized using independent experiments which resulted in 4-fold increase in arginine deiminase specific activity as compared to basal medium. To further enhance arginine deiminase activity in E. faecium sp. GR7 and biomass production including a five-level central composite design (CCD was employed to study the interactive effect of three-process variables. Response surface methodology suggested a quadratic model which was further validated experimentally where it showed approximately 15-fold increase in arginine metabolism (in terms of arginine deiminase specific activity over basal medium. By solving the regression equation and analyzing the response surface cartons, optimal concentrations of the media components (g/L were determined as arginine 20.0; tryptone 15.0; lactose 10.0; K2HPO4 3.0; NaCl 1.0, MnSO4 0.6 mM; Tween 80 1%; pH 6.0 for achieving specific arginine deiminase activity of 4.6 IU/mG with concomitant biomass production of 12.1 mg/L. The model is significant as the coefficient of determination (R2 was 0.87 to 0.90 for all responses. Enhanced arginine deiminase yield from E. faecium, a GRAS lactic acid bacterial strain, is desirable to explore in vitro therapeutic potential of the arginine metabolizing E. faecium sp. GR7.

  20. Impaired nitric oxide production in children with MELAS syndrome and the effect of arginine and citrulline supplementation.

    Science.gov (United States)

    El-Hattab, Ayman W; Emrick, Lisa T; Hsu, Jean W; Chanprasert, Sirisak; Almannai, Mohammed; Craigen, William J; Jahoor, Farook; Scaglia, Fernando

    2016-04-01

    Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most frequent maternally inherited mitochondrial disorders. The pathogenesis of this syndrome is not fully understood and believed to result from several interacting mechanisms including impaired mitochondrial energy production, microvasculature angiopathy, and nitric oxide (NO) deficiency. NO deficiency in MELAS syndrome is likely to be multifactorial in origin with the decreased availability of the NO precursors, arginine and citrulline, playing a major role. In this study we used stable isotope infusion techniques to assess NO production in children with MELAS syndrome and healthy pediatric controls. We also assessed the effect of oral arginine and citrulline supplementations on NO production in children with MELAS syndrome. When compared to control subjects, children with MELAS syndrome were found to have lower NO production, arginine flux, plasma arginine, and citrulline flux. In children with MELAS syndrome, arginine supplementation resulted in increased NO production, arginine flux, and arginine concentration. Citrulline supplementation resulted in a greater increase of these parameters. Additionally, citrulline supplementation was associated with a robust increase in citrulline concentration and flux and de novo arginine synthesis rate. The greater effect of citrulline in increasing NO production is due to its greater ability to increase arginine availability particularly in the intracellular compartment in which NO synthesis takes place. This study, which is the first one to assess NO metabolism in children with mitochondrial diseases, adds more evidence to the notion that NO deficiency occurs in MELAS syndrome, suggests a better effect for citrulline because of its greater role as NO precursor, and indicates that impaired NO production occurs in children as well as adults with MELAS syndrome. Thus, the initiation of treatment with NO precursors may be

  1. ICE1 of Poncirus trifoliata functions in cold tolerance by modulating polyamine levels through interacting with arginine decarboxylase.

    Science.gov (United States)

    Huang, Xiao-San; Zhang, Qinghua; Zhu, Dexin; Fu, Xingzheng; Wang, Min; Zhang, Qian; Moriguchi, Takaya; Liu, Ji-Hong

    2015-06-01

    ICE1 (Inducer of CBF Expression 1) encodes a MYC-like basic helix-loop-helix transcription factor that acts as a central regulator of cold response. In this study, we elucidated the function and underlying mechanisms of PtrICE1 from trifoliate orange [Poncirus trifoliata (L.) Raf.]. PtrICE1 was upregulated by cold, dehydration, and salt, with the greatest induction under cold conditions. PtrICE1 was localized in the nucleus and could bind to a MYC-recognizing sequence. Ectopic expression of PtrICE1 in tobacco and lemon conferred enhanced tolerance to cold stresses at either chilling or freezing temperatures. Yeast two-hybrid screening revealed that 21 proteins belonged to the PtrICE1 interactome, in which PtADC (arginine decarboxylase) was confirmed as a bona fide protein interacting with PtrICE1. Transcript levels of ADC genes in the transgenic lines were slightly elevated under normal growth condition but substantially increased under cold conditions, consistent with changes in free polyamine levels. By contrast, accumulation of the reactive oxygen species, H2O2 and O2 (-), was appreciably alleviated in the transgenic lines under cold stress. Higher activities of antioxidant enzymes, such as superoxide dismutase and catalase, were detected in the transgenic lines under cold conditions. Taken together, these results demonstrated that PtrICE1 plays a positive role in cold tolerance, which may be due to modulation of polyamine levels through interacting with the ADC gene. PMID:25873670

  2. Presenting a foreign antigen on live attenuated Edwardsiella tarda using twin-arginine translocation signal peptide as a multivalent vaccine.

    Science.gov (United States)

    Wang, Yamin; Yang, Weizheng; Wang, Qiyao; Qu, Jiangbo; Zhang, Yuanxing

    2013-12-01

    The twin-arginine translocation (Tat) system is a major pathway for transmembrane translocation of fully folded proteins. In this study, a multivalent vaccine to present foreign antigens on live attenuated vaccine Edwardsiella tarda WED using screened Tat signal peptide was constructed. Because the Tat system increases the yields of folded antigens in periplasmic space or extracellular milieu, it is expected to contribute to the production of conformational epitope-derived specific antibodies. E. tarda Tat signal peptides fused with the green fluorescent protein (GFP) was constructed under the control of an in vivo inducible dps promoter. The resulting plasmids were electroporated into WED and the subcellular localizations of GFP were analyzed with Western blotting. Eight signal peptides with optimized GFP translocation efficiency were further fused to a protective antigen glyceraldehyde-3-phosphate dehydrogenase (GapA) from a fish pathogen Aeromonas hydrophila. Signal peptides of DmsA, NapA, and SufI displayed high efficiency for GapA translocation. The relative percent survival (RPS) of turbot was measured with a co-infection of E. tarda and A. hydrophila, and the strain with DmsA signal peptide showed the maximal protection. This study demonstrated a new platform to construct multivalent vaccines using optimized Tat signal peptide in E. tarda. PMID:23994481

  3. Arginine-vasopressin stimulates the formation of phosphatidic acid in rat Leydig cells

    DEFF Research Database (Denmark)

    Nielsen, J.R.; Hansen, Harald S.; Jensen, B.

