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Sample records for additional arginine residues

  1. Chemical modification of arginine residues in the lactose repressor

    SciTech Connect

    Whitson, P.A.; Matthews, K.S.

    1987-10-06

    The lactose repressor protein was chemically modified with 2,3-butanedione and phenylglyoxal. Arginine reaction was quantitated by either amino aced analysis or incorporation of /sup 14/C-labeled phenylglyoxal. Inducer binding activity was unaffected by the modification of arginine residues, while both operator and nonspecific DNA binding activities were diminished, although to differing degrees. The correlation of the decrease in DNA binding activities with the modification of approx. 1-2 equiv of arginine per monomer suggests increased reactivity of a functionally essential residue(s). For both reagents, operator DNA binding activity was protected by the presence of calf thymus DNA, and the extent of reaction with phenylglyoxal was simultaneously diminished. This protection presumably results from steric restriction of reagent access to an arginine(s) that is (are) essential for DNA binding interactions. These experiments suggest that there is (are) an essential reactive arginine(s) critical for repressor binding to DNA.

  2. Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding.

    PubMed Central

    Lander, M; Pitt, A R; Alefounder, P R; Bardy, D; Abell, C; Battersby, A R

    1991-01-01

    The role of conserved arginine residues in hydroxymethylbilane synthase was investigated by replacing these residues in the enzyme from Escherichia coli with leucine residues by using site-directed mutagenesis. The kinetic parameters for these mutant enzymes and studies on the formation of intermediate enzyme-substrate complexes indicate that several of these arginine residues are involved in binding the carboxylate side chains of the pyrromethane cofactor and the growing oligopyrrole chain. Images Fig. 6. PMID:2025226

  3. Ricin uses arginine 235 as an anchor residue to bind to P-proteins of the ribosomal stalk

    PubMed Central

    Zhou, Yijun; Li, Xiao-Ping; Chen, Brian Y.; Tumer, Nilgun E.

    2017-01-01

    Ricin toxin A chain (RTA) binds to stalk P-proteins to reach the α–sarcin/ricin loop (SRL) where it cleaves a conserved adenine. Arginine residues at the RTA/RTB interface are involved in this interaction. To investigate the individual contribution of each arginine, we generated single, double and triple arginine mutations in RTA. The R235A mutation reduced toxicity and depurination activity more than any other single arginine mutation in yeast. Further reduction in toxicity, depurination activity and ribosome binding was observed when R235A was combined with a mutation in a nearby arginine. RTA interacts with the ribosome via a two-step process, which involves slow and fast interactions. Single arginine mutations eliminated the fast interactions with the ribosome, indicating that they increase the binding rate of RTA. Arginine residues form a positively charged patch to bind to negatively charged residues at the C-termini of P-proteins. When electrostatic interactions conferred by the arginines are lost, hydrophobic interactions are also abolished, suggesting that the hydrophobic interactions alone are insufficient to allow binding. We propose that Arg235 serves as an anchor residue and cooperates with nearby arginines and the hydrophobic interactions to provide the binding specificity and strength in ribosome targeting of RTA. PMID:28230053

  4. Camphorquinone-10-sulfonic acid and derivatives: convenient reagents for reversible modification of arginine residues

    SciTech Connect

    Pande, C.S.; Pelzig, M.; Glass, J.D.

    1980-02-01

    Camphorquinone-10-sulfonic acid hydrate was prepared by the action of selenous acid on camphor-10-sulfonic acid. Camphorquinone-10-sulfonylnorleucine was prepared either from the sulfonic acid via the sulfonyl chloride or by selenous acid oxidation of camphor-10-sulfonylnorleucine. These reagents are useful for specific, reversible modification of the guanidino groups of arginine residues. Camphorquinonsulfonic acid is a crystalline water-soluble reagent that is especially suitable for use with small arginine-containing molecules, because the sulfonic acid group of the reagent is a convenient handle for analytical and preparative separation of products. Camphorquinonesulfonylnorleucine is more useful for work with large polypeptides and proteins, because hydrolysates of modified proteins may be analyzed for norleucine to determine the extent of arginine modification. The adducts of the camphorquinone derivatives with the guanidino group are stable to 0.5 M hydroxylamine solutions at pH 7, the recommended conditions for cleavage of the corresponding cyclohexanedione adducts. At pH 8-9 the adducts of the camphorquinone derivatives with the guanidino group are cleaved by o-phenylenediamine. The modification and regeneration of arginine, of the dipeptide arginylaspartic acid, of ribonuclease S-peptide, and of soybean trypsin inhibitor are presented as demonstrations of the use of the reagents.The use of camphorquinonesulfonyl chloride to prepare polymers containing arginine-specific ligands is discussed.

  5. Immunoglobulin-G glycation by Fructose leads to structural perturbations and drop off in free Lysine and Arginine residues.

    PubMed

    Faisal, Mohammad; Alatar, Abdulrahman; Ahmad, Saheem

    2017-01-17

    Non-enzymatic glycation is the addition of free carbonyl group of reducing sugar to the free amino groups of proteins and leads to the formation of early glycation products and further into advanced glycation end products (AGEs). Fructose reacts rapidly with the free amino groups of proteins to form AGEs. AGEs are believed to be involved in the pathogenesis of several diseases, particularly in diabetic complications. In this study, IgG was glycated with fructose monosaccharide at 10 mM concentration for varying time interval. The reaction mixture was kept at 370C. The early glycation of IgG was done by nitroblue tetrazolium assay (NBT), and the generation of AGEs was done by the extent of side chain modifications (lysine and arginine), Nε-carboxymethyl lysine, pentosidine and carbonyl content. The decrease in free lysine and arginine residues suggests that protein 'IgG' has undergone modification specifically on epsilon amino groups of lysine and arginine. Additionally, their fluorescence and absorbance characteristics were also systematically studied. The results suggest that the maximum Amadori product (ketoamine content) was formed on sixth day of the incubation. The conformational structural perturbation was observed within the glycated IgG protein as studied by using various physicochemical techniques. This study reports structural perturbation, formation of various intermediates and AGEs.

  6. Computational prediction of methylation types of covalently modified lysine and arginine residues in proteins.

    PubMed

    Deng, Wankun; Wang, Yongbo; Ma, Lili; Zhang, Ying; Ullah, Shahid; Xue, Yu

    2016-05-30

    Protein methylation is an essential posttranslational modification (PTM) mostly occurs at lysine and arginine residues, and regulates a variety of cellular processes. Owing to the rapid progresses in the large-scale identification of methylation sites, the available data set was dramatically expanded, and more attention has been paid on the identification of specific methylation types of modification residues. Here, we briefly summarized the current progresses in computational prediction of methylation sites, which provided an accurate, rapid and efficient approach in contrast with labor-intensive experiments. We collected 5421 methyllysines and methylarginines in 2592 proteins from the literature, and classified most of the sites into different types. Data analyses demonstrated that different types of methylated proteins were preferentially involved in different biological processes and pathways, whereas a unique sequence preference was observed for each type of methylation sites. Thus, we developed a predictor of GPS-MSP, which can predict mono-, di- and tri-methylation types for specific lysines, and mono-, symmetric di- and asymmetrical di-methylation types for specific arginines. We critically evaluated the performance of GPS-MSP, and compared it with other existing tools. The satisfying results exhibited that the classification of methylation sites into different types for training can considerably improve the prediction accuracy. Taken together, we anticipate that our study provides a new lead for future computational analysis of protein methylation, and the prediction of methylation types of covalently modified lysine and arginine residues can generate more useful information for further experimental manipulation.

  7. Gas-phase conjugation to arginine residues in polypeptide ions via N-hydroxysuccinimide ester-based reagent ions.

    PubMed

    McGee, William M; Mentinova, Marija; McLuckey, Scott A

    2012-07-18

    Gas-phase conjugation to unprotonated arginine side-chains via N-hydroxysuccinimide (NHS) esters is demonstrated through both charge reduction and charge inversion ion/ion reactions. The unprotonated guanidino group of arginine can serve as a strong nucleophile, resulting in the facile displacement of NHS from NHS esters with concomitant covalent modification of the arginine residue. This reactivity is analogous to that observed with unprotonated primary amines such as the N-terminus or ε-amino group of lysine. In solution, however, the arginine residues tend to be protonated at pH values low enough to prevent hydrolysis of NHS esters, which would render them relatively unreactive with NHS esters. This work demonstrates novel means for gas-phase conjugation to arginine side chains in polypeptide ions.

  8. Chemical modification of lysine and arginine residues of bovine heart 2-oxoglutarate dehydrogenase: effect on the enzyme activity and regulation.

    PubMed

    Ostrovtsova, S A

    1998-01-01

    Chemical modification of arginine and lysine residues of bovine heart 2-oxoglutarate dehydrogenase with phenylglyoxal and pyridoxal 5'-phosphate inactivated the enzyme, indicating the importance of these residues for the catalysis. Inactivation caused by pyridoxal 5'-phosphate was prevented in the presence of thiamine pyrophosphate and Mg2+ allowing the assumption that lysine residues participate in binding of the cofactor.

  9. GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate

    PubMed Central

    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

  10. Two arginine residues suppress the flexibility of nucleosomal DNA in the canonical nucleosome core.

    PubMed

    Kono, Hidetoshi; Shirayama, Kazuyoshi; Arimura, Yasuhiro; Tachiwana, Hiroaki; Kurumizaka, Hitoshi

    2015-01-01

    The dynamics of nucleosomes containing either canonical H3 or its centromere-specific variant CENP-A were investigated using molecular dynamics simulations. The simulations showed that the histone cores were structurally stable during simulation periods of 100 ns and 50 ns, while DNA was highly flexible at the entry and exit regions and partially dissociated from the histone core. In particular, approximately 20-25 bp of DNA at the entry and exit regions of the CENP-A nucleosome exhibited larger fluctuations than DNA at the entry and exit regions of the H3 nucleosome. Our detailed analysis clarified that this difference in dynamics was attributable to a difference in two basic amino acids in the αN helix; two arginine (Arg) residues in H3 were substituted by lysine (Lys) residues at the corresponding sites in CENP-A. The difference in the ability to form hydrogen bonds with DNA of these two residues regulated the flexibility of nucleosomal DNA at the entry and exit regions. Our exonuclease III assay consistently revealed that replacement of these two Arg residues in the H3 nucleosome by Lys enhanced endonuclease susceptibility, suggesting that the DNA ends of the CENP-A nucleosome are more flexible than those of the H3 nucleosome. This difference in the dynamics between the two types of nucleosomes may be important for forming higher order structures in different phases.

  11. Unveiling the Mechanism of Arginine Transport through AdiC with Molecular Dynamics Simulations: The Guiding Role of Aromatic Residues

    PubMed Central

    Krammer, Eva-Maria; Ghaddar, Kassem; André, Bruno

    2016-01-01

    Commensal and pathogenic enteric bacteria have developed several systems to adapt to proton leakage into the cytoplasm resulting from extreme acidic conditions. One such system involves arginine uptake followed by export of the decarboxylated product agmatine, carried out by the arginine/agmatine antiporter (AdiC), which thus works as a virtual proton pump. Here, using classical and targeted molecular dynamics, we investigated at the atomic level the mechanism of arginine transport through AdiC of E. coli. Overall, our MD simulation data clearly demonstrate that global rearrangements of several transmembrane segments are necessary but not sufficient for achieving transitions between structural states along the arginine translocation pathway. In particular, local structural changes, namely rotameric conversions of two aromatic residues, are needed to regulate access to both the outward- and inward-facing states. Our simulations have also enabled identification of a few residues, overwhelmingly aromatic, which are essential to guiding arginine in the course of its translocation. Most of them belong to gating elements whose coordinated motions contribute to the alternating access mechanism. Their conservation in all known E. coli acid resistance antiporters suggests that the transport mechanisms of these systems share common features. Last but not least, knowledge of the functional properties of AdiC can advance our understanding of the members of the amino acid-carbocation-polyamine superfamily, notably in eukaryotic cells. PMID:27482712

  12. Role of pocket flexibility in the modulation of estrogen receptor alpha by key residue arginine 394.

    PubMed

    Mu, Yunsong; Peng, Sufen; Zhang, Aiqian; Wang, Liansheng

    2011-02-01

    Estradiol derivatives, with similar structures as estradiol (E2) or estradiol metabolites, have been recognized to have detrimental health effects on wildlife and humans. However, data at the molecular level about interactions of these compounds with biological targets are still lacking. Herein, a flexible docking approach was used to characterize the molecular interaction of nine estradiol derivatives with estrogen receptor alpha (ERα) in the ligand-binding domain. All ligands were docked in the buried hydrophobic cavity of the steroid hormone pocket. In addition, the plasticity of an active site was also identified by reversing amino acid arginine 394 for better ligand-receptor binding affinity. Finally, bioassays based on genetically modified yeast strains were used to validate the quality of molecular simulation because of their rapidity and high sensitivity. The experimental findings about logarithm values of the median effective concentration (EC50) value had a linear correlation with computational binding affinity from molecular docking, which described a pattern of interaction between estradiol derivatives and ER. The estrogenic activity of all compounds, although more or less lower than E2, was proved to possess high severe environmental risks. Considering the sidechain flexibility in the ligand binding pocket, 17α-ethylestradiol-3-cyclopentylether was reported to correlate highly significantly with known induced fit conformational changes based upon proof-of-principle calculations on human ERα with the preservation of a strong salt bridge between glutamic acid 353 and arginine 394.

  13. Neutralization of a single arginine residue gates open a two-pore domain, alkali-activated K+ channel.

    PubMed

    Niemeyer, María Isabel; González-Nilo, Fernando D; Zúñiga, Leandro; González, Wendy; Cid, L Pablo; Sepúlveda, Francisco V

    2007-01-09

    Potassium channels share a common selectivity filter that determines the conduction characteristics of the pore. Diversity in K+ channels is given by how they are gated open. TASK-2, TALK-1, and TALK-2 are two-pore region (2P) KCNK K+ channels gated open by extracellular alkalinization. We have explored the mechanism for this alkalinization-dependent gating using molecular simulation and site-directed mutagenesis followed by functional assay. We show that the side chain of a single arginine residue (R224) near the pore senses pH in TASK-2 with an unusual pKa of 8.0, a shift likely due to its hydrophobic environment. R224 would block the channel through an electrostatic effect on the pore, a situation relieved by its deprotonation by alkalinization. A lysine residue in TALK-2 fulfills the same role but with a largely unchanged pKa, which correlates with an environment that stabilizes its positive charge. In addition to suggesting unified alkaline pH-gating mechanisms within the TALK subfamily of channels, our results illustrate in a physiological context the principle that hydrophobic environment can drastically modulate the pKa of charged amino acids within a protein.

  14. Neutralization of a single arginine residue gates open a two-pore domain, alkali-activated K+ channel

    PubMed Central

    Niemeyer, María Isabel; González-Nilo, Fernando D.; Zúñiga, Leandro; González, Wendy; Cid, L. Pablo; Sepúlveda, Francisco V.

    2007-01-01

    Potassium channels share a common selectivity filter that determines the conduction characteristics of the pore. Diversity in K+ channels is given by how they are gated open. TASK-2, TALK-1, and TALK-2 are two-pore region (2P) KCNK K+ channels gated open by extracellular alkalinization. We have explored the mechanism for this alkalinization-dependent gating using molecular simulation and site-directed mutagenesis followed by functional assay. We show that the side chain of a single arginine residue (R224) near the pore senses pH in TASK-2 with an unusual pKa of 8.0, a shift likely due to its hydrophobic environment. R224 would block the channel through an electrostatic effect on the pore, a situation relieved by its deprotonation by alkalinization. A lysine residue in TALK-2 fulfills the same role but with a largely unchanged pKa, which correlates with an environment that stabilizes its positive charge. In addition to suggesting unified alkaline pH-gating mechanisms within the TALK subfamily of channels, our results illustrate in a physiological context the principle that hydrophobic environment can drastically modulate the pKa of charged amino acids within a protein. PMID:17197424

  15. HK022 Nun Requires Arginine-Rich Motif Residues Distinct from λ N

    PubMed Central

    Tawk, Caroline S.; Ghattas, Ingrid R.

    2015-01-01

    ABSTRACT Bacteriophage λ N protein binds boxB RNA hairpins in the nut (N utilization) sites of immediate early λ transcripts and interacts with host factors to suppress transcriptional termination at downstream terminators. In opposition to λ N, the Nun protein of HK022 binds the boxBs of coinfecting λ transcripts, interacts with a similar or identical set of host factors, and terminates transcription to suppress λ replication. Comparison of N-boxB and Nun-boxB nuclear magnetic resonance (NMR) structural models suggests similar interactions, though limited mutagenesis of Nun is available. Here, libraries of Nun's arginine-rich motif (ARM) were screened for the ability to exclude λ coinfection, and mutants were assayed for Nun termination with a boxB plasmid reporter system. Several Nun ARM residues appear to be immutable: Asp26, Arg28, Arg29, Arg32, Trp33, and Arg36. Asp26 and Trp33 appear to be unable to contact boxB and are not found at equivalent positions in λ N ARM. To understand if the requirement of Asp26, Trp33, and Arg36 indicated differences between HK022 Nun termination and λ N antitermination complexes, the same Nun libraries were fused to the activation domain of λ N and screened for clones able to complement N-deficient λ. Mutants were assayed for N antitermination. Surprisingly, Asp26 and Trp33 were still essential when Nun ARM was fused to N. Docking suggests that Nun ARM contacts a hydrophobic surface of the NusG carboxy-terminal domain containing residues necessary for Nun function. These findings indicate that Nun ARM relies on distinct contacts in its ternary complex and illustrate how protein-RNA recognition can evolve new regulatory functions. IMPORTANCE λ N protein interacts with host factors to allow λ nut-containing transcripts to elongate past termination signals. A competing bacteriophage, HK022, expresses Nun protein, which causes termination of λ nut transcripts. λ N and HK022 Nun use similar arginine-rich motifs (ARMs) to

  16. Improving fragmentation of poorly fragmenting peptides and phosphopeptides during collision-induced dissociation by malondialdehyde modification of arginine residues.

    PubMed

    Leitner, Alexander; Foettinger, Alexandra; Lindner, Wolfgang

    2007-07-01

    Despite significant technological and methodological advancements in peptide sequencing by mass spectrometry, analyzing peptides that exhibit only poor fragmentation upon collision-induced dissociation (CID) remains a challenge. A major cause for unfavorable fragmentation is insufficient proton 'mobility' due to charge localization at strongly basic sites, in particular, the guanidine group of arginine. We have recently demonstrated that the conversion of the guanidine group of the arginine side chain by malondialdehyde (MDA) is a convenient tool to reduce the basicity of arginine residues and can have beneficial effects for peptide fragmentation. In the present work, we have focused on peptides that typically yield incomplete sequence information in CID-MS/MS experiments. Energy-resolved tandem MS experiments were carried out on angiotensins and arginine-containing phosphopeptides to study in detail the influence of the modification step on the fragmentation process. MDA modification dramatically improved the fragmentation behavior of peptides that exhibited only one or two dominant cleavages in their unmodified form. Neutral loss of phosphoric acid from phosphopeptides carrying phosphoserine and threonine residues was significantly reduced in favor of a higher abundance of fragment ions. Complementary experiments were carried out on three different instrumental platforms (triple-quadrupole, 3D ion trap, quadrupole-linear ion trap hybrid) to ascertain that the observation is a general effect.

  17. Contributions of the substrate-binding arginine residues to maleate-induced closure of the active site of Escherichia coli aspartate aminotransferase.

    PubMed

    Matharu, A; Hayashi, H; Kagamiyama, H; Maras, B; John, R A

    2001-03-01

    Crystallography shows that aspartate aminotransferase binds dicarboxylate substrate analogues by bonds to Arg292 and Arg386, respectively [Jager, J, Moser, M. Sauder, U. & Jansonius, J. N. (1994) J. Mol. Biol., 239, 285-305]. The contribution of each interaction to the conformational change that the enzyme undergoes when it binds ligands via these residues, is assessed by probing mutant forms of the enzyme lacking either or both arginines. The probes used are NaH(3)BCN which reduces the cofactor imine, the reactive substrate analogue, cysteine sulfinate and proteolysis by trypsin. The unreactive substrate analogue, maleate, is used to induce closure. Each single mutant reacted only 2.5-fold more slowly with NaH(3)BCN than the wild-type indicating that charge repulsion by the arginines contributes little to maintaining the open conformation. Maleate lowered the rate of reduction of the wild-type enzyme more than 300-fold but had little effect on the reaction of the mutant enzymes indicating that the ability of this dicarboxylate analogue to bridge the arginines precisely makes the major contribution to closure. The R292L mutant reacted 20 times more rapidly with cysteine sulfinate than R386L but 5 x 10(4) times more slowly than the wild-type enzyme, consistent with the proposal that enzyme's catalytic abilities are not developed unless closure is induced by bridging of the arginines. Proteolysis of the mutants with trypsin showed that, in the wild-type enzyme, the bonds most susceptible to trypsin are those contributed by Arg292 and Arg386. Proteolysis of the next most susceptible bond, at Arg25 in the double mutant, was protected by maleate demonstrating the presence of an additional site on the enzyme for binding dicarboxylates.

  18. New insights into the catalytic mechanism of histidine phosphatases revealed by a functionally essential arginine residue within the active site of the Sts phosphatases.

    PubMed

    San Luis, Boris; Nassar, Nicolas; Carpino, Nick

    2013-07-01

    Sts (suppressor of T-cell receptor signalling)-1 and Sts-2 are HPs (histidine phosphatases) that negatively regulate TCR (T-cell receptor) signalling pathways, including those involved in cytokine production. HPs play key roles in such varied biological processes as metabolism, development and intracellular signalling. They differ considerably in their primary sequence and substrate specificity, but possess a catalytic core formed by an invariant quartet of active-site residues. Two histidine and two arginine residues cluster together within the HP active site and are thought to participate in a two-step dephosphorylation reaction. To date there has been little insight into any additional residues that might play an important functional role. In the present study, we identify and characterize an additional residue within the Sts phosphatases (Sts-1 Arg383 or Sts-2 Arg369) that is critical for catalytic activity and intracellular function. Mutation of Sts-1 Arg383 to an alanine residue compromises the enzyme's activity and renders Sts-1 unable to suppress TCR-induced cytokine induction. Of the multiple amino acids substituted for Arg383, only lysine partially rescues the catalytic activity of Sts-1. Although Sts-1 Arg383 is conserved in all Sts homologues, it is only conserved in one of the two sub-branches of HPs. The results of the present study highlight an essential role for Sts-1 phosphatase activity in regulating T-cell activation and add a new dimension of complexity to our understanding of HP catalytic activity.

  19. Isolation of cucurmoschin, a novel antifungal peptide abundant in arginine, glutamate and glycine residues from black pumpkin seeds.

    PubMed

    Wang, H X; Ng, T B

    2003-07-01

    A novel antifungal peptide, with a molecular mass of 8 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in gel filtration on Superdex 75 and designated cucurmoschin, was isolated from the seeds of the black pumpkin. The peptide was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel. Cucurmoschin inhibited mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella oxysporum. It inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 1.2 microM. The N-terminal sequence of cucurmoschin was rich in arginine, glutamate and glycine residues.

  20. Addition of L-arginine to the fertilization medium enhances subsequent bovine embryo development rates.

    PubMed

    Santana, Priscila P B; da Silva, Bruno B; Silva, Thiago V G; Costa, Nathalia N; Cordeiro, Marcela S; Santos, Simone S D; Ohashi, Otávio M; Miranda, Moysés S

    2016-04-01

    Although L-Arginine (ARG) has been reported as a promising bovine sperm capacitation agent, its effects on embryo development are still poorly understood. Herein, we compared the effects of ARG and/or heparin (HEP) addition to the fertilization medium for bovine oocytes on sperm capacitation and embryo development. We chose 10 mM ARG based on blastocyst development rates in a titration experiment. Addition of ARG and/or HEP to the fertilization medium resulted in similar rates of blastocyst development (P > 0.05). However, when ARG, but not HEP, was combined with a nitric oxide (NO) synthase inhibitor (N-Nitro-L-ARG-methyl ester, 10 mM) blastocyst development was decreased (P < 0.05). To assess the effects on capacitation, bovine sperm were incubated for 0, 3, and 6 hours in fertilization medium containing ARG and/or HEP and/or N-Nitro-L-ARG-methyl esterand acrosomal exocytosis rates were evaluated using fluorescein isothiocyanate conjugated Pisum sativum lectin (FITC-PSA) staining and flow cytometry. With HEP, acrosomal exocytosis rates were highest by 3 hours of incubation; however, by 6 hours, rates were similar for HEP and/or ARG (P > 0.05) and higher than those in control media (P < 0.05). Although both ARG and HEP increased sperm NO production (P < 0.05), combination with L-NAME only precluded acrosomal exocytosis when ARG added alone in the medium (P > 0.05). These results suggest that although both ARG and HEP supported sperm capacitation, only the effects of the former were driven via NO production. Moreover, ARG was also as effective as HEP at improving blastocyst development rates. Therefore, ARG may be used as a low-cost alternative sperm capacitation agent for bovine in vitro embryo production.

  1. Proteolytic Cleavage at Twin Arginine Residues Affects Structural and Functional Transitions of Lupin Seed 11S Storage Globulin

    PubMed Central

    Capraro, Jessica; Sessa, Fabio; Magni, Chiara; Scarafoni, Alessio; Maffioli, Elisa; Tedeschi, Gabriella; Croy, Ron R. D.; Duranti, Marcello

    2015-01-01

    The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed. PMID:25658355

  2. Proteolytic cleavage at twin arginine residues affects structural and functional transitions of lupin seed 11S storage globulin.

    PubMed

    Capraro, Jessica; Sessa, Fabio; Magni, Chiara; Scarafoni, Alessio; Maffioli, Elisa; Tedeschi, Gabriella; Croy, Ron R D; Duranti, Marcello

    2015-01-01

    The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed.

  3. Methylglyoxal-induced modification of arginine residues decreases the activity of NADPH-generating enzymes.

    PubMed

    Morgan, Philip E; Sheahan, Pamela J; Pattison, David I; Davies, Michael J

    2013-08-01

    Inadequate control of plasma and cellular glucose and ketone levels in diabetes is associated with increased generation of reactive aldehydes, including methylglyoxal (MGO). These aldehydes react with protein side chains to form advanced glycation end-products (AGEs). Arg residues are particularly susceptible to MGO glycation and are essential for binding NADP(+) in several enzymes that generate NADPH, a coenzyme for many critical metabolic and antioxidant enzymes. In most animal cells, NADPH is produced predominantly by glucose-6-phosphate dehydrogenase (G6PD) in the oxidative phase of the pentose phosphate pathway and, to a lesser extent, by isocitrate dehydrogenase (IDH) and malic enzyme (ME). In this study, the activities of isolated G6PD, IDH, and ME were inhibited by MGO (0-2.5mM, 2-3h, 37°C), in a dose- and time-dependent manner, with G6PD and IDH more sensitive to modification than ME. Significant inhibition of these two enzymes occurred with MGO levels ≥500μM. Incubation with radiolabeled MGO (0-500µM, 0-3h, 37°C) demonstrated dose- and time-dependent adduction to G6PD and IDH. HPLC analysis provided evidence for AGE formation and particularly the hydroimidazolones MG-H1 and MG-H2 from Arg residues, with corresponding loss of parent Arg residues. Peptide mass mapping studies confirmed hydroimidazolone formation on multiple peptides in G6PD and IDH, including those critical for NADP(+) binding, and substrate binding, in the case of IDH. These results suggest that modification of NADPH-producing enzymes by reactive aldehydes may result in alterations to the cellular redox environment, potentially predisposing cells to further damage by oxidants and reactive aldehydes.

  4. Spectral Envelopes and Additive + Residual Analysis/Synthesis

    NASA Astrophysics Data System (ADS)

    Rodet, Xavier; Schwarz, Diemo

    The subject of this chapter is the estimation, representation, modification, and use of spectral envelopes in the context of sinusoidal-additive-plus-residual analysis/synthesis. A spectral envelope is an amplitude-vs-frequency function, which may be obtained from the envelope of a short-time spectrum (Rodet et al., 1987; Schwarz, 1998). [Precise definitions of such an envelope and short-time spectrum (STS) are given in Section 2.] The additive-plus-residual analysis/synthesis method is based on a representation of signals in terms of a sum of time-varying sinusoids and of a non-sinusoidal residual signal [e.g., see Serra (1989), Laroche et al. (1993), McAulay and Quatieri (1995), and Ding and Qian (1997)]. Many musical sound signals may be described as a combination of a nearly periodic waveform and colored noise. The nearly periodic part of the signal can be viewed as a sum of sinusoidal components, called partials, with time-varying frequency and amplitude. Such sinusoidal components are easily observed on a spectral analysis display (Fig. 5.1) as obtained, for instance, from a discrete Fourier transform.

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

    PubMed

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

    2011-01-01

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

  6. Characterization of a metalloprotease from ovine chromaffin granules which cleaves a proenkephalin fragment (BAM12P) at a single arginine residue.

    PubMed Central

    Tezapsidis, N; Parish, D C

    1994-01-01

    A metalloprotease has been identified in ovine chromaffin granules which cleaves the proenkephalin fragment BAM12P to produce adrenorphin-Gly. This cleavage occurs at a single arginine residue and is an intermediate step in the production of the opiate adrenorphin in vivo. The identity of the product was confirmed by reverse-phase and ion-exchange chromatography. The adrenorphin-Gly-generating enzyme (AGE) was determined by chromatofocusing to have a pI value of 5.2 and bound strongly to a metal-chelate affinity column. After purification by gel-filtration and ion-exchange chromatography AGE was free of contaminating activities, as cleavage of radiolabelled BAM12P generated a single product as judged by reverse-phase and ion-exchange chromatography. The enzyme has a molecular mass of approx. 45 kDa and a pH optimum of 8.6 in Mops, Taps and Hepes buffers, but was inhibited by phosphate buffers. It was inhibited by micromolar concentrations of copper and zinc ions, but not by millimolar concentrations of calcium or manganese ions. The addition of BAM22P, dynorphin 1-13 or dynorphin 1-8 to the incubation mixture inhibited the cleavage of radiolabelled BAM12P. The cleavage was also inhibited by the presence of catecholamines at concentrations similar to those found within the chromaffin granule. This may explain the known effect of reserpine on chromaffin cells of reducing catecholamine levels and simultaneously increasing adrenorphin levels. It may also indicate a function for AGE and adrenorphin as reporters of intragranular conditions. Images Figure 1 PMID:8043007

  7. Role of the conserved arginine 274 and histidine 224 and 228 residues in the NuoCD subunit of complex I from Escherichia coli.

    PubMed

    Belevich, Galina; Euro, Liliya; Wikström, Mårten; Verkhovskaya, Marina

    2007-01-16

    The conserved arginine 274 and histidine 224 and 228 residues in subunit NuoCD of complex I from Escherichia coli were substituted for alanine. The wild-type and mutated NuoCD subunit was expressed on a plasmid in an E. coli strain bearing a nuoCD deletion. Complex I was fully expressed in the H224A and H228A mutants, whereas the R274A mutation yielded approximately 50% expression. Ubiquinone reductase activity of complex I was studied in membranes and with purified enzyme and was 50% and 30% of the wild-type activity in the H224A and H228A mutants, respectively. The activity of R274A was less than 5% of the wild type in membranes but 20% in purified complex I. Rolliniastatin inhibited quinone reductase activity in the mutants with similar affinity as in the wild type, indicating that the quinone-binding site was not significantly altered by the mutations. Ubiquinone-dependent superoxide production by complex I was similar to the wild type in the R274A mutant but slightly higher in the H224A and H228A mutants. The EPR spectra of purified complex I from the H224A and H228A mutants did not differ from the wild type. In contrast, the signals of the N2 cluster and another fast-relaxing [4Fe-4S] cluster, tentatively assigned as N6b, were drastically decreased in the NADH-reduced R274A mutant enzyme but reappeared on further reduction with dithionite. These findings show that the redox potential of the N2 and N6b centers is shifted to more negative values by the R274A mutation. Purified complex I was reconstituted into liposomes, and electric potential was generated across the membrane upon NADH addition in all three mutant enzymes, suggesting that none of the mutations directly affect the proton-pumping machinery.

  8. Insights into the modulation of optimum pH by a single histidine residue in arginine deiminase from Pseudomonas aeruginosa.

    PubMed

    Ding, Hanjing; Liu, Hui; Yin, Yan; Ding, Ying; Jia, Yan; Chen, Qingming; Zou, Guolin; Zheng, Zhongliang

    2012-09-01

    Arginine deiminase (ADI) is a potential antitumor agent for the arginine deprivation treatment of L-arginine auxotrophic tumors. The optimum pH of ADI varies significantly, yet little is known about the origin of this variety. Here, Pseudomonas aeruginosa ADI (PaADI), an enzyme that functions only at acidic pH, was utilized as the model system. The results of UV-pH titration imply that the nucleophilic Cys406 thiol group is protonated in the resting state. The H405R single mutation resulted in an altered pH optimum (from pH 5.5 to 6.5), an increased k(cat) (from 9.8 s(-1) to 101.7 s(-1) at pH 6.5), and a shifted pH rate dependence (ascending limb pK(a) from 3.6 to 4.4). Other mutants were constructed to investigate the effects of hydrogen bonding, charge distribution, and hydrophobicity on the properties of the enzyme. The pH optima of His405 mutants were all shifted to a relatively neutral pH except for the H405E mutant. The results of kinetic characterizations and molecular dynamic simulations revealed that the active site hydrogen bonding network involving Asp280 and His405 plays an important role in controlling the dependence of PaADI activity on pH. Moreover, the H405R variant showed increased cytotoxicity towards arginine auxotrophic cancer cell lines.

  9. Arginine residues on the opposite side of the active site stimulate the catalysis of ribosome depurination by ricin A chain by interacting with the P-protein stalk.

    PubMed

    Li, Xiao-Ping; Kahn, Peter C; Kahn, Jennifer Nielsen; Grela, Przemyslaw; Tumer, Nilgun E

    2013-10-18

    Ricin inhibits protein synthesis by depurinating the α-sarcin/ricin loop (SRL). Ricin holotoxin does not inhibit translation unless the disulfide bond between the A (RTA) and B (RTB) subunits is reduced. Ricin holotoxin did not bind ribosomes or depurinate them but could depurinate free RNA. When RTA is separated from RTB, arginine residues located at the interface are exposed to the solvent. Because this positively charged region, but not the active site, is blocked by RTB, we mutated arginine residues at or near the interface of RTB to determine if they are critical for ribosome binding. These variants were structurally similar to wild type RTA but could not bind ribosomes. Their K(m) values and catalytic rates (k(cat)) for an SRL mimic RNA were similar to those of wild type, indicating that their activity was not altered. However, they showed an up to 5-fold increase in K(m) and up to 38-fold decrease in kcat toward ribosomes. These results suggest that the stalk binding stimulates the catalysis of ribosome depurination by RTA. The mutated arginines have side chains behind the active site cleft, indicating that the ribosome binding surface of RTA is on the opposite side of the surface that interacts with the SRL. We propose that stalk binding stimulates the catalysis of ribosome depurination by orienting the active site of RTA toward the SRL and thereby allows docking of the target adenine into the active site. This model may apply to the translation factors that interact with the stalk.

  10. Chemical modification of a functional arginine residue in diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase I from Saccharomyces cerevisiae.

    PubMed Central

    Robinson, A K; Barnes, L D

    1991-01-01

    Phenylglyoxal, a reagent with high specificity for arginine residues, inactivated Ap4A phosphorylase I from Saccharomyces cerevisiae in a pseudo-first-order manner. The second-order rate constant was 11.5 +/- 2.5 M-1 min-1. The loss of activity was a linear function of the incorporation of [7-14C]phenylglyoxal. The incorporation of 1.9 +/- 0.4 mol of phenylglyoxal/mol of enzyme accounted for complete loss of activity. The specificity of inactivation by phenylglyoxal was tested in the presence of ApnA (n = 2-6), ADP, ATP and Pi. The substrates, Ap4A, Ap5A and Pi protected the enzyme against inactivation, but Ap2A, Ap3A and Ap6A did not. Ap4A, Ap5A and Pi reduced the rate of inactivation by about 70%, 60% and 37% respectively. The Ap4A phosphorolysis products, ADP and ATP, also partially protected the enzyme against inactivation by phenylglyoxal. Thus Ap4A phosphorylase I probably contains an arginine residue in the binding site for Ap4A. Images Fig. 4. PMID:1656937

  11. The bovine mannose 6-phosphate/insulin-like growth factor II receptor. The role of arginine residues in mannose 6-phosphate binding.

    PubMed

    Dahms, N M; Rose, P A; Molkentin, J D; Zhang, Y; Brzycki, M A

    1993-03-15

    The extracytoplasmic region of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) consists of 15 homologous repeating domains, each of which is approximately 147 residues in length. The receptor contains two high affinity mannose 6-phosphate (Man-6-P) binding sites and our recent studies (Westlund, B., Dahms, N. M., and Kornfeld, S. (1991) J. Biol. Chem. 266, 23233-23239) have localized these two binding sites to domains 1-3 and 7-11. To further define the location of the Man-6-P binding sites and to determine the role of specific arginine residues in Man-6-P binding, site-directed mutagenesis was utilized to create truncated soluble forms of the M6P/IGF-II receptor in conjunction with either conservative (Lys) or nonconservative (Ala) replacement of arginine residues. These mutants were expressed transiently in COS-1 cells and assayed for their ability to bind phosphomannosyl residues by affinity chromatography. Analysis of the ligand binding activity of carboxyl-terminal truncated forms of the receptor's extracytoplasmic region demonstrated that the second Man-6-P binding site is contained within domains 7-9. Substitution of Arg435 in domain 3 of the amino-terminal binding site and Arg1334 in domain 9 of the second binding site results in a dramatic loss of ligand binding activity. However, substitutions at positions 435 and/or 1334 did not affect the secretion, glycosylation, or immunoreactivity of these truncated proteins. Taken together, these results indicate that Arg435 and Arg1334 are essential components of the M6P/IGF-II receptor's high affinity Man-6-P binding sites.

  12. In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model.

    PubMed

    Kim, H J; Nishikawa, S; Tokutomi, Y; Takenaka, H; Hamada, M; Kuby, S A; Uesugi, S

    1990-02-06

    Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.

  13. Arginine residues in the C-terminal and their relationship with the analgesic activity of the toxin from the Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU1).

    PubMed

    Wang, Yu; Song, Yong-Bo; Yang, Guang-Zhao; Cui, Yong; Zhao, Yong-Shan; Liu, Yan-Feng; Ma, Yan; Wu, Chun-Fu; Zhang, Jing-Hai

    2012-09-01

    In this study, we investigated the functional role of arginines in the C-terminal (65-67) of BmK AGP-SYPU1, an analgesic peptide from the Chinese scorpion Buthus martensii Karsch. Using site-directed mutagenesis, arginines at the C-terminal (65-66) were deleted or added to the C-terminal (67). The genes for three mutants of BmK AGP-SYPU1 were obtained by PCR. An analgesic activity assay was used to evaluate the role of arginine residues in the analgesic activity. The three-dimensional structure of BmK AGP-SYPU1 was established by homology modeling. As a result, we showed that the arginines in the C-terminal are crucial for the analgesic activity and may be located at analgesic functional sites. Our work has implications for further modification of scorpion toxins to obtain new analgesic peptides with enhanced activity.

  14. Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues.

    PubMed Central

    Mäkinen, K K; Mäkinen, P L

    1978-01-01

    Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin. PMID:743227

  15. Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

    SciTech Connect

    Yu, Jungeun; Shin, Bongjin; Park, Eui-Soon; Yang, Sujeong; Choi, Seunga; Kang, Misun; Rho, Jaerang

    2010-01-01

    Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.

  16. Effect of organic residues addition on the technological properties of clay bricks.

    PubMed

    Demir, Ismail

    2008-01-01

    The objective of this study is to investigate the utilization potential of several organic residues in clay bricks. Sawdust, tobacco residues, and grass are widespread by-products of industrial and agricultural processes in Turkey. These residue materials have long cellulose fibres. Sawdust and tobacco residues generally are used as fuel, and the grass is utilized for agricultural purposes. The insulation capacity of brick increases with the increasing porosity of the clay body. Combustible, organic types of pore-forming additives are most frequently used for this purpose. For this reason, increasing amounts of organic residues (0%, 2.5%, 5% and 10% in wt.) were mixed with raw brick-clay. All samples were fired at 900 degrees C. Effects on shaping, plasticity, density, and mechanical properties were investigated. The organic residue additions were found to be effective for pore-forming in the clay body with the clay maintaining acceptable mechanical properties. It was observed that the fibrous nature of the residues did not create extrusion problems. However, higher residue addition required a higher water content to ensure the right plasticity. As a result, sawdust, tobacco residues, and grass can be utilized in an environmentally safe way as organic pore-forming agents in brick-clay.

  17. MD-2 Residues Tyrosine 42, Arginine 69, Aspartic Acid 122, and Leucine 125 Provide Species Specificity for Lipid IVA*

    PubMed Central

    Meng, Jianmin; Drolet, Joshua R.; Monks, Brian G.; Golenbock, Douglas T.

    2010-01-01

    Lipopolysaccharide (LPS) activates the innate immune response through the Toll-like receptor 4 (TLR4)·MD-2 complex. A synthetic lipid A precursor, lipid IVA, induces an innate immune response in mice but not in humans. Both TLR4 and MD-2 are required for the agonist activity of lipid IVA in mice, with TLR4 interacting through specific surface charges at the dimerization interface. In this study, we used site-directed mutagenesis to identify the MD-2 residues that determine lipid IVA species specificity. A single mutation of murine MD-2 at the hydrophobic pocket entrance, E122K, substantially reduced the response to lipid IVA. Combining the murine MD-2 E122K with the murine TLR4 K367E/S386K/R434Q mutations completely abolished the response to lipid IVA, effectively converting the murine cellular response to a human-like response. In human cells, however, simultaneous mutations of K122E, K125L, Y41F, and R69G on human MD-2 were required to promote a response to lipid IVA. Combining the human MD-2 quadruple mutations with the human TLR4 E369K/Q436R mutations completely converted the human MD-2/human TLR4 receptor to a murine-like receptor. Because MD-2 residues 122 and 125 reside at the dimerization interface near the pocket entrance, surface charge differences here directly affect receptor dimerization. In comparison, residues 42 and 69 reside at the MD-2/TLR4 interaction surface opposite the dimerization interface. Surface charge differences there likely affect the binding angle and/or rigidity between MD-2 and TLR4, exerting an indirect influence on receptor dimerization and activation. Thus, surface charge differences at the two MD-2/TLR4 interfaces determine the species-specific activation of lipid IVA. PMID:20592019

  18. Identification and Characterization of Ana o 3 Modifications on Arginine-111 Residue in Heated Cashew Nuts.

    PubMed

    Mattison, Christopher P; Grimm, Casey C; Li, Yichen; Chial, Heidi J; McCaslin, Darrell R; Chung, Si-Yin; Bren-Mattison, Yvette; Wasserman, Richard L

    2017-01-18

    Raw and roasted cashew nut extracts were evaluated for protein modifications by mass spectrometry. Independent modifications on the Arg-111 residue of Ana o 3 were observed in roasted but not raw cashew nuts. The mass changes of 72.0064 or 53.9529 Da are consistent with the formation of carboxyethyl and hydroimidazolone modifications at the Arg-111 residue. These same modifications were observed in Ana o 3 purified from roasted but not raw cashew nuts, albeit at a relatively low occurrence. Circular dichroism indicated that Ana o 3 purified from raw and roasted cashew nuts had similar secondary structure, and dynamic light scattering analysis indicated there was no observable difference in particle size. The stability of Ana o 3 purified from raw and roasted cashew nuts to trypsin was similar in the absence of or following treatment with a reducing agent. Only minor differences in IgE binding to Ana o 3 were observed by ELISA among a cohort of cashew-allergic patient sera.

  19. Residue cluster additivity of thermodynamic stability in the hydrophobic core of mesophile vs. hyperthermophile rubredoxins.

    PubMed

    LeMaster, David M; Hernández, Griselda

    2007-02-01

    The branched sidechain residues 24 and 33 in the hydrophobic core of rubredoxin differ between the Clostridium pasteurianum (Cp) and Pyrococcus furiosus (Pf) sequences. Their X-ray structures indicate that these two sidechains are in van der Waals contact with each other, while neither appears to significantly interact with the other nonconserved residues. The simultaneous interchange of residues 24 and 33 between the Cp and Pf rubredoxin sequences yield a complementary pair of hybrid proteins for which the sum of their thermodynamic stabilities equals that of the parental rubredoxins. The 1.2 kcal/mol change arising from this two residues interchange accounts for 21% of the differential thermodynamic stability between the mesophile and hyperthermophile proteins. The additional interchange of the sole nonconserved aromatic residue in the hydrophobic core yields a 0.78 kcal/mol deviation from thermodynamic additivity.

  20. Substitution of a single amino acid residue in the aromatic/arginine selectivity filter alters the transport profiles of tonoplast aquaporin homologs.

    PubMed

    Azad, Abul Kalam; Yoshikawa, Naoki; Ishikawa, Takahiro; Sawa, Yoshihiro; Shibata, Hitoshi

    2012-01-01

    Aquaporins are integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray crystallography of aquaporins indicates that four amino acids constitute an aromatic/arginine (ar/R) pore constriction known as the selectivity filter. On the basis of these four amino acids, tonoplast aquaporins called tonoplast intrinsic proteins (TIPs) are divided into three groups in Arabidopsis. Herein, we describe the characterization of two group I TIP1s (TgTIP1;1 and TgTIP1;2) from tulip (Tulipa gesneriana). TgTIP1;1 and TgTIP1;2 have a novel isoleucine in loop E (LE2 position) of the ar/R filter; the residue at LE2 is a valine in all group I TIPs from model plants. The homologs showed mercury-sensitive water channel activity in a fast kinetics swelling assay upon heterologous expression in Pichia pastoris. Heterologous expression of both homologs promoted the growth of P. pastoris on ammonium or urea as sole sources of nitrogen and decreased growth and survival in the presence of H(2)O(2). TgTIP1;1- and TgTIP1;2-mediated H(2)O(2) conductance was demonstrated further by a fluorescence assay. Substitutions in the ar/R selectivity filter of TgTIP1;1 showed that mutants that mimicked the ar/R constriction of group I TIPs could conduct the same substrates that were transported by wild-type TgTIP1;1. In contrast, mutants that mimicked group II TIPs showed no evidence of urea or H(2)O(2) conductance. These results suggest that the amino acid residue at LE2 position is critical for the transport selectivity of the TIP homologs and group I TIPs might have a broader spectrum of substrate selectivity than group II TIPs.

  1. Determination of Bulk Residual Stresses in Electron Beam Additive-Manufactured Aluminum

    NASA Astrophysics Data System (ADS)

    Brice, Craig A.; Hofmeister, William H.

    2013-11-01

    Additive-manufactured aluminum alloy deposits were analyzed using neutron diffraction to characterize the effect of intermediate stress relief anneal heat treatment on bulk residual stresses in the final part. Based on measured interplanar spacing, stresses were calculated at various locations along a single bead, stacked wall deposit. A comparison between an uninterrupted deposited wall and an interrupted, stress-relieved, and annealed deposited wall showed a measureable reduction in residual stress magnitude at the interface with a corresponding shift in stress character into the deposit. This shift changes the interface stresses from purely compressive to partially tensile. The residual stress profile varied along the length of the deposit, and the heat-treatment procedure reduced the overall magnitude of the stress at the interface by 10 through 25 MPa. These results are interpreted in terms of thermal gradients inherent to the process and compared with prior residual stress-characterization studies in additive-manufactured metallic structures.

  2. Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability

    PubMed Central

    Yanagisawa, Tatsuo; Takahashi, Hideyuki; Suzuki, Takehiro; Masuda, Akiko; Dohmae, Naoshi; Yokoyama, Shigeyuki

    2016-01-01

    Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational β-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The β-lysyl EF-P modification pathway is conserved among only 26–28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl–EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli β-lysyl–EF-P. The possible reasons for the unique requirement of rhamnosyl–EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others. PMID:26840407

  3. The secondary structure of apolipoproteins in human HDL3 particles after chemical modification of their tyrosine, lysine, cysteine or arginine residues. A Fourier transform infrared spectroscopy study.

    PubMed

    Herzyk, E; Owen, J S; Chapman, D

    1988-09-02

    Fourier transform infrared spectra of apolipoprotein E-depleted human HDL3 have been obtained in H2O and 2H2O buffers. The absorption bands in the protein amide I and amide II regions (1700-1500 cm-1) were assigned to alpha-helical, disordered and beta-strand/beta-turn structures of apolipoproteins A-I and A-II (apoA-I and apoA-II), the apolipoprotein constituents of HDL3. Modification of HDL3 by tetranitromethane (TNM) treatment, acetylation, reduction plus alkylation and 1,2-cyclohexanedione treatment derivatised tyrosine, lysine, cysteine and arginine residues, respectively, and caused alteration of the secondary structure of the HDL3 apolipoproteins to different extents. Each of the chemical modifications caused changes in the frequency of bands associated with beta-strands/beta-turns, but only TNM treatment of HDL3, as judged by the second- and fourth-derivative spectra, resulted in a shift of the band assigned to the alpha-helical structure of the proteins. In agreement with other workers, only TNM treatment of HDL3 particles was found to inhibit their binding by high-affinity cell membrane receptors. It is proposed, therefore, that receptor recognition of HDL3 particles is dependent on conservation of the alpha-helix structures within apoA-I and apoA-II, and that beta-strand/beta-turn structures are not involved. This conclusion is consistent with the predominance of amphipathic alpha-helical structures in both apolipoproteins and with the relaxed specificity of the receptors which are thought to recognise both apoA-I and apoA-II.

  4. Effects of individual genetic substitutions of arginine residues on the deprotonation and reprotonation kinetics of the Schiff base during the bacteriorhodopsin photocycle.

    PubMed Central

    Lin, G C; el-Sayed, M A; Marti, T; Stern, L J; Mogi, T; Khorana, H G

    1991-01-01

    The rates are determined for the deprotonation and reprotonation of the protonated Schiff base (PSB) as well as of formation and decay of the UV transient in the photocycle of seven bacteriorhodopsin (bR) mutants in which Arg-7, 82, 164, 175, 225, or 227 are replaced by glutamine and Arg-134 by cysteine. The results show that all these mutations increase the rate of deprotonation of the PSB compared to ebR, (wild-type bacteriorhodopsin expressed in Escherichia coli) greatly increase the rate of the reprotonation of the SB (Schiff base) in the case of the Arg-164 and Arg-175 mutations and dramatically decrease this rate in the case of the Arg-227 mutation. Temperature studies on the latter mutant suggest that the observed change in its rate of reprotonation is due to large decrease in the energy and entropy of activation, similar to those observed for Asp-96 mutations (Miller, A. and D. Orsterhelt. 1990. Biochim. Biophys. Acta. 1020:57-64). These results suggest that the reprotonation process is changed to a proton diffusion-controlled mechanism in the Arg-227 mutant due to a change in the structure of the proton channel. The absorption intensity ratio (AUV/AMslow) of each arginine mutant relative to that of ebR is found to be similar to that for native purple membrane (PM) except for the Arg-227 mutant where it is greatly reduced, and for the Arg-82 mutant where it is not observed, suggesting that both Arg-227 and Arg-82 residues somehow play roles in inducing the UV transient absorption. All the above results are discussed in terms of the model for the structure of bR proposed by Henderson, R., J.M. Baldwin, T.A. Ceska, F. Zemlin, E. Beckmann, and K.H. Downing. (1990. J. Mol. Biol. 213:899-929). PMID:1883936

  5. Control of arginine utilization in Neurospora.

    PubMed Central

    Weiss, R L; Davis, R H

    1977-01-01

    The response of Neurospora to changes in the availibility of exogenous arginine was investigated. Upon addition of arginine to the growth medium, catabolism is initiated within minutes. This occurs prior to expansion of the arginine pool or augmentation of catabolic enzyme levels. (Basal levels are approximately 25% of those found during growth in arginine-supplemented medium.) Catabolism of arginine is independent of protein synthesis, indicating that the catabolic enzymes are active but that arginine is not available for catabolism unless present in the medium. Upon exhaustion of the supply of exogenous arginine, catabolism ceases abruptly, despite an expanded arginine pool and induced levels of the catabolic enzymes. The arginine pool supports protein synthesis until the cells regain their normal capacity for endogenous arginine synthesis. These observations, combined with the known small level of induction of arginine catabolic enzymes, non-repressibility of most biosynthetic enzymes, and vesicular localization of the bulk of the arginine pool, suggest that compartmentation plays a significant role in controlling arginine metabolism in Neurospora. PMID:838690

  6. "Keep a low profile": pesticide residue, additives, and freon use in Australian tobacco manufacturing

    PubMed Central

    Chapman, S

    2003-01-01

    Objectives: To review the Australian tobacco industry's knowledge of pesticide residue on Australian tobacco and its policies and practices on resisting calls by tobacco control advocates that consumers should be informed about pesticide residue as well as additives. Methods: Review of previously internal industry documents relevant to pesticides and additives in Australian tobacco located from the Master Settlement Agreement websites. Results: Between 1972 and 1994 Philip Morris Australia was aware that its leaf samples were often contaminated with pesticide residue, sometimes including organochlorine levels described by PM's European laboratories as being "extremely high". Consumers were not advised of the contamination nor products withdrawn. From 1981, the industry also resisted calls to declare fully the extent of use and long term safety data on all additives used in their products. They developed standard public responses that were evasive and misleading and, in 2000, implemented voluntary additive disclosure which allowed the companies to continue to avoid disclosure of any ingredient they deemed to be a trade secret. There was extensive use of ozone depleting freon in Australian tobacco manufacturing. Again, the industry kept this information away from consumers. Conclusions: Australian smokers are unable to make informed decisions about smoking because pesticide and additive disclosure remains voluntary. The Australian government should regulate tobacco to require full disclosure including information on the likely health consequences of inhaling pesticide and additive pyrolysis products. PMID:14645948

  7. Monitoring Residual Solvent Additives and Their Effects in Solution Processed Solar Cells

    NASA Astrophysics Data System (ADS)

    Fogel, Derek M.; Basham, James I.; Engmann, Sebastian; Pookpanratana, Sujitra J.; Bittle, Emily G.; Jurchescu, Oana D.; Gundlach, David J.

    2015-03-01

    High boiling point solvent additives are a widely adopted approach for increasing bulk heterojunction (BHJ) solar cell efficiency. However, experiments show residual solvent can persist for hours after film deposition, and certain common additives are unstable or reactive. We report here on the effects of residual 1,8-diiodooctane on the electrical performance of poly(3-hexylthiophene-2,5-diyl) (P3HT): phenyl-C71-butyric acid methyl ester (PC[71]BM) BHJ photovoltaic cells. We optimized our fabrication process for efficiency at an active layer thickness of 220 nm, and all devices were processed in parallel to minimize unintentional variations between test structures. The one variable in this study is the active layer post spin drying time. Immediately following the cathode deposition, we measured the current-voltage characteristics at one sun equivalent illumination intensity, and performed impedance spectroscopy to quantify charge density, lifetime, and recombination process. Spectroscopic ellipsometry, FTIR, and XPS are also used to monitor residual solvent and correlated with electrical performance. We find that residual additive degrades performance by increasing the series resistance and lowering efficiency, fill factor, and free carrier lifetime.

  8. A novel mutation affecting the arginine-137 residue of AVPR2 in dizygous twins leads to nephrogenic diabetes insipidus and attenuated urine exosome aquaporin-2.

    PubMed

    Hinrichs, Gitte R; Hansen, Louise H; Nielsen, Maria R; Fagerberg, Christina; Dieperink, Hans; Rittig, Søren; Jensen, Boye L

    2016-04-01

    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 inappropriate antidiuresis or congenital X-linked nephrogenic diabetes insipidus. We present a novel mutation in codon 137 within AVPR2 with substitution of glycine for arginine in male dizygotic twins. Nephrogenic diabetes insipidus was demonstrated by water deprivation test and resistance to vasopressin 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.

  9. Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia Coli Alkaline Phosphatase

    SciTech Connect

    O'Brien, P.J.; Lassila, J.K.; Fenn, T.D.; Zalatan, J.G.; Herschlag, D.

    2009-05-22

    Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by {approx}3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 {angstrom} X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state.

  10. Evaluation of an eastern shale oil residue as an asphalt additive

    SciTech Connect

    Thomas, K.P.; Harnsberger, P.M.

    1995-09-01

    An evaluation of eastern shale oil (ESO) residue as an asphalt additive to reduce oxidative age hardening and moisture susceptibility was conducted by Western Research Institute (WRI). The ESO residue, have a viscosity of 23.9 Pa{lg_bullet}s at 60{degree}C (140{degree}F), was blended with three different petroleum-derived asphalts, ASD-1, AAK-1, and AAM-1, which are known to be very susceptible to oxidative aging. Rheological and infrared analyses of the unaged and aged asphalts and the blends were then conducted to evaluate oxidative age hardening. In addition, the petroleum-derived asphalts and the blends were coated onto three different aggregates, Lithonia granite (RA), a low-absorption limestone (RD), and a siliceous Gulf Coast gravel (RL), and compacted into briquettes. Successive freeze-thaw cycling was then conducted to evaluate the moisture susceptibility of the prepared briquettes. The rheological analyses of the unaged petroleum-derived asphalts and their respective blends indicate that the samples satisfy the rutting requirement. However, the aging indexes for the rolling thin film oven (RTFO)-aged and RTFO/pressure aging vessel (PAV)-aged samples indicate that the blends are stiffer than the petroleum-derived asphalts. This means that when in service the blends will be more prone to pavement embrittlement and fatigue cracking than the petroleum-derived asphalts. Infrared analyses were also conducted on the three petroleum-derived asphalts and the blends before and after RTFO/PAV aging. In general, upon RTFO/PAV aging, the amounts of carbonyls and sulfoxides in the samples increase, indicating that the addition of the ESO residue does not mitigate the chemical aging (oxidation) of the petroleum-derived asphalts. This information correlates with the rheological data and the aging indexes that were calculated for the petroleum-derived asphalts and the blends.

  11. Effect of additives on the tensile performance and protein solubility of industrial oilseed residual based plastics.

    PubMed

    Newson, William R; Kuktaite, Ramune; Hedenqvist, Mikael S; Gällstedt, Mikael; Johansson, Eva

    2014-07-16

    Ten chemical additives were selected from the literature for their proposed modifying activity in protein-protein interactions. These consisted of acids, bases, reducing agents, and denaturants and were added to residual deoiled meals of Crambe abyssinica (crambe) and Brassica carinata (carinata) to modify the properties of plastics produced through hot compression molding at 130 °C. The films produced were examined for tensile properties, protein solubility, molecular weight distribution, and water absorption. Of the additives tested, NaOH had the greatest positive effect on tensile properties, with increases of 105% in maximum stress and 200% in strain at maximum stress for crambe and a 70% increase in strain at maximum stress for carinata. Stiffness was not increased by any of the applied additives. Changes in tensile strength and elongation for crambe and elongation for carinata were related to changes in protein solubility. Increased pH was the most successful in improving the protein aggregation and mechanical properties within the complex chemistry of residual oilseed meals.

  12. Evaluation of an eastern shale oil residue as an asphalt additive

    SciTech Connect

    Thomas, K.P.; Harnsberger, P.M.

