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Sample records for acids lysine arginine

  1. Nutritional consequences of interspecies differences in arginine and lysine metabolism.

    PubMed

    Ball, Ronald O; Urschel, Kristine L; Pencharz, Paul B

    2007-06-01

    Differences in lysine and arginine requirements among various species such as omnivores (humans, pigs, rats, dogs), carnivores (cats), herbivores (rabbits, horses), ruminants (cattle), poultry, and fish, are covered in detail in this article. Although lysine is classified as an indispensable amino acid across species, the classification of arginine as either an indispensable or dispensable amino acid is more ambiguous because of differences among species in rates of de novo arginine synthesis. Because lysine is most often the limiting amino acid in the diet, its requirement has been extensively studied. By use of the ideal protein concept, the requirements of the other indispensable amino acids can be extrapolated from the lysine requirement. The successful use of this concept in pigs is compared with potential application of the ideal protein concept in humans. The current dietary arginine requirement varies widely among species, with ruminants, rabbits, and rats having relatively low requirements and carnivores, fish, and poultry having high requirements. Interspecies differences in metabolic arginine utilization and reasons for different rates of de novo arginine synthesis are reviewed in detail, as these are the primary determinants of the dietary arginine requirement. There is presently no dietary requirement for humans of any age, although this needs to be reassessed, particularly in neonates. A thorough understanding of the factors contributing to the lysine and arginine requirements in different species will be useful in our understanding of human amino acid requirements.

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

    PubMed

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

    2011-01-01

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

  3. Quantum Computational Calculations of the Ionization Energies of Acidic and Basic Amino Acids: Aspartate, Glutamate, Arginine, Lysine, and Histidine

    NASA Astrophysics Data System (ADS)

    de Guzman, C. P.; Andrianarijaona, M.; Lee, Y. S.; Andrianarijaona, V.

    An extensive knowledge of the ionization energies of amino acids can provide vital information on protein sequencing, structure, and function. Acidic and basic amino acids are unique because they have three ionizable groups: the C-terminus, the N-terminus, and the side chain. The effects of multiple ionizable groups can be seen in how Aspartate's ionizable side chain heavily influences its preferred conformation (J Phys Chem A. 2011 April 7; 115(13): 2900-2912). Theoretical and experimental data on the ionization energies of many of these molecules is sparse. Considering each atom of the amino acid as a potential departing site for the electron gives insight on how the three ionizable groups affect the ionization process of the molecule and the dynamic coupling between the vibrational modes. In the following study, we optimized the structure of each acidic and basic amino acid then exported the three dimensional coordinates of the amino acids. We used ORCA to calculate single point energies for a region near the optimized coordinates and systematically went through the x, y, and z coordinates of each atom in the neutral and ionized forms of the amino acid. With the calculations, we were able to graph energy potential curves to better understand the quantum dynamic properties of the amino acids. The authors thank Pacific Union College Student Association for providing funds.

  4. Intestinal absorption of amino acids in the Pacific bluefin tuna (Thunnus orientalis): in vitro lysine-arginine interaction using the everted intestine system.

    PubMed

    Martínez-Montaño, Emmanuel; Peña, Emyr; Viana, María Teresa

    2013-04-01

    The interaction between lysine (Lys) and arginine (Arg) in the proximal intestinal region of Pacific bluefin tuna (Thunnus orientalis) was evaluated using the everted intestine method. This in vitro intestinal system has been shown to be an effective tool for studying the nutrient absorption without the need to handle the tuna fish in marine cages as needed for digestibility and amino acid (AA) absorption. We used a factorial design with two sets of variables: low and high Lys concentration (10 and 75 mM) and four different Arg concentrations (3, 10, 20, and 30 mM). Both amino acids were dissolved in marine Ringer solution with a basal amino acidic composition consisting of a tryptone solution (9 mg mL(-1)). No interaction was observed between the absorption of Lys and Arg during the first 10 min of the experiment when low concentration of Lys and Arg was used in the hydrolyzate solution. However, there seemed to be a positive effect on Lys absorption when both amino acids were at high concentrations (30 and 75 mM, respectively). This type of studies will led us to test different formulations and/or additives to better understand the efficiency of AA supplementation as an alternative to in situ studies that are difficult to follow to design with the Pacific Bluefin Tuna.

  5. X-ray studies of crystalline complexes involving amino acids and peptides. XLIV. Invariant features of supramolecular association and chiral effects in the complexes of arginine and lysine with tartaric acid.

    PubMed

    Selvaraj, M; Thamotharan, S; Roy, Siddhartha; Vijayan, M

    2007-06-01

    The tartaric acid complexes with arginine and lysine exhibit two stoichiometries depending upon the ionization state of the anion. The structures reported here are DL-argininium DL-hydrogen tartrate, bis(L-argininium) L-tartrate, bis(DL-lysinium) DL-tartrate monohydrate, L-lysinium D-hydrogen tartrate and L-lysinium L-hydrogen tartrate. During crystallization, L-lysine preferentially interacts with D-tartaric acid to form a complex when DL-tartaric acid is used in the experiment. The anions and the cations aggregate into separate alternating layers in four of the five complexes. In bis(L-argininium) L-tartrate, the amino acid layers are interconnected by individual tartrate ions which do not interact among themselves. The aggregation of argininium ions in the DL- and the L-arginine complexes is remarkably similar, which is in turn similar to those observed in other dicarboxylic acid complexes of arginine. Thus, argininium ions have a tendency to assume similar patterns of aggregation, which are largely unaffected by a change in the chemistry of partner molecules such as the introduction of hydroxyl groups or a change in chirality or stoichiometry. On the contrary, the lysinium ions exhibit fundamentally different aggregation patterns in the DL-DL complexes on the one hand and L-D and L-L complexes on the other. Interestingly, the pattern in the L-D complex is similar to that in the L-L complex. The lysinium ions in the DL-DL complex exhibit an aggregation pattern similar to those observed in the DL-lysine complexes involving other dicarboxylic acids. Thus, the effect of change in the chirality of a subset of the component complexes could be profound or marginal, in an unpredictable manner. The relevant crystal structures appear to indicate that the preference of L-lysine for D-tartaric acid is perhaps caused by chiral discrimination resulting from the amplification of a small energy difference.

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

    PubMed Central

    DeRouchey, Jason; Hoover, Brandon

    2013-01-01

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

  7. 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

  8. 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. PMID:27324284

  9. Converting the Yeast Arginine Can1 Permease to a Lysine Permease*

    PubMed Central

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

    2014-01-01

    Amino acid uptake in yeast cells is mediated by about 16 plasma membrane permeases, most of which belong to the amino acid-polyamine-organocation (APC) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino acid permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Although Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H+-driven arginine-specific permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a three-dimensional Can1 structural model based on the crystal structure of the bacterial APC family arginine/agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q and E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H+ coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport. PMID:24448798

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

    PubMed

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

    2014-03-01

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

  11. Polydiacetylenyl β-cyclodextrin based smart vesicles for colorimetric assay of arginine and lysine.

    PubMed

    Cho, Eunae; Kim, Hwanhee; Choi, Youngjin; Paik, Seung R; Jung, Seunho

    2016-01-01

    Selective visualization of arginine and lysine has been explored among 20 amino acids using the hybrid conjugate of β-cyclodextrin (β-CD) and polydiacetylene (PDA). The mono pentacosa-10,12-diynyl aminomethyl group was successfully coupled to either the primary or the secondary face of β-CD, where mono-6-amino-6-deoxy-β-CD or mono-3-amino-3-deoxy-β-CD reacted with the N-hydroxysuccinimide ester of 10,12-pentacosadiynoic acid. In this combinatorial system, the cylindrical β-cyclodextrin functions as a channel for the introduction of the cationic amino acids to the artificial membrane. The membrane perturbation and aggregation by the target amino acids could be exclusively visualized as a blue to red color change based on the responsive polydiacetylene domain. These interesting findings demonstrated that the developed β-CD conjugated PDA system may offer a new method of cell-penetrating mechanism, a promising vector system, as well as impact the production industry of arginine or lysine. PMID:27502314

  12. Polydiacetylenyl β-cyclodextrin based smart vesicles for colorimetric assay of arginine and lysine.

    PubMed

    Cho, Eunae; Kim, Hwanhee; Choi, Youngjin; Paik, Seung R; Jung, Seunho

    2016-08-09

    Selective visualization of arginine and lysine has been explored among 20 amino acids using the hybrid conjugate of β-cyclodextrin (β-CD) and polydiacetylene (PDA). The mono pentacosa-10,12-diynyl aminomethyl group was successfully coupled to either the primary or the secondary face of β-CD, where mono-6-amino-6-deoxy-β-CD or mono-3-amino-3-deoxy-β-CD reacted with the N-hydroxysuccinimide ester of 10,12-pentacosadiynoic acid. In this combinatorial system, the cylindrical β-cyclodextrin functions as a channel for the introduction of the cationic amino acids to the artificial membrane. The membrane perturbation and aggregation by the target amino acids could be exclusively visualized as a blue to red color change based on the responsive polydiacetylene domain. These interesting findings demonstrated that the developed β-CD conjugated PDA system may offer a new method of cell-penetrating mechanism, a promising vector system, as well as impact the production industry of arginine or lysine.

  13. Polydiacetylenyl β-cyclodextrin based smart vesicles for colorimetric assay of arginine and lysine

    NASA Astrophysics Data System (ADS)

    Cho, Eunae; Kim, Hwanhee; Choi, Youngjin; Paik, Seung R.; Jung, Seunho

    2016-08-01

    Selective visualization of arginine and lysine has been explored among 20 amino acids using the hybrid conjugate of β-cyclodextrin (β-CD) and polydiacetylene (PDA). The mono pentacosa-10,12-diynyl aminomethyl group was successfully coupled to either the primary or the secondary face of β-CD, where mono-6-amino-6-deoxy-β-CD or mono-3-amino-3-deoxy-β-CD reacted with the N-hydroxysuccinimide ester of 10,12-pentacosadiynoic acid. In this combinatorial system, the cylindrical β-cyclodextrin functions as a channel for the introduction of the cationic amino acids to the artificial membrane. The membrane perturbation and aggregation by the target amino acids could be exclusively visualized as a blue to red color change based on the responsive polydiacetylene domain. These interesting findings demonstrated that the developed β-CD conjugated PDA system may offer a new method of cell-penetrating mechanism, a promising vector system, as well as impact the production industry of arginine or lysine.

  14. Polydiacetylenyl β-cyclodextrin based smart vesicles for colorimetric assay of arginine and lysine

    PubMed Central

    Cho, Eunae; Kim, Hwanhee; Choi, Youngjin; Paik, Seung R.; Jung, Seunho

    2016-01-01

    Selective visualization of arginine and lysine has been explored among 20 amino acids using the hybrid conjugate of β-cyclodextrin (β-CD) and polydiacetylene (PDA). The mono pentacosa-10,12-diynyl aminomethyl group was successfully coupled to either the primary or the secondary face of β-CD, where mono-6-amino-6-deoxy-β-CD or mono-3-amino-3-deoxy-β-CD reacted with the N-hydroxysuccinimide ester of 10,12-pentacosadiynoic acid. In this combinatorial system, the cylindrical β-cyclodextrin functions as a channel for the introduction of the cationic amino acids to the artificial membrane. The membrane perturbation and aggregation by the target amino acids could be exclusively visualized as a blue to red color change based on the responsive polydiacetylene domain. These interesting findings demonstrated that the developed β-CD conjugated PDA system may offer a new method of cell-penetrating mechanism, a promising vector system, as well as impact the production industry of arginine or lysine. PMID:27502314

  15. Antagonistic reactions of arginine and lysine against formaldehyde and their relation to cell proliferation, apoptosis, folate cycle and photosynthesis.

    PubMed

    Trézl, Lajos; Hullán, Lehel; Jászay, Zsuzsa M; Szarvas, Tibor; Petneházy, Imre; Szende, Béla; Bocsi, József; Takáts, Zoltán; Vékey, Károly; Töke, László

    2003-02-01

    1H, 13C NMR, ESMS and MS/MS investigations proved that there is an antagonism in the spontaneous reaction of formaldehyde with L-lysine and L-arginine. L-Arginine can only be hydroxymethylated on the guanidino group in a very fast reaction forming mono-, di-, and trihydroxymethyl arginines (HMA). L-Lysine can be methylated on the epsilon-amino group forming mono-, di-, and trimethyl lysine on physiological pH. Hydroxymethyl arginines are relative stable, isolable products, and can also be formed in biological systems, especially in plants. Significant amounts of hydroxymethyl arginines were identified in the aqueous extract of lyophilized kohlrabi, which can be formed in photosynthesis during CO2 fixation. 14C-Formaldehyde formed in a short-term (10, 30 sec) 14CO2 fixation reaction in Zea mays L. (early maturity variety: Szegedi TC 277) was captured by L-arginine, which occurs in leaves in large amount. Formaldehyde formed during photosynthesis can react not only with the arginine, but with ribulose-1,5-diphosphate present in leaves. In model reactions formaldehyde can react with the 'ene diole' group of ribulose-1,5-diphosphate in the absence of Rubisco enzyme, which is a similar reaction to the addition of formaldehyde to L-ascorbic acid. Hydroxymethyl arginines (HMA) are endogenous formaldehyde carrier molecules transferring the bound formaldehyde to thymidylate synthase enzyme system incorporating it into the folate cycle. HMA can also carry the bound formaldehyde to the cells especially to the tumorous cells (HT29 adenocarcinoma), and cause significant inhibition of cell proliferation and causes apoptosis.

  16. Lysine and Arginine Reduce the Effects of Cerebral Ischemic Insults and Inhibit Glutamate-Induced Neuronal Activity in Rats

    PubMed Central

    Kondoh, Takashi; Kameishi, Makiko; Mallick, Hruda Nanda; Ono, Taketoshi; Torii, Kunio

    2010-01-01

    Intravenous administration of arginine was shown to be protective against cerebral ischemic insults via nitric oxide production and possibly via additional mechanisms. The present study aimed at evaluating the neuroprotective effects of oral administration of lysine (a basic amino acid), arginine, and their combination on ischemic insults (cerebral edema and infarction) and hemispheric brain swelling induced by transient middle cerebral artery occlusion/reperfusion in rats. Magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining were performed 2 days after ischemia induction. In control animals, the major edematous areas were observed in the cerebral cortex and striatum. The volumes associated with cortical edema were significantly reduced by lysine (2.0 g/kg), arginine (0.6 g/kg), or their combined administration (0.6 g/kg each). Protective effects of these amino acids on infarction were comparable to the inhibitory effects on edema formation. Interestingly, these amino acids, even at low dose (0.6 g/kg), were effective to reduce hemispheric brain swelling. Additionally, the effects of in vivo microiontophoretic (juxtaneuronal) applications of these amino acids on glutamate-evoked neuronal activity in the ventromedial hypothalamus were investigated in awake rats. Glutamate-induced neuronal activity was robustly inhibited by microiontophoretic applications of lysine or arginine onto neuronal membranes. Taken together, our results demonstrate the neuroprotective effects of oral ingestion of lysine and arginine against ischemic insults (cerebral edema and infarction), especially in the cerebral cortex, and suggest that suppression of glutamate-induced neuronal activity might be the primary mechanism associated with these neuroprotective effects. PMID:20589237

  17. Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures.

    PubMed

    Zhang, Xintao; Tang, Hongping; Sun, Ya-Ting; Liu, Xuping; Tan, Wen-Song; Fan, Li

    2015-08-01

    C-terminal lysine variants are commonly observed in monoclonal antibodies (mAbs) and found sensitive to process conditions, especially specific components in culture medium. The potential roles of media arginine (Arg) and lysine (Lys) in mAb heavy chain C-terminal lysine processing were investigated by monitoring the lysine variant levels under various Arg and Lys concentrations. Both Arg and Lys were found to significantly affect lysine variant level. Specifically, lysine variant level increased from 18.7 to 31.8 % when Arg and Lys concentrations were increased from 2 to 10 mM. Since heterogeneity of C-terminal lysine residues is due to the varying degree of proteolysis by basic carboxypeptidases (Cps), enzyme (basic Cps) level, pH conditions, and product (Arg and Lys) inhibition, which potentially affect the enzymatic reaction, were investigated under various Arg and Lys conditions. Enzyme level and pH conditions were found not to account for the different lysine variant levels, which was evident from the minimal variation in transcription level and intracellular pH. On the other hand, product inhibition effect of Arg and Lys on basic Cps was evident from the notable intracellular and extracellular Arg and Lys concentrations comparable with Ki values (inhibition constant) of basic Cps and further confirmed by cell-free assays. Additionally, a kinetic study of lysine variant level during the cell culture process enabled further characterization of the C-terminal lysine processing.

  18. Complexation des acides aminés basiques arginine, histidine et lysine avec l'ADN plasmidique en solution aqueuse : participation à la capture de radicaux sous irradiation X à 1,5 keV

    NASA Astrophysics Data System (ADS)

    Tariq Khalil, Talat; Taillefumier, Baptiste; Boulanouar, Omar; Mavon, Christophe; Fromm, Michel

    2016-09-01

    L'environnement chimique de l'ADN en situation biologique est complexe notam-ment en raison de la présence d'histones, protéines nucléaires, associées en quantité approximativement égales à l'ADN pour former la chromatine. Les histones possèdent de nombreux radicaux basiques arginine et lysine chargés positivement et dont la majorité se trouve sur les chaînes émergentes, l'ADN présente quant à lui des charges négatives sur ses groupements phosphates localisés tout au long de la double hélice. Dans cette étude, la complexité de la structure de la chromatine nucléaire est dans un premier temps mimée en solution aqueuse par la formation de complexes entre un ADN plasmidique sonde et les trois acides aminés basiques, Arg, His, Lys, qui, mis à part His, sont protonés au pH physiologique. Ces acides aminés libres en solution sont réputés être des capteurs efficaces de radicaux libres, notamment pour le radical hydroxyle, conférant ainsi un pouvoir protecteur vis-à-vis des effets indirects sur l'ADN en situation d'exposition aux rayonnements ionisants. A concentration fixée, les capacités de capture des acides aminés libres, σ, pour le radical hydroxyle sont typiquement les suivantes σHis ≈σArg > σLys (σLys ≈ 0,1 × σArg). Nous avons mesuré les taux de cassures simple brin par plasmide et par Gray (χ) lors d'expositions de solutions aqueuses de complexes [acide aminé - ADN plasmidique] aux rayons X ultra-mous (1,5 keV). A concentrations égales, les trois acides aminés complexés et présents en large excès ne manifestent pas une capacité de protection de l'ADN proportionnelle à leur capacité de capture libre et en solution ; on trouve en effet des taux de cassures dans l'ordre suivant χHis > χArg > χLys (χLys ≈ 0,01 χArg). Après avoir détaillé le mode opératoire de ces mesures, nous analyserons sur des bases bibliographiques, les modes spécifiques d'interaction des acides aminés basiques avec l'ADN. La sp

  19. HDAC inhibitors induce global changes in histone lysine and arginine methylation and alter expression of lysine demethylases.

    PubMed

    Lillico, Ryan; Sobral, Marina Gomez; Stesco, Nicholas; Lakowski, Ted M

    2016-02-01

    Histone deacetylase (HDAC) inhibitors are cancer treatments that inhibit the removal of the epigenetic modification acetyllysine on histones, resulting in altered gene expression. Such changes in expression may influence other histone epigenetic modifications. We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify lysine acetylation and methylation and arginine methylation on histones extracted from cultured cells treated with HDAC inhibitors. The HDAC inhibitors vorinostat, mocetinostat and entinostat induced 400-600% hyperacetylation in HEK 293 and K562 cells. All HDAC inhibitors decreased histone methylarginines in HEK 293 cells but entinostat produced dose dependent reductions in asymmetric dimethylarginine, not observed in K562 cells. Vorinostat produced increases in histone lysine methylation and decreased expression of some lysine demethylases (KDM), measured by quantitative PCR. Entinostat had variable effects on lysine methylation and decreased expression of some KDM while increasing expression of others. Mocetinostat produced dose dependent increases in histone lysine methylation by LC-MS/MS. This was corroborated with a multiplex colorimetric assay showing increases in histone H3 lysine 4, 9, 27, 36 and 79 methylation. Increases in lysine methylation were correlated with dose dependent decreases in the expression of seven KDM. Mocetinostat functions as an HDAC inhibitor and a de facto KDM inhibitor.

  20. Substitution of lysine for arginine in the N-terminal 217th amino acid residue of the H gamma II of Staphylococcal gamma-hemolysin lowers the activity of the toxin.

    PubMed

    Sudo, K; Choorit, W; Asami, I; Kaneko, J; Muramoto, K; Kamio, Y

    1995-09-01

    The staphylococcal toxin gamma-hemolysin consists of two protein components, LukF and H gamma II. Staphylococcus aureus P83 was found to have five components, LukF, LukF-PV, LukM, LukS, and H gamma II for leukocidin or gamma-hemolysin. H gamma II of S. aureus P83 was demonstrated to be a naturally-occurring analogous molecule of H gamma II [H gamma II(P83)], in which the 217th arginine residue was replaced by lysine. The H gamma II(P83) showed about 50% of the hemolytic activity of normal H gamma II in the presence of LukF.

  1. Arginine and lysine reduce the high viscosity of serum albumin solutions for pharmaceutical injection.

    PubMed

    Inoue, Naoto; Takai, Eisuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2014-05-01

    Therapeutic protein solutions for subcutaneous injection must be very highly concentrated, which increases their viscosity through protein-protein interactions. However, maintaining a solution viscosity below 50 cP is important for the preparation and injection of therapeutic protein solutions. In this study, we examined the effect of various amino acids on the solution viscosity of very highly concentrated bovine serum albumin (BSA) and human serum albumin (HSA) at a physiological pH. Among the amino acids tested, l-arginine hydrochloride (ArgHCl) and l-lysine hydrochloride (LysHCl) (50-200 mM) successfully reduced the viscosity of both BSA and HSA solutions; guanidine hydrochloride (GdnHCl), NaCl, and other sodium salts were equally as effective, indicating the electrostatic shielding effect of these additives. Fourier transform infrared spectroscopy showed that BSA is in its native state even in the presence of ArgHCl, LysHCl, and NaCl at high protein concentrations. These results indicate that weakened protein-protein interactions play a key role in reducing solution viscosity. ArgHCl and LysHCl, which are also non-toxic compounds, will be used as additives to reduce the solution viscosity of concentrated therapeutic proteins.

  2. Arginine demethylation is catalysed by a subset of JmjC histone lysine demethylases

    PubMed Central

    Walport, Louise J.; Hopkinson, Richard J.; Chowdhury, Rasheduzzaman; Schiller, Rachel; Ge, Wei; Kawamura, Akane; Schofield, Christopher J.

    2016-01-01

    While the oxygen-dependent reversal of lysine Nɛ-methylation is well established, the existence of bona fide Nω-methylarginine demethylases (RDMs) is controversial. Lysine demethylation, as catalysed by two families of lysine demethylases (the flavin-dependent KDM1 enzymes and the 2-oxoglutarate- and oxygen-dependent JmjC KDMs, respectively), proceeds via oxidation of the N-methyl group, resulting in the release of formaldehyde. Here we report detailed biochemical studies clearly demonstrating that, in purified form, a subset of JmjC KDMs can also act as RDMs, both on histone and non-histone fragments, resulting in formaldehyde release. RDM catalysis is studied using peptides of wild-type sequences known to be arginine-methylated and sequences in which the KDM's methylated target lysine is substituted for a methylated arginine. Notably, the preferred sequence requirements for KDM and RDM activity vary even with the same JmjC enzymes. The demonstration of RDM activity by isolated JmjC enzymes will stimulate efforts to detect biologically relevant RDM activity. PMID:27337104

  3. A kinesin switch I arginine to lysine mutation rescues microtubule function.

    PubMed

    Klumpp, Lisa M; Mackey, Andrew T; Farrell, Christopher M; Rosenberg, John M; Gilbert, Susan P

    2003-10-01

    Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant. PMID:12860992

  4. Why do arginine and lysine organize lipids differently? Insights from coarse-grained and atomistic simulations.

    PubMed

    Wu, Zhe; Cui, Qiang; Yethiraj, Arun

    2013-10-10

    An important puzzle in membrane biophysics is the difference in the behaviors of lysine (Lys) and arginine (Arg) based peptides at the membrane. For example, the translocation of poly-Arg is orders of magnitude faster than that of poly-Lys. Recent experimental work suggests that much of the difference can be inferred from the phase behavior of peptide/lipid mixtures. At similar concentrations, mixtures of phosphatidylethanolamine (PE) and phosphatidylserine (PS) lipids display different phases in the presence of these polypeptides, with a bicontinuous phase observed with poly-Arg peptides and an inverted hexagonal phase observed with poly-Lys peptides. Here we show that simulations with the coarse-grained (CG) BMW-MARTINI model reproduce the experimental results. An analysis using atomistic and CG models reveals that electrostatic and glycerol-peptide interactions play a crucial role in determining the phase behavior of peptide-lipid mixtures, with the difference between Arg and Lys arising from the stronger interactions of the former with lipid glycerols. In other words, the multivalent nature of the guanidinium group allows Arg to simultaneously interact with both phosphate and glycerol groups, while Lys engages solely with phosphate; this feature of amino acid/lipid interactions has not been emphasized in previous studies. The Arg peptides colocalize with PS in regions of high negative Gaussian curvature and stabilize the bicontinuous phase. Decreasing the strength of either the electrostatic interactions or the peptide-glycerol interactions causes the inverted hexagonal phase to become more stable. The results highlight the utility of CG models for the investigation of phase behavior but also emphasize the subtlety of the phenomena, with small changes in specific interactions leading to qualitatively different phases. PMID:24024591

  5. Quantitative microspectral evaluation of the ratio of arginine-rich to lysine-rich histones in neurons and neuroglial cells.

    PubMed

    Pevzner, L Z; Raygorodskaya, T G; Agroskin, L S

    1978-09-01

    Staining of nervous tissue sections with ammoniacal silver according to Black et al. has been confirmed to be a reliable histochemical colour reaction for quantitative evaluation of arginine-rich and lysine-rich histones in cell structures on the basis of determinations of the position of spectral curve maximum. Neurons of several brain nuclei which differed in predominating neurotransmitter did not differ in the ratio of arginine-rich to lysine-rich histones while some differences in this ratio were found out in the glial satelite cells adjacent to the corresponding neurons of these nuclei. Moderate circadian fluctuations were observed in the arginine-rich to lysine-rich histone ratio, these fluctuations being rather similar in the neurons studied and in the cells of perineuronal neuroglia.

  6. Improved synthesis of lysine- and arginine-derived Amadori and Heyns products and in vitro measurement of their angiotensin I-converting enzyme inhibitory activity.

    PubMed

    Srinivas, Sudhanva M; Harohally, Nanishankar V

    2012-02-15

    The L-lysine- and L-arginine-derived Amadori and Heyns products consisting of N-(1-deoxy-d-fructos-1-yl)amino acid and N-(2-deoxy-d-glucos-2-yl)amino acid were prepared by reaction of d-fructose and d-glucose with l-lysine hydrochloride and l-arginine hydrochloride using commercial zinc powder as deprotonating reagent and also as catalyst precursor in a simple synthetic route in high yield. These compounds were screened for angiotensin I-converting enzyme (ACE) inhibitory activity using a high-throughput colorimetric assay (utilizing porcine kidney ACE). The IC(50) values fall in the range of 1030-1175 μM, with N(α)-(1-deoxy-d-fructos-1-yl)arginine showing the best IC(50) value (1030 ± 38 μM). This study demonstrates an improved synthetic method for simple Amadori and Heyns products and their moderate ACE inhibitor activity. PMID:22242891

  7. 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

  8. Exogenous and cell surface glycosaminoglycans alter DNA delivery efficiency of arginine and lysine homopeptides in distinctly different ways.

    PubMed

    Naik, Rangeetha J; Chandra, Pallavi; Mann, Anita; Ganguli, Munia

    2011-05-27

    Glycosaminoglycans (GAGs) expressed ubiquitously on the cell surface are known to interact with a variety of ligands to mediate different cellular processes. However, their role in the internalization of cationic gene delivery vectors such as liposomes, polymers, and peptides is still ambiguous and seems to be controlled by multiple factors. In this report, taking peptides as model systems, we show that peptide chemistry is one of the key factors that determine the dependence on cell surface glycosaminoglycans for cellular internalization and gene delivery. Arginine peptides and their complexes with plasmid DNA show efficient uptake and functional gene transfer independent of the cell surface GAGs. On the other hand, lysine peptides and complexes primarily enter through a GAG-dependent pathway. The peptide-DNA complexes also show differential interaction with soluble GAGs. In the presence of exogenous GAGs under certain conditions, arginine peptide-DNA complexes show increased transfection efficiency that is not observed with lysine. This is attributed to a change in the complex nature that ensures better protection of the compacted DNA in the case of arginine complexes, whereas the lysine complexes get destabilized under these conditions. The presence of a GAG coating also ensures better cell association of arginine complexes, resulting in increased uptake. Our results indicate that the role of both the cell surface and exogenous glycosaminoglycans in gene delivery is controlled by the nature of the peptide and its complex with DNA.

  9. 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

  10. Arginine-to-lysine substitutions influence recombinant horseradish peroxidase stability and immobilisation effectiveness

    PubMed Central

    Ryan, Barry J; Ó'Fágáin, Ciarán

    2007-01-01

    Background Horseradish Peroxidase (HRP) plays important roles in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Often, it is used in immobilised form. With conventional immobilisation techniques, the enzyme adheres in random orientation: the active site may face the solid phase rather than bulk medium, impeding substrate access and leading to sub-optimal catalytic performance. The ability to immobilise HRP in a directional manner, such that the active site would always face outwards from the insoluble matrix, would maximise the immobilised enzyme's catalytic potential and could increase HRP's range of actual and potential applications. Results We have replaced arginine residues on the face of glycan-free recombinant HRP opposite to the active site by lysines. Our strategy differs from previous reports of specific HRP immobilisation via an engineered affinity tag or single reactive residue. These conservative Arg-to-Lys substitutions provide a means of multipoint covalent immobilisation such that the active site will always face away from the immobilisation matrix. One triple and one pentuple mutant were generated by substitution of solvent-exposed arginines on the "back" of the polypeptide (R118, R159 and R283) and of residues known to influence stability (K232 and K241). Orientated HRP immobilisation was demonstrated using a modified polyethersulfone (PES) membrane; the protein was forced to orientate its active site away from the membrane and towards the bulk solution phase. Mutant properties and bioinformatic analysis suggested the reversion of K283R to improve stability, thus generating two additional mutants (K118/R159K and R118K/K232N/K241F/R283K). While most mutants were less stable in free solution than wild type rHRP, the quadruple revertant regained some stability over its mutant counterparts. A greater degree of immobilisation on CNBr-activated Sepharose™ was noted with increased lysine content; however, only marginal

  11. Controlled trial of whole body protein synthesis and plasma amino acid concentrations in yearling horses fed graded amounts of lysine.

    PubMed

    Mastellar, S L; Coleman, R J; Urschel, K L

    2016-10-01

    Lysine has been reported as the first limiting amino acid in typical equine diets. Indicator amino acid oxidation (IAAO) has become the standard method for determining amino acid requirements in other species, but prior to this study, it has not been used to determine equine requirements. The aim of this study was to evaluate whole body protein synthesis and plasma and muscle amino acid concentrations in response to graded levels of lysine intake in yearling horses. Six Thoroughbred colts (358 ± 5 kg) were fed each of six treatment lysine intakes ranging from 76 to 136 mg/kg body weight/day. Blood samples were taken before and 90 min after the morning concentrate meal. Gluteal muscle biopsies were taken ~100 min after the morning concentrate meal. The next day, whole body phenylalanine kinetics were determined using a 2 h primed, constant infusion of [(13)C] sodium bicarbonate followed by a 6 h primed, constant infusion of [1-(13)C] phenylalanine. Plasma lysine concentrations increased linearly (P <0.05) at both the 0 and 90 min time points with increasing lysine intakes. Free muscle asparagine, aspartate, arginine, glutamine, lysine, taurine and tryptophan concentrations responded quadratically to lysine intake (P <0.05). Phenylalanine kinetics did not differ between treatment intakes (P > 0.10). A broken line analysis of lysine intake and phenylalanine oxidation failed to yield a breakpoint from which to determine a lysine requirement. These diets may have been limiting in an amino acid other than lysine, underscoring the lack of data concerning amino acid requirements and bioavailability data in the horse. PMID:27687933

  12. Controlled trial of whole body protein synthesis and plasma amino acid concentrations in yearling horses fed graded amounts of lysine.

    PubMed

    Mastellar, S L; Coleman, R J; Urschel, K L

    2016-10-01

    Lysine has been reported as the first limiting amino acid in typical equine diets. Indicator amino acid oxidation (IAAO) has become the standard method for determining amino acid requirements in other species, but prior to this study, it has not been used to determine equine requirements. The aim of this study was to evaluate whole body protein synthesis and plasma and muscle amino acid concentrations in response to graded levels of lysine intake in yearling horses. Six Thoroughbred colts (358 ± 5 kg) were fed each of six treatment lysine intakes ranging from 76 to 136 mg/kg body weight/day. Blood samples were taken before and 90 min after the morning concentrate meal. Gluteal muscle biopsies were taken ~100 min after the morning concentrate meal. The next day, whole body phenylalanine kinetics were determined using a 2 h primed, constant infusion of [(13)C] sodium bicarbonate followed by a 6 h primed, constant infusion of [1-(13)C] phenylalanine. Plasma lysine concentrations increased linearly (P <0.05) at both the 0 and 90 min time points with increasing lysine intakes. Free muscle asparagine, aspartate, arginine, glutamine, lysine, taurine and tryptophan concentrations responded quadratically to lysine intake (P <0.05). Phenylalanine kinetics did not differ between treatment intakes (P > 0.10). A broken line analysis of lysine intake and phenylalanine oxidation failed to yield a breakpoint from which to determine a lysine requirement. These diets may have been limiting in an amino acid other than lysine, underscoring the lack of data concerning amino acid requirements and bioavailability data in the horse.

  13. Differential mode of antimicrobial actions of arginine-rich and lysine-rich histones against Gram-positive Staphylococcus aureus.

    PubMed

    Morita, Shuu; Tagai, Chihiro; Shiraishi, Takayuki; Miyaji, Kazuyuki; Iwamuro, Shawichi

    2013-10-01

    We previously reported the activities and modes of action of arginine (Arg)-rich histones H3 and H4 against Gram-negative bacteria. In the present study, we investigated the properties of the Arg-rich histones against Gram-positive bacteria in comparison with those of lysine (Lys)-rich histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against Staphylococcus aureus with minimum effective concentration values of 4.0, 4.0, and 5.6 μM, respectively. Laser confocal microscopic analyses revealed that both the Arg-rich and Lys-rich histones associated with the surface of S. aureus. However, while the morphology of S. aureus treated with histone H2B appeared intact, those treated with the histones H3 and H4 closely resembled each other, and the cells were blurred. Electrophoretic mobility shift assay results revealed these histones have binding affinity to lipoteichoic acid (LTA), one of major cell surface components of Gram-positive bacteria. Scanning electron microscopic analyses demonstrated that while histone H2B elicited no obvious changes in cell morphology, histones H3 and H4 disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. Consequently, our results suggest that bacterial cell surface LTA initially attracts both the Arg- and Lys-rich histones, but the modes of antimicrobial action of these histones are different; the former involves cell membrane disruption and the latter involves the cell integrity disruption.

  14. 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.

  15. Characterization of OhS1, an arginine/lysine amidase from the venom of king cobra (Ophiophagus hannah).

    PubMed

    Zhang, Y; Lee, W H; Xiong, Y L; Wang, W Y; Zu, S W

    1994-05-01

    In this paper, we present the results of purification and characterization of an arginine/lysine amidase from the venom of Ophiophagus hannah (OhS1). It was purified by Sephadex G-75 gel filtration and ion-exchange chromatography on DEAE-Sepharose CL-6B. It is a protein of about 43,000, consisting of a single polypeptide chain. It is a minor component in the venom. The purified enzyme was capable of hydrolysing several tripeptidyl-p-nitroanilide substrates having either arginine or lysine as the C-terminal residue. We studied the kinetic parameters of OhS1 on six these chromogenic substrates. OhS1 did not clot fibrinogen. Electrophoresis of fibrinogen degraded with OhS1 revealed the disappearance of the alpha- and beta-chains and the appearance of lower mol. wt fragments. OhS1 had no hemorrhagic activity. It did not hydrolyse casein, nor did it act on blood coagulation factor X, prothrombin and plasminogen. The activity of OhS1 was completely inhibited by NPGB, PMSF, DFP, benzamidine and soybean trypsin inhibitor, suggesting it is a serine protease. Metal chelator (EDTA) had no effect on it.

  16. Effects of Arginine Supplementation on Amino Acid Profiles in Blood and Tissues in Fed and Overnight-Fasted Rats

    PubMed Central

    Holecek, Milan; Sispera, Ludek

    2016-01-01

    Chronic arginine intake is believed to have favorable effects on the body. However, it might be hypothesized that excessive consumption of an individual amino acid exerts adverse effects on distribution and metabolism of other amino acids. We evaluated the effect of chronic intake of arginine on amino acid concentrations in blood plasma, liver, kidneys, and soleus and extensor digitorum longus muscles. Rats were fed a standard diet or a high-arginine diet (HAD) for two months. Half of the animals in each group were sacrificed in the fed state, and the other half after fasting overnight. HAD increased blood plasma concentrations of urea, creatinine, arginine, and ornithine and decreased most other amino acids. Arginine and ornithine also increased in muscles and kidneys; an increase of lysine was observed in both muscle types. Methionine, phenylalanine, threonine, asparagine, glycine, serine, and taurine decreased in most tissues of HAD fed animals. Most of the effects of HAD disappeared after overnight fasting. It is concluded that (i) enhanced dietary arginine intake alters distribution of almost all amino acids; and (ii) to attain a better assessment of the effects of various nutritional interventions, an appropriate number of biochemical measurements must be performed in both postprandial and postabsorptive states. PMID:27070638

  17. Effect of substituting arginine and lysine with alanine on antimicrobial activity and the mechanism of action of a cationic dodecapeptide (CL(14-25)), a partial sequence of cyanate lyase from rice.

    PubMed

    Taniguchi, Masayuki; Takahashi, Nobuteru; Takayanagi, Tomohiro; Ikeda, Atsuo; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Ochiai, Akihito; Tanaka, Takaaki

    2014-01-01

    The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14-25), a cationic α-helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine-substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8-fold and 9.1-fold higher, respectively, than that of CL(14-25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14-25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine-substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine-substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC3 -5 release from P. gingivalis cells and disruption of GUVs induced by the alanine-substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine-substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity).