    1987-01-01

    Arginine-vasopressin (AVP) stimulated the formation of labelled phosphatidic acid (PA) in [C]arachidonic acid-prelabelled rat Leydig cells. After addition of 10 M AVP [C]arachidonoylphosphatidic acid reached a maximum within 2 min. The increase was dose-dependent (10-10 M). No change in labelling...

  4. Arginine-based biodegradable ether-ester polymers with low cytotoxicity as potential gene carriers.

    Science.gov (United States)

    Memanishvili, Tamar; Zavradashvili, Nino; Kupatadze, Nino; Tugushi, David; Gverdtsiteli, Marekh; Torchilin, Vladimir P; Wandrey, Christine; Baldi, Lucia; Manoli, Sagar S; Katsarava, Ramaz

    2014-08-11

    The success of gene therapy depends on safe and effective gene carriers. Despite being widely used, synthetic vectors based on poly(ethylenimine) (PEI), poly(l-lysine) (PLL), or poly(l-arginine) (poly-Arg) are not yet fully satisfactory. Thus, both improvement of established carriers and creation of new synthetic vectors are necessary. A series of biodegradable arginine-based ether-ester polycations was developed, which consists of three main classes: amides, urethanes, and ureas. Compared to that of PEI, PLL, and poly-Arg, much lower cytotoxicity was achieved for the new cationic arginine-based ether-ester polymers. Even at polycation concentrations up to 2 mg/mL, no significant negative effect on cell viability was observed upon exposure of several cell lines (murine mammary carcinoma, human cervical adenocarcinoma, murine melanoma, and mouse fibroblast) to the new polymers. Interaction with plasmid DNA yielded compact and stable complexes. The results demonstrate the potential of arginine-based ether-ester polycations as nonviral carriers for gene therapy applications. PMID:24963693

  5. Evidence for a metabolic shift of arginine metabolism in sickle cell disease

    NARCIS (Netherlands)

    Schnog, JJB; Jager, EH; van der Dijs, FPL; Duits, AJ; Moshage, H; Muskiet, FD; Muskiet, FAJ

    2004-01-01

    Over the last few years, a pivotal role has been ascribed to reduced nitric oxide (NO) availability as a contributing factor to the vaso-occlusive process of sickle cell disease. We investigated whether arginine metabolism in sickle cell patients is different from healthy controls. Blood samples wer

  6. Glutamine supplementation, citrulline production, and de novo arginine synthesis: Is there a relation?

    Science.gov (United States)

    We would like to comment on the recent publications by Buijs et al. The authors hypothesized that a parenteral supplement of glutamine stimulates citrulline formation and enhances de novo arginine synthesis. To test this hypothesis, they conducted an experiment with stable isotopes in patients under...

  7. Watermelon enhances arginine availability in an animal model of type-II diabetes

    Science.gov (United States)

    Watermelon fruit contain lycopene, a red pigment known for its ability to scavenge free hydroxyl radicals. L-Citrulline, an amino acid that acts as a vasodilator and is a precursor of L-arginine, is found in all cucurbits, but is most plentiful in watermelon. In a study with Zucker diabetic fatty ...

  8. Reduced preabsorptive insulin response in aged rats : differential effects of amphetamine and arginine-vasopressin

    NARCIS (Netherlands)

    Buwalda, B.; Strubbe, J.H.; Bohus, B.

    1991-01-01

    The experiments presented here have been designed to investigate whether the age-related attenuation of the vagal reactivity to emotional stressors and its modulation by amphetamine (Amph) or arginine-vasopressin (AVP) can be generalized for other physiological response patterns. We therefore studie

  9. Arginine nutrition and fetal brown adipose tissue development in nutrient-restricted sheep.

    Science.gov (United States)

    Satterfield, M Carey; Dunlap, Kathrin A; Keisler, Duane H; Bazer, Fuller W; Wu, Guoyao

    2013-09-01

    Intrauterine growth restriction is a significant problem worldwide, resulting in increased rates of neonatal morbidity and mortality, as well as increased risks for metabolic and cardiovascular disease. The present study investigated the role of maternal undernutrition and L-arginine administration on fetal growth and development. Embryo transfer was utilized to generate genetically similar singleton pregnancies. On Day 35 of gestation, ewes were assigned to receive either 50 or 100% of their nutritional requirements. Ewes received i.v. injections of either saline or L-arginine three times daily from Day 100 to Day 125. Fetal growth was assessed at necropsy on Day 125. Maternal dietary manipulation altered circulating concentrations of leptin, progesterone, and amino acids in maternal plasma. Fetal weight was reduced in nutrient-restricted ewes on Day 125 compared with 100% fed ewes. Compared with saline-treated underfed ewes, maternal L-arginine administration did not affect fetal weight but increased weight of the fetal pancreas by 32% and fetal peri-renal brown adipose tissue mass by 48%. These results indicate that L-arginine administration enhanced fetal pancreatic and brown adipose tissue development. The postnatal effects of increased pancreatic and brown adipose tissue growth warrant further study.

  10. Levels of arginine-vasopressin in cerebrospinal fluid during passive avoidance behavior in rats

    NARCIS (Netherlands)

    Kloet, E.R. de; Laczi, F.; Gaffori, O.; Fekete, M.; Wied, D. de

    1984-01-01

    The concentration of immunoreactive arginine-vasopressin (IR-AVP) was measured in the cerebrospinal fluid (CSF) during acquisition and retention of passive avoidance behavior. IR-AVP level in CSF of male Wistar rats immediately after the learning trial was increased; the rate of which was related to

  11. Double Strand Break Unwinding and Resection by the Mycobacterial Helicase-Nuclease AdnAB in the Presence of Single Strand DNA-binding Protein (SSB)*

    OpenAIRE

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2010-01-01

    Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3′ to 5′ DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevent...

  12. MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drags as judged by interference with nucleotide trapping

    NARCIS (Netherlands)

    Smith, A.J.; van Helvoort, A.; van Meer, G.; Szabó, K.; Welker, E.; Szakács, G.; Váradi, A.; Sarkadi, B.; Borst, P.