    1995-12-19

    An evaluation of eastern shale oil (ESO) residue as an asphalt additive to reduce oxidative age-hardening and moisture susceptibility was conducted. The ESO residue, having a viscosity of 23.9 Pa{sm_bullet}s at 60{degrees}C (140{degrees}F), was blended with three different petroleum-derived asphalts, AAD-1, AAK-1, and AAM-1, that are known to be very susceptible to oxidative aging. Rheological and infrared analyses of the unaged and aged asphalts and the blends were then conducted to evaluate oxidative age-hardening. In addition, the petroleum-derived asphalts and the blends were coated onto three different aggregates, Lithonia granite (RA), a low-absorption limestone (RD), and a silicious Gulf Coast gravel (RL), and compacted into briquets. Successive freeze-thaw cycling was then conducted to evaluate the moisture susceptibility of the prepared briquets. The abbreviations used above for the asphalts and the aggregates are part of the Strategic Highway Research Program nomenclature.

  13. Hybrid Residual Flexibility/Mass-Additive Method for Structural Dynamic Testing

    NASA Technical Reports Server (NTRS)

    Tinker, M. L.

    2003-01-01

    A large fixture was designed and constructed for modal vibration testing of International Space Station elements. This fixed-base test fixture, which weighs thousands of pounds and is anchored to a massive concrete floor, initially utilized spherical bearings and pendulum mechanisms to simulate Shuttle orbiter boundary constraints for launch of the hardware. Many difficulties were encountered during a checkout test of the common module prototype structure, mainly due to undesirable friction and excessive clearances in the test-article-to-fixture interface bearings. Measured mode shapes and frequencies were not representative of orbiter-constrained modes due to the friction and clearance effects in the bearings. As a result, a major redesign effort for the interface mechanisms was undertaken. The total cost of the fixture design, construction and checkout, and redesign was over $2 million. Because of the problems experienced with fixed-base testing, alternative free-suspension methods were studied, including the residual flexibility and mass-additive approaches. Free-suspension structural dynamics test methods utilize soft elastic bungee cords and overhead frame suspension systems that are less complex and much less expensive than fixed-base systems. The cost of free-suspension fixturing is on the order of tens of thousands of dollars as opposed to millions, for large fixed-base fixturing. In addition, free-suspension test configurations are portable, allowing modal tests to be done at sites without modal test facilities. For example, a mass-additive modal test of the ASTRO-1 Shuttle payload was done at the Kennedy Space Center launch site. In this Technical Memorandum, the mass-additive and residual flexibility test methods are described in detail. A discussion of a hybrid approach that combines the best characteristics of each method follows and is the focus of the study.

  14. Specific arginine modification at the phosphatase site of muscle carbonic anhydrase.

    PubMed

    Pullan, L M; Noltmann, E A

    1985-01-29

    Mammalian carbonic anhydrase III has previously been shown to catalyze the hydrolysis of p-nitrophenyl phosphate in addition to possessing the conventional CO2 hydratase and p-nitrophenylacetate esterase activities. Modification of pig muscle carbonic anhydrase III with the arginine reagent phenylglyoxal yielded two clearly distinctive results. Reaction of the enzyme with phenylglyoxal at concentrations equivalent to those of the enzyme yielded stoichiometric inactivation titration of the enzyme's phosphatase activity, approaching 100% loss of activity with the simultaneous modification of one arginine residue, the latter based on a 1:1 reaction of phenylglyoxal with arginine. At this low ratio of phenylglyoxal to enzyme, neither the CO2 hydratase activity nor the acetate esterase activity was affected. When the modification was performed with a significant excess of phenylglyoxal, CO2 hydratase and acetate esterase activities were diminished as well. That loss of activity was accompanied by the incorporation of an additional half dozen phenylglyoxals and, presumably, the modification of an equal number of arginine residues. The data in their entirety are interpreted to show that the p-nitrophenylphosphatase activity is a unique property of carbonic anhydrase III and that excessive amounts of the arginine-modifying reagent lead to unspecific structural changes of the enzyme as a result of which all of its enzymatic activities are inactivated.

  15. Titration of the bacteriorhodopsin Schiff base involves titration of an additional protein residue.

    PubMed

    Zadok, Uri; Asato, Alfred E; Sheves, Mordechai

    2005-06-14

    The retinal protein protonated Schiff base linkage plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment, the Schiff base (SB) is titrated with a pK(a) of approximately 13, but following light absorption, it experiences a decrease in the pK(a) and undergoes several alterations, including a deprotonation process. We have studied the SB titration using retinal analogues which have intrinsically lower pK(a)'s which allow for SB titrations over a much lower pH range. We found that above pH 9 the channel for the SB titration is perturbed, and the titration rate is considerably reduced. On the basis of studies with several mutants, it is suggested that the protonation state of residue Glu204 is responsible for the channel perturbation. We suggest that above pH 12 a channel for the SB titration is restored probably due to titration of an additional protein residue. The observations may imply that during the bR photocycle and M photointermediate formation the rate of Schiff base protonation from the bulk is decreased. This rate decrease may be due to the deprotonation process of the "proton-releasing complex" which includes Glu204. In contrast, during the lifetime of the O intermediate, the protonated SB is exposed to the bulk. Possible implications for the switch mechanism, and the directionality of the proton movement, are discussed.

  16. Survey of residue levels of organic solvents in "existing food additives" and health food materials by head-space GC.

    PubMed

    Uematsu, Yoko; Ogimoto, Mami; Suzuki, Kumi; Kabashima, Junichirou; Ito, Koichi; Nakazato, Mitsuo

    2008-01-01

    Organic solvent residue levels in "Existing Food Additives" (n=145), health food materials (n=23), and commercial health food products (n=19) were surveyed. Ethanol was the dominant solvent found in the samples, suggesting its use in the manufacturing process. Methanol, acetone, 2-propanol and ethyl acetate was also found. No residual solvent exceeded the limits set by the Food Sanitation Law.

  17. Nitrification in lake sediment with addition of drinking water treatment residuals.

    PubMed

    Wang, Changhui; Liu, Juanfeng; Wang, Zhixin; Pei, Yuansheng

    2014-06-01

    Drinking water treatment residuals (WTRs), non-hazardous by-products generated during potable water production, can effectively reduce the lake internal phosphorus (P) loading and improve water quality in lakes. It stands to reason that special attention regarding the beneficial reuse of WTRs should be given not only to the effectiveness of P pollution control, but also to the effects on the migration and transformation of other nutrients (e.g., nitrogen (N)). In this work, based on laboratory enrichment tests, the effects of WTRs addition on nitrification in lake sediment were investigated using batch tests, fluorescence in situ hybridization, quantitative polymerase chain reaction and phylogenetic analysis techniques. The results indicated that WTRs addition had minor effects on the morphologies of AOB and NOB; however, the addition slightly enhanced the sediment nitrification potential from 12.8 to 13.2 μg-N g(-1)-dry sample h(-1) and also increased the ammonia oxidation bacteria (AOB) and nitrite oxidizing bacteria (NOB) abundances, particularly the AOB abundances (P < 0.05), which increased from 1.11 × 10(8) to 1.31 × 10(8) copies g(-1)-dry sample. Moreover, WTRs addition was beneficial to the enrichment of Nitrosomonas and Nitrosospira multiformis and promoted the emergence of a new Nitrospira cluster, causing the increase in AOB and NOB diversities. Further analysis showed that the variations of nitrification in lake sediment after WTRs addition were primarily due to the decrease of bioavailable P, the introduction of new nitrifiers and the increase of favorable carriers for microorganism attachment in sediments. Overall, these results suggested that WTRs reuse for the control of lake internal P loading would also lead to conditions that are beneficial to nitrification.

  18. Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine, Arginine, and Lysine Residues

    PubMed Central

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-01

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

  19. Isolation and properties of an arginine-binding protein from Saccharomyces cerevisiae.

    PubMed

    Opekarová, M; Kotyk, A; Horák, J; Kholodenko, V P

    1975-11-15

    Transfer of exponentially growing cells of Saccharomyces cerevisiae epsilon 1278 b to a fresh medium (or simply to distilled water) resulted in the loss of ability to transport arginine (and lysine), accompanied by the release of several proteins from the membrane surface or periplasmic space. Fractionation by ultrafiltration, Sephadex G-50 chromatography and freeze-drying yielded a homogeneous protein (55 mg per 100 g dry weight of cells) with specific binding ability for L-arginine (Kd = 3.8 X 10(-1) M) and L-lysine (Ki = 4.2 X 10(-4) M). The protein contains over 40 amino acid residues and has a molecular weight of about 5,000. In solution, it appears to aggregate as its concentration is raised, thereby decreasing the overall binding capacity for arginine. Addition of the protein to a depleted culture does not restore the transport of arginine. It is apparently the recognition protein for the specific arginine-transporting system of Saccharomyces cerevisiae but it occurs in almost identical amounts in the MG 168 mutant with impaired arginine transport.

  20. Determining the Volume of Additive Solution and Residual Plasma in Whole Blood Filtered and Buffy Coat Processed Red Cell Concentrates

    PubMed Central

    Jordan, Andrew; Acker, Jason P.

    2016-01-01

    Summary Background Residual plasma in transfused red cell concentrates (RCCs) has been associated with adverse transfusion outcomes. Despite this, there is no consensus on the standard procedure for measuring residual plasma volume. Methods The volumes of residual plasma and additive solution were measured in RCCs processed using two separation methods: whole blood filtration (WBF) and buffy coat (BC)/RCC filtration. The concentration of mannitol and albumin in RCC components was measured using colorimetric assays. Mannitol concentration was used to calculate additive solution volume. Residual plasma volume was calculated using two methods. Results Calculated RCC supernatant volumes were much lower in BC-processed components compared to WBF-processed components (BC = 97 ± 6 ml, WBF = 109 ± 4 ml; p < 0.05). Calculated additive solution volumes were greater in WBF- than in BC-processed components (BC = 81 ± 4 ml, WBF = 105 ± 2 ml; p < 0.05). Absolute residual plasma volume varied significantly based on the calculation method used. Conclusion Disparity between plasma volume calculation methods was observed. Efforts should be made to standardize residual plasma volume measurement methods in order to accurately assess the impact of residual plasma on transfusion outcomes. PMID:27330533

  1. Survey of the anticoccidial feed additive nicarbazin (as dinitrocarbanilide residues) in poultry and eggs.

    PubMed

    Danaher, Martin; Campbell, Katrina; O'Keeffe, Michael; Capurro, Emiliana; Kennedy, Glenn; Elliott, Christopher T

    2008-01-01

    A survey was carried out on the occurrence of dinitrocarbanilide (DNC), the marker residue for nicarbazin, in poultry produced in Ireland during 2002-2004. Liver (n = 736) and breast muscle samples (n = 342) were tested. DNC residues were found in 40 and 26% of liver and breast muscle samples at levels greater than 12.5 and 5 microg kg(-1), respectively. DNC residues were found at >200 microg kg(-1) in 12 and 0% of liver and muscle samples, respectively. Samples of breast muscle (n = 217) imported from 11 countries were also tested for DNC residues. A lower incidence of DNC residues (6%) was found in imported breast muscle. Egg samples (n = 546) were tested and DNC residues were found in nine samples, with levels ranging between 14 and 122 microg kg(-1). Analysis of poultry, carried out as part of official food inspection in the period 2004-2006, indicated a reduction in the number of broiler liver samples containing DNC at >200 microg kg(-1), to approximately 7%. Low levels of DNC residues continue to be found in <2% of egg samples.

  2. Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia.

    PubMed

    Hoff, R H; Hengge, A C; Wu, L; Keng, Y F; Zhang, Z Y

    2000-01-11

    General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.

  3. Functional analysis of an inosine-guanosine transporter from Leishmania donovani. The role of conserved residues, aspartate 389 and arginine 393.

    PubMed

    Arastu-Kapur, Shirin; Ford, Ethan; Ullman, Buddy; Carter, Nicola S

    2003-08-29

    Equilibrative nucleoside transporters encompass two conserved, charged residues that occur within predicted transmembrane domain 8. To assess the role of these "signature" residues in transporter function, the Asp389 and Arg393 residues within the LdNT2 nucleoside transporter from Leishmania donovani were mutated and the resultant phenotypes evaluated after transfection into Delta ldnt2 parasites. Whereas an R393K mutant retained transporter activity similar to that of wild type LdNT2, the R393L, D389E, and D389N mutations resulted in dramatic losses of transport capability. Tagging the wild type and mutant ldnt2 proteins with green fluorescent protein demonstrated that the D389N and D389E mutants targeted properly to the parasite cell surface and flagellum, whereas the expression of R393L at the cell surface was profoundly compromised. To test whether Asp389 and Arg393 interact, a series of mutants was generated, D389R/R393R, D389D/R393D, and D389R/R393D, within the green fluorescent protein-tagged LdNT2 construct. Although all of these ldnt2 mutants were transport-deficient, D389R/R393D localized properly to the plasma membrane, while neither D389R/R393R nor D389D/R393D could be detected. Moreover, a transport-incompetent D389N/R393N double ldnt2 mutant also localized to the parasite membrane, whereas a D389L/R393L ldnt2 mutant did not, suggesting that an interaction between residues 389 and 393 may be involved in LdNT2 membrane targeting. These studies establish genetically that Asp389 is critical for optimal transporter function and that a positively charged or polar residue at Arg393 is essential for proper expression of LdNT2 at the plasma membrane.

  4. Rmt1 catalyzes zinc-finger independent arginine methylation of the small ribosomal protein Rps2 in Saccharomyces cerevisiae

    PubMed Central

    Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G.

    2010-01-01

    Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells. PMID:20035717

  5. Rmt1 catalyzes zinc-finger independent arginine methylation of ribosomal protein Rps2 in Saccharomyces cerevisiae

    SciTech Connect

    Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G.

    2010-01-22

    Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells.

  6. Associating cooking additives with sodium hydroxide to pretreat bamboo residues for improving the enzymatic saccharification and monosaccharides production.

    PubMed

    Huang, Caoxing; He, Juan; Wang, Yan; Min, Douyong; Yong, Qiang

    2015-10-01

    Cooking additive pulping technique is used in kraft mill to increase delignification degree and pulp yield. In this work, cooking additives were firstly applied in the sodium hydroxide pretreatment for improving the bioconversion of bamboo residues to monosaccharides. Meanwhile, steam explosion and sulfuric acid pretreatments were also carried out on the sample to compare their impacts on monosaccharides production. Results indicated that associating anthraquinone with sodium hydroxide pretreatment showed the best performance in improving the original carbohydrates recovery, delignification, enzymatic saccharification, and monosaccharides production. After consecutive pretreatment and enzymatic saccharification process, 347.49 g, 307.48 g, 142.93 g, and 87.15 g of monosaccharides were released from 1000 g dry bamboo residues pretreated by sodium hydroxide associating with anthraquinone, sodium hydroxide, steam explosion and sulfuric acid, respectively. The results suggested that associating cooking additive with sodium hydroxide is an effective pretreatment for bamboo residues to enhance enzymatic saccharification for monosaccharides production.

  7. Protein Arginine Methylation and Citrullination in Epigenetic Regulation

    PubMed Central

    2015-01-01

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

  8. Histone Arginine Methylation

    PubMed Central

    Lorenzo, Alessandra Di; Bedford, Mark T.

    2012-01-01

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

  9. Targeting a cluster of arginine residues of neuraminidase to avoid oseltamivir resistance in influenza A (H1N1): a theoretical study.

    PubMed

    Gema, L Ramírez-Salinas; Tolentino-Lopez, L E; Martínez-Ramos, F; Padilla-Martínez, I; García-Machorro, J; Correa-Basurto, J

    2015-01-01

    Following the influenza A (H1N1) pandemic in Mexico and around the world in 2009, the numbers of oseltamivir-resistant clinical cases have increased through a mechanism that remains unclear. In this work, we focus on studying the mutated NA structures ADA71175 (GenBank) and 3CKZ (PDB ID). Recently crystallized NA (PDB ID: 3NSS) was used as a wild-type structure and template to construct the three-dimensional (3D) structure of ADA71175. Then, the NA mutants and 3NSS natives as well as their refined monomer structures as determined through MD simulations (snapshots at 50 ns) were used as models to perform a docking study using a set of aryl-oseltamivir derivatives. These aryl-oseltamivir derivatives have better recognition properties than oseltamivir because of cation-π interactions with a cluster of Arg residues (118, 292, and 371) at the binding site. This cluster of Arg residues represents a potential binding site for aryl-oseltamivir derivatives that are potentially new NA inhibitors.

  10. Characterization and comparative study of coal combustion residues from a primary and additional flue gas secondary desulfurization process

    SciTech Connect

    Gomes, S.; Francois, M.; Evrard, O.; Pellissier, C.

    1998-11-01

    An extensive characterization and comparative study was done on two flue gas desulfurization (FGD) residues derived from the same coal. LR residues (originated from Loire/Rhone in the south of Lyon, France) are obtained after a primary desulfurization process (SO{sub 2} is trapped by reaction with CaO at a temperature of about 1100 C), and LM residues (originating from La Maxe, near Metz in the east of France) are obtained after an additional secondary desulfurization process (SO{sub 2} is removed further by reaction with Ca(OH){sub 2} at a temperature of about 120 C). Various and complementary investigation methods were used to determine their chemical, physical, and mineralogical properties: x-ray fluorescence and diffraction, scanning electron microscopy, differential scanning calorimetry, thermogravimetry analysis, granulometric distribution, pycnometric density, BET specific surface area and pH, conductivity measurements, and chemical analysis of their insoluble fraction. The FGD residues contain basically two main components: a silico-aluminous fly ash part and calcic FGD phases. In the LR residues the two components can be considered as independent, whereas they are linked in the LM residues because chemical reactions have occurred, leading to the formation of silico-calcic gel CSH, hydrated aluminate AFm, and AFt phases.

  11. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production

    PubMed Central

    Ng, Ivan H. W.; Chan, Kitti W. K.; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Jans, David A.; Forwood, Jade K.

    2016-01-01

    Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization. PMID:27622521

  12. Multivariate sequence analysis reveals additional function impacting residues in the SDR superfamily.

    PubMed

    Tiwari, Pratibha; Singh, Noopur; Dixit, Aparna; Choudhury, Devapriya

    2014-10-01

    The "extended" type of short chain dehydrogenases/reductases (SDR), share a remarkable similarity in their tertiary structures inspite of being highly divergent in their functions and sequences. We have carried out principal component analysis (PCA) on structurally equivalent residue positions of 10 SDR families using information theoretic measures like Jensen-Shannon divergence and average shannon entropy as variables. The results classify residue positions in the SDR fold into six groups, one of which is characterized by low Shannon entropies but high Jensen-Shannon divergence against the reference family SDR1E, suggesting that these positions are responsible for the specific functional identities of individual SDR families, distinguishing them from the reference family SDR1E. Site directed mutagenesis of three residues from this group in the enzyme UDP-Galactose 4-epimerase belonging to SDR1E shows that the mutants promote the formation of NADH containing abortive complexes. Finally, molecular dynamics simulations have been used to suggest a mechanism by which the mutants interfere with the re-oxidation of NADH leading to the formation of abortive complexes.

  13. Regulation of macrophage functions by L-arginine

    PubMed Central

    1989-01-01

    Sites of inflammation with prominent macrophage infiltration, such as wounds and certain tumors, are uniquely deficient in free arginine. The effects of arginine availability on macrophage physiology were investigated. When cultured in media containing less than 0.1 mM L- arginine, rat resident peritoneal macrophages exhibited enhanced spreading, tumor cytotoxicity, superoxide production, phagocytosis, and protein synthesis. Thus, arginine concentrations similar to those found in sites of inflammation can augment macrophage functions, while those found in plasma (approximately 0.1 mM) and in commonly used culture media (0.4 to 1.2 mM) are inhibitory. Culture in homoarginine, but not D-arginine, ornithine, citrulline, urea, histidine, or lysine also inhibited macrophage tumor cytotoxicity, indicating the specificity of the effect. In contrast to resident macrophages, the tumor cytotoxicity of peritoneal macrophages obtained after C. parvum injection was suppressed by culture in arginine-deficient media. However, L-arginine- deficient media enhanced all other activation-associated functions in C. parvum-elicited macrophages as in resident cells. Arginine-free wound fluid promoted resident macrophage tumoricidal activity when compared with rat serum, and again, the addition of L-arginine was inhibitory. The marked effects of L-arginine availability on macrophage functions, together with the knowledge that these cells modify the extracellular arginine concentration in sites of inflammation through arginase, provide evidence for an autoregulatory mechanism of macrophage activation. PMID:2538541

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

    PubMed

    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.

  15. Supplemental effects and metabolic fate of crystalline arginine in juvenile shrimp Marsupenaeus japonicus.

    PubMed

    Teshima, Shin-ichi; Ishikawa, Manabu; Shah Alam, Md; Koshio, Shunsuke; Michael, Fady Raafat

    2004-02-01

    Feeding experiments with juvenile kuruma shrimp (Marsupenaeus japonicus) were conducted to understand the effects of supplemental levels of crystalline arginine hydrochloride on the growth and assimilation of arginine. In experiment 1 the juvenile shrimp were maintained on diets with and without arginine supplements. The addition of 3.0% arginine to a casein-based diet was slightly effective in improving the growth of the juveniles. In experiment 2, tracer experiments using [14C] arginine were conducted to clarify the ingestion and assimilation of arginine, 9 and 24 h after feeding, at different levels of supplemental arginine. Tracer experiments showed that the leaching rate of arginine 1 h after feeding ranged from 16 to 26% in the diets with different levels (0, 0.1, 1.5, 3.0 and 6.0%) of supplemental arginine; that is, 74-84% of the given arginine was actually ingested by the shrimp fed the diets. However, with increasing levels of supplemental arginine the incorporation rate of arginine into the whole body decreased and the utilization of absorbed arginine for body protein synthesis was reduced, whereas the excretion of absorbed arginine was increased. Thus, the absorbed arginine was not effectively utilized for body protein synthesis when large amounts of arginine were supplemented to the diets.

  16. 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.

    PubMed

    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.

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

    PubMed Central

    2013-01-01

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

  18. Additive effect of waste tire on the hydrogenolysis reaction of coal liquefaction residue

    SciTech Connect

    Motoyuki Sugano; Daigorou Onda; Kiyoshi Mashimo

    2006-12-15

    A numerous amount of waste tire is landfilled or dumped all over the world, which causes environmental problems, such as destruction of natural places and the risk of fires. On the other hand, the coal liquefaction residue (CLR) is produced in 30% yield through the process supporting unit (PSU) of the NEDOL coal liquefaction process. Therefore, the investigation on an effective method for utilization of waste tire and CLR is required. In this study, the simultaneous hydrogenolysis of CLR and pulverized waste tire was carried out by using tetralin. The yields in the simultaneous hydrogenolysis were compared with algebraic sum of the yields of the individual hydrogenolyses of waste tire alone and coal alone. In the simultaneous hydrogenolysis, the synergistic effects to upgrading, such as an increase in the yield of the oil constituent and a decrease in the yield of the asphaltene constituent, occurred because of the stabilization of asphaltenic radicals from CLR with aliphatic radicals from tire. The decrease in asphaltene yield in the simultaneous hydrogenolysis was pronounced with the increase in the tire:CLR ratio because the solvent effects of liquefied tire, such as stabilization of radicals, hydrogen shuttling, and heat transfer, were enhanced. Accordingly, it is estimated that the simultaneous hydrogenolysis of CLR and waste tire is an effective method for processing both materials. 15 refs., 3 figs., 2 tabs.

  19. Impact of addition of amendments on the degradation of DDT and its residues partitioned on soil.

    PubMed

    Singh, Swatantra P; Bose, Purnendu; Guha, Saumyen; Gurjar, Suresh K; Bhalekar, Santosh

    2013-08-01

    Market-grade DDT used for mosquito control and other purposes is a mixture of 4,4-DDT, 2,4-DDT and smaller amounts of 4,4-DDD, 2,4-DDD, 4,4-DDE and 4,4-DDMU. All above components (together known as DDTr) are strongly hydrophobic and hence are present in the environment predominantly in the soil/sediment phases. The persistence of DDTr and the feasibility of attenuation of DDTr concentration in soil matrix through addition of amendments is a subject of ongoing interest. The objective of this study was to compare the decline of soil-partitioned DDTr concentration through, (1) the natural attenuation process, (2) enhanced aerobic and anaerobic biodegradation processes involving addition of acclimatized seed and co-metabolites and (3) Nanoscale Zero Valent Iron (NZVI) addition. The extent of decline in soil DDTr concentration in control experiments, where biodegradation and photolysis were excluded, was around 10-15% in ∼100d. Extent of DDTr decline in natural attenuation experiments was 25-30% and 15-20% under aerobic and anaerobic conditions respectively. In enhanced biodegradation experiments, addition of acclimatized seed and/or co-metabolites did not enhance the extent of DDTr attenuation over and above the natural attenuation rates both in aerobic and anaerobic conditions. It thus appeared that biodegradation of DDTr adsorbed on soil was severely limited and controlled by desorption and consequent bioavailability of DDTr in the aqueous phase. In case of NZVI addition, the rate of DDTr degradation was much faster, with 40% decrease in DDTr concentration within 28h of NZVI addition. Here, the faster DDTr degradation may be through direct electron transfer between NZVI particles and DDTr molecules adsorbed on soil. Increase in the concentration of 4,4-DDD and 2,4-DDD during NZVI addition suggest that these compounds are either intermediate or end products of DDT degradation process.

  20. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  1. Investigation of the causes for the occurrence of residues of the anticoccidial feed additive nicarbazin in commercial poultry.

    PubMed

    O'Keeffe, M; Capurro, E; Danaher, M; Campbell, K; Elliott, C T

    2007-09-01

    Investigations were undertaken to identify causes for the occurrence of high levels of the zootechnical feed additive nicarbazin in broiler liver at slaughter. The first investigation on 32 commercial broiler flocks involved sampling and analysis for nicarbazin (as dinitrocarbanilide, DNC) in liver from birds during a 3-10-day period after withdrawal of nicarbazin from their feed and before commercial slaughter. DNC residues in liver samples of broilers scheduled as being withdrawn from nicarbazin for > or =6 days ranged from 20 to >1600 microg kg(-1) (the specified withdrawal period for nicarbazin is 5 days and the Joint Expert Committee on Food Additives (JECFA) maximum residue limit (MRL) is 200 microg kg(-1) liver). Further on-farm investigations on 12 of these flocks, selected on the basis of the feeding system in use and the levels of DNC residues determined in liver, identified issues in feed management contributing to elevated residues in broiler liver. A significant correlation (0.81, p < 0.01, n = 10) between DNC residues in liver samples and in feed samples from the feeding pans was observed. The second investigation on 12 commercial broiler flocks involved sampling and analysis for DNC in liver samples and feed samples from feeding pans and from the feed mill at the three thinnings of birds for commercial slaughter. In the case of one flock, a clear relationship between nicarbazin in feed from the feed mill (10.5 mg kg(-1) DNC), in feed from the feeding pans (6.6 mg kg(-1) DNC) and in liver (583 microg kg(-1) DNC) at first thinning (9 days scheduled withdrawal from nicarbazin) was observed. Such a clear relationship was not observed in other cases, particularly at second and third thinnings, pointing to re-exposure of birds to nicarbazin late in the flock production cycle, probably from the litter. Guidelines outlining best farm practice to eliminate nicarbazin residues in poultry have been published in booklet and poster format for broiler producers

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

    PubMed Central

    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. Parenteral arginine infusion in humans: nutrient substrate or pharmacologic agent?

    PubMed

    Sigal, R K; Shou, J; Daly, J M

    1992-01-01

    When given as a dietary supplement, arginine enhances lymphocyte mitogenesis and improves nitrogen balance. The purpose of this study was to evaluate arginine's ability to mediate these same effects when given as the sole nitrogen source with minimum additional calories. Thirty patients were randomized to receive 20 g/day arginine hydrochloride or a mixed amino acid solution (Travasol) by intravenous infusion for 7 days after abdominal operations. Mean patient age, body weight, gender ratios, and preoperative degree of weight loss were similar between groups. Mean plasma arginine and ornithine levels rose to 228 +/- 50 mumol/L and 191 +/- 76 mumol/L in the arginine group during infusion. Mean nitrogen balance was -8.8 g/day and -9.2 g/day in the arginine and Travasol groups, respectively. Mean lymphocyte stimulation indices to concanavalin A and phytohemagglutinin fell on postoperative day 1 in both groups. No significant differences in patterns of lymphocyte mitogenesis changes were noted between groups. The mean total number of circulating T cells increased in the arginine group at postoperative day 7. Thus, parenteral arginine infusion in postoperative patients provided comparable nitrogen balance to a balanced amino acid solution but did not increase peripheral blood lymphocyte mitogenesis. When arginine is given parenterally as the sole nitrogen source with minimal additional calories to postoperative patients, no enhancement of mitogen-stimulated lymphocyte proliferation could be demonstrated.

  4. Interaction of free arginine and guanidine with glucose under thermal processing conditions and formation of Amadori-derived imidazolones.

    PubMed

    Zhu, Yuchen; Yaylayan, Varoujan A

    2017-04-01

    To investigate the reactivity of free guanidine and arginine in the formation of imidazolinone derivatives, model systems of guanidine or arginine/glucose or (13)[C-6]-glucose were heated in aqueous solutions at110°C for 3h and the residues were analyzed by ESI/qTOF/MS using MS/MS and isotope labeling techniques. The analysis of the data indicated that guanidine and arginine formed both covalent and non-covalent interaction products. Covalent interactions included Amadori rearrangement at the α-nitrogen with glucose and imidazolinone formation with 3-deoxy-glucosone at the guanidine side-chain. Non-covalent interactions, such as self-interaction and interaction with free guanidine or arginine and glucose, were also observed. Guanidine underwent three sequential Amadori rearrangements and the free and mono-glycated guanidine also formed imidazolinone derivatives and their corresponding dehydration products and at the same time exhibiting various non-covalent interactions. On the other hand, arginine formed free Amadori product, free imidazolinone and Amadori-derived imidazolinone derivative in addition to methylglyoxal-derived hydroimidazolones.

  5. Stabilizing and destabilizing effects of arginine on deoxyribonucleic acid.

    PubMed

    Arakawa, Tsutomu; Hirano, Atsushi; Shiraki, Kentaro; Kita, Yoshiko; Koyama, A Hajime

    2010-03-01

    Aqueous arginine solution now finds a wide range of applications in biotechnology fields, including protein refolding, chromatography and virus inactivation. While progress has been made for mechanistic understanding of the effects of arginine on proteins, we have little understanding on how arginine inactivates viruses. One of the viral components is nucleic acid. We have examined the effects of arginine on the structure and thermal stability of calf thymus deoxyribonucleic acid (DNA) using circular dichroism (CD). Both NaCl and arginine reduced CD intensity. At low concentrations, arginine showed a stronger effect on CD intensity than NaCl. Both NaCl and arginine sharply increased the melting temperature at low concentrations (below 0.25 M). However, they had an opposite effect at higher concentrations. Above this concentration, NaCl gradually increased the melting temperature, leading to the onset melting temperature above 90 degrees C. On the other hand, the thermal stability in the presence of arginine reached a maximum at 0.2-0.5 M, after which further addition of arginine caused decreased melting temperature. It is most likely that the increased melting temperature at low concentration is due to electrostatic stabilization of DNA structure by both NaCl and arginine and that the opposite effects at higher salt concentration are due to salt-specific effects, i.e., stabilizing (salting-out) effects of NaCl and destabilizing (salting-in) effects of arginine. Solubility measurements of nucleic acid bases showed that arginine, but not NaCl, increases the solubilities of the bases, supporting their effects on DNA stability at higher concentration.

  6. Arginine metabolism in wounds

    SciTech Connect

    Albina, J.E.; Mills, C.D.; Barbul, A.; Thirkill, C.E.; Henry, W.L. Jr.; Mastrofrancesco, B.; Caldwell, M.D.

    1988-04-01

    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-/sup 14/C)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.

  7. Influence of Residue and Nitrogen Fertilizer Additions on Carbon Mineralization in Soils with Different Texture and Cropping Histories

    PubMed Central

    Chen, Xianni; Wang, Xudong; Liebman, Matt; Cavigelli, Michel; Wander, Michelle

    2014-01-01

    To improve our ability to predict SOC mineralization response to residue and N additions in soils with different inherent and dynamic organic matter properties, a 330-day incubation was conducted using samples from two long-term experiments (clay loam Mollisols in Iowa [IAsoil] and silt loam Ultisols in Maryland [MDsoil]) comparing conventional grain systems (Conv) amended with inorganic fertilizers with 3 yr (Med) and longer (Long), more diverse cropping systems amended with manure. A double exponential model was used to estimate the size (Ca, Cs) and decay rates (ka, ks) of active and slow C pools which we compared with total particulate organic matter (POM) and occluded-POM (OPOM). The high-SOC IAsoil containing highly active smectite clays maintained smaller labile pools and higher decay rates than the low-SOC MDsoil containing semi-active kaolinitic clays. Net SOC loss was greater (2.6 g kg−1; 8.6%) from the IAsoil than the MDsoil (0.9 g kg−1, 6.3%); fractions and coefficients suggest losses were principally from IAsoil’s resistant pool. Cropping history did not alter SOC pool size or decay rates in IAsoil where rotation-based differences in OPOM-C were small. In MDsoil, use of diversified rotations and manure increased ka by 32% and ks by 46% compared to Conv; differences mirrored in POM- and OPOM-C contents. Residue addition prompted greater increases in Ca (340% vs 230%) and Cs (38% vs 21%) and decreases in ka (58% vs 9%) in IAsoil than MDsoil. Reduced losses of SOC from residue-amended MDsoil were associated with increased OPOM-C. Nitrogen addition dampened CO2-C release. Clay type and C saturation dominated the IAsoil’s response to external inputs and made labile and stable fractions more vulnerable to decay. Trends in OPOM suggest aggregate protection influences C turnover in the low active MDsoil. Clay charge and OPOM-C contents were better predictors of soil C dynamics than clay or POM-C contents. PMID:25078458

  8. Modeling and simulation of cooling-induced residual stresses in heated particulate mixture depositions in additive manufacturing

    NASA Astrophysics Data System (ADS)

    Zohdi, T. I.

    2015-10-01

    One key aspect of many additive manufacturing processes is the deposition of heated mixtures of particulate materials onto surfaces, which then bond and cool, leading to complex microstructures and possible residual stresses. The overall objective of this work is to construct a straightforward computational approach that researchers in the field can easily implement and use as a numerically-efficient simulation and design tool. Specifically because multifield coupling is present, a recursive, staggered, temporally-adaptive, finite difference time domain scheme is developed to resolve the internal microstructural thermal and mechanical fields, accounting for the simultaneous elasto-plasticity and damage. The time step adaptation allows the numerical scheme to iteratively resolve the changing physical fields by refining the time-steps during phases of the process when the system is undergoing large changes on a relatively small time-scale and can also enlarge the time-steps when the processes are relatively slow. The spatial discretization grids are uniform and dense. The deposited microstructure is embedded into spatial discretization. The regular grid allows one to generate a matrix-free iterative formulation which is amenable to rapid computation and minimal memory requirements, making it ideal for laptop computation. Numerical examples are provided to illustrate the approach. This formulation is useful for material scientists who seek ways to deposit such materials while simultaneously avoiding inadvertent excessive residual stresses.

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

    PubMed Central

    Durzan, Don J

    2009-01-01

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

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

    PubMed

    Durzan, Don J

    2009-02-02

    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.

  11. 157 nm photodissociation of dipeptide ions containing N-terminal arginine.

    PubMed

    Webber, Nathaniel; He, Yi; Reilly, James P

    2014-02-01

    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.

  12. Arginine and Lysine Transporters Are Essential for Trypanosoma brucei

    PubMed Central

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

    2017-01-01

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

  13. Protein Arginine Methyltransferases Interact with IFT Particles and Change Location During Flagellar Growth and Resorption.

    PubMed

    Mizuno, Katsutoshi; Sloboda, Roger D

    2017-03-15

    Changes in protein activity driven by post translational modifications comprise an important mechanism for the control of many cellular processes. Several flagellar proteins are methylated on arginine residues during flagellar resorption; however, the function is not understood. To learn more about the role of protein methylation during flagellar dynamics, we have focused on protein arginine methyltransferases (PRMTs) 1, 3, 5, and 10. These PRMTs localize to the tip of flagella and in a punctate pattern along the length, very similar, but not identical, to that of intraflagellar transport (IFT) components. In addition, we found that PRMTs 1 and 3 are also highly enriched at the base of the flagella, and the basal localization of these PRMTs changes during flagellar regeneration and resorption. Proteins with methyl arginine residues are also enriched at the tip and base of flagella, and their localization also changes during flagellar assembly and disassembly. PRMTs are lost from the flagella of fla10-1 cells, which carry a temperature sensitive mutation in the anterograde motor for IFT. The data define the distribution of specific PRMTs and their target proteins in flagella, and demonstrate that PRMTs are cargo for translocation within flagella by the process of IFT.

  14. Asymmetric Arginine dimethylation of Epstein-Barr virus nuclear antigen 2 promotes DNA targeting

    SciTech Connect

    Gross, Henrik; Barth, Stephanie; Mamiani, Alfredo; Zimber-Strobl, Ursula; West, Michelle J.; Kremmer, Elisabeth; Graesser, Friedrich A.

    2010-02-20

    The Epstein-Barr virus (EBV) growth-transforms B-lymphocytes. The virus-encoded nuclear antigen 2 (EBNA2) is essential for transformation and activates gene expression by association with DNA-bound transcription factors such as RBPJkappa (CSL/CBF1). We have previously shown that EBNA2 contains symmetrically dimethylated Arginine (sDMA) residues. Deletion of the RG-repeat results in a reduced ability of the virus to immortalise B-cells. We now show that the RG repeat also contains asymmetrically dimethylated Arginines (aDMA) but neither non-methylated (NMA) Arginines nor citrulline residues. We demonstrate that only aDMA-containing EBNA2 is found in a complex with DNA-bound RBPJkappa in vitro and preferentially associates with the EBNA2-responsive EBV C, LMP1 and LMP2A promoters in vivo. Inhibition of methylation in EBV-infected cells results in reduced expression of the EBNA2-regulated viral gene LMP1, providing additional evidence that methylation is a prerequisite for DNA-binding by EBNA2 via association with the transcription factor RBPJkappa.

  15. Arginine Methylation of hnRNP A2 Does Not Directly Govern Its Subcellular Localization

    PubMed Central

    Nouwens, Amanda S.; Wei, Ying; Rothnagel, Joseph A.; Smith, Ross

    2013-01-01

    The hnRNP A/B paralogs A1, A2/B1 and A3 are key components of the nuclear 40S hnRNP core particles. Despite a high degree of sequence similarity, increasing evidence suggests they perform additional, functionally distinct roles in RNA metabolism. Here we identify and study the functional consequences of differential post-translational modification of hnRNPs A1, A2 and A3. We show that while arginine residues in the RGG box domain of hnRNP A1 and A3 are almost exhaustively, asymmetrically dimethylated, hnRNP A2 is dimethylated at only a single residue (Arg-254) and this modification is conserved across cell types. It has been suggested that arginine methylation regulates the nucleocytoplasmic distribution of hnRNP A/B proteins. However, we show that transfected cells expressing an A2R254A point mutant exhibit no difference in subcellular localization. Similarly, immunostaining and mass spectrometry of endogenous hnRNP A2 in transformed cells reveals a naturally-occurring pool of unmethylated protein but an exclusively nuclear pattern of localization. Our results suggest an alternative role for post-translational arginine methylation of hnRNPs and offer further evidence that the hnRNP A/B paralogs are not functionally redundant. PMID:24098712

  16. Evaluation of a zirconium additive for the mitigation of molten ash formation during combustion of residual fuel oil

    SciTech Connect

    1996-12-01

    Florida Power & Light Company (FP&L) currently fires a residual fuel oil (RFO) containing catalyst fines, which results in a troublesome black aluminosilicate liquid phase that forms on heat-transfer surfaces, remains molten, and flows to the bottom of the boiler. When the unit is shut down for a scheduled outage, this liquid phase freezes to a hard black glass that damages the contracting waterwalls of the boiler. Cleaning the boiler bottom and repairing damaged surfaces increase the boiler downtime, at a significant cost to FP&L. The Energy & Environmental Research Center (EERC) proposed to perform a series of tests for FP&L to evaluate the effectiveness of a zirconium additive to modify the mechanism that forms this liquid phase, resulting in the formation of a dry refractory phase that may be easily handled during cleanup of the boiler.

  17. Modifications of substituted seryl and threonyl residues in phosphopeptides and a polysialoglycoprotein by beta-elimination and nucleophile additions.

    PubMed

    Mega, T; Nakamura, N; Ikenaka, T

    1990-01-01

    The beta-elimination and nucleophile addition reactions of the substituted serine and threonine residues were studied using several synthesized fluorescence-labeled phosphopeptides and a salmon egg polysialoglycoprotein (PSGP). The reagents used were 1 M CH3SH-0.43 M NaOH, 1 M NaBH4-0.1 M NaOH, 1 M CH3NH2-0.1 M NaOH, and 1 M Na2SO3-0.1 M NaOH. The beta-elimination reaction of a phosphoserine peptide, Gly-Ser(PO4)-Glu-AEAP, was about 20 times faster than that of the corresponding phosphothreonine peptide. The carboxyl-side amino acid of the phosphoamino acids in peptides greatly affected the beta-elimination rate. The beta-elimination reaction rates of O-glycosyl serine and threonine in the polysialoglycoprotein were similar and were about a half of that of the phosphoserine peptide. The rates of addition of the three nucleophiles and hydrogen to alpha-aminoacrylic acid (beta-elimination product of substituted serine) in the peptide decreased in the order of CH3SH, Na2SO3, CH3NH2, and H2(NaBH4), and the addition to alpha-aminocrotonic acid (beta-elimination product of substituted threonine) in the order of Na2SO3, CH3NH2, CH3SH, and H2. These results indicated that sulfite is the most recommended nucleophile because of its high addition rate. If sulfite addition is carried out in the presence of NaBH4, sugar chains can be released as alditols, converting the sugar-attaching amino acids to beta-sulfoamino acids.

  18. Structural characterization of the enzymes composing the arginine deiminase pathway in Mycoplasma penetrans.

    PubMed

    Gallego, Pablo; Planell, Raquel; Benach, Jordi; Querol, Enrique; Perez-Pons, Josep A; Reverter, David

    2012-01-01

    The metabolism of arginine towards ATP synthesis has been considered a major source of energy for microorganisms such as Mycoplasma penetrans in anaerobic conditions. Additionally, this pathway has also been implicated in pathogenic and virulence mechanism of certain microorganisms, i.e. protection from acidic stress during infection. In this work we present the crystal structures of the three enzymes composing the gene cluster of the arginine deiminase pathway from M. penetrans: arginine deiminase (ADI), ornithine carbamoyltransferase (OTC) and carbamate kinase (CK). The arginine deiminase (ADI) structure has been refined to 2.3 Å resolution in its apo-form, displaying an "open" conformation of the active site of the enzyme in comparison to previous complex structures with substrate intermediates. The active site pocket of ADI is empty, with some of the catalytic and binding residues far from their active positions, suggesting major conformational changes upon substrate binding. Ornithine carbamoyltransferase (OTC) has been refined in two crystal forms at 2.5 Å and 2.6 Å resolution, respectively, both displaying an identical dodecameric structure with a 23-point symmetry. The dodecameric structure of OTC represents the highest level of organization in this protein family and in M.penetrans it is constituted by a novel interface between the four catalytic homotrimers. Carbamate kinase (CK) has been refined to 2.5 Å resolution and its structure is characterized by the presence of two ion sulfates in the active site, one in the carbamoyl phosphate binding site and the other in the β-phosphate ADP binding pocket of the enzyme. The CK structure also shows variations in some of the elements that regulate the catalytic activity of the enzyme. The relatively low number of metabolic pathways and the relevance in human pathogenesis of Mycoplasma penetrans places the arginine deiminase pathway enzymes as potential targets to design specific inhibitors against this human

  19. Reclamation of acidic mine residues by creation of technosoils with the addition of biochar and marble waste

    NASA Astrophysics Data System (ADS)

    Moreno-Barriga, Fabián; Díaz, Vicente; Acosta, José; Faz, Ángel; Zornoza, Raul

    2016-04-01

    This study reports the short-term effect of biochar and marble waste addition for the reclamation of acidic mine residues. A lab incubation was carried out for 90 days. Biochars derived from pig manure (PM), crop residues (CR) and municipal solid waste (MSW) were added to the soil at a rate of 20 g kg-1. The marble waste (MW) was added at a rate of 200 g kg-1. Bochars and MW were applied independently and combined. A control soil was used without application of amendments. The evolution of different physical, chemical and biochemical properties and availability of heavy metals was periodically monitored. Results showed that original pH (2.8) was increased with all amendments, those samples containing MW being the ones with the highest pH (~8.0). The electrical conductivity (EC) decreased from 6.6 to 3.0-4.5 mS cm-1 in all the treatments receiving MW. Soil organic C (SOC) increased in all samples receiving biochar up to 18-20 g kg-1, with no shifts during the 90 d incubation, indicating the high stability of the C supplied. Recalcitrant organic C accounted for ~90-98% of the SOC. No significant effect of amendment addition was observed for carbohydrates, soluble C, microbial biomass C and β-glucosidase activity. However, arylesterase activity increased with amendments, highly related to pH. The availability of heavy metals decreased up to 90-95% owing to the addition of amendments, mainly in samples containing MW. The MW provided conditions to increase pH and decrease EC and metals mobility. Biochar was an effective strategy to increase SOC, recalcitrant C and AS, essential to create soil structure. However, a labile source of organic matter should be added together with the proposed amendments to promote the activation of microbial communities. Acknowledgement : This work has been funded by Fundación Séneca (Agency of Science and Technology of the Region of Murcia, Spain) by the project 18920/JLI/13

  20. The stabilization effect of dielectric constant and acidic amino acids on arginine-arginine (Arg-Arg) pairings: database survey and computational studies.

    PubMed

    Zhang, Zhengyan; Xu, Zhijian; Yang, Zhuo; Liu, Yingtao; Wang, Jin'an; Shao, Qiang; Li, Shujin; Lu, Yunxiang; Zhu, Weiliang

    2013-05-02

    Database survey in this study revealed that about one-third of the protein structures deposited in the Protein Data Bank (PDB) contain arginine-arginine (Arg-Arg) pairing with a carbon···carbon (CZ···CZ) interaction distance less than 5 Å. All the Arg-Arg pairings were found to bury in a polar environment composed of acidic residues, water molecules, and strong polarizable or negatively charged moieties from binding site or bound ligand. Most of the Arg-Arg pairings are solvent exposed and 68.3% Arg-Arg pairings are stabilized by acidic residues, forming Arg-Arg-Asp/Glu clusters. Density functional theory (DFT) was then employed to study the effect of environment on the pairing structures. It was revealed that Arg-Arg pairings become thermodynamically stable (about -1 kcal/mol) as the dielectric constant increases to 46.8 (DMSO), in good agreement with the results of the PDB survey. DFT calculations also demonstrated that perpendicular Arg-Arg pairing structures are favorable in low dielectric constant environment, while in high dielectric constant environment parallel structures are favorable. Additionally, the acidic residues can stabilize the Arg-Arg pairing structures to a large degree. Energy decomposition analysis of Arg-Arg pairings and Arg-Arg-Asp/Glu clusters showed that both solvation and electrostatic energies contribute significantly to their stability. The results reported herein should be very helpful for understanding Arg-Arg pairing and its application in drug design.

  1. Crystal Structure of the Arginine Repressor Protein in Complex With the DNA Operator From Mycobacterium Tuberculosis

    SciTech Connect

    Cherney, L.T.; Cherney, M.M.; Garen, C.R.; Lu, G.J.; James, M.N.G.

    2009-05-12

    The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the L-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of arginine repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 {angstrom} resolution with bound arginine and at 2.15 {angstrom} resolution in the unliganded form. These structures show that six molecules of MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11{sup o} upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.

  2. Effects of particle size and acid addition on the remediation of chromite ore processing residue using ferrous sulfate.

    PubMed

    Jagupilla, Santhi Chandra; Moon, Deok Hyun; Wazne, Mahmoud; Christodoulatos, Christos; Kim, Min Gyu

    2009-08-30

    A bench-scale treatability study was conducted to assess the effects of particle size and acid addition on the remediation of chromite ore processing residue (COPR) using ferrous sulfate. The remediation scheme entailed the chemical reduction of hexavalent chromium [Cr(VI)] and the mitigation of swell potential. Leaching tests and the EQ3/6 geochemical model were used to estimate the acid dosage required to destabilize Cr(VI)-bearing and swell-causing minerals. The model predicted greater acid dosage than that estimated from the batch leaching tests. This indicated that mass transfer limitation may be playing a significant role in impeding the dissolution of COPR minerals following acid addition and hence hindering the remediation of COPR. Cr(VI) concentrations determined by alkaline digestion for the treated samples were less than the current NJDEP standard. However, Cr(VI) concentrations measured by X-ray absorption near edge structure (XANES) were greater than those measured by alkaline digestion. Greater Cr(VI) percentages were reduced for acid pretreated and also for smaller particle size COPR samples. Upon treatment, brownmillerite content was greatly reduced for the acid pretreated samples. Conversely, ettringite, a swell-causing mineral, was not observed in the treated COPR.

  3. Effect of water treatment additives on lime softening residual trace chemical composition--implications for disposal and reuse.

    PubMed

    Cheng, Weizhi; Roessler, Justin; Blaisi, Nawaf I; Townsend, Timothy G

    2014-12-01

    Drinking water treatment residues (WTR) offer potential benefits when recycled through land application. The current guidance in Florida, US allows for unrestricted land application of lime softening WTR; alum and ferric WTR require additional evaluation of total and leachable concentrations of select trace metals prior to land application. In some cases a mixed WTR is produced when lime softening is accompanied by the addition of a coagulant or other treatment chemical; applicability of the current guidance is unclear. The objective of this research was to characterize the total and leachable chemical content of WTR from Florida facilities that utilize multiple treatment chemicals. Lime and mixed lime WTR samples were collected from 18 water treatment facilities in Florida. Total and leachable concentrations of the WTR were measured. To assess the potential for disposal of mixed WTR as clean fill below the water table, leaching tests were conducted at multiple liquid to solid ratios and under reducing conditions. The results were compared to risk-based soil and groundwater contamination thresholds. Total metal concentrations of WTR were found to be below Florida soil contaminant thresholds with Fe found in the highest abundance at a concentration of 3600 mg/kg-dry. Aluminum was the only element that exceeded the Florida groundwater contaminant thresholds using SPLP (95% UCL = 0.23 mg/L; risk threshold = 0.2 mg/L). Tests under reducing conditions showed elevated concentrations of Fe and Mn, ranging from 1 to 3 orders of magnitude higher than SPLP leachates. Mixed lime WTR concentrations (total and leachable) were lower than the ferric and alum WTR concentrations, supporting that mixed WTR are appropriately represented as lime WTR. Testing of WTR under reducing conditions demonstrated the potential for release of certain trace metals (Fe, Al, Mn) above applicable regulatory thresholds; additional evaluation is needed to assess management options where

  4. Promiscuous modification of the nuclear poly(A)-binding protein by multiple protein-arginine methyltransferases does not affect the aggregation behavior.

    PubMed

    Fronz, Katharina; Otto, Silke; Kölbel, Knut; Kühn, Uwe; Friedrich, Henning; Schierhorn, Angelika; Beck-Sickinger, Annette G; Ostareck-Lederer, Antje; Wahle, Elmar

    2008-07-18

    The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.

  5. Investigation on the eco-toxicity of lake sediments with the addition of drinking water treatment residuals.

    PubMed

    Yuan, Nannan; Wang, Changhui; Pei, Yuansheng

    2016-08-01

    Drinking water treatment residuals (WTRs) have a potential to realize eutrophication control objectives by reducing the internal phosphorus (P) load of lake sediments. Information regarding the ecological risk of dewatered WTR reuse in aquatic environments is generally lacking, however. In this study, we analyzed the eco-toxicity of leachates from sediments with or without dewatered WTRs toward algae Chlorella vulgaris via algal growth inhibition testing with algal cell density, chlorophyll content, malondialdehyde content, antioxidant enzyme superoxide dismutase activity, and subcellular structure indices. The results suggested that leachates from sediments unanimously inhibited algal growth, with or without the addition of different WTR doses (10% or 50% of the sediment in dry weight) at different pH values (8-9), as well as from sediments treated for different durations (10 or 180days). The inhibition was primarily the result of P deficiency in the leachates owing to WTR P adsorption, however, our results suggest that the dewatered WTRs were considered as a favorable potential material for internal P loading control in lake restoration projects, as it shows acceptably low risk toward aquatic plants.

  6. Standard addition method for the determination of pharmaceutical residues in drinking water by SPE-LC-MS/MS.

    PubMed

    Cimetiere, Nicolas; Soutrel, Isabelle; Lemasle, Marguerite; Laplanche, Alain; Crocq, André

    2013-01-01

    The study of the occurrence and fate of pharmaceutical compounds in drinking or waste water processes has become very popular in recent years. Liquid chromatography with tandem mass spectrometry is a powerful analytical tool often used to determine pharmaceutical residues at trace level in water. However, many steps may disrupt the analytical procedure and bias the results. A list of 27 environmentally relevant molecules, including various therapeutic classes and (cardiovascular, veterinary and human antibiotics, neuroleptics, non-steroidal anti-inflammatory drugs, hormones and other miscellaneous pharmaceutical compounds), was selected. In this work, a method was developed using ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) and solid-phase extraction to determine the concentration of the 27 targeted pharmaceutical compounds at the nanogram per litre level. The matrix effect was evaluated from water sampled at different treatment stages. Conventional methods with external calibration and internal standard correction were compared with the standard addition method (SAM). An accurate determination of pharmaceutical compounds in drinking water was obtained by the SAM associated with UPLC-MS/MS. The developed method was used to evaluate the occurrence and fate of pharmaceutical compounds in some drinking water treatment plants in the west of France.

  7. Remediation of hexavalent chromium contamination in chromite ore processing residue by sodium dithionite and sodium phosphate addition and its mechanism.

    PubMed

    Li, Yunyi; Cundy, Andrew B; Feng, Jingxuan; Fu, Hang; Wang, Xiaojing; Liu, Yangsheng

    2017-05-01

    Large amounts of chromite ore processing residue (COPR) wastes have been deposited in many countries worldwide, generating significant contamination issues from the highly mobile and toxic hexavalent chromium species (Cr(VI)). In this study, sodium dithionite (Na2S2O4) was used to reduce Cr(VI) to Cr(III) in COPR containing high available Fe, and then sodium phosphate (Na3PO4) was utilized to further immobilize Cr(III), via a two-step procedure (TSP). Remediation and immobilization processes and mechanisms were systematically investigated using batch experiments, sequential extraction studies, X-ray diffraction (XRD) and X-ray Photoelectron Spectroscopy (XPS). Results showed that Na2S2O4 effectively reduced Cr(VI) to Cr(III), catalyzed by Fe(III). The subsequent addition of Na3PO4 further immobilized Cr(III) by the formation of crystalline CrPO4·6H2O. However, addition of Na3PO4 simultaneously with Na2S2O4 (via a one-step procedure, OSP) impeded Cr(VI) reduction due to the competitive reaction of Na3PO4 and Na2S2O4 with Fe(III). Thus, the remediation efficiency of the TSP was much higher than the corresponding OSP. Using an optimal dosage in the two-step procedure (Na2S2O4 at a dosage of 12× the stoichiometric requirement for 15 days, and then Na3PO4 in a molar ratio (i.e. Na3PO4: initial Cr(VI)) of 4:1 for another 15 days), the total dissolved Cr in the leachate determined via Toxicity Characteristic Leaching Procedure (TCLP Cr) testing of our samples was reduced to 3.8 mg/L (from an initial TCLP Cr of 112.2 mg/L, i.e. at >96% efficiency).

  8. Expanding the yeast protein arginine methylome.

    PubMed

    Plank, Michael; Fischer, Roman; Geoghegan, Vincent; Charles, Philip D; Konietzny, Rebecca; Acuto, Oreste; Pears, Catherine; Schofield, Christopher J; Kessler, Benedikt M

    2015-09-01

    Protein arginine methylation is a PTM involved in various cellular processes in eukaryotes. Recent discoveries led to a vast expansion of known sites in higher organisms, indicating that this modification is more widely spread across the proteome than previously assumed. An increased knowledge of sites in lower eukaryotes may facilitate the elucidation of its functions. In this study, we present the discovery of arginine mono-methylation sites in Saccharomyces cerevisiae by a combination of immunoaffinity enrichment and MS/MS. As detection of methylation is prone to yield false positives, we demonstrate the need for stringent measures to avoid elevated false discovery rates. To this end, we employed MethylSILAC in combination with a multistep data analysis strategy. We report 41 unambiguous methylation sites on 13 proteins. Our results indicate that, while substantially less abundant, arginine methylation follows similar patterns as in higher eukaryotes in terms of sequence context and functions of methylated proteins. The majority of sites occur on RNA-binding proteins participating in processes from transcription and splicing to translation and RNA degradation. Additionally, our data suggest a bias for localization of arginine methylation in unstructured regions of proteins, which frequently involves Arg-Gly-Gly motifs or Asn-rich contexts.

  9. Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

    SciTech Connect

    Kimura, Shuhei; Sawatsubashi, Shun; Ito, Saya; Kouzmenko, Alexander; Suzuki, Eriko; Zhao, Yue; Yamagata, Kaoru; Tanabe, Masahiko; Ueda, Takashi; Fujiyama, Sari; Murata, Takuya; Matsukawa, Hiroyuki; Takeyama, Ken-ichi; Yaegashi, Nobuo

    2008-07-11

    Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressed EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly.

  10. Evaluation of certain veterinary drug residues in food. Seventy-eighth report of the Joint FAO/WHO Expert Committee on Food Additives.

    PubMed

    2014-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of residues of certain veterinary drugs in food and to recommend maximum levels for such residues of food. The first part of the report considers general principles regarding the evaluation of residues of veterinary drugs within the terms of reference of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), including extrapolation of maximum residue limits (MRLs) to minor species, MRLs for veterinary drug residues in honey, MRLs relating to fish and fish species, dietary exposure assessment methodologies, the decision-tree approach to the evaluation of residues of veterinary drugs and guidance for JECFA experts. Summaries follow of the Committee's evaluations of toxicology and residue data on a variety of veterinary drugs: two anthelminthic agents (derquantel, monepantel), three antiparasitic agents (emanectin benzoate, ivermectin, lasalocid sodium), one antibacterial, antifungal and anthelminthic agent (gentian violet), a production aid (recombinant bovine somatotropins) and an adrenoceptor agonist and growth promoter (zilpaterol hydorchloride). Annexed to the report is a summary of the Committee's recommendations on these drugs, including acceptable daily intakes (ADIs)) and proposed MRLs.