  18. Effects of dietary lysine levels on plasma free amino acid profile in late-stage finishing pigs.

    PubMed

    Regmi, Naresh; Wang, Taiji; Crenshaw, Mark A; Rude, Brian J; Wu, Guoyao; Liao, Shengfa F

    2016-01-01

    Muscle growth requires a constant supply of amino acids (AAs) from the blood. Therefore, plasma AA profile is a critical factor for maximizing the growth performance of animals, including pigs. This research was conducted to study how dietary lysine intake affects plasma AA profile in pigs at the late production stage. Eighteen crossbred (Large White × Landrace) finishing pigs (nine barrows and nine gilts; initial BW 92.3 ± 6.9 kg) were individually penned in an environment controlled barn. Pigs were assigned randomly to one of the three dietary treatments according to a randomized complete block design with sex as block and pig as experiment unit (6 pigs/treatment). Three corn- and soybean meal-based diets contained 0.43 % (lysine-deficient, Diet I), 0.71 % (lysine-adequate, Diet II), and 0.98 % (lysine-excess, Diet III) l-lysine, respectively. After a 4-week period of feeding, jugular vein blood samples were collected from the pigs and plasma was obtained for AA analysis using established HPLC methods. The change of plasma lysine concentration followed the same pattern as that of dietary lysine supply. The plasma concentrations of threonine, histidine, phenylalanine, isoleucine, valine, arginine, and citrulline of pigs fed Diet II or III were lower (P < 0.05) than that of pigs fed Diet I. The plasma concentrations of alanine, glutamate, and glycine of pigs fed Diet II or III were higher (P < 0.05) than that of pigs fed Diet I. The change of plasma leucine and asparagine concentrations followed the patterns similar to that of plasma lysine. Among those affected AAs, arginine was decreased (P < 0.05) in the greatest proportion with the lysine-excess diet. We suggest that the skeletal muscle growth of finishing pigs may be further increased with a lysine-excess diet if the plasma concentration of arginine can be increased through dietary supplementation or other practical nutritional management strategies. PMID:27386336

  19. Susceptibility of Gardnerella vaginalis Biofilms to Natural Antimicrobials Subtilosin, ε-Poly-L-Lysine, and Lauramide Arginine Ethyl Ester

    PubMed Central

    Turovskiy, Yevgeniy; Cheryian, Thomson; Algburi, Ammar; Wirawan, Ruth E.; Takhistov, Paul; Sinko, Patrick J.; Chikindas, Michael L.

    2012-01-01

    Bacterial vaginosis is a common vaginal infection associated with numerous gynecological and obstetric complications. This condition is characterized by the presence of thick adherent vaginal biofilms, composed mainly of Gardnerella vaginalis. This organism is thought to be the primary aetiological cause of the infection paving the way for various opportunists to colonize the niche. Previously, we reported that the natural antimicrobials subtilosin, ε-poly-L-lysine, and lauramide arginine ethyl ester selectively inhibit the growth of this pathogen. In this study, we used plate counts to evaluate the efficacy of these antimicrobials against established biofilms of G. vaginalis. Additionally, we validated and compared two rapid methods (ATP viability and resazurin assays) for the assessment of cell viability in the antimicrobial-treated G. vaginalis biofilms. Out of the tested antimicrobials, lauramide arginine ethyl ester had the strongest bactericidal effect, followed by subtilosin, with clindamycin and polylysine showing the weakest effect. In comparison to plate counts, ATP viability and resazurin assays considerably underestimated the bactericidal effect of some antimicrobials. Our results indicate that these assays should be validated for every new application. PMID:23024575

  20. Amphipathicity Determines Different Cytotoxic Mechanisms of Lysine- or Arginine-Rich Cationic Hydrophobic Peptides in Cancer Cells.

    PubMed

    Liu, Xiaoli; Cao, Rui; Wang, Sha; Jia, Junli; Fei, Hao

    2016-06-01

    Cationic amphipathic peptides (CAPs) are known to be able to cause membrane destabilization and induce cell death, yet how the hydrophobicity, amphipathicity, and lysine (K)/arginine (R) composition synergistically affect the peptide activity remains incompletely understood. Here, we designed a panel of peptides based on the well-known anticancer peptide KLA. Increasing hydrophobicity enhanced the cytotoxicities of both the K- and R-rich peptides. Peptides with an intact amphipathic helical interface can cause instant cell death through a membrane lysis mechanism. Interestingly, rearranging the residue positions to minimize amphipathicity caused a great decrease of cytotoxicity to the K-rich peptides but not to the R-rich peptides. The amphipathicity-minimized R-rich peptide 6 (RL2) (RLLRLLRLRRLLRL-NH2) penetrated the cell membrane and induced caspase-3-dependent apoptotic cell death. We found that the modulation of hydrophobicity, amphipathicity, and K/R residues leads to distinct mechanisms of action of cationic hydrophobic peptides. Amphipathicity-reduced, arginine-rich cationic hydrophobic peptides (CHPs) may represent a new class of peptide therapeutics. PMID:27195657

  1. Polygalacturonase-Inhibiting Protein Interacts with Pectin through a Binding Site Formed by Four Clustered Residues of Arginine and Lysine1

    PubMed Central

    Spadoni, Sara; Zabotina, Olga; Di Matteo, Adele; Mikkelsen, Jørn Dalgaard; Cervone, Felice; De Lorenzo, Giulia; Mattei, Benedetta; Bellincampi, Daniela

    2006-01-01

    Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein that inhibits fungal polygalacturonases (PGs) and retards the invasion of plant tissues by phytopathogenic fungi. Here, we report the interaction of two PGIP isoforms from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) with both polygalacturonic acid and cell wall fractions containing uronic acids. We identify in the three-dimensional structure of PvPGIP2 a motif of four clustered arginine and lysine residues (R183, R206, K230, and R252) responsible for this binding. The four residues were mutated and the protein variants were expressed in Pichia pastoris. The ability of both wild-type and mutated proteins to bind pectins was investigated by affinity chromatography. Single mutations impaired the binding and double mutations abolished the interaction, thus indicating that the four clustered residues form the pectin-binding site. Remarkably, the binding of PGIP to pectin is displaced in vitro by PGs, suggesting that PGIP interacts with pectin and PGs through overlapping although not identical regions. The specific interaction of PGIP with polygalacturonic acid may be strategic to protect pectins from the degrading activity of fungal PGs. PMID:16648220

  2. A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades

    SciTech Connect

    Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang

    2015-06-12

    Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions. - Highlights: • Lys27 in NEDD8 is critical for protein neddylation. • NEDD8 K27R mutant impairs the NEDD8 conjugation. • NEDD8 K27R mutant significantly reduces the activity of cullin-RING E3 ligases.

  3. Optimal content and ratio of lysine to arginine in the diet of Pacific white shrimp, Litopenaeus vannamei

    NASA Astrophysics Data System (ADS)

    Feng, Zhengfu; Dong, Chaohua; Wang, Linlin; Hu, Yanjiang; Zhu, Wei

    2013-07-01

    The optimal quantity of dietary lysine (Lys) and arginine (Arg), and the optimal ratio of dietary Lys to Arg for Pacific white shrimp Litopenaeus vannamei were investigated. Coated Lys and Arg were added to a basal diet (37.99% crude protein and 7.28% crude lipid) to provide graded levels of Lys and Arg. The experimental diets contained three Lys levels (2.51%, 2.11%, and 1.70% of total diet), and three Arg levels (1.41%, 1.80%, and 2.21% of total diet) and all combinations of these levels were tested. Pacific white shrimp, with a mean weight of 3.62±0.1 g, were randomly distributed in 36 fiberglass tanks with 30 shrimp per tank and reared on the experimental diets for 50 days. After the feeding trial, the growth performance, survival, feed conversion rate (FCR), body composition and protease and lipase activities in the hepatopancreases of the experimental shrimps were determined. The results show that weight gain (WG), specific growth rate (SGR), FCR, body protein, body Lys and Arg content were significantly affected by dietary Lys and Arg ( P <0.05) and improved when dietary Lys and Arg levels were 2.11% ˜ 2.51% and 1.80%˜2.21%, respectively. Protease and lipase activities in the hepatopancreases of the shrimps appeared higher when dietary Lys and Arg quantities were 2.11% ˜2.51% and 1.80%˜2.21%, although the difference was not statistically significant ( P >0.05). Therefore, according to our results, the optimal Lys and Arg quantities in the diet of Pacific white shrimp, L. vannamei, were considered to be 2.11%-2.51% and 1.80%-2.21%, respectively, and the optimal ratio to be 1:0.88-1:1.05.

  4. Histidine-lysine peptides as carriers of nucleic acids.

    PubMed

    Leng, Qixin; Goldgeier, Lisa; Zhu, Jingsong; Cambell, Patricia; Ambulos, Nicholas; Mixson, A James

    2007-03-01

    With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo. PMID:17440630

  5. Polymerization on the rocks: beta-amino acids and arginine

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  6. Evidence for proximal cysteine and lysine residues at or near the active site of arginine kinase of Stichopus japonicus.

    PubMed

    Guo, Qin; Chen, Baoyu; Wang, Xicheng

    2004-12-01

    Inactivation of arginine kinase (AK) of Stichopus japonicus by o-phthalaldehyde (OPTA) was investigated. The modified enzyme showed an absorption peak at 337 nm and a fluorescent emission peak at 410 nm, which are characteristic of an isoindole derivative formed by OPTA binding to a thiol and an amine group in proximity within the enzyme. Loss of enzymatic activity was concomitant with an increase in fluorescence intensity at 410 nm. Stoichiometry studies by Tsou's method showed that among the cysteine residues available for OPTA modification in the enzyme, only one was essential for the enzyme activity. This cysteine residue is located in a highly hydrophobic environment, presumably near ATP and ADP binding region. This conclusion was verified by 5,5 -dithiobis(2-nitrobenzoic acid) modification. In addition, these results were supported by means of electrophoresis and ultraviolet, fluorescence, circular dichroism spectroscopy and fast performance liquid chromatography. Sequence comparison suggested that this essential cysteine residue maybe the conservative Cys274. PMID:15627388

  7. A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

    PubMed Central

    Kaur, Jagdeep; Olkhova, Elena; Malviya, Viveka Nand; Grell, Ernst; Michel, Hartmut

    2014-01-01

    Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed. PMID:24257746

  8. Effect of Lysine to Arginine Mutagenesis in the V3 Loop of HIV-1 gp120 on Viral Entry Efficiency and Neutralization

    PubMed Central

    Schwalbe, Birco; Schreiber, Michael

    2015-01-01

    HIV-1 infection is characterized by an ongoing replication leading to T-lymphocyte decline which is paralleled by the switch from CCR5 to CXCR4 coreceptor usage. To predict coreceptor usage, several computer algorithms using gp120 V3 loop sequence data have been developed. In these algorithms an occupation of the V3 positions 11 and 25, by one of the amino acids lysine (K) or arginine (R), is an indicator for CXCR4 usage. Amino acids R and K dominate at these two positions, but can also be identified at positions 9 and 10. Generally, CXCR4-viruses possess V3 sequences, with an overall positive charge higher than the V3 sequences of R5-viruses. The net charge is calculated by subtracting the number of negatively charged amino acids (D, aspartic acid and E, glutamic acid) from the number of positively charged ones (K and R). In contrast to D and E, which are very similar in their polar and acidic properties, the characteristics of the R guanidinium group differ significantly from the K ammonium group. However, in coreceptor predictive computer algorithms R and K are both equally rated. The study was conducted to analyze differences in infectivity and coreceptor usage because of R-to-K mutations at the V3 positions 9, 10 and 11. V3 loop mutants with all possible RRR-to-KKK triplets were constructed and analyzed for coreceptor usage, infectivity and neutralization by SDF-1α and RANTES. Virus mutants R9R10R11 showed the highest infectivity rates, and were inhibited more efficiently in contrast to the K9K10K11 viruses. They also showed higher efficiency in a virus-gp120 paired infection assay. Especially V3 loop position 9 was relevant for a switch to higher infectivity when occupied by R. Thus, K-to-R exchanges play a role for enhanced viral entry efficiency and should therefore be considered when the viral phenotype is predicted based on V3 sequence data. PMID:25785610

  9. Effect of lysine to arginine mutagenesis in the V3 loop of HIV-1 gp120 on viral entry efficiency and neutralization.

    PubMed

    Schwalbe, Birco; Schreiber, Michael

    2015-01-01

    HIV-1 infection is characterized by an ongoing replication leading to T-lymphocyte decline which is paralleled by the switch from CCR5 to CXCR4 coreceptor usage. To predict coreceptor usage, several computer algorithms using gp120 V3 loop sequence data have been developed. In these algorithms an occupation of the V3 positions 11 and 25, by one of the amino acids lysine (K) or arginine (R), is an indicator for CXCR4 usage. Amino acids R and K dominate at these two positions, but can also be identified at positions 9 and 10. Generally, CXCR4-viruses possess V3 sequences, with an overall positive charge higher than the V3 sequences of R5-viruses. The net charge is calculated by subtracting the number of negatively charged amino acids (D, aspartic acid and E, glutamic acid) from the number of positively charged ones (K and R). In contrast to D and E, which are very similar in their polar and acidic properties, the characteristics of the R guanidinium group differ significantly from the K ammonium group. However, in coreceptor predictive computer algorithms R and K are both equally rated. The study was conducted to analyze differences in infectivity and coreceptor usage because of R-to-K mutations at the V3 positions 9, 10 and 11. V3 loop mutants with all possible RRR-to-KKK triplets were constructed and analyzed for coreceptor usage, infectivity and neutralization by SDF-1α and RANTES. Virus mutants R9R10R11 showed the highest infectivity rates, and were inhibited more efficiently in contrast to the K9K10K11 viruses. They also showed higher efficiency in a virus-gp120 paired infection assay. Especially V3 loop position 9 was relevant for a switch to higher infectivity when occupied by R. Thus, K-to-R exchanges play a role for enhanced viral entry efficiency and should therefore be considered when the viral phenotype is predicted based on V3 sequence data.

  10. Evaluating Force Fields for the Computational Prediction of Ionized Arginine and Lysine Side-Chains Partitioning into Lipid Bilayers and Octanol.

    PubMed

    Sun, Delin; Forsman, Jan; Woodward, Clifford E

    2015-04-14

    Abundant peptides and proteins containing arginine (Arg) and lysine (Lys) amino acids can apparently permeate cell membranes with ease. However, the mechanisms by which these peptides and proteins succeed in traversing the free energy barrier imposed by cell membranes remain largely unestablished. Precise thermodynamic studies (both theoretical and experimental) on the interactions of Arg and Lys residues with model lipid bilayers can provide valuable clues to the efficacy of these cationic peptides and proteins. We have carried out molecular dynamics simulations to calculate the interactions of ionized Arg and Lys side-chains with the zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayer for 10 widely used lipid/protein force fields: CHARMM36/CHARMM36, SLIPID/AMBER99SB-ILDN, OPLS-AA/OPLS-AA, Berger/OPLS-AA, Berger/GROMOS87, Berger/GROMOS53A6, GROMOS53A6/GROMOS53A6, nonpolarizable MARTINI, polarizable MARTINI, and BMW MARTINI. We performed umbrella sampling simulations to obtain the potential of mean force for Arg and Lys side-chains partitioning from water to the bilayer interior. We found significant differences between the force fields, both for the interactions between side-chains and bilayer surface, as well as the free energy cost for placing the side-chain at the center of the bilayer. These simulation results were compared with the Wimley-White interfacial scale. We also calculated the free energy cost for transferring ionized Arg and Lys side-chains from water to both dry and wet octanol. Our simulations reveal rapid diffusion of water molecules into octanol whereby the equilibrium mole fraction of water in the wet octanol phase was ∼25%. Surprisingly, our free energy calculations found that the high water content in wet octanol lowered the water-to-octanol partitioning free energies for cationic residues by only 0.6 to 0.7 kcal/mol. PMID:26574387

  11. Effect of betaine and arginine in lysine-deficient diets on growth, carcass traits, and pork quality.

    PubMed

    Madeira, M S; Alfaia, C M; Costa, P; Lopes, P A; Martins, S V; Lemos, J P C; Moreira, O; Santos-Silva, J; Bessa, R J B; Prates, J A M

    2015-10-01

    Forty entire male pigs from a commercial crossbreed (Duroc × Large White × Landrace) were used to investigate the individual or combined effects of betaine and Arg supplementation in Lys-deficient diets on growth performance, carcass traits, and pork quality. Pigs with 59.9 ± 1.65 kg BW were randomly assigned to 1 of 5 dietary treatments ( = 8). The 5 dietary treatments were normal Lys and CP diet (0.51% Lys and 16% CP; control), reduced Lys and CP diet (0.35% Lys and 13% CP), reduced Lys and CP diet with betaine supplementation (0.33%), reduced Lys and CP diet with Arg supplementation (1.5%), and reduced Lys and CP diet with betaine and Arg supplementation (0.33% betaine and 1.5% Arg). Pigs were slaughtered at 92.7 ± 2.54 kg BW. The Lys-deficient diets (-35% Lys) increased intramuscular fat (IMF) content by 25% ( = 0.041) and meat juiciness by 12% ( = 0.041) but had a negative effect on growth performance ( < 0.05) of pigs. In addition, Lys-deficient diets increased L* ( = 0.005) and b* ( = 0.010) muscle color parameters and perirenal fat deposition ( < 0.001) and decreased both HCW ( = 0.015) and loin weight ( = 0.023). Betaine and Arg supplementation of Lys-deficient diets had no effect on IMF content but increased ( < 0.05) overall pork acceptability. Arginine supplementation also increased ( = 0.003) meat tenderness. Differences in fatty acid composition of pork were not detected among dietary treatment groups. However, oleic acid was positively correlated ( < 0.05) with IMF content, juiciness, flavor, and overall acceptability of meat. Data confirm that dietary CP reduction enhances pork eating quality but negatively affects pigs' growth performance. Moreover, it is suggested that betaine and Arg supplementation of Lys-deficient diets does not further increase IMF content but improves some pork sensory traits, including overall acceptability.

  12. Utilization of ornithine and arginine as specific precursors of clavulanic acid.

    PubMed Central

    Romero, J; Liras, P; Martín, J F

    1986-01-01

    Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine. PMID:2877616

  13. Distinct Paths for Basic Amino Acid Export in Escherichia coli: YbjE (LysO) Mediates Export of l-Lysine

    PubMed Central

    Pathania, Amit

    2015-01-01

    ABSTRACT In Escherichia coli, argO encodes an exporter for l-arginine (Arg) and its toxic analogue canavanine (CAN), and its transcriptional activation and repression, by Arg and l-lysine (Lys), respectively, are mediated by the regulator ArgP. Accordingly argO and argP mutants are CAN supersensitive (CANss). We report the identification of ybjE as a gene encoding a predicted inner membrane protein that mediates export of Lys, and our results confirm the previous identification with a different approach of YbjE as a Lys exporter, reported by Ueda and coworkers (T. Ueda, Y. Nakai, Y. Gunji, R. Takikawa, and Y. Joe, U.S. patents 7,629,142 B2 [December 2009] and 8,383,363 B1 [February 2013] and European patent 1,664,318 B1 [September 2009]). ybjE was isolated as a multicopy suppressor of the CANss phenotype of a strain lacking ArgO. The absence of YbjE did not confer a CANss phenotype but instead conferred hypersensitivity to the lysine antimetabolite thialysine and led to growth inhibition by the dipeptide lysylalanine, which is associated with elevated cellular Lys content. YbjE overproduction resulted in Lys excretion and syntrophic cross-feeding of a Lys auxotroph. Constitutive overexpression of argO promoted Lys cross-feeding that is indicative of a latent Lys export potential of ArgO. Arg modestly repressed ybjE transcription in an ArgR-dependent manner, and ArgR displayed Arg-sensitive binding to the ybjE promoter region in vitro. Our studies suggest that the reciprocal repression of argO and ybjE, respectively, by Lys and Arg confers the specificity for basic amino acid export by distinct paths and that such cross-repression contributes to maintenance of cytoplasmic Arg/Lys balance. We propose that YbjE be redesignated LysO. IMPORTANCE This work ascribes a lysine export function to the product of the ybjE gene of Escherichia coli, leading to a physiological scenario wherein two proteins, ArgO and YbjE, perform the task of separately exporting arginine and

  14. Effect of betaine and arginine in lysine-deficient diets on growth, carcass traits, and pork quality.

    PubMed

    Madeira, M S; Alfaia, C M; Costa, P; Lopes, P A; Martins, S V; Lemos, J P C; Moreira, O; Santos-Silva, J; Bessa, R J B; Prates, J A M

    2015-10-01

    Forty entire male pigs from a commercial crossbreed (Duroc × Large White × Landrace) were used to investigate the individual or combined effects of betaine and Arg supplementation in Lys-deficient diets on growth performance, carcass traits, and pork quality. Pigs with 59.9 ± 1.65 kg BW were randomly assigned to 1 of 5 dietary treatments ( = 8). The 5 dietary treatments were normal Lys and CP diet (0.51% Lys and 16% CP; control), reduced Lys and CP diet (0.35% Lys and 13% CP), reduced Lys and CP diet with betaine supplementation (0.33%), reduced Lys and CP diet with Arg supplementation (1.5%), and reduced Lys and CP diet with betaine and Arg supplementation (0.33% betaine and 1.5% Arg). Pigs were slaughtered at 92.7 ± 2.54 kg BW. The Lys-deficient diets (-35% Lys) increased intramuscular fat (IMF) content by 25% ( = 0.041) and meat juiciness by 12% ( = 0.041) but had a negative effect on growth performance ( < 0.05) of pigs. In addition, Lys-deficient diets increased L* ( = 0.005) and b* ( = 0.010) muscle color parameters and perirenal fat deposition ( < 0.001) and decreased both HCW ( = 0.015) and loin weight ( = 0.023). Betaine and Arg supplementation of Lys-deficient diets had no effect on IMF content but increased ( < 0.05) overall pork acceptability. Arginine supplementation also increased ( = 0.003) meat tenderness. Differences in fatty acid composition of pork were not detected among dietary treatment groups. However, oleic acid was positively correlated ( < 0.05) with IMF content, juiciness, flavor, and overall acceptability of meat. Data confirm that dietary CP reduction enhances pork eating quality but negatively affects pigs' growth performance. Moreover, it is suggested that betaine and Arg supplementation of Lys-deficient diets does not further increase IMF content but improves some pork sensory traits, including overall acceptability. PMID:26523565

  15. Key chemical factors of arginine finger catalysis of F1-ATPase clarified by an unnatural amino acid mutation.

    PubMed

    Yukawa, Ayako; Iino, Ryota; Watanabe, Rikiya; Hayashi, Shigehiko; Noji, Hiroyuki

    2015-01-20

    A catalytically important arginine, called Arg finger, is employed in many enzymes to regulate their functions through enzymatic hydrolysis of nucleotide triphosphates. F1-ATPase (F1), a rotary motor protein, possesses Arg fingers which catalyze hydrolysis of adenosine triphosphate (ATP) for efficient chemomechanical energy conversion. In this study, we examined the Arg finger catalysis by single-molecule measurements for a mutant of F1 in which the Arg finger is substituted with an unnatural amino acid of a lysine analogue, 2,7-diaminoheptanoic acid (Lyk). The use of Lyk, of which the side chain is elongated by one CH2 unit so that its chain length to the terminal nitrogen of amine is set to be equal to that of arginine, allowed us to resolve key chemical factors in the Arg finger catalysis, i.e., chain length matching and chemical properties of the terminal groups. Rate measurements by single-molecule observations showed that the chain length matching of the side-chain length is not a sole requirement for the Arg finger to catalyze the ATP hydrolysis reaction step, indicating the crucial importance of chemical properties of the terminal guanidinium group in the Arg finger catalysis. On the other hand, the Lyk mutation prevented severe formation of an ADP inhibited state observed for a lysine mutant and even improved the avoidance of inhibition compared with the wild-type F1. The present study demonstrated that incorporation of unnatural amino acids can widely extend with its high "chemical" resolution biochemical approaches for elucidation of the molecular mechanism of protein functions and furnishing novel characteristics.

  16. The combination effect of L-arginine and NaCl on bitterness suppression of amino acid solutions.

    PubMed

    Ogawa, Tazuko; Nakamura, Tomoko; Tsuji, Eriko; Miyanaga, Yohko; Nakagawa, Hiroyo; Hirabayashi, Hitomi; Uchida, Takahiro

    2004-02-01

    The purpose of the present study was to quantify the degree of suppression of the bitterness of two amino acids (L-isoleucine (L-Ile), and L-phenylalanine (L-Phe)) which could be achieved by the addition of various test chemicals, and to examine the mechanism of this bitterness suppression. The test chemicals used were two sweeteners (sucrose, aspartame), NaCl, various acidic (L-aspartic acid, L-glutamic acid), or basic (L-histidine, L-lysine and L-arginine) amino acids, tannic acid and phosphatidic acid. The combination of L-arginine (L-Arg) and NaCl together was the most effective in reducing the bitterness of 100 mM L-Ile and L-Phe solutions in human gustatory sensation tests. Even in bitterness of 0.1 mM quinine solution, L-Arg was also successful in reducing the bitterness. This bitterness-suppression effect was specific to L-Arg and not to the other basic amino acids. No comparable taste-masking effect was observed for the acidic amino acids. The artificial taste sensor failed to predict completely the bitterness-suppressing effect of L-Arg. It seems likely that the bitterness-suppressing effect of L-Arg is mediated not only by binding at the receptor site, but also elsewhere in the process of bitterness perception, such as a direct effect on the sodium channel. It is conjectured that the guanidinium group of L-Arg may interact with sodium channels in taste bud membranes.

  17. Growth and arginine metabolism of the wine lactic acid bacteria Lactobacillus buchneri and Oenococcus oeni at different pH values and arginine concentrations.

    PubMed

    Mira De Orduña, R; Patchett, M L; Liu, S Q; Pilone, G J

    2001-04-01

    During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth of Lactobacillus buchneri CUC-3 and Oenococcus oeni strains MCW and Lo111 in wine were investigated. In contrast to L. buchneri CUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth of L. buchneri CUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF. PMID:11282618

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

    PubMed Central

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

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis. PMID:26808268

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis. PMID:26808268

  1. The Arginine/Lysine-Rich Element within the DNA-Binding Domain Is Essential for Nuclear Localization and Function of the Intracellular Pathogen Resistance 1.

    PubMed

    Yao, Kezhen; Wu, Yongyan; Chen, Qi; Zhang, Zihan; Chen, Xin; Zhang, Yong

    2016-01-01

    The mouse intracellular pathogen resistance 1 (Ipr1) gene plays important roles in mediating host immunity and previous work showed that it enhances macrophage apoptosis upon mycobacterium infection. However, to date, little is known about the regulation pattern of Ipr1 action. Recent studies have investigated the protein-coding genes and microRNAs regulated by Ipr1 in mouse macrophages, but the structure and the functional motif of the Ipr1 protein have yet to be explored. In this study, we analyzed the domains and functional motif of the Ipr1 protein. The resulting data reveal that Ipr1 protein forms a homodimer and that the Sp100-like domain mediates the targeting of Ipr1 protein to nuclear dots (NDs). Moreover, we found that an Ipr1 mutant lacking the classic nuclear localization signal (cNLS) also translocated into the nuclei, suggesting that the cNLS is not the only factor that directs Ipr1 nuclear localization. Additionally, mechanistic studies revealed that an arginine/lysine-rich element within the DNA-binding domain (SAND domain) is critical for Ipr1 binding to the importin protein receptor NPI-1, demonstrating that this element plays an essential role in mediating the nuclear localization of Ipr1 protein. Furthermore, our results show that this arginine/lysine-rich element contributes to the transcriptional regulation and apoptotic activity of Ipr1. These findings highlight the structural foundations of Ipr1 action and provide new insights into the mechanism of Ipr1-mediated resistance to mycobacterium. PMID:27622275

  2. The Arginine/Lysine-Rich Element within the DNA-Binding Domain Is Essential for Nuclear Localization and Function of the Intracellular Pathogen Resistance 1

    PubMed Central

    Yao, Kezhen; Wu, Yongyan; Chen, Qi; Zhang, Zihan; Chen, Xin; Zhang, Yong

    2016-01-01

    The mouse intracellular pathogen resistance 1 (Ipr1) gene plays important roles in mediating host immunity and previous work showed that it enhances macrophage apoptosis upon mycobacterium infection. However, to date, little is known about the regulation pattern of Ipr1 action. Recent studies have investigated the protein-coding genes and microRNAs regulated by Ipr1 in mouse macrophages, but the structure and the functional motif of the Ipr1 protein have yet to be explored. In this study, we analyzed the domains and functional motif of the Ipr1 protein. The resulting data reveal that Ipr1 protein forms a homodimer and that the Sp100-like domain mediates the targeting of Ipr1 protein to nuclear dots (NDs). Moreover, we found that an Ipr1 mutant lacking the classic nuclear localization signal (cNLS) also translocated into the nuclei, suggesting that the cNLS is not the only factor that directs Ipr1 nuclear localization. Additionally, mechanistic studies revealed that an arginine/lysine-rich element within the DNA-binding domain (SAND domain) is critical for Ipr1 binding to the importin protein receptor NPI-1, demonstrating that this element plays an essential role in mediating the nuclear localization of Ipr1 protein. Furthermore, our results show that this arginine/lysine-rich element contributes to the transcriptional regulation and apoptotic activity of Ipr1. These findings highlight the structural foundations of Ipr1 action and provide new insights into the mechanism of Ipr1-mediated resistance to mycobacterium. PMID:27622275

  3. Pathways of Amino Acid Degradation in Nilaparvata lugens (Stål) with Special Reference to Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase (LKR/SDH)

    PubMed Central

    Wan, Pin-Jun; Yuan, San-Yue; Tang, Yao-Hua; Li, Kai-Long; Yang, Lu; Fu, Qiang; Li, Guo-Qing

    2015-01-01

    Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens. PMID:26000452

  4. Voluntary wheel running is beneficial to the amino acid profile of lysine-deficient rats.

    PubMed

    Nagao, Kenji; Bannai, Makoto; Seki, Shinobu; Kawai, Nobuhiro; Mori, Masato; Takahashi, Michio

    2010-06-01

    Rats voluntarily run up to a dozen kilometers per night when their cages are equipped with a running wheel. Daily voluntary running is generally thought to enhance protein turnover. Thus, we sought to determine whether running worsens or improves protein degradation caused by a lysine-deficient diet and whether it changes the utilization of free amino acids released by proteolysis. Rats were fed a lysine-deficient diet and were given free access to a running wheel or remained sedentary (control) for 4 wk. Amino acid levels in plasma, muscle, and liver were measured together with plasma insulin levels and tissue weight. The lysine-deficient diet induced anorexia, skeletal muscle loss, and serine and threonine aminoacidemia, and it depleted plasma insulin and essential amino acids in skeletal muscle. Allowing rats to run voluntarily improved these symptoms; thus, voluntary wheel running made the rats less susceptible to dietary lysine deficiency. Amelioration of the declines in muscular leucine and plasma insulin observed in running rats could contribute to protein synthesis together with the enhanced availability of lysine and other essential amino acids in skeletal muscle. These results indicate that voluntary wheel running under lysine-deficient conditions does not enhance protein catabolism; on the contrary, it accelerates protein synthesis and contributes to the maintenance of muscle mass. The intense nocturnal voluntary running that characterizes rodents might be an adaptation of lysine-deficient grain eaters that allows them to maximize opportunities for food acquisition. PMID:20233939

  5. Structural Insights Into Amino Acid Binding and Gene Control by a Lysine Riboswitch

    SciTech Connect

    Serganov, A.; Huang, L; Patel, D

    2008-01-01

    In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches1, 2, 3, 4. One of the important regulatory circuits involves lysine-specific riboswitches, which direct the biosynthesis and transport of lysine and precursors common for lysine and other amino acids. To understand the molecular basis of amino acid recognition by riboswitches, here we present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the lysine-bound (1.9 A) and free (3.1 A) states. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K+-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogues5, including antibiotics, in an effort to understand the ligand-binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.

  6. A Candida guilliermondii lysine hyperproducer capable of elevated citric acid production.

    PubMed

    West, Thomas P

    2016-05-01

    A mutant of the yeast Candida guilliermondii ATCC 9058 exhibiting elevated citric acid production was isolated based upon its ability to overproduce lysine. This method involved the use of a solid medium containing a combination of lysine analogues to identify a mutant that produced a several-fold higher lysine level compared to its parent strain using glucose or glycerol as a carbon source. The mutant strain was also capable of producing more than a fivefold higher citric acid level on glycerol as a carbon source compared to its parent strain. It was concluded that the screening of yeast lysine hyperproducer strains could provide a rapid approach to isolate yeast citric acid hyperproducer strains. PMID:27038943

  7. A Candida guilliermondii lysine hyperproducer capable of elevated citric acid production.

    PubMed

    West, Thomas P

    2016-05-01

    A mutant of the yeast Candida guilliermondii ATCC 9058 exhibiting elevated citric acid production was isolated based upon its ability to overproduce lysine. This method involved the use of a solid medium containing a combination of lysine analogues to identify a mutant that produced a several-fold higher lysine level compared to its parent strain using glucose or glycerol as a carbon source. The mutant strain was also capable of producing more than a fivefold higher citric acid level on glycerol as a carbon source compared to its parent strain. It was concluded that the screening of yeast lysine hyperproducer strains could provide a rapid approach to isolate yeast citric acid hyperproducer strains.

  8. Influence of wine-like conditions on arginine utilization by lactic acid bacteria.

    PubMed

    Araque, Isabel; Reguant, Cristina; Rozès, Nicolas; Bordons, Albert

    2011-12-01

    Wine can contain trace amounts of ethyl carbamate (EC), a carcinogen formed when ethanol reacts with carbamyl compounds such as citrulline. EC is produced from arginine by lactic acid bacteria (LAB), e.g., Lactobacillus and Pediococcus. Although the amounts of EC in wine are usually negligible, over the last few years there has been a slight but steady increase, as climate change has increased temperatures and alcohol levels have become proportionately higher, both of which favor EC formation. In this study, resting cells of LAB were used to evaluate the effects of ethanol, glucose, malic acid, and low pH on the ability of non-oenococcal strains of these bacteria to degrade arginine and excrete citrulline. Malic acid was found to clearly inhibit arginine consumption in all strains. The relation between citrulline produced and arginine consumed was clearly higher in the presence of ethanol (10-12%) and at low pH (3.0), which is consistent with both the decreased amount of ornithine produced from arginine and the reduction in arginine degradation. In L. brevis and L. buchneri strains isolated from wine and beer, respectively, the synthesis of citrulline from arginine was highest. PMID:22569760

  9. Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance.

    PubMed

    Yang, Qing-Qing; Zhang, Chang-Quan; Chan, Man-Ling; Zhao, Dong-Sheng; Chen, Jin-Zhu; Wang, Qing; Li, Qian-Feng; Yu, Heng-Xiu; Gu, Ming-Hong; Sun, Samuel Sai-Ming; Liu, Qiao-Quan

    2016-07-01

    Rice (Oryza sativa L.), a major staple crop worldwide, has limited levels of the essential amino acid lysine. We previously produced engineered rice with increased lysine content by expressing bacterial aspartate kinase and dihydrodipicolinate synthase and inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase activity. However, the grain quality, field performance, and integration patterns of the transgenes in these lysine-enriched lines remain unclear. In the present study, we selected several elite transgenic lines with endosperm-specific or constitutive regulation of the above key enzymes but lacking the selectable marker gene. All target transgenes were integrated into the intragenic region in the rice genome. Two pyramid transgenic lines (High Free Lysine; HFL1 and HFL2) with free lysine levels in seeds up to 25-fold that of wild type were obtained via a combination of the above two transgenic events. We observed a dramatic increase in total free amino acids and a slight increase in total protein content in both pyramid lines. Moreover, the general physicochemical properties were improved in pyramid transgenic rice, but the starch composition was not affected. Field trials indicated that the growth of HFL transgenic rice was normal, except for a slight difference in plant height and grain colour. Taken together, these findings will be useful for the potential commercialization of high-lysine transgenic rice. PMID:27252467

  10. Effect of Selectively Introducing Arginine and D-Amino Acids on the Antimicrobial Activity and Salt Sensitivity in Analogs of Human Beta-Defensins

    PubMed Central

    Olli, Sudar; Rangaraj, Nandini; Nagaraj, Ramakrishnan

    2013-01-01

    We have examined the antimicrobial activity of C-terminal analogs of human β-defensins HBD-1and-3 wherein lysines have been selectively replaced by L- and D-arginines and L-isoleucine substituted with its D-enantiomer. The analogs exhibited antibacterial and antifungal activities. Physiological concentration of NaCl did not attenuate the activity of the peptides against Gram-negative bacteria considerably, while some attenuation of activity was observed against S. aureus. Variable attenuation of activity was observed in the presence of Ca2+ and Mg2+. Introduction of D-amino acids abrogated the need for a disulfide bridge for exhibiting activity. Confocal images of carboxyfluorescein (CF) labeled peptides indicated initial localization on the membrane and subsequent translocation into the cell. Analogs corresponding to cationic rich segments of human defensins substituted with L- and D-arginine, could be attractive candidates for development as future therapeutic drugs. PMID:24086767

  11. Suppression of human cervical cancer cell lines Hela and DoTc2 4510 by a mixture of lysine, proline, ascorbic acid, and green tea extract.

    PubMed

    Roomi, M W; Ivanov, V; Kalinovsky, T; Niedzwiecki, A; Rath, M

    2006-01-01

    Cervical cancer, the second most common cancer in women, once metastasized, leads to poor prognosis. We investigated the antitumor effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human cervical cancer cells Hela (CCL-2) and DoTc2 4510 by measuring cell proliferation (MTT assay), modulation of matrix metalloproteinases (MMP)-2 and MMP-9) expression (gelatinase zymography), and cancer cell invasive potential (Matrigel). NM showed significant antiproliferative effect on CCL-2 and DoTc2 4510 cancer cells. The NM inhibited CCL-2 expression of MMP-2 and MMP-9 in a dose-dependent fashion, with virtual total inhibition of MMP-2 at 1000 microg/mL and MMP-9 at 500 microg/mL NM. Untreated DoTc2 4510 cells showed MMP-9 expression, which was enhanced with phorbol 12-myristate 13-acetate treatment. NM inhibited MMP-9 expression in a dose-dependent fashion, with virtual inhibition at 500 microg/mL. Invasion of human cervical cancer cells CCL-2 and DoTc2 4510 through Matrigel decreased in a dose-dependent fashion, with 100% inhibition at 500 microg/mL NM (P < 0.0001) and 1000 microg/mL NM (P < 0.0001), respectively. Our results suggest that the mixture of lysine, proline, arginine, ascorbic acid, and green tea extract has potential in the treatment of cervical cancer by inhibiting critical steps in cancer development and spread.