    2000-01-01

    The human MDR3 gene is a member of the multidrug resistance (MDR) gene family. The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine. The MDR1 P-glycoprotein related transports cytotoxic drugs. Its overexpression can make cells resistant to a variety of drugs. Atte

  13. Effect of arginase inhibition on pulmonary L-arginine metabolism in murine Pseudomonas pneumonia.

    Directory of Open Access Journals (Sweden)

    Anne Mehl

    Full Text Available RATIONALE: Infection of the lung with Pseudomonas aeruginosa results in upregulation of nitric oxide synthases (NOS and arginase expression, and both enzymes compete for L-arginine as substrate. Nitric oxide (NO production may be regulated by arginase as it controls L-arginine availability for NOS. We here studied the effect of systemic arginase inhibition on pulmonary L-arginine metabolism in Pseudomonas pneumonia in the mouse. METHODS: Mice (C57BL/6, 8-10 weeks old, female underwent direct tracheal instillation of Pseudomonas (PAO-1-coated agar beads and were treated by repeated intra-peritoneal injections of the arginase inhibitor 2(S-amino-6-boronohexanoic acid (ABH or PBS until lungs were harvested on day 3 of the infection. L-arginine metabolites were quantified using liquid chromatography-tandem mass spectrometry, NO metabolites nitrate and nitrite by Griess reagent and cytokines by ELISA. RESULTS: NO metabolite concentrations (48.5±2.9 vs. 10.9±2.3 µM, p<0.0001, as well as L-ornithine (29.6±1.7 vs 2.3±0.4 µM, p<0.0001, the product of arginase activity, were increased in Pseudomonas infected lungs compared to naïve controls. Concentrations of the NOS inhibitor asymmetric dimethylarginine (ADMA were also increased (0.44±0.02 vs. 0.16±0.01 µM, p<0.0001. Arginase inhibition in the infected animals resulted in a significant decrease in L-ornithine (14.6±1.6 µM, p<0.0001 but increase in L-arginine concentration (p<0.001, L-arginine/ADMA ratio (p<0.001, L-arginine availability for NOS (p<0.001, and NO metabolite concentrations (67.3±5.7 µM, p<0.05. Arginase inhibitor treatment also resulted in an increase in NO metabolite levels in animals following intratracheal injection of LPS (p = 0.015. Arginase inhibition was not associated with an increase in inflammatory markers (IFN-γ, IL-1β, IL-6, MIP-2, KC or TNF-α in lung. Concentrations of the L-ornithine-dependent polyamines putrescine, spermidine and spermine were increased

  14. Glutamine, glutamate, and arginine-based acid resistance in Lactobacillus reuteri.

    Science.gov (United States)

    Teixeira, Januana S; Seeras, Arisha; Sanchez-Maldonado, Alma Fernanda; Zhang, Chonggang; Su, Marcia Shu-Wei; Gänzle, Michael G

    2014-09-01

    This study aimed to determine whether glutamine deamidation improves acid resistance of Lactobacillus reuteri, and to assess whether arginine, glutamine, and glutamate-mediated acid resistance are redundant or complementary mechanisms of acid resistance. Three putative glutaminase genes, gls1, gls2, and gls3, were identified in L. reuteri 100-23. All three genes were expressed during growth in mMRS and wheat sourdough. L. reuteri consistently over-expressed gls3 and the glutamate decarboxylase gadB. L. reuteri 100-23ΔgadB over-expressed gls3 and the arginine deiminase gene adi. Analysis of the survival of L. reuteri in acidic conditions revealed that arginine conversion is effective at pH of 3.5 while glutamine or glutamate conversion were effective at pH of 2.5. Arginine conversion increased the pHin but not ΔΨ; glutamate decarboxylation had only a minor effect on the pHin but increased the ΔΨ. This study demonstrates that glutamine deamidation increases the acid resistance of L. reuteri independent of glutamate decarboxylase activity. Arginine and glutamine/glutamate conversions confer resistance to lactate at pH of 3.5 and phosphate at pH of 2.5, respectively. Knowledge of L. reuteri's acid resistance improves the understanding of the adaptation of L. reuteri to intestinal ecosystems, and facilitates the selection of probiotic and starter cultures.

  15. Arginine deiminase in Staphylococcus epidermidis functions to augment biofilm maturation through pH homeostasis.

    Science.gov (United States)

    Lindgren, J K; Thomas, V C; Olson, M E; Chaudhari, S S; Nuxoll, A S; Schaeffer, C R; Lindgren, K E; Jones, J; Zimmerman, M C; Dunman, P M; Bayles, K W; Fey, P D

    2014-06-01

    Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids. PMID:24727224

  16. The effect of L-Arginine on the brain tissue of stressed rats

    Directory of Open Access Journals (Sweden)

    Batoul Ebadi

    2010-01-01

    Full Text Available   Abstract  Introduction: This study was conducted to determine the possible beneficial results of L-arginine on prefrontal cortex of rats which impressed by immobilization stress to define the synchronous impression of stress and nitric oxide (NO on evolution of prefrontal cortex of rats after birth. Methods: Forty-eight one month, male Wistar rats were divided into two groups: stressed and non-stressed. L-Arginine (200 mg/kg as a NO synthase (NOS inducer and L-NAME (2O mg/kg were injected intraperitonealy (IP and 7- nitroindazde (25 mg/kg as non-specific was injected subcutaneously (S.C. for 4 weeks. The kind of stress was immobilization for 4 weeks, every other day. The brain was removed after this period and each brain divided into two parts in a coronal section manner. Anterior part used for histological studies with H&E staining and posterior part used for measurement of NO production using spectrophotometer at 540 nm wavelengh. Results: Statistical analysis of microscopic and light microscopic finding showed that thickness of prefrontal cortex and NO production were significantly decreased in stressed rats and especially in groups which received 7- nitroindazole and L-NAME and L-arginine could reverse these results. Discussion: According to this research, we could say that L-arginine decreases the cortical damages in stressed rats and 7-nitroindazole and L-NAME increase this damage in non-stressed group. Although in non stressed groups, L-arginine, L-NAME and 7- nitroindazole were all non-protective and damaging.