  11. Arginine production in the neonate

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. Protein arginine methylation facilitates KCNQ channel-PIP2 interaction leading to seizure suppression

    PubMed Central

    Kim, Hyun-Ji; Jeong, Myong-Ho; Kim, Kyung-Ran; Jung, Chang-Yun; Lee, Seul-Yi; Kim, Hanna; Koh, Jewoo; Vuong, Tuan Anh; Jung, Seungmoon; Yang, Hyunwoo; Park, Su-Kyung; Choi, Dahee; Kim, Sung Hun; Kang, KyeongJin; Sohn, Jong-Woo; Park, Joo Min; Jeon, Daejong; Koo, Seung-Hoi; Ho, Won-Kyung; Kang, Jong-Sun; Kim, Seong-Tae; Cho, Hana

    2016-01-01

    KCNQ channels are critical determinants of neuronal excitability, thus emerging as a novel target of anti-epileptic drugs. To date, the mechanisms of KCNQ channel modulation have been mostly characterized to be inhibitory via Gq-coupled receptors, Ca2+/CaM, and protein kinase C. Here we demonstrate that methylation of KCNQ by protein arginine methyltransferase 1 (Prmt1) positively regulates KCNQ channel activity, thereby preventing neuronal hyperexcitability. Prmt1+/- mice exhibit epileptic seizures. Methylation of KCNQ2 channels at 4 arginine residues by Prmt1 enhances PIP2 binding, and Prmt1 depletion lowers PIP2 affinity of KCNQ2 channels and thereby the channel activities. Consistently, exogenous PIP2 addition to Prmt1+/- neurons restores KCNQ currents and neuronal excitability to the WT level. Collectively, we propose that Prmt1-dependent facilitation of KCNQ-PIP2 interaction underlies the positive regulation of KCNQ activity by arginine methylation, which may serve as a key target for prevention of neuronal hyperexcitability and seizures. DOI: http://dx.doi.org/10.7554/eLife.17159.001 PMID:27466704

  13. Arginine deiminase inhibits Porphyromonas gingivalis surface attachment.

    PubMed

    Cugini, Carla; Stephens, Danielle N; Nguyen, Daniel; Kantarci, Alpdogan; Davey, Mary E

    2013-02-01

    The oral cavity is host to a complex microbial community whose maintenance depends on an array of cell-to-cell interactions and communication networks, with little known regarding the nature of the signals or mechanisms by which they are sensed and transmitted. Determining the signals that control attachment, biofilm development and outgrowth of oral pathogens is fundamental to understanding pathogenic biofilm development. We have previously identified a secreted arginine deiminase (ADI) produced by Streptococcus intermedius that inhibited biofilm development of the commensal pathogen Porphyromonas gingivalis through downregulation of genes encoding the major (fimA) and minor (mfa1) fimbriae, both of which are required for proper biofilm development. Here we report that this inhibitory effect is dependent on enzymic activity. We have successfully cloned, expressed and defined the conditions to ensure that ADI from S. intermedius is enzymically active. Along with the cloning of the wild-type allele, we have created a catalytic mutant (ADIC399S), in which the resulting protein is not able to catalyse the hydrolysis of l-arginine to l-citrulline. P. gingivalis is insensitive to the ADIC399S catalytic mutant, demonstrating that enzymic activity is required for the effects of ADI on biofilm formation. Biofilm formation is absent under l-arginine-deplete conditions, and can be recovered by the addition of the amino acid. Taken together, the results indicate that arginine is an important signal that directs biofilm formation by this anaerobe. Based on our findings, we postulate that ADI functions to reduce arginine levels and, by a yet to be identified mechanism, signals P. gingivalis to alter biofilm development. ADI release from the streptococcal cell and its cross-genera effects are important findings in understanding the nature of inter-bacterial signalling and biofilm-mediated diseases of the oral cavity.

  14. Arginine consumption by the intestinal parasite Giardia intestinalis reduces proliferation of intestinal epithelial cells.

    PubMed

    Stadelmann, Britta; Merino, María C; Persson, Lo; Svärd, Staffan G

    2012-01-01

    In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the hosts production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that

  15. Effect of second coagulant addition on coagulation efficiency, floc properties and residual Al for humic acid treatment by Al13 polymer and polyaluminum chloride (PACl).

    PubMed

    Xu, Weiying; Gao, Baoyu; Wang, Yan; Yue, Qinyan; Ren, Haijing

    2012-05-15

    Influence of second dose on coagulation efficiency, floc re-growth, fractal structure and residual Al of the effluent in humic acid (HA) coagulation with Al(13) polymer ([Al(13)O(4)(OH)(24)(H(2)O)(12)](7+)) and PACl were comparatively investigated in this study. Effects of breakage shear on the floc properties generated in the coagulation with and without additional dose were also investigated. The results indicated that additional dose during breakage could essentially improve the HA removal efficiency and floc re-growth in both Al(13) and PACl coagulations. Second doses of Al(13) at 0.5 and 1.0mg/L resulted in better turbidity and UV(254) removal as well as floc re-growth rather than higher additional dose of 1.5 and 2.0mg/L; while in PACl coagulation, more efficient HA removal and better floc re-growth were obtained at higher additional doses (1.0, 1.5 and 2.0mg/L). Small additional Al(13) could apparently increase the D(f) of re-formed flocs while the additional PACl displayed inconspicuous effect on floc D(f). The additional coagulant dose could alleviate the further decrease of re-grown floc size with increased breakage shear for both coagulants. The residual Al analysis implied that two-stage addition contributed to lower residual Al in effluent than one-time addition mode with the same total coagulant concentration.

  16. Arginine mimetic structures in biologically active antagonists and inhibitors.

    PubMed

    Masic, Lucija Peterlin

    2006-01-01

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

  17. Arginine methylation of SmB is required for Drosophila germ cell development.

    PubMed

    Anne, Joël

    2010-09-01

    Sm proteins constitute the common core of spliceosomal small nuclear ribonucleoproteins. Although Sm proteins are known to be methylated at specific arginine residues within the C-terminal arginine-glycine dipeptide (RG) repeats, the biological relevance of these modifications remains unknown. In this study, a tissue-specific function of arginine methylation of the SmB protein was identified in Drosophila. Analysis of the distribution of SmB during oogenesis revealed that this protein accumulates at the posterior pole of the oocyte, a cytoplasmic region containing the polar granules, which are necessary for the formation of primordial germ cells. The pole plasm localisation of SmB requires the methylation of arginine residues in its RG repeats by the Capsuléen-Valois methylosome complex. Functional studies showed that the methylation of these arginine residues is essential for distinct processes of the germline life cycle, including germ cell formation, migration and differentiation. In particular, the methylation of a subset of these arginine residues appears essential for the anchoring of the polar granules at the posterior cortex of the oocyte, whereas the methylation of another subset controls germ cell migration during embryogenesis. These results demonstrate a crucial role of arginine methylation in directing the subcellular localisation of SmB and that this modification contributes specifically to the establishment and development of germ cells.

  18. Evaluation of certain veterinary drug residues in food. Sixty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives.

    PubMed

    2006-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of residues of certain veterinary drugs in food and to recommend maximum levels for such residues in food. The first part of the report considers general principles regarding the evaluation of veterinary drugs within the terms of reference of JECFA, including compounds without an ADI or MRL; recommendations on principles and methods in derivation of MRLs, including a new procedure for estimating chronic dietary intakes; the use of a spreadsheet-based procedure for the statistical evaluation of residue depletion data; a revised approach for the derivation of microbiological ADIs; and the Committee's review of and comments on documents provided by the Codex Committee on Residues of Veterinary Drugs. Summaries follow of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: three antimicrobial agents (colistin, erythromycin, flumequine), two production aids (melengestrol acetate, ractopamine hydrochloride), an insecticide (trichlorfon (metrifonate)) and an anthelminthic (triclabendazole). In addition, the attempt by the Committee to use tylosin as an example to investigate if evaluations are possible based on published data in the absence of data submissions from sponsors is described. Annexed to the report is a summary of the Committee's recommendations on these drugs, including acceptable daily intakes and proposed maximum residue limits.

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

    PubMed

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

    2017-03-01

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

  20. FORMATION OF FINE PARTICLES FROM RESIDUAL OIL COMBUSTION: REDUCING ULTRAFINE NUCLEI THROUGH THE ADDITION OF INORGANIC SORBENT

    EPA Science Inventory

    The paper gives results of an investigation, using an 82-kW-rated laboratory-scale refractory-lined combustor, of the characteristics of particulate matter emitted from residual oil combustion and the reduction of ultrafine nuclei by postflame sorbent injection. Without sorbent a...

  1. Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

    PubMed Central

    de Cima, Sergio; Gil-Ortiz, Fernando; Crabeel, Marjolaine; Fita, Ignacio; Rubio, Vicente

    2012-01-01

    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. PMID:22529931

  2. Evaluation of certain veterinary drug residues in food. Eighty-first report of the Joint FAO/WHO Expert Committee on Food Additives.

    PubMed

    2016-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of residues of certain veterinary drugs in food and to recommend maximum levels for such residues in food. The first part of the report considers general principles regarding the evaluation of residues of veterinary drugs within the terms of reference of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), including MRLs for generic fish species, acute reference doses (ARfDs) for veterinary drugs, an approach for dietary exposure assessment of compounds used for multiple purposes (i.e veterinary drugs and pesticides), dietary exposure assessment for less-than-lifetime exposure, and the assessment of short-term (90-day and 12-month) studies in dogs. Summaries follow of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: two insecticides (diflubenzuron and teflubenzuron), an antiparasitic agent (ivermectin), an ectoparasiticide (sisapronil) and a β2-adrenoceptor agonist (zilpaterol hydrochloride). In addition, the Committee considered issues raised in concern forms from the Codex Committee on Residues of Veterinary Drugs in Foods on lasalocid sodium, an antiparasitic agent. Annexed to the report is a summary of the Committee's recommendations on these drugs, including acceptable daily intakes (ADIs), ARfDs and proposed MRLs.

  3. 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

    PubMed Central

    Schriek, Sarah; Rückert, Christian; Staiger, Dorothee; Pistorius, Elfriede K; Michel, Klaus-Peter

    2007-01-01

    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 cyanobacterial genomes revealed that

  4. Arginine kinase shows nucleoside diphosphate kinase-like activity toward deoxythymidine diphosphate.

    PubMed

    Lopez-Zavala, Alonso A; Sotelo-Mundo, Rogerio R; Hernandez-Flores, Jose M; Lugo-Sanchez, Maria E; Sugich-Miranda, Rocio; Garcia-Orozco, Karina D

    2016-06-01

    Arginine kinase (AK) (ATP: L-arginine phosphotransferase, E.C. 2.7.3.3) catalyzes the reversible transfer of ATP γ-phosphate group to L-arginine to synthetize phospho-arginine as a high-energy storage. Previous studies suggest additional roles for AK in cellular processes. Since AK is found only in invertebrates and it is homologous to creatine kinase from vertebrates, the objective of this work was to demonstrate nucleoside diphosphate kinase-like activity for shrimp AK. For this, AK from marine shrimp Litopenaeus vannamei (LvAK) was purified and its activity was assayed for phosphorylation of TDP using ATP as phosphate donor. Moreover, by using high-pressure liquid chromatography (HPLC) the phosphate transfer reaction was followed. Also, LvAK tryptophan fluorescence emission changes were detected by dTDP titration, suggesting that the hydrophobic environment of Trp 221, which is located in the top of the active site, is perturbed upon dTDP binding. The kinetic constants for both substrates Arg and dTDP were calculated by isothermal titration calorimetry (ITC). Besides, docking calculations suggested that dTDP could bind LvAK in the same cavity where ATP bind, and LvAK basic residues (Arg124, 126 and 309) stabilize the dTDP phosphate groups and the pyrimidine base interact with His284 and Ser122. These results suggest that LvAK bind and phosphorylate dTDP being ATP the phosphate donor, thus describing a novel alternate nucleoside diphosphate kinase-like activity for this enzyme.

  5. Development of an Eastern Shale Oil Residue as an Asphalt Additive - Subtask 2.5: Topical report, February 1, 1994-February 1, 1995

    SciTech Connect

    1997-12-31

    An evaluation of eastern shale oil as an asphalt additive to reduce oxidative age hardening and moisture susceptibility is being conducted. An eastern shale oil residue having a viscosity of 1.30 Pa`s at 60{degrees}C (140{degrees}F) was blended with three different petroleum-derived asphalts that are known to be very susceptible to oxidative aging. In addition, blends of the eastern shale oil residue and the petroleum-derived asphalts are being coated onto three different aggregates that are known to be susceptible to water stripping. The oxidative age hardening portion of this study is not complete at this time. To date, information has been obtained on the unaged samples and two of the aged petroleum-derived asphalts (AAD-1 and AAK-1). When complete, this data will include rheological data on the unaged, RTFO-aged, and the RTFO/PAV-aged samples and infrared data on the unaged and RTFO/PAV-aged samples. With respect to the rheological data, asphalt AAD-1 meets the specifications of a PG 58 asphalt while asphalt AAK-1 does not. In the latter case this indicates that AAK-1 is more appropriately evaluated at a higher temperature range. The infrared spectroscopic data obtained for the eastern shale oil residue show that it contains appreciable amounts of carbonyl and sulfoxide compound types, 0.22 absorbance units and 0. 27 moles/L, respectively. Thus, upon the addition of this residue to the three petroleum-derived asphalts the blends contain increased amounts of these functional groups relative to the petroleum-derived asphalts. This has been observed with other additives and is not considered detrimental. In addition, the data that has been collected to date indicate that the moisture susceptibility of blends of eastern shale oil residue and asphalt AAK-1 are somewhat improved when coated onto Lithonia granite.

  6. Role of arginine in the stabilization of proteins against aggregation.

    PubMed

    Baynes, Brian M; Wang, Daniel I C; Trout, Bernhardt L

    2005-03-29

    The amino acid arginine is frequently used as a solution additive to stabilize proteins against aggregation, especially in the process of protein refolding. Despite arginine's prevalence, the mechanism by which it stabilizes proteins is not presently understood. We propose that arginine deters aggregation by slowing protein-protein association reactions, with only a small concomitant effect on protein folding. The associated rate effect was observed experimentally in association of globular proteins (insulin and a monoclonal anti-insulin) and in refolding of carbonic anhydrase. We suggest that this effect arises because arginine is preferentially excluded from protein-protein encounter complexes but not from dissociated protein molecules. Such an effect is predicted by our gap effect theory [Baynes and Trout (2004) Biophys. J. 87, 1631] for "neutral crowder" additives such as arginine which are significantly larger than water but have only a small effect on the free energies of isolated protein molecules. The effect of arginine on refolding of carbonic anhydrase was also shown to be consistent with this hypothesis.

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

    PubMed

    Arakawa, Junji; Uegaki, Masamichi; Ishimizu, Takeshi

    2011-11-01

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

  8. Characterisation of neuroprotective efficacy of modified poly-arginine-9 (R9) peptides using a neuronal glutamic acid excitotoxicity model.

    PubMed

    Edwards, Adam B; Anderton, Ryan S; Knuckey, Neville W; Meloni, Bruno P

    2017-02-01

    In a recent study, we highlighted the importance of cationic charge and arginine residues for the neuroprotective properties of poly-arginine and arginine-rich peptides. In this study, using cortical neuronal cultures and an in vitro glutamic acid excitotoxicity model, we examined the neuroprotective efficacy of different modifications to the poly-arginine-9 peptide (R9). We compared an unmodified R9 peptide with R9 peptides containing the following modifications: (i) C-terminal amidation (R9-NH2); (ii) N-terminal acetylation (Ac-R9); (iii) C-terminal amidation with N-terminal acetylation (Ac-R9-NH2); and (iv) C-terminal amidation with D-amino acids (R9D-NH2). The three C-terminal amidated peptides (R9-NH2, Ac-R9-NH2, and R9D-NH2) displayed neuroprotective effects greater than the unmodified R9 peptide, while the N-terminal acetylated peptide (Ac-R9) had reduced efficacy. Using the R9-NH2 peptide, neuroprotection could be induced with a 10 min peptide pre-treatment, 1-6 h before glutamic acid insult, or when added to neuronal cultures up to 45 min post-insult. In addition, all peptides were capable of reducing glutamic acid-mediated neuronal intracellular calcium influx, in a manner that reflected their neuroprotective efficacy. This study further highlights the neuroprotective properties of poly-arginine peptides and provides insight into peptide modifications that affect efficacy.

  9. Strategies for the Gas Phase Modification of Cationized Arginine via Ion/ion Reactions.

    PubMed

    Prentice, Boone M; McGee, William M; Stutzman, John R; McLuckey, Scott A

    2013-11-15

    The gas phase acetylation of cationized arginine residues is demonstrated here using ion/ion reactions with sulfosuccinimidyl acetate (sulfo-NHS acetate) anions. Previous reports have demonstrated the gas phase modification of uncharged primary amine (the N-terminus and ε-amino side chain of lysine) and uncharged guanidine (the arginine side chain) functionalities via sulfo-NHS ester chemistry. Herein, charge-saturated arginine-containing peptides that contain sodium ions as the charge carriers, such as [ac-ARAAARA+2Na](2+), are shown to exhibit strong reactivity towards sulfo-NHS acetate whereas the protonated peptide analogues exhibit no such reactivity. This difference in reactivity is attributed to the lower sodium ion (as compared to proton) affinity of the arginine, which results in increased nucleophilicity of the cationized arginine guanidinium functionality. This increased nucleophilicity improves the arginine residue's reactivity towards sulfo-NHS esters and enhances the gas phase covalent modification pathway. No such dramatic increase in reactivity towards sulfo-NHS acetate has been observed upon sodium cationization of lysine amino acid residues, indicating that this behavior appears to be unique to arginine. The sodium cationization process is demonstrated in the condensed phase by simply spiking sodium chloride into the peptide sample solution and in the gas phase by a peptide-sodium cation exchange process with a sulfo-NHS acetate sodium-bound dimer cluster reagent. This methodology demonstrates several ways by which arginine can be covalently modified in the gas phase even when it is charged. Collisional activation of an acetylated arginine product can result in deguanidination of the residue, generating an ornithine. This gas phase ornithination exhibits similar site-specific fragmentation behavior to that observed with peptides ornithinated in solution and may represent a useful approach for inducing selective peptide cleavages.

  10. Arginine deiminase pathway genes and arginine degradation variability in Oenococcus oeni strains.

    PubMed

    Araque, Isabel; Gil, Joana; Carreté, Ramon; Constantí, Magda; Bordons, Albert; Reguant, Cristina

    2016-03-01

    Trace amounts of the carcinogenic ethyl carbamate can appear in wine as a result of a reaction between ethanol and citrulline, which is produced from arginine degradation by some bacteria used in winemaking. In this study, arginine deiminase (ADI) pathway genes were evaluated in 44 Oenococcus oeni strains from wines originating from several locations in order to establish the relationship between the ability of a strain to degrade arginine and the presence of related genes. To detect the presence of arc genes of the ADI pathway in O. oeni, pairs of primers were designed to amplify arcA, arcB, arcC and arcD1 sequences. All strains contained these four genes. The same primers were used to confirm the organization of these genes in an arcABCD1 operon. Nevertheless, considerable variability in the ability to degrade arginine among these O. oeni strains was observed. Therefore, despite the presence of the arc genes in all strains, the expression patterns of individual genes must be strain dependent and influenced by the different wine conditions. Additionally, the presence of arc genes was also determined in the 57 sequenced strains of O. oeni available in GenBank, and the complete operon was found in 83% of strains derived from wine. The other strains were found to lack the arcB, arcC and arcD genes, but all contained sequences homologous to arcA, and some of them had also ADI activity.

  11. Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design.

    PubMed

    Slade, Daniel J; Fang, Pengfei; Dreyton, Christina J; Zhang, Ying; Fuhrmann, Jakob; Rempel, Don; Bax, Benjamin D; Coonrod, Scott A; Lewis, Huw D; Guo, Min; Gross, Michael L; Thompson, Paul R

    2015-04-17

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

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

    PubMed Central

    2015-01-01

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

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

    DOE PAGES

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

    2015-01-26

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

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

    SciTech Connect

    Slade, Daniel J.; Fang, Pengfei; Dreyton, Christina J.; Zhang, Ying; Fuhrmann, Jakob; Rempel, Don; Bax, Benjamin D.; Coonrod, Scott A.; Lewis, Huw D.; Guo, Min; Gross, Michael L.; Thompson, Paul R.

    2015-01-26

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

  15. Effect of lactic acid bacteria inoculant and beet pulp addition on fermentation characteristics and in vitro ruminal digestion of vegetable residue silage.

    PubMed

    Cao, Y; Cai, Y; Takahashi, T; Yoshida, N; Tohno, M; Uegaki, R; Nonaka, K; Terada, F

    2011-08-01

    The objective of this study was to determine the effect of beet pulp (BP) and lactic acid bacteria (LAB) on silage fermentation quality and in vitro ruminal dry matter (DM) digestion of vegetable residues, including white cabbage, Chinese cabbage, red cabbage, and lettuce. Silage was prepared using a small-scale fermentation system, and treatments were designed as control silage without additive or with BP (30% fresh matter basis), LAB inoculant Chikuso-1 (Lactobacillus plantarum, 5mg/kg, fresh matter basis), and BP+LAB. In vitro incubation was performed using rumen fluid mixed with McDougall's artificial saliva (at a ratio of 1:4, vol/vol) at 39°C for 6h to determine the ruminal fermentability of the vegetable residue silages. These vegetable residues contained high levels of crude protein (20.6-22.8% of DM) and moderate levels of neutral detergent fiber (22.7-33.6% of DM). In all silages, the pH sharply decreased and lactic acid increased, and the growth of bacilli, coliform bacteria, molds, and yeasts was inhibited by the low pH at the early stage of ensiling. The silage treated with BP or LAB had a lower pH and a higher lactic acid content than the control silage. After 6h of incubation, all silages had relatively high DM digestibility (38.6-44.9%); in particular, the LAB-inoculated silage had the highest DM digestibility and the lowest methane production. The vegetable residues had high nutritional content and high in vitro DM digestibility. Also, both the addition of a LAB inoculant and moisture adjustment with BP improved the fermentation quality of the vegetable residue silages. In addition, LAB increased DM digestibility and decreased ruminal methane production.

  16. Role of Arginine and Lysine in the Antimicrobial Mechanism of Histone-derived Antimicrobial Peptides

    PubMed Central

    Cutrona, Kara J.; Kaufman, Bethany A.; Figueroa, Dania M.; Elmore, Donald E.

    2015-01-01

    Translocation of cell-penetrating peptides is often promoted by increased content of arginine or other guanidinum groups. However, relatively little research has considered the role of these functional groups on antimicrobial peptide activity. This study compared the activity of three histone-derived antimicrobial peptides—buforin II, DesHDAP1, and parasin— with variants that contain only lysine or arginine cationic residues. These peptides operate via different mechanisms as parasin causes membrane permeabilization while buforin II and DesHDAP1 translocate into bacteria. For all peptides, antibacterial activity increased with increased arginine content. Higher arginine content increased permeabilization for parasin while it improved translocation for buforin II and DesHDAP1. These observations provide insight into the relative importance of arginine and lysine in these antimicrobial peptides. PMID:26555191

  17. Increase in the carbohydrate content of the microalgae Spirulina in culture by nutrient starvation and the addition of residues of whey protein concentrate.

    PubMed

    Vieira Salla, Ana Cláudia; Margarites, Ana Cláudia; Seibel, Fábio Ivan; Holz, Luiz Carlos; Brião, Vandré Barbosa; Bertolin, Telma Elita; Colla, Luciane Maria; Costa, Jorge Alberto Vieira

    2016-06-01

    Non-renewable sources that will end with time are the largest part of world energy consumption, which emphasizes the necessity to develop renewable sources of energy. This necessity has created opportunities for the use of microalgae as a biofuel. The use of microalgae as a feedstock source for bioethanol production requires high yields of both biomass and carbohydrates. With mixotrophic cultures, wastewater can be used to culture algae. The aim of the study was to increase the carbohydrate content in the microalgae Spirulina with the additions of residues from the ultra and nanofiltration of whey protein. The nutrient deficit in the Zarrouk medium diluted to 20% and the addition of 2.5% of both residue types led to high carbohydrate productivity (60 mg L(-1) d(-1)). With these culture conditions, the increase in carbohydrate production in Spirulina indicated that the conditions were appropriate for use with microalgae as a feedstock in the production of bioethanol.

  18. Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is stabilized by additional proline residues and an interdomain salt bridge.

    PubMed

    Kuravsky, Mikhail; Barinova, Kseniya; Marakhovskaya, Aleksandra; Eldarov, Mikhail; Semenyuk, Pavel; Muronetz, Vladimir; Schmalhausen, Elena

    2014-10-01

    Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) exhibits enhanced stability compared to the somatic isoenzyme (GAPD). A comparative analysis of the structures of these isoenzymes revealed characteristic features, which could be important for the stability of GAPDS: six specific proline residues and three buried salt bridges. To evaluate the impact of these structural elements into the stability of this isoenzyme, we obtained two series of mutant GAPDS: 1) six mutants each containing a substitution of one of the specific prolines by alanine, and 2) three mutants each containing a mutation breaking one of the salt bridges. Stability of the mutants was evaluated by differential scanning calorimetry and by their resistance towards guanidine hydrochloride (GdnHCl). The most effect on thermostability was observed for the mutants P326A and P164A: the Tm values of the heat-absorption curves decreased by 6.0 and 3.3°C compared to the wild type protein, respectively. The resistance towards GdnHCl was affected most by the mutation D311N breaking the salt bridge between the catalytic and NAD(+)-binding domains: the inactivation rate constant in the presence of GdnHCl increased six-fold, and the value of GdnHCl concentration corresponding to the protein half-denaturation decreased from 1.83 to 1.35M. Besides, the mutation D311N enhanced the enzymatic activity of the protein two-fold. The results suggest that the residues P164 (β-turn), P326 (first position of α-helix), and the interdomain salt bridge D311-H124 are significant for the enhanced stability of GAPDS. The salt bridge D311-H124 enhances stability of the active site of GAPDS at the expense of the catalytic activity.

  19. Listeria monocytogenes 10403S Arginine Repressor ArgR Finely Tunes Arginine Metabolism Regulation under Acidic Conditions

    PubMed Central

    Cheng, Changyong; Dong, Zhimei; Han, Xiao; Sun, Jing; Wang, Hang; Jiang, Li; Yang, Yongchun; Ma, Tiantian; Chen, Zhongwei; Yu, Jing; Fang, Weihuan; Song, Houhui

    2017-01-01

    Listeria monocytogenes is able to colonize human and animal intestinal tracts and to subsequently cross the intestinal barrier, causing systemic infection. For successful establishment of infection, L. monocytogenes must survive the low pH environment of the stomach. L. monocytogenes encodes a functional ArgR, a transcriptional regulator belonging to the ArgR/AhrC arginine repressor family. We aimed at clarifying the specific functions of ArgR in arginine metabolism regulation, and more importantly, in acid tolerance of L. monocytogenes. We showed that ArgR in the presence of 10 mM arginine represses transcription and expression of the argGH and argCJBDF operons, indicating that L. monocytogenes ArgR plays the classical role of ArgR/AhrC family proteins in feedback inhibition of the arginine biosynthetic pathway. Notably, transcription and expression of arcA (encoding arginine deiminase) and sigB (encoding an alternative sigma factor B) were also markedly repressed by ArgR when bacteria were exposed to pH 5.5 in the absence of arginine. However, addition of arginine enabled ArgR to derepress the transcription and expression of these two genes. Electrophoretic mobility shift assays showed that ArgR binds to the putative ARG boxes in the promoter regions of argC, argG, arcA, and sigB. Reporter gene analysis with gfp under control of the argG promoter demonstrated that ArgR was able to activate the argG promoter. Unexpectedly, deletion of argR significantly increased bacterial survival in BHI medium adjusted to pH 3.5 with lactic acid. We conclude that this phenomenon is due to activation of arcA and sigB. Collectively, our results show that L. monocytogenes ArgR finely tunes arginine metabolism through negative transcriptional regulation of the arginine biosynthetic operons and of the catabolic arcA gene in an arginine-independent manner during lactic acid-induced acid stress. ArgR also appears to activate catabolism as well as sigB transcription by anti

  20. Purification and characterization of arginine kinase from the American cockroach (Periplaneta americana).

    PubMed

    Brown, Ashli E; France, Richard M; Grossman, Steven H

    2004-06-01

    The isolation and characterization of homogeneous arginine kinase from the cockroach is reported. The purification protocol produces 6.6 mg of pure enzyme from 6.8 g of whole cockroach. The purified enzyme cross-reacts with a heterologous antibody and monoclonal antibody against arginine kinase from the shrimp. Both antibody preparations also cross-react with extracts from several species known to contain monomeric arginine kinase, but fail to react with extracts from organisms containing dimeric arginine kinase. Cockroach arginine kinase has a molecular mass of approximately 43,000 determined from measurements by gel filtration and gel electrophoresis. Compared with other arginine kinases, the enzyme from the cockroach is relatively thermostable (50% activity retained at 50 degrees C for 10 min) and has a pH optima of 8.5 and 6.5-7.5, for the forward and reverse reactions, respectively. Treatment with 5,5'dithiobis[2-nitrobenzoic acid] indicates that arginine kinase has a single reactive sulfhydryl group and, interestingly, the reaction is biphasic. The Michaelis constants for the phosphagen substrates, arginine: 0.49 mM, phosphoarginine: 0.94 mM, and nucleotide substrates MgATP: 0.14 mM, MgADP: 0.09 mM, are in the range reported for other arginine kinases. A 1% solution of pure enzyme has an absorbance of 7.0 at 280 nm. Calculations based on circular dichroic spectra indicate that arginine kinase from the cockroach has 12% alpha-helical structure. The intrinsic protein fluorescence emission maximum at 340 nm suggests that tryptophan residues are below the surface of the protein and not exposed to solvent. Arginine kinase from the cockroach and shrimp are known to be deleterious immunogens towards humans. The availability of pure protein, its characterization and potential regulation of activity, will be useful in developing agents to control the cockroach population and its destructive role in agriculture and human health.

  1. Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07.

    PubMed

    Marino, Fabio; Mommen, Geert P M; Jeko, Anita; Meiring, Hugo D; van Gaans-van den Brink, Jacqueline A M; Scheltema, Richard A; van Els, Cécile A C M; Heck, Albert J R

    2017-01-06

    Alterations in protein post-translational modification (PTM) are recognized hallmarks of diseases. These modifications potentially provide a unique source of disease-related human leukocyte antigen (HLA) class I-presented peptides that can elicit specific immune responses. While phosphorylated HLA peptides have already received attention, arginine methylated HLA class I peptide presentation has not been characterized in detail. In a human B-cell line we detected 149 HLA class I peptides harboring mono- and/or dimethylated arginine residues by mass spectrometry. A striking preference was observed in the presentation of arginine (di)methylated peptides for HLA-B*07 molecules, likely because the binding motifs of this allele resemble consensus sequences recognized by arginine methyl-transferases. Moreover, HLA-B*07-bound peptides preferentially harbored dimethylated groups at the P3 position, thus consecutively to the proline anchor residue. Such a proline-arginine sequence has been associated with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in fragmentation spectra, we found most of the peptides to be asymmetrically dimethylated, most likely by CARM1. These data expand our knowledge of the processing and presentation of arginine (di)methylated HLA class I peptides and demonstrate that these types of modified peptides can be presented for recognition by T-cells. HLA class I peptides with mono- and dimethylated arginine residues may therefore offer a novel target for immunotherapy.

  2. Rapid evolution of arginine deiminase for improved anti-tumor activity.

    PubMed

    Ni, Ye; Liu, Yongmei; Schwaneberg, Ulrich; Zhu, Leilei; Li, Na; Li, Lifeng; Sun, Zhihao

    2011-04-01

    Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas. Based on our preliminary results, it was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10% of the activity was retained at pH 7.4 (pH of human plasma). Additionally, the K(m) value for wild-type ADI (WT-ADI) was 2.88 mM (pH 6.0), which is over 20 times of the serum arginine level (100-120 μM). These are two major limitations for PpADI as a potential anti-cancer drug. A highly sensitive and efficient high-throughput screening strategy based on a modified diacetylmonoxime-thiosemicarbazide method was established to isolate ADI mutants with higher activity and lower K(m) under physiological pH. Three improved mutants was selected from 650 variants after one round of ep-PCR, among which mutant 314 (M314: A128T, H404R, I410L) exhibiting the highest activity. Interestingly, sequence alignment shows that three amino acid substitutes in M314 are coincident with corresponding residues in ADI from Mycoplasma arginini. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the K(m) value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties. This work could provide promising drug candidate for curing arginine-auxotrophic cancers.

  3. Arginine kinase: a potential pharmacological target in trypanosomiasis.

    PubMed

    Pereira, Claudio A

    2014-01-01

    Trypanosomatids parasites have complex life cycles which involve a wide diversity of milieus with very different physicochemical properties. Arginine kinase is one of the key enzymes, responsible for the parasites' metabolic plasticity, which maintains the cell energy homeostasis during environment changes. Arginine kinase catalyzes the reversible phosphorylation between phosphoarginine and ADP. The phosphagen phosphoarginine sustains high levels of cellular activity until metabolic events, such as glycolysis and oxidative phosphorylation, are switched on. In different unicellular and multicellular organisms including trypanosomatids, it was demonstrated that arginine kinase is an important component in resistance mechanisms to different stress factors, such as reactive oxygen species, trypanocidal drugs, pH and starvation. In addition, few arginine kinase inhibitors were identified during the lasts years, some of them with trypanocidal activity, such as polyphenolic compounds. All these unique features, in addition to the fact that arginine kinase is completely absent in mammals, make this pathway a favorable start point for rational drug design for the treatment of human trypanosomamiases.

  4. TRPA1 is activated by direct addition of cysteine residues to the N-hydroxysuccinyl esters of acrylic and cinnamic acids.

    PubMed

    Sadofsky, Laura R; Boa, Andrew N; Maher, Sarah A; Birrell, Mark A; Belvisi, Maria G; Morice, Alyn H

    2011-01-01

    The nociceptor TRPA1 is thought to be activated through covalent modification of specific cysteine residues on the N terminal of the channel. The precise mechanism of covalent modification with unsaturated carbonyl-containing compounds is unclear, therefore by examining a range of compounds which can undergo both conjugate and/or direct addition reactions we sought to further elucidate the mechanism(s) whereby TRPA1 can be activated by covalent modification. Calcium signalling was used to determine the mechanism of activation of TRPA1 expressed in HEK293 cells with a series of related compounds which were capable of either direct and/or conjugate addition processes. These results were confirmed using physiological recordings with isolated vagus nerve preparations. We found negligible channel activation with chemicals which could only react with cysteine residues via conjugate addition such as acrylamide, acrylic acid, and cinnamic acid. Compounds able to react via either conjugate or direct addition, such as acrolein, methyl vinyl ketone, mesityl oxide, acrylic acid NHS ester, cinnamaldehyde and cinnamic acid NHS ester, activated TRPA1 in a concentration dependent manner as did compounds only capable of direct addition, namely propionic acid NHS ester and hydrocinnamic acid NHS ester. These compounds failed to activate TRPV1 expressed in HEK293 cells or mock transfected HEK293 cells. For molecules capable of direct or conjugate additions, the results suggest for the first time that TRPA1 may be activated preferentially by direct addition of the thiol group of TRPA1 cysteines to the agonist carbonyl carbon of α,β-unsaturated carbonyl-containing compounds.

  5. The oxidation-state-dependent ATP-binding site of cytochrome c. Implication of an essential arginine residue and the effect of occupancy on the oxidation-reduction potential.

    PubMed Central

    Corthésy, B E; Wallace, C J

    1988-01-01

    Arg-91 is not part of the active site of cytochrome c that mediates binding and electron transfer, yet it is absolutely conserved in eukaryotic cytochromes c, indicating a special function. The physicochemical properties of analogues are unaffected by the modification of this residue, so they can be used with confidence to study the role of Arg-91. We have established limiting conditions under which this residue alone is specifically modified by cyclohexane-1,2-dione, and have subsequently shown that ATP, and to a lesser extent ADP or Pi, protects it from the action of the reagent in an oxidation-state-dependent manner. These observations strongly support the idea that this site exerts a controlling influence on cytochrome c activity in the electron transport or other cellular redox systems, and we have commenced a study of how that influence might operate. We find that the redox potentials of both cytochrome c and analogue are little affected by changing ATP or Pi concentrations. PMID:2843168

  6. Production of l-Arginine by Arginine Hydroxamate-Resistant Mutants of Bacillus subtilis

    PubMed Central

    Kisumi, Masahiko; Kato, Jyoji; Sugiura, Masaki; Chibata, Ichiro

    1971-01-01

    l-Arginine hydroxamate inhibited the growth of various bacteria, and the inhibition was readily reversed by arginine. l-Arginine hydroxamate (10−3m) completely inhibited the growth of Bacillus subtilis. This inhibitory effect was prevented by 2.5 × 10−4ml-arginine, which was the most effective of all the natural amino acids in reversing the inhibition. l-Arginine hydroxamate-resistant mutants of Bacillus subtilis were isolated and found to excrete l-arginine in relatively high yields. One of the mutants, strain AHr-5, produced 4.5 mg of l-arginine per ml in shaken culture in 3 days. PMID:5002904

  7. Purification and characterization of arginine kinase from locust.

    PubMed

    Li, Miao; Wang, Xiao-Yun; Bai, Ji-Gang

    2006-01-01

    L-Arginine kinase (AK; ATP:L-arginine N-phosphotransferase; EC 2.7.3.3) catalyzes the reversible transphosphorylation between N-phospho-L-arginine (PArg) and ATP thus buffering cellular ATP levels. AK was purified from the leg muscle of the locust Migratoria manilensis by Sephacryl S-200 HR gel filtration chromatography and DEAE Sepharose CL-6B fast flow anion exchange chromatography to an apparent homogeneity with a recovery of 80%. The enzyme behaved as monomeric protein with molecular mass of about 40 kD, and had a pH and temperature optimum of 8.6 and 30 degrees C, respectively, and a pI of about 6.3. The Michaelis constants for synthesis of PArg are 0.936 and 1.290 mM for L-arginine and ATP, respectively and k(cat)/K(m)(Arg) 174. The activity of AK required divalent cations such as Mg(2+) and Mn(2+). In the presence of Cu(2+) and Zn(2+), AK activity was greatly inhibited. The intrinsic protein fluorescence emission maximum at 330 nm using the excitation wavelength at 295 nm suggested that tryptophan residues are below the surface of the protein and not exposed to solvent.

  8. The Conserved Arginine Cluster in the Insert of the Third Cytoplasmic Loop of the Long Form of the D₂ Dopamine Receptor (D2L-R) Acts as an Intracellular Retention Signal.

    PubMed

    Kubale, Valentina; Blagotinšek, Kaja; Nøhr, Jane; Eidne, Karin A; Vrecl, Milka

    2016-07-19

    This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D₂ dopamine receptor (D2L-R) confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR) located within the insert could act as an RXR-type endoplasmic reticulum (ER) retention signal. Arginine residues (R) within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E), either individually or in clusters, thus generating single, double, and triple D2L-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA), radioligand binding assay, bioluminescence resonance energy transfer (BRET²) β-arrestin 2 (βarr2) recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E) substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D2L-R mutants preserved their functional integrity regarding ligand binding, agonist-induced βarr2 recruitment and Gαi-mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3) of the D2L-R appears to be the ER retention signal.

  9. Immediate postoperative radiotherapy in residual nonfunctioning pituitary adenoma: Beneficial effect on local control without additional negative impact on pituitary function and life expectancy

    SciTech Connect

    Bergh, Alfons C.M. van den . E-mail: a.c.m.van.den.bergh@rt.umcg.nl; Berg, Gerrit van den; Schoorl, Michiel A.; Sluiter, Wim J.; Vliet, Anton M. van der; Hoving, Eelco W.; Szabo, Ben G.; Langendijk, Johannes A.; Wolffenbuttel, Bruce H.R.; Dullaart, Robin P.F.

    2007-03-01

    Purpose: To demonstrate the benefit of immediate postoperative radiotherapy in residual nonfunctioning pituitary adenoma (NFA) in perspective to the need for hormonal substitution and life expectancy. Methods and Materials: Retrospective cohort analysis of 122 patients, operated for NFA between 1979 and 1998. Recurrence was defined as regrowth on computed tomography or magnetic resonance imaging. The occurrence of hormonal deficiencies was defined as the starting date of hormonal substitution therapy. Results: Seventy-six patients had residual NFA after surgery and received immediate postoperative radiotherapy (Group 1); three patients developed a recurrence, resulting in a 95% local control rate at 10 years. Twenty-eight patients had residual NFA after surgery, but were followed by a wait-and-see policy (Group 2). Sixteen developed a recurrence, resulting in a local control rate of 49% at 5 years and 22% at 10 years (p < 0.001 compared with Group 1). There were no differences between Group 1 and 2 regarding the need for substitution with thyroid hormone, glucocorticoids, and sex hormones before first surgery, directly after surgery and at end of follow-up. There were no differences in hormone substitution free survival between Group 1 and Group 2 during the study period after first surgery. Life expectancy was similar in Group 1 and 2, and their median life expectancy did not differ from median life expectancy in the general population. Conclusions: Immediate postoperative radiotherapy provides a marked improvement of local control among patients with residual NFA compared with surgery alone, without an additional deleterious effect on pituitary function and life expectancy.

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

    PubMed

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

    2014-12-01

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

  11. Protein arginine Methyltransferase 8 gene is expressed in pluripotent stem cells and its expression is modulated by the transcription factor Sox2.

    PubMed

    Solari, Claudia; Echegaray, Camila Vázquez; Luzzani, Carlos; Cosentino, María Soledad; Waisman, Ariel; Petrone, María Victoria; Francia, Marcos; Sassone, Alina; Canizo, Jésica; Sevlever, Gustavo; Barañao, Lino; Miriuka, Santiago; Guberman, Alejandra

    2016-04-22

    Addition of methyl groups to arginine residues is catalyzed by a group of enzymes called Protein Arginine Methyltransferases (Prmt). Although Prmt1 is essential in development, its paralogue Prmt8 has been poorly studied. This gene was reported to be expressed in nervous system and involved in neurogenesis. In this work, we found that Prmt8 is expressed in mouse embryonic stem cells (ESC) and in induced pluripotent stem cells, and modulated along differentiation to neural precursor cells. We found that Prmt8 promoter activity is induced by the pluripotency transcription factors Oct4, Sox2 and Nanog. Moreover, endogenous Prmt8 mRNA levels were reduced in ESC transfected with Sox2 shRNA vector. As a whole, our results indicate that Prmt8 is expressed in pluripotent stem cells and its transcription is modulated by pluripotency transcription factors. These findings suggest that besides its known function in nervous system, Prmt8 could play a role in pluripotent stem cells.

  12. Evaluation of western shale-oil residue as an additive to petroleum asphalt for use as a pavement crack and joint sealant material

    SciTech Connect

    Harnsberger, P.M.; Wolf, J.M.; Robertson, R.E.

    1992-11-01

    The objective of this study was to perform a preliminary evaluation of using a distillation residue from Green River Formation (western) shale oil as an additive to a petroleum asphalt for use as a crack and joint filler material in portland cement concrete and asphaltic pavements. A commercially available rubberized asphalt crack and joint filler material was also tested for comparison. ASTM specification tests for sealant materials used in concrete and asphalt pavements were performed on the sealant materials. Portland cement concrete briquets prepared with an asphalt material sandwiched between two concrete wafers were tested in a stress-relaxation experiment to evaluate the relaxation and recovery properties of the sealant materials. The results show that the shale-oil modified petroleum asphalts and the neat petroleum asphalt do not pass the extension portion of the ASTM test; however, there is indication of improvement in the adhesive properties of the shale-oil modified asphalts. There is also evidence that the addition of shale-oil residue to the petroleum asphalt, especially at the 20% level, improves the relaxation and recovery properties compared with the petroleum asphalt.

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

    PubMed Central

    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

  14. Arginine methylation in yeast proteins during stationary-phase growth and heat shock.

    PubMed

    Lakowski, Ted M; Pak, Magnolia L; Szeitz, András; Thomas, Dylan; Vhuiyan, Mynol I; Clement, Bernd; Frankel, Adam

    2015-12-01

    Arginine methyltransferases (RMTs) catalyze the methylation of arginine residues on proteins. We examined the effects of log-phase growth, stationary-phase growth, and heat shock on the formation of methylarginines on yeast proteins to determine if the conditions favor a particular type of methylation. Utilizing linear ion trap mass spectrometry, we identify methylarginines in wild-type and RMT deletion yeast strains using secondary product ion scans (MS(3)), and quantify the methylarginines using multiple reaction monitoring (MRM). Employing MS(3) and isotopic incorporation, we demonstrate for the first time that Nη1, Nη2-dimethylarginine (sDMA) is present on yeast proteins, and make a detailed structural determination of the fragment ions from the spectra. Nη-monomethylarginine (ηMMA), Nδ-monomethylarginine (δMMA), Nη1, Nη1-dimethylarginine (aDMA), and sDMA were detected in RMT deletion yeast using MS(3) and MRM with and without isotopic incorporation, suggesting that additional RMT enzymes remain to be discovered in yeast. The concentrations of ηMMA and δMMA decreased by half during heat shock and stationary phase compared to log-phase growth of wild-type yeast, whereas sDMA increased by as much as sevenfold and aDMA decreased by 11-fold. Therefore, upon entering stressful conditions like heat shock or stationary-phase growth, there is a net increase in sDMA and decreases in aDMA, ηMMA, and δMMA on yeast proteins.

  15. Dissection of the interaction of the human cytomegalovirus-derived US2 protein with major histocompatibility complex class I molecules: prominent role of a single arginine residue in human leukocyte antigen-A2.

    PubMed

    Thilo, Claudia; Berglund, Peter; Applequist, Steven E; Yewdell, Jonathan W; Ljunggren, Hans-Gustaf; Achour, Adnane

    2006-03-31

    Human cytomegalovirus encodes several proteins that interfere with expression of major histocompatibility complex (MHC) class I molecules on the surface of infected cells. The unique short protein 2 (US2) binds to many MHC class I allomorphs in the endoplasmic reticulum, preventing cell surface expression of the class I molecule in question. The molecular interactions underlying US2 binding to MHC class I molecules and its allele specificity have not been fully clarified. In the present study, we first compared the sequences and the structures of US2 retained versus non-retained human leukocyte antigen (HLA) class I allomorphs to identify MHC residues of potential importance for US2 binding. On the basis of this analysis, 18 individual HLA-A2 mutants were generated and the ability of full-length US2 to bind wild-type and mutated HLA-A2 complexes was assessed. We demonstrate that Arg181 plays a critical role in US2-mediated inhibition of HLA-A2 cell surface expression. The structural comparison of all known crystal structures of HLA-A2 either alone, or in complex with T cell receptor or the CD8 co-receptor, indicates that binding of US2 to HLA-A2 results in a unique, large conformational change of the side chain of Arg181. However, although the presence of Arg181 seems to be a prerequisite for US2 binding to HLA-A2, it is not sufficient for binding to all MHC class I alleles.

  16. New tris-alkoxycarbonyl arginine derivatives for peptide synthesis.

    PubMed

    Izdebski, Jan; Gers, Tomasz; Kunce, Danuta; Markowski, Paweł

    2005-01-01

    alpha-Alkoxycarbonyl protected ornithines were treated with N,N'-[Z(2Cl)](2)-S-methylisothiourea and N,N'-[Z(2Br)](2)-S-methylisothiourea, N,N'-Z(2)-S-methylisothiourea and N,N'-Boc(2)-S-methylisothiourea to form N(alpha, omega, omega')-tris-alkoxycarbonyl arginines. Two of them, Boc-Arg-{omega,omega'-[Z(2Br)](2)}-OH and Boc-Arg-{omega,omega'-[Z(2Cl)](2)}-OH, were used for the synthesis of dermorphin fragments containing two or three arginine residues. Examination of the products by HPLC and ESI-MS revealed that the purity of the materials obtained with the use of the new derivatives was higher than that obtained in concurrent syntheses in which Boc-Arg(Tos) was used.

  17. Critical Role for Arginine Methylation in Adenovirus-Infected Cells▿

    PubMed Central

    Iacovides, Demetris C.; O'Shea, Clodagh C.; Oses-Prieto, Juan; Burlingame, Alma; McCormick, Frank

    2007-01-01

    During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection. PMID:17686851

  18. Influence of commercial and residual sorbents and silicates as additives on the stabilisation/solidification of organic and inorganic industrial waste.

    PubMed

    Coz, A; Andrés, A; Soriano, S; Viguri, J R; Ruiz, M C; Irabien, J A

    2009-05-30

    An environmental problem of the foundry activities is the management of industrial waste generated in different processes. The foundry sludge from gas wet cleaning treatment that contains organic and inorganic compounds and a high content of water is an interesting example. Due to their characteristics, they can be managed using different stabilisation/solidification (S/S) technologies prior to land disposal. The purpose of this work is to study S/S formulations in order to improve the control of the mobility of the pollutants and the ecotoxicity of the samples. Different mixtures of cement or lime as binders and additives (foundry sand, silica fume, sodium silicate, silicic acid, activated carbon and black carbon) have been used in order to reduce the mobility of the chemical and ecotoxicological regulated parameters and to compare the results for commercial and residual additives. The best results have been obtained with sorbents (activated carbon and black carbon) or sodium silicate. The results of the foundry sand ash as additive can conclude that it can be used as replacement in the cement products. However, silica fume in the samples with lime and siliceous resin sand as additives gives products that do not fulfil the regulated limits. Finally, some linear expressions between the chemical parameters and the quantity of material used in the samples have been obtained.

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

    PubMed

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

    1997-07-30

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

  20. Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells.

    PubMed

    Gou, Qing; He, ShuJiao; Zhou, ZeJian

    2017-02-01

    Hepatocellular carcinoma is the most common subtype of liver cancer. Protein arginine N-methyltransferase 1 was shown to be upregulated in various cancers. However, the role of protein arginine N-methyltransferase 1 in hepatocellular carcinoma progression remains incompletely understood. We investigated the clinical and functional significance of protein arginine N-methyltransferase 1 in a series of clinical hepatocellular carcinoma samples and a panel of hepatocellular carcinoma cell lines. We performed suppression analysis of protein arginine N-methyltransferase 1 using small interfering RNA to determine the biological roles of protein arginine N-methyltransferase 1 in hepatocellular carcinoma. In addition, the expression of epithelial-mesenchymal transition indicators was verified by western blotting in hepatocellular carcinoma cell lines after small interfering RNA treatment. Protein arginine N-methyltransferase 1 expression was found to be significantly upregulated in hepatocellular carcinoma cell lines and clinical tissues. Moreover, downregulation of protein arginine N-methyltransferase 1 in hepatocellular carcinoma cells by small interfering RNA could inhibit cell proliferation, migration, and invasion in vitro. These results indicate that protein arginine N-methyltransferase 1 may contribute to hepatocellular carcinoma progression and serves as a promising target for the treatment of hepatocellular carcinoma patients.

  1. Enzymic degradation of plasma arginine using arginine deiminase inhibits nitric oxide production and protects mice from the lethal effects of tumour necrosis factor alpha and endotoxin.

    PubMed Central

    Thomas, J Brandon; Holtsberg, Frederick W; Ensor, C Mark; Bomalaski, John S; Clark, Mike A

    2002-01-01

    Septic shock is mediated in part by nitric oxide (NO) and tumour necrosis factor alpha (TNFalpha). NO is synthesized primarily from extracellular arginine. We tested the ability of an arginine-degrading enzyme to inhibit NO production in mice and to protect mice from the hypotension and lethality that occur after the administration of TNFalpha or endotoxin. Treatment of BALB/c mice with arginine deiminase (ADI) formulated with succinimidyl succinimide polyethylene glycol of M(r) 20000 (ADI-SS PEG(20000)) eliminated all measurable plasma arginine (from normal levels of approximately 155 microM arginine to 2 microM). In addition, ADI-SS PEG(20000) also inhibited the production of NO, as quantified by plasma nitrate+nitrite. Treatment of mice with TNFalpha or endotoxin resulted in a dose-dependent increase in NO production and lethality. Pretreatment of mice with ADI-SS PEG(20000) resulted in increased resistance to the lethal effects of TNFalpha and endotoxin. These observations are consistent with NO production resulting, to some extent, from the metabolism of extracellular arginine. The toxic effects of TNFalpha and endotoxin may be partially inhibited by enzymic degradation of plasma arginine by ADI-SS PEG(20000). Interestingly, pretreatment with ADI-SS PEG(20000) did not inhibit the anti-tumour activity of TNFalpha in vitro or in vivo. This treatment may allow greater amounts of TNFalpha, as well as other cytokines, to be administered while abrogating side effects such as hypotension and death. PMID:11964159

  2. A Potent, Selective and Cell-active Inhibitor of Human Type I Protein Arginine Methyltransferases

    PubMed Central

    Wu, Hong; Senisterra, Guillermo; Li, Fengling; Butler, Kyle V.; Kaniskan, H. Ümit; Speed, Brandon A.; dela Seña, Carlo; Dong, Aiping; Zeng, Hong; Schapira, Matthieu; Brown, Peter J.; Arrowsmith, Cheryl H.; Barsyte-Lovejoy, Dalia; Liu, Jing; Vedadi, Masoud; Jin, Jian

    2015-01-01

    Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, 3, 4, 6 and 8, but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease. PMID:26598975

  3. L-arginine independent macrophage tumor cytotoxicity

    SciTech Connect

    Klostergaard, J.; Leroux, M.E. )

    1989-12-29

    We have investigated the role of L-arginine in macrophage tumor cytotoxicity in coculture. L929, EMT-6, MCA-26, and P815 targets were all susceptible to cytolysis by activated macrophages when cocultured in medium containing L-arginine. When cocultured in arginine-free medium, these targets displayed comparable or even higher levels of lysis. L1210 targets were lytically resistant under either condition. However, 59Fe release from this target did reflect strong dependence on the presence of arginine. The structural analogue, NG-monomethyl-L-arginine, was an effective inhibitor of iron-release from L1210 targets cocultured with activated macrophages, whereas it had minimal inhibitory effects on release of 51Cr from cocultured L929 cells. These results suggest that the L-arginine requiring cytotoxic pathway of activated macrophage is independent of major effector mechanisms involved in tumor cell lysis.

  4. Role of arginine in the binding of thiamin pyrophosphate to Escherichia coli pyruvate oxidase.

    PubMed

    Koland, J G; O'Brien, T A; Gennis, R B

    1982-05-25

    The mode of interaction between Escherichia coli pyruvate oxidase and its cofactor, thiamin pyrophosphate (TPP), was studied with the aid of arginine-directed reagents. The enzyme is rapidly inactivated by either phenylglyoxal or 2,3-butanedione, with the cofactor, TPP, offering partial protection against these reagents. The inactivation by phenylglyoxal was found to be reversible. Experiments with [7-14C]phenylglyoxal showed that while several arginine residues react with this reagent, TPP can prevent the labeling of one such residue. Furthermore, inactivation by 2,3-butanedione is attended by at least a 100-fold decrease in affinity of the enzyme for TPP. These results suggest a direct role for arginine in the binding of the cofactor.

  5. Structural mechanism for the arginine sensing and regulation of CASTOR1 in the mTORC1 signaling pathway

    PubMed Central

    Gai, Zhongchao; Wang, Qian; Yang, Can; Wang, Lei; Deng, Wei; Wu, Geng

    2016-01-01

    The mTOR complex I (mTORC1) signaling pathway controls many metabolic processes and is regulated by amino acid signals, especially arginine. CASTOR1 has been identified as the cytosolic arginine sensor for the mTORC1 pathway, but the molecular mechanism of how it senses arginine is elusive. Here, by determining the crystal structure of human CASTOR1 in complex with arginine, we found that an exquisitely tailored pocket, carved between the NTD and the CTD domains of CASTOR1, is employed to recognize arginine. Mutation of critical residues in this pocket abolished or diminished arginine binding. By comparison with structurally similar aspartate kinases, a surface patch of CASTOR1-NTD on the opposite side of the arginine-binding site was identified to mediate direct physical interaction with its downstream effector GATOR2, via GATOR2 subunit Mios. Mutation of this surface patch disrupted CASTOR1’s recognition and inhibition of GATOR2, revealed by in vitro pull-down assay. Normal mode (NM) analysis revealed an ‘open’-to-‘closed’ conformational change for CASTOR1, which is correlated to the switching between the exposing and concealing of its GATOR2-binding residues, and is most likely related to arginine binding. Interestingly, the GATOR2-binding sites on the two protomers of CASTOR1 dimer face the same direction, which prompted us to propose a model for how dimerization of CASTOR1 relieves the inhibition of GATOR1 by GATOR2. Our study thus provides a thorough analysis on how CASTOR1 recognizes arginine, and describes a possible mechanism of how arginine binding induces the inter-domain movement of CASTOR1 to affect its association with GATOR2. PMID:28066558

  6. Physiological implications of arginine metabolism in plants

    PubMed Central

    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

  7. Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration

    PubMed Central

    Alkaitis, Matthew S.; Nardone, Glenn; Chertow, Jessica H.

    2015-01-01

    Abstract NG,NG‐dimethyl‐l‐arginine (asymmetric dimethylarginine, ADMA),NG‐monomethyl‐l‐arginine (l‐NMMA) and NG,N G’‐dimethyl‐l‐arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l‐NMMA inhibit nitric oxide synthase by competing with l‐arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high‐performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l‐NMMA and SDMA. Our assay requires 12 μL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed‐mode cation‐exchange columns, using synthetic monoethyl‐l‐arginine as an internal standard. Metabolites were derivatized with ortho‐phthaldialdeyhde and 3‐mercaptopropionic acid, separated by reverse‐phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter‐day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l‐NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples. © 2015 The Authors Biomedical Chromatography published by John Wiley & Sons, Ltd. PMID:26130049

  8. Dietary arginine affects energy metabolism through polyamine turnover in juvenile Atlantic salmon (Salmo salar).

    PubMed

    Andersen, Synne M; Holen, Elisabeth; Aksnes, Anders; Rønnestad, Ivar; Zerrahn, Jens-Erik; Espe, Marit

    2013-12-14

    In the present study, quadruplicate groups of juvenile Atlantic salmon (Salmo salar) were fed plant protein-based diets with increasing arginine inclusions (range 28·8-37·4 g/kg DM) to investigate whether arginine supplementation affects growth and lipid accumulation through an elevated polyamine turnover. Dietary lysine was held at a constant concentration, just below the requirement. All other amino acids were balanced and equal in the diets. Arginine supplementation increased protein and fat accretion, without affecting the hepatosomatic or visceralsomatic indices. Dietary arginine correlated with putrescine in the liver (R 0·78, P= 0·01) and with ornithine in the muscle, liver and plasma (P= 0·0002, 0·003 and 0·0002, respectively). The mRNA of ornithine decarboxylase, the enzyme producing putrescine, was up-regulated in the white adipose tissue of fish fed the high-arginine inclusion compared with those fed the low-arginine diet. Concomitantly, spermidine/spermine-(N1)-acetyltransferase, the rate-limiting enzyme for polyamine turnover that consumes acetyl-CoA, showed an increased activity in the liver of fish fed the arginine-supplemented diets. In addition, lower acetyl-CoA concentrations were observed in the liver of fish fed the high-arginine diet, while ATP, which is used in the process of synthesising spermidine and spermine, did not show a similar trend. Gene expression of the rate-limiting enzyme for β-oxidation of long-chain fatty acids, carnitine palmitoyl transferase-1, was up-regulated in the liver of fish fed the high-arginine diet. Taken together, the data support that increased dietary arginine activates polyamine turnover and β-oxidation in the liver of juvenile Atlantic salmon and may act to improve the metabolic status of the fish.

  9. Effects on aflatoxin M1 residues in milk by addition of hydrated sodium calcium aluminosilicate to aflatoxin-contaminated diets of dairy cows.

    PubMed

    Harvey, R B; Phillips, T D; Ellis, J A; Kubena, L F; Huff, W E; Petersen, H D

    1991-09-01

    Hydrated sodium calcium aluminosilicate (HSCAS), an anticaking agent for agricultural feeds, was added to aflatoxin (AF)-contaminated diets of 3 lactating dairy cows and evaluated for its potential to reduce aflatoxin M1 (AFM1) residues in milk. During phase I, cows were fed alternating diets that consisted of 200 micrograms of AF/kg of feed for 7 days, 0.5% HSCAS plus 200 micrograms of AF/kg of feed for 7 days, and feed with the HSCAS removed for a final 7 days. The AFM1 milk concentrations from the intervals with HSCAS added to diets were compared with those times when HSCAS was absent. The presence of 0.5% HSCAS in feed containing 200 micrograms of AF/kg reduced AFM1 secretion into the milk by an average of 0.44 micrograms/L (from pretreatment of 1.85 micrograms/L to 1.41 micrograms/L with HSCAS, a 24% reduction). Following a 10-day period of noncontaminated feed consumption and no AFM1 residues in the milk, phase II of the study was begun. The same experimental design as phase I was used, but the dosages of HSCAS and AF were changed to 1.0% and 100 micrograms/kg of feed, respectively. The addition of 1.0% HSCAS in feed containing 100 micrograms of AF/kg decreased AFM1 content in the milk by an average of 0.40 micrograms/L (from a pretreatment of 0.91 micrograms/L to 0.51 micrograms/L when HSCAS was present, a 44% reduction). These findings suggest that HSCAS, a high-affinity sorbent compound for AF in vitro, is capable of reducing the secretion of AFM1 into milk.