  12. Quantitative comparison between poly(L-arginine) and poly(L-lysine) at each step of polyplex-based gene transfection using a microinjection technique

    NASA Astrophysics Data System (ADS)

    Hashimoto, Tomoko; Kawazu, Takeshi; Nagasaki, Takeshi; Murakami, Akira; Yamaoka, Tetsuji

    2012-02-01

    Among the well-studied polypeptide-type gene carriers, transfection efficiency is empirically known to be higher for poly(L-arginine) (PR) than poly(L-lysine) (PK). The big difference between PR and PK should be determined at one of the intracellular trafficking steps based on the different charge densities, structures or PKa values. However, the endosomal escape and the intranuclear transcription efficiency in living cells have not been clarified yet. In this study, a novel method for quantifying the intranuclear transcription efficiency and the nuclear transport of the polyplex is established based on the nuclear and the cytosolic microinjection technique, and the results for PK and PR with different molecular weights (MWs) are compared in living cells. The intranuclear transcription efficiency is the same in PR and PK and it decreases rapidly with increasing MW, in spite of the commonly measured transfection efficiency. The transcription efficiency is strongly suppressed at high MW and strongly correlates with the polyplex forming ability expressed as a critical ratio of the number of polypeptide cationic groups to the number of pDNA anionic groups. When considered with the results of the cellular uptake and in vitro transfection with or without chloroquine, the rate-limiting step for their gene transfer is the buffering effect-independent endosomal escape.

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

    PubMed

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

    2013-07-01

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

  14. A star-shaped porphyrin-arginine functionalized poly(L-lysine) copolymer for photo-enhanced drug and gene co-delivery.

    PubMed

    Ma, Dong; Lin, Qian-Ming; Zhang, Li-Ming; Liang, Yuan-Yuan; Xue, Wei

    2014-05-01

    The co-delivery of drug and gene has become the primary strategy in cancer and other disease therapy. To co-deliver hydrophobic drug and functional gene efficiently into tumor cells, a star-shaped copolymer (PP-PLLD-Arg) with a photochemical internalization effect consisting of a porphyrin (PP) core and arginine-functionalized poly(L-lysine) dendron (PLLD-Arg) arms has been designed, and used to co-deliver docetaxel (DOC) and MMP-9 shRNA plasmid for nasopharyngeal cancer therapy. It was found that PP-PLLD-Arg/MMP-9 nanocomplex showed the photo-enhanced gene transfection efficiency in vitro, and could mediate a significant reduce of MMP-9 protein expression in HNE-1 cells. For co-delivery analysis, the obtained PP-PLLD-Arg/DOC/MMP-9 complexes could induce a more significant apoptosis than DOC or MMP-9 used only, and decreased invasive capacity of HNE-1 cells. Moreover, the star-shaped copolymer exhibited better blood compatibility and lower cytotoxicity compared to PEI-25k in the hemolysis and MTT assays, and also showed a good biocompatibility in vivo. Therefore, PP-PLLD-Arg with suited irradiation is a promising non-toxic and photo-inducible effective drug and gene delivery strategy, which should be encouraged in tumor therapy.

  15. Structural characterization of arginine-vasopressin and lysine-vasopressin by Fourier- transform ion cyclotron resonance mass spectrometry and infrared multiphoton dissociation.

    PubMed

    Bianco, Giuliana; Battista, Fabio; Buchicchio, Alessandro; Amarena, Concetta G; Schmitt-Kopplin, Philippe; Guerrieri, Antonio

    2015-01-01

    Arginine-vasopressin (AVP) and lysine-vasopressin (LVP) were analyzed by reversed-phase liquid chromatography/mass spectrometry (LC-MS) using Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) electrospray ionization (ESI) in the positive ion mode. LVP and AVP exhibited the protonated adduct [M+H](+) as the predominant ion at m/z 1056.43965 and at m/z 1084.44561, respectively. Infrared multiphoton dissociation (IRMPD), using a CO(2) laser source at a wavelength of 10.6 μm, was applied to protonated vasopressin molecules. The IRMPD mass spectra presented abundant mass fragments essential for a complete structural information. Several fragment ions, shared between two target molecules, are discussed in detail. Some previously unpublished fragments were identified unambiguously utilizing the high resolution and accurate mass information provided by the FT-ICR mass spectrometer. The opening of the disulfide loop and the cleavage of the peptide bonds within the ring were observed even under low-energy fragmentation conditions. Coupling the high-performance FT-ICR mass spectrometer with IRMPD as a contemporary fragmentation technique proved to be very promising for the structural characterization of vasopressin.

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. AMPHOTERIC BEHAVIOR OF COMPLEX SYSTEMS : III. THE CONDUCTIVITY OF SULFANILIC ACID-LYSIN MIXTURES.

    PubMed

    Stearn, A E

    1927-01-20

    Conductivities of sulfanilic acid, lysin, and mixtures of the two were made over a wide pH range, the pH being adjusted by means of phosphate buffers. The actual conductivities of the sulfanilic acid, the lysin, and the mixture were calculated. The difference between the conductivity of the mixture and the sum of the conductivities of the components alone passes through a maximum at a pH theoretically calculable as the isoelectric point of the system. Certain applications of the results are made to the explanation of the behavior of living tissues.

  20. Role of pipecolic acid in the biosynthesis of lysine in Rhodotorula glutinis.

    PubMed

    Kinzel, J J; Bhattacharjee, J K

    1979-05-01

    The role of pipecolic acid in the biosynthesis of lysine was investigated in Rhodotorula glutinis, an aerobic red yeast. Supplementation of pipecolic acid in the minimal medium supported the growth of mutants lys2, lys3, and lys5; alpha-aminoadipic acid supported the growth of lys5; but neither alpha-aminoadipic acid nor pipecolic acid supported the growth of mutants MNNG42 and MNNG37. During the growth of the appropriate mutants, pipecolic acid was removed from the growth medium and the intracellular pool. In tracer experiments, radioactivity from [(14)C]pipecolic acid was selectively incorporated into the cellular lysine of lys5 and the wild-type strain. l-Pipecolic acid-dependent enzyme activity did not require any cofactor and was inhibited by mercuric chloride and potassium cyanide. This activity was present in the wild-type strain and all of the mutants tested and was repressed in mutant lys5 when grown in the presence of higher concentration of lysine. The reaction product of pipecolic acid was converted to saccharopine by lys5 enzyme in the presence of glutamate and reduced nicotin-amide adenine dinucleotide phosphate. Mutant MNNG37 lacked the saccharopine dehydrogenase activity, indicating that this step is involved in the conversion of alpha-aminoadipic acid and pipecolic acid to lysine. Mutants MNNG37 and MNNG42 accumulated a p-dimethylaminobenzaldehyde-reacting product in the culture supernatant and in the intracellular pool. Chromatographic properties of the p-dimethylaminobenzaldehyde adduct and that of the pipecolic acid-dependent reaction product were similar. The reaction product and the accumulation product were characterized on the basis of mass and absorption spectra as alpha-aminoadipic-semialdehyde, which in solution remains in equilibrium with Delta(1)-piperideine-6-carboxylic acid. Since alpha-aminoadipic-semialdehyde is a known intermediate of the alpha-aminoadipic acid pathway for the biosynthesis of lysine, it is concluded that pipecolic

  1. Biological half-lives and organ distribution of tritiated 8-lysine-vasopressin and 1-deamino-8-D-arginine-vasopressin in Brattleboro rats

    SciTech Connect

    Janaky, T.; Laczi, F.; Laszlo, F.A.

    1982-01-01

    The biological half-lives and organ distribution of tritiated 8-lysine-vasopressin and 1-deamino-8-D-arginine-vasopressin were determined in R-Amsterdam rats and in homozygous and heterozygous Brattleboro rats with hereditary central diabetes insipidus. It was found that the biological half-lives of (/sup 3/H)LVP and (/sup 3/H)dDAVP in the Brattleboro rats did not differ significantly from that found in the control R-Amsterdam rats. The half-life of (/sup 3/H)dDAVP proved longer than that of (/sup 3/H)LVP in all three groups of animals. In the case of (/sup 3/H)LVP the highest radioactivities were observed in the neurohypophyses, adenohypophyses, and kidneys of both the R-Amsterdam and Brattleboro rats. The accumulation of tritiated material was higher in the small intestine of the Brattleboro rats than in that of the R-Amsterdam animals. In all three groups of rats, (/sup 3/H)dDAVP was accumulated to the greatest extent in the kidney and the small intestine. The kidney and small intestine contained less radioactivity in homozygous Brattleboro rats than in the controls. There was only a slight radioactivity accumulation in the adenohypophysis and neurohypophysis. From the results it was concluded that the decrease in the rate of enzymatic decomposition may play a role in the increased duration of antidiuretic action of dDAVP. The results have led to the conclusion that the accelerated elimination of vasopressin and its pathologic organ accumulation are probably not involved in the water metabolism disturbance of Brattleboro rats with hereditary diabetes insipidus.

  2. The Natural Antimicrobial Subtilosin A Synergizes with Lauramide Arginine Ethyl Ester (LAE), ε-Poly-L-lysine (Polylysine), Clindamycin Phosphate and Metronidazole, Against the Vaginal Pathogen Gardnerella vaginalis.

    PubMed

    Cavera, Veronica L; Volski, Anna; Chikindas, Michael L

    2015-06-01

    Bacterial vaginosis (BV) is a common, recurrent vaginal infection linked to increased chances of preterm delivery, incidence of sexually transmitted infections and fertility problems. BV is caused by a shift of the vaginal ecosystem from predominately Lactobacillus to a multispecies Actinomyces biofilm with the most common representatives identified as Gardnerella vaginalis and Prevotella spp. Current treatments have been associated with increased resistance as well as negative effects on healthy microbiota. The objective of this study was to evaluate the synergistic potential of ten two-antimicrobial combinations against G. vaginalis and four representative lactobacilli. The four tested antimicrobials were lauramide arginine ethyl ester, ε-poly-L-lysine, clindamycin phosphate, metronidazole and the bacteriocin subtilosin A. The use of bacteriocins as either synergist or alternative treatment positions bacteriocins as an excellent alternative to current antibiotics. The microdilution method was used to determine the minimum inhibitory concentration (MIC) of each of the antimicrobials individually, and the checkerboard assay was used to evaluate these MICs in combination. Clindamycin and subtilosin (CS), and metronidazole and subtilosin were synergistic against G. vaginalis in terms of fractional inhibitory concentration index (FICI). All tested combinations were found to have Bliss synergy. The combination of clindamycin and polylysine (CP) was identified as antagonistic against L. acidophilus in terms of both FICI and Bliss synergy. The combination of clindamycin and metronidazole (CM) was antagonistic against L. vaginalis for both FICI and Bliss synergy. The combinations of CP, clindamycin and LAE, CS, and LAE and polylysine were identified as Bliss antagonistic against L. vaginalis but did not indicate FICI antagonism.

  3. Further ultrastructural research of Chara vulgaris spermiogenesis: endoplasmic reticulum, structure of chromatin, 3H-lysine and 3H-arginine incorporation.

    PubMed

    Kwiatkowska, Maria; Popłońska, Katarzyna

    2002-01-01

    On the basis of morphological features, 10 consecutive structural phases of spermatids were identified in Chara vulgaris spermiogenesis. They were schematically presented. In early and middle spermiogenesis, i.e. during the period preceding formation of fibrillar structure of mature spermatozoid nucleus, a slight remodelling of chromatin, accompanied by proplastid transformation into an amyloplast as well as by development of 2 flagella and a microtubular manchette, is observed. First, condensed chromatin concentrates around the nuclear envelope (phases III-V) and then it transforms into a network-like structure (phase VI). This change in chromatin structure is preceded by nucleolar extrusion to the cytoplasm where nucleoli become degraded (phase IV) and by a dynamic development of rough endoplasmic reticulum (RER) (phase V) which is continuous with the nuclear envelope and with RER of the adjacent spermatids via plasmodesmata. The inner membrane of the nuclear envelope invaginates into the nucleoplasm in which "nuclear reticulum" appears. It all happens during increased 3H-arginine and 3H-lysine incorporation into proteins which are rapidly translocated into the nucleus. In medium-late spermiogenesis (phases VI-VIII), network-like condensed chromatin disappears. Next, the structure of the nucleus changes dramatically. Short, randomly positioned fibrils (phase VII) appear and gradually become longer (phase VIII), thicker (phase IX) and more distinct, lying parallel to the surface of elongating and curling nucleus. Membranes of the nuclear envelope become closer to each other and a distinct dark layer--probably lamin--appears adhering to the inner membrane of the nuclear envelope. Towards the end of spermiogenesis (phase X), very densely packed parallel helices, ca 2 nm in diameter, are visible. The surfaces of flagella and the spermatozoid are covered with diamond-shaped larger and smaller scales, respectively. Helically coiled spermatozoids are liberated from

  4. Glutamine, glutamate, and arginine-based acid resistance in Lactobacillus reuteri.

    PubMed

    Teixeira, Januana S; Seeras, Arisha; Sanchez-Maldonado, Alma Fernanda; Zhang, Chonggang; Su, Marcia Shu-Wei; Gänzle, Michael G

    2014-09-01

    This study aimed to determine whether glutamine deamidation improves acid resistance of Lactobacillus reuteri, and to assess whether arginine, glutamine, and glutamate-mediated acid resistance are redundant or complementary mechanisms of acid resistance. Three putative glutaminase genes, gls1, gls2, and gls3, were identified in L. reuteri 100-23. All three genes were expressed during growth in mMRS and wheat sourdough. L. reuteri consistently over-expressed gls3 and the glutamate decarboxylase gadB. L. reuteri 100-23ΔgadB over-expressed gls3 and the arginine deiminase gene adi. Analysis of the survival of L. reuteri in acidic conditions revealed that arginine conversion is effective at pH of 3.5 while glutamine or glutamate conversion were effective at pH of 2.5. Arginine conversion increased the pHin but not ΔΨ; glutamate decarboxylation had only a minor effect on the pHin but increased the ΔΨ. This study demonstrates that glutamine deamidation increases the acid resistance of L. reuteri independent of glutamate decarboxylase activity. Arginine and glutamine/glutamate conversions confer resistance to lactate at pH of 3.5 and phosphate at pH of 2.5, respectively. Knowledge of L. reuteri's acid resistance improves the understanding of the adaptation of L. reuteri to intestinal ecosystems, and facilitates the selection of probiotic and starter cultures.

  5. Self-assembly and foaming properties of fatty acid-lysine aqueous dispersions.

    PubMed

    Novales, Bruno; Riaublanc, Alain; Navailles, Laurence; Houssou, Bérénice Houinsou; Gaillard, Cédric; Nallet, Frédéric; Douliez, Jean-Paul

    2010-04-20

    We report on dispersions of fatty acid-lysine salts in aqueous solutions which are further used to produce foams. The alkyl chain length is varied from dodecyl to stearic. In aqueous solutions, the lysine salt of the dodecyl chain yields an isotropic solution, probably micelles, whereas for longer alkyl chains, vesicles formed but crystallized upon resting at room temperature or when kept at 4 degrees C. Solid-state NMR showed that in vesicles fatty acids are embedded in a lamellar arrangement passing from a gel to a fluid state upon heating; the transition temperature at which it occurs was determined by DSC. Those results are confirmed by small-angle neutron scattering which also give additional information on the bilayer structure. Incredibly stable foams are obtained using the palmitic acid/Lys salt whereas for other alkyl chain length, poor or no foam is formed. We conclude that the foamability is related to the phase behavior in aqueous solution. PMID:20334439

  6. Arginine-dependent acid-resistance pathway in Shigella boydii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ability to survive the low pH of the human stomach is considered be an important virulent determinant. Acid tolerance of Shigella boydii 18 CDPH, the strain implicated in an outbreak may have played an important role in surviving the acidic food (bean salad). The strain was capable of inducing arg...

  7. Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase.

    PubMed

    Kanjee, Usheer; Gutsche, Irina; Alexopoulos, Eftichia; Zhao, Boyu; El Bakkouri, Majida; Thibault, Guillaume; Liu, Kaiyin; Ramachandran, Shaliny; Snider, Jamie; Pai, Emil F; Houry, Walid A

    2011-03-01

    The Escherichia coli inducible lysine decarboxylase, LdcI/CadA, together with the inner-membrane lysine-cadaverine antiporter, CadB, provide cells with protection against mild acidic conditions (pH∼5). To gain a better understanding of the molecular processes underlying the acid stress response, the X-ray crystal structure of LdcI was determined. The structure revealed that the protein is an oligomer of five dimers that associate to form a decamer. Surprisingly, LdcI was found to co-crystallize with the stringent response effector molecule ppGpp, also known as the alarmone, with 10 ppGpp molecules in the decamer. ppGpp is known to mediate the stringent response, which occurs in response to nutrient deprivation. The alarmone strongly inhibited LdcI enzymatic activity. This inhibition is important for modulating the consumption of lysine in cells during acid stress under nutrient limiting conditions. Hence, our data provide direct evidence for a link between the bacterial acid stress and stringent responses. PMID:21278708

  8. Amino Acid Pools and Metabolism During the Cell Division Cycle of Arginine-Grown Candida utilis

    PubMed Central

    Nurse, P.; Wiemken, A.

    1974-01-01

    Synchronous cultures obtained by isopycnic density gradient centrifugation are used to investigate amino acid metabolism during the cell division cycle of the food yeast Candida utilis. Isotopic labeling experiments demonstrate that the rates of uptake and catabolism of arginine, the sole source of nitrogen, double abruptly during the first half of the cycle, while the cells undergo bud expansion. This is accompanied by a doubling in rate of amino acid biosynthesis, and an accumulation of amino acids. The accumulation probably occurs within the storage pools of the vacuoles. Amino acids derived from protein degradation contribute little to this accumulation. For the remainder of the cell cycle, during cell separation and until the next bud initiation, the rates of uptake and catabolism of arginine and amino acid biosynthesis remain constant. Despite the abrupt doubling in the rate of formation of amino acid pools, their rate of utilization for macromolecular synthesis increases steadily throughout the cycle. The significance of this temporal organization of nitrogen source uptake and amino acid metabolism during the cell division cycle is discussed. Images PMID:4591945

  9. Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation

    PubMed Central

    Gao, Wei-wei; Xiao, Rong-quan; Peng, Bing-ling; Xu, Huan-teng; Shen, Hai-feng; Huang, Ming-feng; Shi, Tao-tao; Yi, Jia; Zhang, Wen-juan; Wu, Xiao-nan; Gao, Xiang; Lin, Xiang-zhi; Dorrestein, Pieter C.; Rosenfeld, Michael G.; Liu, Wen

    2015-01-01

    Although “histone” methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain–containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor β2 (RARβ2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70’s function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control. PMID:26080448

  10. Amino acid metabolism in the piglet. 3. Influence of lysine level in the diet on energy metabolism and in vivo oxidation.

    PubMed

    Chavez, E R; Bayley, H S

    1976-11-01

    1. Supplementing a lysine-deficient diet (5 g lysine/kg) with five increments of lysine, each of 2 g/kg, resulted in increased in growth rate of Yorkshire piglets, aged between 3 and 7 weeks, up to the highest level of lysine (15 g/kg). 2. The free lysine concentration of plasma tended to increase as the dietary lysine level increased from 13 to 15 g/kg, and plasma threonine concentration decreased significantly as the lysine content of the diet was increased from 11 to 15 g/kg indicating that threonine was the second limiting amino acid in the diet. 3. Oxygen consumption and carbon dioxide production of the piglets were not influenced by supplementing the diets with lysine. The heat production was 0-313 kJ/min per kg body-weight in the 6 h experimental period. 4. Supplementation of the diet with lysine had no consistent effect on the recovery of 14C as 14CO2 from a single dose of L-[U-14C]lysine. 5. Adjustment of the determined recoveries of the tracer dose of lysine for the differences in the plasma concentrations of free lysine for the pigs receiving the graded levels of dietary lysine simplified the relationship between recovery and dietary lysine level: it was linear for the first four increments in dietary lysine and then increased sharply for the fifth increment. This indicated that a marked change in the rate of lysine catabolism occurred as the level of dietary lysine was increased from 13 to 15 g/kg. 6. The results of this experiment indicate that the piglets' requirement for lysine is between 13 and 15 g lysine/kg in a diet which contained 181 g crude protein (nitrogen X6-25)/kg.

  11. 6th Amino Acid Assessment Workshop

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The focus of the 6th workshop is on lysine, arginine, and related amino acids. Functions, metabolic pathways, clinical uses, and upper tolerance intakes are emphasized in the articles that follow. Lysine is arguably the most deficient amino acid in the food supply of countries where poverty exists, ...

  12. Arginine-responsive terbium luminescent hybrid sensors triggered by two crown ether carboxylic acids.

    PubMed

    Jiang, Lasheng; Tang, Ke; Ding, Xiaoping; Wang, Qianming; Zhou, Zhan; Xiao, Rui

    2013-12-01

    Crown ether carboxylic acids constitute main building blocks for the synthesis of terbium containing covalent cross-linked luminescent materials. Both the complexes and the hybrid nanomaterials could exhibit remarkable green emissions in pure water. More importantly, they were found to have a profound effect on the luminescence responses to arginine compared with glutamic acid, histidine, tryptophan, threonine, tyrosine and phenylalanine in aqueous environment. The present study provided the possibility of using a host-guest mechanism as a way of signal transduction based on lanthanide supramolecular hybrid materials.

  13. Phase equilibria in a system of aqueous arginine with an octane solution of sulfonic acid

    NASA Astrophysics Data System (ADS)

    Kuvaeva, Z. I.; Koval'chuk, I. V.; Vodop'yanova, L. A.; Soldatov, V. S.

    2013-05-01

    The extraction of arginine (Arg) from aqueous salt (0.1 M NaCl) solutions with a sulfo extractant in a wide range of pH values and amino acid concentrations was studied. The 0.1 M solution of dinonylnaphthalenesulfonic acid (HD) in octane was used as an extractant. The degree of extraction was found to be high at pH 0.8-9.0. This can be explained by the effect of additional intermolecular interactions in the extractant phase involving the guanidine group of Arg.

  14. The 2007 ESPEN Sir David Cuthbertson Lecture: amino acids between and within organs. The glutamate-glutamine-citrulline-arginine pathway.

    PubMed

    Deutz, Nicolaas E P

    2008-06-01

    In daily practice, the plasma concentration of amino acids is usually viewed as a parameter of production. However, both a high production and/or a reduced disposal capacity can result in an increased plasma concentration. In this presentation, I will discuss my research on interorgan relationships of the amino acids glutamate, glutamine, citrulline and arginine to explain the regulation of the plasma arginine level. The reduced glutamine disposal during liver failure is related to enhanced plasma glutamine level without any change in muscle and gut production or consumption rate. In contrast during sepsis, a small reduction in plasma glutamine is related to a substantially enhanced organ glutamate and glutamine production or consumption rate. These observations are a good example that plasma levels are directly related to production or consumption rates. Because glutamine breakdown in the gut produces citrulline, there is a good relation between the amount of metabolically active gut tissue and gut and whole body citrulline production. Arginine is produces from citrulline in the kidney and a reduced gut glutamine to citrulline conversion during sepsis explains the reduced de novo arginine production that is related to the reduced plasma arginine level. The interorgan route between muscle, gut, liver and kidney of the amino acids glutamate, glutamine, citrulline and arginine is a very good example of how complicated the regulation of plasma amino acid levels can be. However, in-depth research is necessary and will give us important clues to new nutritional strategies.

  15. Delivery of siRNA Using Cationic Liposomes Incorporating Stearic Acid-modified Octa-Arginine.

    PubMed

    Yang, Dongsheng; Li, Yuhuan; Qi, Yuhang; Chen, Yongzhen; Yang, Xuewei; Li, Yujing; Liu, Songcai; Lee, Robert J

    2016-07-01

    Cationic liposomes incorporating stearic acid-modified octa-arginine (StA-R8) were evaluated for survivin small interfering RNA (siRNA) delivery. StA-R8 was synthesized and incorporated into liposomes. The composition of liposomes was optimized. Physicochemical properties, cytotoxicity, cellular uptake and gene silencing activity of the liposomes complexed to survivin siRNA were investigated. The results showed that StA-R8-containing liposomes had reduced cytotoxicity and improved delivery efficiency of siRNA into cancer cells compared with StA-R8 by itself.

  16. Delivery of siRNA Using Cationic Liposomes Incorporating Stearic Acid-modified Octa-Arginine.

    PubMed

    Yang, Dongsheng; Li, Yuhuan; Qi, Yuhang; Chen, Yongzhen; Yang, Xuewei; Li, Yujing; Liu, Songcai; Lee, Robert J

    2016-07-01

    Cationic liposomes incorporating stearic acid-modified octa-arginine (StA-R8) were evaluated for survivin small interfering RNA (siRNA) delivery. StA-R8 was synthesized and incorporated into liposomes. The composition of liposomes was optimized. Physicochemical properties, cytotoxicity, cellular uptake and gene silencing activity of the liposomes complexed to survivin siRNA were investigated. The results showed that StA-R8-containing liposomes had reduced cytotoxicity and improved delivery efficiency of siRNA into cancer cells compared with StA-R8 by itself. PMID:27354583

  17. Engineering Corynebacterium glutamicum for fast production of L-lysine and L-pipecolic acid.

    PubMed

    Pérez-García, Fernando; Peters-Wendisch, Petra; Wendisch, Volker F

    2016-09-01

    The Gram-positive Corynebacterium glutamicum is widely used for fermentative production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose uptake and phosphorylation by C. glutamicum mainly occur by the phosphotransferase system (PTS) and to lesser extent by inositol permeases and glucokinases. Heterologous expression of the genes for the high-affinity glucose permease from Streptomyces coelicolor and Bacillus subtilis glucokinase fully compensated for the absence of the PTS in Δhpr strains. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and glucokinase from B. subtilis were overproduced with balanced translation initiation rates using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid or with pVWEx1-IolTBest, 18 to 20 % higher volumetric productivities and 70 to 72 % higher specific productivities as compared to the parental strain resulted. The non-proteinogenic amino acid L-pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics, or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the constructed L-lysine-producing strain, the L-lysine 6-dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were overexpressed as synthetic operon. This enabled C. glutamicum to produce L-PA with a yield of 0.09 ± 0.01 g g(-1) and a volumetric productivity of 0.04 ± 0.01 g L(-1) h(-1).To the best of our knowledge, this is the first fermentative process for the production of L-PA from glucose.

  18. Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

    NASA Technical Reports Server (NTRS)

    Nakashima, T.; Fox, S. W.

    1980-01-01

    The paper examines the synthesis of peptides from aminoacids and ATP with a lysine-rich protenoid. The latter in aqueous solution catalyzes the formation of peptides from free amino acids and ATP; this catalytic activity is not found in acidic protenoids, even though the latter contain a basic aminoacid. The pH optimum for the synthesis is about 11, but it is appreciable below 8 and above 13. Temperature data indicate an optimum at 20 C or above, with little increase in rate up to 60 C. Pyrophosphate can be used instead of ATP, but the yields are lower. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous aminoacids.

  19. NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5'-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly-L-lysine.

    PubMed

    Chan-Huot, Monique; Dos, Alexandra; Zander, Reinhard; Sharif, Shasad; Tolstoy, Peter M; Compton, Shara; Fogle, Emily; Toney, Michael D; Shenderovich, Ilya; Denisov, Gleb S; Limbach, Hans-Heinrich

    2013-12-01

    Using (15)N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5'-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-L-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using (15)N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed (13)C and (15)N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-L-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism. PMID:24147985

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

    PubMed

    Shao, Andrew; Hathcock, John N

    2008-04-01

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

  1. Role of Arginine and Omega-3 Fatty Acids in Wound Healing and Infection.

    PubMed

    Alexander, J Wesley; Supp, Dorothy M

    2014-11-01

    Significance: Only a few decades ago, the primary focus of nutritional supplementation was to prevent deficiencies of essential nutrients. It is now recognized that, at higher than essential levels, selected nutrients can have a pharmacologic effect to prevent or treat disease. Recent Advances: Two of the most important pharmaconutrients, arginine, and the omega-3 polyunsaturated fatty acids in fish oil, have been shown to have profound effects on wound healing and infections. Critical Issues: Both arginine and fish oils have independent benefits, but the combination appears to be much more effective. This combination has been shown to affect outcomes involving wound healing and infections, as reviewed here, and can also affect incidence and outcomes in cardiovascular disease, diabetes, organ transplant rejection, and other inflammatory conditions. These possibilities have not yet progressed to widespread clinical application. Future Directions: The optimal combinations of immunonutrients, timing of administration, and the doses needed for best results need to be determined in preclinical and clinical studies. Also, the mechanisms involved in the administration of pharmaconutrients need to be established.

  2. Favored and disfavored pathways of protein crosslinking by glucose: glucose lysine dimer (GLUCOLD) and crossline versus glucosepane

    PubMed Central

    Nemet, Ina; Strauch, Christopher M.

    2010-01-01

    We describe the isolation and molecular characterization of a novel glucose-lysine dimer crosslink 1,3-bis-(5-amino-5-carboxypentyl)-4-(1′,2′,3′,4′-tetrahydroxybutyl)-3H-imidazolium salt, named GLUCOLD. GLUCOLD was easily formed from the Amadori product (fructose–lysine). However, when BSA was incubated with 100 mM glucose for 25 days, the levels of the lysine-lysine glucose crosslinks GLUCOLD and CROSSLINE were only 21 and <1 pmol/mg, respectively, compared to 611 pmol/mg protein for the lysine-arginine GLUCOSEPANE crosslink, in spite of more than 20 potential lysine-lysine crosslinking sites in the protein. Mechanistic investigation revealed that metal-free phosphate ions catalyzed formation of fructose–lysine and all three crosslinks from amino acids, while cationic MOPS buffer had an opposite effect. This together with the rapid formation of N6-1,4-dideoxy-5,6-dioxoglucosone derivatives by dicarbonyl trapping agents, such as 1,2-diaminobenzene or γ-guanidinobutyric acid, strongly suggests that enolization of the Amadori product and trapping of the 5,6-dioxo derivative by arginine residues constitutes the major pathway for glucose-mediated crosslinking in proteins. PMID:20607325

  3. Accumulation of D-arginine by rat liver mitochondria.

    PubMed

    Villalobos-Molina, R; Pardo, J P; Saavedra-Molina, A; Piña, E

    1987-12-01

    The permeability of the inner mitochondrial membrane from rat liver to D-arginine was studied. By using safranin as a probe of the membrane potential, depolarization of energized liver mitochondria occurred in a dose-dependent fashion starting at 3.3 mmol/L of D- or DL-arginine. When ethidium bromide fluorescence was employed, a decrease in the membrane potential due to D- or DL-arginine was observed. A parallel significant change in succinate-induced respiration in rat liver mitochondria was found in response to osmotic swelling in 125 mmol/L of D-arginine salts. L-Arginine, L-glutamine, L-asparagine, L-ornithine, D-ornithine, and L-lysine did not modify the membrane potential at the concentrations tested. D-Arginine was not transformed into citrulline, but 1.0 mmol/L of the D-amino acid inhibited, by 42%, the state 3 of mitochondrial respiration using succinate as substrate. When D-arginine was used in combination with nigericin, a 40% inhibition of mitochondrial respiration in state 3 was recorded with succinate and with glutamate-malate as substrates. PMID:3454185

  4. Dietary arginine requirement of juvenile hybrid striped bass.

    PubMed

    Griffin, M E; Wilson, K A; Brown, P B

    1994-06-01

    Two experiments were conducted to determine the dietary arginine requirement of juvenile hybrid striped bass (Morone saxatilis x M. chrysops); a third experiment evaluated the interaction of lysine and arginine. Diets in Experiments 1 and 2 were supplemented with graded concentrations of L-arginine-HCl, resulting in eight dietary treatments. Dietary arginine concentrations ranged from 1.0 to 2.4 g/100 g diet in Experiment 1 and from 0.6 to 2.0 g/100 g diet in Experiment 2. Weight gain was not affected by dietary treatments in Experiment 1. Feed efficiency was significantly affected by dietary arginine concentrations, and the data, when subjected to broken-line analysis, resulted in a requirement estimate of 1.53 +/- 0.20 g/100 g diet. Weight gain and feed efficiency were both significantly affected by dietary arginine concentrations in Experiment 2. Broken-line analyses of weight gain and feed efficiency data indicated the dietary arginine requirement to be 1.55 +/- 0.10 and 1.45 +/- 0.12 g/100 g diet, respectively. Diets in Experiment 3 contained lysine and arginine in ratios of 1:1, 1:1.5, 1:2 and 1:2.5 for the previously estimated requirements for both lysine:arginine and arginine:lysine. No differences were observed in weight gain or feed efficiency for fish fed various lysine:arginine ratios, but serum lysine was significantly different among treatment groups.

  5. Identification and Quantitation of the Lipation Product 2-Amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic Acid (MP-Lysine) in Peanuts.

    PubMed

    Globisch, Martin; Deuber, Meike; Henle, Thomas

    2016-08-31

    The lipid peroxidation product acrolein was semiquantitated by GC-MS (EI) in unheated and heated peanut oil, respectively, representing a model system for peanut roasting. Depending on the heating time, acrolein levels significantly increased from 0.2 to 10.7 mg/kg oil. As a result of heating N(α)-acetyl-l-lysine and acrolein, the pyridinium derivative 2-acetamido-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid (MP-acetyl lysine) was identified. In addition, the lysine derivative 2-amino-6-[5-(hydroxymethyl)-3,6-dihydro-2H-pyridin-1-yl]hexanoic acid was identified after reduction and hydrolysis. After preparation of 2-amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid (MP-lysine) as reference material, its amounts were quantitated in acrolein-modified peanut proteins by HPLC-ESI-MS/MS after acid hydrolysis, showing that at low acrolein concentrations, the modification of lysine could be entirely explained by the formation of MP-lysine. Furthermore, for the first time, MP-lysine was quantitated in peanut samples in amounts up to 10.2 mg/kg, showing an increase depending on the roasting time. Thus, MP-lysine might represent a marker to evaluate the extent of food protein lipation by acrolein. PMID:27499313

  6. Kinetics of CO2 Absorption into Aqueous Basic Amino Acid Salt: Potassium Salt of Lysine Solution.

    PubMed

    Shen, Shufeng; Yang, Ya-nan; Bian, Yangyang; Zhao, Yue

    2016-02-16

    Aqueous amino acid salts are considered as an attractive alternative to alkanolamine solvents (e.g., MEA) for carbon dioxide (CO2) absorption. The kinetics of CO2 into unloaded aqueous solutions of potassium lysinate (LysK) was studied using a wetted wall column at concentrations ranging from 0.25 to 2.0 M and temperatures from 298 to 333 K. Physicochemical properties of aqueous LysK solutions such as density, viscosity, and physical solubility of CO2 were measured to evaluate the reaction rate constants. The reaction pathway is described using zwitterion mechanism taking into account the effect of ionic strength on the reaction rate. Under the fast pseudo-first-order regime, the reaction rate parameters were obtained and correlated in a power-law reaction rate expression. LysK shows higher chemical reactivity toward CO2 than the industrial standard MEA and most of amino acid salts. Its reaction rate constants increase considerably with concentration and temperature. The reaction order is found to be an average value of 1.58 with respect to LysK. The forward second-order kinetic rate constant, k2 0 , are obtained as 31615 and 84822 m3 kmol−1 s−1 at 298 and 313 K, respectively with activation energy of 51.0 kJ mol−1. The contribution of water to the zwitterion deprotonation seems to be more significant than that of LysK for the above-mentioned kinetic conditions PMID:26751093

  7. Fatty acid modified octa-arginine for delivery of siRNA.

    PubMed

    Li, Yuhuan; Li, Yujing; Wang, Xinmei; Lee, Robert J; Teng, Lesheng

    2015-11-10

    Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Four fatty acids derivatives of octa-arginine (R8) were synthesized and evaluated for the delivery of siRNA into hepatocellular carcinoma Hep G2 and human lung adenocarcinoma A549 cells. The results showed that the long chain acid oleic acid or stearic acid derivatives of R8, OA-R8 and StA-R8, were more efficient in siRNA complexation and form nanoparticles with greater stability compared to the native R8. Cellular uptake of fluorescence-labeled siRNA delivered by OA-R8 and StA-R8 in Hep G2 and A549 cells was substantially 40-50 times higher than unmodified R8. A significant reduction in siRNA cellular uptake was observed in the presence of sucrose and cytochalasin D, indicating endocytosis as a primary mechanism of cellular entry. A survivin siRNA was used to prepare nanoparticles with OA-R8 or StA-R8 and evaluated for silencing of survivin mRNA and protein in A549 cells, and the inhibition efficiencies of survivin protein reached to 50.3% and 54.6%, respectively. The results showed greater effectiveness with the derivatized R8. Taken together, these findings showed that long chain fatty acid derivatives of R8 are efficient delivery agents for siRNA and may facilitate its therapeutic application. PMID:26386137

  8. Arginine as a general acid catalyst in serine recombinase-mediated DNA cleavage.

    PubMed

    Keenholtz, Ross A; Mouw, Kent W; Boocock, Martin R; Li, Nan-Sheng; Piccirilli, Joseph A; Rice, Phoebe A

    2013-10-01

    Members of the serine family of site-specific DNA recombinases use an unusual constellation of amino acids to catalyze the formation and resolution of a covalent protein-DNA intermediate. A recent high resolution structure of the catalytic domain of Sin, a particularly well characterized family member, provided a detailed view of the catalytic site. To determine how the enzyme might protonate and stabilize the 3'O leaving group in the strand cleavage reaction, we examined how replacing this oxygen with a sulfur affected the cleavage rate by WT and mutant enzymes. To facilitate direct comparison of the cleavage rates, key experiments used suicide substrates that prevented religation after cleavage. The catalytic defect associated with mutation of one of six highly conserved arginine residues, Arg-69 in Sin, was partially rescued by a 3' phosphorothiolate substrate. We conclude that Arg-69 has an important role in stabilizing the 3'O leaving group and is the prime candidate for the general acid that protonates the 3'O, in good agreement with the position it occupies in the high resolution structure of the active site of Sin.

  9. Mobility of lysozyme in poly(l-lysine)/hyaluronic acid multilayer films.

    PubMed

    Velk, Natalia; Uhlig, Katja; Vikulina, Anna; Duschl, Claus; Volodkin, Dmitry

    2016-11-01

    The spatial and temporal control over presentation of protein-based biomolecules such as growth factors and hormones is crucial for in vitro applications to mimic the complex in vivo environment. We investigated the interaction of a model protein lysozyme (Lys) with poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films. We focused on Lys diffusion as well as adsorption and retention within the film as a function of the film deposition conditions and post-treatment. Additionally, an effect of Lys concentration on its mobility was probed. A combination of confocal fluorescence microscopy, fluorescence recovery after photobleaching, and microfluidics was employed for this investigation. Our main finding is that adsorption of PLL and HA after protein loading induces acceleration and reduction of Lys mobility, respectively. These results suggest that a charge balance in the film to a high extent governs the protein-film interaction. We believe that control over protein mobility is a key to reach the full potential of the PLL/HA films as reservoirs for biomolecules depending on the application demand. PMID:27552029

  10. Heterologous Production of Hyaluronic Acid in an ε-Poly-l-Lysine Producer, Streptomyces albulus

    PubMed Central

    Yoshimura, Tomohiro; Shibata, Nobuyuki; Hamano, Yoshimitsu

    2015-01-01

    Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo. PMID:25795665

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

    PubMed

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

    2016-10-01

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

  12. L-arginine augments the antioxidant effect of garlic against acetic acid-induced ulcerative colitis in rats.

    PubMed

    Harisa, Gamal Eldin I; Abo-Salem, Osama M; El-Sayed, El-Sayed M; Taha, Ehab I; El-Halawany, Nermin

    2009-10-01

    Garlic contains many sulfhydryl compounds that act as antioxidants. However, the role of nitric oxide (NO) in inflammation is controversial. The aim of the present study is to investigate the possible protective effect of garlic against acetic acid-induced ulcerative colitis in rats, as well as the probable modulatory effect of L-arginine (NO precursor) on garlic activity. Intra-rectal inoculation of rats with 4% acetic acid for 3 consecutive days caused a significant increase in the colon weight and marked decrease in the colon length. In addition, acetic acid induced a significant increase in serum levels of nitrate as well as colonic tissue content of malondialdehyde (MDA). Moreover, colonic tissue contents of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were markedly reduced. On the other hand, pre-treatment of rats with garlic (0.25 g/kgbwt, orally) for 4 consecutive weeks and 3 days during induction of colitis significantly reduced the increase in the colon weight induced by acetic acid and ameliorated alterations in oxidant and antioxidant parameters. Interestingly, oral co-administration of garlic (0.25 g/kgbwt) and L-arginine (625 mg/kgbwt) for the same period of garlic administration mitigated the changes in both colon weight and length induced by acetic acid and increased garlic effect on colon tissue contents of MDA and GSH. In conclusion, L-arginine can augment the protective effect of garlic against ulcerative colitis; an effect that might be mainly attributed to its NO donating property resulting in enhancement of garlic antioxidant effect. Further studies will be needed to determine which one of the active ingredients of garlic has the main antioxidant effect to be used with L-arginine. PMID:19783514

  13. The roles of selected arginine and lysine residues of TAFI (Pro-CPU) in its activation to TAFIa by the thrombin-thrombomodulin complex.