  17. Comparison of the structure and function of phospholamban and the arginine-14 deficient mutant associated with dilated cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Eleri Hughes

    Full Text Available Phospholamban (PLB is a pentameric protein that plays an important role in regulating cardiac contractility via a reversible inhibitory association with the sarcoplasmic reticulum Ca2+ATPase (SERCA, the enzyme responsible for maintaining correct calcium homeostasis. Here we study the functional and biophysical characteristics of a PLB mutant associated with human dilated cardiomyopathy (DCM, with a deletion of arginine at position 14 (PLBR14Δ. In agreement with recent findings, we find that PLBR14Δ has a reduced inhibitory effect on SERCA compared to wild type PLB (PLBWT when reconstituted into lipid membranes. The mutation also leads to a large reduction in the protein kinase A-catalysed phosphorylation of Ser-16 in the cytoplasmic domain of PLBR14Δ. Measurements on SERCA co-reconstituted with an equimolar mixture of PLBWT and PLBR14Δ (representing the lethal heterozygous state associated with DCM indicates that the loss-of-function mutation has a dominant effect on PLBWT functionality and phosphorylation capacity, suggesting that mixed PLBWT/PLBR14Δ pentamers are formed that have characteristics typical of the mutant protein. Structural and biophysical analysis of PLBR14Δ indicates that the mutation perturbs slightly the helical structure of the PLB cytoplasmic domain and reduces its affinity for the phospholipid bilayer surface, thereby altering the orientation of the cytoplasmic domain relative to the wild-type protein. These results indicate that the structure and function consequences of the R14 deletion have profound effects on the regulation of SERCA which may contribute to the aetiology of DCM.

  18. Site-directed mutation of arginine 282 to glutamate uncouples the movement of peptides and protons by the rabbit proton-peptide cotransporter PepT1.

    Science.gov (United States)

    Meredith, David

    2004-04-16

    A conserved positive residue in the seventh transmembrane domain of the mammalian proton-coupled di- and tripeptide transporter PepT1 has been shown by site-directed mutagenesis to be a key residue for protein function. Substitution of arginine 282 with a glutamate residue (R282E-PepT1) gave a protein at the plasma membrane of Xenopus laevis oocytes that was able to transport the non-hydrolyzable dipeptide [3H]d-Phe-l-Gln, although unlike the wild type, the rate of transport by R282E-PepT1 was independent of the extracellular pH level, and the substrate could not be accumulated above equilibrium. The binding affinity of the mutant transport protein was unchanged from the wild type. Thus, R282E-Pept1 appears to have been changed from a proton-driven to a facilitated transporter for peptides. In addition, peptide transport by R282E-PepT1 still induced depolarization as measured by microelectrode recordings of membrane potential. A more detailed study by two-electrode voltage clamping revealed that R282E-PepT1 behaved as a peptide-gated non-selective cation channel with the ion selectivity series lithium > sodium > N-methyl-d-glucamine at pH 7.4. There was also a proton conductance (comparing pH 7.4 and 8.4), and at pH 5.5 the predominant conductance was for potassium ions. Therefore, it can be concluded that changing arginine 282 to a glutamate not only uncouples the cotransport of protons and peptides of the wild-type PepT1 but also creates a peptide-gated cation channel in the protein.

  19. Regulation of intestinal protein metabolism by amino acids.

    Science.gov (United States)

    Bertrand, Julien; Goichon, Alexis; Déchelotte, Pierre; Coëffier, Moïse

    2013-09-01

    Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50% per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.

  20. Preventive oral supplementation with glutamine and arginine has beneficial effects on the intestinal mucosa and inflammatory cytokines in endotoxemic rats.

    Science.gov (United States)

    Zhou, Xihong; Wu, Xin; Yin, Yulong; Zhang, Cui; He, Liuqin

    2012-08-01

    The objective of this study was to evaluate the effect of oral supplementation with a combination of arginine and glutamine on the intestinal mucosa and inflammatory cytokines of lipopolysaccharide (LPS)-induced adult rats. Fifty Sprague-Dawley rats (average weight of 185 ± 15 g) were randomly divided into five groups: control group A (CA) and control group B (CB), both orally supplemented with 0.9% saline; group Arg, supplemented with 300 mg/kg day(-1) arginine; group Gln, supplemented with 300 mg/kg day(-1) glutamine; group AG, supplemented with 150 mg/kg day(-1) arginine and 150 mg/kg day(-1) glutamine. The experiment lasted for 2 weeks. Food intake and body weight were measured during the experiment. At 10.00 h of day 15, animals were injected with 4 mg/kg LPS (group CB, Arg, Gln, and AG) or sterile saline (group CA) after supplementation. Then at 14.00 h, all animals were killed and blood and tissue collected. The results showed that compared with group CB, arginine concentration tended to be increased (P > 0.05) in group Arg and AG, while there was no significant difference in glutamine concentration among the groups challenged with LPS. Oral supplementation with arginine or/and glutamine mitigated morphology impairment (lower villus height, P Arginine only significantly decreased TNF-α mRNA abundance in the ileum, while glutamine significantly decreased both TNF-α and IL-10 mRNA in the ileum. A combination of arginine and glutamine significantly decreased TNF-α and IL-1β mRNA abundance in both the jejunum and ileum, while they also significantly decreased anti-inflammatory IL-10 in the ileum. These results revealed that an oral supply of combined arginine and glutamine had more favorable effects on the intestinal mucosa and inflammatory cytokines than a supply of arginine or glutamine alone.

  1. Effect of arginase II on L-arginine depletion and cell growth in murine cell lines of renal cell carcinoma

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    Patterson John R

    2008-09-01

    Full Text Available Abstract Background L-arginine is the common substrate for the two isoforms of arginase. Arginase I, highly expressed in the liver and arginase II mainly expressed in the kidney. Arginase I-producing myeloid derived suppressor cells have been shown to inhibit T-cell function by the depletion of L-arginine. On the other hand, arginase II has been detected in patients with cancer and is thought to metabolize L-arginine to L-ornithine needed to sustain rapid tumor growth; however its role in L-arginine depletion is unclear. Thus, in tumor biology, L-arginine metabolism may play a dual role in tumor growth and in the induction of T cell dysfunction. Therefore, we studied in murine renal cell carcinoma (RCC cell lines, the effect of arginase II on tumor cell proliferation and L-arginine depletion. The effect of arginase inhibitors on cell proliferation was also tested. Methods Three murine renal cell carcinoma (mRCC cell lines were tested for the presence of arginase. nor-NOHA, an arginase inhibitor was used to substantiate the effect of arginase on cell growth and L-arginine depletion. Amino acid levels were tested by HPLC. Results Our results show that mRCC cell lines express only arginase II and were able to deplete L-arginine from the medium. Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01 reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity. The depletion of L-arginine by mRCC induced the decrease expression of CD3ζ a key element for T-cell function. Conclusion The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3ζ. These results indicate that RCC cell lines expressing arginase II can modulate the L-arginine metabolic pathway to regulate both cell growth and T-cell function. Blocking arginase may lead to a decrease in RCC cell