  10. Rate limiting domain and loop motions in arginine kinase

    PubMed Central

    Davulcu, Omar; Skalicky, Jack J.; Chapman, Michael S.

    2011-01-01

    Arginine kinase catalyzes the reversible transfer of a phosphoryl group between ATP and arginine. It is the arthropod homolog of creatine kinase, buffering cellular ATP levels. Crystal structures of arginine kinase, in substrate-free and substrate-bound forms, have revealed large conformational changes associated with the catalytic cycle. Recent NMR identified movements of the N-terminal domain and a loop comprising residues I182-G209 with conformational exchange rates in the substrate-free enzyme similar to the turnover rate. Here, to understand whether these motions might be rate-limiting, activation barriers for both the intrinsic dynamics and enzyme turnover are determined using measurements over a temperature range of 15 to 30°C. 15N transverse relaxation dispersion yields activation barriers of 46 ± 8 and 34 ± 12 kJ/mol for of the N-terminal domain and I182-G209 loop, respectively. An activation barrier of 34 ± 13 kJ/mol was obtained for enzyme turnover from steady-state kinetics. The similarity between the activation barriers is indeed consistent with turnover being limited by backbone conformational dynamics, and pinpoints the locations of potentially rate limiting motions. PMID:21425868

  11. Arginine metabolism in developing soybean cotyledons

    SciTech Connect

    Micallef, B.J.; Shelp, B.J. )

    1989-09-01

    Tracerkinetic experiments were performed using L-(guanidino-{sup 14}C)arginine, L-(U-{sup 14}C)arginine, L-(ureido-{sup 14}C)citrulline, and L-(1-{sup 14}C)ornithine to investigate arginine utilization in developing cotyledons of Gycine max (L.) Merrill. Excised cotyledons were injected with carrier-free {sup 14}C compounds and incubated in sealed vials containing a CO{sub 2} trap. The free and protein amino acids were analyzed using higher performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the {sup 14}C metabolized from (guanidino-{sup 14}C)arginine and (U-{sup 14}C)arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours {sup 14}CO{sub 2} was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra {sup 14}CO{sub 2} was evolved during the catabolism of ornithine-derived glutamate; {sup 14}C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.

  12. Differential responses of needle and branch order-based root decay to nitrogen addition: dominant effects of acid-unhydrolyzable residue and microbial enzymes

    NASA Astrophysics Data System (ADS)

    Kou, Liang; Chen, Weiwei; Zhang, Xinyu; Gao, Wenlong; Yang, Hao; Li, Dandan; Li, Shenggong

    2016-04-01

    Both chemical differences between foliage and different orders of fine roots and their contrasting decomposing microenvironments may affect their decomposition. However, little is known about how foliage and branch order-based root decomposition responds to increased N availability and the response mechanisms behind. The effects of different doses of N addition on the decomposition of needles and order-based roots of Pinus elliottii (slash pine) were monitored using the litterbag method for 524 days in a subtropical slash pine plantation in south China. The acid-unhydrolyzable residue (AUR) concentration and microbial extracellular enzymatic activities (EEA) in decomposing needles and roots were also determined. Our results indicate that the responses of needle and order-based root decomposition were N-dose-specific. The decomposition of both needles and lower-order roots was inhibited under the high N dose rate. The retarded decomposition of lower-order roots could be explained more by the increased binding of AUR to inorganic N ions, while the retarded decomposition of needles could be explained more by the reduced microbial EEA. Further, in contrast to lower-order roots, N addition had no effect on the decomposition of higher-order roots. We conclude that the decomposition of foliage and fine roots may fail to mirror each other at ambient conditions or in response to N deposition due to their contrasting decomposition microenvironments and tissue chemistry. Given the differential effects of N addition on order-based roots, our findings highlight the need to consider the tissue chemistry heterogeneity within branching fine root systems when predicting the responses of root decomposition to N loading.

  13. Triple therapy with pyridoxine, arginine supplementation and dietary lysine restriction in pyridoxine-dependent epilepsy: Neurodevelopmental outcome.

    PubMed

    Coughlin, Curtis R; van Karnebeek, Clara D M; Al-Hertani, Walla; Shuen, Andrew Y; Jaggumantri, Sravan; Jack, Rhona M; Gaughan, Sommer; Burns, Casey; Mirsky, David M; Gallagher, Renata C; Van Hove, Johan L K

    2015-01-01

    Pyridoxine-dependent epilepsy (PDE) is an epileptic encephalopathy characterized by response to pharmacologic doses of pyridoxine. PDE is caused by deficiency of α-aminoadipic semialdehyde dehydrogenase resulting in impaired lysine degradation and subsequent accumulation of α-aminoadipic semialdehyde. Despite adequate seizure control with pyridoxine monotherapy, 75% of individuals with PDE have significant developmental delay and intellectual disability. We describe a new combined therapeutic approach to reduce putative toxic metabolites from impaired lysine metabolism. This approach utilizes pyridoxine, a lysine-restricted diet to limit the substrate that leads to neurotoxic metabolite accumulation and L-arginine to compete for brain lysine influx and liver mitochondrial import. We report the developmental and biochemical outcome of six subjects who were treated with this triple therapy. Triple therapy reduced CSF, plasma, and urine biomarkers associated with neurotoxicity in PDE. The addition of arginine supplementation to children already treated with dietary lysine restriction and pyridoxine further reduced toxic metabolites, and in some subjects appeared to improve neurodevelopmental outcome. Dietary lysine restriction was associated with improved seizure control in one subject, and the addition of arginine supplementation increased the objective motor outcome scale in two twin siblings, illustrating the contribution of each component of this treatment combination. Optimal results were noted in the individual treated with triple therapy early in the course of the disease. Residual disease symptoms could be related to early injury suggested by initial MR imaging prior to initiation of treatment or from severe epilepsy prior to diagnosis. This observational study reports the use of triple therapy, which combines three effective components in this rare condition, and suggests that early diagnosis and treatment with this new triple therapy may ameliorate the

  14. Arginine Metabolism in Developing Soybean Cotyledons 1

    PubMed Central

    Micallef, Barry J.; Shelp, Barry J.

    1989-01-01

    The free and protein amino acid composition of Glycine max (L.) Merrill cotyledons was determined for the entire developmental period using high performance liquid chromatography. Arginine constituted 18% of the total protein nitrogen throughout development, and there was a linear arginine nitrogen accumulation rate of 1212 nanomoles per cotyledon per day between 16 and 58 days after anthesis. Arginine and asparagine were major constituents of the free amino acid pool, constituting 14 to 62% and 2 to 41% of the total free amino acid nitrogen, respectively. The urea cycle intermediates, citrulline, ornithine, and argininosuccinate were also detected in the free pool. A comparison of the amino acid composition of cotyledonary protein and of seedcoat exudate suggested that 72% of the cotyledon's arginine requirement is satisfied by in situ biosynthesis, and that 20% of the transformed nitrogen is incorporated into arginine. Also, [1-14C]glutamate and [U-14C]glutamine were fed to excised cotyledons. After 4 hours, 14C was incorporated into protein and released as 14CO2, but none was incorporated into the C-1 and C-6 positions of free and protein arginine, determined using arginine-specific enzyme-linked assays. It is not currently known whether arginine biosynthesis in the cotyledon involves glutamate delivered from the mother plant or glutamate derived in situ. PMID:16666818

  15. PSEUDOMONAS PYOCYANEA AND THE ARGININE DIHYDROLASE SYSTEM.

    PubMed

    TAYLOR, J J; WHITBY, J L

    1964-03-01

    Non-pigmented strains of Pseudomonas pyocyanea occur frequently and this organism has only limited activity in conventional biochemical tests; 50 strains were tested for the presence of arginine dihydrolase and found positive whereas only Salmonella sp. and Enterobacter sp. among other Gram-negative species were positive. The test for arginine dihydrolase is rapid and simple and suitable for routine use.

  16. Protein arginine methylation/demethylation and cancer

    PubMed Central

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

    2016-01-01

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

  17. Chromium remediation or release? Effect of iron(II) sulfate addition on chromium(VI) leaching from columns of chromite ore processing residue.

    PubMed

    Geelhoed, Jeanine S; Meeussen, Johannes C L; Roe, Martin J; Hillier, Stephen; Thomas, Rhodri P; Farmer, John G; Paterson, Edward

    2003-07-15

    Chromite ore processing residue (COPR), derived from the so-called high lime processing of chromite ore, contains high levels of Cr(III) and Cr(VI) and has a pH between 11 and 12. Ferrous sulfate, which is used for remediation of Cr(VI) contamination in wastewater and soils via reduction to Cr(III) and subsequent precipitation of iron(III)/chromium(III) hydroxide, has also been proposed for remediation of Cr(VI) in COPR. Instead, however, addition of FeSO4 to the infiltrating solution in column experiments with COPR greatly increased leaching of Cr(VI). Leached Cr(VI) increased from 3.8 to 12.3 mmol kg(-1) COPR in 25 pore volumes with 20 mM FeSO4, reaching solution concentrations as high as 1.6 mM. Fe(II) was ineffective in reducing Cr(VI) to Cr(III) because it precipitated when it entered the column due to the high pH of COPR, while Cr(VI) in solution was transported away with the infiltrating solution. The large increase in leaching of Cr(VI) upon infiltration of sulfate, either as FeSO4 or Na2SO4, was caused by anion exchange of sulfate for chromate in the layered double hydroxide mineral hydrocalumite, a process for which scanning electron microscopy with energy-dispersive X-ray microanalysis provided direct evidence.

  18. Enteral Glutamine Administration in Critically Ill Nonseptic Patients Does Not Trigger Arginine Synthesis

    PubMed Central

    Vermeulen, Mechteld A. R.; Brinkmann, Saskia J. H.; Buijs, Nikki; Beishuizen, Albertus; Bet, Pierre M.; Houdijk, Alexander P. J.; van Goudoever, Johannes B.; van Leeuwen, Paul A. M.

    2016-01-01

    Glutamine supplementation in specific groups of critically ill patients results in favourable clinical outcome. Enhancement of citrulline and arginine synthesis by glutamine could serve as a potential mechanism. However, while receiving optimal enteral nutrition, uptake and enteral metabolism of glutamine in critically ill patients remain unknown. Therefore we investigated the effect of a therapeutically relevant dose of L-glutamine on synthesis of L-citrulline and subsequent L-arginine in this group. Ten versus ten critically ill patients receiving full enteral nutrition, or isocaloric isonitrogenous enteral nutrition including 0.5 g/kg L-alanyl-L-glutamine, were studied using stable isotopes. A cross-over design using intravenous and enteral tracers enabled splanchnic extraction (SE) calculations. Endogenous rate of appearance and SE of glutamine citrulline and arginine was not different (SE controls versus alanyl-glutamine: glutamine 48 and 48%, citrulline 33 versus 45%, and arginine 45 versus 42%). Turnover from glutamine to citrulline and arginine was not higher in glutamine-administered patients. In critically ill nonseptic patients receiving adequate nutrition and a relevant dose of glutamine there was no extra citrulline or arginine synthesis and glutamine SE was not increased. This suggests that for arginine synthesis enhancement there is no need for an additional dose of glutamine when this population is adequately fed. This trial is registered with NTR2285. PMID:27200186

  19. Supplementation with apple enriched with L-arginine may improve metabolic control and survival rate in alloxan-induced diabetic rats.

    PubMed

    Escudero, Andrea; Petzold, Guillermo; Moreno, Jorge; Gonzalez, Marcelo; Junod, Julio; Aguayo, Claudio; Acurio, Jesenia; Escudero, Carlos

    2013-01-01

    Supplementation with L-arginine or fresh food with high content of this amino acid is associated with favorable effects in the metabolic control of diabetes. We aimed to determine whether supplementation with apples enriched with L-arginine offer additional benefits compared to L-arginine by itself in a preclinical study of diabetes. This study combines food-engineer technologies with in vivo and in vitro analysis. In vitro experiments show that cells derived from non-diabetic animals and exposed to high glucose (25 mM, 12 H) and cells isolated from alloxan-induced diabetic animals exhibited a reduction (∼50%) in the L-arginine uptake. This effect was reverted by L-arginine pretreatment (12 H) in both the normal and diabetes-derived cells. In preclinical studies, normoglycemic (n = 25) and diabetic groups (n = 50) were divided into subgroups that received either L-arginine (375 mg/kg per 10 days) or apple enriched with L-arginine or vehicle (control). In a preliminary analysis, supplementation with L-arginine by itself (50%) or apple enriched with L-arginine (100%) improve survival rate in the diabetic group compared to control (0%) at the end of the follow up (17 days). This phenomenon was associated with a partial but sustained high plasma level of L-arginine, as well as plasma concentration of nitrites and insulin in the L-arginine or apple + L-arginine groups after supplementation. Apple + L-arginine supplementation in diabetic animals induced the highest and longest effects in the level of these three markers among the studied groups. Therefore, apple enriched by L-arginine offers more benefits than L-arginine by itself in this preclinical study.

  20. Regulatory role for L-arginine in the utilization of amino acids by pig small-intestinal bacteria.

    PubMed

    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.

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

    PubMed Central

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

    2012-01-01

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

  2. A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein.

    PubMed

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

    2012-01-01

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

  3. Engineering an arginine catabolizing bioconjugate: Biochemical and pharmacological characterization of PEGylated derivatives of arginine deiminase from Mycoplasma arthritidis.

    PubMed

    Wang, Maoliang; Basu, Amartya; Palm, Thomas; Hua, Jack; Youngster, Stephen; Hwang, Lisa; Liu, Hsien-Ching; Li, Xiguang; Peng, Ping; Zhang, Yue; Zhao, Hong; Zhang, Zhihua; Longley, Clifford; Mehlig, Mary; Borowski, Virna; Sai, Prakash; Viswanathan, Manickam; Jang, Eun; Petti, Gerald; Liu, Sam; Yang, Karen; Filpula, David

    2006-01-01

    Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivation has been investigated in animal models and human clinical trials for its effects on inhibition of tumor growth, angiogenesis, or nitric oxide synthesis. In order to design an optimal arginine-catabolizing enzyme bioconjugate, a novel recombinant arginine deiminase (ADI) from Mycoplasma arthritidis was prepared, and multi-PEGylated derivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic and pharmacodynamic behavior in rats and mice. ADI bioconjugates constructed with 12 kDa or 20 kDa monomethoxy-poly(ethylene glycol) polymers with linear succinimidyl carbonate linkers were investigated via intravenous, intramuscular, or subcutaneous administration in rodents. The selected PEG-ADI compounds have 22 +/- 2 PEG strands per protein dimer, providing an additional molecular mass of about 0.2-0.5 x 10(6) Da and prolonging the plasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation was demonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallel measurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with the pharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginine to undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneous and intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growth inhibition of several cultured tumor lines that are deficient in the anabolic enzyme, argininosuccinate synthetase. Investigations of structure-activity optimization for PEGylated ADI compounds revealed a benefit to constraining the PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of the experimental therapeutics

  4. The arginine finger of the Bloom syndrome protein: its structural organization and its role in energy coupling

    PubMed Central

    Ren, Hua; Dou, Shuo-Xing; Rigolet, Pascal; Yang, Ye; Wang, Peng-Ye; Amor-Gueret, Mounira; Xi, Xu Guang

    2007-01-01

    RecQ family helicases are essential in maintaining chromosomal DNA stability and integrity. Despite extensive studies, the mechanisms of these enzymes are still poorly understood. Crystal structures of many helicases reveal a highly conserved arginine residue located near the γ-phosphate of ATP. This residue is widely recognized as an arginine finger, and may sense ATP binding and hydrolysis, and transmit conformational changes. We investigated the existence and role of the arginine finger in the Bloom syndrome protein (BLM), a RecQ family helicase, in ATP hydrolysis and energy coupling. Our studies by combination of structural modelling, site-directed mutagenesis and biochemical and biophysical approaches, demonstrate that mutations of residues interacting with the γ-phosphate of ATP or surrounding the ATP-binding sites result in severe impairment in the ATPase activity of BLM. These mutations also impair BLM's DNA-unwinding activities, but do not affect its ATP and DNA-binding abilities. These data allow us to identify R982 as the residue that functions as a BLM arginine finger. Our findings further indicate how the arginine finger is precisely positioned by the conserved motifs with respect to the γ-phosphate. PMID:17766252

  5. Occurrence of Arginine Deiminase Pathway Enzymes in Arginine Catabolism by Wine Lactic Acid Bacteria

    PubMed Central

    Liu, S.; Pritchard, G. G.; Hardman, M. J.; Pilone, G. J.

    1995-01-01

    l-Arginine, an amino acid found in significant quantities in grape juice and wine, is known to be catabolized by some wine lactic acid bacteria. The correlation between the occurrence of arginine deiminase pathway enzymes and the ability to catabolize arginine was examined in this study. The activities of the three arginine deiminase pathway enzymes, arginine deiminase, ornithine transcarbamylase, and carbamate kinase, were measured in cell extracts of 35 strains of wine lactic acid bacteria. These enzymes were present in all heterofermentative lactobacilli and most leuconostocs but were absent in all the homofermentative lactobacilli and pediococci examined. There was a good correlation among arginine degradation, formation of ammonia and citrulline, and the occurrence of arginine deiminase pathway enzymes. Urea was not detected during arginine degradation, suggesting that the catabolism of arginine did not proceed via the arginase-catalyzed reaction, as has been suggested in some earlier studies. Detection of ammonia with Nessler's reagent was shown to be a simple, rapid test to assess the ability of wine lactic acid bacteria to degrade arginine, although in media containing relatively high concentrations (>0.5%) of fructose, ammonia formation is inhibited. PMID:16534912

  6. Dysregulated arginine metabolism and cardiopulmonary dysfunction in patients with thalassaemia.

    PubMed

    Morris, Claudia R; Kim, Hae-Young; Klings, Elizabeth S; Wood, John; Porter, John B; Trachtenberg, Felicia; Sweeters, Nancy; Olivieri, Nancy F; Kwiatkowski, Janet L; Virzi, Lisa; Hassell, Kathryn; Taher, Ali; Neufeld, Ellis J; Thompson, Alexis A; Larkin, Sandra; Suh, Jung H; Vichinsky, Elliott P; Kuypers, Frans A

    2015-06-01

    Pulmonary hypertension (PH) commonly develops in thalassaemia syndromes, but is poorly characterized. The goal of this study was to provide a comprehensive description of the cardiopulmonary and biological profile of patients with thalassaemia at risk for PH. A case-control study of thalassaemia patients at high versus low PH-risk was performed. A single cross-sectional measurement for variables reflecting cardiopulmonary status and biological pathophysiology were obtained, including Doppler-echocardiography, 6-min-walk-test, Borg Dyspnoea Score, New York Heart Association functional class, cardiac magnetic resonance imaging (MRI), chest-computerized tomography, pulmonary function testing and laboratory analyses targeting mechanisms of coagulation, inflammation, haemolysis, adhesion and the arginine-nitric oxide pathway. Twenty-seven thalassaemia patients were evaluated, 14 with an elevated tricuspid-regurgitant-jet-velocity (TRV) ≥ 2·5 m/s. Patients with increased TRV had a higher frequency of splenectomy, and significantly larger right atrial size, left atrial volume and left septal-wall thickness on echocardiography and/or MRI, with elevated biomarkers of abnormal coagulation, lactate dehydrogenase (LDH) levels and arginase concentration, and lower arginine-bioavailability compared to low-risk patients. Arginase concentration correlated significantly to several echocardiography/MRI parameters of cardiovascular function in addition to global-arginine-bioavailability and biomarkers of haemolytic rate, including LDH, haemoglobin and bilirubin. Thalassaemia patients with a TRV ≥ 2·5 m/s have additional echocardiography and cardiac-MRI parameters suggestive of right and left-sided cardiac dysfunction. In addition, low arginine bioavailability may contribute to cardiopulmonary dysfunction in β-thalassaemia.

  7. Arginine metabolism: nitric oxide and beyond.

    PubMed Central

    Wu, G; Morris, S M

    1998-01-01

    Arginine is one of the most versatile amino acids in animal cells, serving as a precursor for the synthesis not only of proteins but also of nitric oxide, urea, polyamines, proline, glutamate, creatine and agmatine. Of the enzymes that catalyse rate-controlling steps in arginine synthesis and catabolism, argininosuccinate synthase, the two arginase isoenzymes, the three nitric oxide synthase isoenzymes and arginine decarboxylase have been recognized in recent years as key factors in regulating newly identified aspects of arginine metabolism. In particular, changes in the activities of argininosuccinate synthase, the arginases, the inducible isoenzyme of nitric oxide synthase and also cationic amino acid transporters play major roles in determining the metabolic fates of arginine in health and disease, and recent studies have identified complex patterns of interaction among these enzymes. There is growing interest in the potential roles of the arginase isoenzymes as regulators of the synthesis of nitric oxide, polyamines, proline and glutamate. Physiological roles and relationships between the pathways of arginine synthesis and catabolism in vivo are complex and difficult to analyse, owing to compartmentalized expression of various enzymes at both organ (e.g. liver, small intestine and kidney) and subcellular (cytosol and mitochondria) levels, as well as to changes in expression during development and in response to diet, hormones and cytokines. The ongoing development of new cell lines and animal models using cDNA clones and genes for key arginine metabolic enzymes will provide new approaches more clearly elucidating the physiological roles of these enzymes. PMID:9806879

  8. Antifungal properties of peptidomimetics with an arginine-[β-(2,5,7-tri-tert-butylindol-3-yl)alanine]-arginine motif against Saccharomyces cerevisiae and Zygosaccharomyces bailii.

    PubMed

    Larsen, Camilla Eggert; Larsen, Camilla Josephine; Franzyk, Henrik; Regenberg, Birgitte

    2015-05-01

    Due to increased occurrence of infections and food spoilage caused by yeast, there is an unmet need for new antifungal agents. The arginine-β-(2,5,7-tri-tert-butylindol-3-yl) alanine-arginine (R-Tbt-R) motif was previously proved useful in the design of an antifungal tripeptide. Here, an array of peptidomimetics based on this motif was investigated for antifungal and hemolytic activity. The five most promising modified tetrapeptide analogues ( 6: and 9-12: contain an additional C-terminal hydrophobic residue, and these were found to exhibit antifungal activity against Saccharomyces cerevisiae (MIC 6 and 12 μg mL(-1)) and Zygosaccharomyces bailii (MIC 6-25 μg mL(-1)). Four compounds ( 6: and 9-11: , had limited hemolytic activity (<10% hemolysis at 8 × MIC). Determination of their killing kinetics revealed that compound 9: displayed fungicidal effect. Testing against cells from an S. cerevisiae deletion mutant library indicated that interaction with yeast-specific fungal sphingolipids, most likely constitutes a crucial step in the mode of action. Interestingly, a lack of activity of peptidomimetics 6: and 9-11: towards Candida spp. was shown to be due to degradation or sequestering by the yeast. Due to their ultrashort nature, antifungal activity and low toxicity, the four compounds may have potential as leads for novel preservatives.

  9. Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF.

    PubMed

    Lazar, G; Schaal, T; Maniatis, T; Goodman, H M

    1995-08-15

    We show that the higher plant Arabidopsis thaliana has a serine-arginine-rich (SR) protein family whose members contain a phosphoepitope shared by the animal SR family of splicing factors. In addition, we report the cloning and characterization of a cDNA encoding a higher-plant SR protein from Arabidopsis, SR1, which has striking sequence and structural homology to the human splicing factor SF2/ASF. Similar to SF2/ASF, the plant SR1 protein promotes splice site switching in mammalian nuclear extracts. A novel feature of the Arabidopsis SR protein is a C-terminal domain containing a high concentration of proline, serine, and lysine residues (PSK domain), a composition reminiscent of histones. This domain includes a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2.

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

    PubMed Central

    Haskins, Nantaporn; Panglao, Maria; Qu, Qiuhao; Majumdar, Himani; Cabrera-Luque, Juan; Morizono, Hiroki; Tuchman, Mendel; Caldovic, Ljubica

    2008-01-01

    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 inhibition of NAGS to activation

  11. Small molecule regulators of protein arginine methyltransferases.

    PubMed

    Cheng, Donghang; Yadav, Neelu; King, Randall W; Swanson, Maurice S; Weinstein, Edward J; Bedford, Mark T

    2004-06-04

    Here we report the identification of small molecules that specifically inhibit protein arginine N-methyltransferase (PRMT) activity. PRMTs are a family of proteins that either monomethylate or dimethylate the guanidino nitrogen atoms of arginine side chains. This common post-translational modification is implicated in protein trafficking, signal transduction, and transcriptional regulation. Most methyltransferases use the methyl donor, S-adenosyl-L-methionine (AdoMet), as a cofactor. Current methyltransferase inhibitors display limited specificity, indiscriminately targeting all enzymes that use AdoMet. In this screen we have identified a primary compound, AMI-1, that specifically inhibits arginine, but not lysine, methyltransferase activity in vitro and does not compete for the AdoMet binding site. Furthermore, AMI-1 prevents in vivo arginine methylation of cellular proteins and can modulate nuclear receptor-regulated transcription from estrogen and androgen response elements, thus operating as a brake on certain hormone actions.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  13. Arginine requirement of starting broiler chicks.

    PubMed

    Cuca, M; Jensen, L S

    1990-08-01

    Three experiments were conducted to estimate the arginine requirement of male broiler chicks from 0 to 3 wk of age. The experiments were conducted in battery brooders with wires floors, and the birds received water and feed ad libitum. In the first experiment, chicks were fed a diet based on corn, soybean meal, casein, and corn-gluten meal containing 3,200 kcal ME per kg and either 20 or 23% crude protein. Regression analysis indicated an arginine requirement of 1.22% for maximum growth rate and feed efficiency with the 20% protein diet. For chicks fed the 23% protein diet, neither growth rate nor feed efficiency was significantly different among the diets containing arginine ranging from 1.13 to 1.43%. In the second experiment, a basal diet was used containing 17.5% casein and 22.5% protein with arginine ranging from 1.03 to 1.43%. An arginine requirement of 1.18% for maximum body weight gain was estimated by regression analysis, but no significant response to arginine above the basal level was observed for feed efficiency. Performance of chicks fed the basal diet was somewhat reduced because of a difficulty with adherence of feed to the beaks. In a third experiment, three basal diets containing 21, 22, or 23% protein were formulated from practical ingredients without use of casein. The requirement for maximum growth rate and feed efficiency was estimated to be 1.24 to 1.28% for the three diets. The results of these investigations indicate that the arginine requirement for starting chicks suggested by the National Research Council in 1984 of 1.44% in diets containing 3,200 kcal ME per kg is too high for practical diets. The data presented here support an arginine requirement of 1.25%.

  14. Arginine deprivation by arginine deiminase of Streptococcus pyogenes controls primary glioblastoma growth in vitro and in vivo.

    PubMed

    Fiedler, Tomas; Strauss, Madlen; Hering, Silvio; Redanz, Ulrike; William, Doreen; Rosche, Yvonne; Classen, Carl Friedrich; Kreikemeyer, Bernd; Linnebacher, Michael; Maletzki, Claudia

    2015-01-01

    Arginine auxotrophy constitutes a weak point of several tumors, among them glioblastoma multiforme (GBM). Hence, those tumors are supposed to be sensitive for arginine-depleting substances, such as arginine deiminase (ADI). Here we elucidated the sensitivity of patient-individual GBM cell lines toward Streptococcus pyogenes-derived ADI. To improve therapy, ADI was combined with currently established and pre-clinical cytostatic drugs. Additionally, effectiveness of local ADI therapy was determined in xenopatients. Half of the GBM cell lines tested responded well toward ADI monotherapy. In those cell lines, viability decreased significantly (up to 50%). Responding cell lines were subjected to combination therapy experiments to test if any additive or even synergistic effects may be achieved. Such promising results were obtained in 2/3 cases. In cell lines HROG02, HROG05 and HROG10, ADI and Palomid 529 combinations were most effective yielding more than 70% killing after 2 rounds of treatment. Comparable boosted antitumoral effects were observed after adding chloroquine to ADI (>60% killing). Apoptosis, as well as cell cycle dysregulation were found to play a minor role. In some, but clearly not all cases, (epi-) genetic silencing of arginine synthesis pathway genes (argininosuccinate synthetase 1 and argininosuccinate lyase) explained obtained results. In vivo, ADI as well as the combination of ADI and SAHA efficiently controlled HROG05 xenograft growth, whereas adding Palomid 529 to ADI did not further increase the strong antitumoral effect of ADI. The cumulative in vitro and in vivo results proved ADI as a very promising candidate therapeutic, especially for development of adjuvant GBM combination treatments.

  15. Impact of the addition of different plant residues on nitrogen mineralization-immobilization turnover and carbon content of a soil incubated under laboratory conditions

    NASA Astrophysics Data System (ADS)

    Kaleeem Abbasi, M.; Tahir, M. Mahmood; Sabir, N.; Khurshid, M.

    2015-02-01

    Application of plant residues as soil amendment may represent a valuable recycling strategy that affects carbon (C) and nitrogen (N) cycling in soil-plant systems. The amount and rate of nutrient release from plant residues depend on their quality characteristics and biochemical composition. A laboratory incubation experiment was conducted for 120 days under controlled conditions (25 °C and 58% water-filled pore space) to quantify initial biochemical composition and N mineralization of leguminous and non-leguminous plant residues, i.e., the roots, shoots and leaves of Glycine max, Trifolium repens, Zea mays, Populus euramericana, Robinia pseudoacacia and Elaeagnus umbellata, incorporated into the soil at the rate of 200 mg residue N kg-1 soil. The diverse plant residues showed a wide variation in total N, C, lignin, polyphenols and C / N ratio with higher polyphenol content in the leaves and higher lignin content in the roots. The shoot of Glycine max and the shoot and root of Trifolium repens displayed continuous mineralization by releasing a maximum of 109.8, 74.8 and 72.5 mg N kg-1 and representing a 55, 37 and 36% recovery of N that had been released from these added resources. The roots of Glycine max and Zea mays and the shoot of Zea mays showed continuous negative values throughout the incubation. After an initial immobilization, leaves of Populus euramericana, Robinia pseudoacacia and Elaeagnus umbellata exhibited net mineralization by releasing a maximum of 31.8, 63.1 and 65.1 mg N kg-1, respectively, and representing a 16, 32 and 33% N recovery, respectively. Nitrogen mineralization from all the treatments was positively correlated with the initial residue N contents (r = 0.89; p ≤ 0.01) and negatively correlated with lignin content (r = -0.84; p ≤ 0.01), C / N ratio (r = -0.69; p ≤ 0.05), lignin / N ratio (r = -0.68; p ≤ 0.05), polyphenol / N ratio (r = -0.73; p ≤ 0.05) and (lignin + polyphenol) : N ratio (r = -0.70; p ≤ 0.05) indicating a

  16. Impact of the addition of different plant residues on carbon-nitrogen content and nitrogen mineralization-immobilization turnover in a soil incubated under laboratory conditions

    NASA Astrophysics Data System (ADS)

    Abbasi, M. K.; Tahir, M. M.; Sabir, N.; Khurshid, M.

    2014-10-01

    Application of plant residues as soil amendment may represent a valuable recycling strategy that affects on carbon (C) and nitrogen (N) cycling, soil properties improvement and plant growth promotion. The amount and rate of nutrient release from plant residues depend on their quality characteristics and biochemical composition. A laboratory incubation experiment was conducted for 120 days under controlled conditions (25 °C and 58% water filled pore space (WFPS)) to quantify initial biochemical composition and N mineralization of leguminous and non-leguminous plant residues i.e. the roots, shoots and leaves of Glycine max, Trifolium repens, Zea mays, Poplus euramericana, Rubinia pseudoacacia and Elagnus umbellate incorporated into the soil at the rate of 200 mg residue N kg-1 soil. The diverse plant residues showed wide variation in total N, carbon, lignin, polyphenols and C/N ratio with higher polyphenol content in the leaves and higher lignin content in the roots. The shoot of G. max and the shoot and root of T. repens displayed continuous mineralization by releasing a maximum of 109.8, 74.8 and 72.5 mg N kg-1 and representing a 55, 37 and 36% of added N being released from these resources. The roots of G. max and Z. mays and the shoot of Z. mays showed continuous negative values throughout the incubation showing net immobilization. After an initial immobilization, leaves of P. euramericana, R. pseudoacacia and E. umbellate exhibited net mineralization by releasing a maximum of 31.8, 63.1 and 65.1 mg N kg-1, respectively and representing a 16, 32 and 33% of added N being released. Nitrogen mineralization from all the treatments was positively correlated with the initial residue N contents (r = 0.89; p ≤ 0.01), and negatively correlated with lignin content (r = -0.84; p ≤ 0.01), C/N ratio (r = -0.69; p ≤ 0.05), lignin/N ratio (r = -0.68; p ≤ 0.05), polyphenol/N ratio (r = -0.73; p ≤ 0.05) and ligin + polyphenol/N ratio (r = -0.70; p ≤ 0.05) indicating

  17. Effects of NG-nitro-L-arginine and L-arginine on regional cerebral blood flow in the cat.

    PubMed Central

    Kovách, A G; Szabó, C; Benyó, Z; Csáki, C; Greenberg, J H; Reivich, M

    1992-01-01

    1. We studied the effects of NG-nitro-L-arginine (NOLA), a potent inhibitor of the L-arginine-nitric oxide pathway, and L-arginine, the precursor of nitric oxide, on regional cerebral blood flow, electrocortical activity and ex vivo cerebrovascular reactivity in the cat. Flow was measured via radiolabelled microspheres, and vascular responses were studied by measuring isometric tension of isolated middle cerebral arterial rings. 2. NOLA (30 mg kg-1 bolus followed by 1 mg kg-1 min-1 infusion) caused an approximately 40 mmHg elevation in the mean arterial blood pressure, a regionally heterogenous increase of the regional cerebrovascular resistance and a decrease in the regional cerebral blood flow 15 and 40 min after the start of its administration. In contrast L-arginine (30 mg kg-1 bolus followed by 10 mg kg-1 min-1 infusion) did not alter blood pressure, cerebrovascular resistance nor regional cerebral blood flow 15 min after the start of its administration. The NOLA-induced changes in tissue flow were the most pronounced in the cerebellum, pituitary and medulla oblongata, whereas there was no decrease in the flow of the cortex and white matter. 3. NOLA caused characteristic changes in total fronto-occipital EEG power and in power spectra which were unlikely to have been due to cerebral ischaemia. In addition, the ex vivo reactivity of the middle cerebral arteries showed signs of impaired endothelial nitric oxide synthesis: there were enhanced noradrenaline-induced contractions and N-ethoxycarbonyl-3-morpholino-sydnonimine (SIN-1)-induced relaxations and markedly attenuated acetylcholine- and ATP-induced relaxations after NOLA treatment. 4. The present data indicate that resting cerebral blood flow and cerebrovascular resistance are regulated by nitric oxide derived from L-arginine in a regionally heterogenous way and that exogenous L-arginine availability is not a limiting factor in this nitric oxide generation. Possibly, both the vascular endothelium and the

  18. Arginine Relieves the Inflammatory Response and Enhances the Casein Expression in Bovine Mammary Epithelial Cells Induced by Lipopolysaccharide

    PubMed Central

    Wu, Tianyou; Wang, Chao; Ding, Luoyang; Shen, Yizhao; Cui, Huihui; Wang, Mengzhi; Wang, Hongrong

    2016-01-01

    As one of functional active amino acids, L-arginine holds a key position in immunity. However, the mechanism that arginine modulates cow mammary inflammatory response in ruminant is unclear. Therefore, this study was conducted to investigate the effects of L-arginine on inflammatory response and casein expression after challenging the bovine mammary epithelial cells (BMECs) with lipopolysaccharide (LPS). The cells were divided into four groups, stimulated with or without LPS (10 μg/mL) and treated with or without arginine (100 μg/mL) for 12 h. The concentration of proinflammatory cytokines, inducible nitric oxide synthase (iNOS), mammalian target of rapamycin (mTOR), and Toll-like receptor 4 (TLR4) signaling pathways as well as the casein was determined. The results showed that arginine reduced the LPS-induced production like IL-1β, IL-6, TNF-α, and iNOS. Though the expression of NF-κB was attenuated and the mTOR signaling pathway was upregulated, arginine had no effect on TLR4 expression. In addition, our results show that the content of β-casein and the total casein were enhanced after arginine was supplemented in LPS-induced BMECs. In conclusion, arginine could relieve the inflammatory reaction induced by LPS and enhance the concentration of β-casein and the total casein in bovine mammary epithelial cells. PMID:27110069

  19. Arginine promotes Proteus mirabilis motility and fitness by contributing to conservation of the proton gradient and proton motive force.

    PubMed

    Armbruster, Chelsie E; Hodges, Steven A; Smith, Sara N; Alteri, Christopher J; Mobley, Harry L T

    2014-10-01

    Swarming contributes to Proteus mirabilis pathogenicity by facilitating access to the catheterized urinary tract. We previously demonstrated that 0.1-20 mmol/L arginine promotes swarming on normally nonpermissive media and that putrescine biosynthesis is required for arginine-induced swarming. We also previously determined that arginine-induced swarming is pH dependent, indicating that the external proton concentration is critical for arginine-dependent effects on swarming. In this study, we utilized survival at pH 5 and motility as surrogates for measuring changes in the proton gradient (ΔpH) and proton motive force (μH(+) ) in response to arginine. We determined that arginine primarily contributes to ΔpH (and therefore μH(+) ) through the action of arginine decarboxylase (speA), independent of the role of this enzyme in putrescine biosynthesis. In addition to being required for motility, speA also contributed to fitness during infection. In conclusion, consumption of intracellular protons via arginine decarboxylase is one mechanism used by P. mirabilis to conserve ΔpH and μH(+) for motility.

  20. L-Arginine enhances the triglyceride-lowering effect of simvastatin in patients with elevated plasma triglycerides.

    PubMed

    Schulze, Friedrich; Glos, Sabrina; Petruschka, Dörte; Altenburg, Christiane; Maas, Renke; Benndorf, Ralf; Schwedhelm, Edzard; Beil, Ulrich; Böger, Rainer H

    2009-05-01

    We recently noticed a possible triglyceride-lowering effect during dietary supplementation with L-arginine. The major limitation of prior studies on L-arginine, however, was that triglyceride levels were not the primary end point, and patients were not necessarily hypertriglyceridemic. Therefore, we conducted a 2-arm, randomized, double-blind study in 33 hypertriglyceridemic patients to investigate the hypothesis that oral L-arginine may lower serum triglyceride levels in hypertriglyceridemic patients on and off statins. The study consisted of a 6-week run-in phase, 6 weeks of treatment with L-arginine (n = 22, 1.5 g bid) or placebo (n = 11), and a 6-week extension period where simvastatin (20 mg qd) was added. All patients received dietary advice during each study visit. Routine and lipid laboratory parameters were determined in the local routine clinical laboratory. Treatment with L-arginine alone had no effects on serum lipids compared to placebo. The combination of L-arginine with simvastatin led to a significantly stronger reduction in triglycerides compared to placebo plus simvastatin (-140.5 +/- 149.2 mg/dL vs -56.1 +/- 85.0 mg/dL; P = .048). In addition, we found simvastatin-induced increases in aspartate transaminase and fibrinogen to be attenuated by L-arginine as compared to placebo. We conclude from our data that L-arginine enhances the effects of simvastatin on lipid metabolism, but it has no triglyceride-lowering effects when given alone.

  1. Improved method for expression and isolation of the Mycoplasma hominis arginine deiminase from the recombinant strain of Escherichia coli.

    PubMed

    Fayura, Lyubov R; Boretsky, Yuriy R; Pynyaha, Yuriy V; Wheatley, Denys N; Sibirny, Andriy A

    2013-09-20

    Arginine deiminase is a promising anticancer drug active against melanoma, hepatocarcinoma and other tumors. Recombinant strains of Escherichia coli that express arginine deiminase from pathogenic bacteria Mycoplasma have been developed. However, production costs of heterologous arginine deiminase are high due to use of an expensive inducer and extraction buffer, as well as using diluted culture for enzyme induction. We report on a new advanced protocol for Mycoplasma hominis arginine deiminase expression, extraction and renaturation. The main improvements include manipulation with dense suspensions of E. coli, use of lactose instead of isopropyl β-D-1-thiogalactopyranoside as an inducer and a cheaper but not less efficient buffer for solubilization of arginine deiminase inclusion bodies. In addition, supplementation of the storage culture medium with glucose and substrate (arginine) significantly stabilized the recombinant arginine deiminase producer. Homogenous preparations of recombinant arginine deiminase were obtained using anion-exchange and hydrophobic chromatography. The purified enzyme retained a specific activity of 30-34 U/mg for 12 months when stored at 4°C in 20 mM sodium phosphate buffer pH 7.2 containing 1 M NaCl.

  2. Impacts of arginine nutrition on embryonic and fetal development in mammals.

    PubMed

    Wu, Guoyao; Bazer, Fuller W; Satterfield, M Carey; Li, Xilong; Wang, Xiaoqiu; Johnson, Gregory A; Burghardt, Robert C; Dai, Zhaolai; Wang, Junjun; Wu, Zhenlong

    2013-08-01

    Embryonic loss and intrauterine growth restriction (IUGR) are significant problems in humans and other animals. Results from studies involving pigs and sheep have indicated that limited uterine capacity and placental insufficiency are major factors contributing to suboptimal reproduction in mammals. Our discovery of the unusual abundance of the arginine family of amino acids in porcine and ovine allantoic fluids during early gestation led to the novel hypothesis that arginine plays an important role in conceptus (embryo and extra-embryonic membranes) development. Arginine is metabolized to ornithine, proline, and nitric oxide, with each having important physiological functions. Nitric oxide is a vasodilator and angiogenic factor, whereas ornithine and proline are substrates for uterine and placental synthesis of polyamines that are key regulators of gene expression, protein synthesis, and angiogenesis. Additionally, arginine activates the mechanistic (mammalian) target of rapamycin cell signaling pathway to stimulate protein synthesis in the placenta, uterus, and fetus. Thus, dietary supplementation with 0.83 % L-arginine to gilts consuming 2 kg of a typical gestation diet between either days 14 and 28 or between days 30 and 114 of pregnancy increases the number of live-born piglets and litter birth weight. Similar results have been reported for gestating rats and ewes. In sheep, arginine also stimulates development of fetal brown adipose tissue. Furthermore, oral administration of arginine to women with IUGR has been reported to enhance fetal growth. Collectively, enhancement of uterine as well as placental growth and function through dietary arginine supplementation provides an effective solution to improving embryonic and fetal survival and growth.

  3. Biocatalytic synthesis, antimicrobial properties and toxicity studies of arginine derivative surfactants.

    PubMed

    Fait, M Elisa; Garrote, Graciela L; Clapés, Pere; Tanco, Sebastian; Lorenzo, Julia; Morcelle, Susana R

    2015-07-01

    Two novel arginine-based cationic surfactants were synthesized using as biocatalyst papain, an endopeptidase from Carica papaya latex, adsorbed onto polyamide. The classical substrate N (α)-benzoyl-arginine ethyl ester hydrochloride for the determination of cysteine and serine proteases activity was used as the arginine donor, whereas decyl- and dodecylamine were used as nucleophiles for the condensation reaction. Yields higher than 90 and 80 % were achieved for the synthesis of N (α)-benzoyl-arginine decyl amide (Bz-Arg-NHC10) and N (α)-benzoyl-arginine dodecyl amide (Bz-Arg-NHC12), respectively. The purification process was developed in order to make it more sustainable, by using water and ethanol as the main separation solvents in a single cationic exchange chromatographic separation step. Bz-Arg-NHC10 and Bz-Arg-NHC12 proved antimicrobial activity against both Gram-positive and Gram-negative bacteria, revealing their potential use as effective disinfectants as they reduced 99 % the initial bacterial population after only 1 h of contact. The cytotoxic effect towards different cell types of both arginine derivatives was also measured. Bz-Arg-NHCn demonstrated lower haemolytic activity and were less eye-irritating than the commercial cationic surfactant cetrimide. A similar trend could also be observed when cytotoxicity was tested on hepatocytes and fibroblast cell lines: both arginine derivatives were less toxic than cetrimide. All these properties would make the two novel arginine compounds a promising alternative to commercial cationic surfactants, especially for their use as additives in topical formulations.

  4. The Conserved Arginine Cluster in the Insert of the Third Cytoplasmic Loop of the Long Form of the D2 Dopamine Receptor (D2L-R) Acts as an Intracellular Retention Signal

    PubMed Central

    Kubale, Valentina; Blagotinšek, Kaja; Nøhr, Jane; Eidne, Karin A.; Vrecl, Milka

    2016-01-01

    This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D2 dopamine receptor (D2L-R) confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR) located within the insert could act as an RXR-type endoplasmic reticulum (ER) retention signal. Arginine residues (R) within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E), either individually or in clusters, thus generating single, double, and triple D2L-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA), radioligand binding assay, bioluminescence resonance energy transfer (BRET2) β-arrestin 2 (βarr2) recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E) substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D2L-R mutants preserved their functional integrity regarding ligand binding, agonist-induced βarr2 recruitment and Gαi-mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3) of the D2L-R appears to be the ER retention signal. PMID:27447620

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

    PubMed

    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.

  6. Chemical modification of cationic residues in toxin a from king cobra (Ophiophagus hannah) venom.

    PubMed

    Lin, S R; Chi, S H; Chang, L S; Kuo, K W; Chang, C C

    1996-01-01

    The cationic groups of arginine and lysine residues in alpha-neurotoxin, Toxin a, isolated from king cobra (Ophiophagus hannah) venom were subjected to modification with trinitrobenzene sulfonate (TNBS) and p-hydroxyphenylglyoxal (HPG), respectively. The trinitrophenylated (TNP) derivatives of Toxin a at Lys-10, 56, or 71 showed approximately 25% residual lethality, and modifications on Lys-10 and 56 or Lys-10 and 50 resulted in a decrease of lethality by 84% and 86%, respectively. Modifications on Arg-34, 37, and 70 and Arg-34, 37, and 72 in Toxin a caused a decrease in lethality by 92% and 93%, respectively, and it almost completely lost its lethality and binding activity to nicotinic acetylcholine receptor (nAChR) when all four arginine residues were modified. These results indicate that in addition to the cationic residues on loop II (Arg-34, 37), loop III (Lys-50, 56), and the C-terminal tail (Arg-70, 72; Lys-71), Lys-10 on loop I is also related to the neurotoxicity of Toxin a.

  7. Targeting Arginine-Dependent Cancers with Arginine-Degrading Enzymes: Opportunities and Challenges

    PubMed Central

    Phillips, Melissa M.; Sheaff, Michael T.

    2013-01-01

    Arginine deprivation is a novel antimetabolite strategy for the treatment of arginine-dependent cancers that exploits differential expression and regulation of key urea cycle enzymes. Several studies have focused on inactivation of argininosuccinate synthetase 1 (ASS1) in a range of malignancies, including melanoma, hepatocellular carcinoma (HCC), mesothelial and urological cancers, sarcomas, and lymphomas. Epigenetic silencing has been identified as a key mechanism for loss of the tumor suppressor role of ASS1 leading to tumoral dependence on exogenous arginine. More recently, dysregulation of argininosuccinate lyase has been documented in a subset of arginine auxotrophic glioblastoma multiforme, HCC and in fumarate hydratase-mutant renal cancers. Clinical trials of several arginine depletors are ongoing, including pegylated arginine deiminase (ADI-PEG20, Polaris Group) and bioengineered forms of human arginase. ADI-PEG20 is furthest along the path of clinical development from combinatorial phase 1 to phase 3 trials and is described in more detail. The challenge will be to identify tumors sensitive to drugs such as ADI-PEG20 and integrate these agents into multimodality drug regimens using imaging and tissue/fluid-based biomarkers as predictors of response. Lastly, resistance pathways to arginine deprivation require further study to optimize arginine-targeted therapies in the oncology clinic. PMID:24453997

  8. Arginine deprivation using pegylated arginine deiminase has activity against primary acute myeloid leukemia cells in vivo.

    PubMed

    Miraki-Moud, Farideh; Ghazaly, Essam; Ariza-McNaughton, Linda; Hodby, Katharine A; Clear, Andrew; Anjos-Afonso, Fernando; Liapis, Konstantinos; Grantham, Marianne; Sohrabi, Fareeda; Cavenagh, Jamie; Bomalaski, John S; Gribben, John G; Szlosarek, Peter W; Bonnet, Dominique; Taussig, David C

    2015-06-25

    The strategy of enzymatic degradation of amino acids to deprive malignant cells of important nutrients is an established component of induction therapy of acute lymphoblastic leukemia. Here we show that acute myeloid leukemia (AML) cells from most patients with AML are deficient in a critical enzyme required for arginine synthesis, argininosuccinate synthetase-1 (ASS1). Thus, these ASS1-deficient AML cells are dependent on importing extracellular arginine. We therefore investigated the effect of plasma arginine deprivation using pegylated arginine deiminase (ADI-PEG 20) against primary AMLs in a xenograft model and in vitro. ADI-PEG 20 alone induced responses in 19 of 38 AMLs in vitro and 3 of 6 AMLs in vivo, leading to caspase activation in sensitive AMLs. ADI-PEG 20-resistant AMLs showed higher relative expression of ASS1 than sensitive AMLs. This suggests that the resistant AMLs survive by producing arginine through this metabolic pathway and ASS1 expression could be used as a biomarker for response. Sensitive AMLs showed more avid uptake of arginine from the extracellular environment consistent with their auxotrophy for arginine. The combination of ADI-PEG 20 and cytarabine chemotherapy was more effective than either treatment alone resulting in responses in 6 of 6 AMLs tested in vivo. Our data show that arginine deprivation is a reasonable strategy in AML that paves the way for clinical trials.

  9. Mechanism of the intrinsic arginine finger in heterotrimeric G proteins

    PubMed Central

    Mann, Daniel; Teuber, Christian; Tennigkeit, Stefan A.; Schröter, Grit; Gerwert, Klaus

    2016-01-01

    Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine “finger.” We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis. PMID:27911799

  10. Conformational profile of a proline-arginine hybrid

    PubMed Central

    Revilla-López, Guillermo; Jiménez, Ana I.; Cativiela, Carlos; Nussinov, Ruth; Alemán, Carlos; Zanuy, David

    2010-01-01

    The intrinsic conformational preferences of a new non-proteinogenic amino acid have been explored by computational methods. This tailored molecule, named (βPro)Arg, is conceived as a replacement for arginine in bioactive peptides when the stabilization of folded turn-like conformations is required. The new residue features a proline skeleton that bears the guanidilated side chain of arginine at the Cβ position of the five-membered pyrrolidine ring, either in a cis or a trans orientation with respect to the carboxylic acid. The conformational profile of the N-acetyl-N'-methylamide derivatives of the cis and trans isomers of (βPro)Arg has been examined in the gas phase and in solution by B3LYP/6–31+G(d,p) calculations and molecular dynamics simulations. The main conformational features of both isomers represent a balance between geometric restrictions imposed by the five-membered pyrrolidine ring and the ability of the guanidilated side chain to interact with the backbone through hydrogen-bonds. Thus, both cis and trans (βPro)Arg exhibit a preference for the αL conformation as a consequence of the interactions established between the guanidinium moiety and the main-chain amide groups. PMID:20886854

  11. Mechanism of the intrinsic arginine finger in heterotrimeric G proteins.

    PubMed

    Mann, Daniel; Teuber, Christian; Tennigkeit, Stefan A; Schröter, Grit; Gerwert, Klaus; Kötting, Carsten

    2016-12-13

    Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.

  12. Risk-based approach to developing a national residue sampling plan for testing under European Union regulation for veterinary medicinal products and coccidiostat feed additives in domestic animal production.

    PubMed

    Danaher, Martin; Shanahan, Conor; Butler, Francis; Evans, Rhodri; O'Sullivan, Dan; Glynn, Denise; Camon, Tim; Lawlor, Peadar; O'Keeffe, Michael

    2016-07-01

    A ranking system for veterinary medicinal products and coccidiostat feed additives has been developed as a tool to be applied in a risk-based approach to the residue testing programme for foods of animal origin in the Irish National Residue Control Plan (NRCP). Three characteristics of substances that may occur as residues in food are included in the developed risk ranking system: Potency, as measured by the acceptable daily intake assigned by the European Medicines Agency Committee for Medicinal Products for Veterinary Use, to each substance; Usage, as measured by the three factors of Number of Doses, use on Individual animals or for Group treatment, and Withdrawal Period; and Residue Occurrence, as measured by the number of Non-Compliant Samples in the NRCP. For both Number of Doses and Non-Compliant Samples, data for the 5-year period 2008-12 have been used. The risk ranking system for substances was developed for beef cattle, sheep and goats, pigs, chickens and dairy cattle using a scoring system applied to the various parameters described above to give an overall score based on the following equation: Potency × Usage (Number of Doses + Individual/Group Use + Withdrawal Period) × Residue Occurrence. Applying this risk ranking system, the following substances are ranked very highly: antimicrobials such as amoxicillin (for all species except pigs), marbofloxacillin (for beef cattle), oxytetracycline (for all species except chickens), sulfadiazine with trimethoprim (for pigs and chickens) and tilmicosin (for chickens); antiparasitic drugs, such as the benzimidazoles triclabendazole (for beef and dairy cattle), fenbendazole/oxfendazole (for sheep/goats and dairy cattle) and albendazole (for dairy cattle), the avermectin ivermectin (for beef cattle), and anti-fluke drugs closantel and rafoxanide (for sheep/goats); the anticoccidials monensin, narasin, nicarbazin and toltrazuril (for chickens). The risk ranking system described is a relatively simple system

  13. Arginine Deiminase Enzyme Evolving As A Potential Antitumor Agent.

    PubMed

    Somani, Rakesh; Chaskar, Pratip K

    2016-08-17

    Some melanomas and hepatocellular carcinomas have been shown to be auxotrophic for arginine. Arginine deiminase (ADI), an arginine degrading enzyme isolated from Mycoplasma, can inhibit the growth of these tumors. It is a catabolizing enzyme which catabolizes arginine to citrulline. Tumor cells do not express an enzyme called arginosuccinate synthetase (ASS) and hence, these cells becomes auxotrophic for arginine. It is found that ADI is specific for arginine and did not degrade other amino acid. This review covers various aspects of ADIs like origin, properties and chemical modifications for better antitumor activity.

  14. Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells.

    PubMed

    Sipos, Adrienn; Iván, Judit; Bécsi, Bálint; Darula, Zsuzsanna; Tamás, István; Horváth, Dániel; Medzihradszky, Katalin F; Erdődi, Ferenc; Lontay, Beáta

    2017-01-11

    Myosin phosphatase (MP) holoenzyme is a protein phosphatase-1 (PP1) type Ser/Thr specific enzyme that consists of a PP1 catalytic (PP1c) and a myosin phosphatase target subunit-1 (MYPT1). MYPT1 is an ubiquitously expressed isoform and it targets PP1c to its substrates. We identified the protein arginine methyltransferase 5 (PRMT5) enzyme of the methylosome complex as a MYPT1-binding protein uncovering the nuclear MYPT1-interactome of hepatocellular carcinoma cells. It is shown that PRMT5 is regulated by phosphorylation at Thr80 by RhoA-associated protein kinase and MP. Silencing of MYPT1 increased the level of the PRMT5-specific symmetric dimethylation on arginine residues of histone 2 A/4, a repressing gene expression mark, and it resulted in a global change in the expression of genes affecting cellular processes like growth, proliferation and cell death, also affecting the expression of the retinoblastoma protein and c-Myc. The phosphorylation of the MP inhibitory MYPT1(T850) and the regulatory PRMT5(T80) residues as well as the symmetric dimethylation of H2A/4 were elevated in human hepatocellular carcinoma and in other types of cancers. These changes correlated positively with the grade and state of the tumors. Our results suggest the tumor suppressor role of MP via inhibition of PRMT5 thereby regulating gene expression through histone arginine dimethylation.

  15. Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

    PubMed Central

    Sipos, Adrienn; Iván, Judit; Bécsi, Bálint; Darula, Zsuzsanna; Tamás, István; Horváth, Dániel; Medzihradszky, Katalin F.; Erdődi, Ferenc; Lontay, Beáta

    2017-01-01

    Myosin phosphatase (MP) holoenzyme is a protein phosphatase-1 (PP1) type Ser/Thr specific enzyme that consists of a PP1 catalytic (PP1c) and a myosin phosphatase target subunit-1 (MYPT1). MYPT1 is an ubiquitously expressed isoform and it targets PP1c to its substrates. We identified the protein arginine methyltransferase 5 (PRMT5) enzyme of the methylosome complex as a MYPT1-binding protein uncovering the nuclear MYPT1-interactome of hepatocellular carcinoma cells. It is shown that PRMT5 is regulated by phosphorylation at Thr80 by RhoA-associated protein kinase and MP. Silencing of MYPT1 increased the level of the PRMT5-specific symmetric dimethylation on arginine residues of histone 2 A/4, a repressing gene expression mark, and it resulted in a global change in the expression of genes affecting cellular processes like growth, proliferation and cell death, also affecting the expression of the retinoblastoma protein and c-Myc. The phosphorylation of the MP inhibitory MYPT1T850 and the regulatory PRMT5T80 residues as well as the symmetric dimethylation of H2A/4 were elevated in human hepatocellular carcinoma and in other types of cancers. These changes correlated positively with the grade and state of the tumors. Our results suggest the tumor suppressor role of MP via inhibition of PRMT5 thereby regulating gene expression through histone arginine dimethylation. PMID:28074910

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

    DOE PAGES

    Yue, Yufei; CHu, Yuzhuo; Guo, Hong

    2015-01-01

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

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

    SciTech Connect

    Yue, Yufei; CHu, Yuzhuo; Guo, Hong

    2015-01-01

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

  18. Identification of key residues involved in Si transport by the aquaglyceroporins

    PubMed Central

    Carpentier, Gabriel A.; Garneau, Alexandre P.; Marcoux, Andrée-Anne; Noël, Micheline; Frenette-Cotton, Rachelle

    2016-01-01

    We recently demonstrated that the aquaglyceroporins (AQGPs) could act as potent transporters for orthosilicic acid (H4SiO4). Although interesting, this finding raised the question of whether water and H4SiO4, the transportable form of Si, permeate AQGPs by interacting with the same region of the pore, especially in view of the difference in molecular radius between the two substrates. Here, our goal was to identify residues that endow the AQGPs with the ability to facilitate Si diffusion by examining the transport characteristics of mutants in which residues were interchanged between a water-permeable but Si-impermeable channel (aquaporin 1 [AQP1]) and a Si-permeable but water-impermeable channel (AQP10). Our results indicate that the composition of the arginine filter (XX/R), known to include three residues that play an important role in water transport, may also be involved in Si selectivity. Interchanging the identities of the nonarginine residues within this filter causes Si transport to increase by approximately sevenfold in AQP1 and to decrease by approximately threefold in AQP10, whereas water transport and channel expression remain unaffected. Our results further indicate that two additional residues in the AQP arginine filter may be involved in substrate selectivity: replacing one of the residues has a profound effect on water permeability, and replacing the other has a profound effect on Si permeability. This study has thus led to the identification of residues that could play a key role in Si transport by the AQGPs and shown that substrate selectivity is likely ensured by more than one checkpoint within or near the pore. PMID:27527099

  19. Arginine: Its pKa value revisited

    PubMed Central

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

    2015-01-01

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

  20. Arginine: Its pKa value revisited.

    PubMed

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

    2015-05-01

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

  1. A novel prokaryotic L-arginine:glycine amidinotransferase is involved in cylindrospermopsin biosynthesis.

    PubMed

    Muenchhoff, Julia; Siddiqui, Khawar S; Poljak, Anne; Raftery, Mark J; Barrow, Kevin D; Neilan, Brett A

    2010-09-01

    We report the first characterization of an L-arginine:glycine amidinotransferase from a prokaryote. The enzyme, CyrA, is involved in the pathway for biosynthesis of the polyketide-derived hepatotoxin cylindrospermopsin from Cylindrospermopsis raciborskii AWT205. CyrA is phylogenetically distinct from other amidinotransferases, and structural alignment shows differences between the active site residues of CyrA and the well-characterized human L-arginine:glycine amidinotransferase (AGAT). Overexpression of recombinant CyrA in Escherichia coli enabled biochemical characterization of the enzyme, and we confirmed the predicted function of CyrA as an L-arginine:glycine amidinotransferase by (1) H NMR. As compared with AGAT, CyrA showed narrow substrate specificity when presented with substrate analogs, and deviated from regular Michaelis-Menten kinetics in the presence of the non-natural substrate hydroxylamine. Studies of initial reaction velocities and product inhibition, and identification of intermediate reaction products, were used to probe the kinetic mechanism of CyrA, which is best described as a hybrid of ping-pong and sequential mechanisms. Differences in the active site residues of CyrA and AGAT are discussed in relation to the different properties of both enzymes. The enzyme had maximum activity and maximum stability at pH 8.5 and 6.5, respectively, and an optimum temperature of 32 °C. Investigations into the stability of the enzyme revealed that an inactivated form of this enzyme retained an appreciable amount of secondary structure elements even on heating to 94 °C, but lost its tertiary structure at low temperature (T(max) of 44.5 °C), resulting in a state reminiscent of a molten globule. CyrA represents a novel group of prokaryotic amidinotransferases that utilize arginine and glycine as substrates with a complex kinetic mechanism and substrate specificity that differs from that of the eukaryotic L-arginine:glycine amidinotransferases.