    PubMed

    Wu, Chengliang; Kim, Paul Y; Manuel, Reg; Seto, Marian; Whitlow, Marc; Nagashima, Mariko; Morser, John; Gils, Ann; Declerck, Paul; Nesheim, Michael E

    2009-03-13

    Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

  14. Arginine modifications by methylglyoxal: discovery in a recombinant monoclonal antibody and contribution to acidic species.

    PubMed

    Chumsae, Chris; Gifford, Kathreen; Lian, Wei; Liu, Hongcheng; Radziejewski, Czeslaw H; Zhou, Zhaohui Sunny

    2013-12-01

    Heterogeneity is common among protein therapeutics. For example, the so-called acidic species (charge variants) are typically observed when recombinant monoclonal antibodies (mAbs) are analyzed by weak-cation exchange chromatography (WCX). Several protein post-translational modifications have been established as contributors but still cannot completely account for all heterogeneity. As reported herein, an unexpected modification by methylglyoxal (MGO) was identified, for the first time, in a recombinant monoclonal antibody expressed in Chinese hamster ovary (CHO) cells. Modifications of arginine residues by methylglyoxal lead to two adducts (dihydroxyimidazolidine and hydroimidazolone) with increases of molecular weights of 72 and 54 Da, respectively. In addition, the modification by methylglyoxal causes the antibody to elute earlier in the weak cation exchange chromatogram. Consequently, the extent to which an antibody was modified at multiple sites corresponds to the degree of shift in elution time. Furthermore, cell culture parameters also affected the extent of modifications by methylglyoxal, a highly reactive metabolite that can be generated from glucose or lipids or other metabolic pathways. Our findings again highlight the impact that cell culture conditions can have on the product quality of recombinant protein pharmaceuticals.

  15. Arginine Modifications by Methylglyoxal: Discovery in a Recombinant Monoclonal Antibody and Contribution to Acidic Species

    PubMed Central

    Chumsae, Chris; Gifford, Kathreen; Lian, Wei; Liu, Hongcheng; Radziejewski, Czeslaw H.; Zhou, Zhaohui Sunny

    2013-01-01

    Heterogeneity is common among protein therapeutics. For example, the so-called acidic species (charge variants) are typically observed when recombinant monoclonal antibodies (mAbs) are analyzed by weak-cation exchange chromatography (WCX). Several protein post-translational modifications have been established as contributors, but still cannot completely account for all heterogeneity. As reported herein, an unexpected modification by methylglyoxal (MGO) was identified, for the first time, in a recombinant monoclonal antibody expressed in Chinese hamster ovary (CHO) cells. Modifications of arginine residues by methylglyoxal lead to two adducts (dihydroxyimidazolidine and hydroimidazolone) with increase of molecular weights of 72 and 54 Daltons, respectively. In addition, the modification by methylglyoxal causes the antibody to elute earlier in the weak cation exchange chromatogram. Consequently, the extent to which an antibody was modified at multiple sites corresponds to the degree of shift in elution time. Furthermore, cell culture parameters also affected the extent of modifications by methylglyoxal, a highly reactive metabolite that can be generated from glucose or lipids or other metabolic pathways. Our findings again highlight the impact that cell culture conditions can have on the product quality of recombinant protein pharmaceuticals. PMID:24168114

  16. NO synthesis from arginine is favored by α-linolenic acid in mice fed a high-fat diet.

    PubMed

    Hermier, Dominique; Guelzim, Najoua; Martin, Pascal G P; Huneau, Jean-François; Mathé, Véronique; Quignard-Boulangé, Annie; Lasserre, Frédéric; Mariotti, François

    2016-09-01

    Alterations in NO availability and signaling play a pivotal role at early stages of the metabolic syndrome (MetSynd). We hypothesized that dietary α-linolenic acid (ALA, 18:3 n-3) favors NO availability by modulating amino acid metabolism, with a specific impact on the arginine-NO pathway. Mice were fed a hyperlipidic diet (285 g lipid/kg, 51.1 % energy), rich in either saturated fatty acids (SFA, provided by palm oil, PALM group) or ALA (provided by linseed oil, LIN group). We measured whole-body NO synthesis and systemic arginine hydrolysis with a tracer-based method, plasma concentration of related metabolites, and hepatic mRNA level of related enzymes, and the study was completed by a transcriptomic analysis in the liver. As expected with this model, hyperlipidic diets resulted in increased adiposity and glycemia after 5 weeks. As compared to PALM mice, LIN mice had a higher plasma nitrite and nitrate concentration, a higher whole-body conversion of arginine into NO vs urea, and a similar plasma concentration of asymmetric dimethylarginine (ADMA), despite a higher expression of the liver dimethylargininase-1. In LIN mice, there was a higher expression of genes involved in PPARα signaling, but a little impact on gene expression related to amino acids and arginine metabolism. This effect cannot be directly ascribed to changes in arginase activity in the liver or ADMA metabolism, nor to direct regulation of the related target genes. In conclusion, dietary ALA favors NO synthesis, which could contribute to rescue NO availability when jeopardized by the nutritional conditions in relation with the initiation of the MetSynd. PMID:27178023

  17. Citrulline Protects Streptococcus pyogenes from Acid Stress Using the Arginine Deiminase Pathway and the F1Fo-ATPase

    PubMed Central

    Cusumano, Zachary T.

    2015-01-01

    ABSTRACT A common stress encountered by both pathogenic and environmental bacteria is exposure to a low-pH environment, which can inhibit cell growth and lead to cell death. One major defense mechanism against this stress is the arginine deiminase (ADI) pathway, which catabolizes arginine to generate two ammonia molecules and one molecule of ATP. While this pathway typically relies on the utilization of arginine, citrulline has also been shown to enter into the pathway and contribute to protection against acid stress. In the pathogenic bacterium Streptococcus pyogenes, the utilization of citrulline has been demonstrated to contribute to pathogenesis in a murine model of soft tissue infection, although the mechanism underlying its role in infection is unknown. To gain insight into this question, we analyzed a panel of mutants defective in different steps in the ADI pathway to dissect how arginine and citrulline protect S. pyogenes in a low-pH environment. While protection provided by arginine utilization occurred through the buffering of the extracellular environment, citrulline catabolism protection was pH independent, requiring the generation of ATP via the ADI pathway and a functional F1Fo-ATP synthase. This work demonstrates that arginine and citrulline catabolism protect against acid stress through distinct mechanisms and have unique contributions to virulence during an infection. IMPORTANCE An important aspect of bacterial pathogenesis is the utilization of host-derived nutrients during an infection for growth and virulence. Previously published work from our lab identified a unique role for citrulline catabolism in Streptococcus pyogenes during a soft tissue infection. The present article probes the role of citrulline utilization during this infection and its contribution to protection against acid stress. This work reveals a unique and concerted action between the catabolism of citrulline and the F1Fo-ATPase that function together to provide protection for

  18. The Supplementation of Branched-Chain Amino Acids, Arginine, and Citrulline Improves Endurance Exercise Performance in Two Consecutive Days

    PubMed Central

    Cheng, I-Shiung; Wang, Yi-Wen; Chen, I-Fan; Hsu, Gi-Sheng; Hsueh, Chun-Fang; Chang, Chen-Kang

    2016-01-01

    The central nervous system plays a crucial role in fatigue during endurance exercise. Branched-chain amino acids (BCAA) could reduce cerebral serotonin synthesis by competing with its precursor tryptophan for crossing the blood brain barrier. Arginine and citrulline could prevent excess hyperammonemia accompanied by BCAA supplementation. This study investigated the combination of BCAA, arginine, and citrulline on endurance performance in two consecutive days. Seven male and three female endurance runners ingested 0.17 g·kg-1 BCAA, 0.05 g·kg-1 arginine and 0.05 g·kg-1 citrulline (AA trial) or placebo (PL trial) in a randomized cross-over design. Each trial contained a 5000 m time trial on the first day, and a 10000 m time trial on the second day. The AA trial had significantly better performance in 5000 m (AA: 1065.7 ± 33.9 s; PL: 1100.5 ± 40.4 s) and 10000 m (AA: 2292.0 ± 211.3 s; PL: 2375.6 ± 244.2 s). The two trials reported similar ratings of perceived exertion. After exercise, the AA trial had significantly lower tryptophan/BCAA ratio, similar NH3, and significantly higher urea concentrations. In conclusion, the supplementation could enhance time-trial performance in two consecutive days in endurance runners, possibly through the inhibition of cerebral serotonin synthesis by BCAA and the prevention of excess hyperammonemia by increased urea genesis. Key points The combined supplementation of BCAA, arginine, and citrulline could enhance performance in 5000 m and 10000 m in 2 consecutive days in competitive runners. The supplementation may be helpful in multi-day competitions. The supplemented BCAA may alleviate central fatigue, allowing the subjects to run faster at the same degree of perceived exertion. The hyperammonemia that is usually accompanied with BCAA supplementation may be prevented by arginine and citrulline through increased urea genesis. PMID:27803630

  19. First hyperpolarizability of the natural aromatic amino acids tryptophan, tyrosine, and phenylalanine and the tripeptide lysine-tryptophan-lysine determined by hyper-Rayleigh scattering.

    PubMed

    Duboisset, J; Matar, G; Russier-Antoine, I; Benichou, E; Bachelier, G; Jonin, Ch; Ficheux, D; Besson, F; Brevet, P F

    2010-11-01

    We report the first hyperpolarizability of tryptophan (Trp) and tyrosine (Tyr) and an upper limit for that of phenylalanine (Phe), three natural aromatic amino acids. The measurements were performed with hyper-Rayleigh scattering in an aqueous Tris buffer solution at a pH of 8.5 and 150 mM salt concentration with a fundamental wavelength of 780 nm. A value of (4.7 ± 0.7) × 10(-30) esu is found for Trp and (4.1 ± 0.7) × 10(-30) esu for Tyr whereas the upper limit of 1.4 × 10(-30) esu is found for that of Phe due to its limited solubility. The influence of the presence of lysine (Lys) in close vicinity of Trp is investigated with a measurement of the first hyperpolarizabilty of Trp in an excess of Lys and compared to the first hyperpolarizability obtained for the tripeptide Lys-Trp-Lys. The clear decrease of the values measured in these two cases indicates that the first hyperpolarizabilty of Trp is very sensitive to its local environment.

  20. 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.

  1. Creatine, arginine alpha-ketoglutarate, amino acids, and medium-chain triglycerides and endurance and performance.

    PubMed

    Little, Jonathan P; Forbes, Scott C; Candow, Darren G; Cornish, Stephen M; Chilibeck, Philip D

    2008-10-01

    Creatine (Cr) supplementation increases muscle mass, strength, and power. Arginine a-ketoglutarate (A-AKG) is a precursor for nitric oxide production and has the potential to improve blood flow and nutrient delivery (i.e., Cr) to muscles. This study compared a commercial dietary supplement of Cr, A-AKG, glutamine, taurine, branched-chain amino acids, and medium-chain triglycerides with Cr alone or placebo on exercise performance and body composition. Thirty-five men (approximately 23 yr) were randomized to Cr + A-AKG (0.1 g . kg(-1) . d(-1) Cr + 0.075 g . kg(-1) . d(-1)A-AKG, n = 12), Cr (0.1 g . kg(-1) . d(-1), n = 11), or placebo (1 g . kg(-1) . d(-1) sucrose, n = 12) for 10 d. Body composition, muscle endurance (bench press), and peak and average power (Wingate tests) were measured before and after supplementation. Bench-press repetitions over 3 sets increased with Cr + A-AKG (30.9 +/- 6.6 +/- 34.9 +/- 8.7 reps; p < .01) and Cr (27.6 +/- 5.9 +/- 31.0 +/- 7.6 reps; p < .01), with no change for placebo (26.8 +/- 5.0 +/- 27.1 +/- 6.3 reps). Peak power significantly increased in Cr + A-AKG (741 +/- 112 +/- 794 +/- 92 W; p < .01), with no changes in Cr (722 +/- 138 +/- 730 +/- 144 W) and placebo (696 +/- 63 +/- 705 +/- 77 W). There were no differences in average power between groups over time. Only the Cr-only group increased total body mass (79.9 +/- 13.0 +/- 81.1 +/- 13.8 kg; p < .01), with no significant changes in lean-tissue or fat mass. These results suggest that Cr alone and in combination with A-AKG improves upper body muscle endurance, and Cr + A-AKG supplementation improves peak power output on repeated Wingate tests. PMID:19033611

  2. Development of a wound dressing composed of a hyaluronic acid sponge containing arginine.

    PubMed

    Matsumoto, Yasuhiro; Arai, Kiwako; Momose, Hitomi; Kuroyanagi, Yoshimitsu

    2009-01-01

    Spongy sheets composed of cross-linked high-molecular-weight (HMW) hyaluronic acid (HA) were prepared by freeze-drying an aqueous HMW-HA solution containing cross-linking agent (Group I). The Group I sheet was immersed into an aqueous low-molecular-weight (LMW) HA solution with or without L-arginine (Arg) and was then freeze-dried to prepare several types of spongy sheets (Groups II-V). The amount of Arg was 1.0 g, 0.5 g, 0.2 g and 0 g in Groups III, IV, V and II, respectively. In the first experiment, each spongy sheet was applied to a full-thickness skin defect with a diameter of 35 mm in the abdominal region of SD rats, with intact skin in the central area measuring 15 mm in diameter. Commercially available polyurethane film dressing was applied over each spongy sheet as a covering material. The control group was covered with polyurethane film dressing alone. All spongy sheets promoted epithelization, as well as angiogenesis, as compared with controls. These findings indicate that HA and Arg are essential for wound healing. Re-epithelizaion was particularly active in Groups IV and V. In the second experiment, each spongy sheet was applied to a full-thickness burn injury measuring 35 mm in diameter in the abdominal region of SD rats, after necrotic skin was surgically removed. Groups II-V showed decreased wound size when compared with Group I and controls. The present findings indicate that the release of LMW-HA and Arg from a cross-linked HMW-HA spongy sheet effectively stimulates wound healing.

  3. Ornithine is a novel amino acid and a marker of arginine damage by oxoaldehydes in senescent proteins.

    PubMed

    Sell, David R; Monnier, Vincent M

    2005-06-01

    Long-lived proteins undergo age-related postsynthetic modifications by glycation and advanced glycation end products (AGEs), which destabilize them by altering their conformation and charge. It was accidentally discovered that ornithine (orn) increased with age in acid hydrolyzates of human skin collagen and lens crystallins which led us to investigate the source of orn. Here, we detected such modifications of orn in these proteins. Acid hydrolysis of arginine (arg)-base AGE standards produced orn at different yields. The data provide unequivocal evidence for the in vivo formation of orn and its own AGEs in aging proteins, and suggest that arg-based AGEs serve as precursors of orn.

  4. Comparative analysis of some essential amino acids and available lysine in Acacia colei and A. tumida seeds using chemical methods and an amino acid analyzer.

    PubMed

    Falade, Olumuyiwa S; Adewusi, Steve R A

    2013-01-01

    Methionine, cysteine, tryptophan, and available lysine were determined in Acacia colei and A. tumida seeds and some cereals using chemical methods, and the results were compared to those obtained using an amino acid analyzer. Ba(OH)2 hydrolysis gave the best result of the three methods of hydrolysis (acid, base, and enzyme) tried. Oxidized methionine, cysteine, and tryptophan were not detected, but S-carboxyethylcysteine was estimated as cysteine by the chemical methods, thus overestimating cysteine's content in Acacia seeds. Tryptophan and methionine were higher in cereals than in Acacia seeds, while the level of cysteine and available lysine was higher in Acacia seeds than in cereals. These results agreed with values obtained using the amino acid analyzer and could therefore be used in low budget laboratories.

  5. The distribution of lysine vasopressin (lysipressin) in placental mammals: a reinvestigation of the Hippopotamidae (Hippopotamus amphibius) and Tayassuidae (Tayassu angulatus) families.

    PubMed

    Rouille, Y; Chauvet, M T; Chauvet, J; Acher, R; Hadley, M E

    1988-09-01

    The neurohypophyseal hormones of the hippopotamus (Hippopotamus amphibius) and collared peccary (Tayassu angulatus) were isolated by molecular sieving and preparative high-pressure liquid chromatography (HPLC). Oxytocin and arginine vasopressin have been identified by their amino acid compositions and their retention times in HPLC. Lysipressin (lysine vasopressin) was not detected in posterior pituitaries of two hippopotami and nine peccaries (less than 2% of arginine vasopressin in molar ratios). Among the suborder Suiformes of Artiodactyla, the families Hippopotamidae and Tayassuidae do not seem to possess lysipressin, in contrast to the family Suidae in which the pig has lysipressin in place of arginine vasopressin.

  6. Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster.

    PubMed

    Gatto, Gregory J; Boyne, Michael T; Kelleher, Neil L; Walsh, Christopher T

    2006-03-22

    Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner. PMID:16536560

  7. KDP crystal doped with L-arginine amino acid: growth, structure perfection, optical and strength characteristics

    NASA Astrophysics Data System (ADS)

    Pritula, I. M.; Kostenyukova, E. I.; Bezkrovnaya, O. N.; Kolybaeva, M. I.; Sofronov, D. S.; Dolzhenkova, E. F.; Kanaev, A.; Tsurikov, V.

    2016-07-01

    Potassium Dihydrogen Phosphate (KDP) crystal doped with L-arginine (L-arg) amino acid with 1.4 wt% concentration in the solution was grown onto a point seed by the method of temperature reduction. For the first time an attempt was made to grow large-size (7 × 6 × 8 cm3) optically transparent crystals, which allowed to analyze the effect of L-arg additive on the physical properties of the different growth sectors ({100} and {101}) of KDP. The incorporation of L-arg into both growth sectors of the crystal was confirmed by the methods of optical and IR spectroscopy and found to be caused by the ability of the amino acid to form hydrogen bonds with the face {100} and electrostatically interact with the positively charged face {101} of KDP crystal. A slight variation in the unit cell parameters was reported, the elementary cell volume of KDP:L-arg crystal increased in comparison with the one of pure KDP by 2·10-2 and 2.07·10-2 Å3 in the sectors {100} and {101}, respectively. It was found that the doping of L-arg enhanced the SHG efficiency of KDP and depended on the crystal growth sectors. The SHG efficiency of KDP:L-arg was by a factor 2.53 and 3.95 higher in comparison with those of pure KDP for {101} and {100} growth sector, respectively. The doping was found to lead to softening of both faces by ∼3-10% and ∼14-17% in the sectors {101} and {100}, respectively. Investigation of the influence of L-arg molecules on the bulk laser damage threshold of the crystals showed that the bulk laser damage threshold of the samples of KDP:L-arg crystal was higher than the one of the pure crystal in the sector {101} and lower in the sector {100}. The correlation between microhardness and laser damage threshold were discussed. The study is helpful for further searching, designing and simulation of hybrid NLO materials.

  8. Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies

    PubMed Central

    Kaur, Randeep; Chitanda, Jackson M; Michel, Deborah; Maley, Jason; Borondics, Ferenc; Yang, Peng; Verrall, Ronald E; Badea, Ildiko

    2012-01-01

    Purpose: Detonation nanodiamonds (NDs) are carbon-based nanomaterials that, because of their size (4–5 nm), stable inert core, alterable surface chemistry, fluorescence, and biocompatibility, are emerging as bioimaging agents and promising tools for the delivery of biochemical molecules into cellular systems. However, diamond particles possess a strong propensity to aggregate in liquid formulation media, restricting their applicability in biomedical sciences. Here, the authors describe the covalent functionalization of NDs with lysine in an attempt to develop nanoparticles able to act as suitable nonviral vectors for transferring genetic materials across cellular membranes. Methods: NDs were oxidized and functionalized by binding lysine moieties attached to a three-carbon-length linker (1,3-diaminopropane) to their surfaces through amide bonds. Raman and Fourier transform infrared spectroscopy, zeta potential measurement, dynamic light scattering, atomic force microscopic imaging, and thermogravimetric analysis were used to characterize the lysine-functionalized NDs. Finally, the ability of the functionalized diamonds to bind plasmid DNA and small interfering RNA was investigated by gel electrophoresis assay and through size and zeta potential measurements. Results: NDs were successfully functionalized with the lysine linker, producing surface loading of 1.7 mmol g−1 of ND. These modified NDs formed highly stable aqueous dispersions with a zeta potential of 49 mV and particle size of approximately 20 nm. The functionalized NDs were found to be able to bind plasmid DNA and small interfering RNA by forming nanosized “diamoplexes”. Conclusion: The lysine-substituted ND particles generated in this study exhibit stable aqueous formulations and show potential for use as carriers for genetic materials. PMID:22904623

  9. Role of amino acid insertions on intermolecular forces between arginine peptide condensed DNA helices: implications for protamine-DNA packaging in sperm.

    PubMed

    DeRouchey, Jason E; Rau, Donald C

    2011-12-01

    In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short range repulsion while only slightly affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads.

  10. Hydroxyapatite formation on graphene oxide modified with amino acids: arginine versus glutamic acid.

    PubMed

    Tavafoghi, M; Brodusch, N; Gauvin, R; Cerruti, M

    2016-01-01

    Hydroxyapatite (HA, Ca5(PO4)3OH) is the main inorganic component of hard tissues, such as bone and dentine. HA nucleation involves a set of negatively charged phosphorylated proteins known as non-collagenous proteins (NCPs). These proteins attract Ca(2+) and PO4(3-) ions and increase the local supersaturation to a level required for HA precipitation. Polar and charged amino acids (AAs) are highly expressed in NCPs, and seem to be responsible for the mineralizing effect of NCPs; however, the individual effect of these AAs on HA mineralization is still unclear. In this work, we investigate the effect of a negatively charged (Glu) and positively charged (Arg) AA bound to carboxylated graphene oxide (CGO) on HA mineralization in simulated body fluids (SBF). Our results show that Arg induces HA precipitation faster and in larger amounts than Glu. We attribute this to the higher stability of the complexes formed between Arg and Ca(2+) and PO4(3-) ions, and also to the fact that Arg exposes both carboxyl and amino groups on the surface. These can electrostatically attract both Ca(2+) and PO4(3-) ions, thus increasing local supersaturation more than Glu, which exposes carboxyl groups only.

  11. 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. PMID:23656796

  12. 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.

  13. Development of a wound dressing composed of hyaluronic acid sponge containing arginine and epidermal growth factor.

    PubMed

    Matsumoto, Yasuhiro; Kuroyanagi, Yoshimitsu

    2010-01-01

    Hyaluronic acid (HA) has the ability to promote wound healing. Epidermal growth factor (EGF) is able to promote the proliferation of various cell types, in addition to epidermal cells. A novel wound dressing was designed using high-molecular-weight hyaluronic acid (HMW-HA) and low-molecular-weight hyaluronic acid (LMW-HA). Spongy sheets composed of cross-linked high-molecular-weight hyaluronic acid (c-HMW-HA) were prepared by freeze-drying an aqueous solution of HMW-HA containing a crosslinking agent. Each spongy sheet was immersed into an aqueous solution of LMW-HA containing arginine (Arg) alone or both Arg and epidermal growth factor (EGF), and were then freeze-dried to prepare two types of product. One was a wound dressing composed of c-HMW-HA sponge containing LMW-HA and Arg (c-HMW-HA/LMW-HA + Arg; Group I). The other was a wound dressing composed of c-HMW-HA sponge containing LMW-HA, Arg and EGF (c-HMW-HA/LMW-HA + Arg + EGF; Group II). The efficacy of these products was evaluated in animal tests using rats. In the first experiment, each wound dressing was applied to a full-thickness skin defect with a diameter of 35 mm in the abdominal region of Sprague-Dawley (SD) rats, leaving an intact skin island measuring 15 mm in diameter in the central area of this skin defect. Commercially available polyurethane film dressing was then applied to each wound dressing as a covering material. In the control group, the wound surface was covered with polyurethane film dressing alone. Both wound dressings (Group I and Group II) potently decreased the size of the full-thickness skin defect and increased the size of the intact skin island, when compared with the control group. The wound dressing in Group II showed particularly potent activity in increasing the distance of epithelization from the intact skin island. This suggests that EGF release from the spongy sheet serves to promote epithelization. The wound dressing in Group II enhanced early-stage inflammation after 1 week

  14. 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.

  15. Identification and characterization of lysine-methylated sites on histones and non-histone proteins.

    PubMed

    Lee, Tzong-Yi; Chang, Cheng-Wei; Lu, Cheng-Tzung; Cheng, Tzu-Hsiu; Chang, Tzu-Hao

    2014-06-01

    Protein methylation is a kind of post-translational modification (PTM), and typically takes place on lysine and arginine amino acid residues. Protein methylation is involved in many important biological processes, and most recent studies focused on lysine methylation of histones due to its critical roles in regulating transcriptional repression and activation. Histones possess highly conserved sequences and are homologous in most species. However, there is much less sequence conservation among non-histone proteins. Therefore, mechanisms for identifying lysine-methylated sites may greatly differ between histones and non-histone proteins. Nevertheless, this point of view was not considered in previous studies. Here we constructed two support vector machine (SVM) models by using lysine-methylated data from histones and non-histone proteins for predictions of lysine-methylated sites. Numerous features, such as the amino acid composition (AAC) and accessible surface area (ASA), were used in the SVM models, and the predictive performance was evaluated using five-fold cross-validations. For histones, the predictive sensitivity was 85.62% and specificity was 80.32%. For non-histone proteins, the predictive sensitivity was 69.1% and specificity was 88.72%. Results showed that our model significantly improved the predictive accuracy of histones compared to previous approaches. In addition, features of the flanking region of lysine-methylated sites on histones and non-histone proteins were also characterized and are discussed. A gene ontology functional analysis of lysine-methylated proteins and correlations of lysine-methylated sites with other PTMs in histones were also analyzed in detail. Finally, a web server, MethyK, was constructed to identify lysine-methylated sites. MethK now is available at http://csb.cse.yzu.edu.tw/MethK/.

  16. Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level.

    PubMed

    Duan, Xiangke; Li, Yunsong; Du, Qinglin; Huang, Qinqin; Guo, Siyao; Xu, Mengmeng; Lin, Yanping; Liu, Zhidong; Xie, Jianping

    2016-01-01

    Bacterial persisters, usually slow-growing, non-replicating cells highly tolerant to antibiotics, play a crucial role contributing to the recalcitrance of chronic infections and treatment failure. Understanding the molecular mechanism of persister cells formation and maintenance would obviously inspire the discovery of new antibiotics. The significant upregulation of Mycobacterium tuberculosis Rv3290c, a highly conserved mycobacterial lysine ε-aminotransferase (LAT) during hypoxia persistent model, suggested a role of LAT in persistence. To test this, a lat deleted Mycobacterium smegmatis was constructed. The expression of transcriptional regulator leucine-responsive regulatory protein (LrpA) and the amino acids abundance in M. smegmatis lat deletion mutants were lowered. Thus, the persistence capacity of the deletion mutant was impaired upon norfloxacin exposure under nutrient starvation. In summary, our study firstly reported the involvement of mycobacterium LAT in persister formation, and possibly through altering the intracellular amino acid metabolism balance. PMID:26806099

  17. The Coding Properties of Lysine-accepting Transfer Ribonucleic Acids from Black-eyed Peas 1

    PubMed Central

    Hague, Donald R.; Kofoid, Eric C.

    1971-01-01

    Lysine-accepting transfer RNA from ungerminated and germinated embryo axes of black-eyed peas (Vigna sinensis L. Savi) was fractionated on benzoylated diethylaminoethyl cellulose and reverse phase Freon columns. Cochromatography indicated the presence of two similar lysyl transfer RNA fractions in each tissue. Ribosome binding studies revealed that the larger of the two fractions in each case is specific for the AAG codon, while the smaller one recognizes AAA and AAG. Possible implications of this difference in quantities of isoacceptors in translation of genetic information are discussed. PMID:16657787

  18. The Coding Properties of Lysine-accepting Transfer Ribonucleic Acids from Black-eyed Peas.

    PubMed

    Hague, D R; Kofoid, E C

    1971-09-01

    Lysine-accepting transfer RNA from ungerminated and germinated embryo axes of black-eyed peas (Vigna sinensis L. Savi) was fractionated on benzoylated diethylaminoethyl cellulose and reverse phase Freon columns. Cochromatography indicated the presence of two similar lysyl transfer RNA fractions in each tissue. Ribosome binding studies revealed that the larger of the two fractions in each case is specific for the AAG codon, while the smaller one recognizes AAA and AAG. Possible implications of this difference in quantities of isoacceptors in translation of genetic information are discussed.

  19. The lysine-ketoglutarate reductase-saccharopine dehydrogenase is involved in the osmo-induced synthesis of pipecolic acid in rapeseed leaf tissues.

    PubMed

    Moulin, M; Deleu, C; Larher, F; Bouchereau, A

    2006-01-01

    Higher plant responses to abiotic stresses are associated with physiological and biochemical changes triggering a number of metabolic adjustments. We focused on L-lysine catabolism, and have previously demonstrated that degradation of this amino acid is osmo-regulated at the level of lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) in Brassica napus. LKR and SDH activities are enhanced by decreasing osmotic potential and decrease when the upshock osmotic treatment is followed by a downshock osmotic one. Moreover we have shown that the B. napus LKR/SDH gene is up-regulated in osmotically-stressed tissues. The LKR/SDH activity produces alpha-aminoadipate semialdehyde which could be further converted into alpha-aminoadipate and acetyl CoA. Alternatively alpha-aminoadipate could behave as a precursor for pipecolic acid. Pipecolic acid is described as an osmoprotectant in bacteria and is co-accumulated with proline in halophytic plants. We suggest that osmo-induction of the LKR/SDH activity could be partly responsible for pipecolic acid accumulation. This proposal has been assessed in this study through pipecolic acid amounts determination in rape leaf discs subjected to various upshift and downshift osmotic treatments. Changes in pipecolic acid level actually behave as those observed for LKR and SDH activities, since it increases or decreases in rape leaf discs treated under hyper- or hypo-osmotic conditions, respectively. In addition we show that pipecolic acid level is positively correlated with the external osmotic potential as well as with the duration of the applied treatment. On the other hand pipecolic acid level is related to the availability of L-lysine and not to that of D-lysine. Collectively the results obtained demonstrate that lysine catabolism through LKR/SDH activity is involved in osmo-induced synthesis of pipecolic acid.

  20. Preparation, characterization, and biocompatibility evaluation of poly(Nɛ-acryloyl-L-lysine)/hyaluronic acid interpenetrating network hydrogels.

    PubMed

    Cui, Ning; Qian, Junmin; Xu, Weijun; Xu, Minghui; Zhao, Na; Liu, Ting; Wang, Hongjie

    2016-01-20

    In the present study, poly(Nɛ-acryloyl-L-lysine)/hyaluronic acid (pLysAAm/HA) interpenetrating network (IPN) hydrogels were successfully fabricated through the combination of hydrazone bond crosslinking and photo-crosslinking reactions. The HA hydrogel network was first synthesized from 3,3'-dithiodipropionate hydrazide-modified HA and polyethylene glycol dilevulinate by hydrazone bond crosslinking. The pLysAAm hydrogel network was prepared from Nɛ-acryloyl-L-lysine and N,N'-bis(acryloyl)-(L)-cystine by photo-crosslinking. The resultant pLysAAm/HA hydrogels had a good shape recovery property after loading and unloading for 1.5 cycles (up to 90%) and displayed a highly porous microstructure. Their compressive moduli were at least 5 times higher than that of HA hydrogels. The pLysAAm/HA hydrogels had an equilibrium swelling ratio of up to 37.9 and displayed a glutathione-responsive degradation behavior. The results from in vitro biocompatibility evaluation with pre-osteoblasts MC3T3-E1 cells revealed that the pLysAAm/HA hydrogels could support cell viability and proliferation. Hematoxylin and eosin staining indicated that the pLysAAm/HA hydrogels allowed cell and tissue infiltration, confirming their good in vivo biocompatibility. Therefore, the novel pLysAAm/HA IPN hydrogels have great potential for bone tissue engineering applications.

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

    PubMed

    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

  2. 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.

  3. 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

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

    PubMed Central

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

    2013-01-01

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

  5. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1.

    PubMed

    Saxton, Robert A; Chantranupong, Lynne; Knockenhauer, Kevin E; Schwartz, Thomas U; Sabatini, David M

    2016-08-11

    The mechanistic Target of Rapamycin Complex 1 (mTORC1) is a major regulator of eukaryotic growth that coordinates anabolic and catabolic cellular processes with inputs such as growth factors and nutrients, including amino acids. In mammals arginine is particularly important, promoting diverse physiological effects such as immune cell activation, insulin secretion, and muscle growth, largely mediated through activation of mTORC1 (refs 4, 5, 6, 7). Arginine activates mTORC1 upstream of the Rag family of GTPases, through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting Cellular Arginine Sensor for mTORC1 (CASTOR1). However, the mechanism by which the mTORC1 pathway detects and transmits this arginine signal has been elusive. Here, we present the 1.8 Å crystal structure of arginine-bound CASTOR1. Homodimeric CASTOR1 binds arginine at the interface of two Aspartate kinase, Chorismate mutase, TyrA (ACT) domains, enabling allosteric control of the adjacent GATOR2-binding site to trigger dissociation from GATOR2 and downstream activation of mTORC1. Our data reveal that CASTOR1 shares substantial structural homology with the lysine-binding regulatory domain of prokaryotic aspartate kinases, suggesting that the mTORC1 pathway exploited an ancient, amino-acid-dependent allosteric mechanism to acquire arginine sensitivity. Together, these results establish a structural basis for arginine sensing by the mTORC1 pathway and provide insights into the evolution of a mammalian nutrient sensor. PMID:27487210

  6. Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences.

    PubMed

    Suzuki, T; Inoue, N; Higashi, T; Mizobuchi, R; Sugimura, N; Yokouchi, K; Furukohri, T

    2000-12-01

    Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a Km value of 0.6 mM for arginine and a Vmax value of 13 micromole Pi min(-1) mg protein(-1) for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1,608 and 1,239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66-73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63-71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55-57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered.

  7. Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

    PubMed Central

    de la Fuente, A.; Martín, J. F.; Rodríguez-García, A.; Liras, P.

    2004-01-01

    The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations. PMID:15375131

  8. Inhibition of corneal neovascularization with a nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract.

    PubMed

    Shakiba, Yadollah; Mostafaie, Ali

    2007-10-01

    Corneal neovascularization is a significant, sight-threatening complication of many ocular surface disorders. Various growth factors and proteinases are involved in corneal neovascularization. The data supporting a causal role for vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) are extensive. Inhibition of VEGF and MMPs is a main strategy for treating corneal neovascularization. Several findings have shown that corneal neovascularization can be reduced by using anti-VEGF and anti-MMPs agents. Efficacy of a nutrient mixture (NM) containing lysine, proline, ascorbic acid, and green tea extract has been demonstrated for reducing VEGF and MMPs secretion by various cells. Moreover, NM can inhibit endothelial cell migration and capillary tube formation. We herein note that topical application of NM is potentially useful for inhibiting corneal neovascularization and restoration of corneal clarity. Further investigations in animal models are needed to place NM alongside corneal neovascularization therapeutics.

  9. CROP/Luc7A, a novel serine/arginine-rich nuclear protein, isolated from cisplatin-resistant cell line.

    PubMed

    Nishii, Y; Morishima, M; Kakehi, Y; Umehara, K; Kioka, N; Terano, Y; Amachi, T; Ueda, K

    2000-01-14

    A novel putative SR protein, designated cisplatin resistance-associated overexpressed protein (CROP), has been cloned from cisplatin-resistant cell lines by differential display. The N-half of the deduced amino acid sequence of 432 amino acids of CROP contains cysteine/histidine motifs and leucine zipper-like repeats. The C-half consists mostly of charged and polar amino acids: arginine (58 residues or 25%), glutamate (36 residues or 16%), serine (35 residues or 15%), lysine (30 residues, 13%), and aspartate (20 residues or 9%). The C-half is extremely hydrophilic and comprises domains rich in lysine and glutamate residues, rich in alternating arginine and glutamate residues, and rich in arginine and serine residues. The arginine/serine-rich domain is dominated by a series of 8 amino acid imperfect repetitive motif (consensus sequence, Ser-Arg-Ser-Arg-Asp/Glu-Arg-Arg-Arg), which has been found in RNA splicing factors. The RNase protection assay and Western blotting analysis indicate that the expression of CROP is about 2-3-fold higher in mRNA and protein levels in cisplatin-resistant ACHN/CDDP cells than in host ACHN cells. CROP is the human homologue of yeast Luc7p, which is supposed to be involved in 5'-splice site recognition and is essential for vegetative growth. PMID:10631324

  10. A phosphonic acid appended naphthalene diimide motif for self-assembly into tunable nanostructures through molecular recognition with arginine in water.

    PubMed

    Nandre, Kamalakar P; Bhosale, Sheshanath V; Rama Krishna, K V S; Gupta, Akhil; Bhosale, Sidhanath V

    2013-06-18

    A naphthalene diimide motif bearing phosphonic acid functionalities has been found to be self-assembled with L- and D-arginine through chirality induced molecular recognitions and leads to the formation of micrometre long nanobelts and spherical aggregates at pH 9 in water, respectively. PMID:23589823

  11. A single lysine of the two-lysine recognition motif of the D3 domain of receptor-associated protein is sufficient to mediate endocytosis by low-density lipoprotein receptor-related protein.