  2. Cloning of Adenine Nucleotide Translocase Gene From Bombyx mori Based on EST Database%利用EST库资源克隆家蚕腺苷酸转移酶基因

    Institute of Scientific and Technical Information of China (English)

    陈大福; 牛宝龙; 翁宏飚; 孟智启; 吕顺霖

    2004-01-01

    根据物种间同源基因相对保守的特点,利用生物信息学方法以果蝇(Drosophila melanogaster)腺苷酸转移酶基因(adenine nucleotide translocase, ant) cDNA序列作为模板,对家蚕(Bombyx mori) EST数据库进行同源检索筛选,克隆了家蚕腺苷酸转移酶基因的cDNA序列(GenBank登录号为AY227000),全长为1 936 bp,并经RT-PCR克隆、序列分析验证,结果与电子克隆序列完全一致.该cDNA序列具有完整的开放阅读框架(ORF, 207~1 109 bp),推测编码蛋白为300个氨基酸,通过与烟草天蛾(Manduca sexta)、蜜蜂(Apis mellifera)、绿蝇(Lucilia cuprina)、果蝇、蚊子(Anopheles gambiae)等昆虫的腺苷酸转移酶蛋白序列比较,发现该基因具有高度的保守性.表明根据物种间同源基因序列,对跨物种间EST数据库进行同源检索筛选、拼接,是基因克隆的一条有效途径.

  3. Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway.

    Science.gov (United States)

    Henrich, Erik; Ma, Yi; Engels, Ina; Münch, Daniela; Otten, Christian; Schneider, Tanja; Henrichfreise, Beate; Sahl, Hans-Georg; Dötsch, Volker; Bernhard, Frank

    2016-01-29

    Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.

  4. Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway.

    Science.gov (United States)

    Henrich, Erik; Ma, Yi; Engels, Ina; Münch, Daniela; Otten, Christian; Schneider, Tanja; Henrichfreise, Beate; Sahl, Hans-Georg; Dötsch, Volker; Bernhard, Frank

    2016-01-29

    Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin. PMID:26620564

  5. MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drags as judged by interference with nucleotide trapping

    OpenAIRE

    Smith, A.J.; van Helvoort, A.; van Meer, G; Szabó, K.; Welker, E; Szakács, G; Váradi, A; Sarkadi, B.; Borst, P

    2000-01-01

    The human MDR3 gene is a member of the multidrug resistance (MDR) gene family. The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine. The MDR1 P-glycoprotein related transports cytotoxic drugs. Its overexpression can make cells resistant to a variety of drugs. Attempts to show that MDR3 P-glycoprotein can cause MDR have been unsuccessful thus far. Here, we report an increased directional transport of several MDR1 P-glycoprotein substrates, such as digoxin, p...

  6. Anti-stress and Adaptogenic Activity of l-Arginine Supplementation

    Directory of Open Access Journals (Sweden)

    Vanita Gupta

    2005-01-01

    Full Text Available In the present study, oral supplementation of l-arginine in rats was evaluated for its anti-stress and adaptogenic activity using the cold (5°C–hypoxia (428 mmHg–restraint (C-H-R animal model. A dose-dependent study of l-arginine was carried out at doses of 12.5, 25.0, 50.0, 100.0, 200.0 and 500.0 mg/kg body weight, administered orally 30 min prior to C-H-R exposure. The time taken by the rat to attain a rectal temperature of 23°C (Trec 23°C during C-H-R exposure and its recovery to Trec 37°C at normal atmospheric pressure and 32 ± 1°C were used as biomarkers of anti-stress and adaptogenic activity. Biochemical parameters related to lipid peroxidation, anti-oxidants, cell membrane permeability, nitric oxide and stress, with and without administration of the least effective l-arginine dose, were measured in rats on attaining Trec 23°C and Trec 37°C. The least effective adaptogenic dose of l-arginine was 100.0 mg/kg body weight. The C-H-R exposure of control rats, on attaining Trec 23°C, resulted in a significant increase in plasma malondialdehyde (MDA, blood lactate dehydrogenase (LDH and a decrease in blood catalase (CAT and plasma testosterone levels. On recovery (Trec 37°C of control rats, there was a further decrease in CAT and plasma testosterone, and an increase in LDH. l-Arginine supplementation resulted in a significant decrease in plasma MDA, an increase in blood superoxide dismutase (SOD, CAT levels maintained at control values and a lower increase in LDH compared with controls (45.3 versus 58.5% and 21.5 versus 105.2% on attaining Trec 23°C during C-H-R exposure and on recovery to Trec 37°C. The results suggested that l-arginine possesses potent anti-stress activity during C-H-R exposure and recovery from C-H-R-induced hypothermia.

  7. Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity

    Directory of Open Access Journals (Sweden)

    Sarker SR

    2013-04-01

    Full Text Available Satya Ranjan Sarker, Yumiko Aoshima, Ryosuke Hokama, Takafumi Inoue, Keitaro Sou, Shinji Takeoka Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns, Tokyo, Japan Background: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt in the arginine head group. Methods: Cationic lipids were hydrated in 4-(2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000. Results: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the

  8. Insight on an arginine synthesis metabolon from the tetrameric structure of yeast acetylglutamate kinase.

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    Sergio de Cima

    Full Text Available N-acetyl-L-glutamate kinase (NAGK catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS, which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK has, in addition to the amino acid kinase (AAK domain found in other NAGKs, a ~150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the -110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.