  2. Understanding the synergistic effect of arginine and glutamic acid mixtures on protein solubility.

    PubMed

    Shukla, Diwakar; Trout, Bernhardt L

    2011-10-20

    Understanding protein solubility is a key part of physical chemistry. In particular, solution conditions can have a major effect, and the effect of multiple cosolutes is little understood. It has been shown that the simultaneous addition of L-arginine hydrochloride and L-glutamic acid enhances the maximum achievable solubility of several poorly soluble proteins up to 4-8 times (Golovanov et. al, J. Am. Chem. Soc., 2004, 126, 8933-8939) and reduces the intermolecular interactions between proteins. The observed solubility enhancement is negligible for arginine and glutamic acid solutions as compared to the equimolar mixtures. In this study, we have established the molecular mechanism behind this observed synergistic effect of arginine and glutamic acid mixtures using preferential interaction theory and molecular dynamics simulations of Drosophilia Su(dx) protein (ww34). It was found that the protein solubility enhancement is related to the relative increase in the number of arginine and glutamic acid molecules around the protein in the equimolar mixtures due to additional hydrogen bonding interactions between the excipients on the surface of the protein when both excipients are present. The presence of these additional molecules around the protein leads to enhanced crowding, which suppresses the protein association. These results highlight the role of additive-additive interaction in tuning the protein-protein interactions. Furthermore, this study reports a unique behavior of additive solutions, where the presence of one additive in solution affects the concentration of another on the protein surface.

  3. Arginine: A Potent Prey Attractant to Predatory Newts in Mountain Streams

    NASA Astrophysics Data System (ADS)

    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.

  4. Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase

    PubMed Central

    Bowles, Tawnya L.; Kim, Randie; Galante, Joseph; Parsons, Colin M.; Virudachalam, Subbulakshmi; Kung, Hsing-Jien; Bold, Richard J.

    2009-01-01

    Eukaryotic cells can synthesize the non-essential amino acid arginine from aspartate and citrulline using the enzyme argininosuccinate synthetase (ASS). It has been observed that ASS is under-expressed in various types of cancers ASS, for which arginine become auxotrophic. Arginine deiminase (ADI) is a prokaryotic enzyme that metabolizes arginine to citrulline and has been found to inhibit melanoma and hepatoma cancer cells deficient of ASS. We tested the hypothesis that pancreatic cancers have low ASS expression and therefore arginine deprivation by ADI will inhibit cell growth. ASS expression was examined in 47 malignant and 20 non-neoplastic pancreatic tissues as well as a panel of human pancreatic cancer cell lines. Arginine deprivation was achieved by treatment with a recombinant form of ADI formulated with polyethylene glycol (PEG-ADI). Effects on caspase activation, cell growth and cell death were examined. Furthermore, the effect of PEG-ADI on the in vivo growth of pancreatic xenografts was examined. Eighty-seven percent of the tumors lacked ASS expression; 5 of 7 cell lines similarly lacked ASS expression. PEG-ADI specifically inhibited growth of those cell lines lacking ASS. PEG-ADI treatment induced caspase activation and induction of apoptosis. PEG-ADI was well tolerated in mice despite complete elimination of plasma arginine; tumor growth was inhibited by ∼50%. Reduced expression of ASS occurs in pancreatic cancer and predicts sensitivity to arginine deprivation achieved by PEG-ADI treatment. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of pancreatic cancer, a malignancy in which new therapy is desperately needed. PMID:18661517

  5. Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase.

    PubMed

    Bowles, Tawnya L; Kim, Randie; Galante, Joseph; Parsons, Colin M; Virudachalam, Subbulakshmi; Kung, Hsing-Jien; Bold, Richard J

    2008-10-15

    Eukaryotic cells can synthesize the non-essential amino acid arginine from aspartate and citrulline using the enzyme argininosuccinate synthetase (ASS). It has been observed that ASS is underexpressed in various types of cancers ASS, for which arginine become auxotrophic. Arginine deiminase (ADI) is a prokaryotic enzyme that metabolizes arginine to citrulline and has been found to inhibit melanoma and hepatoma cancer cells deficient of ASS. We tested the hypothesis that pancreatic cancers have low ASS expression and therefore arginine deprivation by ADI will inhibit cell growth. ASS expression was examined in 47 malignant and 20 non-neoplastic pancreatic tissues as well as a panel of human pancreatic cancer cell lines. Arginine deprivation was achieved by treatment with a recombinant form of ADI formulated with polyethylene glycol (PEG-ADI). Effects on caspase activation, cell growth and cell death were examined. Furthermore, the effect of PEG-ADI on the in vivo growth of pancreatic xenografts was examined. Eighty-seven percent of the tumors lacked ASS expression; 5 of 7 cell lines similarly lacked ASS expression. PEG-ADI specifically inhibited growth of those cell lines lacking ASS. PEG-ADI treatment induced caspase activation and induction of apoptosis. PEG-ADI was well tolerated in mice despite complete elimination of plasma arginine; tumor growth was inhibited by approximately 50%. Reduced expression of ASS occurs in pancreatic cancer and predicts sensitivity to arginine deprivation achieved by PEG-ADI treatment. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of pancreatic cancer, a malignancy in which new therapy is desperately needed.

  6. [L-arginine and male infertility].

    PubMed

    Scibona, M; Meschini, P; Capparelli, S; Pecori, C; Rossi, P; Menchini Fabris, G F

    1994-12-01

    The clinical efficacy and acceptance of L-arginina HCL was tested in 40 infertile men. All of these men had a normal number of spermatozoa (> 20 million/ml), but a decreased motility; this decreased motility was not due to infection or to immunological disorders. The treatment consisted of 80 ml of 10% L-arginine HCL administered daily per os for 6 months. L-arginine HCL showed to be able to improve the motility of spermatozoa without any side-effects.

  7. Anti-tumor activity of arginine deiminase via arginine deprivation in retinoblastoma.

    PubMed

    Kim, Jeong Hun; Kim, Jin Hyoung; Yu, Young Suk; Kim, Dong Hun; Min, Bon-Hong; Kim, Kyu-Won

    2007-12-01

    In spite of recent advances in the treatment of retinoblastoma, chemotherapy is still challenging in high-stage intraocular retinoblastoma or metastatic retinoblastoma. Here, we investigated whether arginine deprivation via arginine deiminase (ADI) could be a new anti-tumor therapy in retinoblastoma cells. Expression of argininosuccinate synthetase (ASS) was detected in human retinoblastoma tissues. Even with a high expression of ASS, ADI effectively inhibited the proliferation of retinoblastoma cells and induced retinoblastoma cell death in a dose-dependent manner. These results indicate that arginine deprivation via ADI could be another treatment option for retinoblastoma due to low ASS activity in retinoblastoma cells.

  8. l-Arginine Modifies the Exopolysaccharide Matrix and Thwarts Streptococcus mutans Outgrowth within Mixed-Species Oral Biofilms

    PubMed Central

    He, Jinzhi; Hwang, Geelsu; Liu, Yuan; Gao, Lizeng; Kilpatrick-Liverman, LaTonya; Santarpia, Peter; Zhou, Xuedong

    2016-01-01

    ABSTRACT l-Arginine, a ubiquitous amino acid in human saliva, serves as a substrate for alkali production by arginolytic bacteria. Recently, exogenous l-arginine has been shown to enhance the alkalinogenic potential of oral biofilm and destabilize its microbial community, which might help control dental caries. However, l-arginine exposure may inflict additional changes in the biofilm milieu when bacteria are growing under cariogenic conditions. Here, we investigated how exogenous l-arginine modulates biofilm development using a mixed-species model containing both cariogenic (Streptococcus mutans) and arginolytic (Streptococcus gordonii) bacteria in the presence of sucrose. We observed that 1.5% (wt/vol) l-arginine (also a clinically effective concentration) exposure suppressed the outgrowth of S. mutans, favored S. gordonii dominance, and maintained Actinomyces naeslundii growth within biofilms (versus vehicle control). In parallel, topical l-arginine treatments substantially reduced the amounts of insoluble exopolysaccharides (EPS) by >3-fold, which significantly altered the three-dimensional (3D) architecture of the biofilm. Intriguingly, l-arginine repressed S. mutans genes associated with insoluble EPS (gtfB) and bacteriocin (SMU.150) production, while spxB expression (H2O2 production) by S. gordonii increased sharply during biofilm development, which resulted in higher H2O2 levels in arginine-treated biofilms. These modifications resulted in a markedly defective EPS matrix and areas devoid of any bacterial clusters (microcolonies) on the apatitic surface, while the in situ pH values at the biofilm-apatite interface were nearly one unit higher in arginine-treated biofilms (versus the vehicle control). Our data reveal new biological properties of l-arginine that impact biofilm matrix assembly and the dynamic microbial interactions associated with pathogenic biofilm development, indicating the multiaction potency of this promising biofilm disruptor. IMPORTANCE

  9. [The synthesis of RGD peptide derivatives containing glutaric and adipic residues].

    PubMed

    Vigorov, A Iu; Demin, A M; Nizova, I A; Krasnov, V P

    2014-01-01

    A method of the synthesis of RGD peptide derivatives containing glutaric or adipic residues linked with α-amino group of L-arginine and allowing carrying out their coupling with other biomolecules and nanoparticles.

  10. 21 CFR 582.5145 - Arginine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Arginine. 582.5145 Section 582.5145 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  11. 21 CFR 582.5145 - Arginine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Arginine. 582.5145 Section 582.5145 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  12. 21 CFR 582.5145 - Arginine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Arginine. 582.5145 Section 582.5145 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  13. 21 CFR 582.5145 - Arginine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Arginine. 582.5145 Section 582.5145 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  14. 21 CFR 582.5145 - Arginine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Arginine. 582.5145 Section 582.5145 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  15. Altered brain arginine metabolism in schizophrenia

    PubMed Central

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

    2016-01-01

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

  16. Structural insight into the arginine-binding specificity of CASTOR1 in amino acid-dependent mTORC1 signaling

    PubMed Central

    Xia, Jing; Wang, Rong; Zhang, Tianlong; Ding, Jianping

    2016-01-01

    The mechanistic Target Of Rapamycin Complex 1 (mTORC1) is central to the cellular response to changes in nutrient signals such as amino acids. CASTOR1 is shown to be an arginine sensor, which plays an important role in the activation of the mTORC1 pathway. In the deficiency of arginine, CASTOR1 interacts with GATOR2, which together with GATOR1 and Rag GTPases controls the relocalization of mTORC1 to lysosomes. The binding of arginine to CASTOR1 disrupts its association with GATOR2 and hence activates the mTORC1 signaling. Here, we report the crystal structure of CASTOR1 in complex with arginine at 2.5 Å resolution. CASTOR1 comprises of four tandem ACT domains with an architecture resembling the C-terminal allosteric domains of aspartate kinases. ACT1 and ACT3 adopt the typical βαββαβ topology and function in dimerization via the conserved residues from helices α1 of ACT1 and α5 of ACT3; whereas ACT 2 and ACT4, both comprising of two non-sequential regions, assume the unusual ββαββα topology and contribute an arginine-binding pocket at the interface. The bound arginine makes a number of hydrogen-bonding interactions and extensive hydrophobic contacts with the surrounding residues of the binding pocket. The functional roles of the key residues are validated by mutagenesis and biochemical assays. Our structural and functional data together reveal the molecular basis for the arginine-binding specificity of CASTOR1 in the arginine-dependent activation of the mTORC1 signaling. PMID:27648300

  17. L-arginine pretreatment reduces intestinal mucositis as induced by 5-FU in mice.

    PubMed

    Leocádio, Paola C L; Antunes, Maísa M; Teixeira, Lílian G; Leonel, Alda J; Alvarez-Leite, Jacqueline I; Machado, Denise C C; Generoso, Simone V; Cardoso, Valbert N; Correia, Maria Isabel T D

    2015-01-01

    Beneficial effects of L-arginine on immune responses and bowel function have been reported. Mucositis is a side effect of chemotherapy treatment that affects approximately 40% of patients. This complication is characterized by inflammation that affects the gastrointestinal tract, increasing permeability and causing abdominal pain, nausea, vomiting, and diarrhea, which worsen the patient's nutritional status and increases morbimortality. The aim of this study was to evaluate the effect of pretreating with 2% L-arginine supplementation in water on mucositis as induced by 5-fluorouracil (5-FU; a single dose of 200 mg/kg body weight) in Swiss male mice. The effect of L-arginine on weight, intestinal permeability, morphology, and the histopathological score of the small intestine (from 0 to 12), oxidative stress, myeloperoxidase (MPO), and N-acetylglucosaminidase (NAG) activities were evaluated. Intestinal length improvement was observed, in addition to the partial recovery of the mucosal architecture. L-arginine attenuated the histopathological score and MPO activity. There was also an improvement in intestinal permeability, despite weight loss after 5-FU administration. In conclusion, L-arginine can positively impact intestinal mucositis by promoting partial mucosal recovery, reducing inflammation and improving intestinal permeability.

  18. Structure-based rational design and introduction of arginines on the surface of an alkaline α-amylase from Alkalimonas amylolytica for improved thermostability.

    PubMed

    Deng, Zhuangmei; Yang, Haiquan; Shin, Hyun-dong; Li, Jianghua; Liu, Long

    2014-11-01

    In this study, the thermostability of an alkaline α-amylase from Alkalimonas amylolytica was significantly improved through structure-based rational and the introduction of multiple arginines (Arg) on the protein surface. Based on an analysis of the tertiary structure, seven residues (glutamine (Gln) 166, Gln 169, serine (Ser) 270, lysine (Lys) 315, Gln 327, asparagine (Asn) 346, and Asn 423) were selected as engineering targets and individually replaced with arginine. Five of the seven single-mutated enzymes-S270R, K315R, Q327R, N346R, and N423R-showed enhanced thermostability. Multiple arginines were subsequently introduced on the protein surface, and the quintuple-mutated enzyme S270R/K315R/Q327R/N346R/N423R showed a 6.4-fold improvement in half-life at 60 and a 5.4 °C increase in melting temperature (T m) compared with those of wild-type enzyme. Concomitantly, the optimal temperature, optimal pH, and catalytic efficiency of this mutated enzyme also improved. The mutated enzyme displayed a large shift in optimal pH from 9.5 to 11.0. In addition, the optimum temperature increased from 50 to 55 °C, and the catalytic efficiency (k cat/K m) increased from 1.8 × 10(4) to 3.6 × 10(4) L/(g · min). The intramolecular interactions of mutated enzymes that contributed to increased thermostability were examined through comparative analysis of the model structures of wild-type and mutated enzymes. The thermostable mutated enzymes generated in this study have potential applications in the textile industry.

  19. Dual Role of Arginine Metabolism in Establishing Pathogenesis

    PubMed Central

    Gogoi, Mayuri; Datey, Akshay; Wilson, Keith T; Chakravortty, Dipshikha

    2015-01-01

    Arginine is an integral part of host defense when invading pathogens are encountered. The arginine metabolite nitric oxide (NO) confers antimicrobial properties, whereas the metabolite ornithine is utilized for polyamine synthesis. Polyamines are crucial to tissue repair and anti-inflammatory responses. iNOS/arginase balance can determine Th1/Th2 response. Furthermore, the host arginine pool and its metabolites are utilized as energy sources by various pathogens. Apart from its role as an immune modulator, recent studies have also highlighted the therapeutic effects of arginine. This article sheds light upon the roles of arginine metabolism during pathological conditions and its therapeutic potential. PMID:26610300

  20. Dual role of arginine metabolism in establishing pathogenesis.

    PubMed

    Gogoi, Mayuri; Datey, Akshay; Wilson, Keith T; Chakravortty, Dipshikha

    2016-02-01

    Arginine is an integral part of host defense when invading pathogens are encountered. The arginine metabolite nitric oxide (NO) confers antimicrobial properties, whereas the metabolite ornithine is utilized for polyamine synthesis. Polyamines are crucial to tissue repair and anti-inflammatory responses. iNOS/arginase balance can determine Th1/Th2 response. Furthermore, the host arginine pool and its metabolites are utilized as energy sources by various pathogens. Apart from its role as an immune modulator, recent studies have also highlighted the therapeutic effects of arginine. This article sheds light upon the roles of arginine metabolism during pathological conditions and its therapeutic potential.

  1. Arginine Metabolism in Myeloid Cells Shapes Innate and Adaptive Immunity

    PubMed Central

    Rodriguez, Paulo C.; Ochoa, Augusto C.; Al-Khami, Amir A.

    2017-01-01

    Arginine metabolism has been a key catabolic and anabolic process throughout the evolution of the immune response. Accruing evidence indicates that arginine-catabolizing enzymes, mainly nitric oxide synthases and arginases, are closely integrated with the control of immune response under physiological and pathological conditions. Myeloid cells are major players that exploit the regulators of arginine metabolism to mediate diverse, although often opposing, immunological and functional consequences. In this article, we focus on the importance of arginine catabolism by myeloid cells in regulating innate and adaptive immunity. Revisiting this matter could result in novel therapeutic approaches by which the immunoregulatory nodes instructed by arginine metabolism can be targeted. PMID:28223985

  2. Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2*

    PubMed Central

    Hadjikyriacou, Andrea; Yang, Yanzhong; Espejo, Alexsandra; Bedford, Mark T.; Clarke, Steven G.

    2015-01-01

    Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides. PMID:25979344

  3. Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2.

    PubMed

    Hadjikyriacou, Andrea; Yang, Yanzhong; Espejo, Alexsandra; Bedford, Mark T; Clarke, Steven G

    2015-07-03

    Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.

  4. Reengineering of the feedback-inhibition enzyme N-acetyl-L-glutamate kinase to enhance L-arginine production in Corynebacterium crenatum.

    PubMed

    Zhang, Jingjing; Xu, Meijuan; Ge, Xiaoxun; Zhang, Xian; Yang, Taowei; Xu, Zhenghong; Rao, Zhiming

    2017-02-01

    N-acetyl-L-glutamate kinase (NAGK) catalyzes the second step of L-arginine biosynthesis and is inhibited by L-arginine in Corynebacterium crenatum. To ascertain the basis for the arginine sensitivity of CcNAGK, residue E19 which located at the entrance of the Arginine-ring was subjected to site-saturated mutagenesis and we successfully illustrated the inhibition-resistant mechanism. Typically, the E19Y mutant displayed the greatest deregulation of L-arginine feedback inhibition. An equally important strategy is to improve the catalytic activity and thermostability of CcNAGK. For further strain improvement, we used site-directed mutagenesis to identify mutations that improve CcNAGK. Results identified variants I74V, F91H and K234T display higher specific activity and thermostability. The L-arginine yield and productivity of the recombinant strain C. crenatum SYPA-EH3 (which possesses a combination of all four mutant sites, E19Y/I74V/F91H/K234T) reached 61.2 and 0.638 g/L/h, respectively, after 96 h in 5 L bioreactor fermentation, an increase of approximately 41.8% compared with the initial strain.

  5. Protein Arginine Methyltransferase 6 Enhances Polyglutamine-Expanded Androgen Receptor Function and Toxicity in Spinal and Bulbar Muscular Atrophy

    PubMed Central

    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

    Summary 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. PMID:25569348

  6. Evolution of phosphagen kinase V. cDNA-derived amino acid sequences of two molluscan arginine kinases from the chiton Liolophura japonica and the turbanshell Battilus cornutus.

    PubMed

    Suzuki, T; Ban, T; Furukohri, T

    1997-06-20

    The cDNAs of arginine kinases from the chiton Liolophura japonica (Polyplacophora) and the turbanshell Battilus cornutus (Gastropoda) were amplified by polymerase chain reaction (PCR), and the complete nucleotide sequences of 1669 and 1624 bp, respectively, were determined. The open reading frame for Liolophura arginine kinase is 1050 nucleotides in length and encodes a protein with 349 amino acid residues, and that for Battilus is 1077 nucleotides and 358 residues. The validity of the cDNA-derived amino acid sequence was supported by chemical sequencing of internal tryptic peptides. The molecular masses were calculated to be 39,057 and 39,795 Da, respectively. The amino acid sequence of Liolophura arginine kinase showed 65-68% identity with those of Battilus and Nordotis (abalone) arginine kinases, and the homology between Battilus and Nordotis was 79%. Molluscan arginine kinases also show lower, but significant homology (38-43%) with rabbit creatine kinase. The sequences of arginine kinases could be used as a molecular clock to elucidate the phylogeny of Mollusca, one of the most diverse animal phyla.

  7. Influence of carbohydrate starvation and arginine on culturability and amino acid utilization of lactococcus lactis subsp. lactis.

    PubMed

    Stuart, M R; Chou, L S; Weimer, B C

    1999-02-01

    Two strains of Lactococcus lactis subsp. lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L. lactis ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for L. lactis 11454. Without lactose or arginine, L. lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However, L. lactis 11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of L. lactis during carbohydrate starvation.

  8. The effects of fuel composition and ammonium sulfate addition on PCDD, PCDF, PCN and PCB concentrations during the combustion of biomass and paper production residuals.

    PubMed

    Lundin, Lisa; Jansson, Stina

    2014-01-01

    The use of waste wood as an energy carrier has increased during the last decade. However, the higher levels of alkali metals and chlorine in waste wood compared to virgin biomass can promote the formation of deposits and organic pollutants. Here, the effect of fuel composition and the inhibitory effects of ammonium sulfate, (NH4)2SO4, on the concentrations of persistent organic pollutants (POPs) in the flue gas of a lab-scale combustor was investigated. Ammonium sulfate is often used as a corrosion-preventing additive and may also inhibit formation of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). In addition to PCDDs and PCDFs, polychlorinated naphthalenes (PCN) and biphenyls (PCB) were also analyzed. It was found that the flue gas composition changed dramatically when (NH4)2SO4 was added: CO, SO2, and NH3 levels increased, while those of HCl decreased to almost zero. However, the additive's effects on POP formation were less pronounced. When (NH4)2SO4 was added to give an S:Cl ratio of 3, only the PCDF concentration was reduced, indicating that this ratio was not sufficient to achieve a general reduction in POP emissions. Conversely, at an S:Cl ratio of 6, significant reductions in the WHO-TEQ value and the PCDD and PCDF contents of the flue gas were observed. The effect on the PCDF concentration was especially pronounced. PCN formation seemed to be promoted by the elevated CO concentrations caused by adding (NH4)2SO4.

  9. Arginine dipeptides affect insulin aggregation in a pH- and ionic strength-dependent manner.

    PubMed

    Nuhu, Mariam M; Curtis, Robin

    2015-03-01

    Solutions containing arginine or mixtures of arginine and other amino acids are commonly used for protein liquid formulations to overcome problems such as high viscosities, aggregation, and phase separation. The aim of this work is to examine whether the stabilizing properties of arginine can be improved by incorporating the amino acid into a dipeptide. A series of arginine-containing dipeptides have been tested for their ability to suppress insulin aggregation over a range of pH and ionic strength. The aggregation is monitored at room temperature using a combination of turbidimetry and light scattering for solutions at pH 5.5 or 3.7, whereas thermal-induced aggregation is measured at pH 7.5. In addition, intrinsic fluorescence has been used to quantify additive binding to insulin. The dipeptide diArg is the most effective additive in solutions at pH 5.5 and 3.7, whereas the dipeptide Arg-Phe almost completely eliminates thermally-induced aggregation of insulin at pH 7.5 up to temperature of 90°C. Insulin has been chosen as a model system because the molecular forces controlling its aggregation are well known. From this understanding, we are able to provide a molecular basis for how the various dipeptides affect insulin aggregation.

  10. Interaction of arginine oligomer with model membrane

    SciTech Connect

    Yi, Dandan . E-mail: yi_dandan@yahoo.com.cn; Guoming, Li; Gao, Li; Wei, Liang

    2007-08-10

    Short oligomers of arginine (R8) have been shown to cross readily a variety of biological barriers. A hypothesis was put forward that inverted micelles form in biological membranes in the presence of arginine oligomer peptides, facilitating their transfer through the membranes. In order to define the role of peptide-lipid interaction in this mechanism, we prepared liposomes as the model membrane to study the ability of R8 inducing calcein release from liposomes, the fusion of liposomes, R8 binding to liposomes and membrane disturbing activity of the bound R8. The results show that R8 binding to liposome membrane depends on lipid compositions, negative surface charge density and interior water phase pH values of liposomes. R8 has no activity to induce the leakage of calcein from liposomes or improve liposome fusion. R8 does not permeabilize through the membrane spontaneously. These peptides delivering drugs through membranes may depend on receptors and energy.

  11. Direct observation of an anisotropic in-plane residual stress induced by B addition as an origin of high magnetic anisotropy field of Ru/FeCoB film

    SciTech Connect

    Hirata, Ken-ichiro; Gomi, Shunsuke; Mashiko, Yasuhiro; Nakagawa, Shigeki

    2010-05-15

    Although boron-free FeCo films prepared on a Ru underlayer exhibits isotropic in-plane magnetic property, boron added FeCoB films prepared on Ru underlayer revealed large in-plane magnetic anisotropy with a high anisotropy field of 500 Oe. The effect of boron addition on the in-plane anisotropic residual stress in FeCoB film was investigated using sin{sup 2} {psi} method of x-ray diffraction analysis. Large isotropic compressive stress was observed in Ru/FeCo film. In contrast, anisotropic in-plane residual stress was observed in Ru/FeCoB film. The compressive stress along the easy axis of Ru/FeCoB film is released more than that along the hard axis. Such anisotropic residual stress is regarded as an origin of the in-plane magnetic anisotropy through inverse magnetostriction effect. Owing to the configuration of the facing targets sputtering system, boron atoms are sputtered and deposited anisotropically, and so they penetrate FeCo crystals and release the compressive stress along the incidence direction.

  12. Effects of arginine on multimodal anion exchange chromatography.

    PubMed

    Hirano, Atsushi; Arakawa, Tsutomu; Kameda, Tomoshi

    2015-12-01

    The effects of arginine on binding and elution properties of a multimodal anion exchanger, Capto adhere, were examined using bovine serum albumin (BSA) and a monoclonal antibody against interleukin-8 (mAb-IL8). Negatively charged BSA was bound to the positively charged Capto adhere and was readily eluted from the column with a stepwise or gradient elution using 1M NaCl at pH 7.0. For heat-treated BSA, small oligomers and remaining monomers were also eluted using a NaCl gradient, whereas larger oligomers required arginine for effective elution. The positively charged mAb-IL8 was bound to Capto adhere at pH 7.0. Arginine was also more effective for elution of the bound mAb-IL8 than was NaCl. The results imply that arginine interacts with the positively charged Capto adhere. The mechanism underlying the interactions of arginine with Capto adhere was examined by calculating the binding free energy between an arginine molecule and a Capto adhere ligand in water through molecular dynamics simulations. The overall affinity of arginine for Capto adhere is attributed to the hydrophobic and π-π interactions between an arginine side chain and the aromatic moiety of the ligand as well as hydrogen bonding between arginine and the ligand hydroxyl group, which may account for the characteristics of protein elution using arginine.

  13. Molecular characterization of arginine deiminase pathway in Laribacter hongkongensis and unique regulation of arginine catabolism and anabolism by multiple environmental stresses.

    PubMed

    Xiong, Lifeng; Teng, Jade L L; Watt, Rory M; Liu, Cuihua; Lau, Susanna K P; Woo, Patrick C Y

    2015-11-01

    The betaproteobacterium Laribacter hongkongensis is associated with invasive bacteremic infections and gastroenteritis. Its genome contains two adjacent arc gene cassettes (arc1 and arc2) under independent transcriptional control, which are essential for acid resistance. Laribacter hongkongensis also encodes duplicate copies of the argA and argB genes from the arginine biosynthesis pathway. We show that arginine enhances the transcription of arcA2 but suppresses arcA1 expression. We demonstrate that ArgR acts as a transcriptional regulator of the two arc operons through binding to ARG operator sites (ARG boxes). Upon temperature shift from 20°C to 37°C, arcA1 transcription is upregulated while arcA2, argA2, argB2 and argG are downregulated. The transcription of arcA1 and arcA2 are augmented under anaerobic and acidic conditions. The transcription levels of argA1, argA2, argB1, argB2 and argG are significantly increased under anaerobic and acidic conditions but are repressed by the addition of arginine. Deletion of argR significantly decreases bacterial survival in macrophages, while expression of both arc operons, argR and all five of the anabolic arg genes increases 8 h post-infection. Our results show that arginine catabolism in L. hongkongensis is finely regulated by controlling the transcription of two arc operons, whereas arginine anabolism is controlled by two copies of argA and argB.

  14. Exogenous L-arginine and HDL can alter LDL and ox-LDL-mediated platelet activation: using platelet P-selectin receptor numbers.

    PubMed

    Sener, Azize; Enc, Elif; Ozsavci, Derya; Vanizor-Kural, Birgul; Yanikkaya-Demirel, Gulderen; Oba, Rabia; Uras, Fikriye; Demir, Muzaffer

    2011-01-01

    The aim of this study is to investigate the effects of exogenous L-arginine and HDL on LDL and oxidized LDL (ox-LDL)-mediated platelet activation. Adenosine diphosphate (ADP)-activated platelets have been incubated with lipoproteins with or without L-arginine. P-selectin receptor numbers per platelet have been measured by flow cytometry. After incubation with only L-arginine (without lipoproteins), platelet nitric oxide (NO) levels and P-selectin receptor numbers significantly increased compared to the controls (P < .05). After incubation with LDL or ox-LDL, receptor numbers of P-selectin significantly increased (P < .001). However, P-selectin receptor numbers in platelets treated with L-arginine + LDL or L-arginine + ox-LDL decreased compared to the levels in platelets treated with only LDL or ox-LDL (P < .01, P < .001, respectively). Addition of HDL to L-arginine + ox-LDL caused significant reduction in P-selectin receptor numbers as in the control values (P < .001).We have concluded that L-arginine causes enhanced platelet NO levels and blocks the effects of LDL or ox-LDL on platelet P-selectin receptor numbers and HDL also strengthens this effect of L-arginine.

  15. The role of basic residues in the adsorption of blood proteins onto the graphene surface

    NASA Astrophysics Data System (ADS)

    Gu, Zonglin; Yang, Zaixing; Wang, Lingle; Zhou, Hong; Jimenez-Cruz, Camilo A.; Zhou, Ruhong

    2015-06-01

    With its many unique properties, graphene has shown great potential in various biomedical applications, while its biocompatibility has also attracted growing concerns. Previous studies have shown that the formation of protein-graphene corona could effectively reduce its cytotoxicity; however, the underlying molecular mechanism remains not well-understood. Herein, we use extensive molecular dynamics simulations to demonstrate that blood proteins such as bovine fibrinogen (BFG) can absorb onto the graphene surface quickly and tightly to form a corona complex. Aromatic residues contributed significantly during this adsorption process due to the strong π-π stacking interactions between their aromatic rings and the graphene sp2-carbons. Somewhat surprisingly, basic residues like arginine, also played an equally or even stronger role during this process. The strong dispersion interactions between the sidechains of these solvent-exposed basic residues and the graphene surface provide the driving force for a tight binding of these basic residues. To the best of our knowledge, this is the first study with blood proteins to show that, in addition to the aromatic residues, the basic residues also play an important role in the formation of protein-graphene corona complexes.

  16. Addition of microbially-treated sugar beet residue and a native bacterium increases structural stability in heavy metal-contaminated Mediterranean soils.

    PubMed

    Carrasco, L; Caravaca, F; Azcón, R; Kohler, J; Roldán, A

    2009-10-15

    A mesocosm experiment was conducted to investigate the effect of the addition of Aspergillus niger-treated sugar beet waste, in the presence of rock phosphate, and inoculation with a native, metal-tolerant bacterium, Bacillus thuringiensis, on the stabilisation of soil aggregates of two mine tailings, with differing pH values, from a semiarid Mediterranean area and on the stimulation of growth of Piptatherum miliaceum. Bacterium combined with organic amendment enhanced structural stability (38% in acidic soil and 106% in neutral soil compared with their corresponding controls). Only the organic amendment increased pH, electrical conductivity, water-soluble C, water-soluble carbohydrates and plant growth, in both soils. While in neutral soil both organic amendment and bacterium increased dehydrogenase activity, only organic amendment had a significant effect in acidic soil. This study demonstrates that the use of P. miliaceum in combination with organic amendment and bacterium is a suitable tool for the stabilisation of the soil structure of degraded mine tailings, although its effectiveness is dependent on soil pH.

  17. The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase.

    PubMed

    Bachmann, Julie; Bauer, Brigitte; Zwicker, Klaus; Ludwig, Bernd; Anderka, Oliver

    2006-11-01

    The Rieske [2Fe-2S] protein (ISP) is an essential subunit of cytochrome bc(1) complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram-negative soil bacterium, is inserted into the cytoplasmic membrane via the twin-arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin-arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h-region compared with generic Tat signals and the absence of an otherwise well-conserved '+5'-consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and/or cytochrome bc(1) stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe-2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.

  18. Carbamoyl Phosphate Synthetase Subunit MoCpa2 Affects Development and Pathogenicity by Modulating Arginine Biosynthesis in Magnaporthe oryzae

    PubMed Central

    Liu, Xinyu; Cai, Yongchao; Zhang, Xi; Zhang, Haifeng; Zheng, Xiaobo; Zhang, Zhengguang

    2016-01-01

    Arginine is a semi-essential amino acid that affects physiological and biochemical functions. The CPA2 gene in yeast encodes a large subunit of arginine-specific carbamoyl phosphate synthetase (CPS) and is involved in arginine biosynthesis. Here, an ortholog of yeast CPA2 was identified in the rice blast fungus Magnaporthe oryzae, and was named MoCPA2. MoCpa2 is an 1180-amino acid protein which contains an ATP grasp domain and two CPSase domains. Targeted deletion of MoCPA2 supported its role in de novo arginine biosynthesis in M. oryzae as mutant phenotypes were complemented by arginine but not ornithine. The ΔMocpa2 mutant exhibited defects in asexual development and pathogenicity but not appressorium formation. Further examination revealed that the invasive hyphae of the ΔMocpa2 mutant were restricted mainly to the primary infected cells. In addition, the ΔMocpa2 mutant was unable to induce a plant defense response and had the ability to scavenge ROS during pathogen-plant interactions. Structure analysis revealed that the ATP grasp domain and each CPS domain were indispensable for the proper localization and full function of MoCpa2. In summary, our results indicate that MoCpa2 plays an important role in arginine biosynthesis, and affects growth, conidiogenesis, and pathogenicity. These results suggest that research into metabolism and processes that mediate amino acid synthesis are valuable for understanding M. oryzae pathogenesis. PMID:28066349

  19. Arginine methylation regulates antibody responses through modulating cell division and isotype switching in B cells.

    PubMed

    Hata, Kikumi; Mizuguchi, Junichiro

    2013-03-01

    Protein arginine methylation plays crucial roles, including signal transduction, transcriptional control, cell proliferation and/or differentiation. B cells undergo clonal division, isotype switching and differentiate into antibody forming cells following stimulation with Toll-like receptor-ligand, lipopolysaccharide (LPS) and T cell-derived signals, including CD40-ligand (CD40-L) and interleukin 4 (IL-4). Whether protein arginine methylation affects B cell division and/or isotype switching to IgG1 in response to LPS, IL-4, and CD40-L was examined using the arginine methyl transferase inhibitor adenosine-2',3'-dialdehyde (AdOx). Addition of AdOx substantially reduced the number of division cycles of stimulated B cells, whereas cell viability remained intact. Upon stimulation with LPS/IL-4/CD40-L, the proportion of surface IgG1 positive cells in each division cycle was slightly diminished by AdOx. However, the degree of expression of γ1 germ line transcript and activation-induced cytidine deaminase (AID) in response to LPS/IL-4/CD40-L were unaffected by addition of AdOx, suggesting that AdOx influences class switch recombination independent of AID expression through transcriptional control. Taken together, arginine methylation appears to be involved in B cell isotype switching, as well as in clonal expansion of B cells in response to LPS/IL-4/CD40-L.

  20. Metabolic engineering of Corynebacterium glutamicum for L-arginine production.

    PubMed

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

    2014-08-05

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

  1. Characterization of the Effects of Arginine and Glucose on Glucagon and Insulin Release from the Perfused Rat Pancreas

    PubMed Central

    Gerich, John E.; Charles, M. Arthur; Grodsky, Gerold M.

    1974-01-01

    To characterize the mechanisms by which arginine and glucose affect pancreatic alpha and beta cell function, the effects of these agents over their full dose response, both alone and in various combinations, were studied using the perfused rat pancreas. Arginine (0-38 mM), in the absence of glucose, stimulated biphasic glucagon (IRG) secretion (Km≃3-4 mM) at concentrations less than 1 mM and caused nonphasic insulin (IRI) release (Km≃12-13 mM) but only at concentrations greater than 6 mM. Glucose (0-27.5 mM) alone stimulated biphasic IRI release (Km≃9-10 mM) at concentrations in excess of 5.5 mM and caused nonphasic inhibition of IRG secretion (Kt≃5-6 mM) at concentrations as low as 4.1 mM. These results demonstrate fundamental differences in pancreatic alpha and beta cell secretory patterns in response to glucose and arginine and suggest that glucagon secretion is more sensitive to the effect of both glucose and arginine. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated biphasic IRG and IRI release: IRG responses were diminished and IRI responses were enhanced compared with those seen with arginine in the absence of glucose. Glucose (0-27.5 mM) in the presence of 3.2 or 19.2 mM arginine caused similar inhibition of IRG secretion (Km≃5-6 mM) and stimulation of IRI release (Km≃9-10 mM) as that seen with glucose alone, although greater IRG and IRI release occurred. This augmentation of IRI secretion was greater than that expected from mere additive effects of glucose and arginine. Classical Lineweaver-Burk analysis of these results indicates that glucose is a non-competitive inhibitor arginine-stimulated glucagon secretion and suggests that glucose and arginine affect pancreatic alpha and beta cell function via different mechanisms. In addition, comparison of simultaneous insulin and glucagon secretion patterns under various conditions suggests that endogenous insulin per se has little or no direct effect on IRG secretion

  2. A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

    PubMed Central

    Debler, Erik W.; Jain, Kanishk; Warmack, Rebeccah A.; Feng, You; Clarke, Steven G.; Blobel, Günter; Stavropoulos, Pete

    2016-01-01

    Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs. PMID:26858449

  3. The electrostatic driving force for nucleophilic catalysis in L-arginine deiminase: a combined experimental and theoretical study.

    PubMed

    Li, Ling; Li, Zhimin; Wang, Canhui; Xu, Dingguo; Mariano, Patrick S; Guo, Hua; Dunaway-Mariano, Debra

    2008-04-22

    L-arginine deiminase (ADI) catalyzes the hydrolysis of L-arginine to form L-citrulline and ammonia via two partial reactions. A working model of the ADI catalytic mechanism assumes nucleophilic catalysis by a stringently conserved active site Cys and general acid-general base catalysis by a stringently conserved active site His. Accordingly, in the first partial reaction, the Cys attacks the substrate guanidino C zeta atom to form a tetrahedral covalent adduct, which is protonated by the His at the departing ammonia group to facilitate the formation of the Cys- S-alkylthiouronium intermediate. In the second partial reaction, the His activates a water molecule for nucleophilic addition at the thiouronium C zeta atom to form the second tetrahedral intermediate, which eliminates the Cys in formation of the L-citrulline product. The absence of a basic residue near the Cys thiol suggested that the electrostatic environment of the Cys thiol, in the enzyme-substrate complex, stabilizes the Cys thiolate anion. The studies described in this paper explore the mechanism of stabilization of the Cys thiolate. First, the log(k(cat)/K(m)) and log k(cat) pH rate profiles were measured for several structurally divergent ADIs to establish the pH range for ADI catalysis. All ADIs were optimally active at pH 5, which suggested that the Cys pKa is strongly perturbed by the prevailing electrostatics of the ADI active site. The p K a of the Bacillus cereus ADI (BcADI) was determined by UV-pH titration to be 9.6. In contrast, the pKa determined by iodoacetamide Cys alkylation is 6.9. These results suggest that the negative electrostatic field from the two opposing Asp carboxylates perturbs the Cys pKa upward in the apoenzyme and that the binding of the iodoacetamide (a truncated analogue of the citrulline product) between the Cys thiol and the two Asp carboxylates shields the Cys thiol, thereby reducing its pKa. It is hypothesized that the bound positively charged guanidinium group of the

  4. Leishmania metacaspase: an arginine-specific peptidase.

    PubMed

    Martin, Ricardo; Gonzalez, Iveth; Fasel, Nicolas

    2014-01-01

    The purpose of this chapter is to give insights into metacaspase of Leishmania protozoan parasites as arginine-specific cysteine peptidase. The physiological role of metacaspase in Leishmania is still a matter of debate, whereas its peptidase enzymatic activity has been well characterized. Among the different possible expression systems, metacaspase-deficient yeast cells (Δyca1) have been instrumental in studying the activity of Leishmania major metacaspase (LmjMCA). Here, we describe techniques for purification of LmjMCA and its activity measurement, providing a platform for further identification of LmjMCA substrates.

  5. [First fixed dose combination perindopril arginine-indapamide-amlodipine: new approach in combination therapy in hypertension].

    PubMed

    Jr, Jiří Widimský

    2014-09-01

    Use of fixed combination of antihypertensive drugs clearly improves compliance to the pharmacological therapy, control of hypertension and prognosis. Based on the current guidelines triple antihypertensive therapy with RAS-blocker, calcium channel blocker (CCB) and diuretic represents the standard and best option. The article introduces first and innovative fixed triple combination of perindopril arginine + indapamide + amlodipine (Triplixam®). This type of therapy is suitable for patients already treated with free combinations of three antihypertensive drugs or in those hypertensives with uncontrolled hypertension on two antihypertensive molecules (approx. 60% of all hypertensive population). Fixed combination of perindopril arginine + indapamid + amlodipin is indicated also in severe hypertension (approx. 30% of pts). Large clinical data from various morbidity-mortality studies related to each of these substances are discussed as well as basic pharmacological characteristics. Based on the results from ADVANCE-CCB study combination of perindopril arginine + indapamide + CCB decreases total mortality in hypertension by 28%. Another discussed study-PIANIST confirmed significant antihypertensive effect of Triplixam® on large sample of patients with various stages of hypertension. Triplixam® in addition to that has very good tolerance with low side effects profile, flexibility of the dosages and large body of evidence of positive impact on prognosis of hypertensive patients. Use of Triplixam® may improve control of hypertension in the Czech Republic.Key words: amlodipine - fixed combination - hypertension - indapamide - perindopril arginine - therapy.

  6. Overlapping transport and chaperone-binding functions within a bacterial twin-arginine signal peptide.

    PubMed

    Grahl, Sabine; Maillard, Julien; Spronk, Chris A E M; Vuister, Geerten W; Sargent, Frank

    2012-03-01

    The twin-arginine translocation (Tat) pathway is a protein targeting system present in many prokaryotes. The physiological role of the Tat pathway is the transmembrane translocation of fully-folded proteins, which are targeted by N-terminal signal peptides bearing conserved SRRxFLK 'twin-arginine' amino acid motifs. In Escherichia coli the majority of Tat targeted proteins bind redox cofactors and it is important that only mature, cofactor-loaded precursors are presented for export. Cellular processes have been unearthed that sequence these events, for example the signal peptide of the periplasmic nitrate reductase (NapA) is bound by a cytoplasmic chaperone (NapD) that is thought to regulate assembly and export of the enzyme. In this work, genetic, biophysical and structural approaches were taken to dissect the interaction between NapD and the NapA signal peptide. A NapD binding epitope was identified towards the N-terminus of the signal peptide, which overlapped significantly with the twin-arginine targeting motif. NMR spectroscopy revealed that the signal peptide adopted a α-helical conformation when bound by NapD, and substitution of single residues within the NapA signal peptide was sufficient to disrupt the interaction. This work provides an increased level of understanding of signal peptide function on the bacterial Tat pathway.

  7. KAP1 is a Novel Substrate for the Arginine Methyltransferase PRMT5.

    PubMed

    di Caprio, Roberta; Ciano, Michela; Montano, Giorgia; Costanzo, Paola; Cesaro, Elena

    2015-01-09

    KRAB-associated protein 1 (KAP1), the transcriptional corepressor of Kruppel-associated box zinc finger proteins (KRAB-ZFPs), is subjected to multiple post-translational modifications that are involved in fine-tuning of the multiple biological functions of KAP1. In previous papers, we analyzed the KAP1-dependent molecular mechanism of transcriptional repression mediated by ZNF224, a member of the KRAB-ZFP family, and identified the protein arginine methyltransferase PRMT5 as a component of the ZNF224 repression complex. We demonstrated that PRMT5-mediated histone arginine methylation is required to elicit ZNF224 transcriptional repression. In this study, we show that KAP1 interacts with PRMT5 and is a novel substrate for PRMT5 methylation. Also, we present evidence that the methylation of KAP1 arginine residues regulate the KAP1-ZNF224 interaction, thus suggesting that this KAP1 post-translational modification could actively contribute to the regulation of ZNF224-mediated repression.

  8. KAP1 is a Novel Substrate for the Arginine Methyltransferase PRMT5

    PubMed Central

    di Caprio, Roberta; Ciano, Michela; Montano, Giorgia; Costanzo, Paola; Cesaro, Elena

    2015-01-01

    KRAB-associated protein 1 (KAP1), the transcriptional corepressor of Kruppel-associated box zinc finger proteins (KRAB-ZFPs), is subjected to multiple post-translational modifications that are involved in fine-tuning of the multiple biological functions of KAP1. In previous papers, we analyzed the KAP1-dependent molecular mechanism of transcriptional repression mediated by ZNF224, a member of the KRAB-ZFP family, and identified the protein arginine methyltransferase PRMT5 as a component of the ZNF224 repression complex. We demonstrated that PRMT5-mediated histone arginine methylation is required to elicit ZNF224 transcriptional repression. In this study, we show that KAP1 interacts with PRMT5 and is a novel substrate for PRMT5 methylation. Also, we present evidence that the methylation of KAP1 arginine residues regulate the KAP1-ZNF224 interaction, thus suggesting that this KAP1 post-translational modification could actively contribute to the regulation of ZNF224-mediated repression. PMID:25585209

  9. Arginine methylation promotes translation repression activity of eIF4G-binding protein, Scd6

    PubMed Central

    Poornima, Gopalakrishna; Shah, Shanaya; Vignesh, Venkadasubramanian; Parker, Roy; Rajyaguru, Purusharth I.

    2016-01-01

    Regulation of translation plays a critical role in determining mRNA fate. A new role was recently reported for a subset of RGG-motif proteins in repressing translation initiation by binding eIF4G1. However the signaling mechanism(s) that leads to spatial and temporal regulation of repression activity of RGG-motif proteins remains unknown. Here we report the role of arginine methylation in regulation of repression activity of Scd6, a conserved RGG-motif protein. We demonstrate that Scd6 gets arginine methylated at its RGG-motif and Hmt1 plays an important role in its methylation. We identify specific methylated arginine residues in the Scd6 RGG-motif in vivo. We provide evidence that methylation augments Scd6 repression activity. Arginine methylation defective (AMD) mutant of Scd6 rescues the growth defect caused by overexpression of Scd6, a feature of translation repressors in general. Live-cell imaging of the AMD mutant revealed that it is defective in inducing formation of stress granules. Live-cell imaging and pull-down results indicate that it fails to bind eIF4G1 efficiently. Consistent with these results, a strain lacking Hmt1 is also defective in Scd6-eIF4G1 interaction. Our results establish that arginine methylation augments Scd6 repression activity by promoting eIF4G1-binding. We propose that arginine methylation of translation repressors with RGG-motif could be a general modulator of their repression activity. PMID:27613419

  10. Deceleration of arginine kinase refolding by induced helical structures.

    PubMed

    Li, Hai-Long; Zhou, Sheng-Mei; Park, Daeui; Jeong, Hyoung Oh; Chung, Hae Young; Yang, Jun-Mo; Meng, Fan-Guo; Hu, Wei-Jiang

    2012-04-01

    Arginine kinase (AK) is a key metabolic enzyme for keeping energy balance in invertebrates. Therefore, regulation of the enzymatic activity and the folding studies of AK from the various invertebrates have been the focus of investigation. We studied the effects of helical structures by using hexafluoroisopropanol (HFIP) on AK folding. Folding kinetic studies showed that the folding rates of the urea-denatured AKs were significantly decelerated after being induced in various concentrations of HFIP. AK lost its activity completely at concentrations greater than 60%. The results indicated that the HFIP-induced helical structures in the denatured state play a negative role in protein folding, and the helical structures induced in 5% (v/v) HFIP act as the most effective barrier against AK taking its native structure. The computational docking simulations (binding energies for -2.19 kcal/mol for AutoDock4.2 and -20.47 kcal/mol for Dock6.3) suggested that HFIP interacts with the several important residues that are predicted by both programs. The excessively pre-organized helical structures not only hampered the folding process, but also ultimately brought about changes in the three-dimensional conformation and biological function of AK.

  11. Depletion of arginine by recombinant arginine deiminase induces nNOS-activated neurotoxicity in neuroblastoma cells.

    PubMed

    Lin, Shan-Erh; Wu, Fe-Lin Lin; Wei, Ming-Feng; Shen, Li-Jiuan

    2014-01-01

    The abnormal regulation of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) is associated with neurodegenerative disorders. Recombinant arginine deiminase (rADI) is a selective NO modulator of iNOS and eNOS in endothelial cells, and it also exhibits neuroprotective activity in an iNOS-induced neuron-microglia coculture system. However, the effect of rADI on nNOS remains unknown. Addressing this issue is important for evaluating the potential application of rADI in neurodegenerative diseases. SH-SY5Y cells were treated with N-methyl-D-aspartic acid (NMDA) to activate nNOS. NMDA increased NO production by 39.7 ± 3.9% via nNOS under arginine-containing conditions, but there was no significant increase in both arginine-free and rADI pretreated arginine-containing (citrulline) buffer. Subsequently, neither NMDA nor rADI alone caused cytotoxicity, whereas cotreatment with NMDA and rADI resulted in dissipation of the cell mitochondrial membrane potential and decreased cell viability. The mechanism of rADI cytotoxicity in the presence of NMDA is caused by the inhibition of NO production via nNOS mediated by the NMDA receptor, which was abolished when extracellular arginine was absent, even in the presence of citrulline. rADI not only reduced NO production but also caused cellular toxicity in nNOS-activated SH-SY5Y cells, suggesting a dual role for rADI in NOS-mediated neurotoxicity.

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

    PubMed

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

    2017-02-01

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

  13. Overview of existing European food consumption databases: critical aspects in relation to their use for the assessment of dietary exposure to additives, flavourings and residues of food contact materials.

    PubMed

    Le Donne, Cinzia; Piccinelli, Raffaela; Sette, Stefania; Leclercq, Catherine

    2011-03-01

    A critical analysis of existing food consumption databases was performed with particular regard for their current and potential use for the assessment of dietary exposure to additives, flavourings and residues of food contact materials. Within the European Food Consumption Validation project (EFCOVAL), a questionnaire on critical aspects of such datasets was developed and administered to researchers responsible for the collection/analysis of national food consumption data in European countries. Information collected was complemented through a review of the literature and of grey publications in order to provide an inventory of the main food consumption surveys performed in Europe from 1994 to 2007, for a total of 23 countries and 37 surveys. It appeared that existing European food consumption surveys have as a main objective the assessment of nutrient intake in the population. On the other hand, most of the databases were shown to be used also for the purpose of dietary exposure assessment.

  14. The Kruppel-like zinc finger protein ZNF224 recruits the arginine methyltransferase PRMT5 on the transcriptional repressor complex of the aldolase A gene.

    PubMed

    Cesaro, Elena; De Cegli, Rossella; Medugno, Lina; Florio, Francesca; Grosso, Michela; Lupo, Angelo; Izzo, Paola; Costanzo, Paola

    2009-11-20

    Gene transcription in eukaryotes is modulated by the coordinated recruitment of specific transcription factors and chromatin-modulating proteins. Indeed, gene activation and/or repression is/are regulated by histone methylation status at specific arginine or lysine residues. In this work, by co-immunoprecipitation experiments, we demonstrate that PRMT5, a type II protein arginine methyltransferase that monomethylates and symmetrically dimethylates arginine residues, is physically associated with the Kruppel-like associated box-zinc finger protein ZNF224, the aldolase A gene repressor. Moreover, chromatin immunoprecipitation assays show that PRMT5 is recruited to the L-type aldolase A promoter and that methylation of the nucleosomes that surround the L-type promoter region occurs in vivo on the arginine 3 of histone H4. Consistent with its association to the ZNF224 repressor complex, the decrease of PRMT5 expression produced by RNA interference positively affects L-type aldolase A promoter transcription. Finally, the alternating occupancy of the L-type aldolase A promoter by the ZNF224-PRMT5 repression complex in proliferating and growth-arrested cells suggests that these regulatory proteins play a significant role during the cell cycle modulation of human aldolase A gene expression. Our data represent the first experimental evidence that protein arginine methylation plays a role in ZNF224-mediated transcriptional repression and provide novel insight into the chromatin modifications required for repression of gene transcription by Kruppel-like associated box-zinc finger proteins.

  15. L-arginine-induced experimental pancreatitis

    PubMed Central

    Hegyi, Péter; Jr, Zoltán Rakonczay; Sári, Réka; Góg, Csaba; Lonovics, János; Takács, Tamás; Czakó, László

    2004-01-01

    Despite medical treatment, the lethality of severe acute pancreatitis is still high (20%-30%). Therefore, it is very important to find good animal models to characterise the events of this severe disease. In 1984, Mizunuma et al[1] developed a new type of experimental necrotizing pancreatitis by intraperitoneal administration of a high dose of L-arginine in rats. This non-invasive model is highly reproducible and produces selective, dose-dependent acinar cell necrosis. Not only is this a good model to study the pathomechanisms of acute necrotizing pancreatitis, but it is also excellent to observe and influence the time course changes of the disease. By writing this review we iluminate some new aspects of cell physiology and pathology of acute necrotizing pancreatitis. Unfortunately, the reviews about acute experimental pancreatitis usually did not discuss this model. Therefore, the aim of this manuscript was to summarise the observations and address some challenges for the future in L-arginine-induced pancreatitis. PMID:15237423

  16. Arginine Decarboxylase Is Localized in Chloroplasts.

    PubMed Central

    Borrell, A.; Culianez-Macia, F. A.; Altabella, T.; Besford, R. T.; Flores, D.; Tiburcio, A. F.

    1995-01-01

    Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC. PMID:12228631

  17. Small Molecule Inhibitors of Protein Arginine Methyltransferases

    PubMed Central

    Hu, Hao; Qian, Kun; Ho, Meng-Chiao; Zheng, Y. George

    2016-01-01

    Introduction Arginine methylation is an abundant posttranslational modification occurring in mammalian cells and catalyzed by protein arginine methyltransferases (PRMTs). Misregulation and aberrant expression of PRMTs are associated with various disease states, notably cancer. PRMTs are prominent therapeutic targets in drug discovery. Areas covered The authors provide an updated review of the research on the development of chemical modulators for PRMTs. Great efforts are seen in screening and designing potent and selective PRMT inhibitors, and a number of micromolar and submicromolar inhibitors have been obtained for key PRMT enzymes such as PRMT1, CARM1, and PRMT5. The authors provide a focus on their chemical structures, mechanism of action, and pharmacological activities. Pros and cons of each type of inhibitors are also discussed. Expert opinion Several key challenging issues exist in PRMT inhibitor discovery. Structural mechanisms of many PRMT inhibitors remain unclear. There lacks consistency in potency data due to divergence of assay methods and conditions. Physiologically relevant cellular assays are warranted. Substantial engagements are needed to investigate pharmacodynamics and pharmacokinetics of the new PRMT inhibitors in pertinent disease models. Discovery and evaluation of potent, isoform-selective, cell-permeable and in vivo-active PRMT modulators will continue to be an active arena of research in years ahead. PMID:26789238

  18. Arginine Kinase. Joint Crystallographic & NMR RDC Analyses link Substrate-Associated Motions to Intrinsic Flexibility

    PubMed Central

    Niu, Xiaogang; Brüschweiler-Li, Lei; Davulcu, Omar; Skalicky, Jack J.; Brüschweiler, Rafael; Chapman, Michael S.