    PubMed

    van den Biggelaar, Maartje; Sellink, Erica; Klein Gebbinck, Jacqueline W T M; Mertens, Koen; Meijer, Alexander B

    2011-03-01

    Ligand binding of the low-density lipoprotein (LDL) receptor family is mediated by complement-type repeats (CR) each comprising a binding pocket for a single basic amino acid residue. It has been proposed that at least two CRs are required for high-affinity interaction by utilising two spatially distinct lysine residues on the ligand surface. LDL receptor-related protein (LRP) mediates the cellular uptake of a multitude of ligands, some of which bind LRP with a relatively low affinity suggesting a suboptimal positioning of the two critical lysines. We now addressed the role of the two critical lysines not only in LRP binding but also in LRP-dependent endocytosis. Variants of the third domain (D3) of receptor-associated protein (RAP) were created carrying lysine to alanine or arginine replacements at the putative contact residues K253, K256 and K270. Surface plasmon resonance revealed that replacement of K253 did not affect high-affinity LRP binding at all, whereas replacement of either K256 or K270 markedly reduced the affinity by approximately 10-fold. Binding was abolished when both lysines were replaced. Substitution by either alanine or arginine exerted an almost identical effect on LRP binding. This suggests that despite their positive charge, arginine residues do not support receptor binding at all. Confocal microscopy and flow cytometry studies surprisingly revealed that the single mutants were still taken up and still competed for the uptake of full length RAP despite their receptor binding defect. We therefore propose that the presence of only one of the two critical lysines is sufficient to drive endocytosis. PMID:21144910

  12. Peptide nucleic acids tagged with four lysine residues for amperometric genosensors

    PubMed Central

    Zanardi, Chiara; Terzi, Fabio; Seeber, Renato; Baldoli, Clara; Licandro, Emanuela; Maiorana, Stefano

    2012-01-01

    A homothymine PNA decamer bearing four lysine residues has been synthesized as a probe for the development of amperometric sensors. On one hand, the four amino groups introduced make this derivative nine times more soluble than the corresponding homothymine PNA decamer and, on the other hand, allow the stable anchoring of this molecule on Au nanostructured surface through the terminal -NH2 moieties. In particular, XPS and electrochemical investigations performed with hexylamine, as a model molecule, indicate that the stable deposition of primary amine derivatives on such a nanostructured surface is possible and involves the free electron doublet on the nitrogen atom. This finding indicates that this PNA derivative is suitable to act as the probe molecule for the development of amperometric sensors.   Thanks to the molecular probe chosen and to the use of a nanostructured surface as the substrate for the sensor assembly, the device proposed makes possible the selective recognition of the target oligonucleotide sequence with very high sensitivity. PMID:22772036

  13. Poly(L-diaminopropionic acid), a novel non-proteinic amino acid oligomer co-produced with poly(ε-L-lysine) by Streptomyces albulus PD-1.

    PubMed

    Xia, Jun; Xu, Hong; Feng, Xiaohai; Xu, Zhaoxian; Chi, Bo

    2013-09-01

    Poly(ε-L-lysine) (ε-PL) producer strain Streptomyces albulus PD-1 secreted a novel polymeric substance into its culture broth along with ε-PL. The polymeric substance was purified to homogeneity and identified. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance spectroscopy as well as other analytical techniques revealed that the substance was poly(L-diaminopropionic acid) (PDAP). PDAP is an L-α,β-diaminopropionic acid oligomer linking between amino and carboxylic acid functional groups. The molecular weight of PDAP ranged from 500 to 1500 Da, and no co-polymers composed of L-diaminopropionic acid and L-lysine were present in the culture broth. Compared with ε-PL, PDAP exhibited stronger inhibitory activities against yeasts but weaker activities against bacteria. ε-PL and PDAP co-production was also investigated. Both ε-PL and PDAP were synthesized during the stationary phase of growth, and the final ε-PL and PDAP concentration reached 21.7 and 4.8 g L(-1), respectively, in fed-batch fermentation. Citric acid feeding resulted in a maximum ε-PL concentration of 26.1 g L(-1) and a decrease in the final concentration of PDAP to 3.8 g L(-1). No studies on ε-PL and PDAP co-production in Streptomyces albulus have been reported previously, and inhibition of by-products such as PDAP is potentially useful in ε-PL production. PMID:23775267

  14. Fortifying plants with the essential amino acids lysine and methionine to improve nutritional quality.

    PubMed

    Galili, Gad; Amir, Rachel

    2013-02-01

    Humans, as well as farm animals, cannot synthesize a number of essential amino acids, which are critical for their survival. Hence, these organisms must obtain these essential amino acids from their diets. Cereal and legume crops, which represent the major food and feed sources for humans and livestock worldwide, possess limiting levels of some of these essential amino acids, particularly Lys and Met. Extensive efforts were made to fortify crop plants with these essential amino acids using traditional breeding and mutagenesis. However, aside from some results obtained with maize, none of these approaches was successful. Therefore, additional efforts using genetic engineering approaches concentrated on increasing the synthesis and reducing the catabolism of these essential amino acids and also on the expression of recombinant proteins enriched in them. In the present review, we discuss the basic biological aspects associated with the synthesis and accumulation of these amino acids in plants and also describe recent developments associated with the fortification of crop plants with essential amino acids by genetic engineering approaches.

  15. Evolution of Substrate Specificity within a Diverse Family of [beta/alpha]-Barrel-fold Basic Amino Acid Decarboxylases X-ray Structure Determination of Enzymes with Specificity for L-Arginine and Carboxynorspermidine

    SciTech Connect

    Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J.; Tomchick, Diana R.; Goldsmith, Elizabeth J.; Phillips, Margaret A.

    2010-08-26

    Pyridoxal 5{prime}-phosphate (PLP)-dependent basic amino acid decarboxylases from the {beta}/{alpha}-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-{angstrom} resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the {beta}/{alpha}-barrel domain of one subunit and the {beta}-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the {beta}-barrel domain we refer to as the 'specificity helix.' Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu{sup 314}, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5{prime}-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.

  16. FXR-induced lysine-specific histone demethylase, LSD1, reduces hepatic bile acid levels and protects the liver against bile acid toxicity

    PubMed Central

    Kim, Young-Chae; Fang, Sungsoon; Byun, Sangwon; Seok, Sunmi; Kemper, Byron; Kemper, Jongsook Kim

    2015-01-01

    Bile acids (BAs) function as endocrine signaling molecules that activate multiple nuclear and membrane receptor signaling pathways to control fed-state metabolism. Since the detergent-like property of BAs causes liver damage at high concentrations, hepatic BA levels must be tightly regulated. BA homeostasis is regulated largely at the level of transcription by nuclear receptors, particularly the primary bile acid receptor, farnesoid X receptor (FXR), and small heterodimer partner (SHP) that inhibits BA synthesis by recruiting repressive histone-modifying enzymes. Although histone modifiers have been shown to regulate BA-responsive genes, their in vivo functions remain unclear. Here we show that lysine-specific histone demethylase1 (LSD1) is directly induced by BA-activated FXR, is recruited to BA synthetic genes, Cyp7a1 and Cyp8b1, and the BA uptake transporter gene, Ntcp, and removes a gene-activation mark, tri-methylated histone H3 lysine-4, leading to gene repression. LSD1 recruitment was dependent on SHP, and LSD1-mediated demethylation of H3K4-me3 was required for additional repressive histone modifications, H3K9/K14 deacetylation and H3K9 methylation. BA overload, feeding 0.5% cholic acid chow for 6 days, resulted in adaptive responses of altered expression of hepatic genes involved in BA synthesis, transport, and detoxification/conjugation. In contrast, adenoviral-mediated downregulation of hepatic LSD1 blunted these responses, which led to substantial increases in liver and serum BA levels, serum AST/ALT levels, and hepatic inflammation. This study identifies LSD1 as a novel histone-modifying enzyme in the orchestrated regulation mediated by the FXR and SHP that reduces hepatic BA levels and protects the liver against BA toxicity. PMID:25545350

  17. Adsorption of monomers on microspherical structures of thermal heterocomplex molecules from amino ACIDS

    NASA Astrophysics Data System (ADS)

    Honda, Hajime; Sakurazawa, Shigeru; Dekikimura, H.; Imai, Eiichi; Matsuno, Koichiro

    1995-10-01

    The surface of a microspherical structure formed in the aqueous suspension of thermal heterocomplex molecules made by heating aspartic acid and proline can adsorb basic amino acids such as histidine, lysine and arginine. It can also adsorb adenine, cytosine, adenosine and cytidine. Electrostatic interactions acting between those monomers to be adsorbed and the adsorbing surface are responsible for the adsorption.

  18. Amino acids improve acid tolerance and internal pH maintenance in Bacillus cereus ATCC14579 strain.

    PubMed

    Senouci-Rezkallah, Khadidja; Schmitt, Philippe; Jobin, Michel P

    2011-05-01

    This study investigated the involvement of glutamate-, arginine- and lysine-dependent systems in the Acid Tolerance Response (ATR) of Bacillus cereus ATCC14579 strain. Cells were grown in a chemostat at external pH (pH(e)) 7.0 and 5.5. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted) compared with cells grown at pH 7.0 (unadapted), indicating that B. cereus cells grown at low pH(e) were able to induce a marked ATR. Glutamate, arginine and lysine enhanced the resistance of unadapted cells to pH 4.0 acid shock of 1-log or 2-log populations, respectively. Amino acids had no detectable effect on acid resistance in acid-adapted cells. An acid shock at pH 4.0 resulted in a marked drop in internal pH (pH(i)) in unadapted cells compared with acid-adapted cells. When acid shock was achieved in the presence of glutamate, arginine or lysine, pH(i) was maintained at higher values (6.31, 6.69 or 6.99, respectively) compared with pH(i) in the absence of amino acids (4.88). Acid-adapted cells maintained their pH(i) at around 6.4 whatever the condition. Agmatine (a competitive inhibitor of arginine decarboxylase) had a negative effect on the ability of B. cereus cells to survive and maintain their pH(i) during acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. This adaptation depends on pH(i) homeostasis and is enhanced in the presence of glutamate, arginine and lysine. Hence evaluations of the pathogenicity of B. cereus must take into account its ability to adapt to acid stress.

  19. Arginine transport in catabolic disease states.

    PubMed

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

    2004-10-01

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

  20. Poly(L-lysine)-g-poly(D,L-lactic-co-glycolic acid) micelles for low cytotoxic biodegradable gene delivery carriers.

    PubMed

    Jeong, Ji Hoon; Park, Tae Gwan

    2002-07-18

    Poly(lactic-co-glycolic acid) (PLGA)-grafted poly(L-lysine) (PLL) (PLL-g-PLGA) was synthesized to demonstrate its micelle-forming property in an aqueous solution. The micelles were used as a gene delivery carrier. The hydrodynamic diameter of PLL-g-PLGA micelles in an aqueous solution was ca. 149 nm with a narrow size distribution. Critical micelle concentration (cmc) was 9.6 mg/l. The PLL-g-PLGA micelles could be used to produce compact nanoparticulate complexes with plasmid DNA, which could efficiently protect the complexed DNA from enzymatic degradation by DNase I. The micelle/DNA complexes had highly compacted structure sized between 200-300 nm with a positive surface charge value. The PLL-g-PLGA micelles exhibited much higher transfection efficiency with lower cytotoxicity than PLL. Here, we demonstrated that biodegradable and cationic PLL-g-PLGA micelles could be used as an effective DNA condensation carrier for gene delivery system.

  1. Functional impact of polar and acidic substitutions in the lactose repressor hydrophobic monomer.monomer interface with a buried lysine.

    PubMed

    Zhan, Hongli; Sun, Zhifei; Matthews, Kathleen Shive

    2009-02-17

    Despite predicted energetic penalties, the charged K84 side chains of tetrameric lactose repressor protein (LacI) are found buried within the highly hydrophobic monomer.monomer interface that includes side chains of V94 and V96. Once inducer binding has occurred, these K84 side chains move to interact with the more solvent-exposed side chains of D88 and E100'. Previous studies demonstrated that hydrophobic substitutions for K84 increased protein stability and significantly impaired the allosteric response. These results indicated that enhanced hydrophobic interactions at the monomer.monomer interface remove the energetic driving force of the buried charges, decreasing the likelihood of a robust conformational change and stabilizing the structure. We hypothesized that creating a salt bridge network with the lysine side chains by including nearby negatively charged residues might result in a similar outcome. To that end, acidic residues, D and E, and their neutral amides, N and Q, were substituted for the valines at positions 94 and 96. These variants exhibited one or more of the following functional changes: weakened inducer binding, impaired allosteric response, and diminished protein stability. For V96D and V96E, ion pair formation with K84 appears optimal, and the loss of inducer response exceeds that of the hydrophobic K84A and -L variants. However, impacts on functional properties indicate that stabilizing the buried positive charge with polar or ion pair interactions is not functionally equivalent to structural stabilization via hydrophobic enhancement. PMID:19166325

  2. Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization in Entamoeba histolytica.

    PubMed

    Perdomo, Doranda; Manich, Maria; Syan, Sylvie; Olivo-Marin, Jean-Christophe; Dufour, Alexandre C; Guillén, Nancy

    2016-08-01

    The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well-defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER-Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin-rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica. PMID:26857352

  3. A lysine- and glutamic acid-rich protein, KERP1, from Entamoeba histolytica binds to human enterocytes.

    PubMed

    Seigneur, Marie; Mounier, Joelle; Prevost, Marie-Christine; Guillén, Nancy

    2005-04-01

    Contact-dependent cytolysis of host cells by Entamoeba histolytica is an important hallmark of amoebiasis that points out the importance of molecules involved in the interaction between the parasite and the human cells. To decipher the molecular and cellular mechanisms supporting the invasion of the intestinal epithelium by E. histolytica, we analysed proteins involved in the interaction of the parasite with enterocytes. Affinity chromatography revealed several amoebic proteins interacting with purified brush border of differentiated Caco2 cells. Among them were found the intermediate subunit of the Gal/GalNAc lectin, an alpha-actinin-like protein and two new proteins KERP1 and KERP2 rich in lysine and glutamic acid. In silico analysis revealed the presence of KERP2 in the closely related non-pathogenic amoeba species Entamoeba dispar but not of KERP1. In additon, polymerase chain reaction analysis allowed to suggest the absence of kerp1 homologous gene in E. dispar. Therefore, we concentrated on the cellular analysis of KERP1. Cloning of the KERP1-encoding gene, production of a recombinant protein in Escherichia coli and production of a specific antibody allowed us to show the following properties: (i) purified KERP1 binds to epithelial cell surface, (ii) KERP1 is located on the plasma membrane and in vesicles of trophozoites and (iii) KERP1 is delivered in the interstitial area between the trophozoites and the intestinal cells.

  4. A leptin derived 30-amino-acid peptide modified pegylated poly-L-lysine dendrigraft for brain targeted gene delivery.

    PubMed

    Liu, Yang; Li, Jianfeng; Shao, Kun; Huang, Rongqin; Ye, Liya; Lou, Jinning; Jiang, Chen

    2010-07-01

    The blood-brain barrier is the major obstacle that prevents diagnostic and therapeutic drugs being delivered to the central nervous systems in order to exert their effects. Specific ligand-receptor binding mediated endocytosis is one of the possible strategies to cross this barrier. A 30-amino-acid peptide (leptin30) derived from an endogenic hormone-leptin is exploited as brain-targeting ligand as it is reported to possess the same brain accumulation efficiency after intravenous injection. Dendrigraft poly-L-lysine (DGL) is used as non-viral gene vector in this study. DGL-PEG-Leptin30 was complexed with plasmid DNA yielding nanoparticles (NPs). The cellular uptake characteristic and mechanism were explored in brain capillary endothelial cells (BCECs) which express leptin receptors. Furthermore, brain parenchyma microglia cells such as BV-2 cells expressing leptin receptors could promote ligand-receptor mediated endocytosis leading to enhanced gene transfection ability of DGL-PEG-Leptin30/DNA NPs. The targeted NPs were proved to be transported across in vitro BBB model effectively and accumulate more in brains after i.v. resulting in a relatively high gene transfection efficiency both in vitro and in vivo. Besides, the NPs showed low cytotoxicity after in vitro transfection. Thus, DGL-PEG-Leptin30 provides a safe and noninvasive approach for the delivery of gene across the blood-brain barrier.

  5. Proline-glutamic acid-proline-lysine repetition peptide as an antigen for the serological diagnosis of strangles.

    PubMed

    Hobo, S; Niwa, H; Anzai, T

    2008-04-12

    The reactivity of the proline-glutamic acid-proline-lysine (PEPK) repetition peptide antigen in 3176 serum samples was investigated to evaluate its utility as an antigen for the serological diagnosis of strangles. The reactivity of the sera of horses infected with Streptococcus equi subspecies equi was high when the peptide had several PEPK repetitions. However, as the number of PEPK repetitions increased, the reactivity of the antigen with the sera of horses infected with Streptococcus equi subspecies zooepidemicus also increased. In horses infected experimentally with S equi, the reactivity of the PEPK antigen with five repetitions increased one week after inoculation and continued to increase during the following four weeks. The optical density (OD) values of test sera from horses infected experimentally with S equi and sera from horses that had recovered from strangles were high. The od values of sera from horses that had recovered from an experimental infection with S zooepidemicus and of sera from healthy horses were comparatively low.

  6. Proline-glutamic acid-proline-lysine peptide set as a specific antigen for the serological diagnosis of strangles.

    PubMed

    Hobo, S; Niwa, H; Anzai, T

    2006-11-01

    The reactivity of synthesised peptide sets for the M-like proteins SeM and SzPSe with sera from horses infected with Streptococcus equi or Streptococcus zooepidemicus, or control horses, was investigated by an ELISA. Seventeen horses were infected experimentally with S equi or S zooepidemicus, convalescent sera were obtained from 25 horses and control sera were obtained from 1945 horses. The serum antibody responses of individual horses to the peptide sets were highly variable. Some of the peptide sets for SeM reacted strongly with the sera from the horses infected experimentally with S equi, but also reacted with sera from some of the horses infected experimentally with S zooepidemicus. However, the proline-glutamic acid-proline-lysine (PEPK) repeats peptide set, synthesised from the PEPK repeats areas of SzPSe, reacted most strongly with the sera from the horses infected experimentally with S equi and the horses convalescing from strangles, and reacted only minimally with the sera from the horses infected experimentally with S zooepidemicus and the control horses.

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

    PubMed

    Ren, Wenkai; Yin, Jie; Wu, Miaomiao; Liu, Gang; Yang, Guan; Xion, Yan; Su, Dingding; Wu, Li; Li, Tiejun; Chen, Shuai; Duan, Jielin; Yin, Yulong; Wu, Guoyao

    2014-01-01

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

  8. Indispensable but insufficient role of renal D-amino acid oxidase in chiral inversion of NG-nitro-D-arginine.

    PubMed

    Xin, Yan-Fei; Li, Xin; Hao, Bin; Gong, Nian; Sun, Wen-Qiang; Konno, Ryuichi; Wang, Yong-Xiang

    2010-06-01

    Unidirectionally chiral inversion of N(G)-nitro-D-arginine (D-NNA) to its L-enantiomer (L-NNA) occurred in rats, and it was blocked markedly (ca. 80%) by renal vascular ligation, and entirely (100%) by the D-amino acid oxidase (DAO) inhibitor sodium benzoate, suggesting that renal DAO is essential for the inversion. However, the doses of sodium benzoate administrated were extremely high (e.g., 400 mg/kg) due to its low potency. It is thus possible that sodium benzoate-mediated blockade of D-NNA inversion might be due to its nonspecific (or non-DAO-related) effects. In addition, after D-NNA was incubated with the pure enzyme of DAO in vitro without tissue homogenates, L-NNA was not produced, even though D-NNA was disposed. We propose that this occurred because D-NNA was first converted to its corresponding alpha-keto acid by DAO and then to L-NNA by transaminase(s); however, there was no direct evidence for this process. The goal of this study is to further elucidate the process of D-NNA chiral inversion both in vivo and in in vitro tissue homogenates by comparing mutant ddY/DAO(-/-) mice that lack DAO activity entirely compared to normal ddY/DAO(+/+) mice and Swiss mice. Furthermore, the ability to produce L-NNA from D-NNA-corresponding alpha-keto acids (N(G)-nitroguanidino-2-oxopentanoic acid) produced by porcine kidney-derived DAO (pkDAO) was also studied in the DAO inhibitor-pretreated rats. We found that D-NNA chiral inversion occurred in Swiss mice and ddY/DAO(+/+) mice both in vivo and in in vitro kidney homogenates, but not in ddY/DAO(-/-) mice, correlated to their DAO activities. The alpha-keto acid (N(G)-nitro-guanidino-2-oxopentanoic acid) from D-NNA was able to produce L-NNA, and subsequent vasoconstriction and pressor responses. These results indicate that the role of renal DAO is indispensible but insufficient for chiral inversion of D-NNA and other neutral and polar D-amino acids, and unidentified aminotransferase(s) are involved in a subsequent

  9. Indispensable but insufficient role of renal D-amino acid oxidase in chiral inversion of NG-nitro-D-arginine.

    PubMed

    Xin, Yan-Fei; Li, Xin; Hao, Bin; Gong, Nian; Sun, Wen-Qiang; Konno, Ryuichi; Wang, Yong-Xiang

    2010-06-01

    Unidirectionally chiral inversion of N(G)-nitro-D-arginine (D-NNA) to its L-enantiomer (L-NNA) occurred in rats, and it was blocked markedly (ca. 80%) by renal vascular ligation, and entirely (100%) by the D-amino acid oxidase (DAO) inhibitor sodium benzoate, suggesting that renal DAO is essential for the inversion. However, the doses of sodium benzoate administrated were extremely high (e.g., 400 mg/kg) due to its low potency. It is thus possible that sodium benzoate-mediated blockade of D-NNA inversion might be due to its nonspecific (or non-DAO-related) effects. In addition, after D-NNA was incubated with the pure enzyme of DAO in vitro without tissue homogenates, L-NNA was not produced, even though D-NNA was disposed. We propose that this occurred because D-NNA was first converted to its corresponding alpha-keto acid by DAO and then to L-NNA by transaminase(s); however, there was no direct evidence for this process. The goal of this study is to further elucidate the process of D-NNA chiral inversion both in vivo and in in vitro tissue homogenates by comparing mutant ddY/DAO(-/-) mice that lack DAO activity entirely compared to normal ddY/DAO(+/+) mice and Swiss mice. Furthermore, the ability to produce L-NNA from D-NNA-corresponding alpha-keto acids (N(G)-nitroguanidino-2-oxopentanoic acid) produced by porcine kidney-derived DAO (pkDAO) was also studied in the DAO inhibitor-pretreated rats. We found that D-NNA chiral inversion occurred in Swiss mice and ddY/DAO(+/+) mice both in vivo and in in vitro kidney homogenates, but not in ddY/DAO(-/-) mice, correlated to their DAO activities. The alpha-keto acid (N(G)-nitro-guanidino-2-oxopentanoic acid) from D-NNA was able to produce L-NNA, and subsequent vasoconstriction and pressor responses. These results indicate that the role of renal DAO is indispensible but insufficient for chiral inversion of D-NNA and other neutral and polar D-amino acids, and unidentified aminotransferase(s) are involved in a subsequent

  10. Two amino acid-based superlow fouling polymers: poly(lysine methacrylamide) and poly(ornithine methacrylamide).

    PubMed

    Liu, Qingsheng; Li, Wenchen; Singh, Anuradha; Cheng, Gang; Liu, Lingyun

    2014-07-01

    We developed and investigated two new antifouling zwitterionic polymers, poly(lysine methacrylamide) (pLysAA) and poly(ornithine methacrylamide) (pOrnAA), both derived from natural amino acids - lysine and ornithine, respectively. The pLysAA and pOrnAA brushes were grafted on gold via the surface-initiated photoiniferter-mediated polymerization, with the polymer film thickness controlled by the UV-irradiation time. Nonspecific adsorption from human blood serum and plasma was investigated by surface plasmon resonance. Results show that the adsorption level decreased with the increasing film thickness. With the thin films of ∼14.5 nm, the minimal adsorption on pLysAA was 3.9 ng cm(-2) from serum and 5.4 ng cm(-2) from plasma, whereas the lowest adsorption on pOrnAA was 1.8 and 3.2 ng cm(-2), from serum and plasma, respectively. Such protein resistance is comparable to other widely reported antifouling surfaces such as poly(sulfobetaine methacrylate) and polyacrylamide, with a much thinner polymer film thickness. Both pLysAA and pOrnAA showed better protein resistance than the previously reported serine-based poly(serine methacrylate), whereas the pOrnAA is the best among three. The pLysAA- and pOrnAA-grafted surfaces also highly resisted the endothelial cell attachment and Escherichia coli K12 bacterial adhesion. Nanogels made of pLysAA and pOrnAA were found to be ultrastable in undiluted serum, with no aggregation observed after culturing for 24h. Dextran labeled with fluorescein isothiocyanate (FITC-dextran) was encapsulated in nanogels as a model drug. The encapsulated FITC-dextran exhibited controlled release from the pOrnAA nanogels. The superlow fouling, biomimetic and multifunctional properties of pLysAA and pOrnAA make them promising materials for a wide range of applications, such as implant coating, drug delivery and biosensing. PMID:24613545

  11. Possible Evidence of Amide Bond Formation Between Sinapinic Acid and Lysine-Containing Bacterial Proteins by Matrix-Assisted Laser Desorption/Ionization (MALDI) at 355 nm

    NASA Astrophysics Data System (ADS)

    Fagerquist, Clifton K.; Sultan, Omar; Carter, Michelle Q.

    2012-12-01

    We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, Hde, and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight tandem mass spectrometry (TOF-TOF-MS/MS) and post-source decay (PSD). We also reported the absence of adduct formation when using α-cyano-4-hydroxycinnamic acid (CHCA) matrix. Further mass spectrometric analysis of disulfide-intact and disulfide-reduced over-expressed HdeA and HdeB proteins from lysates of gene-inserted E. coli plasmids suggests covalent attachment of SA occurs not at cysteine residues but at lysine residues. In this revised hypothesis, the attachment of SA is preceded by formation of a solid phase ammonium carboxylate salt between SA and accessible lysine residues of the protein during sample preparation under acidic conditions. Laser irradiation at 355 nm of the dried sample spot results in equilibrium retrogradation followed by nucleophilic attack by the amine group of lysine at the carbonyl group of SA and subsequent amide bond formation and loss of water. The absence of CHCA adducts suggests that the electron-withdrawing effect of the α-cyano group of this matrix may inhibit salt formation and/or amide bond formation. This revised hypothesis is supported by dissociative loss of SA (-224 Da) and the amide-bound SA (-206 Da) from SA-adducted HdeA and HdeB ions by MS/MS (PSD). It is proposed that cleavage of the amide-bound SA from the lysine side-chain occurs via rearrangement involving a pentacyclic transition state followed by hydrogen abstraction/migration and loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal (-206 Da).

  12. Titanium dioxide nanoparticles modified by salicylic acid and arginine: Structure, surface properties and photocatalytic decomposition of p-nitrophenol

    NASA Astrophysics Data System (ADS)

    Li, Lei; Feng, Yujie; Liu, Youzhi; Wei, Bing; Guo, Jiaxin; Jiao, Weizhou; Zhang, Zhaohan; Zhang, Qiaoling

    2016-02-01

    In this study, titanium dioxide (TiO2) nanoparticles were surface-modified with salicylic acid (SA) and arginine (Arg) using an environmentally friendly and convenient method, and the bonding structure, surface properties and degradation efficiency of p-nitrophenol (PNP) were investigated. X-ray diffractometry (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brunauer-Emmett-Teller (BET), X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FT-IR), water contact angle (WCA) measurements, ζ-potentiometric analysis, UV/visible diffuse reflectance spectroscopy (UV-vis DRS), and thermogravimetric analysis (TGA) were performed to evaluate the modification effect. The degradation rates were determined by high-performance liquid chromatography (HPLC). The results show that bidentate or bridging bonds are most likely formed between SA/Arg and TiO2 surface. Surface modification with SA, Arg, or both can improve the lipophilic properties and decrease the zeta potential, and also result in a red shift of the absorption wavelength. TiO2 nanoparticles modified by Arg or both SA and Arg show a large specific surface area and pore volume. Further, degradation experiments under visible light show that Arg modification is most efficient. This simple and versatile synthetic method to produce TiO2 nanoparticles surface-modified with various organic capping agents can be used for novel multifunctional photocatalysts as required for various applications in energy saving and environmental protection.

  13. Effect of l-lysine-assisted surface grafting for nano-hydroxyapatite on mechanical properties and in vitro bioactivity of poly(lactic acid-co-glycolic acid).

    PubMed

    Liuyun, Jiang; Lixin, Jiang; Chengdong, Xiong; Lijuan, Xu; Ye, Li

    2016-01-01

    It is promising and challenging to study surface modification for nano-hydroxyapatite to improve the dispersion and enhance the mechanical properties and bioactivity of poly(lactic acid-co-glycolic acid). In this paper, we designed an effective new surface grafting with the assist of l-lysine for nano-hydroxyapatite, and the nano-hydroxyapatite surface grafted with the assist of l-lysine (g-nano-hydroxyapatite) was incorporated into poly(lactic acid-co-glycolic acid) to develop a series of g-nano-hydroxyapatite/poly(lactic acid-co-glycolic acid) nano-composites. The surface modification reaction for nano-hydroxyapatite, the mechanical properties, and in vitro human osteoblast-like cell (MG-63) response were characterized and investigated by Fourier transformation infrared, thermal gravimetric analysis, dispersion test, electromechanical universal tester, differential scanning calorimeter measurements, and in vitro cells culture experiment. The results showed that the grafting amount on the surface of nano-hydroxyapatite was enhanced with the increase of l-lysine, and the dispersion of nano-hydroxyapatite was improved more, so that it brought about better promotion crystallization and more excellent mechanical enhancement effect for poly(lactic acid-co-glycolic acid), comparing with the unmodified nano-hydroxyapatite. Moreover, the cells' attachment and proliferation results confirmed that the incorporation of the g-nano-hydroxyapatite into poly(lactic acid-co-glycolic acid) exhibited better biocompatibility than poly(lactic acid-co-glycolic acid). The above results indicated that the new surface grafting with the assist of l-lysine for nano-hydroxyapatite was an ideal novel surface modification method, which brought about better mechanical enhancement effect and in vitro bioactivity for poly(lactic acid-co-glycolic acid) with adding higher g-nano-hydroxyapatite content, suggesting it had a great potential to be used as bone fracture internal fixation materials

  14. Activation of l-arginine transport by protein kinase C in rabbit, rat and mouse alveolar macrophages

    PubMed Central

    Racké, Kurt; Hey, Claudia; Mössner, Jutta; Hammermann, Rainer; Stichnote, Christina; Wessler, Ignaz

    1998-01-01

    The role of protein kinase C in controlling L-arginine transport in alveolar macrophages was investigated. L-[3H]Arginine uptake in rabbit alveolar macrophages declined by 80 % after 20 h in culture. 4β-Phorbol 12-myristate 13-acetate (PMA), but not 4α-phorbol 12-myristate 13-acetate (α-PMA), present during 20 h culture, enhanced L-[3H]arginine uptake more than 10-fold. Staurosporine and chelerythrine opposed this effect. L-[3H]Arginine uptake was saturable and blockable by L-lysine. After PMA treatment Vmax was increased more than 5-fold and Km was reduced from 0.65 to 0.32 mM. Time course experiments showed that PMA increased L-[3H]arginine uptake almost maximally within 2 h. This short-term effect was not affected by cycloheximide or actinomycin D. L-[3H]Arginine uptake and its stimulation by PMA was also observed in sodium-free medium. L-Leucine (0.1 mM) inhibited L-[3H]arginine uptake by 50 % in sodium-containing medium, but not in sodium-free medium. At 1 mM, L-leucine caused significant inhibition in sodium-free medium also. L-Leucine showed similar effects on PMA-treated cells. N-Ethylmaleimide (200 μm, 10 min) reduced L-[3H]arginine uptake by 70 % in control cells, but had no effect on PMA-treated (20 or 2 h) cells. In alveolar macrophages, multiple transport systems are involved in L-arginine uptake, which is markedly stimulated by protein kinase C, probably by modulation of the activity of already expressed cationic amino acid transporters. PMID:9714862

  15. 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...

  16. Nic1 inactivation enables stable isotope labeling with 13C615N4-arginine in Schizosaccharomyces pombe.

    PubMed

    Carpy, Alejandro; Patel, Avinash; Tay, Ye Dee; Hagan, Iain M; Macek, Boris

    2015-01-01

    Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, (13)C(6) (15)N(4)-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of (13)C(6) (15)N(4)-arginine is catabolized by arginase and urease activity to (15)N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni(2+)-dependent urease activity, through deletion of the sole Ni(2+) transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable (13)C(6) (15)N(4)-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe.

  17. The regulatory effect of citric acid on the co-production of poly(ε-lysine) and poly(L-diaminopropionic acid) in Streptomyces albulus PD-1.

    PubMed

    Xia, Jun; Xu, Zhaoxian; Xu, Hong; Feng, Xiaohai; Bo, Fangfang

    2014-10-01

    Streptomyces albulus PD-1 can co-produce antimicrobial homo-polymers poly(ε-lysine) (ε-PL) and poly(L-diaminopropionic acid) (PDAP). In this study, a novel feeding strategy of citric acid coupled with glucose-(NH4)2SO4 feeding was employed to S. albulus PD-1. When the pH of the culture broth dropped to 4.0, the feeding solution was added continuously to maintain the concentrations of glucose and citric acid at 10 and 4 g L(-1), respectively. As a result, the final concentration of ε-PL increased from 21.7 to 29.7 g L(-1) and the final concentration of PDAP decreased from 4.8 to 3.2 g L(-1). Assays on intracellular nucleotide levels and key enzyme activities were performed to elucidate the underlying regulation mechanism. The addition of citric acid increased NADH/NAD(+) ratio and decreased intracellular ATP level; meanwhile, the activities of pyruvate kinase, citrate synthase and isocitrate dehydrogenase decreased while aspartate aminotransferase activity increased. Therefore, we deduced that citric acid feeding resulted in metabolic flux redistribution at the node of phosphoenolpyruvate; the metabolic pathway from phosphoenolpyruvate directed into tricarboxylic acid cycle was weakened and thus PDAP production was inhibited. On the other hand, the metabolic pathway from phosphoenolpyruvate directed into oxaloacetate and L-aspartate was enhanced, thereby improving ε-PL production. This fermentation strategy may be potentially useful in ε-PL production because it can effectively inhibit the formation of by-products, such as PDAP.

  18. The regulatory effect of citric acid on the co-production of poly(ε-lysine) and poly(L-diaminopropionic acid) in Streptomyces albulus PD-1.

    PubMed

    Xia, Jun; Xu, Zhaoxian; Xu, Hong; Feng, Xiaohai; Bo, Fangfang

    2014-10-01

    Streptomyces albulus PD-1 can co-produce antimicrobial homo-polymers poly(ε-lysine) (ε-PL) and poly(L-diaminopropionic acid) (PDAP). In this study, a novel feeding strategy of citric acid coupled with glucose-(NH4)2SO4 feeding was employed to S. albulus PD-1. When the pH of the culture broth dropped to 4.0, the feeding solution was added continuously to maintain the concentrations of glucose and citric acid at 10 and 4 g L(-1), respectively. As a result, the final concentration of ε-PL increased from 21.7 to 29.7 g L(-1) and the final concentration of PDAP decreased from 4.8 to 3.2 g L(-1). Assays on intracellular nucleotide levels and key enzyme activities were performed to elucidate the underlying regulation mechanism. The addition of citric acid increased NADH/NAD(+) ratio and decreased intracellular ATP level; meanwhile, the activities of pyruvate kinase, citrate synthase and isocitrate dehydrogenase decreased while aspartate aminotransferase activity increased. Therefore, we deduced that citric acid feeding resulted in metabolic flux redistribution at the node of phosphoenolpyruvate; the metabolic pathway from phosphoenolpyruvate directed into tricarboxylic acid cycle was weakened and thus PDAP production was inhibited. On the other hand, the metabolic pathway from phosphoenolpyruvate directed into oxaloacetate and L-aspartate was enhanced, thereby improving ε-PL production. This fermentation strategy may be potentially useful in ε-PL production because it can effectively inhibit the formation of by-products, such as PDAP. PMID:24752482

  19. Efficacy of a Complex of 5-Aminolevulinic Acid and Glycyl-Histidyl-Lysine Peptide on Hair Growth

    PubMed Central

    Sim, Hyun Bo; Jang, Yong Hyun; Lee, Seok-Jong; Kim, Do Won; Yim, Soon-Ho

    2016-01-01

    Background Pattern hair loss is a very common problem. Although effective therapeutics for the treatment of pattern hair loss have been used, novel therapeutic modalities are still required to enhance hair growth. Objective We investigated the efficacy and safety of a complex (ALAVAX) of 5-aminolevulinic acid (5-ALA) and glycyl-histidyl-lysine (GHK) peptide for the treatment of pattern hair loss. Methods Forty-five patients with male pattern hair loss were treated with ALAVAX 100 mg/ml (group A), ALAVAX 50 mg/ml (group B) or placebo (group C) once a day for 6 months. Total hair count, hair length, hair thickness, patient's assessment and adverse events were evaluated at month 1, 3, and 6. Results An increase in hair count for 6 months was 52.6 (p<0.05) in group A, 71.5 (p<0.05) in group B, and 9.6 in group C. The ratio of changes in hair count between group B (2.38) and group C (1.21) at 6 months showed a statistically significant difference (p<0.05). The proportion above good satisfaction was higher in group A (26.7%) than in the other groups (group B: 14.3%, group C: 7.1%). There was no statistically significant difference in hair length and hair thickness among 3 groups at 6 months. There was no adverse event in 3 groups. Conclusion Our study showed that a complex of 5-ALA and GHK peptide may be considered as one of the complementary agents for the treatment of male pattern hair loss. PMID:27489425

  20. The catabolic function of the alpha-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine-oxoglutarate reductase, but not saccharopine dehydrogenase.

    PubMed Central

    Zhu, X; Tang, G; Galili, G

    2000-01-01

    Whereas plants and animals use the alpha-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine-oxoglutarate reductase (lysine-ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the alpha-amino adipic acid pathway in plants. PMID:10998364

  1. Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation.