  9. The structure and IR signatures of the arginine-glutamate salt bridge. Insights from the classical MD simulations.

    Science.gov (United States)

    Vener, M V; Odinokov, A V; Wehmeyer, C; Sebastiani, D

    2015-06-01

    Salt bridges and ionic interactions play an important role in protein stability, protein-protein interactions, and protein folding. Here, we provide the classical MD simulations of the structure and IR signatures of the arginine (Arg)-glutamate (Glu) salt bridge. The Arg-Glu model is based on the infinite polyalanine antiparallel two-stranded β-sheet structure. The 1 μs NPT simulations show that it preferably exists as a salt bridge (a contact ion pair). Bidentate (the end-on and side-on structures) and monodentate (the backside structure) configurations are localized [Donald et al., Proteins 79, 898-915 (2011)]. These structures are stabilized by the short (+)N-H⋯O(-) bonds. Their relative stability depends on a force field used in the MD simulations. The side-on structure is the most stable in terms of the OPLS-AA force field. If AMBER ff99SB-ILDN is used, the backside structure is the most stable. Compared with experimental data, simulations using the OPLS all-atom (OPLS-AA) force field describe the stability of the salt bridge structures quite realistically. It decreases in the following order: side-on > end-on > backside. The most stable side-on structure lives several nanoseconds. The less stable backside structure exists a few tenth of a nanosecond. Several short-living species (solvent shared, completely separately solvated ionic groups ion pairs, etc.) are also localized. Their lifetime is a few tens of picoseconds or less. Conformational flexibility of amino acids forming the salt bridge is investigated. The spectral signature of the Arg-Glu salt bridge is the IR-intensive band around 2200 cm(-1). It is caused by the asymmetric stretching vibrations of the (+)N-H⋯O(-) fragment. Result of the present paper suggests that infrared spectroscopy in the 2000-2800 frequency region may be a rapid and quantitative method for the study of salt bridges in peptides and ionic interactions between proteins. This region is usually not considered in spectroscopic

  10. The structure and IR signatures of the arginine-glutamate salt bridge. Insights from the classical MD simulations

    Energy Technology Data Exchange (ETDEWEB)

    Vener, M. V., E-mail: mikhail.vener@gmail.com [Mendeleev University of Chemical Technology, Moscow (Russian Federation); Odinokov, A. V. [Photochemistry Center of the Russian Academy of Sciences, Moscow (Russian Federation); Wehmeyer, C. [Free University, Berlin (Germany); Sebastiani, D. [Martin-Luther-Universität Halle-Wittenberg, Halle (Germany)

    2015-06-07

    Salt bridges and ionic interactions play an important role in protein stability, protein-protein interactions, and protein folding. Here, we provide the classical MD simulations of the structure and IR signatures of the arginine (Arg)–glutamate (Glu) salt bridge. The Arg-Glu model is based on the infinite polyalanine antiparallel two-stranded β-sheet structure. The 1 μs NPT simulations show that it preferably exists as a salt bridge (a contact ion pair). Bidentate (the end-on and side-on structures) and monodentate (the backside structure) configurations are localized [Donald et al., Proteins 79, 898–915 (2011)]. These structures are stabilized by the short {sup +}N–H⋯O{sup −} bonds. Their relative stability depends on a force field used in the MD simulations. The side-on structure is the most stable in terms of the OPLS-AA force field. If AMBER ff99SB-ILDN is used, the backside structure is the most stable. Compared with experimental data, simulations using the OPLS all-atom (OPLS-AA) force field describe the stability of the salt bridge structures quite realistically. It decreases in the following order: side-on > end-on > backside. The most stable side-on structure lives several nanoseconds. The less stable backside structure exists a few tenth of a nanosecond. Several short-living species (solvent shared, completely separately solvated ionic groups ion pairs, etc.) are also localized. Their lifetime is a few tens of picoseconds or less. Conformational flexibility of amino acids forming the salt bridge is investigated. The spectral signature of the Arg-Glu salt bridge is the IR-intensive band around 2200 cm{sup −1}. It is caused by the asymmetric stretching vibrations of the {sup +}N–H⋯O{sup −} fragment. Result of the present paper suggests that infrared spectroscopy in the 2000–2800 frequency region may be a rapid and quantitative method for the study of salt bridges in peptides and ionic interactions between proteins. This region is

  11. Improved Muscle Function in Duchenne Muscular Dystrophy through L-Arginine and Metformin: An Investigator-Initiated, Open-Label, Single-Center, Proof-Of-Concept-Study.

    Directory of Open Access Journals (Sweden)

    Patricia Hafner

    Full Text Available Altered neuronal nitric oxide synthase function in Duchenne muscular dystrophy leads to impaired mitochondrial function which is thought to be one cause of muscle damage in this disease. The study tested if increased intramuscular nitric oxide concentration can improve mitochondrial energy metabolism in Duchenne muscular dystrophy using a novel therapeutic approach through the combination of L-arginine with metformin. Five ambulatory, genetically confirmed Duchenne muscular dystrophy patients aged between 7–10 years were treated with L-arginine (3 x 2.5 g/d and metformin (2 x 250 mg/d for 16 weeks. Treatment effects were assessed using mitochondrial protein expression analysis in muscular biopsies, indirect calorimetry, Dual-Energy X-Ray Absorptiometry, quantitative thigh muscle MRI, and clinical scores of muscle performance. There were no serious side effects and no patient dropped out. Muscle biopsy results showed pre-treatment a significantly reduced mitochondrial protein expression and increased oxidative stress in Duchenne muscular dystrophy patients compared to controls. Post-treatment a significant elevation of proteins of the mitochondrial electron transport chain was observed as well as a reduction in oxidative stress. Treatment also decreased resting energy expenditure rates and energy substrate use shifted from carbohydrates to fatty acids. These changes were associated with improved clinical scores. In conclusion pharmacological stimulation of the nitric oxide pathway leads to improved mitochondria function and clinically a slowing of disease progression in Duchenne muscular dystrophy. This study shall lead to further development of this novel therapeutic approach into a real alternative for Duchenne muscular dystrophy patients.ClinicalTrials.gov NCT02516085.

  12. Synergies between vaccination and dietary arginine and glutamine supplementation improve the immune response of channel catfish against Edwardsiella ictaluri.

    Science.gov (United States)

    Pohlenz, Camilo; Buentello, Alejandro; Criscitiello, Michael F; Mwangi, Waithaka; Smith, Roger; Gatlin, Delbert M

    2012-09-01

    Channel catfish was used to investigate the enhancement of vaccine efficacy following dietary supplementation with arginine (ARG, 4% of diet), glutamine (GLN, 2% of diet), or a combination of both. After vaccination against Edwardsiella ictaluri, humoral and cellular immune responses, along with lymphoid organ responses were evaluated. E. ictaluri-specific antibody titers in plasma were higher (P < 0.05) in fish fed the supplemented diets compared to those fed the basal diet as early as 7 d post-vaccination (dpv). B-cell proportion in head-kidney was higher (P < 0.05) at 14 dpv in vaccinated fish fed the GLN diet. The responsiveness of spleen and head-kidney lymphocytes against E. ictaluri was enhanced (P < 0.05) by dietary supplementation of ARG or GLN at 14 dpv. Additionally, at 7 dpv, vaccinated fish fed the GLN diet had higher (P < 0.05) head kidney weights relative to the other dietary treatments, and vaccinated fish fed ARG-supplemented diets had higher (P < 0.05) protein content in this tissue. Results from this study suggest that dietary supplementation of ARG and GLN may improve specific cellular and humoral mechanisms, enhancing the acquired immunity in vaccinated channel catfish.