    2010-01-01

    The phosphagen kinase family, including creatine and arginine kinases, catalyze the reversible transfer of a “high energy” phosphate between ATP and a phospho-guanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of arginine kinase structures was interpreted as a plastic deformation. Here, the structure of Limulus substrate-free arginine kinase is refined against high resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa), and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.2 Å transition state analog complex shows large substrate-induced domain motions which can be broken down into movements of smaller quasi-rigid bodies. The solution state structure of substrate-free arginine kinase is most consistent with an equilibrium of substrate-free and –bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, “substrate-induced” motions are along modes that are intrinsically flexible in the substrate-free enzyme, and likely involve some degree of conformational selection. PMID:21075117

  19. Esophagogastric anastomosis in rats: Improved healing by BPC 157 and L-arginine, aggravated by L-NAME

    PubMed Central

    Djakovic, Zeljko; Djakovic, Ivka; Cesarec, Vedran; Madzarac, Goran; Becejac, Tomislav; Zukanovic, Goran; Drmic, Domagoj; Batelja, Lovorka; Zenko Sever, Anita; Kolenc, Danijela; Pajtak, Alen; Knez, Nikica; Japjec, Mladen; Luetic, Kresimir; Stancic-Rokotov, Dinko; Seiwerth, Sven; Sikiric, Predrag

    2016-01-01

    AIM To cure typically life-threatening esophagogastric anastomosis in rats, lacking anastomosis healing and sphincter function rescue, in particular. METHODS Because we assume esophagogastric fistulas represent a particular NO-system disability, we attempt to identify the benefits of anti-ulcer stable gastric pentadecapeptide BPC 157, which was in trials for ulcerative colitis and currently for multiple sclerosis, in rats with esophagocutaneous fistulas. Previously, BPC 157 therapies have promoted the healing of intestinal anastomosis and fistulas, and esophagitis and gastric lesions, along with rescued sphincter function. Additionally, BPC 157 particularly interacts with the NO-system. In the 4 d after esophagogastric anastomosis creation, rats received medication (/kg intraperitoneally once daily: BPC 157 (10 μg, 10 ng), L-NAME (5 mg), or L-arginine (100 mg) alone and/or combined or BPC 157 (10 μg, 10 ng) in drinking water). For rats underwent esophagogastric anastomosis, daily assessment included progressive stomach damage (sum of the longest diameters, mm), esophagitis (scored 0-5), weak anastomosis (mL H2O before leak), low pressure in esophagus at anastomosis and in the pyloric sphincter (cm H2O), progressive weight loss (g) and mortality. Immediate effect assessed blood vessels disappearance (scored 0-5) at the stomach surface immediately after anastomosis creation. RESULTS BPC 157 (all regimens) fully counteracted the perilous disease course from the very beginning (i.e., with the BPC 157 bath, blood vessels remained present at the gastric surface after anastomosis creation) and eliminated mortality. Additionally, BPC 157 treatment in combination with L-NAME nullified any effect of L-NAME that otherwise intensified the regular course. Consistently, with worsening (with L-NAME administration) and amelioration (with L-arginine), either L-arginine amelioration prevails (attenuated esophageal and gastric lesions) or they counteract each other (L-NAME + L-arginine

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

    PubMed

    Marini, Juan C; Didelija, Inka Cajo

    2015-01-01

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

  1. Arginine Deprivation as a Targeted Therapy for Cancer

    PubMed Central

    Feun, L.; You, M.; Wu, C.J.; Kuo, M.T.; Wangpaichitr, M.; Spector, S.; Savaraj, N.

    2011-01-01

    Certain cancers may be auxotrophic for a particular amino acid and amino acid deprivation is one method to treat these tumors. Arginine deprivation is a novel approach to target tumors which lack argininosuccinate synthetase (ASS) expression. ASS is a key enzyme which converts citrulline to arginine. Tumors which usually do not express ASS include melanoma, hepatocellular carcinoma, some mesotheliomas and some renal cell cancers. Arginine can be degraded by several enzymes including arginine deiminase (ADI). Although ADI is a microbial enzyme from mycoplasma, it has high affinity to arginine and catalyzes arginine to citrulline and ammonia. Citrulline can be recycled back to arginine in normal cells which express ASS, whereas ASS(−) tumor cells cannot. A pegylated form of ADI (ADI-PEG20) has been formulated and has shown in vitro and in vivo activity against melanoma and hepatocellular carcinoma. ADI-PEG20 induces apoptosis in melanoma cell lines. However, arginine deprivation can also induce ASS expression in certain melanoma cell lines which can lead to in-vitro drug resistance. Phase I and II clinical trials with ADI-PEG20 have been conducted in patients with melanoma and hepatocellular carcinoma and antitumor activity has been demonstrated in both cancers. This article reviews our laboratory and clinical experience as well as others with ADI-PEG20 as an antineoplastic agent. Future direction in utilizing this agent is also discussed. PMID:18473854

  2. Arginine kinase from Litopenaeus vannamei: cloning, expression and catalytic properties.

    PubMed

    Yao, Cui-Luan; Ji, Pei-Feng; Kong, Peng; Wang, Zhi-Yong; Xiang, Jian-Hai

    2009-03-01

    Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. In this paper, the full-length cDNA of AK was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Escherichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps.

  3. Argininosuccinate synthase: at the center of arginine metabolism.

    PubMed

    Haines, Ricci J; Pendleton, Laura C; Eichler, Duane C

    2011-01-01

    The levels of L-arginine, a cationic, semi-essential amino acid, are often controlled within a cell at the level of local availability through biosynthesis. The importance of this temporal and spatial control of cellular L-arginine is highlighted by the tissue specific roles of argininosuccinate synthase (argininosuccinate synthetase) (EC 6.3.4.5), as the rate-limiting step in the conversion of L-citrulline to L-arginine. Since its discovery, the function of argininosuccinate synthase has been linked almost exclusively to hepatic urea production despite the fact that alternative pathways involving argininosuccinate synthase were defined, such as its role in providing arginine for creatine and for polyamine biosynthesis. However, it was the discovery of nitric oxide that meaningfully extended our understanding of the metabolic importance of non-hepatic argininosuccinate synthase. Indeed, our knowledge of the number of tissues that manage distinct pools of arginine under the control of argininosuccinate synthase has expanded significantly.

  4. Purification of free arginine from chickpea (Cicer arietinum) seeds.

    PubMed

    Cortés-Giraldo, Isabel; Megías, Cristina; Alaiz, Manuel; Girón-Calle, Julio; Vioque, Javier

    2016-02-01

    Chickpea is a grain legume widely consumed in the Mediterranean region and other parts of the world. Chickpea seeds are rich in proteins but they also contain a substantial amount of free amino acids, especially arginine. Hence chickpea may represent a useful source of free amino acids for nutritional or pharmaceutical purposes. Arginine is receiving great attention in recent years because it is the substrate for the synthesis of nitric oxide, an important signaling molecule involved in numerous physiological and pathological processes in mammals. In this work we describe a simple procedure for the purification of arginine from chickpea seeds, using nanofiltration technology and an ion-exchange resin, Amberlite IR-120. Arginine was finally purified by precipitation or crystallization, yielding preparations with purities of 91% and 100%, respectively. Chickpea may represent an affordable green source of arginine, and a useful alternative to production by fermentation or protein hydrolysis.

  5. Pegylated arginine deiminase: a novel anticancer enzyme agent

    PubMed Central

    Feun, Lynn; Savaraj, Niramol

    2011-01-01

    Pegylated arginine deiminase (ADI-PEG20) is a novel anticancer enzyme that produces depletion of arginine, which is a nonessential amino acid in humans. Certain tumours, such as malignant melanoma and hepatocellular carcinoma, are auxotrophic for arginine. These tumours that are sensitive to arginine depletion do not express argininosuccinate synthetase, a key enzyme in the synthesis of arginine from citrulline. ADI-PEG20 inhibits human melanomas and hepatocellular carcinomas in vitro and in vivo. Phase I – II trials in patients with melanoma and hepatocellular carcinomas have shown the drug to have antitumour activity and tolerable side effects. Large Phase II trials and randomised, controlled Phase III trials are needed to determine its overall efficacy in the treatment of these malignancies and others. PMID:16787144

  6. Effects of arginine on hair damage via oxidative coloring process.

    PubMed

    Oshimura, Eiko; Ino, Masahiro

    2004-01-01

    The purpose of this study was to measure the protective effects of arginine in oxidative coloring or bleaching process. Contact angle measurement, tensile measurement and amino acid analysis were employed. As the first step, it was shown that oxidative coloring or bleaching process decreases hair surface hydrophobicity and tensile strength in wet condition. Next the study has been conducted with coloring agents in which part of the ammonia was replaced with arginine, to find that arginine reduced the oxidative change in contact angle and tensile strength. These results suggest that arginine prevents the undesirable attack by hydrogen peroxide on hair proteins and hair surface lipids. Furthermore, it is also suggested from amino acid analysis that a considerable amount of arginine is deposited on, or in hair fibers from coloring agents.

  7. Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas.

    PubMed

    Cheng, Feng; Zhu, Leilei; Lue, Hongqi; Bernhagen, Jürgen; Schwaneberg, Ulrich

    2015-02-01

    Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, ADI's application as anticancer drug is hampered by its low activity for arginine under physiological conditions mainly due to its high "K M" (S₀.₅) values which are often 1 magnitude higher than the arginine concentration in blood (0.10-0.12 mM arginine in human plasma). Previous evolution campaigns were directed by us with the aim of boosting activity of PpADI (ADI from Pseudomonas plecoglossicida, k cat = 0.18 s(-1); S₀.₅ = 1.30 mM), and yielded variant M6 with slightly reduced S₀.₅ values and enhanced k cat (S₀.₅ = 0.81 mM; k cat = 11.64 s(-1)). In order to further reduce the S₀.₅ value and to increase the activity of PpADI at physiological arginine concentration, a more sensitive screening system based on ammonia detection in 96-well microtiter plate to reliably detect ≥0.005 mM ammonia was developed. After screening ~5,500 clones with the ammonia detection system (ADS) in two rounds of random mutagenesis and site-directed mutagenesis, variant M19 with increased k cat value (21.1 s(-1); 105.5-fold higher compared to WT) and reduced S₀.₅ value (0.35 mM compared to 0.81 mM (M6) and 1.30 mM (WT)) was identified. Improved performance of M19 was validated by determining IC₅₀ values for two melanoma cell lines. The IC₅₀ value for SK-MEL-28 dropped from 8.67 (WT) to 0.10 (M6) to 0.04 μg/mL (M19); the IC₅₀ values for G361 dropped from 4.85 (WT) to 0.12 (M6) to 0.05 μg/mL (M19).

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dietary arginine is the main dietary precursor for citrulline synthesis, but it is not known if other precursors can compensate for when arginine is absent in the diet. To address this question, the contribution of plasma and dietary precursors were determined, utilizing multitracer protocols in con...

  9. Analysis of arginine and lysine methylation utilizing peptide separations at neutral pH and electron transfer dissociation mass spectrometry.

    PubMed

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

    2010-01-01

    Arginine and lysine methylation are widespread protein post-translational modifications. Peptides containing these modifications are difficult to retain using traditional reversed-phase liquid chromatography because they are intrinsically basic/hydrophilic and often fragment poorly during collision induced fragmentation (CID). Therefore, they are difficult to analyze using standard proteomic workflows. To overcome these caveats, we performed peptide separations at neutral pH, resulting in increased retention of the hydrophilic/basic methylated peptides before identification using MS/MS. Alternatively trifluoroacetic acid (TFA) was used for increased trapping of methylated peptides. Electron-transfer dissociation (ETD) mass spectrometry was then used to identify and characterize methylated residues. In contrast to previous reports utilizing ETD for arginine methylation, we observed significant amount of side-chain fragmentation. Using heavy methyl stable isotope labeling with amino acids in cell culture it was shown that, similar to CID, a loss of monomethylamine or dimethylamine from the arginine methylated side-chain during ETD can be used as a diagnostic to determine the type of arginine methylation. CID of lysine methylated peptides does not lead to significant neutral losses, but ETD is still beneficial because of the high charge states of such peptides. The developed LC MS/MS methods were successfully applied to tryptic digests of a number of methylated proteins, including splicing factor proline-glutamine-rich protein (SFPQ), RNA and export factor-binding protein 2 (REF2-I) and Sul7D, demonstrating significant advantages over traditional LC MS/MS approaches.

  10. How to improve fertility of African soils? Leguminous fallows (Cameroon), addition of farmyard manure and mineral fertilizer (Kenya), organic residues management and introduction of N2 fixing species in forest plantations (Congo).

    NASA Astrophysics Data System (ADS)

    Koutika, Lydie-Stella; Mareschal, Louis; Mouanda, Cadeau; Epron, Daniel

    2014-05-01

    Most of African soils are inherently infertile and poor in nutrients mainly nitrogen and phosphorus. Several practices are used to improve soil fertility, increase productivity and ensure their sustainability. Soil fertility in the leguminous fallows was evaluated through particulate organic matter (POM), the more active part of soil organic matter (SOM) in Cameroon. The combination of mineral and organic (manure) fertilizers increased microbial P biomass allowing the release of P along the plant growing period in the Kenyan soils. Organic residues management and introduction of nitrogen fixing species (Acacia) were used to improve soil fertility and sustain forest productivity on the coastal plains of Congo. SOM fractionation was made under Pueraria, Mucuna fallows and natural regrowth mainly Chromolaena and under 3 forest plantation treatments installed in previous savanna: 1) no input, 2) normal input, and 3) double input of organic residues. Microbial P biomass and sequential P fractionation were evaluated in high and low P fixing soils. N, C, available P and pH were determined on soil sampled in acacia (100A), eucalypt (100E) and mixed-species (50A:50E) stands. N and P were determined in aboveground litters and in leaves, bark and wood of trees. The two leguminous fallows increased N content in POM fractions i.e., N >1% for Pueraria and Mucuna against N<1% for natural regrowth in the 0-0.10m depth, probably through N input from N2 fixation from the atmosphere (Cameroon).The addition of mineral fertilizers and farmyard manure increases P biomass (4.8 after 2 weeks to 15.2 after 16 weeks), and then decreased to 9.7 mg P g-1 soil (week 32). It also changes the P Hedley fractions partition in the high P fixing Kenyan soil (0-0.10m). After two rotations (14 years), SOM mineralization was the highest in the double input of organic residues treatment (low coarse POM 5.6 g kg-1 of soil and high organo-mineral fraction (OMF) 115 g kg-1 of soil). The introduction of A

  11. Drug evaluation: ADI-PEG-20--a PEGylated arginine deiminase for arginine-auxotrophic cancers.

    PubMed

    Shen, Li-Jiuan; Shen, Wei-Chiang

    2006-06-01

    Pheonix is developing ADI-PEG-20, a PEGylated arginine deiminase for the potential treatment of hepatocellular carcinoma, for which the Food and Drug Administration (FDA) and the European Agency for the Evaluation of Medicinal Products have granted the drug Orphan Drug status, and melanoma, for which the FDA has also awarded ADI-PEG-20 Orphan Drug status. ADI-PEG-20 is also being investigated for the potential treatment of influenza virus infection and hepatitis C virus infection.

  12. Arginine Vasopressin and Copeptin in Perinatology

    PubMed Central

    Evers, Katrina Suzanne; Wellmann, Sven

    2016-01-01

    Arginine vasopressin (AVP) plays a major role in the homeostasis of fluid balance, vascular tonus, and the regulation of the endocrine stress response. The measurement of AVP levels is difficult due to its short half-life and laborious method of detection. Copeptin is a more stable peptide derived from the same precursor molecule, is released in an equimolar ratio to AVP, and has a very similar response to osmotic, hemodynamic, and stress-related stimuli. In fact, copeptin has been propagated as surrogate marker to indirectly determine circulating AVP concentrations in various conditions. Here, we present an overview of the current knowledge on AVP and copeptin in perinatology with a particular focus on the baby’s transition from placenta to lung breathing. We performed a systematic review of the literature on fetal stress hormone levels, including norepinephrine, cortisol, AVP, and copeptin, in regard to birth stress. Finally, diagnostic and therapeutic options for copeptin measurement and AVP functions are discussed. PMID:27532032

  13. L-Arginine and Alzheimer's Disease

    PubMed Central

    Yi, Jing; Horky, Laura L.; Friedlich, Avi L.; Shi, Ying; Rogers, Jack T.; Huang, Xudong

    2009-01-01

    Alzheimer's disease (AD), the most common form of dementia, is characterized by progressive neurodegeneration and loss of cognitive and memory functions. Although the exact causes of AD are still unclear, evidence suggests that atherosclerosis, redox stress, inflammation, neurotransmitter dysregulation, and impaired brain energy metabolism may all be associated with AD pathogenesis. Herein, we explore a possible role for L-arginine (L-arg) in AD, taking into consideration known functions for L-arg in atherosclerosis, redox stress and the inflammatory process, regulation of synaptic plasticity and neurogenesis, and modulation of glucose metabolism and insulin activity. L-arg, a precursor of nitric oxide and polyamine, exhibits multiple functions in human health and may play a prominent role in age-related degenerative diseases such as AD. PMID:19079617

  14. Infrared Spectroscopy of Cationized Arginine in the Gas Phase: Direct Evidence for the Transition from Nonzwitterionic to Zwitterionic Structure

    PubMed Central

    Bush, Matthew F.; O'Brien, Jeremy T.; Prell, James S.; Saykally, Richard J.; Williams, Evan R.

    2009-01-01

    The gas-phase structures of protonated and alkali metal cationized arginine (Arg) and arginine methyl ester (ArgOMe) are investigated with infrared spectroscopy and ab initio calculations. Infrared spectra, measured in the hydrogen-stretch region, provide compelling evidence that arginine changes from its nonzwitterionic to zwitterionic form with increasing metal ion size, with the transition in structure occurring between lithium and sodium. For sodiated arginine, evidence for both forms is obtained from spectral deconvolution, although the zwitterionic form is predominant. Comparisons of the photodissociation spectra with spectra calculated for low-energy candidate structures provide additional insights into the detailed structures of these ions. Arg•Li+, ArgOMe•Li+, and ArgOMe•Na+ exist in nonzwitterionic forms in which the metal ion is tricoordinated with the amino acid, whereas Arg•Na+ and Arg•K+ predominately exist in a zwitterionic form where the protonated side chain donates one hydrogen bond to the N terminus of the amino acid and the metal ion is bicoordinated with the carboxylate group. Arg•H+ and ArgOMe•H+ have protonated side chains that form the same interaction with the N terminus as zwitterionic, alkali metal cationized arginine, yet both are unambiguously determined to be nonzwitterionic. Calculations indicate that for clusters with protonated side chains, structures with two strong hydrogen bonds are lowest in energy, in disagreement with these experimental results. This study provides new detailed structural assignments and interpretations of previously observed fragmentation patterns for these ions. PMID:17249666

  15. Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis.

    PubMed

    Delage, B; Luong, P; Maharaj, L; O'Riain, C; Syed, N; Crook, T; Hatzimichael, E; Papoudou-Bai, A; Mitchell, T J; Whittaker, S J; Cerio, R; Gribben, J; Lemoine, N; Bomalaski, J; Li, C-F; Joel, S; Fitzgibbon, J; Chen, L-T; Szlosarek, P W

    2012-07-05

    Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.

  16. Arginine feeding modifies cyclosporine nephrotoxicity in rats.

    PubMed Central

    De Nicola, L; Thomson, S C; Wead, L M; Brown, M R; Gabbai, F B

    1993-01-01

    Glycine (G) infusion causes renal vasodilation mediated by nitric oxide (NO). Cyclosporine A (CsA) nephrotoxicity is characterized by preglomerular vasoconstriction and decreased efferent arteriolar tone probably related to reduced NO and angiotensin II, respectively. L-Arginine (ARG) is a precursor to NO. To test the hypothesis that chronic CsA decreases renal NO activity, we compared the glomerular hemodynamic response to glycine infusion in rats after 8 d of CsA (30 mg/kg per d s.c.), CsA and ARG (1.6 g/kg per d p.o.) (A/CsA), and in two groups of pair-fed controls (CON, A/CON). Single nephron GFR (SNGFR), single nephron plasma flow (SNPF), glomerular capillary hydrostatic pressure gradient (delta P), proximal tubular reabsorption (APR), and kidney tissue angiotensin II (AIIk) were measured before and during G. CsA was associated with baseline decrements in SNGFR, SNPF, delta P, and AIIk, and with a blunted hemodynamic response to G. In CON, ARG did not affect baseline hemodynamics or modify the response to G. In CsA, ARG decreased baseline preglomerular resistance and restored the glomerular hemodynamic response to G. G was associated with a significant increase in AIIk in both CON and CsA. These findings suggest that (a) CsA is associated with decreased AIIk, and (b) CsA may diminish NO activity within the kidney, and that this capacity may be partially restored by arginine feeding. PMID:8408638

  17. Theoretical study of the mechanism of protein arginine deiminase 4 (PAD4) inhibition by F-amidine.

    PubMed

    Li, Dongmei; Liu, Cui; Lin, Jianping

    2015-02-01

    Protein arginine deiminase 4 (PAD4) catalyzes the hydrolysis of a peptidylarginine residue to form a citrulline residue and ammonia during posttranslational modification. This process plays a pivotal role in rheumatoid arthritis (RA) and gene regulation. F-amidine belongs to a series of haloacetamidine compounds that are the most potent PAD4 inhibitors described to date. F-amidine acts as a mechanism-based inhibitor of PAD4, inactivating PAD4 by the covalent modification of the active site Cys645. In this manuscript, the fundamental mechanism of PAD4 inhibition by F-amidine is investigated using a QM/MM approach. Our simulations show that in the PAD4-F-amidine reactant complex, the active site Cys645 exists as a thiolate and His471 is protonated. This is consistent with the reverse protonation mechanism wherein the active site nucleophile, Cys645, in PAD4 exists as a thiolate in the active form of the enzyme. Inhibition of PAD4 by F-amidine is initiated by the nucleophilic addition of Sγ to the Cζ of F-amidine, leading to the formation of a tetrahedral intermediate. His471 serves as a proton donor, helping F to leave the fluoroacetamidine moiety of F-amidine; meanwhile, Sγ forms a three-membered ring with Cζ and Cη of F-amidine. Subsequently, the three-membered sulfonium ring collapses and rearranges to the final thioether product. His471 acts as a proton donor in the transition state and facilitates the inhibition reaction of PAD4.

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

    PubMed

    Chen, Heng-Li; Su, Pei-Yi; Chang, Ya-Shu; Wu, Szu-Yao; Liao, You-Di; Yu, Hui-Ming; Lauderdale, Tsai-Ling; Chang, Kaichih; Shih, Chiaho

    2013-01-01

    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 HBc ARD

  19. Identification of a Novel Antimicrobial Peptide from Human Hepatitis B Virus Core Protein Arginine-Rich Domain (ARD)

    PubMed Central

    Chen, Heng-Li; Su, Pei-Yi; Chang, Ya-Shu; Wu, Szu-Yao; Liao, You-Di; Yu, Hui-Ming; Lauderdale, Tsai-Ling; Chang, Kaichih; Shih, Chiaho

    2013-01-01

    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

  20. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit.

    PubMed Central

    Cooke, J P; Singer, A H; Tsao, P; Zera, P; Rowan, R A; Billingham, M E

    1992-01-01

    The purpose of this study was to determine if chronic administration of L-arginine, the precursor of endothelium-derived relaxing factor (EDRF), normalizes endothelium-dependent relaxation and decreases atherosclerosis in hypercholesterolemic animals. Male rabbits were fed (a) normal rabbit chow; (b) 1% cholesterol diet; or (c) 1% cholesterol diet supplemented by 2.25% L-arginine HCl in drinking water. Arginine supplementation doubled plasma arginine levels without affecting serum cholesterol values. After 10 wk, the thoracic aorta was harvested for studies of vascular reactivity and histomorphometry. Endothelium-dependent relaxations (to acetylcholine and calcium ionophore A23187) were significantly impaired in thoracic aortae from animals fed a 1% cholesterol diet. By contrast, vessels from hypercholesterolemic animals receiving L-arginine supplementation exhibited significantly improved endothelium-dependent relaxations. Responses to norepinephrine or nitroglycerin were not affected by either dietary intervention. Histomorphometric analysis revealed a reduction in lesion surface area and intimal thickness in thoracic aortae from arginine-supplemented animals compared to those from untreated hypercholesterolemic rabbits. This is the first study to demonstrate that supplementation of dietary L-arginine, the EDRF precursor, improves endothelium-dependent vasorelaxation. More importantly, we have shown that this improvement in EDRF activity is associated with a reduction in atherogenesis. PMID:1522225

  1. Improved anaerobic use of arginine by Saccharomyces cerevisiae.

    PubMed

    Martin, Olga; Brandriss, Marjorie C; Schneider, Gisbert; Bakalinsky, Alan T

    2003-03-01

    Anaerobic arginine catabolism in Saccharomyces cerevisiae was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine. This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead. A pro3 ure2 strain expressing a PGK1 promoter-driven PUT2 allele encoding Delta(1)-pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.

  2. Pentadecapeptide BPC 157 Reduces Bleeding and Thrombocytopenia after Amputation in Rats Treated with Heparin, Warfarin, L-NAME and L-Arginine

    PubMed Central

    Stupnisek, Mirjana; Kokot, Antonio; Drmic, Domagoj; Hrelec Patrlj, Masa; Zenko Sever, Anita; Kolenc, Danijela; Radic, Bozo; Suran, Jelena; Bojic, Davor; Vcev, Aleksandar; Seiwerth, Sven; Sikiric, Predrag

    2015-01-01

    Background BPC 157 is a stable gastric pentadecapeptide recently implicated with a role in hemostasis. While NO is largely implicated in hemostatic mechanisms, in tail-amputation-models under heparin- and warfarin-administration, both the NO-synthase (NOS)-blocker, L-NAME (prothrombotic) and the NOS-substrate L-arginine (antithrombotic), were little investigated. Objective. To investigate the effect of L-NAME and L-arginine on hemostatic parameters, and to reveal the effects of BPC 157 on the L-NAME- and L-arginine-induced hemostatic actions under different pathological condition: tail amputation without or with anticoagulants, heparin or warfarin. Methods Tail amputation, and/or i.v.-heparin (10 mg/kg), i.g.-warfarin (1.5 mg/kg/day for 3 days) were used in rats. Treatment includes BPC 157, L-NAME, L-arginine, per se and their combination. Results After (tail) amputation, with or without i.v.-heparin or i.g.-warfarin, BPC 157 (10 μg/kg, 10 ng/kg, i.p., i.v. (heparin), 10 μg/kg i.g. (warfarin)) always reduced bleeding time and/or haemorrhage and counteracted thrombocytopenia. As for L-NAME and/or L-arginine, we noted: L-arginine (100 mg/kg i.p.)–rats: more bleeding, less/no thrombocytopenia; L-NAME (5 mg/kg i.p.)-rats: less bleeding (amputation only), but present thrombocytopenia; L-NAME+L-arginine-rats also exhibited thrombocytopenia: L-NAME counteracted L-arginine-increased bleeding, L-arginine did not counteract L-NAME-thrombocytopenia. All animals receiving BPC 157 in addition (BPC 157μg+L-NAME; BPC 157μg+L-arginine, BPC 157μg+L-NAME+L-arginine), exhibited decreased haemorrhage and markedly counteracted thrombocytopenia. Conclusions L-NAME (thrombocytopenia), L-arginine (increased haemorrhage) counteraction and BPC 157 (decreased haemorrhage, counteracted thrombocytopenia) with rescue against two different anticoagulants, implicate a BPC 157 modulatory and balancing role with rescued NO-hemostatic mechanisms. PMID:25897838

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

    PubMed

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

    2015-06-01

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

  4. Convergent evolution of the arginine deiminase pathway: the ArcD and ArcE arginine/ornithine exchangers.

    PubMed

    Noens, Elke E E; Lolkema, Juke S

    2017-02-01

    The arginine deiminase (ADI) pathway converts L-arginine into L-ornithine and yields 1 mol of ATP per mol of L-arginine consumed. The L-arginine/L-ornithine exchanger in the pathway takes up L-arginine and excretes L-ornithine from the cytoplasm. Analysis of the genomes of 1281 bacterial species revealed the presence of 124 arc gene clusters encoding the pathway. About half of the clusters contained the gene encoding the well-studied L-arginine/L-ornithine exchanger ArcD, while the other half contained a gene, termed here arcE, encoding a membrane protein that is not a homolog of ArcD. The arcE gene product of Streptococcus pneumoniae was shown to take up L-arginine and L-ornithine with affinities of 0.6 and 1 μmol/L, respectively, and to catalyze metabolic energy-independent, electroneutral exchange. ArcE of S. pneumoniae could replace ArcD in the ADI pathway of Lactococcus lactis and provided the cells with a growth advantage. In contrast to ArcD, ArcE catalyzed translocation of the pathway intermediate L-citrulline with high efficiency. A short version of the ADI pathway is proposed for L-citrulline catabolism and the presence of the evolutionary unrelated arcD and arcE genes in different organisms is discussed in the context of the evolution of the ADI pathway.

  5. Effect of Exercise Training and L-arginine on Oxidative Stress and Left Ventricular Function in the Post-ischemic Failing Rat Heart.

    PubMed

    Ranjbar, Kamal; Nazem, Farzad; Nazari, Afshin

    2016-04-01

    The aim of the present study was to evaluate the effect of exercise training (ET) and L-arginine on oxidative stress and ventricular function in rat with myocardial infarction (MI). Four weeks after the surgical procedures, 40 Wistar male rats were randomized to the following groups: MI-sedentary (Sed); MI-exercise (Ex); MI-sedentary + L-arginine (Sed + LA); and MI-exercise + L-arginine (Ex + LA); the rats were subjected to aerobic training in the form of treadmill running. Rats in the L-arginine-treated groups drank water containing 4 % L-arginine. Before and after the training program, all subjects underwent resting echocardiography. Catalase (CAT) glutathione peroxidase (GPx), malondialdehyde (MDA) and myeloperoxidase (MPO) were measured. Cardiac output, stroke volume and fractional shortening in Ex and Ex + LA groups significantly increased in comparison with the Sed group. Cardiac systolic function indices in Ex + LA group were significantly greater than Ex group. Also, GPx activity and MDA, respectively, increased and decreased in response to ET, but no change was observed in MPO and CAT. These results suggest that ET increased LV function by decreasing oxidative stress and increasing antioxidant defense system in rats with MI. In addition in response to training, L-arginine appears to have additive effect on cardiac function, but have no effect on oxidative stress indices.

  6. Comparative effects of sildenafil, phentolamine, yohimbine and L-arginine on the rabbit corpus cavernosum.

    PubMed

    Sharabi, F M; Daabees, T T; El-Metwally, M A; Senbel, A M

    2004-04-01

    , phentolamine was found to possess the highest potency in inducing relaxation of RCC proving that its action is independent on the stimulated neurogenic system. In addition, the combination of less effective doses of sildenafil and L-arginine has a potential advantage on erectile functions. The importance of this combination remains to be solved clinically.

  7. Mitochondrial dysfunction in brain cortex mitochondria of STZ-diabetic rats: effect of l-Arginine.

    PubMed

    Ortiz, M Del Carmen; Lores-Arnaiz, Silvia; Albertoni Borghese, M Florencia; Balonga, Sabrina; Lavagna, Agustina; Filipuzzi, Ana Laura; Cicerchia, Daniela; Majowicz, Monica; Bustamante, Juanita

    2013-12-01

    Mitochondrial dysfunction has been implicated in many diseases, including diabetes. It is well known that oxygen free radical species are produced endogenously by mitochondria, and also nitric oxide (NO) by nitric oxide synthases (NOS) associated to mitochondrial membranes, in consequence these organelles constitute main targets for oxidative damage. The aim of this study was to analyze mitochondrial physiology and NO production in brain cortex mitochondria of streptozotocin (STZ) diabetic rats in an early stage of diabetes and the potential effect of L-arginine administration. The diabetic condition was characterized by a clear hyperglycaemic state with loose of body weight after 4 days of STZ injection. This hyperglycaemic state was associated with mitochondrial dysfunction that was evident by an impairment of the respiratory activity, increased production of superoxide anion and a clear mitochondrial depolarization. In addition, the alteration in mitochondrial physiology was associated with a significant decrease in both NO production and nitric oxide synthase type I (NOS I) expression associated to the mitochondrial membranes. An increased level of thiobarbituric acid-reactive substances (TBARS) in brain cortex homogenates from STZ-diabetic rats indicated the presence of lipid peroxidation. L-arginine treatment to diabetic rats did not change blood glucose levels but significantly ameliorated the oxidative stress evidenced by lower TBARS and a lower level of superoxide anion. This effect was paralleled by improvement of mitochondrial respiratory function and a partial mitochondrial repolarization.In addition, the administration of L-arginine to diabetic rats prevented the decrease in NO production and NOSI expression. These results could indicate that exogenously administered L-arginine may have beneficial effects on mitochondrial function, oxidative stress and NO production in brain cortex mitochondria of STZ-diabetic rats.

  8. Isotope labeling studies on the formation of multiple addition products of alanine in the pyrolysis residue of glucose/alanine mixtures by high-resolution ESI-TOF-MS.

    PubMed

    Chu, Fong Lam; Sleno, Lekha; Yaylayan, Varoujan A

    2011-11-09

    Pyrolysis was used as a microscale sample preparation tool to generate glucose/alanine reaction products to minimize the use of expensive labeled precursors in isotope labeling studies. The residue remaining after the pyrolysis at 250 °C was analyzed by electrospray time-of-flight mass spectrometry (ESI-TOF-MS). It was observed that a peak at m/z 199.1445 in the ESI-TOF-MS spectrum appeared only when the model system contained at least 2-fold excess alanine. The accurate mass determination indeed indicated the presence of two nitrogen atoms in the molecular formula (C(10)H(18)N(2)O(2)). To verify the origin of the carbon atoms in this unknown compound, model studies with [(13)U(6)]glucose, [(13)C-1]alanine, [(13)C-2]alanine, [(13)C-3]alanine, and [(15)N]alanine were also performed. Glucose furnished six carbon atoms, and alanine provides four carbon (2 × C-2 and 2 × C-3) and two nitrogen atoms. When commercially available fructosylalanine (N-attached to C-1) was reacted with only 1 mol of alanine, a peak at m/z 199.1445 was once again observed. In addition, when 3-deoxyglucosone (3-DG) was reacted with a 2-fold excess of alanine, a peak at m/z 199.1433 was also generated, confirming the points of attachment of the two amino acids at C-1 and C-2 atoms of 3-DG. These studies have indicated that amino acids can undergo multiple addition reactions with 1,2-dicarbonyl compounds such as 3-deoxyglucosone and eventually form a tetrahydropyrazine moiety.

  9. The glucose intolerance of acute pancreatitis: hormonal response to arginine.

    PubMed

    Solomon, S S; Duckworth, W C; Jallepalli, P; Bobal, M A; Iyer, R

    1980-01-01

    Patients with acute pancreatitis were studied by arginine infusion at 48--72 h. 7--10 days, and 18--21 days after onset of their illness. Plasma glucose, insulin, and glucagon values were determined. Acute pancreatitis was characterized by fasting hyperglycemia and hyperglucagonemia, associated with relative hyoinsulinemia. Arginine stimulation early in the disease (48--72 h) demonstrated hyperglycemia and hyperglucagonemia, which normalized by 18--21 days. Both phases of the normal biphasic insulin response to arginine were decreased during the initial arginine infusion. By 18--21 days, although the first phase was completely normal, the second phase of insulin secretion remained depressed. Acute pancreatitis is associated with damage to both the endocrine and exocrine pancreas. Glucose intolerance seen with this disease appears to be the result of hyperglucagonemia and relative hypoinsulinemia. Although the healing process at 3 wk is associated with return of plasma glucose and glucagon concentrations to normal, the impaired second phase insulin secretion persists.

  10. Regulation of immune responses by L-arginine metabolism.

    PubMed

    Bronte, Vincenzo; Zanovello, Paola

    2005-08-01

    L-Arginine is an essential amino acid for birds and young mammals, and it is a conditionally essential amino acid for adult mammals, as it is important in situations in which requirements exceed production, such as pregnancy. Recent findings indicate that increased metabolism of L-arginine by myeloid cells can result in the impairment of lymphocyte responses to antigen during immune responses and tumour growth. Two enzymes that compete for L-arginine as a substrate - arginase and nitric-oxide synthase - are crucial components of this lymphocyte-suppression pathway, and the metabolic products of these enzymes are important moderators of T-cell function. This Review article focuses on the relevance of L-arginine metabolism by myeloid cells for immunity under physiological and pathological conditions.

  11. Modulating the activity of short arginine-tryptophan containing antibacterial peptides with N-terminal metallocenoyl groups

    PubMed Central

    Albada, H Bauke; Chiriac, Alina-Iulia; Wenzel, Michaela; Penkova, Maya; Bandow, Julia E; Sahl, Hans-Georg

    2012-01-01

    Summary A series of small synthetic arginine and tryptophan containing peptides was prepared and analyzed for their antibacterial activity. The effect of N-terminal substitution with metallocenoyl groups such as ferrocene (FcCO) and ruthenocene (RcCO) was investigated. Antibacterial activity in different media, growth inhibition, and killing kinetics of the most active peptides were determined. The toxicity of selected derivatives was determined against erythrocytes and three human cancer cell lines. It was shown that the replacement of an N-terminal arginine residue with a metallocenoyl moiety modulates the activity of WRWRW-peptides against Gram-positive and Gram-negative bacteria. MIC values of 2–6 µM for RcCO-W(RW)2 and 1–11 µM for (RW)3 were determined. Interestingly, W(RW)2-peptides derivatized with ferrocene were significantly less active than those derivatized with ruthenocene which have similar structural but different electronic properties, suggesting a major influence of the latter. The high activities observed for the RcCO-W(RW)2- and (RW)3-peptides led to an investigation of the origin of activity of these peptides using several important activity-related parameters. Firstly, killing kinetics of the RcCO-W(RW)2-peptide versus killing kinetics of the (RW)3 derivative showed faster reduction of the colony forming units for the RcCO-W(RW)2-peptide, although MIC values indicated higher activity for the (RW)3-peptide. This was confirmed by growth inhibition studies. Secondly, hemolysis studies revealed that both peptides did not lead to significant destruction of erythrocytes, even up to 500 µg/mL for (RW)3 and 250 µg/mL for RcCO-W(RW)2. In addition, toxicity against three human cancer cell lines (HepG2, HT29, MCF7) showed that the (RW)3-peptide had an IC50 value of ~140 µM and the RcW(RW)2 one of ~90 µM, indicating a potentially interesting therapeutic window. Both the killing kinetics and growth inhibition studies presented in this work point to

  12. No effect of short-term arginine supplementation on nitric oxide production, metabolism and performance in intermittent exercise in athletes.

    PubMed

    Liu, Tsung-Han; Wu, Ching-Lin; Chiang, Chi-Wei; Lo, Yu-Wei; Tseng, Hung-Fu; Chang, Chen-Kang

    2009-06-01

    Arginine supplementation has been shown to alleviate endothelial dysfunction and improve exercise performance through increasing nitric oxide production in patients with cardiopulmonary diseases. In addition, arginine supplementation could decrease accumulations of lactate and ammonia, metabolites involved in development of muscular fatigue. The aim of this study was to investigate the effect of short-term arginine supplementation on performance in intermittent anaerobic exercise and the underlying mechanism in well-trained male athletes. Ten elite male college judo athletes participated with a randomized crossover, placebo-controlled design. The subjects consumed 6 g/day arginine (ARG trial) or placebo (CON trial) for 3 days then performed an intermittent anaerobic exercise test on a cycle ergometer. Blood samples were collected before supplementation, before and during exercise and 0, 3, 6, 10, 30 and 60 min after exercise. ARG trial had significantly higher arginine concentrations than CON trial at the same time point before, during and after exercise. In both trials, nitrate and nitrite concentration was significantly higher during and 6 min after exercise comparing to the basal concentration. The increase in nitrate and nitrite concentration during exercise in both trials was parallel to the increase in plasma citrulline concentrations. There was no significant difference between the 2 trials in plasma nitrate and nitrite, lactate and ammonia concentrations and peak and average power in the exercise. The results of this study suggested that short-term arginine supplementation had no effect on nitric oxide production, lactate and ammonia metabolism and performance in intermittent anaerobic exercise in well-trained male athletes.

  13. Dietary L-arginine supplementation increases muscle gain and reduces body fat mass in growing-finishing pigs.

    PubMed

    Tan, Bie; Yin, Yulong; Liu, Zhiqiang; Li, Xinguo; Xu, Haijun; Kong, Xiangfeng; Huang, Ruilin; Tang, Wenjie; Shinzato, Izuru; Smith, Stephen B; Wu, Guoyao

    2009-05-01

    Obesity in humans is a major public health crisis worldwide. In addition, livestock species exhibit excessive subcutaneous fat at market weight. However, there are currently few means of reducing adiposity in mammals. This study was conducted with a swine model to test the hypothesis that dietary L-arginine supplementation may increase muscle gain and decrease fat deposition. Twenty-four 110-day-old barrows were assigned randomly into two treatments, representing supplementation with 1.0% L-arginine or 2.05% L-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Growth performance was measured based on weight gain and food intake. After a 60-day period of supplementation, carcass and muscle composition were measured. Serum triglyceride concentration was 20% lower (P < 0.01) but glucagon level was 36% greater (P < 0.05) in arginine-supplemented than in control pigs. Compared with the control, arginine supplementation increased (P < 0.05) body weight gain by 6.5% and carcass skeletal-muscle content by 5.5%, while decreasing (P < 0.01) carcass fat content by 11%. The arginine treatment enhanced (P < 0.05) longissimus dorsi muscle protein, glycogen, and fat contents by 4.8, 42, and 70%, respectively, as well as muscle pH at 45 min post-mortem by 0.32, while reducing muscle lactate content by 37%. These results support our hypothesis that dietary arginine supplementation beneficially promotes muscle gain and reduces body fat accretion in growing-finishing pigs. The findings have a positive impact on development of novel therapeutics to treat human obesity and enhance swine lean-tissue growth.

  14. An Arginine Finger Regulates the Sequential Action of Asymmetrical Hexameric ATPase in the Double-Stranded DNA Translocation Motor

    PubMed Central

    Zhao, Zhengyi; De-Donatis, Gian Marco; Schwartz, Chad; Fang, Huaming; Li, Jingyuan

    2016-01-01

    Biological motors are ubiquitous in living systems. Currently, how the motor components coordinate the unidirectional motion is elusive in most cases. Here, we report that the sequential action of the ATPase ring in the DNA packaging motor of bacteriophage ϕ29 is regulated by an arginine finger that extends from one ATPase subunit to the adjacent unit to promote noncovalent dimer formation. Mutation of the arginine finger resulted in the interruption of ATPase oligomerization, ATP binding/hydrolysis, and DNA translocation. Dimer formation reappeared when arginine mutants were mixed with other ATPase subunits that can offer the arginine to promote their interaction. Ultracentrifugation and virion assembly assays indicated that the ATPase was presenting as monomers and dimer mixtures. The isolated dimer alone was inactive in DNA translocation, but the addition of monomer could restore the activity, suggesting that the hexameric ATPase ring contained both dimer and monomers. Moreover, ATP binding or hydrolysis resulted in conformation and entropy changes of the ATPase with high or low DNA affinity. Taking these observations together, we concluded that the arginine finger regulates sequential action of the motor ATPase subunit by promoting the formation of the dimer inside the hexamer. The finding of asymmetrical hexameric organization is supported by structural evidence of many other ATPase systems showing the presence of one noncovalent dimer and four monomer subunits. All of these provide clues for why the asymmetrical hexameric ATPase gp16 of ϕ29 was previously reported as a pentameric configuration by cryo-electron microscopy (cryo-EM) since the contact by the arginine finger renders two adjacent ATPase subunits closer than other subunits. Thus, the asymmetrical hexamer would appear as a pentamer by cryo-EM, a technology that acquires the average of many images. PMID:27457616

  15. Environmental pH determines citrulline and ornithine release through the arginine deiminase pathway in Lactobacillus fermentum IMDO 130101.

    PubMed

    Vrancken, G; Rimaux, T; Weckx, S; De Vuyst, L; Leroy, F

    2009-11-15

    Sourdough lactic acid bacteria (LAB) need to be adapted to a highly acidic and, therefore, challenging environment. Different mechanisms are employed to enhance competitiveness, among which conversion of arginine into ornithine through the arginine deiminase (ADI) pathway is an important one. A combined molecular and kinetic approach of the ADI pathway in Lactobacillus fermentum IMDO 130101, a highly competitive sourdough LAB strain, identified mechanisms with advantageous technological effects and quantified the impact of these effects. First, molecular analysis of the arcBCAD operon of 4.8 kb revealed the genes encoding the enzymes ornithine transcarbamoylase, carbamate kinase, arginine deiminase, and an arginine/ornithine (A/O) antiporter, respectively, with an additional A/O antiporter 702.5 kb downstream of the ADI operon. The latter could play a role in citrulline transport. Second, pH-controlled batch fermentations were carried out, generating data for the development of a mathematical model to describe the temporal evolution of the three amino acids involved in the ADI pathway (arginine, citrulline, and ornithine) as a result of the activity of these enzymes and transporter(s). Free arginine in the medium was converted completely into a mixture of citrulline and ornithine under all conditions tested. However, the ratio between these end-products and the pattern of their formation showed variation as a function of environmental pH. Under optimal pH conditions for growth, citrulline release and some further conversion into ornithine was observed. When growing under sub-optimal pH conditions, ornithine was the main product of the ADI pathway. These kinetic data suggest a role in adaptation of L. fermentum IMDO 130101 to growth under sub-optimal conditions.

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

    PubMed

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

    2017-04-01

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

  17. Localization of arginine decarboxylase in tobacco plants.

    PubMed

    Bortolotti, Cristina; Cordeiro, Alexandra; Alcázar, Rubén; Borrell, Antoni; Culiañez-Macià, Francisco A.; Tiburcio, Antonio F.; Altabella, Teresa

    2004-01-01

    The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.

  18. Fluorometric enzymatic assay of L-arginine

    NASA Astrophysics Data System (ADS)

    Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo

    2017-01-01

    The enzymes of L-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a limit of detection of 34 nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

  19. Geometry of guanidinium groups in arginines.

    PubMed

    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.

  20. Peptidyl Arginine Deiminase from Porphyromonas gingivalis Abolishes Anaphylatoxin C5a Activity*

    PubMed Central

    Bielecka, Ewa; Scavenius, Carsten; Kantyka, Tomasz; Jusko, Monika; Mizgalska, Danuta; Szmigielski, Borys; Potempa, Barbara; Enghild, Jan J.; Prossnitz, Eric R.; Blom, Anna M.; Potempa, Jan

    2014-01-01

    Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis. PMID:25324545

  1. Peptidyl arginine deiminase from Porphyromonas gingivalis abolishes anaphylatoxin C5a activity.

    PubMed

    Bielecka, Ewa; Scavenius, Carsten; Kantyka, Tomasz; Jusko, Monika; Mizgalska, Danuta; Szmigielski, Borys; Potempa, Barbara; Enghild, Jan J; Prossnitz, Eric R; Blom, Anna M; Potempa, Jan

    2014-11-21

    Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis.

  2. Human leukocyte-derived arginine aminopeptidase. The third member of the oxytocinase subfamily of aminopeptidases.

    PubMed

    Tanioka, Toshihiro; Hattori, Akira; Masuda, Shinako; Nomura, Yoshihiro; Nakayama, Hiroshi; Mizutani, Shigehiko; Tsujimoto, Masafumi

    2003-08-22

    In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid protein with significant homology to placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase. The predicted L-RAP contains the HEXXH(X)18E zinc-binding motif, which is characteristic of the M1 family of zinc metallopeptidases. Phylogenetic analysis indicates that L-RAP forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family. Immunocytochemical analysis indicates that L-RAP is located in the lumenal side of the endoplasmic reticulum. Among various synthetic substrates tested, L-RAP revealed a preference for arginine, establishing that the enzyme is a novel arginine aminopeptidase with restricted substrate specificity. In addition to natural hormones such as angiotensin III and kallidin, L-RAP cleaved various N-terminal extended precursors to major histocompatibility complex class I-presented antigenic peptides. Like other proteins involved in antigen presentation, L-RAP is induced by interferon-gamma. These results indicate that L-RAP is a novel aminopeptidase that can trim the N-terminal extended precursors to antigenic peptides in the endoplasmic reticulum.

  3. Effects of cadmium on the cuttlefish Sepia pharaonis' arginine kinase: unfolding kinetics integrated with computational simulations.

    PubMed

    Si, Yue-Xiu; Lee, Jinhyuk; Zhao, Feng; Yin, Shang-Jun; Park, Yong-Doo; Qian, Guo-Ying; Jiang, Xia-Min

    2016-08-01

    Arginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd(2+) on the cuttlefish Sepia pharaonis' arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band on SDS-PAGE. Cd(2+) effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd(2+) induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd(2+) induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd(2+) showed that Cd(2+) partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd(2+)-induced inactivation of SPAK. These results demonstrate the effect of Cd(2+) on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd(2+) in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefish.

  4. Hot Melt Extrusion and Spray Drying of Co-amorphous Indomethacin-Arginine With Polymers.

    PubMed

    Lenz, Elisabeth; Löbmann, Korbinian; Rades, Thomas; Knop, Klaus; Kleinebudde, Peter

    2017-01-01

    Co-amorphous drug-amino acid systems have gained growing interest as an alternative to common amorphous formulations which contain polymers as stabilizers. Several preparation methods have recently been investigated, including vibrational ball milling on a laboratory scale or spray drying in a larger scale. In this study, the feasibility of hot melt extrusion for continuous manufacturing of co-amorphous drug-amino acid formulations was examined, challenging the fact that amino acids melt with degradation at high temperatures. Furthermore, the need for an addition of a polymer in this process was evaluated. After a polymer screening via the solvent evaporation method, co-amorphous indomethacin-arginine was prepared by a melting-solvent extrusion process without and with copovidone. The obtained products were characterized with respect to their solid-state properties, non-sink dissolution behavior, and stability. Results were compared to those of spray-dried formulations with the same compositions and to spray-dried indomethacin-copovidone. Overall, stable co-amorphous systems could be prepared by extrusion without or with copovidone, which exhibited comparable molecular interaction properties to the respective spray-dried products, while phase separation was detected by differential scanning calorimetry in several cases. The formulations containing indomethacin in combination with arginine and copovidone showed enhanced dissolution behavior over the formulations with only copovidone or arginine.

  5. Influence of chemical modification of N alpha-cocoyl-L-arginine ethyl ester on its hepatitis B surface antigen-inactivating effect.

    PubMed Central

    Sugimoto, Y; Toyoshima, S

    1980-01-01

    We have reported previously that N alpha-cocoyl-L-arginine ethyl ester (CAE) strongly inactivates hepatitis B surface antigen (HBsAg; Sugimoto and Toyoshima, Antimicrob. Agents Chemother. 16:329--332, 1979). Replacement of the L-arginine moiety of CAE by L-lysine did not decrease the HBsAg-inactivating effect of CAE, whereas replacement by some neutral amino acids and L-ornithine decreased it. Esterification of the carboxyl group of N alpha-acyl-L-arginine enhanced its inactivating effect. When the ethyl ester of CAE was converted to an amide group, the effect was appreciably decreased. Modification of the carboxyl group was essential for the inactivation. The effectiveness of N alpha-acyl-L-arginine ethyl ester depends upon the length of the acyl group, with the optimum length for the inactivation of HBsAg being C12 to C14. In addition to CAE, N alpha-lauroyl-L-lysine ethyl ester and N alpha-cocoyl-L-arginine amide were found to be strong inactivators of HBsAg. Significant inactivating effects on HBsAg were not observed in many anionic detergents containing an amino acid. These results suggest that for strongly inactivating HBsAg, a compound should contain a special amino acid, such as L-arginine, and a long acyl group and exhibit a cationic property. PMID:7447415

  6. Arginine mutations within a transmembrane domain of Tar, an Escherichia coli aspartate receptor, can drive homodimer dissociation and heterodimer association in vivo

    PubMed Central

    2004-01-01

    The interactions between the TM (transmembrane) domains of many membrane proteins are important for their proper functioning. Mutations of residues into positively charged ones within TM domains were reported to be involved in many genetic diseases, possibly because these mutations affect the self- and/or hetero-assembly of the corresponding proteins. To our knowledge, despite significant progress in understanding the role of various amino acids in TM–TM interactions in vivo, the direct effect of positively charged residues on these interactions has not been studied. To address this issue, we employed the N-terminal TM domain of the aspartate receptor (Tar-1) as a dimerization model system. We expressed within the ToxR TM assembly system several Tar-1 constructs that dimerize via polar- or non-polar amino acid motifs, and mutated these by replacement with a single arginine residue. Our results have revealed that a mutation in each of the motifs significantly reduced the ability of the TMs to dimerize. Furthermore, a Tar-1 construct that contained two arginine residues was unable to correctly integrate itself into the membrane. Nevertheless, an exogenous synthetic Tar-1 peptide containing these two arginine residues was able to inhibit in vivo the marked dimerization of a mutant Tar-1 construct that contained two glutamate residues at similar positions. This indicates that hetero-assembly of TM domains can be mediated by the interaction of two oppositely charged residues, probably by formation of ion pairs. This study broadens our knowledge regarding the effect of positively charged residues on TM–TM interactions in vivo, and provides a potential therapeutic approach to inhibit uncontrolled dimerization of TM domains caused by mutations of polar amino acids. PMID:15330757

  7. Arginine metabolism and nutrition in growth, health and disease

    PubMed Central

    Bazer, Fuller W.; Davis, Teresa A.; Kim, Sung Woo; Li, Peng; Rhoads, J. Marc; Satterfield, M. Carey; Smith, Stephen B.; Spencer, Thomas E.; Yin, Yulong

    2009-01-01

    l-Arginine (Arg) is synthesised from glutamine, glutamate, and proline via the intestinal-renal axis in humans and most other mammals (including pigs, sheep and rats). Arg degradation occurs via multiple pathways that are initiated by arginase, nitric-oxide synthase, Arg:glycine amidinotransferase, and Arg decarboxylase. These pathways produce nitric oxide, polyamines, proline, glutamate, creatine, and agmatine with each having enormous biological importance. Arg is also required for the detoxification of ammonia, which is an extremely toxic substance for the central nervous system. There is compelling evidence that Arg regulates interorgan metabolism of energy substrates and the function of multiple organs. The results of both experimental and clinical studies indicate that Arg is a nutritionally essential amino acid (AA) for spermatogenesis, embryonic survival, fetal and neonatal growth, as well as maintenance of vascular tone and hemodynamics. Moreover, a growing body of evidence clearly indicates that dietary supplementation or intravenous administration of Arg is beneficial in improving reproductive, cardiovascular, pulmonary, renal, gastrointestinal, liver and immune functions, as well as facilitating wound healing, enhancing insulin sensitivity, and maintaining tissue integrity. Additionally, Arg or l-citrulline may provide novel and effective therapies for obesity, diabetes, and the metabolic syndrome. The effect of Arg in treating many developmental and health problems is unique among AAs, and offers great promise for improved health and wellbeing of humans and animals. PMID:19030957

  8. A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase

    PubMed Central

    Huang, Qi; Alcock, Felicity; Kneuper, Holger; Deme, Justin C.; Rollauer, Sarah E.; Berks, Ben C.

    2017-01-01

    The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA–YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase. PMID:28223511

  9. Cell compatible arginine containing cationic polymer: one-pot synthesis and preliminary biological assessment.

    PubMed

    Zavradashvili, Nino; Memanishvili, Tamar; Kupatadze, Nino; Baldi, Lucia; Shen, Xiao; Tugushi, David; Wandrey, Christine; Katsarava, Ramaz

    2014-01-01

    Synthetic cationic polymers are of interest as both nonviral vectors for intracellular gene delivery and antimicrobial agents. For both applications synthetic polymers containing guanidine groups are of special interest since such kind of organic compounds/polymers show a high transfection potential along with antibacterial activity. It is important that the delocalization of the positive charge of the cationic group in guanidine significantly decreases the toxicity compared to the ammonium functionality. One of the most convenient ways for incorporating guanidine groups is the synthesis of polymers composed of the amino acid arginine (Arg) via either application of Arg-based monomers or chemical modification of polymers with derivatives of Arg. It is also important to have biodegradable cationic polymers that will be cleared from the body after their function as transfection or antimicrobial agent is fulfilled. This chapter deals with a two-step/one-pot synthesis of a new biodegradable cationic polymer-poly(ethylene malamide) containing L-arginine methyl ester covalently attached to the macrochains in β-position of the malamide residue via the α-amino group. The goal cationic polymer was synthesized by in situ interaction of arginine methyl ester dihydrochloride with intermediary poly(ethylene epoxy succinimide) formed by polycondensation of di-p-nitrophenyl-trans-epoxy succinate with ethylenediamine. The cell compatibility study with Chinese hamster ovary (CHO) and insect Schneider 2 cells (S2) within the concentration range of 0.02-500 mg/mL revealed that the new polymer is not cytotoxic. It formed nanocomplexes with pDNA (120-180 nm in size) at low polymer/DNA weight ratios (WR = 5-10). A preliminarily transfection efficiency of the Arg-containing new cationic polymer was assessed using CHO, S2, H5, and Sf9 cells.

  10. Novel arginine deiminase-based method to assay L-arginine in beverages.

    PubMed

    Stasyuk, N Ye; Gayda, G Z; Fayura, L R; Boretskyy, Y R; Gonchar, M V; Sibirny, A A

    2016-06-15

    A highly selective and sensitive enzymatic method for the quantitative determination of L-arginine (Arg) has been developed. The method is based on the use of recombinant bacterial arginine deiminase (ADI) isolated from the cells of a recombinant strain Escherichia coli and o-phthalaldehyde (OPA) as a chemical reagent. Ammonia, the product of the enzymatic digestion of Arg by ADI, reacts with OPA and forms in the presence of sulfite a product, which can be detected by spectrophotometry (S) and fluorometry (F). The linear concentration range for Arg assay in the final reaction mixture varies for ADI-OPA-F variant of the method from 0.35 μM to 24 μM with the detection limit of 0.25 μM. For ADI-OPA-S variant of the assay, the linearity varies from 0.7 μM to 50 μM with the detection limit of 0.55 μM. The new method was tested on real samples of wines and juices. A high correlation (R=0.978) was shown for the results obtained with the proposed and the reference enzymatic method.

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

    SciTech Connect

    Wang, H.-C.; Kao, Y.-C.; Chang, T-J.; Wong, M.-L. . E-mail: mlwong@dragon.nchu.edu.tw

    2005-08-26

    Pseudorabies virus (PRV) is a member of Alphahepesviruses; it is an enveloped virus with a double-stranded DNA genome. Polyamines (such as spermine and spermidine) are ubiquitous in animal cells and participate in cellular proliferation and differentiation. Previous results of our laboratory showed that the PRV can accomplish lytic infection either in the presence of exogenous spermine (or spermidine) or depletion of cellular polyamines. The amino acid arginine is a precursor of polyamine biosynthesis. In this work, we investigated the role of arginine in PRV infection. It was found that the plaque formation of PRV was inhibited by arginase (enzyme 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.

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

    SciTech Connect

    Nakadate, Yusuke; Uchida, Koji; Shikata, Keiji; Yoshimura, Saori; Azuma, Masayuki; Hirata, Tatsumi; Konishi, Hiroyuki; Kiyama, Hiroshi; Tachibana, Taro

    2009-01-09

    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.

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

    PubMed Central

    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

  14. Arginine side chains as a dispersant for individual single-wall carbon nanotubes.

    PubMed

    Hirano, Atsushi; Tanaka, Takeshi; Kataura, Hiromichi; Kameda, Tomoshi

    2014-04-22

    Charged peptides and proteins disperse single-wall carbon nanotubes (SWCNTs) in aqueous solutions. However, little is known about the role of their side chains in their interactions with SWCNTs. Homopolypeptide-SWCNT systems are ideal for investigating the mechanisms of such interactions. In this study, we demonstrate that SWCNTs are individually dispersed by poly-L-arginine (PLA). The debundled SWCNTs exhibited a distinct fluorescence. The dispersibility of SWCNTs with PLA was greater than that of SWCNTs with poly-L-lysine (PLL). Molecular dynamics simulations suggest that the side chains of PLA have stronger interactions with the sidewalls of SWCNTs compared with those of PLL. The guanidinium group at the end of the side chain of an arginine residue plays an important role in the interaction with SWCNTs, likely through hydrophobic, van der Waals, and π-π interactions. PLA can be useful as a tool for the dispersion of SWCNTs and can be used to non-covalently anchor materials to SWCNTs with strong binding.

  15. Insights into the Mechanism of Streptonigrin-Induced Protein Arginine Deiminase Inactivation

    PubMed Central

    Dreyton, Christina J.; Anderson, Erin D.; Subramanian, Venkataraman; Boger, Dale L.; Thompson, Paul R.