    PubMed

    Colak, Gozde; Pougovkina, Olga; Dai, Lunzhi; Tan, Minjia; Te Brinke, Heleen; Huang, He; Cheng, Zhongyi; Park, Jeongsoon; Wan, Xuelian; Liu, Xiaojing; Yue, Wyatt W; Wanders, Ronald J A; Locasale, Jason W; Lombard, David B; de Boer, Vincent C J; Zhao, Yingming

    2015-11-01

    The protein substrates of sirtuin 5-regulated lysine malonylation (Kmal) remain unknown, hindering its functional analysis. In this study, we carried out proteomic screening, which identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts. Increased malonyl-CoA levels in malonyl-CoA decarboxylase (MCD)-deficient cells induces Kmal levels in substrate proteins. We identified 461 Kmal sites showing more than a 2-fold increase in response to MCD deficiency as well as 1452 Kmal sites detected only in MCD-/- fibroblast but not MCD+/+ cells, suggesting a pathogenic role of Kmal in MCD deficiency. Cells with increased lysine malonylation displayed impaired mitochondrial function and fatty acid oxidation, suggesting that lysine malonylation plays a role in pathophysiology of malonic aciduria. Our study establishes an association between Kmal and a genetic disease and offers a rich resource for elucidating the contribution of the Kmal pathway and malonyl-CoA to cellular physiology and human diseases. PMID:26320211

  2. Preventive effect of a high fluoride toothpaste and arginine-carbonate toothpaste on dentinal tubules exposure followed by acid challenge: a dentine permeability evaluation

    PubMed Central

    2014-01-01

    Background Considering the current high use of high fluoride toothpastes, the aim of the study was to quantify alterations in the root dentine permeability submitted to treatment with a high fluoride toothpaste and 8% arginine, calcium carbonate, sodium monofluorophosphate toothpaste as a preventive treatment for dentinal tubules exposure followed by acid challenge. Methods Thirty-third molars were sectioned below the cementoenamel. The root segments were connected to a hydraulic pressure apparatus to measure dentine permeability after the following sequential steps (n = 10 per group): I) Baseline; II) treatment with phosphoric acid for 30 s (maximum permeability); III) Toothbrushing (1 min) according to the experimental groups (G1- control; G2- 5000 ppm fluoride toothpaste; G3- 8% arginine-calcium carbonate toothpaste); IV) acid challenge for 5 min (orange juice). The data were converted into percentage, considering stage II as 100%. Results The results have shown a statistically significant decreasing on dentine permeability after treatment with toothpaste (Friedman test and Dunn’s post hoc test). Comparison among groups demonstrated a high increasing on dentine permeability when acid challenge was performed after toothbrushing with distilled water (control group) (Kruskal-Wallis and Dunn’s post hoc test). Conclusion The toothpaste treatment may provide sufficient resistance on dentine surface, preventing dentinal tubules exposure after acid challenge. PMID:24958423

  3. Linkages in thermal copolymers of lysine

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Suzuki, F.

    1975-01-01

    The thermal copolymerization of lysine with other alpha-amino acids was studied. The identity of the second amino acid influences various properties of the polymer obtained, including the proportion of alpha and epsilon linkages of lysine. A review of linkages in proteinoids indicates alpha and beta linkages for aspartic acid, alpha and gamma linkages for glutamic acid, alpha and epsilon linkages for lysine, and alpha linkages for other amino acids. Thermal proteinoids are thus more complex in types of linkage than are proteins.

  4. 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. PMID:27003128

  5. 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.

  6. Cationic Amino Acid Uptake Constitutes a Metabolic Regulation Mechanism and Occurs in the Flagellar Pocket of Trypanosoma cruzi

    PubMed Central

    Bouvier, León A.; Cámara, María de los Milagros; Montserrat, Javier; Pereira, Claudio A.

    2012-01-01

    Trypanosomatids' amino acid permeases are key proteins in parasite metabolism since they participate in the adaptation of parasites to different environments. Here, we report that TcAAP3, a member of a Trypanosoma cruzi multigene family of permeases, is a bona fide arginine transporter. Most higher eukaryotic cells incorporate cationic amino acids through a single transporter. In contrast, T. cruzi can recognize and transport cationic amino acids by mono-specific permeases since a 100-fold molar excess of lysine could not affect the arginine transport in parasites that over-express the arginine permease (TcAAP3 epimastigotes). In order to test if the permease activity regulates downstream processes of the arginine metabolism, the expression of the single T. cruzi enzyme that uses arginine as substrate, arginine kinase, was evaluated in TcAAP3 epimastigotes. In this parasite model, intracellular arginine concentration increases 4-folds and ATP level remains constant until cultures reach the stationary phase of growth, with decreases of about 6-folds in respect to the controls. Interestingly, Western Blot analysis demonstrated that arginine kinase is significantly down-regulated during the stationary phase of growth in TcAAP3 epimastigotes. This decrease could represent a compensatory mechanism for the increase in ATP consumption as a consequence of the displacement of the reaction equilibrium of arginine kinase, when the intracellular arginine concentration augments and the glucose from the medium is exhausted. Using immunofluorescence techniques we also determined that TcAAP3 and the specific lysine transporter TcAAP7 co-localize in a specialized region of the plasma membrane named flagellar pocket, staining a single locus close to the flagellar pocket collar. Taken together these data suggest that arginine transport is closely related to arginine metabolism and cell energy balance. The clinical relevance of studying trypanosomatids' permeases relies on the

  7. The same substitution, glutamic acid----lysine at position 501, occurs in three alloalbumins of Asiatic origin: albumins Vancouver, Birmingham, and Adana.

    PubMed Central

    Huss, K; Madison, J; Ishioka, N; Takahashi, N; Arai, K; Putnam, F W

    1988-01-01

    A strategy is described for identifying structural changes in genetic variants of human serum albumin (alloalbumins). By use of this strategy we have determined an amino acid substitution in three alloalbumins of Asiatic origin. The same amino acid exchange, glutamic acid----lysine at position 501, occurs in albumins Vancouver and Birmingham, both from families that migrated from northern India, and also in albumin Adana from Turkey. This exchange corresponds to a single base mutation in the codon GAG to AAG and accords with the slow mobility of the three albumins at pH 8.6. Each of the three alloalbumins had been reported to be a new variant, yet they have the same substitution. These results emphasize the need for structural study of genetic variants that have been differentiated only by nonspecific physical criteria such as dye binding and electrophoretic mobility. We know of no other description of the substitution involved in an alloalbumin originating from the Indian subcontinent. However, the same change of glutamic acid----lysine at position 501 may be present in several other named variants reported for populations in north India and the surrounding regions. Images PMID:2901102

  8. Plasma arginine correlations in trauma and sepsis.

    PubMed

    Chiarla, C; Giovannini, I; Siegel, J H

    2006-02-01

    Arginine (ARG) is an amino acid (AA) with unique properties and with a key-role in the metabolic, immune and reparative response to trauma and sepsis. This study has been performed to characterize the correlations between plasma levels of ARG, of other AA and of multiple metabolic variables in trauma and sepsis. Two-hundred and sixty-three plasma amino-acidograms with a large series of additional biochemical and blood variables were obtained consecutively in 9 trauma patients who developed sepsis, undergoing total parenteral nutrition with dextrose, fat and a mixed AA solution containing 10.4% arginine. ARG was low soon after trauma, then it increased with increasing distance from trauma and with the development of sepsis. ARG was also directly related to the AA infusion rate (AAIR) and for any given AAIR, was lower after trauma than after the development of sepsis. ARG was also related directly to the plasma levels of most of the other AA, the best correlation being that with lysine (r(2) = 0.81, p < 0.001). These correlations were often shifted downwards (showing lower ARG for any given level of the other AA) in measurements performed after trauma, compared to those performed after development of sepsis; this effect was more pronounced for the correlations with branched chain AA. Correlations between ARG and non-AA variables were not particularly relevant. The best simultaneous correlates of ARG, among variables involved in plasma ARG availability, were citrulline level, AAIR and urinary 3-methylhistidine excretion (accounting for the effect of endogenous proteolysis) (multiple r(2) = 0.70, p < 0.001). Plasma ornithine (ORN), the AA more specifically linked to ARG metabolism, correlated with AAIR better than ARG and, for any given AAIR, was lower after trauma than after the development of sepsis. Correlations of ORN with other AA levels were poorer than those found for ARG, however ORN was directly related to white blood cell and platelet count, fibrinogen

  9. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    PubMed

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  10. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    PubMed

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  11. 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

  12. An Arginine to Lysine Mutation in the Vicinity of the Heme Propionates Affects the Redox Potentials of the Hemes and Associated Electron and Proton Transfer in Cytochrome c Oxidase

    PubMed Central

    Mills, Denise A.; Geren, Lois; Hiser, Carrie; Schmidt, Bryan; Durham, Bill; Millett, Francis; Ferguson-Miller, Shelagh

    2008-01-01

    Cytochrome c oxidase pumps protons across a membrane using energy from electron transfer and reduction of oxygen to water. It is postulated that an element of the energy transduction mechanism is the movement of protons to the vicinity of the hemes upon reduction, to favor charge neutrality. Possible sites on which protons could reside, in addition to the conserved carboxylate E286, are the propionate groups of heme a and/or heme a3. A highly conserved pair of arginines (R481/R482) interact with these propionates through ionic and hydrogen bonds. This study shows that the conservative mutant, R481K, although fully as active as wild-type under many conditions, exhibits a significant decrease in the midpoint redox potential of heme a relative to CuA of Em ≅ 40 mV, has lowered activity under conditions of high pH or in the presence of a membrane potential and has a slowed heme a3 reduction with dithionite. Another mutant, D132A, that strongly inhibits proton uptake from the internal side of the membrane, has <4 % the activity of wild-type and appears dependent on proton uptake from the outside. A double mutation, D132A/R481K, is even more strongly inhibited (~1 % wild-type). The more-than-additive effect supports the concept that R481K not only lowers the midpoint potential of heme a but also limits a supply route for protons from the outside of the membrane used by the D132 mutant. The results are consistent with an important role of R481 and heme a/a3 propionates in proton movement in a reversible exit path. PMID:16060654

  13. A combination of both arginine- and lysine-specific gingipain activity of Porphyromonas gingivalis is necessary for the generation of the micro-oxo bishaem-containing pigment from haemoglobin.

    PubMed Central

    Smalley, John W; Thomas, Michael F; Birss, Andrew J; Withnall, Robert; Silver, Jack

    2004-01-01

    The black pigment of Porphyromonas gingivalis is composed of the mu-oxo bishaem complex of Fe(III) protoporphyrin IX (mu-oxo oligomer, dimeric haem), namely [Fe(III)PPIX]2O. P. gingivalis W50 and Rgp (Arg-gingipain)- and Kgp (Lys-gingipain)-deficient mutants K1A, D7, E8 and W501 [Aduse-Opoku, Davies, Gallagher, Hashim, Evans, Rangarajan, Slaney and Curtis (2000) Microbiology 146, 1933-1940] were grown on horse blood/agar for 14 days and examined for the production of mu-oxo bishaem. Mu-oxo Bishaem was detected by UV-visible, Mössbauer and Raman spectroscopies in wild-type W50 and in the black-pigmented RgpA- and RgpB-deficient mutants (W501 and D7 respectively), whereas no haem species were detected in the straw-coloured colonies of Kgp-deficient strain K1A. The dark brown pigment of the double RgpA/RgpB knockout mutant (E8) was not composed of mu-oxo bishaem, but of a high-spin monomeric Fe(III) protoporphyrin IX species (possibly a haem-albumin complex). In vitro incubation of oxyhaemoglobin with cells of the W50 strain and the RgpA- and RgpB-deficient mutants (W501 and D7) resulted in the formation of mu-oxo bishaem via methaemoglobin as an intermediate. Although the Kgp-deficient strain K1A converted oxyhaemoglobin into methaemoglobin, this was not further degraded into mu-oxo bishaem. The double RgpA/RgpB knockout was also not capable of producing mu-oxo bishaem from oxyhaemoglobin, but instead generated a haemoglobin haemichrome. Inhibition of Arg-X protease activity of W50, W501, D7 and K1A with leupeptin, under conditions where Lys-X protease activity was unaffected, prevented the production of mu-oxo bishaem from oxyhaemoglobin, but resulted in the formation of a haemoglobin haemichrome. These results show that one or both of RgpA and RgpB gingipains, in addition to the lysine-specific gingipain, is necessary for the production of mu-oxo bishaem from haemoglobin by whole cells of P. gingivalis. PMID:14741050

  14. [Amino acid composition and quaternary structure of glucosoisomerase (d-xylose-ketol-isomerase) from Actinomyces olivocinereus 154].

    PubMed

    Rezchikov, A A; Ulezlo, I V; Ananichev, A V; Bezborodov, A M

    1980-01-01

    The amino acid composition of glucosoisomerase from Actinomyces olivocinereus 154 was investigated. The content of dicarboxylic acids--aspartic and glutamic--was found to be greater than that of basic acids--lysine, arginine and histidine. Hydrophobic acids were also detected to occur on appreciable quantities. No cysteine was seen in the enzyme. The experimental data on the effect of sodium dodecyl sulfate and urea suggests that the enzyme has a quaternary structure consisting of four nonidentical subunits. PMID:7220510

  15. Amino acids (L-arginine and L-alanine) passivated CdS nanoparticles: Synthesis of spherical hierarchical structure and nonlinear optical properties

    NASA Astrophysics Data System (ADS)

    Talwatkar, S. S.; Tamgadge, Y. S.; Sunatkari, A. L.; Gambhire, A. B.; Muley, G. G.

    2014-12-01

    CdS nanoparticles (NPs) passivated with amino acids (L-alanine and L-arginine) having spherical hierarchical morphology were synthesized by room temperature wet chemical method. Synthesized NPs were characterized by ultraviolet-visible (UV-vis) spectroscopy to study the variation of band gaps with concentration of surface modifying agents. Increase in band gap has been observed with the increase in concentration of surface modifying agents and was found more prominent for CdS NPs passivated with L-alanine. Powder X-ray diffraction (XRD) and transmission electron microscopy (TEM) analysis were carried out for the study of crystal structure and morphology of CdS NPs. The average particle size of CdS NPs calculated from Debye-Scherer formula was found to less than 5 nm and agrees well with those determined from UV-vis spectra and TEM data. Fourier transform infrared (FT-IR) spectroscopy was performed to know the functional groups of the grown NPs. Peaks in FT-IR spectra indicate the formation of CdS NPs and capping with L-alanine and L-arginine. Photoluminescence spectra of these NPs were also studied. Finally, colloidal solution of CdS-PVAc was subjected to Z-scan experiment under low power cw laser illumination to characterize them for third order nonlinear optical properties. CdS-PVAc colloidal solution shows enhanced nonlinear absorption due to RSA and weak FCA on account of two photon absorption processes triggered by thermal effect.

  16. L-arginine

    MedlinePlus

    ... muscle and nervous system problems). There is some interest in using L-arginine to improve symptoms associated ... might also increase potassium in the body. In theory, taking L-arginine along with some "water pills" ...

  17. Lysine and novel hydroxylysine lipids in soil bacteria: amino acid membrane lipid response to temperature and pH in Pseudopedobacter saltans

    PubMed Central

    Moore, Eli K.; Hopmans, Ellen C.; Rijpstra, W. Irene C.; Sánchez-Andrea, Irene; Villanueva, Laura; Wienk, Hans; Schoutsen, Frans; Stams, Alfons J. M.; Sinninghe Damsté, Jaap S.

    2015-01-01

    Microbial decomposition of organic matter is an essential process in the global carbon cycle. The soil bacteria Pseudopedobacter saltans and Flavobacterium johnsoniae are both able to degrade complex organic molecules, but it is not fully known how their membrane structures are adapted to their environmental niche. The membrane lipids of these species were extracted and analyzed using high performance liquid chromatography-electrospray ionization/ion trap/mass spectrometry (HPLC-ESI/IT/MS) and high resolution accurate mass/mass spectrometry (HRAM/MS). Abundant unknown intact polar lipids (IPLs) from P. saltans were isolated and further characterized using amino acid analysis and two dimensional nuclear magnetic resonance (NMR) spectroscopy. Ornithine IPLs (OLs) with variable (hydroxy) fatty acid composition were observed in both bacterial species. Lysine-containing IPLs (LLs) were also detected in both species and were characterized here for the first time using HPLC-MS. Novel LLs containing hydroxy fatty acids and novel hydroxylysine lipids with variable (hydroxy) fatty acid composition were identified in P. saltans. The confirmation of OL and LL formation in F. johnsoniae and P. saltans and the presence of OlsF putative homologs in P. saltans suggest the OlsF gene coding protein is possibly involved in OL and LL biosynthesis in both species, however, potential pathways of OL and LL hydroxylation in P. saltans are still undetermined. Triplicate cultures of P. saltans were grown at three temperature/pH combinations: 30°C/pH 7, 15°C/pH 7, and 15°C/pH 9. The fractional abundance of total amino acid containing IPLs containing hydroxylated fatty acids was significantly higher at higher temperature, and the fractional abundance of lysine-containing IPLs was significantly higher at lower temperature and higher pH. These results suggest that these amino acid-containing IPLs, including the novel hydroxylysine lipids, could be involved in temperature and pH stress

  18. Estimation of the optimal standardized ileal digestible lysine requirement for primiparous lactating sows fed diets supplemented with crystalline amino acids.

    PubMed

    Shi, Meng; Zang, Jianjun; Li, Zhongchao; Shi, Chuanxin; Liu, Ling; Zhu, Zhengpeng; Li, Defa

    2015-10-01

    This experiment was conducted to determine the optimal standardized ileal digestible lysine (SID Lys) level in diets fed to primiparous sows during lactation. A total of 150 (Landrace × Large White) crossbred gilts (weighing 211.1 ± 3.5 kg with a litter size of 11.1 ± 0.2) were fed lactation diets (3325 kcal metabolizable energy (ME)/kg) containing SID Lys levels of 0.76, 0.84, 0.94, 1.04 or 1.14%, through 28 days lactation. Gilts were allocated to treatments based on their body weight and backfat thickness 48 h after farrowing. Gilt body weight loss was significantly (P < 0.05) decreased by increasing dietary SID Lys levels. Fitted broken-line (P < 0.05) and quadratic plot (P < 0.05) analysis of body weight loss indicated that the optimal SID Lys for primiparous sows was 0.85 and 1.01%, respectively. Average daily feed intake (ADFI), weaning-to-estrus interval and subsequent conception rate were not affected by dietary SID Lys levels. Increasing dietary lysine had no effect on litter performances. Protein content in milk was increased by dietary SID Lys (P < 0.05). Dietary SID Lys tended to increase concentrations of serum insulin-like growth factor I (P = 0.066). These results of this experiment indicate that the optimal dietary SID Lys for lactating gilts was at least 0.85%, which approaches the recommendation of 0.84% that is estimated by the National Research Council (2012).

  19. Lysine catabolism in Rhizoctonia leguminicola and related fungi.

    PubMed Central

    Guengerich, F P; Broquist, H P

    1976-01-01

    The catabolism of lysine was studied in several yeasts and fungi. Results with cell-free extracts of Rhizoctonia leguminicola support a proposed pathway involving (D- and L-) EPSILON-N-acetyllysine, alpha-keto-epsilon-acetamidohexanoic acid, delta-acetamidovaleric acid, and delta-aminovaleric acid in the conversion of L-lysine to shortchain organic acids. Label from radioactive L-lysine was found to accumulate in D- and L-epsilon-N-acetyllysine, delta-acetamidovaleric acid, delta-aminovaleric acid, and glutaric acid in cultures of R. leguminicola, Neurospora crassa, Saccharomyces cerevisiae, and Hansenula saturnus, suggesting that the proposed omega-acetyl pathway of lysine catabolism is generalized among yeasts and fungi. In N. crassa, as is the case in R. leguminicola, the major precursor of L-pipecolic acid was the L-isomer of lysine; 15N experiments were consistent with delta1-piperideine-2-carboxylic acid as an intermediate in the transformation. PMID:131119

  20. Arginine-glycine-aspartic acid-polyethylene glycol-polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture.

    PubMed

    Chen, Zhanfei; Lian, Fen; Wang, Xiaoqian; Chen, Yanling; Tang, Nanhong

    2016-01-01

    The polyamidoamine (PAMAM) dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine-glycine-aspartic acid (RGD) peptide. Our studies demonstrate that RGD-polyethylene glycol (PEG)-PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT-MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system. PMID:27621619

  1. Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1): arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen.

    PubMed

    Chang, Hsin-Hou; Shyu, Huey-Fen; Wang, Yo-Ming; Sun, Der-Shan; Shyu, Rong-Hwa; Tang, Shiao-Shek; Huang, Yao-Shine

    2002-09-15

    Dengue virus infection causes life-threatening hemorrhagic fever. Increasing evidence implies that dengue viral nonstructural protein 1 (NS1) exhibits a tendency to elicit potentially hazardous autoantibodies, which show a wide spectrum of specificity against extracellular matrix and platelet antigens. How NS1 elicits autoantibodies remains unclear. To address the hypothesis that NS1 and matrix proteins may have structural and functional similarity, cell-matrix and cell-NS1 interactions were evaluated using a cell-adhesion assay. The present study showed that dengue NS1 immobilized on coverslips resulted in more cell adhesion than did the control proteins. This cell adhesion was inhibited by peptides containing arginine-glycine-aspartic acid (RGD), a motif important for integrin-mediated cell adhesion. In addition, anti-NS1 antibodies blocked RGD-mediated cell adhesion. Although there is no RGD motif in the NS1 protein sequence, these data indicate that RGD structural mimicry exists within the NS1 antigen.

  2. Effects of acetic acid and arginine on pH elevation and growth of Bacillus licheniformis in an acidified cucumber juice medium.

    PubMed

    Yang, Zhenquan; Meng, Xia; Breidt, Frederick; Dean, Lisa L; Arritt, Fletcher M

    2015-04-01

    Bacillus licheniformis has been shown to cause pH elevation in tomato products having an initial pH below 4.6 and metabiotic effects that can lead to the growth of pathogenic bacteria. Because of this, the organism poses a potential risk to acidified vegetable products; however, little is known about the growth and metabolism of this organism in these products. To clarify the mechanisms of pH change and growth of B. licheniformis in vegetable broth under acidic conditions, a cucumber juice medium representative of a noninhibitory vegetable broth was used to monitor changes in pH, cell growth, and catabolism of sugars and amino acids. For initial pH values between pH 4.1 to 6.0, pH changes resulted from both fermentation of sugar (lowering pH) and ammonia production (raising pH). An initial pH elevation occurred, with starting pH values of pH 4.1 to 4.9 under both aerobic and anaerobic conditions, and was apparently mediated by the arginine deiminase reaction of B. licheniformis. This initial pH elevation was prevented if 5 mM or greater acetic acid was present in the brine at the same pH. In laboratory media, under favorable conditions for growth, data indicated that growth of the organism was inhibited at pH 4.6 with protonated acetic acid concentrations of 10 to 20 mM, corresponding to 25 to 50 mM total acetic acid; however, growth inhibition required greater than 300 mM citric acid (10-fold excess of the amount in processed tomato products) products under similar conditions. The data indicate that growth and pH increase by B. licheniformis may be inhibited by the acetic acid present in most commercial acidified vegetable products but not by the citric acid in many tomato products.

  3. The effect of an arginine-glycine-aspartic acid peptide and hyaluronate synthetic matrix on epithelialization of meshed skin graft interstices.

    PubMed

    Cooper, M L; Hansbrough, J F; Polarek, J W

    1996-01-01

    Keratinocytes and fibroblasts interact with proteins of the extracellular matrix such as fibronectin and vitronectin through RGD (arginine-glycine-aspartic acid) cell-attachment sequences. This study evaluated the ability of a provisional synthetic matrix composed of an RGD peptide and hyaluronic acid to accelerate the epithelialization of the interstices of meshed, human, split-thickness skin when placed on full-thickness wounds of athymic mice. Full-thickness skin defects, sparing the panniculus carnosus, were created on athymic mice and 3:1 meshed, human skin was placed on them. The grafts had four central, isolated interstices, which epithelialized by migration of human keratinocytes. Conditions were either the addition to the wound of the synthetic matrix or a matrix of hyaluronic acid alone. The time to closure of the graft interstices was decreased (p < 0.02) in the wounds treated with the RGD peptide-hyaluronic acid provisional matrix. The resultant epithelium of the closed interstices was significantly thicker 8 days after surgery for the RGD-treated wounds. Basement membrane proteins (laminin and type IV collagen) were also found to be present at the dermoepidermal junction earlier in the RGD-treated wounds. These results imply that use of the RGD peptide conjugate to effect cell-matrix interactions may have clinical significance in the field of wound healing.

  4. Amino acids and immune response: a role for cysteine, glutamine, phenylalanine, tryptophan and arginine in T-cell function and cancer?

    PubMed

    Sikalidis, Angelos K

    2015-01-01

    While proteins are critical for immunity, T-cells constitute a critical component of adaptive immunity by clearing cancerous cells among other abnormal cells. However, cancer cells exhibit a potential to escape T-cell control by employing mechanisms not completely delineated. Interesting work has investigated how certain amino acids affect the proliferation rate of T-cells as well as their effectiveness in clearing tumors. The role of amino acids cysteine, glutamine, phenylalanine, tryptophan and arginine in immunomodulation and particularly regarding T-cell proliferation and activation is discussed. The redox balance is reported to affect T-cell proliferation via modulation of cysteine availability. In addition antigen presenting cells (APCs), similar to myeloid cells determine the availability of amino acids in the extracellular microenvironment affecting T-cell proliferation and activation. A better mechanistic understanding of T-cell function modulation via amino acid signaling or metabolic properties may be helpful towards optimization of adaptive immunity with implications for cancer prognosis and treatment.

  5. Granulocyte functions are independent of arginine availability.

    PubMed

    Kapp, Katharina; Prüfer, Steve; Michel, Christian S; Habermeier, Alice; Luckner-Minden, Claudia; Giese, Thomas; Bomalaski, John; Langhans, Claus-Dieter; Kropf, Pascale; Müller, Ingrid; Closs, Ellen I; Radsak, Markus P; Munder, Markus

    2014-12-01

    Arginine depletion via myeloid cell arginase is critically involved in suppression of the adaptive immune system during cancer or chronic inflammation. On the other hand, arginine depletion is being developed as a novel anti-tumor metabolic strategy to deprive arginine-auxotrophic cancer cells of this amino acid. In human immune cells, arginase is mainly expressed constitutively in PMNs. We therefore purified human primary PMNs from healthy donors and analyzed PMN function as the main innate effector cell and arginase producer in the context of arginine deficiency. We demonstrate that human PMN viability, activation-induced IL-8 synthesis, chemotaxis, phagocytosis, generation of ROS, and fungicidal activity are not impaired by the absence of arginine in vitro. Also, profound pharmacological arginine depletion in vivo via ADI-PEG20 did not inhibit PMN functions in a mouse model of pulmonary invasive aspergillosis; PMN invasion into the lung, activation, and successful PMN-dependent clearance of Aspergillus fumigatus and survival of mice were not impaired. These novel findings add to a better understanding of immunity during inflammation-associated arginine depletion and are also important for the development of therapeutic arginine depletion as anti-metabolic tumor therapy. PMID:25104794

  6. Molecular recognition of arginine by supramolecular complexation with calixarene crown ether based on surface plasmon resonance.

    PubMed

    Chen, Hongxia; Gu, Limin; Yin, Yongmei; Koh, Kwangnak; Lee, Jaebeom

    2011-01-01

    Arginine plays an important role in cell division and the functioning of the immune system. We describe a novel method by which arginine can be identified using an artificial monolayer based on surface plasmon resonance (SPR). The affinity of arginine binding its recognition molecular was compared to that of lysine. In fabrication of an arginine sensing interface, a calix[4]crown ether monolayer was anchored onto a gold surface and then characterized by Fourier Transform infrared reflection absorption spectroscopy, atomic force microscopy, and cyclic voltammetry. The interaction between arginine and its host compound was investigated by SPR. The calix[4]crown ether was found to assemble as a monolayer on the gold surface. Recognition of calix[4]crown monolayer was assessed by the selective binding of arginine. Modification of the SPR chip with the calix[4]crown monolayer provides a reliable and simple experimental platform for investigation of arginine under aqueous conditions.

  7. Bioavailability of lysine in Maillard browned protein as determined by plasma lysine response in rainbow trout (Salmo gairdneri).

    PubMed

    Plakas, S M; Lee, T C; Wolke, R E

    1988-01-01

    The bioavailability of lysine in Maillard browned protein was investigated by plasma lysine response in rainbow trout (Salmo gairdneri). The concentrations of free lysine in the plasma were measured after feeding control and browned protein diets supplemented with graded levels of lysine. Bioavailability of lysine was estimated based on the amounts of supplemental lysine in the diets that resulted in rapid increases in plasma lysine. An approximately 80% loss in bioavailable lysine content was determined by this method in a fish protein isolate subjected to the Maillard browning reaction under mild conditions (40 d incubation at 37 degrees C). The nutritional damage to lysine determined by plasma lysine response was similar to that estimated in vitro by enzymatic hydrolysis and fluorodinitrobenzene reagent, but was underestimated by acid hydrolysis and trinitrobenzene sulfonic acid reagent. Rainbow trout are similar to other animals in their inability to utilize the deoxyketosyl (Amadori) compound of lysine formed in early Maillard reaction, and in their plasma response to dietary levels of essential amino acids. PMID:3121813

  8. Bioavailability of free lysine and protein-bound lysine from casein and fishmeal in juvenile turbot (Psetta maxima).

    PubMed

    Kroeckel, Saskia; Dietz, Carsten; Schulz, Carsten; Susenbeth, Andreas

    2015-03-14

    In the present study, a linear regression analysis between lysine intake and lysine retention was conducted to investigate the efficiency of lysine utilisation (k(Lys)) at marginal lysine intake of either protein-bound or free lysine sources in juvenile turbot (Psetta maxima). For this purpose, nine isonitrogenous and isoenergetic diets were formulated to contain 2·25-4·12 g lysine/100 g crude protein (CP) to ensure that lysine was the first-limiting amino acid in all diets. The basal diet contained 2·25 g lysine/100 g CP. Graded levels of casein (Cas), fishmeal (FM) and L-lysine HCl (Lys) were added to the experimental diets to achieve stepwise lysine increments. A total of 240 fish (initial weight 50·1 g) were hand-fed all the experimental diets once daily until apparent satiation over a period of 56 d. Feed intake was significantly affected by dietary lysine concentration rather than by dietary lysine source. Specific growth rate increased significantly at higher lysine concentrations (P< 0·001). CP, crude lipid and crude ash contents in the whole body were affected by the dietary treatments. The linear regression slope between lysine retention and lysine intake (k(Lys)) was similar between all the dietary lysine sources. The k(Lys) values for the diets supplemented with Cas, Lys or FM were 0·833, 0·857 and 0·684, respectively. The bioavailability of lysine from the respective lysine sources was determined by a slope-ratio approach. The bioavailability of lysine (relative to the reference lysine source Cas) from FM and Lys was 82·1 and 103 %, respectively. Nutrient requirement for maintenance was in the range of 16·7-23·4 mg/kg(0·8) per d, and did not differ between the treatments. There were no significant differences in lysine utilisation efficiency or bioavailability of protein-bound or crystalline lysine from the respective sources observed when lysine was confirmed to be the first-limiting nutrient.

  9. Effect of Maillard browning reaction on protein utilization and plasma amino acid response by rainbow trout (Salmo gairdneri).

    PubMed

    Plakas, S M; Lee, T C; Wolke, R E; Meade, T L

    1985-12-01

    The effect of the Maillard browning reaction in the diet of rainbow trout (Salmo gairdneri) on growth and amino acid availability was investigated. Chemical and enzymatic hydrolysis methods were applied for the detection of the losses of amino acids in a model protein browning system. Arginine and lysine exhibited the greatest losses in the mixture of fish protein isolate and glucose stored for 40 d at 37 degrees C. The apparent digestibility and absorption of individual amino acids, particularly lysine, was lower in trout fed browned protein than in those fed the control protein. Plasma lysine levels were significantly depressed, while the plasma levels of glucose and most other amino acids were elevated in relation to the loss in nutritive value of dietary protein after browning. The early Maillard reaction derivative of lysine, epsilon-deoxy-fructosyl-lysine, was recovered from browned protein (by using the in vitro enzymatic hydrolysis procedure) and from the plasma of trout fed browned protein. Analysis of plasma free amino acids provided an indication of lysine bioavailability and identified lysine as the first-limiting amino acid in the diets containing browned protein. PMID:3934350

  10. Enzymatic Modification of Soluble Cyanophycin Using the Type II Peptidyl Arginine Deiminase from Oryctolagus cuniculus.

    PubMed

    Wiefel, Lars; Steinbüchel, Alexander

    2016-07-01

    An increased structural variety expands the number of putative applications for cyanophycin (multi-l-arginyl-poly-[l-aspartic acid], CGP). Therefore, structural modifications of CGP are of major interest; these are commonly obtained by modification and optimization of the bacterial producing strain or by chemical modification. In this study, an enzymatic modification of arginine side chains from lysine-rich CGP is demonstrated using the peptidyl arginine deiminase from Oryctolagus cuniculus, purified from Escherichia coli after heterologous expression. About 10% of the arginine side chains are converted to citrulline which corresponds to 4% of the polymer's total side chains. An inhibition of the reaction in the presence of small amounts of l-citrulline is observed, thereby explaining the low conversion rate. CGP dipeptides can be modified with about 7.5 mol% of the Asp-Arg dipeptides being converted to Asp-Cit. These results show that the enzymatic modification of CGP is feasible, opening up a whole new area of possible CGP modifications for further research. PMID:26953800

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

    PubMed Central

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

    1996-01-01

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

  12. Detection of non-protein amino acids in the presence of protein amino acids. II.

    NASA Technical Reports Server (NTRS)

    Shapshak, P.; Okaji, M.

    1972-01-01

    Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

  13. Exogenous arginine in sepsis.

    PubMed

    Luiking, Yvette C; Deutz, Nicolaas E P

    2007-09-01

    Sepsis is a severe condition in critically ill patients and is considered an arginine deficiency state. The rationale for arginine deficiency in sepsis is mainly based on the reduced arginine levels in sepsis that are associated with the specific changes in arginine metabolism related to endothelial dysfunction, severe catabolism, and worse outcome. Exogenous arginine supplementation in sepsis shows controversial results with only limited data in humans and variable results in animal models of sepsis. Since in these studies the severity of sepsis varies but also the route, timing, and dose of arginine, it is difficult to draw a definitive conclusion for sepsis in general without considering the influence of these factors. Enhanced nitric oxide production in sepsis is related to suggested detrimental effects on hemodynamic instability and enhanced oxidative stress. Potential mechanisms for beneficial effects of exogenous arginine in sepsis include enhanced (protein) metabolism, improved microcirculation and organ function, effects on immune function and antibacterial effects, improved gut function, and an antioxidant role of arginine. We recently performed a study indicating that arginine can be given to septic patients without major effects on hemodynamics, suggesting that more studies can be conducted on the effects of arginine supplementation in septic patients.

  14. Genetic identification of ACC-RESISTANT2 reveals involvement of LYSINE HISTIDINE TRANSPORTER1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in Arabidopsis thaliana.

    PubMed

    Shin, Kihye; Lee, Sumin; Song, Won-Yong; Lee, Rin-A; Lee, Inhye; Ha, Kyungsun; Koo, Ja-Choon; Park, Soon-Ki; Nam, Hong-Gil; Lee, Youngsook; Soh, Moon-Soo

    2015-03-01

    1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene, a gaseous plant hormone which controls a myriad of aspects of development and stress adaptation in higher plants. Here, we identified a mutant in Arabidopsis thaliana, designated as ACC-resistant2 (are2), displaying a dose-dependent resistance to exogenously applied ACC. Physiological analyses revealed that mutation of are2 impaired various aspects of exogenous ACC-induced ethylene responses, while not affecting sensitivity to other plant hormones during seedling development. Interestingly, the are2 mutant was normally sensitive to gaseous ethylene, compared with the wild type. Double mutant analysis showed that the ethylene-overproducing mutations, eto1 or eto3, and the constitutive ethylene signaling mutation, ctr1 were epistatic to the are2 mutation. These results suggest that the are2 mutant is not defective in ethylene biosynthesis or ethylene signaling per se. Map-based cloning of ARE2 demonstrated that LYSINE HISTIDINE TRANSPORTER1 (LHT1), encoding an amino acid transporter, is the gene responsible. An uptake experiment with radiolabeled ACC indicated that mutations of LHT1 reduced, albeit not completely, uptake of ACC. Further, we performed an amino acid competition assay and found that two amino acids, alanine and glycine, known as substrates of LHT1, could suppress the ACC-induced triple response in a LHT1-dependent way. Taken together, these results provide the first molecular genetic evidence supporting that a class of amino acid transporters including LHT1 takes part in transport of ACC, thereby influencing exogenous ACC-induced ethylene responses in A. thaliana. PMID:25520403

  15. Arginine and citrulline and the immune response in sepsis.

    PubMed

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

    2015-02-18

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

  16. Arginine and Citrulline and the Immune Response in Sepsis

    PubMed Central

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

    2015-01-01

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

  17. Diminished L-arginine bioavailability in hypertension.

    PubMed

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

    2004-10-01

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

  18. Diminished L-arginine bioavailability in hypertension.

    PubMed

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

    2004-10-01

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

  19. Differential trace labeling of calmodulin: investigation of binding sites and conformational states by individual lysine reactivities. Effects of beta-endorphin, trifluoperazine, and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid

    SciTech Connect

    Giedroc, D.P.; Sinha, S.K.; Brew, K.; Puett, D.

    1985-11-05

    The CaS -dependent association of beta-endorphin and trifluoperazine with porcine testis calmodulin, as well as the effects of removing CaS by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) treatment, were investigated by the procedure of differential kinetic labeling. This technique permitted determination of the relative rates of acylation of each of the epsilon-amino groups of the seven lysyl residues on calmodulin by (TH)acetic anhydride under the different conditions. In all cases, less than 0.52 mol of lysyl residue/mol of calmodulin was modified, thus ensuring that the labeling pattern reflects the microenvironments of these groups in the native protein. Lysines 75 and 94 were found to be the most reactive amino groups in CaS -saturated calmodulin. In the presence of CaS and under conditions where beta-endorphin and calmodulin were present at a molar ratio of 2.5:1, the amino groups of lysines 75 and 148 were significantly reduced in reactivity compared to calmodulin alone. At equimolar concentrations of peptides and proteins, essentially the same result was obtained except that the magnitudes of the perturbation of these two lysines were less pronounced. With trifluoperazine, at a molar ratio to calmodulin of 2.5:1, significant perturbations of lysines 75 and 148, as well as Lys 77, were also found. These results further substantiate previous observations of a commonality between phenothiazine and peptide binding sites on calmodulin. Lastly, an intriguing difference in CaS -mediated reactivities between lysines 75 and 77 of calmodulin is demonstrated. In the CaS -saturated form of the protein, both lysines are part of the long connecting helix between the two homologous halves of the protein.

  20. Economical production of poly(ε-l-lysine) and poly(l-diaminopropionic acid) using cane molasses and hydrolysate of streptomyces cells by Streptomyces albulus PD-1.

    PubMed

    Xia, Jun; Xu, Zhaoxian; Xu, Hong; Liang, Jinfeng; Li, Sha; Feng, Xiaohai

    2014-07-01

    Poly(ε-L-lysine) (ε-PL) and poly(L-diaminopropionic acid) (PDAP) co-production by Streptomyces albulus PD-1 from cane molasses and hydrolysate of strepyomyces cells (HSC) was investigated for the first time in this study. The optimal initial total sugar concentration of the cane molasses pretreated with sulfuric acid was determined to be 20 g L(-1), and HSC could substitute for yeast extract for ε-PL and PDAP co-production. When fed-batch fermentation was performed in 1t fermentor with pretreated cane molasses and HSC, 20.6 ± 0.5 g L(-1) of ε-PL and 5.2 ± 0.6 g L(-1) of PDAP were obtained. The amount of strepyomyces cells obtained in one fed-batch fermentation is sufficient to prepare the HSC to satisfy the demand of subsequent fermentations, thus the self-cycling of organic nitrogen source becomes available. These results suggest that the low-cost cane molasses and HSC can be used for the economical production of ε-PL and PDAP by S. albulus PD-1.