  13. Nanoscale characterization of effect of L-arginine on Streptococcus mutans biofilm adhesion by atomic force microscopy.

    Science.gov (United States)

    Sharma, Shivani; Lavender, Stacey; Woo, JungReem; Guo, Lihong; Shi, Wenyuan; Kilpatrick-Liverman, LaTonya; Gimzewski, James K

    2014-07-01

    A major aetiological factor of dental caries is the pathology of the dental plaque biofilms. The amino acid L-arginine (Arg) is found naturally in saliva as a free molecule or as a part of salivary peptides and proteins. Plaque bacteria metabolize Arg to produce alkali and neutralize glycolytic acids, promoting a less cariogenous oral microbiome. Here, we explored an alternative and complementary mechanism of action of Arg using atomic force microscopy. The nanomechanical properties of Streptococcus mutans biofilm extracellular matrix were characterized under physiological buffer conditions. We report the effect of Arg on the adhesive behaviour and structural properties of extracellular polysaccharides in S. mutans biofilms. High-resolution imaging of biofilm surfaces can reveal additional structural information on bacterial cells embedded within the surrounding extracellular matrix. A dense extracellular matrix was observed in biofilms without Arg compared to those grown in the presence of Arg. S. mutans biofilms grown in the presence of Arg could influence the production and/or composition of extracellular membrane glucans and thereby affect their adhesion properties. Our results suggest that the presence of Arg in the oral cavity could influence the adhesion properties of S. mutans to the tooth surface. PMID:24763427

  14. Conductometric and volumetric studies of atorvastatin in aqueous solution of arginine from 298.15 to 313.15 K

    Directory of Open Access Journals (Sweden)

    M. M. R. Meor Mohd Affandi

    2016-01-01

    Full Text Available Categorized as a Biopharmaceutics Classification System Class II drugs, atorvastatin (ATV exhibits low aqueous solubility and bioavailability thus presenting an obstacle and great challenge to formulation researchers. Numerous studies are available in regard to the solubility enhancement of ATV, but very few actually describe this phenomenon in terms of thermodynamics and the solute-solvent interaction. Arginine (ARG is an amino acid that has been reported to enhance the solubility of the highly insoluble wheat protein gluten through hydrogen bonding and π electron-cation interaction. To our knowledge, ARG has never been investigated as a solubility enhancement agent of aqueous insoluble drugs. Thus, this study aimed to elucidate the solute-solvent and solute-cosolute interactions and derive thermodynamic parameters that bolstered the solubility of ATV in the presence of ARG. We examined the electrolytic conductance and densities of ATV-ARG binary system covering the temperature ranging from 298.15 K to 313.15 K. Conductometric and volumetric parameters such as limiting molar conductance, association constants, limiting partial molar volumes, and expansibility values were calculated. Additionally, thermodynamic parameters (ΔG0, ΔH0, ΔS0, and Es involved in the association process of the solute in the aqueous solution of ARG were also determined.

  15. Role of distal arginine in early sensing intermediates in the heme domain of the oxygen sensor FixL.

    Science.gov (United States)

    Jasaitis, Audrius; Hola, Klara; Bouzhir-Sima, Latifa; Lambry, Jean-Christophe; Balland, Veronique; Vos, Marten H; Liebl, Ursula

    2006-05-16

    FixL is a bacterial heme-based oxygen sensor, in which release of oxygen from the sensing PAS domain leads to activation of an associated kinase domain. Static structural studies have suggested an important role of the conserved residue arginine 220 in signal transmission at the level of the heme domain. To assess the role of this residue in the dynamics and properties of the initial intermediates in ligand release, we have investigated the effects of R220X (X = I, Q, E, H, or A) mutations in the FixLH heme domain on the dynamics and spectral properties of the heme upon photolysis of O(2), NO, and CO using femtosecond transient absorption spectroscopy. Comparison of transient spectra for CO and NO dissociation with steady-state spectra indicated less strain on the heme in the ligand dissociation species for all mutants compared to the wild type (WT). For CO and NO, the kinetics were similar to those of the wild type, with the exception of (1) a relatively low yield of picosecond NO rebinding to R220A, presumably related to the increase in the free volume of the heme pocket, and (2) substantial pH-dependent picosecond to nanosecond rebinding of CO to R220H, related to formation of a hydrogen bond between CO and histidine 220. Upon excitation of the complex bound with the physiological sensor ligand O(2), a 5-8 ps decay phase and a nondecaying (>4 ns) phase were observed for WT and all mutants. The strong distortion of the spectrum associated with the decay phase in WT is substantially diminished in all mutant proteins, indicating an R220-induced role of the heme in the primary intermediate in signal transmission. Furthermore, the yield of dissociated oxygen after this phase ( approximately 10% in WT) is increased in all mutants, up to almost unity in R220A, indicating a key role of R220 in caging the oxygen near the heme through hydrogen bonding. Molecular dynamics simulations corroborate these findings and suggest motions of O(2) and arginine 220 away from the heme

  16. Promoting and avoiding recombination: contrasting activities of the Escherichia coli RuvABC Holliday junction resolvase and RecG DNA translocase.

    Science.gov (United States)

    Zhang, Jing; Mahdi, Akeel A; Briggs, Geoffrey S; Lloyd, Robert G

    2010-05-01

    RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalyzing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam, or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb potentially pathological replication initiated via PriA protein at sites remote from oriC. PMID:20157002

  17. One-Pot Green Synthesis and Bioapplication of l-Arginine-Capped Superparamagnetic Fe3O4 Nanoparticles

    Science.gov (United States)

    Lai, Yongchao; Yin, Weiwei; Liu, Jinting; Xi, Rimo; Zhan, Jinhua

    2010-02-01

    Water-soluble l-arginine-capped Fe3O4 nanoparticles were synthesized using a one-pot and green method. Nontoxic, renewable and inexpensive reagents including FeCl3, l-arginine, glycerol and water were chosen as raw materials. Fe3O4 nanoparticles show different dispersive states in acidic and alkaline solutions for the two distinct forms of surface binding l-arginine. Powder X-ray diffraction and X-ray photoelectron spectroscopy were used to identify the structure of Fe3O4 nanocrystals. The products behave like superparamagnetism at room temperature with saturation magnetization of 49.9 emu g-1 and negligible remanence or coercivity. In the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride, the anti-chloramphenicol monoclonal antibodies were connected to the l-arginine-capped magnetite nanoparticles. The as-prepared conjugates could be used in immunomagnetic assay.