    2014-01-01

    Protein citrullination is just one of more than 200 known PTMs. This modification, catalyzed by the Protein Arginine Deiminases (PADs 1–4 and PAD6 in humans), converts the positively charged guanidinium group of an arginine residue into a neutral ureido-group. Given the strong links between dysregulated PAD activity and human disease, we initiated a program to develop PAD inhibitors as potential therapeutics for these and other diseases in which the PADs are thought to play a role. Streptonigrin which possesses both anti-tumor and anti-bacterial activity was later identified as a highly potent PAD4 inhibitor. In an effort to understand why streptonigrin is such a potent and selective PAD4 inhibitor, we explored its structure-activity relationships by examining the inhibitory effects of several analogues that mimic the A, B, C, and/or D rings of streptonigrin. We report the identification of the 7-amino-quinoline-5,8-dione core of streptonigrin as a highly potent pharmacophore that acts as a pan-PAD inhibitor. PMID:24440480

  16. Fragment based discovery of Arginine isosteres through REPLACE: towards non-ATP competitive CDK inhibitors

    PubMed Central

    Premnath, Padmavathy Nandha; Liu, Shu; Perkins, Tracy; Abbott, Jennifer; Anderson, Erin; McInnes, Campbell

    2013-01-01

    In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating binding to protein-protein interaction sites. Further to this, potential arginine isosteres predicted using the validated LigandFit docking method were ligated to the truncated C-terminal peptide, RLIF using solid phase synthesis and evaluated in a competitive binding assay. After testing, identified fragments were shown to represent not only appropriate mimics for a critical arginine residue but also to interact effectively with a minor hydrophobic pocket present in the binding groove. Further evaluation of binding modes was undertaken to optimize the potency of these compounds. Through further application of the REPLACE strategy in this study, peptide-small molecule hybrid CDK2 inhibitors were identified that are more drug-like and suitable for further optimization as anti-tumor therapeutics. PMID:24286762

  17. Selective inhibition of protein arginine methyltransferase 5 blocks initiation and maintenance of B-cell transformation

    PubMed Central

    Alinari, Lapo; Mahasenan, Kiran V.; Yan, Fengting; Karkhanis, Vrajesh; Chung, Ji-Hyun; Smith, Emily M.; Quinion, Carl; Smith, Porsha L.; Kim, Lisa; Patton, John T.; Lapalombella, Rosa; Yu, Bo; Wu, Yun; Roy, Satavisha; De Leo, Alessandra; Pileri, Stefano; Agostinelli, Claudio; Ayers, Leona; Bradner, James E.; Chen-Kiang, Selina; Elemento, Olivier; Motiwala, Tasneem; Majumder, Sarmila; Byrd, John C.; Jacob, Samson; Sif, Said; Li, Chenglong

    2015-01-01

    Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)–induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV+ lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas. PMID:25742700

  18. Insights into the mechanism of streptonigrin-induced protein arginine deiminase inactivation.

    PubMed

    Dreyton, Christina J; Anderson, Erin D; Subramanian, Venkataraman; Boger, Dale L; Thompson, Paul R

    2014-02-15

    Protein citrullination is just one of more than 200 known PTMs. This modification, catalyzed by the protein arginine deiminases (PADs 1-4 and PAD6 in humans), converts the positively charged guanidinium group of an arginine residue into a neutral ureido-group. Given the strong links between dysregulated PAD activity and human disease, we initiated a program to develop PAD inhibitors as potential therapeutics for these and other diseases in which the PADs are thought to play a role. Streptonigrin which possesses both anti-tumor and anti-bacterial activity was later identified as a highly potent PAD4 inhibitor. In an effort to understand why streptonigrin is such a potent and selective PAD4 inhibitor, we explored its structure-activity relationships by examining the inhibitory effects of several analogues that mimic the A, B, C, and/or D rings of streptonigrin. We report the identification of the 7-amino-quinoline-5,8-dione core of streptonigrin as a highly potent pharmacophore that acts as a pan-PAD inhibitor.

  19. Arginine-phosphate salt bridges between histones and DNA: Intermolecular actuators that control nucleosome architecture

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir I.; Li, Yun; Singh, Gautam; Olson, Wilma K.

    2014-10-01

    Structural bioinformatics and van der Waals density functional theory are combined to investigate the mechanochemical impact of a major class of histone-DNA interactions, namely, the formation of salt bridges between arginine residues in histones and phosphate groups on the DNA backbone. Principal component analysis reveals that the configurational fluctuations of the sugar-phosphate backbone display sequence-specific directionality and variability, and clustering of nucleosome crystal structures identifies two major salt-bridge configurations: a monodentate form in which the arginine end-group guanidinium only forms one hydrogen bond with the phosphate, and a bidentate form in which it forms two. Density functional theory calculations highlight that the combination of sequence, denticity, and salt-bridge positioning enables the histones to apply a tunable mechanochemical stress to the DNA via precise and specific activation of backbone deformations. The results suggest that selection for specific placements of van der Waals contacts, with high-precision control of the spatial distribution of intermolecular forces, may serve as an underlying evolutionary design principle for the structure and function of nucleosomes, a conjecture that is corroborated by previous experimental studies.

  20. Protein arginine methyltransferase 1 is a novel regulator of MYCN in neuroblastoma

    PubMed Central

    Eberhardt, Allison; Hansen, Jeanne N.; Koster, Jan; Lotta, Louis T.; Wang, Simeng; Livingstone, Emmett; Qian, Kun; Valentijn, Linda J.; Zheng, Yujun George; Schor, Nina F.; Li, Xingguo

    2016-01-01

    Amplification or overexpression of MYCN is associated with poor prognosis of human neuroblastoma. We have recently defined a MYCN-dependent transcriptional signature, including protein arginine methyltransferase 1 (PRMT1), which identifies a subgroup of patients with high-risk disease. Here we provide several lines of evidence demonstrating PRMT1 as a novel regulator of MYCN and implicating PRMT1 as a potential therapeutic target in neuroblastoma pathogenesis. First, we observed a strong correlation between MYCN and PRMT1 protein levels in primary neuroblastoma tumors. Second, MYCN physically associates with PRMT1 by direct protein-protein interaction. Third, depletion of PRMT1 through siRNA knockdown reduced neuroblastoma cell viability and MYCN expression. Fourth, we showed that PRMT1 regulates MYCN stability and identified MYCN as a novel substrate of PRMT1. Finally, we demonstrated that mutation of putatively methylated arginine R65 to alanine decreased MYCN stability by altering phosphorylation at residues serine 62 and threonine 58. These results provide mechanistic insights into the modulation of MYCN oncoprotein by PRMT1, and suggest that targeting PRMT1 may have a therapeutic impact on MYCN-driven oncogenesis. PMID:27571165

  1. Anaerobic arginine metabolism of Mycobacterium tuberculosis is mediated by arginine deiminase (arcA), but is not essential for chronic persistence in an aerogenic mouse model of infection.

    PubMed

    Sürken, Michael; Keller, Christine; Röhker, Claudia; Ehlers, Stefan; Bange, Franz-Christoph

    2008-10-01

    In many pathogens, degradation of arginine via the arginine deiminase pathway supports anaerobic metabolism. Here we show by deletion of Rv1001 (arcA) in Mycobacterium tuberculosis that this gene functions as an arginine deiminase. Arginine metabolism in the presence of oxygen was not affected by the mutation, indicating a separate pathway for arginine degradation under aerobic conditions. Following aerosol infection in mice, the DeltaarcA mutant and wild-type strain of M. tuberculosis multiplied and persisted in infected organs in a similar fashion.

  2. Thermodynamic characterization of the DmsD binding site for the DmsA twin-arginine motif.

    PubMed

    Winstone, Tara M L; Turner, Raymond J

    2015-03-24

    The system specific chaperone DmsD interacts with the twin-arginine leader peptide of its substrate, DmsA, allowing for proper folding and assembly of the DmsA catalytic subunit of dimethyl sulfoxide reductase prior to translocation by the twin-arginine translocase. DmsD residues important for binding the complete 45-amino acid sequence of the DmsA leader (DmsAL) peptide were previously identified and found to cluster in a pocket of the DmsD structure. In this study, we have utilized isothermal titration calorimetry (ITC) to determine the dissociation constant and thermodynamic parameters of 15 single-substitution DmsD variant proteins and a synthetic DmsAL peptide consisting of 27 amino acids (DmsAL₁₅₋₄₁). The stoichiometry values were determined via ITC, and the multimeric compositions of the DmsD variants in the absence and presence of peptide were characterized via size exclusion chromatography and native polyacrylamide gel electrophoresis. An up to 4-fold change in affinity was observed for DmsD variant proteins relative to that of wild-type DmsD, and variation of the entropic contribution to binding divided the binding site into two clusters: residues with either more or less favorable entropy. Substitution of hydrophobic residues along one helix face (helix 5) or prolines found on adjacent loops caused reduced binding affinity because of the increased entropic cost, which suggests that the twin-arginine motif of the DmsAL peptide binds to a preformed site on DmsD. Most DmsD variants were more than 90% monomeric in solution and bound a single peptide per protein molecule. The DmsD variant with the largest dimer population showed increased affinity and induced the formation of tetramers in the presence of peptide, suggesting that dimeric DmsD or an alternatively folded form of DmsD may play an as yet undefined role in binding.

  3. Arginine Metabolism in Bacterial Pathogenesis and Cancer Therapy

    PubMed Central

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

    2016-01-01

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

  4. Dietary L-arginine and cutaneous wound healing.

    PubMed

    Naderpour, Masoud; Rad, Jafar Soleimani; Ayat, Esmail; Mesgari, Mehran; Farahani, Ramin M; Roshangar, Leila; Tubbs, R Shane; Shoja, Mohammadali M

    2008-01-01

    Skin wound healing has been the subject of extensive studies and various drugs have been used in an attempt to improve wound healing. There are conflicting data regarding the effects of L-arginine, the substrate of nitric oxide, on wound healing. We examined the 1-week rate of cutaneous wound healing and collagen deposition in three groups of rats who received a (1) L-arginine (2% in drinking water)-supplemented diet from three days before until the seventh day following injury (Group 1), (2) L-arginine-supplemented diet for three days before injury (Group 2), and (3) a standard diet without L-arginine supplementation (Group 3). The wound length and width were measured each day and then the open wound area and cumulative percentage of open wound area reduction were calculated. Wound biopsy samples were examined with Trichrome-Masson stain in a subgroup of animals. Results showed that Group 1 rats had a significantly lower cumulative percentage of open wound area reduction on day 7 compared to other two groups (Mann-Whitney U test, P < 0.05). Relatively higher degrees of wound collagen deposit (day 7) were noted in groups 2 and 3. It may be concluded that L-arginine (2% in water) administered three days before until the seventh day following skin wound induction may diminish the rate of skin wound healing and collagen deposition.

  5. Increased mitochondrial arginine metabolism supports bioenergetics in asthma

    PubMed Central

    Xu, Weiling; Ghosh, Sudakshina; Comhair, Suzy A.A.; Asosingh, Kewal; Janocha, Allison J.; Mavrakis, Deloris A.; Bennett, Carole D.; Gruca, Lourdes L.; Graham, Brian B.; Queisser, Kimberly A.; Kao, Christina C.; Wedes, Samuel H.; Petrich, John M.; Tuder, Rubin M.; Kalhan, Satish C.; Erzurum, Serpil C.

    2016-01-01

    High levels of arginine metabolizing enzymes, including inducible nitric oxide synthase (iNOS) and arginase (ARG), are typical in asthmatic airway epithelium; however, little is known about the metabolic effects of enhanced arginine flux in asthma. Here, we demonstrated that increased metabolism sustains arginine availability in asthmatic airway epithelium with consequences for bioenergetics and inflammation. Expression of iNOS, ARG2, arginine synthetic enzymes, and mitochondrial respiratory complexes III and IV was elevated in asthmatic lung samples compared with healthy controls. ARG2 overexpression in a human bronchial epithelial cell line accelerated oxidative bioenergetic pathways and suppressed hypoxia-inducible factors (HIFs) and phosphorylation of the signal transducer for atopic Th2 inflammation STAT6 (pSTAT6), both of which are implicated in asthma etiology. Arg2-deficient mice had lower mitochondrial membrane potential and greater HIF-2α than WT animals. In an allergen-induced asthma model, mice lacking Arg2 had greater Th2 inflammation than WT mice, as indicated by higher levels of pSTAT6, IL-13, IL-17, eotaxin, and eosinophils and more mucus metaplasia. Bone marrow transplants from Arg2-deficient mice did not affect airway inflammation in recipient mice, supporting resident lung cells as the drivers of elevated Th2 inflammation. These data demonstrate that arginine flux preserves cellular respiration and suppresses pathological signaling events that promote inflammation in asthma. PMID:27214549

  6. Oral administration of both tetrahydrobiopterin and L-arginine prevents endothelial dysfunction in rats with chronic renal failure.

    PubMed

    Yamamizu, Kohei; Shinozaki, Kazuya; Ayajiki, Kazuhide; Gemba, Munekazu; Okamura, Tomio

    2007-03-01

    We examined the mechanism of endothelial dysfunction in chronic renal failure (CRF), with reference to NO synthase. CRF was induced by 5/6 nephrectomy in rats. Either L-arginine (1.25 g/L in drinking water), tetrahydrobiopterin (BH4, 10 mg/kg per day in food), or a combination of the 2 were orally administered to CRF rats for 9 weeks. CRF rats showed elevation of systolic blood pressure compared with sham-operated rats. Endothelium-dependent relaxation induced by acetylcholine or A23187 in the isolated aorta was significantly reduced, and in vitro treatment with L-arginine, BH4, or superoxide dismutase restored the relaxation. Aortic segments from CRF rats showed significantly higher superoxide production in response to A23187, which was inhibited by L-NAME. Plasma concentrations of asymmetric dimethylarginine and symmetric dimethylarginine were higher in CRF rats. These changes in CRF rats were totally or partially decreased by L-arginine or BH4 supplementation in vivo. Interestingly, the combined treatment showed additive effects in certain parameters. These results suggest that vascular disorders in CRF rats may be partly due to NOS uncoupling caused by a relative deficiency of BH4 and partially due to accumulation of endogenous inhibitors of NOS and L-arginine uptake, resulting in the decrease of NO production and the increase of reactive oxygen species.

  7. The arginine deiminase pathway of Lactobacillus fermentum IMDO 130101 responds to growth under stress conditions of both temperature and salt.

    PubMed

    Vrancken, G; Rimaux, T; Wouters, D; Leroy, F; De Vuyst, L

    2009-10-01

    The arginine deiminase (ADI) pathway is a means by which certain sourdough lactic acid bacteria (LAB) convert arginine into ornithine via citrulline while producing ammonia and ATP, thereby coping with acid stress and gaining an energetic advantage. Lactobacillus fermentum IMDO 130101, an isolate from a spontaneous laboratory rye sourdough, possesses an ADI pathway which is modulated by environmental pH. In the present study, a broader view of the activity of the ADI pathway in response to growth under two other commonly encountered stress factors, temperature and added salt, was obtained. In both cases, an increase in ornithine production was observed as a response to growth under both temperature and salt stress conditions. Biokinetic parameters were obtained to describe the kinetics of the ADI pathway as a function of temperature and added salt. The arginine conversion rate increased as a function of added NaCl concentrations but was hardly affected by temperature. In addition, arginine-into-citrulline conversion rate was not affected by temperature but increased with increasing NaCl concentrations. Citrulline-into-ornithine conversion rate increased with increasing temperature, while it dropped to zero with added salt. These findings suggest a more pronounced adaptation of the strain through the ADI pathway to added salt, as compared with different constant temperatures. Furthermore, these results suggest that the ADI pathway in L. fermentum IMDO 130101 is active in adapting to non-optimal growth conditions.

  8. PEGylation and pharmacological characterization of a potential anti-tumor drug, an engineered arginine deiminase originated from Pseudomonas plecoglossicida.

    PubMed

    Zhang, Long; Liu, Menghan; Jamil, Serwanja; Han, Ruizhi; Xu, Guochao; Ni, Ye

    2015-02-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for arginine-auxotrophic tumors. PEGylation is one of the best methods to formulate a bioconjugated protein with extended physical stability and reduced immunogenicity. Here, PEGylation and pharmacological properties of an engineered ADI originated from Pseudomonas plecoglossicida were studied. Among polyethylene glycol (PEG) reagents with succinimidyl ester groups varying in size and linkers, three PEGylated products with high yield and catalytic activity were further characterized, named ADI-SS(20 kDa), ADI-SC(20 kDa), and ADI-SPA(20 kDa). In the pharmacodynamic/pharmacokinetic (PD/PK) studies with ADI-SPA(20 kDa), a remarkable improvement in circulating half-life compared with native ADI was observed. ADI-SPA(20 kDa) injections via intravenous, intramuscular and subcutaneous routes all exhibited superior efficacy than native ADI on depleting serum arginine. Additionally, our results demonstrated that single ADI-SPA(20 kDa) administration of 5 U/mouse via intravenous injection could maintain serum arginine at an undetectable level for 5 days with a half-life of 53.2 h, representing 11-fold improvement in half-life than that of the native ADI. In a mice H22 hepatocarcinoma model, ADI-SPA(20 kDa) dosage of 5 U per 5 days showed an inhibition rate of 95.02% on tumor growth during 15-day treatments.

  9. Determination of l-arginine and NG, NG - and NG, NG' -dimethyl-L-arginine in plasma by liquid chromatography as AccQ-Fluor fluorescent derivatives.

    PubMed

    Heresztyn, Tamila; Worthley, Matthew I; Horowitz, John D

    2004-06-15

    A new HPLC assay for the detection of L-arginine, NG, NG-dimethyl-L-arginine (ADMA) and NG, NG' -dimethyl-L-arginine (SDMA) in plasma using the derivatisation reagent AccQ-Fluor (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) is described. The fluorescent derivatives produced are extremely stable enabling routine processing of large numbers of samples. Arginine and its metabolites are extracted from plasma on strong cation exchange (SCX) cartridges with NG-monomethyl-L-arginine (NMMA) as internal standard, derivatised and separated on a C18 column with acetonitrile in 0.1M sodium acetate buffer pH 6. Separation of the stereoisomers ADMA and SDMA was excellent and improvements to the solid phase extraction (SPE) procedure enabled good recovery (>80%) of arginine, ADMA and SDMA. The utility of the method is exemplified by comparison of plasma concentrations of ADMA, SDMA and arginine in healthy volunteers and diabetic/ischaemic patients.

  10. Crystallization and preliminary X-ray diffraction analysis of the arginine repressor ArgR from Bacillus halodurans

    PubMed Central

    Kang, Jina; Park, Young Woo; Yeo, Hyun Ku; Lee, Jae Young

    2015-01-01

    The arginine repressor (ArgR) is a transcriptional regulator which regulates genes encoding proteins involved in arginine biosynthesis and the arginine catabolic pathway. ArgR from the alkaliphilic bacterium Bacillus halodurans was cloned and overexpressed in Escherichia coli. ArgR (Bh2777) from B. halodurans is composed of 149 amino-acid residues with a molecular mass of 16 836 Da. ArgR was crystallized at 296 K using 1,2-propanediol as a precipitant. Crystals of N-terminally His-tagged ArgR were obtained by the sitting-drop vapour-diffusion method. Dehydrated crystals showed a dramatic improvement in diffraction quality and diffracted to 2.35 Å resolution. The crystals belonged to the cubic space group I23, with unit-cell parameters a = b = c = 104.68 Å. The asymmetric unit contained one monomer of ArgR, which generates a trimer by the threefold axis of the space group, giving a crystal volume per mass (V M) of 2.98 Å3 Da−1 and a solvent content of 56.8%. PMID:25760703

  11. Reengineering of a Corynebacterium glutamicum l-Arginine and l-Citrulline Producer▿

    PubMed Central

    Ikeda, Masato; Mitsuhashi, Satoshi; Tanaka, Kenji; Hayashi, Mikiro

    2009-01-01

    Toward the creation of a robust and efficient producer of l-arginine and l-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92up, and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (ΔargR), the production was further increased. The best mutation set, ΔargR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical l-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefold-increased production without retardation, revealing that the limitation in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance, thus

  12. L-arginine destabilizes oral multi-species biofilm communities developed in human saliva.

    PubMed

    Kolderman, Ethan; Bettampadi, Deepti; Samarian, Derek; Dowd, Scot E; Foxman, Betsy; Jakubovics, Nicholas S; Rickard, Alexander H

    2015-01-01

    The amino acid L-arginine inhibits bacterial coaggregation, is involved in cell-cell signaling, and alters bacterial metabolism in a broad range of species present in the human oral cavity. Given the range of effects of L-arginine on bacteria, we hypothesized that L-arginine might alter multi-species oral biofilm development and cause developed multi-species biofilms to disassemble. Because of these potential biofilm-destabilizing effects, we also hypothesized that L-arginine might enhance the efficacy of antimicrobials that normally cannot rapidly penetrate biofilms. A static microplate biofilm system and a controlled-flow microfluidic system were used to develop multi-species oral biofilms derived from pooled unfiltered cell-containing saliva (CCS) in pooled filter-sterilized cell-free saliva (CFS) at 37° C. The addition of pH neutral L-arginine monohydrochloride (LAHCl) to CFS was found to exert negligible antimicrobial effects but significantly altered biofilm architecture in a concentration-dependent manner. Under controlled flow, the biovolume of biofilms (μm(3)/μm(2)) developed in saliva containing 100-500 mM LAHCl were up to two orders of magnitude less than when developed without LAHCI. Culture-independent community analysis demonstrated that 500 mM LAHCl substantially altered biofilm species composition: the proportion of Streptococcus and Veillonella species increased and the proportion of Gram-negative bacteria such as Neisseria and Aggregatibacter species was reduced. Adding LAHCl to pre-formed biofilms also reduced biovolume, presumably by altering cell-cell interactions and causing cell detachment. Furthermore, supplementing 0.01% cetylpyridinium chloride (CPC), an antimicrobial commonly used for the treatment of dental plaque, with 500 mM LAHCl resulted in greater penetration of CPC into the biofilms and significantly greater killing compared to a non-supplemented 0.01% CPC solution. Collectively, this work demonstrates that LAHCl moderates multi

  13. Analogues of arginine vasopressin modified in position 2 and 3 with conformationally constrained dipeptide fragments.

    PubMed

    Łempicka, Elzbieta; Derdowska, Izabela; Kowalczyk, Wioleta; Dawidowska, Olga; Prahl, Adam; Janecki, Marcin; Jasiński, Tomasz; Trzeciak, Henryk I; Lammek, Bernard

    2005-02-01

    This study describes the synthesis and some pharmacological properties of ten new analogues of arginine vasopressin (AVP) containing a conformationally constrained dipeptide fragment in the N-terminal part of their molecules. Amino acid residues in positions 2 and 3 of AVP and some of its agonistic analogues were replaced with -Phe-Phe and D-Phe-D-Phe, dipeptides having a -CH2-CH2- link bridging two nitrogens. All the new peptides were tested for vasopressor and antidiuretic activities. Four peptides with pA2 values ranging from 5.96 to 7.21 turned out to be weak or moderately potent V1a antagonists. The results supplied new information about the structure-activity relationship of AVP analogues. As some of these were unexpected, they point to the need for caution when extrapolating previously known effects of modifications to analogues having conformationally constrained fragments in their molecules.

  14. L-arginine and arginine analogues: effects on isolated blood vessels and cultured endothelial cells.

    PubMed Central

    Schmidt, H. H.; Baeblich, S. E.; Zernikow, B. C.; Klein, M. M.; Böhme, E.

    1990-01-01

    1. The present study examined effects of arginine (Arg) and various Arg analogues on the vascular tone of rabbit and rat aortic rings, the release of nitrite from cultured bovine aortic endothelial cells and the metabolism of L-Arg in bovine and porcine endothelial cell homogenates. The respective D-enantiomers or N-alpha-benzoyl-L-arginine ethyl ester did not substitute for L-Arg. 2. In bovine aortic endothelial cells, the release of nitrite was only observed in the presence of L-Arg or L-Arg methyl ester in the cell culture medium. 3. In dialyzed homogenates of porcine and bovine aortic endothelial cells, L-Arg was metabolized independently of NADPH and Ca2+ to yield L-ornithine (L-Orn) and L-citrulline (L-Cit). No concomitant nitrite formation was detected. 4. Pretreatment of rabbit and rat aortic rings with L-canavanine (L-Can) or NG-monomethyl-L-Arg (L-NMMA) inhibited ATP- and acetylcholine-induced relaxations (endothelium-dependent) but not glyceryltrinitrate-induced relaxations (endothelium-independent). 5. In rabbit aortic rings, Arg and monomeric Arg analogues induced endothelium-independent relaxations. L-Arg methyl ester induced an endothelium-independent contraction, and L-NMMA induced a relaxation in the absence of endothelium and a contraction in the presence of endothelium. Polymeric basic amino acids such as poly L-Arg induced endothelium-dependent relaxations (inhibited by L-Can), a subsequent refractoriness to endothelium-dependent vasodilators (not prevented by L-Can) and endothelial cell death.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2282457

  15. Residue 82 of the Chikungunya Virus E2 Attachment Protein Modulates Viral Dissemination and Arthritis in Mice

    PubMed Central

    Ashbrook, Alison W.; Burrack, Kristina S.; Silva, Laurie A.; Montgomery, Stephanie A.; Heise, Mark T.; Morrison, Thomas E.

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has reemerged to cause profound epidemics of fever, rash, and arthralgia throughout sub-Saharan Africa, Southeast Asia, and the Caribbean. Like other arthritogenic alphaviruses, mechanisms of CHIKV pathogenesis are not well defined. Using the attenuated CHIKV strain 181/25 and virulent strain AF15561, we identified a residue in the E2 viral attachment protein that is a critical determinant of viral replication in cultured cells and pathogenesis in vivo. Viruses containing an arginine at E2 residue 82 displayed enhanced infectivity in mammalian cells but reduced infectivity in mosquito cells and diminished virulence in a mouse model of CHIKV disease. Mice inoculated with virus containing an arginine at this position exhibited reduced swelling at the site of inoculation with a concomitant decrease in the severity of necrosis in joint-associated tissues. Viruses containing a glycine at E2 residue 82 produced higher titers in the spleen and serum at early times postinfection. Using wild-type and glycosaminoglycan (GAG)-deficient Chinese hamster ovary (CHO) cell lines and soluble GAGs, we found that an arginine at residue 82 conferred greater dependence on GAGs for infection of mammalian cells. These data suggest that CHIKV E2 interactions with GAGs diminish dissemination to lymphoid tissue, establishment of viremia, and activation of inflammatory responses early in infection. Collectively, these results suggest a function for GAG utilization in regulating CHIKV tropism and host responses that contribute to arthritis. IMPORTANCE CHIKV is a reemerging alphavirus of global significance with high potential to spread into new, immunologically naive populations. The severity of CHIKV disease, particularly its propensity for chronic musculoskeletal manifestations, emphasizes the need for identification of genetic determinants that dictate CHIKV virulence in the host. To better understand mechanisms of

  16. Lotus hairy roots expressing inducible arginine decarboxylase activity.

    PubMed

    Chiesa, María A; Ruiz, Oscar A; Sánchez, Diego H

    2004-05-01

    Biotechnological uses of plant cell-tissue culture usually rely on constitutive transgene expression. However, such expression of transgenes may not always be desirable. In those cases, the use of an inducible promoter could be an alternative approach. To test this hypothesis, we developed two binary vectors harboring a stress-inducible promoter from Arabidopsis thaliana, driving the beta-glucuronidase reporter gene and the oat arginine decarboxylase. Transgenic hairy roots of Lotus corniculatus were obtained with osmotic- and cold-inducible beta-glucuronidase and arginine decarboxylase activities. The increase in the activity of the latter was accompanied by a significant rise in total free polyamines level. Through an organogenesis process, we obtained L. corniculatus transgenic plants avoiding deleterious phenotypes frequently associated with the constitutive over-expression of arginine decarboxylation and putrescine accumulation.

  17. Renal cell carcinoma does not express argininosuccinate synthetase and is highly sensitive to arginine deprivation via arginine deiminase.

    PubMed

    Yoon, Cheol-Yong; Shim, Young-Jun; Kim, Eun-Ho; Lee, Ju-Han; Won, Nam-Hee; Kim, Jeong-Hun; Park, In-Sun; Yoon, Duck-Ki; Min, Bon-Hong

    2007-02-15

    Recently, pegylated arginine deiminase (ADI; EC 3.5.3.6) has been used to treat the patients with hepatocellular carcinoma or melanoma, in which the level of argininosuccinate synthetase (ASS) activity is low or undetectable. The efficacy of its antitumor activity largely depends on the level of intracellular ASS, which enables tumor cells to recycle citrulline to arginine. Thus, we examined the expression levels of ASS in various cancer cells and found that it is low in renal cell carcinoma (RCC) cells, rendering the cells highly sensitive to arginine deprivation by ADI treatment. Immunohistochemical analysis revealed that in biopsy specimens from RCC patients (n = 98), the expression of ASS is highly demonstrated in the epithelium of normal proximal tubule but not seen in tumor cells. Furthermore, RCC cells treated with ADI showed remarkable growth retardation in a dose dependent manner. ADI also exerted in vivo antiproliferative effect on the allografted renal cell carcinoma (RENCA) tumor cells and prolonged the survival of tumor-bearing mice. Histological examination of the tumors revealed that tumor angiogenesis and vascular endothelial growth factor (VEGF) expression were significantly diminished by ADI administration. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of RCC in ways of inhibitions of arginine availability and neovascularization.

  18. Protective effect of L-arginine on gentamicin-induced nephrotoxicity in rats

    PubMed Central

    Başhan, İbrahim; Başhan, Perihan; Seçilmiş, Mehmet Ata; Şingirik, Ergin

    2014-01-01

    Introduction: L-arginine has a protective effect on gentamicin-induced renal failure and it may decrease the tubular reabsorption of another cationic substance, gentamicin due to its cationic structure. The aim of this study is to compare the possible protective effects of L-arginine and its inactive isomer D-arginine on gentamicin-induced nephrotoxicity in rats. Materials and Methods: Wistar albino rats were housed in metabolic cages and assigned to six groups as: control group, gentamicin (100 mg/kg), gentamicin + L-arginine (2 g/l), gentamicin + D-arginine (2 g/l), gentamicin + L-arginine + Nv-nitro-L-arginine methyl ester (L-NAME) (100 mg/l) and gentamicin + D-arginine + L-NAME. Gentamicin was administered by subcutaneous injections and the other drugs were added in drinking water for seven consecutive days. The animals were killed by decapitation and intracardiac blood and urine samples were obtained on the seventh day. Blood urea nitrogen, serum creatinine, sodium, potassium, urine gamma glutamyl transferase, creatinine, sodium, potassium and gentamicin levels were measured using High Performance Liquid Chromatography (HPLC) technique. Results: Gentamicin treated group had significant increase in blood urea nitrogen, serum creatinine, fractional Na excretion and urine gamma glutamyl transferase levels, and significant decrease in creatinine clearance compared to the control group. L-arginine and D-arginine reversed these findings. L-NAME abolished the nephroprotective effect of L-arginine. The urinary levels of gentamicin were significantly increased in rats treated with L-arginine or D-arginine compared to those treated with gentamicin. L-arginine and D-arginine reversed the advanced degenerative changes due to gentamicin administration in histopathological examination. Conclusion: Our study revealed the protective effect of L-arginine on gentamicin-induced nephrotoxicity, the contribution of the cationic feature of L-arginine, and the major role of NO in

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

    PubMed

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

    2015-06-29

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

  20. Inactivation of microbial arginine deiminases by L-canavanine.

    PubMed

    Li, Ling; Li, Zhimin; Chen, Danqi; Lu, Xuefeng; Feng, Xiaohua; Wright, Elizabeth C; Solberg, Nathan O; Dunaway-Mariano, Debra; Mariano, Patrick S; Galkin, Andrey; Kulakova, Liudmila; Herzberg, Osnat; Green-Church, Kari B; Zhang, Liwen

    2008-02-13

    Arginine deiminase (ADI) catalyzes the hydrolytic conversion of L-arginine to ammonia and L-citrulline as part of the energy-producing L-arginine degradation pathway. The chemical mechanism for ADI catalysis involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate. The structure of the Pseudomonas aeruginosa ADI-(L-arginine) complex guided the design of arginine analogs that might react with the ADIs to form inactive covalent adducts during catalytic turnover. One such candidate is L-canavanine, in which an N-methylene of L-arginine is replaced by an N-O. This substance was shown to be a slow substrate-producing O-ureido-L-homoserine. An in depth kinetic and mass spectrometric analysis of P. aeruginosa ADI inhibition by L-canavanine showed that two competing pathways are followed that branch at the Cys-alkylthiouronium ion intermediate. One pathway leads to direct formation of O-ureido-L-homoserine via a reactive thiouronium intermediate. The other pathway leads to an inactive form of the enzyme, which was shown by chemical model and mass spectrometric studies to be a Cys-alkylisothiourea adduct. This adduct undergoes slow hydrolysis to form O-ureido-L-homoserine and regenerated enzyme. In contrast, kinetic and mass spectrometric investigations demonstrate that the Cys-alkylthiouronium ion intermediate formed in the reaction of L-canavanine with Bacillus cereus ADI partitions between the product forming pathway (O-ureido-L-homoserine and free enzyme) and an inactivation pathway that leads to a stable Cys-alkylthiocarbamate adduct. The ADIs from Escherichia coli, Burkholderia mallei, and Giardia intestinalis were examined in order to demonstrate the generality of the L-canavanine slow substrate inhibition and to distinguish the kinetic behavior that defines the irreversible inhibition observed with the B. cereus ADI from the time controlled inhibition observed with the P. aeruginosa, E. coli, B. mallei, and G. intestinalis ADIs.

  1. Peptidomimetics as protein arginine deiminase 4 (PAD4) inhibitors.

    PubMed

    Trabocchi, Andrea; Pala, Nicolino; Krimmelbein, Ilga; Menchi, Gloria; Guarna, Antonio; Sechi, Mario; Dreker, Tobias; Scozzafava, Andrea; Supuran, Claudiu T; Carta, Fabrizio

    2015-06-01

    The protein arginine deiminase 4 (PAD4) is a calcium-dependent enzyme, which catalyses the irreversible conversion of peptidyl-arginines into peptidyl-citrullines and plays an important role in several diseases such as in the rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, Creutzfeldt-Jacob's disease and cancer. In this study, we report the inhibition profiles and computational docking toward the PAD4 enzyme of a series of 1,2,3-triazole peptidomimetic-based derivatives incorporating the β-phenylalanine and guanidine scaffolds. Several effective, low micromolar PAD4 inhibitors are reported in this study.

  2. Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1

    PubMed Central

    Castellano, Sabrina; Spannhoff, Astrid; Milite, Ciro; Dal Piaz, Fabrizio; Cheng, Donghang; Tosco, Alessandra; Viviano, Monica; Yamani, Abdellah; Cianciulli, Agostino; Sala, Marina; Cura, Vincent; Cavarelli, Jean; Novellino, Ettore; Mai, Antonello; Bedford, Mark T.; Sbardella, Gianluca

    2012-01-01

    Arginine methylation is a common post-translational modification that is crucial in modulating gene expression at multiple critical levels. The arginine methyltransferases (PRMTs) are envisaged as promising druggable targets but their role in physiological and pathological pathways is far from being clear, due to the limited number of modulators reported to date. In this effort, enzyme activators can be invaluable tools useful as gain-of-function reagents to interrogate the biological roles in cells and in vivo of PRMTs. Yet the identification of such molecules is rarely pursued. Herein we describe a series of aryl ureido acetamido indole carboxylates (dubbed “uracandolates”), able to increase the methylation of histone- (H3) or non-histone (polyadenylate-binding protein 1, PABP1) substrates induced by coactivator-associated arginine methyltransferase 1 (CARM1), both in in vitro and cellular settings. To the best of our knowledge, this is the first report of compounds acting as CARM1 activators. PMID:23095008

  3. Chimpanzee Personality and the Arginine Vasopressin Receptor 1A Genotype.

    PubMed

    Wilson, V A D; Weiss, A; Humle, T; Morimura, N; Udono, T; Idani, G; Matsuzawa, T; Hirata, S; Inoue-Murayama, M

    2017-03-01

    Polymorphisms of the arginine vasopressin receptor 1a (AVPR1a) gene have been linked to various measures related to human social behavior, including sibling conflict and agreeableness. In chimpanzees, AVPR1a polymorphisms have been associated with traits important for social interactions, including sociability, joint attention, dominance, conscientiousness, and hierarchical personality dimensions named low alpha/stability, disinhibition, and negative emotionality/low dominance. We examined associations between AVPR1a and six personality domains and hierarchical personality dimensions in 129 chimpanzees (Pan troglodytes) living in Japan or in a sanctuary in Guinea. We fit three linear and three animal models. The first model included genotype, the second included sex and genotype, and the third included genotype, sex, and sex × genotype. All personality phenotypes were heritable. Chimpanzees possessing the long form of the allele were higher in conscientiousness, but only in models that did not include the other predictors; however, additional analyses suggested that this may have been a consequence of study design. In animal models that included sex and sex × genotype, chimpanzees homozygous for the short form of the allele were higher in extraversion. Taken with the findings of previous studies of chimpanzees and humans, the findings related to conscientiousness suggest that AVPR1a may be related to lower levels of impulsive aggression. The direction of the association between AVPR1a genotype and extraversion ran counter to what one would expect if AVPR1a was related to social behaviors. These results help us further understand the genetic basis of personality in chimpanzees.

  4. A model study for tethering of (bio)active molecules to biomaterial surfaces through arginine.

    PubMed

    Taraballi, F; Russo, L; Battocchio, C; Polzonetti, G; Nicotra, F; Cipolla, L

    2014-06-28

    A new approach for tethering of bioactive molecules via arginine is proposed and validated on collagen 2D matrices. The method involves the introduction of a methyl ketone on arginine side-chains, followed by reaction with model alkoxyamino derivatives.

  5. Salt bridge stabilization of charged zwitterionic arginine aggregates in the gas phase.

    PubMed

    Julian, R R; Hodyss, R; Beauchamp, J L

    2001-04-18

    The discovery of several new unusually stable aggregates of arginine that are intermolecularly bound by salt bridges is reported. Quadrupole ion-trap mass spectrometry provides evidence for the stability of arginine in the zwitterionic state, where the protonated guanidinium group of one arginine interacts strongly with the carboxylate of another to form stable noncovalent complexes, coordinated to either a cation or anion. Clusters of arginine with itself, sodium, potassium, lithium, magnesium, chloride, fluoride, bromide, iodide, and nitrate are observed. DFT calculations at the B3LYP/6-31G level are used to assess the structures and energetics of particularly prominent clusters. An examination of mixtures of D-arginine with isotopically labeled L-arginine indicates that the stability of these clusters does not depend on arginine enantiomeric purity. The cyclic trimers of arginine, capped with either Cl(-) or NO(3)(-), possess exceptional stability.

  6. L-arginine does not improve biochemical and hormonal response in trained runners after 4 weeks of supplementation.

    PubMed

    Alvares, Thiago Silveira; Conte-Junior, Carlos Adam; Silva, Joab Trajano; Paschoalin, Vânia Margaret Flosi

    2014-01-01

    It has been hypothesized that L-arginine improves exercise performance by increasing nitric oxide synthesis and levels of insulin and growth hormone (GH). Metabolic and hormonal responses to chronic L-arginine supplementation may clarify the mechanisms underlying its putative physiologic effects on physical performance. Therefore, the aim of this study was to investigate the effects that 4 weeks of supplementation with L-arginine would have on metabolic and hormonal parameters at rest and in response to exercise. Fifteen healthy runners were divided into treatment (ARG; 6 g L-arginine) and placebo (PLA; 6 g cornstarch) groups. On the first visit, blood samples were collected for baseline, and the supplement or placebo was provided. After 4 weeks of supplementation (second visit), blood samples were collected at the following intervals: at rest, immediately after the first 5-km time-trial running test (5km-TT), immediately after the second 5km-TT, and after 20 minutes of recovery (+20). In addition to exercise performance (total running time), plasma nitrate, nitrite, nitrate plus nitrite, cyclic guanosine monophosphate, lactate, ammonia and serum insulin, GH, insulin-like growth factor 1, and cortisol concentrations were evaluated. There were significant increases in plasma nitrite, cyclic guanosine monophosphate, lactate, ammonia and serum GH, and cortisol at the first 5km-TT, immediately after the second 5km-TT, and +20 in both ARG and PLA. Nitrate plus nitrite and nitrate increased only at +20. No significant change was observed in serum insulin and insulin-like growth factor 1 in any sample period. Total running time did not differ significantly between the 2 tests, in either ARG or PLA. Thus, according to our results, 4 weeks of L-arginine supplementation did not cause beneficial changes in metabolic and hormonal parameters, beyond those achieved with exercise alone.

  7. Combined effects of aerobic exercise and l-arginine ingestion on blood pressure in normotensive postmenopausal women: A crossover study.

    PubMed

    Puga, Guilherme M; de P Novais, Iane; Katsanos, Christos S; Zanesco, Angelina

    2016-04-15

    After menopause the incidence of cardiovascular diseases increases in women. A decrease in nitric oxide (NO) bioavailability has been pointed out to play a major role in this phenomenon. Since it is believed that l-arginine administration could improve NO bioavailability, the aim of this study was to examine the effects of acute l-arginine administration associated with aerobic exercise on blood pressure (BP), redox state and inflammatory biomarkers in normotensive postmenopausal women (NPW). Sixteen volunteers (57±6yr) were subjected to four experimental sessions (crossover design): arginine+exercise (A-E); arginine (ARG); exercise+placebo (EXE); control (CON). Each session was initiated with either 9g of l-arginine ingestion (ARG or A-E days), placebo (EXE day), or nothing (CON day). The participants performed 30min of aerobic exercise (A-E and EXE days) or sitting rest (CON and ARG days). Blood samples were collected before each session and 45min after the intervention. Office BP and ambulatory blood pressure monitoring (ABPM) were evaluated. NO/cGMP pathway, redox state and inflammatory biomarkers were measured. Systolic BP decreased during the 24-hour in A-E and EXE sessions. However, diastolic BP reduced only in A-E session. No changes were found in the biomarkers concentrations. In conclusion, the association was effective in lowering diastolic BP in NPW. Additionally, physical exercise alone promoted a long lasting effect on systolic BP measured by ABPM in this population, although this beneficial effect was not associated with changes in the cardio-inflammatory biomarkers. Possibly, other factors such as neural influences could be mediating this effect.

  8. Effects of a food supplement rich in arginine in patients with smear positive pulmonary tuberculosis--a randomised trial.

    PubMed

    Schön, T; Idh, J; Westman, A; Elias, D; Abate, E; Diro, E; Moges, F; Kassu, A; Ayele, B; Forslund, T; Getachew, A; Britton, S; Stendahl, O; Sundqvist, T

    2011-09-01

    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 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 and secondary outcomes were sputum smear conversion, weight gain, sedimentation rate, reduction of cough and chest X-ray improvement as well as levels of NO in urine (uNO) or exhaled air (eNO) at two months. There was no effect of the intervention on the primary outcome (OR 1.44, 95% CI: 0.69-3.0, p = 0.39) 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 < 0.01). A low baseline eNO (<10 ppb) in HIV+/TB patients was associated with a decreased cure rate. We conclude that nutritional supplementation with a food supplement rich in arginine did not have any overall clinical effect. In the subgroup of HIV positive TB patients, it significantly increased the cure rate and as an additional finding in this subgroup, low initial levels of NO in exhaled air were associated with a poor clinical outcome but this needs to be confirmed in further studies.

  9. Effects of acute supplementation of L-arginine and nitrate on endurance and sprint performance in elite athletes.

    PubMed

    Sandbakk, Silvana Bucher; Sandbakk, Øyvind; Peacock, Oliver; James, Philip; Welde, Boye; Stokes, Keith; Böhlke, Nikolai; Tjønna, Arnt Erik

    2015-08-01

    This study examined the effects of acute supplementation with L-arginine and nitrate on running economy, endurance and sprint performance in endurance-trained athletes. In a randomised cross-over, double-blinded design we compared the effects of combined supplementation with 6 g L-arginine and 614 mg nitrate against 614 mg nitrate alone and placebo in nine male elite cross-country skiers (age 18 ± 0 years, VO2max 69.3 ± 5.8 ml ⋅ min(-1) ⋅ kg(-1)). After a 48-hour standardisation of nutrition and exercise the athletes were tested for plasma nitrate and nitrite concentrations, blood pressure, submaximal running economy at 10 km ⋅ h(-1) and 14 km ⋅ h(-1) at 1% incline and 180 m as well as 5-km time-trial running performances. Plasma nitrite concentration following L-arginine + nitrate supplementation (319 ± 54 nmol ⋅ L(-1)) did not differ from nitrate alone (328 ± 107 nmol ⋅ L(-1)), and both were higher than placebo (149 ± 64 nmol ⋅ L(-1), p < 0.01). There were no differences in physiological responses during submaximal running or in 5-km performance between treatments. The plasma nitrite concentrations indicate greater nitric oxide availability both following acute supplementation of L-arginine + nitrate and with nitrate alone compared to placebo, but no additional effect was revealed when L-arginine was added to nitrate. Still, there were no effects of supplementation on exercise economy or endurance running performance in endurance-trained cross-country skiers.

  10. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food additives, bromide ion and residual bromine, may be present in water, potable in accordance with...

  11. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food additives, bromide ion and residual bromine, may be present in water, potable in accordance with...

  12. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food additives, bromide ion and residual bromine, may be present in water, potable in accordance with...

  13. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food additives, bromide ion and residual bromine, may be present in water, potable in accordance with...

  14. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food additives, bromide ion and residual bromine, may be present in water, potable in accordance with...

  15. Physico-chemical changes during storage and sensory acceptance of low sodium probiotic Minas cheese added with arginine.

    PubMed

    Felicio, T L; Esmerino, E A; Vidal, V A S; Cappato, L P; Garcia, R K A; Cavalcanti, R N; Freitas, M Q; Conte Junior, C A; Padilha, M C; Silva, M C; Raices, R S L; Arellano, D B; Bollini, H M A; Pollonio, M A R; Cruz, A G

    2016-04-01

    The partial substitution of sodium chloride by potassium chloride (0%, 25%, and 50%) and addition of arginine (1% w/w) in probiotic Minas cheese was investigated. Microbiological (Lactococcus lactis and Lactobacillus acidophilus counts, and functionality of the prebiotics L. acidophilus), physicochemical (pH, proteolysis, organic acids, fatty acids, and volatile profiles), rheological (uniaxial compression) and sensory (hedonic test with 100 consumers) characterizations were carried out. The sodium reduction and addition of arginine did not constitute a hurdle to lactic and probiotic bacteria survival, with presented values of about 9 log CFU/g, ranging from 7.11 to 9.21 log CFU/g, respectively. In addition, lower pH values, higher proteolysis, and a decrease in toughness, elasticity and firmness were observed, as well as an increase in lactic, citric, and acetic acid contents. In contrast, no change was observed in the fatty acid profile. With respect to the sensory acceptance, the probiotic low-sodium Minas cheese presented scores above 6.00 (liked slightly) for the attributes flavor and overall acceptance. The addition of arginine can be a potential alternative for the development of probiotic dairy products with reduced sodium content.

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

    PubMed Central

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

    2015-01-01

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

  17. The Arginine/ADMA Ratio Is Related to the Prevention of Atherosclerotic Plaques in Hypercholesterolemic Rabbits When Giving a Combined Therapy with Atorvastatine and Arginine

    PubMed Central

    Brinkmann, Saskia J. H.; Wörner, Elisabeth A.; Buijs, Nikki; Richir, Milan; Cynober, Luc; van Leeuwen, Paul A. M.; Couderc, Rémy

    2015-01-01

    Supplementation with arginine in combination with atorvastatin is more efficient in reducing the size of an atherosclerotic plaque than treatment with a statin or arginine alone in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits. We evaluated the mechanism behind this feature by exploring the role of the arginine/asymmetric dimethylarginine (ADMA) ratio, which is the substrate and inhibitor of nitric oxide synthase (NOS) and thereby nitric oxide (NO), respectively. Methods: Rabbits were fed either an arginine diet (group A, n = 9), standard rabbit chow plus atorvastatin (group S, n = 8), standard rabbit chow plus an arginine diet with atorvastatin (group SA, n = 8) or standard rabbit chow (group C, n = 9) as control. Blood was sampled and the aorta was harvested for topographic and histological analysis. Plasma levels of arginine, ADMA, cholesterol and nitric oxide were determined and the arginine/ADMA ratio was calculated. Results: The decrease in ADMA levels over time was significantly correlated to fewer aortic lesions in the distal aorta and total aorta. The arginine/ADMA ratio was correlated to cholesterol levels and decrease in cholesterol levels over time in the SA group. A lower arginine/ADMA ratio was significantly correlated to lower NO levels in the S and C group. Discussion: A balance between arginine and ADMA is an important indicator in the prevention of the development of atherosclerotic plaques. PMID:26035753

  18. Metabolic Stress Induced by Arginine Deprivation Induces Autophagy Cell Death in Prostate Cancer

    DTIC Science & Technology

    2009-08-01

    death. Inhibition of autophagy appears to stimulate the induction of cell death. 15. SUBJECT TERMS Prostate cancer, autophagy, arginine deiminase ... arginine deiminase (ADI). Both of these enzymes shuttle arginine away from the urea cycle and have been demonstrated to reduce intracellular...F.Y.S. Chuang, R.J. Bold, and H-J. Kung. Arginine deiminase as a novel therapy for prostate cancer induces autophagy and caspase-independent

  19. The Effect of Selective D- or Nα-Methyl Arginine Substitution on the Activity of the Proline-Rich Antimicrobial Peptide, Chex1-Arg20

    PubMed Central

    Li, Wenyi; Sun, Zhe; O'Brien-Simpson, Neil M.; Otvos, Laszlo; Reynolds, Eric C.; Hossain, Mohammed A.; Separovic, Frances; Wade, John D.

    2017-01-01

    In vivo pharmacokinetics studies have shown that the proline-rich antimicrobial peptide, A3-APO, which is a discontinuous dimer of the peptide, Chex1-Arg20, undergoes degradation to small fragments at positions Pro6-Arg7 and Val19-Arg20. With the aim of minimizing or abolishing this degradation, a series of Chex1-Arg20 analogs were prepared via Fmoc/tBu solid phase peptide synthesis with D-arginine or, in some cases, peptide backbone Nα-methylated arginine, substitution at these sites. All the peptides were tested for antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae. The resulting activity of position-7 substitution of Chex1-Arg20 analogs showed that arginine-7 is a crucial residue for maintaining activity against K. pneumoniae. However, arginine-20 substitution had a much less deleterious effect on the antibacterial activity of the peptide. Moreover, none of these peptides displayed any cytotoxicity to HEK and H-4-II-E mammalian cells. These results will aid the development of more effective and stable PrAMPs via judicious amino acid substitutions. PMID:28154813

  20. Intestinal trophic effect of enteral arginine is independent of blood flow in neonatal piglets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arginine is an indispensable amino acid in neonates. Arginine is synthesized by gut epithelial cells and may have a protective role in preventing necrotizing enterocolitis. We hypothesized our method included that enteral arginine is a stimulus for intestinal blood flow and subsequent mucosal growth...

  1. Identification and Characterization of Ana o 3 Modifications on Arginine-111 Residue in Heated Cashew Nuts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heating foods can alter the physical, chemical, and biological characteristics of the proteins we consume. Raw and roasted cashew nut extracts were evaluated for allergen modifications by mass-spectrometry. We did not identify modifications on Ana o 1 or Ana o 2, but we observed two independent mo...

  2. Identification of critical amino acid residues and functional conservation of the Neurospora crassa and Rattus norvegicus orthologues of neuronal calcium sensor-1.

    PubMed

    Gohain, Dibakar; Deka, Rekha; Tamuli, Ranjan

    2016-12-01

    Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca(2+)) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca(2+) binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca(2+) stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca(2+) binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.

  3. Arginine metabolism and nutrition in growth, health and disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. Metabolic and immune effects of dietary arginine supplementation after burn.

    PubMed

    Saito, H; Trocki, O; Wang, S L; Gonce, S J; Joffe, S N; Alexander, J W

    1987-07-01

    The effect of supplemental dietary arginine on metabolism and immunity was studied in 36 burned guinea pigs (30% of total body surface area) with previously placed catheter gastrostomies. The animals were randomized into four groups. After an initial three-day adaptation period, all groups received continuous isonitrogenous, isocaloric (175 kcal [735 kJ]/kg/d), and isovolemic intragastric tube feedings until postburn day (PBD) 14. Groups A, B, C, and D received 0%, 1%, 2%, and 4%, respectively, of total energy intake as arginine given in the form of crystalline arginine hydrochloride with 22%, 21%, 20%, and 18%, respectively, of total energy as whey protein. The average body weight after burn decreased equally in all groups. Resting metabolic expenditure on PBD 6 was higher in groups B (151% +/- 6% of preburn) and C (156% +/- 7%) than in groups A (131% +/- 4%) and D (136% +/- 3%). Ear-thickness response to dinitrofluorobenzene challenge on PBD 12 showed the best response in group C. The mortality rates of groups A, B, C, and D were 56%, 29%, 22%, and 56%, respectively. This study suggests that oral dietary arginine supplementation up to 2% of energy intake may be beneficial after burn injury.

  5. The tribulations of toothpaste trials: Unethical arginine dentifrice research.

    PubMed

    Shaw, D; Naimi-Akbar, A; Astvaldsdottir, A

    2015-12-18

    Arginine toothpaste is being promoted as being more efficacious than conventional fluoride-only toothpaste. Recent revelations concerning the design and conduct of the clinical trials conducted on schoolchildren in China and Thailand cast serious doubt on these claims. This paper describes and analyses the ethical and design flaws affecting these studies.

  6. Arginine utilization of citrulline synthesis in arginase II knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The synthesis of citrulline (Cit) from arginine (Arg) in the small intestine depends on the activity of arginase II (ARG2). To test the hypothesis that Arg is the main dietary precursor for Cit synthesis, despite the lack of ARG2, tracer studies were conducted in WT and ARG2 ko conscious mice. WT mi...

  7. Poly-arginine conjugated triarylmethyl radical as intracellular spin label.

    PubMed

    Driesschaert, Benoit; Bobko, Andrey A; Eubank, Timothy D; Samouilov, Alexandre; Khramtsov, Valery V; Zweier, Jay L

    2016-04-01

    Stable triarylmethyl radicals are ideal spin labels used for biomedical electron paramagnetic resonance applications. Previously reported structures exhibit polar charged functions for water solubilization preventing them from crossing the cell membrane. We report the synthesis of a triarylmethyl radical conjugated to poly-arginine peptide allowing intracellular delivery of the paramagnetic label.

  8. RESIDUAL RISK ASSESSMENTS - RESIDUAL RISK ...

    EPA Pesticide Factsheets

    This source category previously subjected to a technology-based standard will be examined to determine if health or ecological risks are significant enough to warrant further regulation for Coke Ovens. These assesments utilize existing models and data bases to examine the multi-media and multi-pollutant impacts of air toxics emissions on human health and the environment. Details on the assessment process and methodologies can be found in EPA's Residual Risk Report to Congress issued in March of 1999 (see web site). To assess the health risks imposed by air toxics emissions from Coke Ovens to determine if control technology standards previously established are adequately protecting public health.

  9. Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin.

    PubMed

    Lu, Bangmin; Zhang, Bin; Qi, Wei; Zhu, Yanan; Zhao, Yan; Zhou, Nan; Sun, Rong; Bao, Jinku; Wu, Chuanfang

    2014-11-01

    Clematis montana lectin (CML), a novel mannose-binding lectin purified from C. montana Buch.-Ham stem (Ranunculaceae), has been proved to have hemagglutinating activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells. However, the biochemical properties of CML have not revealed and its structural information still needs to be elucidated. In this study, it was found that CML possessed quite good thermostability and alkaline resistance, and its hemagglutinating activity was bivalent metal cation dependent. In addition, hemagglutination test and fluorescence spectroscopy proved that GuHCl, urea, and sodium dodecyl sulfate could change the conformation of CML and further caused the loss of hemagglutination activity. Moreover, the changes of fluorescence spectrum indicated that the tryptophan (Trp) microenvironment conversion might be related to the conformation and bioactivities of CML. In addition, it was also found that Trp residues, arginine (Arg) residues, and sulfhydryl were important for the hemagglutinating activity of CML, but only Trp was proved to be crucial for the CML conformation. Furthermore, the Trp, Arg, and sulfhydryl-modified CML exhibited 97.17%, 76.99%, and 49.64% loss of its anti-proliferative activity, respectively, which was consistent with the alterations of its hemagglutinating activity. Given these findings, Trp residues on the surface of CML are essential for the active center to form substrate-accessible conformation and suitable environment for carbohydrate binding.

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

    PubMed

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

    2014-08-01

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

  11. Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by /sup 15/N NMR using magnetization transfer and indirect detection via protons

    SciTech Connect

    Live, D.H.; Cowburn, D.

    1987-10-06

    NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, /sup 15/N labeling being used to identify specific backbone /sup 15/N and /sup 1/H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence for hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neutrophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of /sup 15/N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone. The results suggest significant conformational alteration in neurophysin-hormone complexes at low pH possibly associated with protonation of the carboxyl group of the hormone-protein salt bridge.

  12. Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by 15N NMR using magnetization transfer and indirect detection via protons.

    PubMed

    Live, D H; Cowburn, D; Breslow, E

    1987-10-06

    NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, 15N labeling being used to identify specific backbone 15N and 1H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence of hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neurophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of 15N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Enzymatic production of l-citrulline by hydrolysis of the guanidinium group of l-arginine with recombinant arginine deiminase.

    PubMed

    Song, Wei; Sun, Xia; Chen, Xiulai; Liu, Dongxu; Liu, Liming

    2015-08-20

    In this study, a simple, efficient enzymatic production process for the environmentally friendly synthesis of l-citrulline from l-arginine was developed using arginine deiminase (ADI) from Lactococcus lactis. Following overexpression of L. lactis ADI in Escherichia. coli BL21 (DE3) and experimental evolution using error-prone PCR, mutant FMME106 was obtained with a Km for l-arginine of 3.5mM and a specific activity of 195.7U/mg. This mutant exhibited a maximal conversion of 92.6% and achieved a final l-citrulline concentration of 176.9g/L under optimal conditions (190g/L l-arginine, 15g/L whole-cell biocatalyst treated with 2% isopropanol for 30min, 50°C, pH 7.2, 8h). The average l-citrulline synthesis rate of 22.1g/L/h is considerably higher than that reported for other similar biocatalytic approaches, therefore the process developed in the present work has great potential for large-scale production of l-citrulline.

  14. Identification of tropomyosin and arginine kinase as major allergens of Portunus pelagicus (blue swimming crab).

    PubMed

    Rosmilah, M; Shahnaz, M; Zailatul, H M Y; Noormalin, A; Normilah, I

    2012-09-01

    Crab is an important source of food allergen. Tropomyosin represents the main crab allergen and is responsible for IgE cross-reactivity between various species of crustaceans. Recently, other new crab allergens including arginine kinase have been identified. However, information on allergens of the local Portunidcrab is not available. Thus, the aim of this study was to identify the major allergens of Portunus pelagicus (blue swimming crab) using the allergenomics approach. Raw and cooked extracts of the crab were prepared from the crab meat. Protein profile and IgE binding pattern were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from 30 patients with crab allergy. The major allergens of the crab were then identified by two-dimensional electrophoresis (2-DE), followed by mass spectrometry analysis of the peptide digests. The SDS-PAGE of raw extract revealed approximately 20 protein fractions over a wide molecular weight range, while cooked extract demonstrated fewer protein bands. The raw extract also demonstrated a higher number of IgE reactive bands than the cooked extract. A heat-resistant protein of 36 kDa has been identified as the major allergen in both raw and cooked extracts. In addition, a heat-sensitive protein of 41 kDa was also recognized as a major allergen in raw crab. The 2-DE gel profile of the raw extract demonstrated about >100 distinct proteins spots and immunoblotting of the 2-DE profile demonstrated at least 12 different major IgE reactive spots with molecular masses between 13 to 250 kDa and isoelectric point (pI) values ranging from 4.0 to 7.0. The 36 and 41 kDa proteins were identified as the crab tropomyosin and arginine kinase, respectively by mass spectrometry. Therefore, this study confirmed that tropomyosin and arginine kinase are the major allergens of the local Portunid crab, P. pelagicus.