  1. The role of arginine in infection and sepsis.

    PubMed

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

    2005-01-01

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

  2. Influence of betaine and arginine supplementation of reduced protein diets on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of cross-bred pigs.

    PubMed

    Madeira, Marta S; Rolo, Eva S; Alfaia, Cristina M; Pires, Virgínia R; Luxton, Richard; Doran, Olena; Bessa, Rui J B; Prates, José A M

    2016-03-28

    The isolated or combined effects of betaine and arginine supplementation of reduced protein diets (RPD) on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig m. longissimus lumborum and subcutaneous adipose tissue (SAT) were assessed. The experiment was performed on forty intact male pigs (Duroc×Large White×Landrace cross-breed) with initial and final live weights of 60 and 93 kg, respectively. Pigs were randomly assigned to one of the following five diets (n 8): 16·0 % of crude protein (control), 13·0 % of crude protein (RPD), RPD supplemented with 0·33 % of betaine, RPD supplemented with 1·5 % of arginine and RPD supplemented with 0·33 % of betaine and 1·5 % of arginine. Data confirmed that RPD increase intramuscular fat (IMF) content and total fat content in SAT. The increased total fat content in SAT was accompanied by higher GLUT type 4, lipoprotein lipase and stearoyl-CoA desaturase mRNA expression levels. In addition, the supplementation of RPD with betaine and/or arginine did not affect either IMF or total fat in SAT. However, dietary betaine supplementation slightly affected fatty acid composition in both muscle and SAT. This effect was associated with an increase of carnitine O-acetyltransferase mRNA levels in SAT but not in muscle, which suggests that betaine might be involved in the differential regulation of some key genes of lipid metabolism in pig muscle and SAT. Although the arginine-supplemented diet decreased the mRNA expression level of PPARG in muscle and SAT, it did not influence fat content or fatty acid composition in any of these pig tissues.

  3. Effects of glycine-arginine-α-ketoisocaproic acid supplementation in college-age trained females during multi-bouts of resistance exercise.

    PubMed

    Wax, Benjamin; Hilton, Laura; Vickers, Brad; Gilliland, Katherine; Conrad, Mandy

    2013-03-01

    Glycine-arginine-α-ketoisocaproic acid (GAKIC) has been proposed to increase anaerobic high-intensity exercise performance in male subjects. However, the effects of GAKIC ingestion in female subjects have not been studied. Therefore, the purpose of this study was to investigate the effects of GAKIC supplementation on total load volume (i.e., mass lifted) and metabolic parameters during repeated bouts of submaximal leg extensions in college-age females. Nine resistance-trained females participated in a randomized, counterbalanced, double blind study. Subjects were randomly assigned to placebo or GAKIC (10.2 g) and performed six sets of 50% of one repetition maximum leg extensions (two legs simultaneously) to failure. One week later, subjects ingested the other supplement and performed the same exercise protocol. Furthermore, blood lactic acid, blood glucose, and heart rate were also measured preexercise and 5 s after the completion of the exercise protocol (postexercise). GAKIC supplementation significantly increased leg extension total load volume (GAKIC = 1721.7 ± 479.9 kg; placebo = 1479.1 ± 396.8 kg, p < .01). Heart rate and blood lactic acid were significantly increased (p < .01 for both measures) postexercise compared to preexercise, but were not significantly different between GAKIC and placebo (p = .40 for heart rate; p = .88 for lactic acid). Blood glucose was significantly decreased (p = .03) postexercise compared to preexercise, but was not significantly different (p = .78) between GAKIC and placebo. Collectively, these findings suggest that GAKIC increased lower body resistance performance in trained college-age females; however, these findings are not necessarily generalizable.

  4. Bacterial Lysine Decarboxylase Influences Human Dental Biofilm Lysine Content, Biofilm Accumulation and Sub-Clinical Gingival Inflammation

    PubMed Central

    Lohinai, Z.; Keremi, B.; Szoko, E.; Tabi, T.; Szabo, C.; Tulassay, Z.; Levine, M.

    2012-01-01

    Background Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study were to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR), and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for a week. Methods Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm and saliva before OHR and in dental biofilm after OHR. Results Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After a week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine post-OHR, unless biofilm lysine exceeded the minimal blood plasma content in which case PI was further increased but GCF exudation was reduced. Conclusions After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Clinical Relevance Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis. PMID:22141361

  5. Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases.

    PubMed

    Duncan, Anna L; Robinson, Alan J; Walker, John E

    2016-08-01

    The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme. PMID:27382158

  6. Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases

    PubMed Central

    Duncan, Anna L.

    2016-01-01

    The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme. PMID:27382158

  7. The Arabidopsis LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE3 Regulates the Cross Talk between Immunity and Abscisic Acid Responses1[W][OPEN

    PubMed Central

    Paparella, Chiara; Savatin, Daniel Valentin; Marti, Lucia; De Lorenzo, Giulia; Ferrari, Simone

    2014-01-01

    Transmembrane receptor-like kinases characterized by the presence of one or more lysin motif (LysM) domains in the extracytoplasmic portion (LysM-containing receptor-like kinases [LYKs]) mediate recognition of symbiotic and pathogenic microorganisms in plants. The Arabidopsis (Arabidopsis thaliana) genome encodes five putative LYKs; among them, AtLYK1/CHITIN ELICITOR RECEPTOR KINASE1 is required for response to chitin and peptidoglycan, and AtLYK4 contributes to chitin perception. More recently, AtLYK3 has been shown to be required for full repression, mediated by Nod factors, of Arabidopsis innate immune responses. In this work, we show that AtLYK3 also negatively regulates basal expression of defense genes and resistance to Botrytis cinerea and Pectobacterium carotovorum infection. Enhanced resistance of atlyk3 mutants requires PHYTOALEXIN-DEFICIENT3, which is crucial for camalexin biosynthesis. The expression of AtLYK3 is strongly repressed by elicitors and fungal infection and is induced by the hormone abscisic acid (ABA), which has a negative impact on resistance against B. cinerea and P. carotovorum. Plants lacking a functional AtLYK3 also show reduced physiological responses to ABA and are partially resistant to ABA-induced inhibition of PHYTOALEXIN-DEFICIENT3 expression. These results indicate that AtLYK3 is important for the cross talk between signaling pathways activated by ABA and pathogens. PMID:24639336

  8. Biocompatibility Assessment of Polyethylene Glycol-Poly L-Lysine-Poly Lactic-Co-Glycolic Acid Nanoparticles In Vitro and In Vivo.

    PubMed

    Guo, Liting; Chen, Baoan; Liu, Ran; Xia, Guohua; Wang, Yonglu; Li, Xueming; Wei, Chen; Wang, Xuemei; Jiang, Hulin

    2015-05-01

    The present study was designed to evaluate the biocompatibility of nanoparticles polyethylene glycol (PEG)-poly L-lysine (PLL)-poly lactic-co-glycolic acid copolymer (PLGA) (PEG-PLL-PLGA) before clinical application. We applied some tests to assess the safety of PEG-PLL-PLGA nanoparticles (NPs). There was low cytotoxicity of PEG-PLL-PLGA NPs in vitro as detected by MTT assay. Cell apoptosis and intracellular accumulation of PEG-PLL-PLGA were determined by FCM assay. The apoptotic rate induced by nanoparticles and the fluorescence intensity of intracellular daunorubicin (DNR) demonstrated that DNR-PEG-PLL-PLGA could be taken up by the mouse fibroblast cells (L929 cells). Hemolysis test and micronucleus (MN) assay demonstrated that the nanoparticles have no obviously blood toxicity and genotoxicity. DNR-PEG-PLL-PLGA NPs were injected into mice through tail vein to calculate the median lethal dose (LD50), the results showed that they had a wide safe scale. Blood was taken by removing the eyeball of mice to study the influence of DNR-PEG-PLL-PLGA in hepatic and renal functions. The results revealed that there was no significant difference as compared with the control group. Interestingly, the pathologic changes of heart, liver, spleen, lung and kidney were observed in nanoparticles treated mice. Thus, this study demonstrates that PEG-PLL-PLGA NPs appear to be highly biocompatible and safe nanoparticles that can be suitable for further application in the treatment of tumor.

  9. Influence of assembling pH on the stability of poly(L-glutamic acid) and poly(L-lysine) multilayers against urea treatment.

    PubMed

    Zhou, Jie; Wang, Bo; Tong, Weijun; Maltseva, Elena; Zhang, Gang; Krastev, Rumen; Gao, Changyou; Möhwald, Helmuth; Shen, Jiacong

    2008-04-01

    Polyelectrolyte multilayers of poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) were built up using the layer-by-layer (LbL) technique in low pH (3.6, PM3.6) and in neutral pH (7.4, PM7.4) solutions. The multilayers were then treated with a concentrated urea (one kind of denaturant for proteins and polypeptides) solution (8M) and rinsed with corresponding buffer. The buildup and treatment processes were investigated by ultraviolet visible spectroscopy and ellipsometry. The surface morphology was observed by scanning force microscopy (SFM). The inner structures were determined by X-ray reflectometry and circular dichroism spectroscopy (CD). An exponential growth of the optical mass and the layer thickness was observed for both PM3.6 and PM7.4. After urea treatment, a significant mass loss for PM3.6 was found, while no mass change was recorded for PM7.4. The dominant driving force for PM7.4 is electrostatic interaction, resulting in multilayers with an abundant beta-sheet structure, which has higher stability against urea treatment. By contrast, the dominant driving force for PM3.6 is hydrogen bonding and hydrophobic interaction, which are sensitive to the urea treatment. The mechanism is substantiated by molecular mechanics calculation. This has offered a convenient pathway to mediate the multilayer properties, which is of great importance for potential applications.

  10. The Arabidopsis LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE3 regulates the cross talk between immunity and abscisic acid responses.

    PubMed

    Paparella, Chiara; Savatin, Daniel Valentin; Marti, Lucia; De Lorenzo, Giulia; Ferrari, Simone

    2014-05-01

    Transmembrane receptor-like kinases characterized by the presence of one or more lysin motif (LysM) domains in the extracytoplasmic portion (LysM-containing receptor-like kinases [LYKs]) mediate recognition of symbiotic and pathogenic microorganisms in plants. The Arabidopsis (Arabidopsis thaliana) genome encodes five putative LYKs; among them, AtLYK1/CHITIN ELICITOR RECEPTOR KINASE1 is required for response to chitin and peptidoglycan, and AtLYK4 contributes to chitin perception. More recently, AtLYK3 has been shown to be required for full repression, mediated by Nod factors, of Arabidopsis innate immune responses. In this work, we show that AtLYK3 also negatively regulates basal expression of defense genes and resistance to Botrytis cinerea and Pectobacterium carotovorum infection. Enhanced resistance of atlyk3 mutants requires PHYTOALEXIN-DEFICIENT3, which is crucial for camalexin biosynthesis. The expression of AtLYK3 is strongly repressed by elicitors and fungal infection and is induced by the hormone abscisic acid (ABA), which has a negative impact on resistance against B. cinerea and P. carotovorum. Plants lacking a functional AtLYK3 also show reduced physiological responses to ABA and are partially resistant to ABA-induced inhibition of PHYTOALEXIN-DEFICIENT3 expression. These results indicate that AtLYK3 is important for the cross talk between signaling pathways activated by ABA and pathogens.

  11. Role of arginine 285 in the active site of Rhodotorula gracilis D-amino acid oxidase. A site-directed mutagenesis study.

    PubMed

    Molla, G; Porrini, D; Job, V; Motteran, L; Vegezzi, C; Campaner, S; Pilone, M S; Pollegioni, L

    2000-08-11

    Arg(285), one of the very few conserved residues in the active site of d-amino acid oxidases, has been mutated to lysine, glutamine, aspartate, and alanine in the enzyme from the yeast Rhodotorula gracilis (RgDAAO). The mutated proteins are all catalytically competent. Mutations of Arg(285) result in an increase ( approximately 300-fold) of K(m) for the d-amino acid and in a large decrease ( approximately 500-fold) of turnover number. Stopped-flow analysis shows that the decrease in turnover is paralleled by a similar decrease in the rate of flavin reduction (k(2)), the latter still being the rate-limiting step of the reaction. In agreement with data from the protein crystal structure, loss of the guanidinium group of Arg(285) in the mutated DAAOs drastically reduces the binding of several carboxylic acids (e.g. benzoate). These results highlight the importance of this active site residue in the precise substrate orientation, a main factor in this redox reaction. Furthermore, Arg(285) DAAO mutants have spectral properties similar to those of the wild-type enzyme, but show a low degree of stabilization of the flavin semiquinone and a change in the redox properties of the free enzyme. From this, we can unexpectedly conclude that Arg(285) in the free enzyme form is involved in the stabilization of the negative charge on the N(1)-C(2)=O locus of the isoalloxazine ring of the flavin. We also suggest that the residue undergoes a conformational change in order to bind the carboxylate portion of the substrate/ligand in the complexed enzyme. PMID:10821840

  12. Copper utilization in humans as affected by amino acid supplements

    SciTech Connect

    Kies, C.; Chuang, J.H.; Fox, H.M. )

    1989-02-09

    Earlier work suggests that absorption of copper as well as several other mineral nutrients may be promoted, inhibited or unaffected by the formation of mineral-amino acid complexes. The objective of the current project was to determine effects of low level supplements of selected amino acids on copper utilization. In a series of studies, healthy, human adult subjected received a basal diet with or without test supplements in separate 14-day periods which were arranged according to a randomized, cross-over design. Test amino acids and amounts given per subject per day were as follows; L-arginine, 1.2 g; L-lysine, 1.0 g; L-cystine, 1.0 g and L-methionine, 1.0 g. Subjects made complete collections of urine and stools. Fasting blood samples were drawn. Food, urine, feces and blood were analyzed for copper contents using a carbon rod attachment on a Varian atomic absorption spectrophotometer. Fecal copper losses were unaffected by used of lysine, tryptophan and methionine supplements but were reduced with use of the arginine and cystine supplements. Urine losses of copper were reduced with used of the lysine and tryptophan supplements, were increased with the methionine and cystine supplements and were unaffected when the arginine supplements were employed. Blood serum copper levels were not significantly affected by use of these supplement although some trends were noted.

  13. Acid-base interactions and secondary structures of poly-L-lysine probed by 15N and 13C solid state NMR and Ab initio model calculations.

    PubMed

    Dos, Alexandra; Schimming, Volkmar; Tosoni, Sergio; Limbach, Hans-Heinrich

    2008-12-11

    The interactions of the 15N-labeled amino groups of dry solid poly-L-lysine (PLL) with various halogen and oxygen acids HX and the relation to the secondary structure have been studied using solid-state 15N and 13C CPMAS NMR spectroscopy (CP = cross polarization and MAS = magic angle spinning). For comparison, 15N NMR spectra of an aqueous solution of PLL were measured as a function of pH. In order to understand the effects of protonation and hydration on the 15N chemical shifts of the amino groups, DFT and chemical shielding calculations were performed on isolated methylamine-acid complexes and on periodic halide clusters of the type (CH3NH3(+)X(-))n. The combined experimental and computational results reveal low-field shifts of the amino nitrogens upon interaction with the oxygen acids HX = HF, H2SO4, CH3COOH, (CH3)2POOH, H3PO4, HNO3, and internal carbamic acid formed by reaction of the amino groups with gaseous CO2. Evidence is obtained that only hydrogen-bonded species of the type (Lys-NH2***H-X)n are formed in the absence of water. 15N chemical shifts are maximum when H is located in the hydrogen bond center and then decrease again upon full protonation, as found for aqueous solution at low pH. By contrast, halogen acids interact in a different way. They form internal salts of the type (Lys-NH3(+)X(-))n via the interaction of many acid-base pairs. This salt formation is possible only in the beta-sheet conformation. By contrast, the formation of hydrogen-bonded complexes can occur both in beta-sheet domains as well as in alpha-helical domains. The 15N chemical shifts of the protonated ammonium groups increase when the size of the interacting halogen anions is increased from chloride to iodide and when the number of the interacting anions is increased. Thus, the observed high-field 15N shift of ammonium groups upon hydration is the consequence of replacing interacting halogen atoms by oxygen atoms.

  14. 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.

  15. Accumulation, selection and covariation of amino acids in sieve tube sap of tansy (Tanacetum vulgare) and castor bean (Ricinus communis): evidence for the function of a basic amino acid transporter and the absence of a γ-amino butyric acid transporter.

    PubMed

    Bauer, Susanne N; Nowak, Heike; Keller, Frank; Kallarackal, Jose; Hajirezaei, Mohamad-Reza; Komor, Ewald

    2014-09-01

    Sieve tube sap was obtained from Tanacetum by aphid stylectomy and from Ricinus after apical bud decapitation. The amino acids in sieve tube sap were analyzed and compared with those from leaves. Arginine and lysine accumulated in the sieve tube sap of Tanacetum more than 10-fold compared to the leaf extracts and they were, together with asparagine and serine, preferably selected into the sieve tube sap, whereas glycine, methionine/tryptophan and γ-amino butyric acid were partially or completely excluded. The two basic amino acids also showed a close covariation in sieve tube sap. The acidic amino acids also grouped together, but antagonistic to the other amino acids. The accumulation ratios between sieve tube sap and leaf extracts were smaller in Ricinus than in Tanacetum. Arginine, histidine, lysine and glutamine were enriched and preferentially loaded into the phloem, together with isoleucine and valine. In contrast, glycine and methionine/tryptophan were partially and γ-amino butyric acid almost completely excluded from sieve tube sap. The covariation analysis grouped arginine together with several neutral amino acids. The acidic amino acids were loaded under competition with neutral amino acids. It is concluded from comparison with the substrate specificities of already characterized plant amino acid transporters, that an AtCAT1-like transporter functions in phloem loading of basic amino acids, whereas a transporter like AtGAT1 is absent in phloem. Although Tanacetum and Ricinus have different minor vein architecture, their phloem loading specificities for amino acids are relatively similar.

  16. 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. PMID:25896651

  17. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors

    PubMed Central

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  18. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

    PubMed

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  19. Effects of a subconvulsive dose of kainic acid on the gene expressions of the arginine vasopressin, oxytocin and neuronal nitric oxide synthase in the rat hypothalamus.

    PubMed

    Yoshimura, Mitsuhiro; Ohkubo, Jun-ichi; Hashimoto, Hirofumi; Matsuura, Takanori; Maruyama, Takashi; Onaka, Tatsushi; Suzuki, Hideaki; Ueta, Yoichi

    2015-10-01

    Arginine vasopressin (AVP) synthesis in the hypothalamo-neurohypophysial system (HNS) is up-regulated by kainic acid (KA)-induced seizure in rats. However, it remains unknown whether a subconvulsive dose of KA affects the HNS. Here we examined the effects of subcutaneous (s.c.) administration of a low dose of KA (4 mg/kg) on the gene expressions of the AVP, oxytocin (OXT) and neuronal nitric oxide synthase (nNOS) in the supraoptic (SON) and paraventricular nuclei (PVN) of the rat hypothalamus, using in situ hybridization histochemistry. The expression of the AVP gene in the SON and PVN was judged to be up-regulated in KA-treated rats in comparison with saline-treated rats as controls. Next, the expression of the OXT gene was significantly increased in the SON at 6-24h and in the PVN at 6 and 12h after s.c. administration of KA. Finally, the expression of the nNOS gene was significantly increased in the SON and PVN at 3 and 6h after s.c. administration of KA. These results suggest that up-regulation of the gene expressions of the AVP, OXT and nNOS in the rat hypothalamus may be differentially affected by peripheral administration of a subconvulsive dose of KA.

  20. Biochemical constraints in a protobiotic earth devoid of basic amino acids: the "BAA(-) world".

    PubMed

    McDonald, Gene D; Storrie-Lombardi, Michael C

    2010-12-01

    It has been hypothesized in this journal and elsewhere, based on surveys of published data from prebiotic synthesis experiments and carbonaceous meteorite analyses, that basic amino acids such as lysine and arginine were not abundant on prebiotic Earth. If the basic amino acids were incorporated only rarely into the first peptides formed in that environment, it is important to understand what protobiotic chemistry is possible in their absence. As an initial test of the hypothesis that basic amino acid negative [BAA(-)] proteins could have performed at least a subset of protobiotic chemistry, the current work reports on a survey of 13 archaeal and 13 bacterial genomes that has identified 61 modern gene sequences coding for known or putative proteins not containing arginine or lysine. Eleven of the sequences found code for proteins whose functions are well known and important in the biochemistry of modern microbial life: lysine biosynthesis protein LysW, arginine cluster proteins, copper ion binding proteins, bacterial flagellar proteins, and PE or PPE family proteins. These data indicate that the lack of basic amino acids does not prevent peptides or proteins from serving useful structural and biochemical functions. However, as would be predicted from fundamental physicochemical principles, we see no fossil evidence of prebiotic BAA(-) peptide sequences capable of interacting directly with nucleic acids.

  1. Biochemical Constraints in a Protobiotic Earth Devoid of Basic Amino Acids: The "BAA(-) World"

    NASA Astrophysics Data System (ADS)

    McDonald, Gene D.; Storrie-Lombardi, Michael C.

    2010-12-01

    It has been hypothesized in this journal and elsewhere, based on surveys of published data from prebiotic synthesis experiments and carbonaceous meteorite analyses, that basic amino acids such as lysine and arginine were not abundant on prebiotic Earth. If the basic amino acids were incorporated only rarely into the first peptides formed in that environment, it is important to understand what protobiotic chemistry is possible in their absence. As an initial test of the hypothesis that basic amino acid negative [BAA(-)] proteins could have performed at least a subset of protobiotic chemistry, the current work reports on a survey of 13 archaeal and 13 bacterial genomes that has identified 61 modern gene sequences coding for known or putative proteins not containing arginine or lysine. Eleven of the sequences found code for proteins whose functions are well known and important in the biochemistry of modern microbial life: lysine biosynthesis protein LysW, arginine cluster proteins, copper ion binding proteins, bacterial flagellar proteins, and PE or PPE family proteins. These data indicate that the lack of basic amino acids does not prevent peptides or proteins from serving useful structural and biochemical functions. However, as would be predicted from fundamental physicochemical principles, we see no fossil evidence of prebiotic BAA(-) peptide sequences capable of interacting directly with nucleic acids.

  2. Effect of acidity on the equilibria of formation of mixed Co2+ complexes with heparin and arginine in aqueous solutions at 37°C

    NASA Astrophysics Data System (ADS)

    Feofanova, M. A.; Frantseva, Yu. V.; Semenov, A. N.; Baranova, N. V.; Zhuravlev, E. V.

    2014-02-01

    Results from studying interactions in the heparin-Co2+ ion-arginine system are presented. The constants of formation of mixed Co2+ complexes with heparin and arginine in aqueous solutions in a broad pH range at 37°C are determined potentiometrically. The chemical equilibria in the system are simulated and the stoichiometry of formation of the complex forms is determined.

  3. Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae.

    PubMed

    Kumar, Vidya Prasanna; Thomas, Leonard M; Bobyk, Kostyantyn D; Andi, Babak; Cook, Paul F; West, Ann H

    2012-01-31

    Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α-aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-kg) using NAD⁺ as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 Å resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 Å, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E(t) and >10³-fold increases in V₂/K(Lys)E(t) and V₂/K(α-kg)E(t) (the double mutation gives >10⁵-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V₂/K(Lys). A value of 2.0 was also observed for (D)(V₂/K(Lys))(D₂O) when the primary deuterium kinetic isotope effect was repeated in D₂O, consistent with a

  4. Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae.

    PubMed

    Kumar, Vidya Prasanna; Thomas, Leonard M; Bobyk, Kostyantyn D; Andi, Babak; Cook, Paul F; West, Ann H

    2012-01-31

    Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α-aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-kg) using NAD⁺ as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 Å resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 Å, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E(t) and >10³-fold increases in V₂/K(Lys)E(t) and V₂/K(α-kg)E(t) (the double mutation gives >10⁵-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V₂/K(Lys). A value of 2.0 was also observed for (D)(V₂/K(Lys))(D₂O) when the primary deuterium kinetic isotope effect was repeated in D₂O, consistent with a

  5. Thermodynamics of Ion Pair Formations Between Charged Poly(Amino Acid)s.

    PubMed

    Petrauskas, Vytautas; Maximowitsch, Eglė; Matulis, Daumantas

    2015-09-17

    Electrostatic interactions between the positively and negatively charged amino acids in proteins play an important role in macromolecular stability, binding, and recognition. Numerous amino acids in proteins are ionizable and may exist in negatively (e.g., Glu, Asp, Cys, Tyr) or positively (e.g., Arg, Lys, His, Orn) charged form dependent on pH and their pKas. In this work, isothermal titration calorimetry was used to determine the average standard values of thermodynamic parameters (the Gibbs free energy, enthalpy, entropy, and the heat capacity) of interaction between the positively charged amino acid homopolymers (polyarginine, polylysine, and polyornithine) and the negatively charged homopolymers (polyaspartic and polyglutamic acids). These values are of potential use in the computational models of interacting proteins and other biological macromolecules. The study showed that oppositely charged poly(amino acid)s bound each other with the stoichiometry of one positive to one negative charge. Arginine bound to the negatively charged amino acids with exothermic enthalpy and higher affinity than lysine. This result also suggests that positive charges in proteins should not be considered entirely equivalent if carried by lysine or arginine. The difference in binding energy of arginine and lysine association with the negatively charged amino acids was attributed to the enthalpy of the second ionic hydrogen bond formation between the guanidine and carboxylic groups. Despite the favorable enthalpic contribution, all such ion pair formation reactions were largely entropy-driven. Consistent with previously observed ionic interactions, the positive heat capacity was always observed during the amino acid ion pair formation.

  6. Isolation, structural characterization and biological function of a lysine-conopressin in the central nervous system of the pharyngobdellid leech Erpobdella octoculata.

    PubMed

    Salzet, M; Bulet, P; Van Dorsselaer, A; Malecha, J

    1993-11-01

    Several neuropeptides are suspected to act on the control of hydric balance in leeches. One of these peptides, a peptide immunoreactive to an antibody against oxytocin, was previously characterized from the central nervous system of the leech Erpobdella octoculata [Salzet, M., Wattez, C., Verger-Bocquet, M., Beauvillain, J.-C. & Malecha, J. (1993) Brain Res. 601, 173-184]. This paper reports the isolation from the central nervous system of E. octoculata of another peptide of the oxytocin/vasopressin family; a lysine-vasopressin-like molecule. Its purification was performed by reverse-phase high-performance liquid chromatography combined with both dot immunobinding assay and enzyme-linked immunosorbent assay for lysine-vasopressin. The amino acid sequence was established by Edman degradation and confirmed by electrospray-mass-spectrometry measurement. The nonapeptide obtained corresponded to the lysine-conopressin previously isolated from the venom of the mollusc Conus geographus [Cruz, L. L., de Santos, V., Zafaralla, G. C., Ramilo, C. A., Zeikus, R., Gray, W. R. & Olivera, B. M. (1987) J. Biol. Chem. 262, 15821-15824]. In leeches, synthetic lysine-conopressin exerts a diuretic effect which can be compared to that of the arginine-vasopressin-like peptide isolated in the Insect Locusta migratoria [Proux, J., Miller, C. A., Li, J. P., Carney, R. L., Girardie, A., Delaage, M. & Schooley, D. A. (1987) Biochem. Biophys. Res. Commun. 149, 180-186].

  7. Quinone-amino acid conjugates targeting Leishmania amino acid transporters.

    PubMed

    Prati, Federica; Goldman-Pinkovich, Adele; Lizzi, Federica; Belluti, Federica; Koren, Roni; Zilberstein, Dan; Bolognesi, Maria Laura

    2014-01-01

    The aim of the present study was to investigate the feasibility of targeting Leishmania transporters via appropriately designed chemical probes. Leishmania donovani, the parasite that causes visceral leishmaniasis, is auxotrophic for arginine and lysine and has specific transporters (LdAAP3 and LdAAP7) to import these nutrients. Probes 1-15 were originated by conjugating cytotoxic quinone fragments (II and III) with amino acids (i.e. arginine and lysine) by means of an amide linkage. The toxicity of the synthesized conjugates against Leishmania extracellular (promastigotes) and intracellular (amastigotes) forms was investigated, as well their inhibition of the relevant amino acid transporters. We observed that some conjugates indeed displayed toxicity against the parasites; in particular, 7 was identified as the most potent derivative (at concentrations of 1 µg/mL and 2.5 µg/mL residual cell viability was reduced to 15% and 48% in promastigotes and amastigotes, respectively). Notably, 6, while retaining the cytotoxic activity of quinone II, displayed no toxicity against mammalian THP1 cells. Transport assays indicated that the novel conjugates inhibited transport activity of lysine, arginine and proline transporters. Furthermore, our analyses suggested that the toxic conjugates might be translocated by the transporters into the cells. The non-toxic probes that inhibited transport competed with the natural substrates for binding to the transporters without being translocated. Thus, it is likely that 6, by exploiting amino acid transporters, can selectively deliver its toxic effects to Leishmania cells. This work provides the first evidence that amino acid transporters of the human pathogen Leishmania might be modulated by small molecules, and warrants their further investigation from drug discovery and chemical biology perspectives. PMID:25254495

  8. Limiting amino acid for protein synthesis with mammary cells in tissue culture.

    PubMed

    Park, C S; Chandler, P T; Norman, A W

    1976-05-01

    To identify the limiting amino acid in the minimal essential medium as published by Eagle (Science 130:432, 1959) for milk protein synthesis in rat mammary cells in tissue culture, two different experimental approaches were used. The first study involved the reduction of amino acids singly from the total amino acid complement of the medium for milk protein synthesis. The second study was to investigate the effect on milk protein synthesis of single amino acid addition to the basic complement of amino acids. Order of limiting amino acids was lysine (first) and possible methionine, valine, or arginine (second).

  9. Effect of steaming, blanching, and high temperature/high pressure processing on the amino Acid contents of commonly consumed korean vegetables and pulses.

    PubMed

    Kim, Su-Yeon; Kim, Bo-Min; Kim, Jung-Bong; Shanmugavelan, Poovan; Kim, Heon-Woong; Kim, So-Young; Kim, Se-Na; Cho, Young-Sook; Choi, Han-Seok; Park, Ki-Moon

    2014-09-01

    In the present report, the effects of blanching, steaming, and high temperature/high pressure processing (HTHP) on the amino acid contents of commonly consumed Korean root vegetables, leaf vegetables, and pulses were evaluated using an Automatic Amino Acid Analyzer. The total amino acid content of the samples tested was between 3.38 g/100 g dry weight (DW) and 21.32 g/100 g DW in raw vegetables and between 29.36 g/100 g DW and 30.55 g/100 g DW in raw pulses. With HTHP, we observed significant decreases in the lysine and arginine contents of vegetables and the lysine, arginine, and cysteine contents of pulses. Moreover, the amino acid contents of blanched vegetables and steamed pulses were more similar than the amino acid contents of the HTHP vegetables and HTHP pulses. Interestingly, lysine, arginine, and cysteine were more sensitive to HTHP than the other amino acids. Partial Least Squares-Discriminate Analyses were also performed to discriminate the clusters and patterns of amino acids.

  10. Functional relationship between cationic amino acid transporters and beta-defensins: implications for dry skin diseases and the dry eye.

    PubMed

    Jäger, Kristin; Garreis, Fabian; Posa, Andreas; Dunse, Matthias; Paulsen, Friedrich P

    2010-04-20

    The ocular surface, constantly exposed to environmental pathogens, is particularly vulnerable to infection. Hence an advanced immune defence system is essential to protect the eye from microbial attack. Antimicrobial peptides, such as beta-defensins, are essential components of the innate immune system and are the first line of defence against invaders of the eye. High concentrations of L-arginine and L-lysine are necessary for the expression of beta-defensins. These are supplied by epithelial cells in inflammatory processes. The limiting factor for initiation of beta-defensin production is the transport of L-arginine and L-lysine into the cell. This transport is performed to 80% by only one transporter system in the human, the y(+)-transporter. This group of proteins exclusively transports the cationic amino acids L-arginine, L-lysine and L-ornithine and is also known under the term cationic amino acid transporter proteins (CAT-proteins). Various infections associated with L-arginine deficiency (for example psoriasis, keratoconjuctivitis sicca) are also associated with an increase in beta-defensin production. For the first time, preliminary work has shown the expression of human CATs in ocular surface epithelia and tissues of the lacrimal apparatus indicating their relevance for diseases of the ocular surface. In this review, we summarize current knowledge on the human CATs that appear to be integrated in causal regulation cascades of beta-defensins, thereby offering novel concepts for therapeutic perspectives.

  11. Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

    PubMed

    Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

    2016-03-10

    Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

  12. Detection and characterization of carrier-mediated cationic amino acid transport in lysosomes of normal and cystinotic human fibroblasts. Role in therapeutic cystine removal

    SciTech Connect

    Pisoni, R.L.; Thoene, J.G.; Christensen, H.N.

    1985-04-25

    The discovery of a trans-stimulation property associated with lysine exodus from lysosomes of human fibroblasts has enabled us to characterize a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. The cationic amino acids arginine, lysine, ornithine, diaminobutyrate, histidine, 2-aminoethylcysteine, and the mixed disulfide of cysteine and cysteamine all caused trans-stimulation of the exodus of radiolabeled lysine from the lysosomal fraction of human fibroblasts at pH 6.5. In contrast, neutral and acidic amino acids did not affect the rate of lysine exodus. Trans-stimulation of lysine exodus was observed over the pH range from 5.5 to 7.6, was specific for the L-isomer of the cationic amino acid, and was intolerant to methylation of the alpha-amino group of the amino acid. The lysosomotropic amine, chloroquine, greatly retarded lysine exodus, whereas the presence of sodium ion was without effect. The specificity and lack of Na+ dependence of this lysosomal transport system is similar to that of System y+ present on the plasma membrane of human fibroblasts. An important mechanism by which cysteamine treatment of cystinosis allows cystine escape from lysosomes may be the ability of the mixed disulfide of cysteine and cysteamine formed by sulfhydryl-disulfide exchange to migrate by this newly discovered system mediating cationic amino acid transport.

  13. Autologous aldrithiol-2-inactivated HIV-1 combined with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose as a vaccine platform for therapeutic dendritic cell immunotherapy.

    PubMed

    Miller, Elizabeth; Spadaccia, Meredith; Sabado, Rachel; Chertova, Elena; Bess, Julian; Trubey, Charles Mac; Holman, Rose Marie; Salazar, Andres; Lifson, Jeffrey; Bhardwaj, Nina

    2015-01-01

    Therapeutic interventions for HIV-1 that successfully augment adaptive immunity to promote killing of infected cells may be a requisite component of strategies to reduce latent cellular reservoirs. Adoptive immunotherapies utilizing autologous monocyte-derived dendritic cells (DCs) that have been activated and antigen loaded ex vivo may serve to circumvent defects in DC function that are present during HIV infection in order to enhance adaptive immune responses. Here we detail the clinical preparation of DCs loaded with autologous aldrithiol-2 (AT-2)-inactivated HIV that have been potently activated with the viral mimic, Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (Poly-ICLC). HIV is first propagated from CD4+ T cells from HIV-infected donors and then rendered non-replicative by chemical inactivation with aldrithiol-2 (AT-2), purified, and quantified. Viral inactivation is confirmed through measurement of Tat-regulated β-galactosidase reporter gene expression following infection of TZM-bl cells. In-process testing for sterility, mycoplasma, LPS, adventitious agents, and removal of AT-2 is performed on viral preparations. Autologous DCs are generated and pulsed with autologous AT-2-inactivated virus and simultaneously stimulated with Poly-ICLC to constitute the final DC vaccine product. Phenotypic identity, maturation, and induction of HIV-specific adaptive immune responses are confirmed via flow cytometric analysis of DCs and cocultured autologous CD4+ and CD8+ T cells. Lot release criteria for the DC vaccine have been defined in accordance with Good Manufacturing Practice (GMP) guidelines. The demonstrated feasibility of this approach has resulted in approval by the FDA for investigational use in antiretroviral (ART) suppressed individuals. We discuss how this optimized DC formulation may enhance the quality of anti-HIV adaptive responses beyond what has been previously observed during DC immunotherapy trials for HIV infection.

  14. Effect of L-lysine on expression of selected genes, serum concentration of amino acids, muscle growth and performance of growing pigs.

    PubMed

    Morales, A; García, H; Arce, N; Cota, M; Zijlstra, R T; Araiza, B A; Cervantes, M

    2015-08-01

    Lysine (Lys) is the first limiting amino acid (AA) in most feed formulations for pigs and most abundant, along with leucine, in muscle proteins. An experiment was conducted with 17 pigs (17.7 ± 0.05 kg initial BW) to identify a role of dietary Lys in the control of protein synthesis in pigs. Fourteen pigs were randomly assigned to one of the two wheat-based dietary treatments: Lys-deficient, 3.0 g/kg (DEF) and Lys-adequate, 10.8 g/kg (ADE). Samples from jejunum mucosa, liver, Longissumus and Semitendinosus muscles, and blood were collected. The other three pigs were sacrificed at the beginning of the trial to measure basal carcass composition. Weight gain, gain:feed ratio, Lys intake and loin eye area were greater in ADE than in DEF pigs (p < 0.01). Muscle-related carcass characteristics were better, and myosin heavy chain IIb expression (MyHC IIb) in Semitendinosus was higher in ADE than in DEF pigs. Expression of AA transporters CAT-1 was lower (p < 0.05), serum Lys was higher and serum Val was lower in pigs fed the ADE diet. The higher muscularity, MyHC IIb expression in Semitendinosus muscle and Lys serum of pigs fed the ADE diet suggest that Lys increases growth rate not only by functioning as protein construction unit but also as potential control of the protein synthesis process.

  15. 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

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

    PubMed

    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

  17. Effect of feeding guanidinoacetic acid and L-arginine on the fertility rate and sperm penetration in the perivitelline layer of aged broiler breeder hens.

    PubMed

    Sharideh, H; Esmaeile Neia, L; Zaghari, M; Zhandi, M; Akhlaghi, A; Lotfi, L

    2016-04-01

    Two experiments were conducted to evaluate the effects of feeding guanidinoacetic acid (GAA) and L-arginine (ARG) on fertility and sperm penetration (SP) rate of broiler breeder hens. In the first experiment, a total of 200 broiler breeder hens (Ross 308) aged 53 weeks were randomly allotted to four dietary treatments (0, 0.6, 1.2 and 1.8 g GAA/kg diet) with five replicates of 10 birds each. In the second experiment, 320 broiler breeder hens (Ross 308) were used from 53 to 62 weeks of age in a 2 × 4 factorial arrangement (0 or 1.2 g GAA/kg diet along with 0, 3, 6 or 9 g ARG/kg diet). The hens received a diet containing 2800 kcal ME/kg and 14% CP. Sixteen sexually mature Ross 308 breeder roosters (34 weeks old) were used to artificially inseminate the hens. Fertility of the hens was determined in 61 and 62 weeks of age. The sperm penetration holes in the inner perivitelline layer (IPL) overlying the germinal disc were enumerated on days 3 and 7 following each insemination. Adding GAA to the breeder diet increased the number of SPs in the IPL and fertility in both experiments (p < 0.01). The interactive effect of ARG and GAA on the SP and fertility was significant. Supplementary ARG increased the SP rate in the IPL (p < 0.01). In conclusion, dietary supplementation of GAA and ARG might be potentially used to improve the fertility of broiler breeder hens at the later phase of the egg production period.