  18. Effects of a food supplement rich in arginine in patients with smear positive pulmonary tuberculosis--a randomised trial

    DEFF Research Database (Denmark)

    Schön, T; Idh, J; Westman, A;

    2011-01-01

    In tuberculosis (TB), the production of nitric oxide (NO) is confirmed but its importance in host defense is debated. Our aim was to investigate whether a food supplement rich in arginine could enhance clinical improvement in TB patients by increased NO production. Smear positive TB patients from.......39) or secondary outcomes. In the subgroup analysis according to HIV status, peanut supplemented HIV+/TB patients showed increased cure rate (83.8% (31/37) vs 53.1% (17/32), p ... Gondar, Ethiopia (n = 180) were randomized to a food supplementation rich in arginine (peanuts, equivalent to 1 g of arginine/day) or with a low arginine content (wheat crackers, locally called daboqolo) during four weeks. The primary outcome was cure rate according to the WHO classification...

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

    Science.gov (United States)

    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.

  20. Atomic-Resolution Structure of an N(5) Flavin Adduct in D-Arginine Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Guoxing; Yuan, Hongling; Wang, Siming; Gadda, Giovanni; Weber, Irene T. (GSU)

    2011-09-06

    D-Arginine dehydrogenase (DADH) catalyzes the flavin-dependent oxidative deamination of D-arginine and other D-amino acids to the corresponding imino acids. The 1.07 {angstrom} atomic-resolution structure of DADH crystallized with D-leucine unexpectedly revealed a covalent N(5) flavin adduct, instead of the expected iminoleucine product in the active site. This acyl adduct has been successfully reproduced by photoreduction of DADH in the presence of 4-methyl-2-oxopentanoic acid (ketoleucine). The iminoleucine may be released readily because of weak interactions in the binding site, in contrast to iminoarginine, converted to ketoleucine, which reacts with activated FAD to form the covalently linked acyl adduct.

  1. Atomic-resolution structure of an N5 flavin adduct in D-arginine dehydrogenase.

    Science.gov (United States)

    Fu, Guoxing; Yuan, Hongling; Wang, Siming; Gadda, Giovanni; Weber, Irene T

    2011-07-26

    D-Arginine dehydrogenase (DADH) catalyzes the flavin-dependent oxidative deamination of D-arginine and other D-amino acids to the corresponding imino acids. The 1.07 Å atomic-resolution structure of DADH crystallized with D-leucine unexpectedly revealed a covalent N(5) flavin adduct, instead of the expected iminoleucine product in the active site. This acyl adduct has been successfully reproduced by photoreduction of DADH in the presence of 4-methyl-2-oxopentanoic acid (ketoleucine). The iminoleucine may be released readily because of weak interactions in the binding site, in contrast to iminoarginine, converted to ketoleucine, which reacts with activated FAD to form the covalently linked acyl adduct. PMID:21707047

  2. Zinc induces unfolding and aggregation of dimeric arginine kinase by trapping reversible unfolding intermediate.

    Science.gov (United States)

    Liu, Taotao; Wang, Xicheng

    2010-11-01

    Arginine kinase plays an important role in the cellular energy metabolism of invertebrates. Dimeric arginine kinase (dAK) is unique in some marine invertebrates. The effects of Zn²(+) on the unfolding and aggregation of dAK from the sea cucumber Stichopus japonicus were investigated. Our results indicated that Zn²(+) caused dAK inactivation accompanied by conformational unfolding, the exposure of hydrophobic surface, and aggregation. Kinetic studies showed the inactivation and unfolding of dAK followed biphasic kinetic courses. Zn²(+) can affect unfolding and refolding of dAK by trapping the reversible intermediate. Our study provides important information regarding the effect of Zn²(+) on metabolic enzymes in marine invertebrates.

  3. 157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

    Science.gov (United States)

    Webber, Nathaniel; He, Yi; Reilly, James P.

    2013-12-01

    Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m2 and m2+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

  4. Topology independent protein structural alignment

    Directory of Open Access Journals (Sweden)

    DasGupta Bhaskar

    2007-10-01

    Full Text Available Abstract Background Identifying structurally similar proteins with different chain topologies can aid studies in homology modeling, protein folding, protein design, and protein evolution. These include circular permuted protein structures, and the more general cases of non-cyclic permutations between similar structures, which are related by non-topological rearrangement beyond circular permutation. We present a method based on an approximation algorithm that finds sequence-order independent structural alignments that are close to optimal. We formulate the structural alignment problem as a special case of the maximum-weight independent set problem, and solve this computationally intensive problem approximately by iteratively solving relaxations of a corresponding integer programming problem. The resulting structural alignment is sequence order independent. Our method is also insensitive to insertions, deletions, and gaps. Results Using a novel similarity score and a statistical model for significance p-value, we are able to discover previously unknown circular permuted proteins between nucleoplasmin-core protein and auxin binding protein, between aspartate rasemase and 3-dehydrogenate dehydralase, as well as between migration inhibition factor and arginine repressor which involves an additional strand-swapping. We also report the finding of non-cyclic permuted protein structures existing in nature between AML1/core binding factor and ribofalvin synthase. Our method can be used for large scale alignment of protein structures regardless of the topology. Conclusion The approximation algorithm introduced in this work can find good solutions for the problem of protein structure alignment. Furthermore, this algorithm can detect topological differences between two spatially similar protein structures. The alignment between MIF and the arginine repressor demonstrates our algorithm's ability to detect structural similarities even when spatial

  5. Amino acids composition and protein quality evaluation of marine species and meals for feed formulations in cephalopods

    OpenAIRE

    Cerezo Valverde, Jesús; Martínez-Llorens, Silvia; TOMÁS VIDAL, ANA; Jover Cerda, Miguel; Rodriguez, Carmen; Estefanell, Juan; Gairin, Joan I.; Domingues, Pedro Miguel; Rodriguez, Carlos J.; Garcia Garcia, Benjamin

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