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

    SciTech Connect

    Shin, Soyoung; Mohan, Srinidi; Fung, Ho-Leung

    2011-11-04

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

  16. Selective Intracellular Delivery of Recombinant Arginine Deiminase (ADI) Using pH-Sensitive Cell Penetrating Peptides To Overcome ADI Resistance in Hypoxic Breast Cancer Cells.

    PubMed

    Yeh, Tzyy-Harn; Chen, Yun-Ru; Chen, Szu-Ying; Shen, Wei-Chiang; Ann, David K; Zaro, Jennica L; Shen, Li-Jiuan

    2016-01-04

    Arginine depletion strategies, such as pegylated recombinant arginine deiminase (ADI-PEG20), offer a promising anticancer treatment. Many tumor cells have suppressed expression of a key enzyme, argininosuccinate synthetase 1 (ASS1), which converts citrulline to arginine. These tumor cells become arginine auxotrophic, as they can no longer synthesize endogenous arginine intracellularly from citrulline, and are therefore sensitive to arginine depletion therapy. However, since ADI-PEG20 only depletes extracellular arginine due to low internalization, ASS1-expressing cells are not susceptible to treatment since they can synthesize arginine intracellularly. Recent studies have found that several factors influence ASS1 expression. In this study, we evaluated the effect of hypoxia, frequently encountered in many solid tumors, on ASS1 expression and its relationship to ADI-resistance in human MDA-MB-231 breast cancer cells. It was found that MDA-MB-231 cells developed ADI resistance in hypoxic conditions with increased ASS1 expression. To restore ADI sensitivity as well as achieve tumor-selective delivery under hypoxia, we constructed a pH-sensitive cell penetrating peptide (CPP)-based delivery system to carry ADI inside cells to deplete both intra- and extracellular arginine. The delivery system was designed to activate the CPP-mediated internalization only at the mildly acidic pH (6.5-7) associated with the microenvironment of hypoxic tumors, thus achieving better selectivity toward tumor cells. The pH sensitivity of the CPP HBHAc was controlled by recombinant fusion to a histidine-glutamine (HE) oligopeptide, generating HBHAc-HE-ADI. The tumor distribution of HBHAc-HE-ADI was comparable to ADI-PEG20 in a mouse xenograft model of human breast cancer cells in vivo. In addition, HBHAc-HE-ADI showed increased in vitro cellular uptake in cells incubated in a mildly acidic pH (hypoxic conditions) compared to normal pH (normoxic conditions), which correlated with p

  17. Arginine-deficient diets alter plasma and tissue amino acids in young and aged rats.

    PubMed

    Gross, K L; Hartman, W J; Ronnenberg, A; Prior, R L

    1991-10-01

    Blood and urine metabolites were measured in two experiments for young (2-mo-old) and aged (20-mo-old) male Sprague-Dawley rats fed arginine-devoid diets made isonitrogenous to a control 1.12% arginine diet by adding alanine or glycine. Diet, fed for 7 or 13 d, had little effect on urinary or plasma ammonia and urea. Urinary orotate excretion was more than 40-fold higher in rats fed the arginine-deficient diets (P less than 0.01) in both experiments. Source of nonessential N (alanine or glycine) in the arginine-deficient diets did not alter orotic acid excretion or plasma or urine ammonia or urea. Changes in plasma arginine, alanine and glycine concentrations reflected the levels of these amino acids in the diet. Tissue ornithine levels reflected dietary arginine level, but tissue citrulline was unaffected by dietary arginine. Glutamate and glutamine were greater in the plasma and liver of rats fed arginine-deficient diets. Plasma concentrations of glutamate and glutamine were positively correlated with urinary orotic acid excretion (P less than 0.05) and ornithine and arginine were negatively correlated with orotic acid excretion (P less than 0.01). Increased tissue glutamine may be related to the greater orotate excretion in rats fed arginine-devoid diets. The metabolic responses to dietary arginine deficiency were similar in young and aged rats. In general, concentrations of amino acids in plasma, liver and spleen were higher in aged rats.

  18. Arginine Starvation Impairs Mitochondrial Respiratory Function in ASS1-Deficient Breast Cancer Cells

    PubMed Central

    Liu, Xiyong; Chu, Cheng-Ying; Shen, Li-Jiuan; Xu, Jinghong; Gaur, Shikha; Forman, Henry Jay; Zhang, Hang; Zheng, Shu; Yen, Yun; Huang, Jian; Kung, Hsing-Jien; Ann, David K.

    2014-01-01

    Autophagy is the principal catabolic response to nutrient starvation and is necessary to clear dysfunctional or damaged organelles, but excessive autophagy can be cytotoxic or cytostatic and contributes to cell death. Depending on the abundance of enzymes involved in molecule biosynthesis, cells can be dependent on uptake of exogenous nutrients to provide these molecules. Argininosuccinate synthetase 1 (ASS1) is a key enzyme in arginine biosynthesis, and its abundance is reduced in many solid tumors, making them sensitive to external arginine depletion. We demonstrated that prolonged arginine starvation by exposure to ADI-PEG20 (pegylated arginine deiminase) induced autophagy-dependent death of ASS1-deficient breast cancer cells, because these cells are arginine auxotrophs (dependent on uptake of extracellular arginine). Indeed, these breast cancer cells died in culture when exposed to ADI-PEG20 or cultured in the absence of arginine. Arginine starvation induced mitochondrial oxidative stress, which impaired mitochondrial bioenergetics and integrity. Furthermore, arginine starvation killed breast cancer cells in vivo and in vitro only if they were autophagy-competent. Thus, a key mechanism underlying the lethality induced by prolonged arginine starvation was the cytotoxic autophagy that occurred in response to mitochondrial damage. Last, ASS1 was either low in abundance or absent in more than 60% of 149 random breast cancer bio-samples, suggesting that patients with such tumors could be candidates for arginine starvation therapy. PMID:24692592

  19. Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency.

    PubMed

    Burrage, Lindsay C; Sun, Qin; Elsea, Sarah H; Jiang, Ming-Ming; Nagamani, Sandesh C S; Frankel, Arthur E; Stone, Everett; Alters, Susan E; Johnson, Dale E; Rowlinson, Scott W; Georgiou, George; Lee, Brendan H

    2015-11-15

    Arginase deficiency is caused by deficiency of arginase 1 (ARG1), a urea cycle enzyme that converts arginine to ornithine. Clinical features of arginase deficiency include elevated plasma arginine levels, spastic diplegia, intellectual disability, seizures and growth deficiency. Unlike other urea cycle disorders, recurrent hyperammonemia is typically less severe in this disorder. Normalization of plasma arginine levels is the consensus treatment goal, because elevations of arginine and its metabolites are suspected to contribute to the neurologic features. Using data from patients enrolled in a natural history study conducted by the Urea Cycle Disorders Consortium, we found that 97% of plasma arginine levels in subjects with arginase deficiency were above the normal range despite conventional treatment. Recently, arginine-degrading enzymes have been used to deplete arginine as a therapeutic strategy in cancer. We tested whether one of these enzymes, a pegylated human recombinant arginase 1 (AEB1102), reduces plasma arginine in murine models of arginase deficiency. In neonatal and adult mice with arginase deficiency, AEB1102 reduced the plasma arginine after single and repeated doses. However, survival did not improve likely, because this pegylated enzyme does not enter hepatocytes and does not improve hyperammonemia that accounts for lethality. Although murine models required dosing every 48 h, studies in cynomolgus monkeys indicate that less frequent dosing may be possible in patients. Given that elevated plasma arginine rather than hyperammonemia is the major treatment challenge, we propose that AEB1102 may have therapeutic potential as an arginine-reducing agent in patients with arginase deficiency.

  20. Detection of local polarity and conformational changes at the active site of rabbit muscle creatine kinase with a new arginine-specific fluorescent probe.

    PubMed

    Wang, Shujuan; Wang, Xiaochun; Shi, Wen; Wang, Ke; Ma, Huimin

    2008-02-01

    A new polarity-sensitive fluorescent probe, 3-(4-chloro-6-p-glyoxal-phenoxy-1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CGTDP), is synthesized for selective labeling of active-site arginine residues. The probe comprises a neutral red moiety as a polarity-sensitive fluorophore and a phenylglyoxal unit as an arginine-specific labeling group. The probe exhibits a sensitive response of shift of fluorescence maximum emission wavelength to solvent polarity only instead of pH or temperature, which leads to the use of the probe in detecting the local polarity and conformational changes of the active site of rabbit muscle creatine kinase (CK) denatured by pH or temperature. The polarity of the active site domain has been first found to correspond to a dielectric constant of about 44, and the conformational change of the active site directly revealed by CGTDP occurs far before that of CK as a whole disclosed by the intrinsic tryptophan fluorescence during acid or thermal denaturation. The present strategy may provide a useful method to detect the local polarity and conformational changes of the active sites of many enzymes that employ arginine residues as anion recognition sites under different denaturation conditions.

  1. Identification of an arginine452 to histidine substitution in the erythroid 5-aminolaevulinate synthetase gene in a large pedigree with X-linked hereditary sideroblastic anaemia.

    PubMed

    Edgar, A J; Losowsky, M S; Noble, J S; Wickramasinghe, S N

    1997-01-01

    The coding region of the erythroid 5-aminolaevulinate synthetase gene (ALAS2) from a large pedigree with pyridoxine-responsive X-linked hereditary sideroblastic anaemia was examined for mutations. In three affected males from this pedigree, single strand conformational polymorphism (SSCP) analysis showed anomalous migration of a PCR product spanning exon 9. Sequencing of amplified genomic DNA from one of these affected males revealed a guanine to adenine transition at nucleotide 1407 of the cDNA sequence in exon 9 of the gene. This mutation results in the loss of an HhaI restriction enzyme digest site. An HhaI digest assay demonstrated the presence of this mutation in other affected males but not in unaffected males and unrelated individuals. The point mutation results in an arginine to histidine substitution at amino acid residue 452. The arginine residue is conserved in both the erythroid and housekeeping ALAS genes in all known vertebrate sequences. This arginine is located in the middle of a predicted alpha-helix.

  2. Development and cytotoxicity of Schiff base derivative as a fluorescence probe for the detection of L-Arginine

    NASA Astrophysics Data System (ADS)

    Shang, Xuefang; Li, Jie; Guo, Kerong; Ti, Tongyu; Wang, Tianyun; Zhang, Jinlian

    2017-04-01

    Inspired from biological counter parts, chemical modification of Schiff base derivatives with function groups may provide a highly efficient method to detect amino acids. Therefore, a fluorescent probe involving Schiff base and hydroxyl group has been designed and prepared, which showed high response and specificity for Arginine (Arg) among normal eighteen standard kinds of amino acids (Alanine, Valine, Leucine, Isoleucine, Methionine, Asparticacid, Glutamicacid, Arginine, Glycine, Serine, Threonine, Asparagine, Phenylalanine, Histidine, Tryptophan, Proline, Lysine, Glutamine, Tyrosine and Cysteine). Furthermore, theoretical investigation further illustrated the possible binding mode in the host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. In addition, the synthesized fluorescent probe exhibited high binding ability for Arg and low cytotoxicity to MCF-7 cells over a concentration range of 0-200 μg mL-1 which can be also used as a biosensor for the Arg detection in vivo.

  3. Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation

    PubMed Central

    Yang, Jen-Hao; Chiou, Yi-Ying; Fu, Shu-Ling; Shih, I-Yun; Weng, Tsai-Hsuan; Lin, Wey-Jinq; Lin, Chao-Hsiung

    2014-01-01

    Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA/DNA-binding protein involved in chromatin remodeling, RNA processing and the DNA damage response. In addition, increased hnRNPK expression has been associated with tumor development and progression. A variety of post-translational modifications of hnRNPK have been identified and shown to regulate hnRNPK function, including phosphorylation, ubiquitination, sumoylation and methylation. However, the functional significance of hnRNPK arginine methylation remains unclear. In the present study, we demonstrated that the methylation of two essential arginines, Arg296 and Arg299, on hnRNPK inhibited a nearby Ser302 phosphorylation that was mediated through the pro-apoptotic kinase PKCδ. Notably, the engineered U2OS cells carrying an Arg296/Arg299 methylation-defective hnRNPK mutant exhibited increased apoptosis upon DNA damage. While such elevated apoptosis can be diminished through addition with wild-type hnRNPK, we further demonstrated that this increased apoptosis occurred through both intrinsic and extrinsic pathways and was p53 independent, at least in part. Here, we provide the first evidence that the arginine methylation of hnRNPK negatively regulates cell apoptosis through PKCδ-mediated signaling during DNA damage, which is essential for the anti-apoptotic role of hnRNPK in apoptosis and the evasion of apoptosis in cancer cells. PMID:25104022

  4. Adaptation to a long term (4 weeks) arginine- and precursor (glutamate, proline and aspartate)-free diet

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It is not known whether arginine homeostasis is negatively affected by a "long-term" dietary restriction of arginine and its major precursors in healthy adults. To assess the effects of a 4-week arginine- and precursor-free dietary intake on the regulatory mechanisms of arginine homeostasis in healt...

  5. Arginine in membranes: the connection between molecular dynamics simulations and translocon-mediated insertion experiments.

    PubMed

    Schow, Eric V; Freites, J Alfredo; Cheng, Philip; Bernsel, Andreas; von Heijne, Gunnar; White, Stephen H; Tobias, Douglas J

    2011-01-01

    Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths.

  6. Arginine kinase from the Tardigrade, Macrobiotus occidentalis: molecular cloning, phylogenetic analysis and enzymatic properties.

    PubMed

    Uda, Kouji; Ishida, Mikako; Matsui, Tohru; Suzuki, Tomohiko

    2010-10-01

    Arginine kinase (AK), which catalyzes the reversible transfer of phosphate from ATP to arginine to yield phosphoarginine and ADP, is widely distributed throughout the invertebrates. We determined the cDNA sequence of AK from the tardigrade (water bear) Macrobiotus occidentalis, cloned the sequence into pET30b plasmid, and expressed it in Escherichia coli as a 6x His-tag—fused protein. The cDNA is 1377 bp, has an open reading frame of 1080 bp, and has 5′- and 3′-untranslated regions of 116 and 297 bp, respectively. The open reading frame encodes a 359-amino acid protein containing the 12 residues considered necessary for substrate binding in Limulus AK. This is the first AK sequence from a tardigrade. From fragmented and non-annotated sequences available from DNA databases, we assembled 46 complete AK sequences: 26 from arthropods (including 19 from Insecta), 11 from nematodes, 4 from mollusks, 2 from cnidarians and 2 from onychophorans. No onychophoran sequences have been reported previously. The phylogenetic trees of 104 AKs indicated clearly that Macrobiotus AK (from the phylum Tardigrada) shows close affinity with Epiperipatus and Euperipatoides AKs (from the phylum Onychophora), and therefore forms a sister group with the arthropod AKs. Recombinant 6x His-tagged Macrobiotus AK was successfully expressed as a soluble protein, and the kinetic constants (K(m), K(d), V(ma) and k(cat)) were determined for the forward reaction. Comparison of these kinetic constants with those of AKs from other sources (arthropods, mollusks and nematodes) indicated that Macrobiotus AK is unique in that it has the highest values for k(cat) and K(d)K(m) (indicative of synergistic substrate binding) of all characterized AKs.

  7. Peptidyl arginine deiminase-4 activation exacerbates kidney ischemia-reperfusion injury.

    PubMed

    Ham, Ahrom; Rabadi, May; Kim, Mihwa; Brown, Kevin M; Ma, Zhe; D'Agati, Vivette; Lee, H Thomas

    2014-11-01

    Peptidyl arginine deiminase (PAD)4 is a nuclear enzyme that catalyzes the posttranslational conversion of arginine residues to citrulline. Posttranslational protein citrullination has been implicated in several inflammatory autoimmune diseases, including rheumatoid arthritis, colitis, and multiple sclerosis. Here, we tested the hypothesis that PAD4 contributes to ischemic acute kidney injury (AKI) by exacerbating the inflammatory response after renal ischemia-reperfusion (I/R). Renal I/R injury in mice increased PAD4 activity as well as PAD4 expression in the mouse kidney. After 30 min of renal I/R, vehicle-treated mice developed severe AKI with large increases in plasma creatinine. In contrast, mice pretreated with PAD4 inhibitors (2-chloroamidine or streptonigrin) had significantly reduced renal I/R injury. Further supporting a critical role for PAD4 in generating ischemic AKI, mice pretreated with recombinant human PAD4 (rPAD4) protein and subjected to mild (20 min) renal I/R developed exacerbated ischemic AKI. Consistent with the hypothesis that PAD4 regulates renal tubular inflammation after I/R, mice treated with a PAD4 inhibitor had significantly reduced renal neutrophil chemotactic cytokine (macrophage inflammatory protein-2 and keratinocyte-derived cytokine) expression and had decreased neutrophil infiltration. Furthermore, mice treated with rPAD4 had significantly increased renal tubular macrophage inflammatory protein-2 and keratinocyte-derived cytokine expression as well as increased neutrophil infiltration and necrosis. Finally, cultured mouse kidney proximal tubules treated with rPAD4 had significantly increased proinflammatory chemokine expression compared with vehicle-treated cells. Taken together, our results suggest that PAD4 plays a critical role in renal I/R injury by increasing renal tubular inflammatory responses and neutrophil infiltration after renal I/R.

  8. Peptidyl arginine deiminase-4 activation exacerbates kidney ischemia-reperfusion injury

    PubMed Central

    Ham, Ahrom; Rabadi, May; Kim, Mihwa; Brown, Kevin M.; Ma, Zhe; D'Agati, Vivette

    2014-01-01

    Peptidyl arginine deiminase (PAD)4 is a nuclear enzyme that catalyzes the posttranslational conversion of arginine residues to citrulline. Posttranslational protein citrullination has been implicated in several inflammatory autoimmune diseases, including rheumatoid arthritis, colitis, and multiple sclerosis. Here, we tested the hypothesis that PAD4 contributes to ischemic acute kidney injury (AKI) by exacerbating the inflammatory response after renal ischemia-reperfusion (I/R). Renal I/R injury in mice increased PAD4 activity as well as PAD4 expression in the mouse kidney. After 30 min of renal I/R, vehicle-treated mice developed severe AKI with large increases in plasma creatinine. In contrast, mice pretreated with PAD4 inhibitors (2-chloroamidine or streptonigrin) had significantly reduced renal I/R injury. Further supporting a critical role for PAD4 in generating ischemic AKI, mice pretreated with recombinant human PAD4 (rPAD4) protein and subjected to mild (20 min) renal I/R developed exacerbated ischemic AKI. Consistent with the hypothesis that PAD4 regulates renal tubular inflammation after I/R, mice treated with a PAD4 inhibitor had significantly reduced renal neutrophil chemotactic cytokine (macrophage inflammatory protein-2 and keratinocyte-derived cytokine) expression and had decreased neutrophil infiltration. Furthermore, mice treated with rPAD4 had significantly increased renal tubular macrophage inflammatory protein-2 and keratinocyte-derived cytokine expression as well as increased neutrophil infiltration and necrosis. Finally, cultured mouse kidney proximal tubules treated with rPAD4 had significantly increased proinflammatory chemokine expression compared with vehicle-treated cells. Taken together, our results suggest that PAD4 plays a critical role in renal I/R injury by increasing renal tubular inflammatory responses and neutrophil infiltration after renal I/R. PMID:25164081

  9. A Selective V1A Receptor Agonist, Selepressin, Is Superior to Arginine Vasopressin and to Norepinephrine in Ovine Septic Shock*

    PubMed Central

    He, Xinrong; Su, Fuhong; Taccone, Fabio Silvio; Laporte, Régent; Kjølbye, Anne Louise; Zhang, Jing; Xie, Keliang; Moussa, Mouhamed Djahoum; Reinheimer, Torsten Michael

    2016-01-01

    Objective: Selective vasopressin V1A receptor agonists may have advantages over arginine vasopressin in the treatment of septic shock. We compared the effects of selepressin, a selective V1A receptor agonist, arginine vasopressin, and norepinephrine on hemodynamics, organ function, and survival in an ovine septic shock model. Design: Randomized animal study. Setting: University hospital animal research laboratory. Subjects: Forty-six adult female sheep. Interventions: Fecal peritonitis was induced in the anesthetized, mechanically ventilated, fluid-resuscitated sheep, and they were randomized in two successive phases. Three late-intervention groups (each n = 6) received IV selepressin (1 pmol/kg/min), arginine vasopressin (0.25 pmol [0.1 mU]/kg/min), or norepinephrine (3 nmol [0.5 μg]/kg/min) when mean arterial pressure remained less than 70 mm Hg despite fluid challenge; study drugs were thereafter titrated to keep mean arterial pressure at 70–80 mm Hg. Three early-intervention groups (each n = 7) received selepressin, arginine vasopressin, or norepinephrine at the same initial infusion rates as for the late intervention, but already when mean arterial pressure had decreased by 10% from baseline; doses were then titrated as for the late intervention. A control group (n = 7) received saline. All animals were observed until death or for a maximum of 30 hours. Measurements and Main Results: In addition to hemodynamic and organ function assessment, plasma interleukin-6 and nitrite/nitrate levels were measured. In the late-intervention groups, selepressin delayed the decrease in mean arterial pressure and was associated with lower lung wet/dry weight ratios than in the other two groups. In the early-intervention groups, selepressin maintained mean arterial pressure and cardiac index better than arginine vasopressin or norepinephrine, slowed the increase in blood lactate levels, and was associated with less lung edema, lower cumulative fluid balance, and lower

  10. Effects of arginine vasopressin on musical working memory.

    PubMed

    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 < 0.05) with no main Session effect nor Group × Session interaction. In the Gordon test there was a main Session effect (p < 0.05) with scores higher in the second as compared to the first session, a marginal main Group 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.

  11. Effects of arginine vasopressin on musical working memory

    PubMed Central

    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 < 0.05) with no main Session effect nor Group × Session interaction. In the Gordon test there was a main Session effect (p < 0.05) with scores higher in the second as compared to the first session, a marginal main Group 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

  12. Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

    PubMed Central

    Defeo-Jones, D; Tai, J Y; Vuocolo, G A; Wegrzyn, R J; Schofield, T L; Riemen, M W; Oliff, A

    1989-01-01

    Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction. PMID:2506441

  13. Folding forms of Escherichia coli DmsD, a twin-arginine leader binding protein.

    PubMed

    Sarfo, Kwabena J; Winstone, Tara L; Papish, Andriyka L; Howell, Jenika M; Kadir, Hakan; Vogel, Hans J; Turner, Raymond J

    2004-03-05

    Escherichia coli DmsD interacts with the twin-arginine leader sequence of the catalytic sub-unit (DmsA) of DMSO reductase. DmsD was purified as a mixture of a number of different folding forms including: dimer (A); monomer (B); a minor thiol oxidized form; a heterogeneously folded or multi-conformational monomer form which displayed a ladder of bands on native-PAGE (D); and proteolytically degraded and aggregated forms. Polyacrylamide gel electrophoresis (PAGE), under denaturing and non-denaturing conditions, was used to examine the folding and stability of DmsD. Additionally, the biophysical methods of dynamic light scattering, circular dichroism, fluorescence, and mass spectroscopy were also used. Form D could be converted to form B by treatment with 4M urea, which is the concentration at which form B begins to denature. Forms A/B could be converted to D by incubation at pH 5.0. Forms A/B and D all had twin-arginine leader binding activity.

  14. Characterization of the activity and expression of arginine decarboxylase in human and animal Chlamydia pathogens.

    PubMed

    Bliven, Kimberly A; Fisher, Derek J; Maurelli, Anthony T

    2012-12-01

    Chlamydia pneumoniae encodes a functional arginine decarboxylase (ArgDC), AaxB, that activates upon self-cleavage and converts l-arginine to agmatine. In contrast, most Chlamydia trachomatis serovars carry a missense or nonsense mutation in aaxB abrogating activity. The G115R missense mutation was not predicted to impact AaxB functionality, making it unclear whether AaxB variations in other Chlamydia species also result in enzyme inactivation. To address the impact of gene polymorphism on functionality, we investigated the activity and production of the Chlamydia AaxB variants. Because ArgDC plays a critical role in the Escherichia coli acid stress response, we studied the ability of these Chlamydia variants to complement an E. coli ArgDC mutant in an acid shock assay. Active AaxB was detected in four additional species: Chlamydia caviae, Chlamydia pecorum, Chlamydia psittaci, and Chlamydia muridarum. Of the C. trachomatis serovars, only E appears to encode active enzyme. To determine when functional enzyme is present during the chlamydial developmental cycle, we utilized an anti-AaxB antibody to detect both uncleaved and cleaved enzyme throughout infection. Uncleaved enzyme production peaked around 20 h postinfection, with optimal cleavage around 44 h. While the role ArgDC plays in Chlamydia survival or virulence is unclear, our data suggest a niche-specific function.

  15. Effect of dietary lysine restriction and arginine supplementation in two patients with pyridoxine-dependent epilepsy.

    PubMed

    Yuzyuk, Tatiana; Thomas, Amanda; Viau, Krista; Liu, Aiping; De Biase, Irene; Botto, Lorenzo D; Pasquali, Marzia; Longo, Nicola

    2016-07-01

    Pyridoxine-Dependent Epilepsy (PDE) is a recessive disorder caused by deficiency of α-aminoadipic semialdehyde dehydrogenase in the catabolic pathway of lysine. It is characterized by intractable seizures controlled by the administration of pharmacological doses of vitamin B6. Despite seizure control with pyridoxine, intellectual disability and developmental delays are still observed in some patients with PDE, likely due to the accumulation of toxic intermediates in the lysine catabolic pathway: alpha-aminoadipic semialdehyde (AASA), delta-1-piperideine-6-carboxylate (P6C), and pipecolic acid. Here we evaluate biochemical and clinical parameters in two PDE patients treated with a lysine-restricted diet and arginine supplementation (100-150mg/kg), aimed at reducing the levels of PDE biomarkers. Lysine restriction resulted in decreased accumulation of PDE biomarkers and improved development. Plasma lysine but not plasma arginine, directly correlated with plasma levels of AASA-P6C (p<0.001, r(2)=0.640) and pipecolic acid (p<0.01, r(2)=0.484). In addition, plasma threonine strongly correlated with the levels of AASA-P6C (p<0.0001, r(2)=0.732) and pipecolic acid (p<0.005, r(2)=0.527), suggesting extreme sensitivity of threonine catabolism to pyridoxine availability. Our results further support the use of dietary therapies in combination with pyridoxine for the treatment of PDE.

  16. Evolution of the Maillard Reaction in Glutamine or Arginine-Dextrinomaltose Model Systems

    PubMed Central

    Pastoriza, Silvia; Rufián-Henares, José Ángel; García-Villanova, Belén; Guerra-Hernández, Eduardo

    2016-01-01

    Enteral formulas are foods designed for medical uses to feed patients who are unable to eat normally. They are prepared by mixing proteins, amino acids, carbohydrates and fats and submitted to sterilization. During thermal treatment, the Maillard reaction takes place through the reaction of animo acids with reducing sugars. Thus, although glutamine and arginine are usually added to improve the nutritional value of enteral formulas, their final concentration may vary. Thus, in the present paper the early, intermediate, and advanced states of the Maillard reaction were studied in model systems by measuring loss of free amino acids through the decrease of fluorescence intensity with o-phtaldialdehyde (OPA), 5-Hydroximethylfurfural (HMF), furfural, glucosylisomaltol, fluorescence, and absorbance at 420 nm. The systems were prepared by mixing glutamine or arginine with dextrinomaltose (similar ingredients to those used in special enteral formula), and heated at 100 °C, 120 °C and 140 °C for 0 to 30 min. The recorded changes in the concentration of furanic compounds was only useful for longer heating times of high temperatures, while absorbance and fluorescence measurements were useful in all the assayed conditions. In addition, easiness and sensitivity of absorbance and fluorescence make them useful techniques that could be implemented as indicators for monitoring the manufacture of special enteral formulas. Glucosylisomaltol is a useful indicator to monitor the manufacture of glutamine-enriched enteral formulas. PMID:28231180

  17. Synthesis of Mycoplasma arginine deiminase in E. coli using stress-responsive proteins.

    PubMed

    Ahn, Keum-Young; Lee, Boram; Han, Kyung-Yeon; Song, Jong-Am; Lee, Doo Sung; Lee, Jeewon

    2014-09-01

    We found Escherichia coli proteins, elongation factor Ts (Tsf), and malate dehydrogenase (Mdh) that can exist in the form of native and soluble proteins even under stress situation such as heat shock and protein denaturing condition. To examine their property as solubility enhancers, aggregation-prone Mycoplasma arginine deiminase (mADI), which has been suggested as anti-cancer agent, was fused to the C-terminal of each of them and cloned into pET28a to be expressed in the E. coli cytoplasm. When mADI was fused to fusion partners (Mdh, Tsf), a significant portion of the recombinant mADI was expressed in a soluble fraction (>90%) whereas the directly expressed mADI was aggregated to the inclusion body. In addition, recombinant mADI released from the fusion tag retained its soluble form and presented its specific enzymatic activity of converting l-arginine into citrulline (>10 U/mg). These results show that Tsf and Mdh could serve as effective solubility enhancers for aggregation-prone proteins (e.g. mADI in this case) when used as fusion expression partners in bacterial expression systems.

  18. Arginine decarboxylase as the source of putrescine for tobacco alkaloids

    NASA Technical Reports Server (NTRS)

    Tiburcio, A. F.; Galston, A. W.

    1986-01-01

    The putrescine which forms a part of nicotine and other pyrrolidine alkaloids is generally assumed to arise through the action of ornithine decarboxylase (ODC). However, we have previously noted that changes in the activity of arginine decarboxylase (ADC), an alternate source of putrescine, parallel changes in tissue alkaloids, while changes in ODC activity do not. This led us to undertake experiments to permit discrimination between ADC and ODC as enzymatic sources of putrescine destined for alkaloids. Two kinds of evidence presented here support a major role for ADC in the generation of putrescine going into alkaloids: (a) A specific 'suicide inhibitor' of ADC effectively inhibits the biosynthesis of nicotine and nornicotine in tobacco callus, while the analogous inhibitor of ODC is less effective, and (b) the flow of 14C from uniformly labelled arginine into nicotine is much more efficient than that from ornithine.

  19. Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src.

    PubMed Central

    Kamps, M P; Sefton, B M

    1986-01-01

    All 15 protein kinases whose amino acid sequence is known contain a lysine residue at a position homologous to that of lysine-295 in p60src, the transforming protein of Rous sarcoma virus. The ATP analog p-fluorosulfonyl 5'-benzoyl adenosine inactivates both p60src and the catalytic subunit of the cyclic AMP-dependent protein kinase by modification of this lysine. We used oligonucleotide-directed mutagenesis to examine the possible functions of this residue. Lysine-295 in p60src was replaced with a glutamic acid, an arginine, or a histidine residue, and mutant p60src proteins were characterized in chicken cells infected by mutant viruses. None of these three mutant p60src proteins had tyrosine protein kinase activity in vitro, and none induced morphological transformation of infected cells. Since neither a histidine nor an arginine residue can replace the function of lysine-295, we suggest that it carries out the specialized function of proton transfer in the phosphotransferase reaction. All three mutant viruses underwent reversion to wild type during passage in tissue culture. Because the rate with which this occurred differed significantly among the mutants, reversion appears to have resulted from errors in transcription, rather than from recombination with the cellular src gene. Images PMID:2430174

  20. Arginoplexes: an arginine-anchored nanoliposomal carrier for gene delivery

    NASA Astrophysics Data System (ADS)

    Patel, Ketan; Tyagi, Monica; Monpara, Jasmin; Vora, Lalit; Gupta, Sanjay; Vavia, Pradeep

    2014-04-01

    There is a need of an efficient and safe non-viral gene delivery carrier due to promising future of nucleic acid-based therapeutics in the treatment of intractable diseases. Cytotoxicity and cost are the major concerns with current quaternary ammonium-based cationic liposomes. The major aim of current research work was development and in vitro evaluation of arginine-anchored nanoliposomes for gene delivery. l-Arginine-fatty acid conjugate was synthesized and characterized using IR, NMR, and mass spectroscopy. Synthesized conjugate—lauroyl arginine ethyl ester (LAE) was successfully incorporated into liposomes. Effect of nanocarrier composition on DNA binding was evaluated by preparing solid lipid nanoparticle (SLN) and self nanoemulsifying system (SNES) using same LAE concentration. Effect of cationic head on DNA binding was also evaluated. Arginine-anchored nanoliposomes—arginoplexes (APX) showed superior DNA-binding affinity. Surface PEG was expected to cause hindrance in DNA binding in SLNs and SNES. Guanidino group was found to be a better cationic head for DNA binding compared to primary amine or quaternary amine. Gel retardation assay was performed to optimize the ratio of DNA to LAE in nanocarrier. Serum stability, haemolysis, cytotoxicity, and transfection studies were carried out to evaluate APX. Binding of DNA to APX was found to be stable in the presence of serum, and no degradation of DNA was observed. APX containing 2 mg/ml LAE which exhibited particle size of 72 nm with zeta potential of +57.5 mV, showed lower cytotoxicity and better transfection. APX can be a promising carrier for gene delivery.

  1. Characterization of the arginine deiminase of Streptococcus equi subsp. zooepidemicus.

    PubMed

    Hong, Kyongsu

    2006-09-01

    Streptococcus equi subsp. zooepidemicus is an important cause of infectious diseases in horses and rarely humans. Little is known about the virulence factors or protective antigens of S. equi subsp. zooepidemicus. In the present study, I designed original primers based on an alignment of the gene sagp(arcA) from Streptococcus pyogenes encoding streptococcal acid glycoprotein-arginine deiminase (SAGP/AD) to amplify the S. equi subsp. zooepidemicus counterpart sequence by polymerase chain reaction, and I analyzed the sagp(arcA) gene of the organism. Using chromosomal walking steps, I identified a contiguous eight-gene locus involved in SAGP/AD production. Their open reading frames were found to share significant homologies and to correspond closely in molecular mass to previously sequenced arc genes of S. pyogenes, thus they were designated ahrC.2 (arginine repressor), arcR (CRP/FNR transcription regulator), sagp(arcA) (streptococcal acid glycoprotein-arginine deiminase), putative acetyltransferase gene, arcB (ornithine carbamyl transferase), arcD (arginine-ornithine antiporter), arcT (Xaa-His peptidase), and arcC (carbamate kinase). The SAGP homologue of S. equi subsp. zooepidemicus (SzSAGP), encoded by arcA gene of the bacteria (arcA(SZ)), was successfully expressed in Escherichia coli and purified to homogeneity. When in vitro growth inhibitory activity of the recombinant SzSAGP was tested against MOLT-3 cells, it inhibited the growth of the cells during the 3 days of culture in a dose-dependent manner, accompanied by the induction of apoptotic cell death. The recombinant protein also possessed AD activity. By immunoblot analysis using both anti-SzSAGP-SfbI(H8) and anti-SfbI(H8) sera, I was able to demonstrate that the SzSAGP protein is expressed on the streptococcal surface.

  2. Food additives

    PubMed Central

    Spencer, Michael

    1974-01-01

    Food additives are discussed from the food technology point of view. The reasons for their use are summarized: (1) to protect food from chemical and microbiological attack; (2) to even out seasonal supplies; (3) to improve their eating quality; (4) to improve their nutritional value. The various types of food additives are considered, e.g. colours, flavours, emulsifiers, bread and flour additives, preservatives, and nutritional additives. The paper concludes with consideration of those circumstances in which the use of additives is (a) justified and (b) unjustified. PMID:4467857

  3. PRMT1-mediated arginine methylation controls ATXN2L localization

    SciTech Connect

    Kaehler, Christian; Guenther, Anika; Uhlich, Anja; Krobitsch, Sylvia

    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.

  4. Arginine Methylation by PRMT1 Regulates Muscle Stem Cell Fate.

    PubMed

    Blanc, Roméo Sébastien; Vogel, Gillian; Li, Xing; Yu, Zhenbao; Li, Shawn; Richard, Stéphane

    2017-02-01

    Quiescent muscle stem cells (MSCs) become activated in response to skeletal muscle injury to initiate regeneration. Activated MSCs proliferate and differentiate to repair damaged fibers or self-renew to maintain the pool and ensure future regeneration. The balance between self-renewal, proliferation, and differentiation is a tightly regulated process controlled by a genetic cascade involving determinant transcription factors such as Pax7, Myf5, MyoD, and MyoG. Recently, there have been several reports about the role of arginine methylation as a requirement for epigenetically mediated control of muscle regeneration. Here we report that the protein arginine methyltransferase 1 (PRMT1) is expressed in MSCs and that conditional ablation of PRMT1 in MSCs using Pax7(CreERT2) causes impairment of muscle regeneration. Importantly, PRMT1-deficient MSCs have enhanced cell proliferation after injury but are unable to terminate the myogenic differentiation program, leading to regeneration failure. We identify the coactivator of Six1, Eya1, as a substrate of PRMT1. We show that PRMT1 methylates Eya1 in vitro and that loss of PRMT1 function in vivo prevents Eya1 methylation. Moreover, we observe that PRMT1-deficient MSCs have reduced expression of Eya1/Six1 target MyoD due to disruption of Eya1 recruitment at the MyoD promoter and subsequent Eya1-mediated coactivation. These findings suggest that arginine methylation by PRMT1 regulates muscle stem cell fate through the Eya1/Six1/MyoD axis.

  5. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

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

    PubMed Central

    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

  7. Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

    PubMed

    Almeida, A M; Queiroz, J A; Sousa, F; Sousa, A

    2015-01-26

    The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26512645

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

    PubMed

    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.

  10. The Inhibitory Effects of Cu(2+) on Exopalaemon carinicauda Arginine Kinase via Inhibition Kinetics and Molecular Dynamics Simulations.

    PubMed

    Si, Yue-Xiu; Lee, Jinhyuk; Yin, Shang-Jun; Gu, Xiao-Xu; Park, Yong-Doo; Qian, Guo-Ying

    2015-06-01

    We studied the Cu(2+)-mediated inhibition and aggregation of Exopalaemon carinicauda arginine kinase (ECAK). We found that Cu(2+) significantly inactivated ECAK activity and double-reciprocal kinetics demonstrated that Cu(2+) induced noncompetitive inhibition of arginine and ATP (IC50 = 2.27 ± 0.16 μM; K i for arginine = 13.53 ± 3.76; K i for ATP = 4.02 ± 0.56). Spectrofluorometry results showed that Cu(2+) induced ECAK tertiary structural changes including the exposure of hydrophobic surfaces that directly induced ECAK aggregation. The addition of osmolytes such as glycine and proline successfully blocked ECAK aggregation induced by Cu(2+) and recovered ECAK activity. We built a 3D structure for ECAK using the ECAK ORF gene sequence. Molecular dynamics (MD) and docking simulations between ECAK and Cu(2+) were conducted to elucidate the binding mechanisms. The results showed that Cu(2+) blocked the entrance to the ATP active site; these results are consistent with the experimental result that Cu(2+) induced ECAK inactivation. Since arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrates, our study can provide new information about the effect of Cu(2+) on ECAK enzymatic function and unfolding, including aggregation, and the protective effects of osmolytes on ECAK folding to better understand the role of the invertebrate ECAK metabolic enzyme in marine environments.

  11. Arginine, soy isoflavone and hydroxypropylmethylcellulose have protective effects against obesity in broiler breeder hens fed on high-energy diets.

    PubMed

    Khalaji, S; Zaghari, M; Ganjkhanloo, M; Ghaziani, F

    2013-01-01

    1. The present study was undertaken to determine the effects of arginine, soy isoflavone (ISF) and hydroxypropylmethylcellulose (HPMC) on obesity in broiler breeder hens. 2. A total of 320 Cobb 500 hens, 45 weeks of age, were assigned to 64 floor pens. The experiment was conducted as a completely randomised design in a factorial arrangement (2 × 2 × 2 × 2) with 4 replicates of 5 hens in each pen. Factors included two concentrations of HPMC (0 and 1%), two concentrations of arginine (8.4 and 12 g/kg), two concentrations of ISF (zero and three times more than that present in basal diets) and two contents of energy (11.7 and 14.6 MJ/kg). Performance criteria and blood characteristics of hens were measured during the experimental period. Expression of genes involved in lipid metabolism was determined in the liver at 55 weeks of age. 3. Hens given high-energy diets showed increased BW (body weight), ovary weight and abdominal fat pad and enhanced plasma glucose, triglyceride (TG), cholesterol, haemoglobin, haematocrit and low lymphocyte percentages. The expression of malic enzyme, peroxisome proliferator-activated receptor-α (PPARα), peroxisome proliferator-activated receptor-γ (PPARγ) and inducible nitric oxide (iNOS) increased and sterol regulatory element binding protein-1c (SREBP1c) decreased with increasing energy content of diets. Arginine addition decreased TG, cholesterol and A1-c haemoglobin concentration and increased PPARα, PPARγ and iNOS expression. Inclusion of ISF and HPMC decreased BW, egg weight, plasma TG, cholesterol and increased egg production and also enhanced PPARγ and iNOS expression. Significant interactions were observed between energy concentration and ISF and HPMC on BW. 4. The results of the current study revealed that ISF, HPMC and arginine have beneficial effects on controlling the metabolism of obese broiler breeder hens and using a mix of these products minimises the harmful effects of obesity.

  12. Effects of exercise and L-arginine intake on inflammation in aorta of high-fat diet induced obese rats

    PubMed Central

    Kim, Hee-jae; Son, Junseok; Jin, Eunhee; Lee, Jin; Park, Sok

    2016-01-01

    [Purpose] In the present study, we investigated the effect of exercise and arginine on the inflammatory makers and Cu-Mn superoxide dismutase (SOD) expression in the aortas of high-fat-induced obese rats. [Methods] Fifty 6-month-old male Sprague-Dawley rats were randomly assigned as follows: HF-Con: high-fat diet, HF-Ex: high-fat diet and exercise, HF-Ex+A: high-fat diet and combined exercise and arginine, HF-A: high-fat diet and arginine. The high-fat diet was fed for 12 weeks following 1 week of environmental adaptation with mixed solid chow. The rats performed treadmill exercise 6 times per week for 12 weeks at20 m/min for 60 min. L-argininewas mixed with saline and orally administered at 150 mg/kg once a day. Expressions of inflammatory markers (including NF- κB, TNF-α, COX-2) and SOD were evaluated using western blotting. [Results] NF-κB expression decreased significantly (p<0.05) in the HF-Ex group compared with HF-Con group, and we found additional effects(p<0.01) on NF-κB expression in HF-EX+A compared withHF-Ex. TNF-α expression decreased significantly (p<0.01) in HF-Ex, FH-Ex+A, and FH-A compared with HF-Con. In a similar trend with NF-κB expression, COX-2 expression decreased significantly in HF-Ex compared withHF-Con. In Cu-Mn SOD expression, there was no difference between HF and HF-Ex, but significant increases (p<0.01) inCu-Mn SOD werefound in HF-Ex+A and HF-A. [Conclusion] Based on our results, treatment that combines exercise and arginine might be effective for modulatingvascular inflammation and oxidative stress in obesity PMID:27298811

  13. Identification of essential amino acid residues of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

    PubMed

    Takahashi, T; Hiramoto, S; Wato, S; Nishimoto, T; Wada, Y; Nagai, K; Yamaguchi, H

    1999-11-01

    Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue.

  14. Evaluation of two year treatment outcome and limited impact of arginine restriction in a patient with GAMT deficiency.

    PubMed

    Mercimek-Mahmutoglu, Saadet; Dunbar, Mary; Friesen, Andrea; Garret, Susan; Hartnett, Carol; Huh, Linda; Sinclair, Graham; Stockler, Sylvia; Wellington, Stephen; Pouwels, Petra J W; Salomons, Gajja S; Jakobs, Cornelis

    2012-01-01

    A 4-year-old female with history of developmental regression and autistic features was diagnosed with guanidinoacetate methyltransferase deficiency at age 21 months. Upon treatment, she showed improvements in her developmental milestones, sensorial-neural hearing loss and brain atrophy on cranial-MRI. The creatine/choline ratio increased 82% in basal ganglia and 88% in white matter on cranial MR-spectroscopy. The CSF guanidinoacetate decreased 80% after six months of ornithine and creatine supplementation and an additional 8% after 18 months of additional arginine restricted diet. We report the most favorable clinical and biochemical outcome on treatment in our patient.

  15. Hepatic albumin and urea synthesis: The mathematical modelling of the dynamics of [14C]carbonate-derived guanidine-labelled arginine in the isolated perfused rat liver.

    PubMed Central

    Tavill, A S; Nadkarni, D; Metcalfe, J; Black, E; Hoffenberg, R; Carson, E R

    1975-01-01

    A mathematical model was constructed to define the dynamics of incorporation of radioactivity into urea carbon and the guanidine carbon of arginine in plasma albumin after the rapid intraportal-venous administration of Na214CO3 in the isolated perfused rat liver. 2. The model was formulated in terms of compartmental analysis and additional experiments were designed to provide further information on subsystem dynamics and to discriminate between alternative model structures. 3. Evidence for the rapid-time-constant of labelling of intracellular arginine was provided by precursor-product analysis of precursor [14C]carboante and product [14C]urea in the perfusate. 4. Compartmental analysis of the dynamics of newly synthesized urea was based on the fate of exogenous [13C]urea, endogenous [14C]urea and the accumulation of [12C]urea in perfusate water, confirming the early completion of urea carbon labelling, the absence of continuing synthesis of labelled urea, and the presence of a small intrahepatic urea-delay pool. 5. Analysis of the perfusate dynamics of endogenously synthesized and exogenously administered [6-14C]arginine indicated that although the capacity for extrahepatic formation of [14C]-urea exists, little or no arginine formed within the intrahepatic urea cycle was transported out of the liver. However, the presence of a rapidly turning-over intrahepatic arginine pool was confirmed. 6. On the basis of these subsystem analyses it was possible to offer feasible estimations for the parameters of the mathematical model. However, it was not possible to stimulate the form and magnitude of the dynamics of newly synthesized labelled urea and albumin which were simultaneously observed after administration of [14C]carbonate on the basis of a preliminary model which postulated that both products were derived from a single hepatic pool of [16-14C]arginine. On the other hand these observed dynamics could be satisfied to a two-compartment arginine model, which also

  16. Critical amino acid residues involved in the electrogenic sodium-bicarbonate cotransporter kNBC1-mediated transport.

    PubMed

    Abuladze, Natalia; Azimov, Rustam; Newman, Debra; Sassani, Pakan; Liu, Weixin; Tatishchev, Sergei; Pushkin, Alexander; Kurtz, Ira

    2005-06-15

    We have previously reported a topological model of the electrogenic Na(+)-HCO(3)(-) cotransporter (NBC1) in which the cotransporter spans the plasma membrane 10 times with N- and C-termini localized intracellularly. An analysis of conserved amino acid residues among members of the SLC4 superfamily in both the transmembrane segments (TMs) and intracellular/extracellular loops (ILs/ELs) provided the basis for the mutagenesis approach taken in the present study to determine amino acids involved in NBC1-mediated ion transport. Using large-scale mutagenesis, acidic and basic amino acids putatively involved in ion transport mediated by the predominant variant of NBC1 expressed in the kidney (kNBC1) were mutated to neutral and/or oppositely charged amino acids. All mutant kNBC1 cotransporters were expressed in HEK-293T cells and the Na(+)-dependent base flux of the mutants was determined using intracellular pH measurements with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Critical glutamate, aspartate, lysine, arginine and histidine residues in ILs/ELs and TMs were detected that were essential for kNBC1-mediated Na(+)-dependent base transport. In addition, critical phenylalanine, serine, tyrosine, threonine and alanine residues in TMs and ILs/ELs were detected. Furthermore, several amino acid residues in ILs/ELs and TMs were shown to be essential for membrane targeting. The data demonstrate asymmetry of distribution of kNBC1 charged amino acids involved in ion recognition in putative outward-facing and inward-facing conformations. A model summarizing key amino acid residues involved in kNBC1-mediated ion transport is presented.

  17. Critical amino acid residues involved in the electrogenic sodium–bicarbonate cotransporter kNBC1-mediated transport

    PubMed Central

    Abuladze, Natalia; Azimov, Rustam; Newman, Debra; Sassani, Pakan; Liu, Weixin; Tatishchev, Sergei; Pushkin, Alexander; Kurtz, Ira

    2005-01-01

    We have previously reported a topological model of the electrogenic Na+–HCO3− cotransporter (NBC1) in which the cotransporter spans the plasma membrane 10 times with N- and C-termini localized intracellularly. An analysis of conserved amino acid residues among members of the SLC4 superfamily in both the transmembrane segments (TMs) and intracellular/extracellular loops (ILs/ELs) provided the basis for the mutagenesis approach taken in the present study to determine amino acids involved in NBC1-mediated ion transport. Using large-scale mutagenesis, acidic and basic amino acids putatively involved in ion transport mediated by the predominant variant of NBC1 expressed in the kidney (kNBC1) were mutated to neutral and/or oppositely charged amino acids. All mutant kNBC1 cotransporters were expressed in HEK-293T cells and the Na+-dependent base flux of the mutants was determined using intracellular pH measurements with 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Critical glutamate, aspartate, lysine, arginine and histidine residues in ILs/ELs and TMs were detected that were essential for kNBC1-mediated Na+-dependent base transport. In addition, critical phenylalanine, serine, tyrosine, threonine and alanine residues in TMs and ILs/ELs were detected. Furthermore, several amino acid residues in ILs/ELs and TMs were shown to be essential for membrane targeting. The data demonstrate asymmetry of distribution of kNBC1 charged amino acids involved in ion recognition in putative outward-facing and inward-facing conformations. A model summarizing key amino acid residues involved in kNBC1-mediated ion transport is presented. PMID:15817634

  18. Combination of Insecticide Treated Nets and Indoor Residual Spraying in Northern Tanzania Provides Additional Reduction in Vector Population Density and Malaria Transmission Rates Compared to Insecticide Treated Nets Alone: A Randomised Control Trial

    PubMed Central

    Protopopoff, Natacha; Wright, Alexandra; West, Philippa A; Tigererwa, Robinson; Mosha, Franklin W; Kisinza, William; Kleinschmidt, Immo; Rowland, Mark

    2015-01-01

    Indoor residual spraying (IRS) combined with insecticide treated nets (ITN) has been implemented together in several sub-Saharan countries with inconclusive evidence that the combined intervention provides added benefit. The impact on malaria transmission was evaluated in a cluster randomised trial comparing two rounds of IRS with bendiocarb plus universal coverage ITNs, with ITNs alone in northern Tanzania. From April 2011 to December 2012, eight houses in 20 clusters per study arm were sampled monthly for one night with CDC light trap collections. Anopheles gambiae s.l. were identified to species using real time PCR Taq Man and tested for the presence of Plasmodium falciparum circumsporozoite protein. ITN and IRS coverage was estimated from household surveys. IRS coverage was more than 85% in two rounds of spraying in January and April 2012. Household coverage with at least one ITN per house was 94.7% after the universal coverage net campaign in the baseline year and the proportion of household with all sleeping places covered by LLIN was 50.1% decreasing to 39.1% by the end of the intervention year. An.gambiae s.s. comprised 80% and An.arabiensis 18.3% of the anopheline collection in the baseline year. Mean An.gambiae s.l. density in the ITN+IRS arm was reduced by 84% (95%CI: 56%-94%, p = 0.001) relative to the ITN arm. In the stratum of clusters categorised as high anopheline density at baseline EIR was lower in the ITN+IRS arm compared to the ITN arm (0.5 versus 5.4 per house per month, Incidence Rate Ratio: 0.10, 95%CI: 0.01–0.66, p-value for interaction <0.001). This trial provides conclusive evidence that combining carbamate IRS and ITNs produces major reduction in Anopheles density and entomological inoculation rate compared to ITN alone in an area of moderate coverage of LLIN and high pyrethroid resistance in An.gambiae s.s. PMID:26569492

  19. Combination of Insecticide Treated Nets and Indoor Residual Spraying in Northern Tanzania Provides Additional Reduction in Vector Population Density and Malaria Transmission Rates Compared to Insecticide Treated Nets Alone: A Randomised Control Trial.

    PubMed

    Protopopoff, Natacha; Wright, Alexandra; West, Philippa A; Tigererwa, Robinson; Mosha, Franklin W; Kisinza, William; Kleinschmidt, Immo; Rowland, Mark

    2015-01-01

    Indoor residual spraying (IRS) combined with insecticide treated nets (ITN) has been implemented together in several sub-Saharan countries with inconclusive evidence that the combined intervention provides added benefit. The impact on malaria transmission was evaluated in a cluster randomised trial comparing two rounds of IRS with bendiocarb plus universal coverage ITNs, with ITNs alone in northern Tanzania. From April 2011 to December 2012, eight houses in 20 clusters per study arm were sampled monthly for one night with CDC light trap collections. Anopheles gambiae s.l. were identified to species using real time PCR Taq Man and tested for the presence of Plasmodium falciparum circumsporozoite protein. ITN and IRS coverage was estimated from household surveys. IRS coverage was more than 85% in two rounds of spraying in January and April 2012. Household coverage with at least one ITN per house was 94.7% after the universal coverage net campaign in the baseline year and the proportion of household with all sleeping places covered by LLIN was 50.1% decreasing to 39.1% by the end of the intervention year. An.gambiae s.s. comprised 80% and An.arabiensis 18.3% of the anopheline collection in the baseline year. Mean An.gambiae s.l. density in the ITN+IRS arm was reduced by 84% (95%CI: 56%-94%, p = 0.001) relative to the ITN arm. In the stratum of clusters categorised as high anopheline density at baseline EIR was lower in the ITN+IRS arm compared to the ITN arm (0.5 versus 5.4 per house per month, Incidence Rate Ratio: 0.10, 95%CI: 0.01-0.66, p-value for interaction <0.001). This trial provides conclusive evidence that combining carbamate IRS and ITNs produces major reduction in Anopheles density and entomological inoculation rate compared to ITN alone in an area of moderate coverage of LLIN and high pyrethroid resistance in An.gambiae s.s.

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

    PubMed Central

    Bakshi, Nitya; Morris, Claudia R

    2016-01-01

    Sickle cell disease (SCD) is the most common hemoglobinopathy in the US, affecting approximately 100,000 individuals in the US and millions worldwide. Pain is the hallmark of SCD, and a subset of patients experience pain virtually all of the time. Of interest, the arginine metabolome is associated with several pain mechanisms highlighted in this review. Since SCD is an arginine deficiency syndrome, the contribution of the arginine metabolome to acute and chronic pain in SCD is a topic in need of further attention. Normal arginine metabolism is impaired in SCD through various mechanisms that contribute to endothelial dysfunction, vaso-occlusion, pulmonary complications, risk of leg ulcers, and early mortality. Arginine is a semiessential amino acid that serves as a substrate for protein synthesis and is the precursor to nitric oxide (NO), polyamines, proline, glutamate, creatine, and agmatine. Since arginine is involved in multiple metabolic processes, a deficiency of this amino acid has the potential to disrupt many cellular and organ functions. NO is a potent vasodilator that is depleted in SCD and may contribute to vaso-occlusive pain. As the obligate substrate for NO production, arginine also plays a mechanistic role in SCD-related pain, although its contribution to pain pathways likely extends beyond NO. Low global arginine bioavailability is associated with pain severity in both adults and children with SCD as well as other non-SCD pain syndromes. Preliminary clinical studies of arginine therapy in SCD demonstrate efficacy in treating acute vaso-occlusive pain, as well as leg ulcers and pulmonary hypertension. Restoration of arginine bioavailability through exogenous supplementation of arginine is, therefore, a promising therapeutic target. Phase II clinical trials of arginine therapy for sickle-related pain are underway and a Phase III randomized controlled trial is anticipated in the near future. PMID:27099528

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

    PubMed Central

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

    2014-01-01

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

  2. Effects of dietary L-arginine on the reactivity of canine coronary arteries.

    PubMed

    Ray, E C; Landis, M E; Miller, V M

    1999-01-01

    Experiments were designed to determine the effects of supplemental dietary L-arginine on the endothelial and smooth muscle function of canine coronary arteries. One group of dogs was fed the standard laboratory chow while another group was supplemented with 250 mg/kg per day L-arginine. All dogs had undergone bilateral reversed interposition saphenous vein grafting and received 325 mg/day oral aspirin. After 5 weeks of arginine feeding, left circumflex coronary arteries were removed, cut into rings, and suspended for the measurement of isometric force in organ chambers. Concentration-response curves were obtained to L-arginine, UK-14,304 (alpha2-adrenergic agonist) and A23187 (calcium ionophore) in the absence and presence of N(G)-monomethyl-L-arginine (L-NMMA) and tetraethylammonium (TEA) alone or in combination. Serum concentrations of L-arginine increased by about 20% following 2 weeks of arginine feeding and remained elevated throughout the study. In rings with and without endothelium contracted with prostaglandin F2alpha, L-arginine caused concentration-dependent contractions in rings from control animals but no significant change in tension in rings from arginine-fed animals. Contractions to L-arginine in control animals were reduced by either L-NMMA or TEA. Endothelium-dependent relaxations to the alpha2-adrenergic agonist were decreased with arginine feeding while relaxations to the calcium ionophore and the endothelium-derived factor nitric oxide were similar among groups. Relaxations to UK-14,304 were reduced by L-NMMA in both groups but by TEA only in rings from control animals. These results suggest that dietary supplementation with L-arginine modifies reactivity of endothelium and smooth muscle by at least two mechanisms: one associated with activation of potassium channels and the other with receptor-coupled release of nitric oxide.

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

    PubMed

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

    2007-06-12

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

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

    PubMed

    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.

  5. Metabolic Stress Induced by Arginine Deprivation Induces Autophagy Cell Death in Prostate Cancer

    DTIC Science & Technology

    2010-08-01

    Arginine deiminase as a novel therapy for prostate cancer induces autophagy and caspase-independent apoptosis. Cancer Research, 69(2):700-708...TITLE: Metabolic stress induced by arginine deprivation induces autophagy cell death in prostate cancer PRINCIPAL INVESTIGATOR: Richard Bold, MD...4. TITLE AND SUBTITLE Metabolic stress induced by arginine deprivation induces autophagy cell 5a. CONTRACT NUMBER death in prostate cancer 5b

  6. The Role of Autophagy with Arginine Deiminase as a Novel Prostate Cancer Therapy

    DTIC Science & Technology

    2009-07-01

    Summary 3. DATES COVERED (From - To) 1 July 2008 – 30 June 2009 4. TITLE AND SUBTITLE The Role of Autophagy with Arginine Deiminase as a Novel... arginine deiminase ; autophagy; caspase-independent apoptosis 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES...deprivation as an anti-cancer therapy has historically been met with limited success. The development of pegylated arginine deiminase (ADI-PEG20

  7. Metabolic Stress Induced by Arginine Deprivation Induces Autophagy Cell Death in Prostate Cancer

    DTIC Science & Technology

    2011-08-01

    arginine deiminase W81XWH-08-1-0385 1 AUG 2010 - 31 JUL 2011Annual01-08-2011 University of California, Davis Davis, CA 95618 Metabolic Stress Induced...J Coates, T Bowles, J Sutcliffe, R Jung, R Gandour-Edwards, R Bold, HJ Kung. Arginine deiminase : a novel therapy for prostate cancer and a tool to...R Jung, R Gandour-Edwards, R Bold, HJ Kung. Arginine deiminase induces autophagic cell death in human prostate cancer. EMBO Conference: Autophagy