  18. Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

    PubMed Central

    Bharathi, Sivakama S.; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E.; Rardin, Matthew J.; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W.; Hirschey, Matthew D.; Goetzman, Eric S.

    2013-01-01

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  19. Sirtuin 3 (SIRT3) protein regulates long-chain acyl-CoA dehydrogenase by deacetylating conserved lysines near the active site.

    PubMed

    Bharathi, Sivakama S; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E; Rardin, Matthew J; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W; Hirschey, Matthew D; Goetzman, Eric S

    2013-11-22

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  20. 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

  1. 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

  2. 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.

  3. Evaluation of protein content, lysine and sulfur-containing amino acids content and electrophoretic patterns of soluble proteins for gamma-irradiated semolina before and after milling of durum wheat

    NASA Astrophysics Data System (ADS)

    Azzeh, F. S.; Amr, A. S.

    2009-11-01

    Influenced of gamma irradiation (0, 0.25, 1, 2.5, 5 and 10 kGy) on total nitrogen, lysine and sulfur-containing amino acids content and electrophoretic patterns of soluble proteins of semolina was studied. The effect of irradiation before and after milling on previous parameters was also investigated. Protein content of semolina was not affected with gamma irradiation before and after milling. Up to 10 kGy dose, cystine and methionine were not significantly changed, although they increased slightly with increasing irradiation dose. Lysine content decreased significantly ( P≤0.05) at irradiation dose higher than 5 kGy. At 10 kGy dose, lysine decreased 5% and 14% for irradiated semolina and that obtained from irradiated wheat grains, respectively. The bands number and intensity of soluble proteins decreased with increasing irradiation dose higher than 5 kGy, as shown on SDS-PAGE electrophoresis. Irradiated semolina and semolina obtained from irradiated wheat grains at 10 kGy showed 13 and 15 bands, respectively. Unirradiated sample showed 19 bands.

  4. Significance of lysine/glycine cluster structure in gastric H+,K+-ATPase.

    PubMed

    Asano, S; Miwa, K; Yashiro, H; Tabuchi, Y; Takeguchi, N

    2000-08-01

    Gastric H+,K+-ATPase consists of alpha- and beta-subunits. The catalytic alpha-subunit contains a very unique structure consisting of lysine and glycine clusters, KKK(or KKKK)AG(G/R)GGGK-(K/R)K, in the amino-terminal cytoplasmic region. This structure is well conserved in all gastric H+,K+-ATPases from different animal species, and was postulated to be the site controlling the access of cations (or proton) to its binding site. In this report, we studied the role of this unique structure by expressing several H+,K+-ATPase mutants of the alpha-subunit together with the wild-type beta-subunit in HEK-293 cells. Even after replacing all the positively-charged amino acid residues (six lysines and one arginine) in the cluster with alanine or removing all the glycine residues in the cluster, the mutants preserved the H+,K+-ATPase activity, and showed similar affinity for ATP and K+ as well as similar pH profiles as those of wild-type H+,K+-ATPase, indicating that the cluster is not indispensable for H+,K+-ATPase activity and not directly involved in determination of the affinity for cation (proton).

  5. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  6. 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.

  7. Prolonged incubation time in sheep with prion protein containing lysine at position 171

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sheep scrapie susceptibility or resistance is a function of genotype with polymorphisms at codon 171 in the sheep prion gene playing a major role. Glutamine (Q) at 171 contributes to scrapie susceptibility while arginine (R) is associated with resistance. In some breeds, lysine (K) occurs at codon 1...

  8. In vitro degradation of lysine by ruminal fluid-based fermentations and by Fusobacterium necrophorum.

    PubMed

    Elwakeel, E A; Amachawadi, R G; Nour, A M; Nasser, M E A; Nagaraja, T G; Titgemeyer, E C

    2013-01-01

    The objective of these studies was to characterize some factors affecting lysine degradation by mixed ruminal bacteria and by ruminal Fusobacterium necrophorum. Mixed ruminal bacteria degraded lysine, and addition of pure cultures of F. necrophorum did not increase lysine degradation. Addition of acetic or propionic acid strikingly reduced NH(3) production from lysine by mixed ruminal bacteria at pH 6, but not at pH 7. Although typical ruminal environments with acidic pH and normal concentrations of volatile fatty acids might inhibit lysine degradation by F. necrophorum, ruminal fluid contained enough bacteria with a lysine-degrading capacity to ferment 50 mM lysine in vitro. Of 7 strains of ruminal F. necrophorum tested, all grew on both lactate and lysine as the primary energy source. Both subspecies of ruminal F. necrophorum (necrophorum and funduliforme) used lysine as a primary C and energy source. Lysine and glutamic acid were effectively fermented by F. necrophorum, but alanine and tryptophan were not, and histidine and methionine were fermented only to a minor extent. The end products of lactate fermentation by F. necrophorum were propionate and acetate, and those of lysine degradation were butyrate and acetate. Fermentation of glutamic acid by F. necrophorum yielded acetate and butyrate in a ratio near to 2:1. The minimum inhibitory concentration of tylosin for F. necrophorum was not dependent on whether bacteria were grown with lactate or lysine, but F. necrophorum was more susceptible to monensin when grown on lysine than on lactate. Although F. necrophorum is generally resistant to monensin, the ionophore may reduce lysine degradation by F. necrophorum in the rumen. The essential oil components limonene, at 20 or 100 μg/mL, and thymol, at 100 μg/mL, inhibited F. necrophorum growth, whereas eugenol, guaiacol, and vanillin had no effect. Our findings may lead to ways to minimize ruminal lysine degradation and thus increase its availability to the animal

  9. A Biocompatible Arginine-based Polycation

    PubMed Central

    Zern, Blaine J.; Chu, Hunghao; Osunkoya, Adeboye O.; Gao, Jin; Wang, Yadong

    2013-01-01

    Self assembly between cations and anions is ubiquitous throughout nature. Important biological structures such as chromatin often use polyvalent assembly between a polycation and a polyaninon. Biomedical importance of synthetic polycations arises from their affinity to polyanions such as nucleic acid and heparan sulfate. However, the limited biocompatibility of synthetic polycations hampers the realization of their immense potential. By examining biocompatible cationic peptides, we hypothesize that a biocompatible polycation should be biodegradable and made from endogenous cations. We designed an arginine-based biodegradable polycation and demonstrated that it was orders of magnitude more compatible than conventional polycations in vitro and in vivo. This biocompatibility diminishes when L-arginine is substituted with D-arginine or when the biodegradable ester linker changes to a biostable ether linker. We believe this design can lead to many biocompatible polycations that can significantly advance a wide range of applications including controlled release, tissue engineering, biosensing, and medical devices. PMID:23393493

  10. 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

  11. Amino acids

    MedlinePlus

    ... amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan , and valine. Nonessential amino acids "Nonessential" means that our bodies produce an amino ...

  12. Removal of acidic or basic α-amino acids in water by poorly water soluble scandium complexes.

    PubMed

    Hayashi, Nobuyuki; Jin, Shigeki; Ujihara, Tomomi

    2012-11-01

    To recognize α-amino acids with highly polar side chains in water, poorly water soluble scandium complexes with both Lewis acidic and basic portions were synthesized as artificial receptors. A suspension of some of these receptor molecules in an α-amino acid solution could remove acidic and basic α-amino acids from the solution. The compound most efficient at preferentially removing basic α-amino acids (arginine, histidine, and lysine) was the receptor with 7,7'-[1,3-phenylenebis(carbonylimino)]bis(2-naphthalenesulfonate) as the ligand. The neutral α-amino acids were barely removed by these receptors. Removal experiments using a mixed amino acid solution generally gave results similar to those obtained using solutions containing a single amino acid. The results demonstrated that the scandium complex receptors were useful for binding acidic and basic α-amino acids. PMID:23050492

  13. Removal of acidic or basic α-amino acids in water by poorly water soluble scandium complexes.

    PubMed

    Hayashi, Nobuyuki; Jin, Shigeki; Ujihara, Tomomi

    2012-11-01

    To recognize α-amino acids with highly polar side chains in water, poorly water soluble scandium complexes with both Lewis acidic and basic portions were synthesized as artificial receptors. A suspension of some of these receptor molecules in an α-amino acid solution could remove acidic and basic α-amino acids from the solution. The compound most efficient at preferentially removing basic α-amino acids (arginine, histidine, and lysine) was the receptor with 7,7'-[1,3-phenylenebis(carbonylimino)]bis(2-naphthalenesulfonate) as the ligand. The neutral α-amino acids were barely removed by these receptors. Removal experiments using a mixed amino acid solution generally gave results similar to those obtained using solutions containing a single amino acid. The results demonstrated that the scandium complex receptors were useful for binding acidic and basic α-amino acids.

  14. 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

  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. Arginine residues within the DNA binding domain of STAT3 promote intracellular shuttling and phosphorylation of STAT3.

    PubMed

    Ginter, Torsten; Fahrer, Jörg; Kröhnert, Ulrike; Fetz, Verena; Garrone, Alessio; Stauber, Roland H; Reichardt, Werner; Müller-Newen, Gerhard; Kosan, Christian; Heinzel, Thorsten; Krämer, Oliver H

    2014-08-01

    Acetylation-dependent inactivation of STAT1 can be mimicked by the exchange of its lysine residues K410 and K413 to glutamine residues. STAT3 harbors non-acetylatable arginine moieties at the corresponding sites R414 and R417. It is unclear whether the mutation of these sites to glutamine residues antagonizes STAT3 activation. Here, we show that an arginine-glutamine-exchange at the STAT3 moieties R414 and R417 (R414Q and R417Q) reduces cytokine-dependent tyrosine phosphorylation of STAT3. This inhibitory effect can be partially rescued by phosphatase inhibition. In addition, the R414Q and R417Q mutations enhance the nuclear accumulation of unphosphorylated STAT3. STAT3 R414Q and STAT3 R417Q show a reduced response to cytokine stimulation emanating from the plasma membrane. Moreover, these STAT3 mutants have no direct inhibitory effect on the cytokine-induced activation of STAT1/STAT3-mediated gene expression. Since the mutations R414Q and R417Q reside within the STAT3 DNA binding domain (DBD), the STAT3 R414Q and R417Q mutants also lack intrinsic activity as transcription factors. Furthermore, in contrast to wild-type STAT3 they cannot compensate for a loss of STAT1 and they cannot promote STAT1/STAT3-dependent transcriptional activation. We further analyzed a STAT3 arginine-lysine-exchange mutant (R414K/R417K). This molecule mimics corresponding lysine residues found within the DBD of STAT1. Compared to wild-type STAT3, the STAT3 R414K/R417K mutant shows attenuated tyrosine phosphorylation and it is a less active transcription factor. In addition, STAT3 R414K/R417K is not activated by deacetylase inhibition. On the other hand, C-terminal acetylation of STAT3 is intact in STAT3 R414K/R417K. Our results suggest that the exchange of amino acid residues within the DBDs of STAT1/STAT3 affects their phosphorylation as well as their intracellular shuttling. PMID:24721162

  17. Molecular recognition of amino acids with some fluorescent ditopic pyrylium- and pyridinium-based crown ether receptors

    NASA Astrophysics Data System (ADS)

    Moghimi, A.; Maddah, B.; Yari, A.; Shamsipur, M.; Boostani, M.; Fall Rastegar, M.; Ghaderi, A. R.

    2005-10-01

    The molecular recognition of L-amino acids such as asparagine, glutamine, lysine and arginine with some crownpyryliums, CP's, and a crownpyridinium compound, as receptors, were examined in methanol. 1H NMR spectroscopy was used to examine the structural stability of the receptors in the presence of the amino acids. The fluorimetric titration of the receptors by specified amino acids, other than arginine, was followed within a few minutes and the stoichiometry and stability of the resulting amino acid complexes were evaluated. The data analysis clearly demonstrated the critical role of the terminal amino group to carboxylic acid distance of amino acids for their proper fixation on the receptor molecules. Ion pairing for the two oppositely charged carboxylate anion and pyrylium (or pyridinium) cation, as well as the hydrogen bonding between crown ethers' oxygens and ammonium hydrogens are expected as the main interaction sources in the host-guest complexations.

  18. 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.

  19. Determination of lysine modification product epsilon-N-pyrrolylnorleucine in hydrolyzed proteins and trout muscle microsomes by micellar electrokinetic capillary chromatography.

    PubMed

    Zamora, R; Navarro, J L; Hidalgo, F J

    1995-06-01

    epsilon-N-Pyrrolylnorleucine (Pnl) is a product of the reaction between the lipid peroxidation product 4,5(E)-epoxy-2(E)-heptenal (EH) and the epsilon-amino group of lysine. Because Pnl might also be produced in proteins, a specific method to determine this compound in protein hydrolysates has been developed. Homoarginine, added as the internal standard, and Pnl are derivatized with diethyl ethoxymethylenemalonate and analyzed by micellar electrokinetic capillary chromatography. The method also analyzes lysine and arginine, and these analyses were useful in determining losses of these amino acids after treatment with EH. The lowest concentration of Pnl detected with acceptable reproducibility is 5 nmol/mL, and the coefficient of variation was determined from four standard curves assayed on separate days. Detector response was linear for samples containing 1.6 to 74 nmol/mL of Pnl. The assay was applied in investigations of Pnl production in bovine serum albumin (BSA) and trout muscle microsomes treated with EH. When BSA was incubated overnight with 30 mM EH, 76% of lysine residues were modified, and a part of these residues were detected as Pnl (12%). Pnl formation was also detected when trout muscle microsomes were incubated for three hours with 1 or 10 mM EH. These results show that Pnl is produced in vitro in proteins treated with the lipid peroxidation product EH, and suggest that Pnl might also be constituent of in vivo damaged proteins by their reaction with oxidized lipids.

  20. An engineered l-arginine sensor of Chlamydia pneumoniae enables arginine-adjustable transcription control in mammalian cells and mice

    PubMed Central

    Hartenbach, Shizuka; Daoud-El Baba, Marie; Weber, Wilfried; Fussenegger, Martin

    2007-01-01

    For optimal compatibility with biopharmaceutical manufacturing and gene therapy, heterologous transgene control systems must be responsive to side-effect-free physiologic inducer molecules. The arginine-inducible interaction of the ArgR repressor and the ArgR-specific ARG box, which synchronize arginine import and synthesis in the intracellular human pathogen Chlamydia pneumoniae, was engineered for arginine-regulated transgene (ART) expression in mammalian cells. A synthetic arginine-responsive transactivator (ARG), consisting of ArgR fused to the Herpes simplex VP16 transactivation domain, reversibly adjusted transgene transcription of chimeric ARG box-containing mammalian minimal promoters (PART) in an arginine-inducible manner. Arginine-controlled transgene expression showed rapid induction kinetics in a variety of mammalian cell lines and was adjustable and reversible at concentrations which were compatible with host cell physiology. ART variants containing different transactivation domains, variable spacing between ARG box and minimal promoter and several tandem ARG boxes showed modified regulation performance tailored for specific expression scenarios and cell types. Mice implanted with microencapsulated cells engineered for ART-inducible expression of the human placental secreted alkaline phosphatase (SEAP) exhibited adjustable serum phosphatase levels after treatment with different arginine doses. Using a physiologic inducer, such as the amino acid l-arginine, to control heterologous transgenes in a seamless manner which is devoid of noticeable metabolic interference will foster novel opportunities for precise expression dosing in future gene therapy scenarios as well as the manufacturing of difficult-to-produce protein pharmaceuticals. PMID:17947334

  1. Metabolite proofreading in carnosine and homocarnosine synthesis: molecular identification of PM20D2 as β-alanyl-lysine dipeptidase.

    PubMed

    Veiga-da-Cunha, Maria; Chevalier, Nathalie; Stroobant, Vincent; Vertommen, Didier; Van Schaftingen, Emile

    2014-07-11

    Carnosine synthase is the ATP-dependent ligase responsible for carnosine (β-alanyl-histidine) and homocarnosine (γ-aminobutyryl-histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a "metabolite repair" enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic β-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈ 200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with β-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine, and γ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on β-alanyl-arginine and γ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from β-alanine or γ-aminobutyrate, PM20D2 also acted at lower rates on some "classic dipeptides" like α-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was ∼ 100-200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.

  2. Changes in amino acids and lipids during embryogenesis of European lobster, Homarus gammarus (Crustacea: Decapoda).

    PubMed

    Rosa, R; Calado, R; Andrade, A M; Narciso, L; Nunes, M L

    2005-02-01

    We studied the amino acid and lipid dynamics during embryogenesis of Homarus gammarus. Major essential amino acids (EAA) in the last stage of embryonic development were arginine, lysine and leucine; major nonessential amino acids (NEAA) were glutamic acid, aspartic acid, valine and glycine. The highest percent of utilization occurred in respect to EAA (27.8%), mainly due to a significant decrease (p<0.05) of methionine (38.3%) and threonine (36.0%). NEAA also decreased significantly (p<0.05, 11.4%), namely serine (38.1%), tyrosine (26.4%) and glutamic acid (25.7%). In contrast, the free amino acid content increased significantly (p<0.05) during embryonic development, especially the free nonessential amino acids (FNEAA). In the last stage, the most abundant FNEAA were glycine, proline, alanine and taurine, and the major free essential amino acids (FEAA) were arginine, lysine and leucine. Lipid content decreased significantly (p<0.05) during embryonic development. A substantial decrease in all neutral lipid classes was observed (>80% of utilization). Major fatty acids were 16:0, 18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3. Unsaturated (UFA) and saturated fatty acids (SFA) were used up at similar rates (76.5% and 76.3%, respectively). Within UFA, monounsaturates (MUFA) were consumed more than polyunsaturates (PUFA) (82.9% and 67.5%, respectively). PMID:15649771

  3. Changes in the metabolome of rats after exposure to arginine and N-carbamylglutamate in combination with diquat, a compound that causes oxidative stress, assessed by 1H NMR spectroscopy.

    PubMed

    Liu, Guangmang; Xiao, Liang; Cao, Wei; Fang, Tingting; Jia, Gang; Chen, Xiaoling; Zhao, Hua; Wu, Caimei; Wang, Jing

    2016-02-01

    Numerous factors can induce oxidative stress in animal production and lead to growth retardation, disease, and even death. Arginine and N-carbamylglutamate can alleviate the effects of oxidative stress. However, the systematic changes in metabolic biochemistry linked to oxidative stress and arginine and N-carbamylglutamate treatment remain largely unknown. This study aims to examine the effects of arginine and N-carbamylglutamate on rat metabolism under oxidative stress. Thirty rats were randomly divided into three dietary groups (n = 10 each). The rats were fed a basal diet supplemented with 0 (control), 1% arginine, or 0.1% N-carbamylglutamate for 30 days. On day 28, the rats in each treatment were intraperitoneally injected with diquat at 12 mg per kg body weight or sterile solution. Urine and plasma samples were analyzed by metabolomics. Compared with the diquat group, the arginine + diquat group had significantly lower levels of acetamide, alanine, lysine, pyruvate, tyrosine, α-glucose, and β-glucose in plasma; N-carbamylglutamate + diquat had higher levels of 3-hydroxybutyrate, 3-methylhistidine, acetone, allantoin, asparagine, citrate, phenylalanine, trimethylamine-N-oxide, and tyrosine, and lower levels of low density lipoprotein, lipid, lysine, threonine, unsaturated lipid, urea, and very low density lipoprotein (P < 0.05) in plasma. Compared with the diquat group, the arginine + diquat group had significantly higher levels of citrate, creatinine, homogentisate, and α-ketoglutarate while lower levels of acetamide, citrulline, ethanol, glycine, isobutyrate, lactate, malonate, methymalonate, N-acetylglutamate, N-methylnicotinamide, propionate, and β-glucose (P < 0.05) in urine. Compared with the diquat group, the N-carbamylglutamate + diquat group had significantly higher levels of allantoin, citrate, homogentisate, phenylacetylglycine, α-ketoglutarate, and β-glucose while lower levels of acetamide, acetate, acetone, benzoate, citrulline, ethanol

  4. The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence

    PubMed Central

    Gottig, Natalia; Ottado, Jorgelina

    2015-01-01

    Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family’s response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction. PMID:25961560

  5. The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence.

    PubMed

    Ficarra, Florencia A; Garofalo, Cecilia G; Gottig, Natalia; Ottado, Jorgelina

    2015-01-01

    Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family's response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction.

  6. The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence.

    PubMed

    Ficarra, Florencia A; Garofalo, Cecilia G; Gottig, Natalia; Ottado, Jorgelina

    2015-01-01

    Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family's response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction. PMID:25961560

  7. Synthesis of lysine methyltransferase inhibitors

    PubMed Central

    Hui, Chunngai; Ye, Tao

    2015-01-01

    Lysine methyltransferase which catalyze methylation of histone and non-histone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery. PMID:26258118

  8. Structural basis of arginine asymmetrical dimethylation by PRMT6.

    PubMed

    Wu, Hong; Zheng, Weihong; Eram, Mohammad S; Vhuiyan, Mynol; Dong, Aiping; Zeng, Hong; He, Hao; Brown, Peter; Frankel, Adam; Vedadi, Masoud; Luo, Minkui; Min, Jinrong

    2016-10-01

    PRMT6 is a type I protein arginine methyltransferase, generating the asymmetric dimethylarginine mark on proteins such as histone H3R2. Asymmetric dimethylation of histone H3R2 by PRMT6 acts as a repressive mark that antagonizes trimethylation of H3 lysine 4 by the MLL histone H3K4 methyltransferase. PRMT6 is overexpressed in several cancer types, including prostate, bladder and lung cancers; therefore, it is of great interest to develop potent and selective inhibitors for PRMT6. Here, we report the synthesis of a potent bisubstrate inhibitor GMS [6'-methyleneamine sinefungin, an analog of sinefungin (SNF)], and the crystal structures of human PRMT6 in complex, respectively, with S-adenosyl-L-homocysteine (SAH) and the bisubstrate inhibitor GMS that shed light on the significantly improved inhibition effect of GMS on methylation activity of PRMT6 compared with SAH and an S-adenosyl-L-methionine competitive methyltransferase inhibitor SNF. In addition, we also crystallized PRMT6 in complex with SAH and a short arginine-containing peptide. Based on the structural information here and available in the PDB database, we proposed a mechanism that can rationalize the distinctive arginine methylation product specificity of different types of arginine methyltransferases and pinpoint the structural determinant of such a specificity. PMID:27480107

  9. Insights into the regulatory landscape of the lysine riboswitch

    PubMed Central

    Garst, Andrew D.; Porter, Ely B.; Batey, Robert T.

    2012-01-01

    A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, the relative contributions of the polar functional groups to binding affinity and the regulatory response have been determined. Our results reveal that the lysine riboswitch has >1,000-fold specificity for lysine over other amino acids. To achieve this specificity, the aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main chain functional groups. At low NTP concentrations, we observe good agreement between the half-maximal regulatory activity (T50) and the affinity of the receptor for lysine (KD) as well many of its analogs. However, above 400 µM [NTP] the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that under physiologically relevant conditions riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell. PMID:22771573

  10. Insights into the regulatory landscape of the lysine riboswitch.

    PubMed

    Garst, Andrew D; Porter, Ely B; Batey, Robert T

    2012-10-12

    A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors, these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main-chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, we have determined the relative contributions of the polar functional groups to binding affinity and the regulatory response. Our results reveal that the lysine riboswitch has >1000-fold specificity for lysine over other amino acids. The aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main-chain functional groups in order to achieve this specificity. At low nucleotide triphosphate (NTP) concentrations, we observe good agreement between the half-maximal regulatory activity (T(50)) and the affinity of the receptor for lysine (K(d)), as well as many of its analogs. However, above 400 μM [NTP], the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that, under physiologically relevant conditions, riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell. PMID:22771573

  11. Cyclization increases the antimicrobial activity and selectivity of arginine- and tryptophan-containing hexapeptides.

    PubMed

    Dathe, Margitta; Nikolenko, Heike; Klose, Jana; Bienert, Michael

    2004-07-20

    Arginine- and tryptophan-rich motifs have been identified in antimicrobial peptides with various secondary structures. We synthesized a set of linear hexapeptides derived from the sequence AcRRWWRF-NH(2) by substitution of tryptophan (W) by tyrosine (Y) or naphthylalanine (Nal) and by replacement of arginine (R) by lysine (K) to investigate the role of cationic charge and aromatic residues in membrane activity and selectivity. A second set of corresponding head-to-tail cyclic analogues was prepared to analyze the role of conformational constraints. The biological activity of the linear peptides followed the order Nal- > W- > Y-containing compounds and slightly decreased upon R-K substitution. A pronounced activity-improving and bacterial selectivity-enhancing effect was found upon cyclization of the R- and W-bearing parent peptide, whereas the activity-modifying effect of cyclization of Y- and Nal-containing peptides was low. The analysis of the driving forces of peptide interaction with model membranes showed that the activities correlated with the partition coefficients and the depths of peptide insertion into neutral and negatively charged lipid bilayers. Spectroscopic studies, RP-HPLC, and titration calorimetry implied that the combination of cationic and aromatic amino acid composition and conformational rigidity afforded a membrane-active, amphipathic structure with a highly charged face opposed by a cluster of aromatic side chains. However, threshold values of low and high hydrophobicity seemed to exist beyond which the activity-enhancing effect of cyclization was negligible. The results suggest that cyclization of small peptides of an appropriate amino acid composition may serve as a promising strategy in the design of antimicrobial peptides.

  12. The dietary lysine requirement of juvenile hybrid striped bass.

    PubMed

    Griffin, M E; Brown, P B; Grant, A L

    1992-06-01

    Two experiments were conducted to determine the dietary lysine requirement of juvenile hybrid striped bass (Morone saxatilis x M. chrysops). In both experiments the diets contained 35 g crude protein/100 g diet (10 g crude protein supplied by casein and gelatin and 25 g crude protein supplied by crystalline L-amino acids) and contained graded levels of L-lysine.HCl resulting in eight dietary treatments. Diets were fed to triplicate groups of fish and ranged in dietary lysine concentration from 1.2 to 2.6 g/100 g of the dry diet in Experiment 1 and from 0.8 to 2.2 g/100 g of the dry diet in Experiment 2. Weight gain and food efficiency data from Experiment 1 indicated the dietary lysine requirement to be between 1.2 and 1.4 g/100 g of the dry diet. Weight gain, food efficiency and serum lysine data from Experiment 2 confirmed the requirement to be between 1.2 and 1.4 g/100 g of the dry diet. Broken-line analysis of weight gain and food efficiency data from Experiment 2 indicated the dietary lysine requirement to be 1.4 +/- 0.2% of the dry diet, or 4.0 g/100 g of the dietary protein. Changes in the relative proportions of dietary lipid and carbohydrate between the two experiments, although maintaining similar gross energy levels, did not alter the lysine requirement estimate of juvenile hybrid striped bass.

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

    PubMed

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

    2016-04-01

    Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade. PMID:27043018

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

    PubMed Central

    Strasser, Rona; Zeituni-Molad, Michal; Bendelak, Keren; Rentsch, Doris; Ephros, Moshe; Wiese, Martin; Jardim, Armando; Myler, Peter J.; Zilberstein, Dan

    2016-01-01

    Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade. PMID:27043018

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

    PubMed

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

    2013-06-01

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

  16. Effects of charge-carrying amino acids on the gelatinization and retrogradation properties of potato starch.

    PubMed

    Chen, Wenting; Zhou, Hongxian; Yang, Hong; Cui, Min

    2015-01-15

    The objective of this study was to evaluate the effects of charge-carrying amino acids (lysine (Lys), arginine (Arg), aspartic acid (Asp) and glutamic acid (Glu)) on the gelatinization and retrogradation properties of potato starch. Acidic amino acids (Asp and Glu) showed a decreasing trend in swelling power and granule size of potato starch, but increased amylose leaching and gelatinization temperature. Alkaline amino acid (Arg) showed an increasing trend in swelling power and granule size of potato starch, but decreasing amylose leaching and gelatinization temperature. Lys had no effect on the swelling power of potato starch, except at a high content (0.2 mol/kg). Like other two acidic amino acids, Lys also increased gelatinization temperature. Moreover, the addition of alkaline amino acids (Arg) decreased syneresis value of potato starch but acidic amino acids (Asp and Glu) increased it. Compared to Arg, the syneresis of potato starch with Lys was similar to that of its native starch.

  17. Arginine deiminase inhibits Porphyromonas gingivalis surface attachment

    PubMed Central

    Cugini, Carla; Stephens, Danielle N.; Nguyen, Daniel; Kantarci, Alpdogan

    2013-01-01

    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. PMID:23242802

  18. Arginine: New Insights into Growth Performance and Urinary Metabolomic Profiles of Rats.

    PubMed

    Liu, Guangmang; Wu, Xianjian; Jia, Gang; Chen, Xiaoling; Zhao, Hua; Wang, Jing; Wu, Caimei; Cai, Jingyi

    2016-01-01

    Arginine regulates growth performance, nutrient metabolism and health effects, but the underlying mechanism remains unknown. This study aims to investigate the effect of dietary arginine supplementation on rat growth performance and urinary metabolome through ¹H-NMR spectroscopy. Twenty rats were randomly assigned to two groups supplemented with 0% or 1.0% l-arginine for 4 weeks. Urine samples were analyzed through NMR-based metabolomics. Arginine supplementation significantly increased the urine levels of 4-aminohippurate, acetate, creatine, creatinine, ethanolamine, formate, hippurate, homogentisate, indoxyl sulfate, and phenylacetyglycine. Conversely, arginine decreased the urine levels of acetamide, β-glucose, cirtulline, ethanol, glycine, isobutyrate, lactate, malonate, methymalonate, N-acetylglutamate, N-methylnicotinamide, and propionate. Results suggested that arginine can alter common systemic metabolic processes, including energy metabolism, amino acid metabolism, and gut microbiota metabolism. Moreover, the results also imply a possible physiological role of the metabolism in mediating the arginine supplementation-supported growth of rats. PMID:27589702

  19. Arginine depletion increases susceptibility to serious infections in preterm newborns

    PubMed Central

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

    2015-01-01

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

  20. 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

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

    PubMed

    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

  2. L-arginine transport is increased in macrophages generating nitric oxide.

    PubMed Central

    Bogle, R G; Baydoun, A R; Pearson, J D; Moncada, S; Mann, G E

    1992-01-01

    Transport of L-arginine and nitrite production were examined in the murine macrophage cell line J774. Bacterial lipopolysaccharide (LPS) induced a dose- and time-dependent stimulation of nitrite production, which was further increased in the presence of interferon-gamma. Nitrite synthesis was absolutely dependent on extracellular L-arginine and inhibited in the presence of L-lysine or L-ornithine. In unactivated J774 cells L-arginine transport was saturable, with an apparent Km of 0.14 +/- 0.04 mM and Vmax. of 15 +/- 2 nmol/h per 10(6) cells. LPS (1 microgram/ml) induced a time-dependent stimulation of L-arginine transport, and after 24 h the Vmax. increased to 34 +/- 2 nmol/h per 10(6) cells. These findings indicate that activation of J774 cells with LPS produces an increase in both L-arginine transport and nitrite synthesis. The elevated rate of L-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization of L-arginine for the generation of NO. PMID:1599394

  3. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    SciTech Connect

    Dwyer, B.P. )

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  4. Biofortification of rice with lysine using endogenous histones.

    PubMed

    Wong, H W; Liu, Q; Sun, S S M

    2015-02-01

    Rice is the most consumed cereal grain in the world, but deficient in the essential amino acid lysine. Therefore, people in developing countries with limited food diversity who rely on rice as their major food source may suffer from malnutrition. Biofortification of stable crops by genetic engineering provides a fast and sustainable method to solve this problem. In this study, two endogenous rice lysine-rich histone proteins, RLRH1 and RLRH2, were over-expressed in rice seeds to achieve lysine biofortification. Their protein sequences passed an allergic sequence-based homology test. Their accumulations in rice seeds were raised to a moderate level by the use of a modified rice glutelin 1 promoter with lowered expression strength to avoid the occurrence of physiological abnormalities like unfolded protein response. The expressed proteins were further targeted to protein storage vacuoles for stable storage using a glutelin 1 signal peptide. The lysine content in the transgenic rice seeds was enhanced by up to 35 %, while other essential amino acids remained balanced, meeting the nutritional standards of the World Health Organization. No obvious unfolded protein response was detected. Different degrees of chalkiness, however, were detected in the transgenic seeds, and were positively correlated with both the levels of accumulated protein and lysine enhancement. This study offered a solution to the lysine deficiency in rice, while at the same time addressing concerns about food safety and physiological abnormalities in biofortified crops.

  5. Role of the tissue free amino acids in adaptation of medicinal leeches Hirudo medicinalis L., 1758 to extreme climatic conditions.

    PubMed

    Chernaya, L V; Kovalchuk, L A; Nokhrina, E S

    2016-01-01

    The first comparison of the spectra of free amino acids in tissues of the medicinal leeches H. medicinalis from different climatic and geographical Eurasian areas has been performed. Adaptation of H. medicinalis to extreme climatic conditions occurs via intensification of the amino acid metabolism resulting from a significant increase in the content of essential amino acids. Accumulation of arginine, histidine, and lysine (3.6-, 3.9-, and 2.0-fold increases, respectively) has proved to play a special protective role in adaptation of H. medicinalis to the low positive temperatures.

  6. 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...

  7. Modeling of cellular arginine uptake by more than one transporter.

    PubMed

    Nel, Marietha J; Woodiwiss, Angela J; Candy, Geoffrey P

    2012-01-01

    Determining the kinetic constants of arginine uptake by endothelial cells mediated by more than one transporter from linearization of data as Eadie-Hofstee plots or modeling which does not include the concentration of trace radiolabeled amino acid used to measure uptake may not be correct. The initial rate of uptake of trace [³H]L-arginine by HUVECs and ECV₃₀₄ cells in the presence of a range of unlabeled arginine and modifiers was used in nonlinear models to calculate the constants of arginine uptake using GraphPad Prism. Theoretical plots of uptake derived from constants determined from Eadie-Hofstee graphs overestimated uptake, whereas those from the nonlinear modeling approach agreed with experimental data. The contribution of uptake by individual transporters could be modeled and showed that leucine inhibited the individual transporters differently and not necessarily competitively. N-Ethylmaleimide inhibited only y⁺ transport, and BCH may be a selective inhibitor of y⁺L transport. The absence of sodium reduced arginine uptake by y⁺L transport and reduced the K(m)', whereas reducing sodium decreased arginine uptake by y⁺ transport without affecting the K (m)'. The nonlinear modeling approach using raw data avoided the errors inherent in methods deriving constants from the linearization of the uptake processes following Michaelian kinetics. This study provides explanations for discrepancies in the literature and suggests that a nonlinear modeling approach better characterizes the kinetics of amino acid uptake into cells by more than one transporter.

  8. Discrimination between citrulline and arginine transport in activated murine macrophages: inefficient synthesis of NO from recycling of citrulline to arginine.

    PubMed Central

    Baydoun, A. R.; Bogle, R. G.; Pearson, J. D.; Mann, G. E.

    1994-01-01

    1. The kinetics, specificity, pH- and Na(+)-dependency of L-citrulline transport were examined in unstimulated and lipopolysaccharide (LPS)-activated murine macrophage J774 cells. The dependency of nitric oxide production on extracellular arginine or citrulline was investigated in cells activated with LPS (1 microgram ml-1) for 24 h. 2. In unstimulated J774 cells, transport of citrulline was saturable (Kt = 0.16 mM and Vmax = 32 pmol micrograms-1 protein min-1), pH-insensitive and partially Na(+)-dependent. In contrast to arginine, transport of citrulline was unchanged in LPS-activated (1 microgram ml-1, 24 h) cells. 3. Kinetic inhibition experiments revealed that arginine was a relatively poor inhibitor of citrulline transport, whilst citrulline was a more potent inhibitor (Ki = 3.4 mM) of arginine transport but only in the presence of extracellular Na+. Neutral amino acids inhibited citrulline transport (Ki = 0.2-0.3 mM), but were poor inhibitors of arginine transport. 4. Activated J774 cells did not release nitrite in the absence of exogenous arginine. Addition of citrulline (0.01-10 mM), in the absence of exogenous arginine, could only partially restore the ability of cells to synthesize nitrite, which was abolished by 100 microM NG-nitro-L-arginine methyl ester or NG-iminoethyl-L-ornithine. 5. Intracellular metabolism of L-[14C]-citrulline to L-[14C]-arginine was detected in unstimulated J774 cells and was increased further in cells activated with LPS and interferon-gamma. 6. We conclude that J774 macrophage cells transport citrulline via a saturable but nonselective neutral carrier which is insensitive to induction by LPS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8075867

  9. Biodegradable tri-block copolymer poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)(PLA-PEG-PLL) as a non-viral vector to enhance gene transfection.

    PubMed

    Fu, Chunhua; Sun, Xiaoli; Liu, Donghua; Chen, Zhijing; Lu, Zaijun; Zhang, Na

    2011-02-23

    Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid)-poly(ethylene glycol)-poly(l-lysine) (PLA-PEG-PLL) copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z)-NCA onto amine-terminated NH(2)-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs) possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ɛ-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs) were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells) than PEI/DNA complexes (14.01% and 24.22%). These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo.

  10. Thermodynamical characteristics of the reaction of pyridoxal-5'-phosphate with L-amino acids in aqueous buffer solution

    NASA Astrophysics Data System (ADS)

    Barannikov, V. P.; Badelin, V. G.; Venediktov, E. A.; Mezhevoi, I. N.; Guseinov, S. S.

    2011-01-01

    The reaction of pyridoxal-5'-phosphate with L-isomers of alanine, lysine, arginine, aspartic acid, glutamic acid, and glycine in phosphate buffer solution was studied by absorption spectroscopy and the calorimetry of dissolution at physiological acidity of the medium (pH 7.35). The formation constants of Schiff bases during reactions and changes in Gibbs energy, enthalpy, and entropy were determined. It was shown that the formation constant of the Schiff base and its spectral properties depend on the nature of the bound amino acid. The progress of the reaction with a majority of amino acids is governed by the entropy factor due to the predominant role of the dehydration effect of the reaction center of amino acids during chemical reactions. The intramolecular electrostatic interaction of an ionized phosphate group with the positively charged amino group on the end of the chain of amino acid residue stabilizes the Schiff bases formed by lysine and arginine. The extinction coefficient of the base, equilibrium constant, and the exothermic effect of the reaction then increase. The excess negative charge on the end of the chain of amino acid residues of aspartic and glutamic acids destabilizes the molecule of the Schiff base. In this case, the equilibrium constant decreases and the endothermic effect of the reaction increases.

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

    PubMed Central

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

    2013-01-01

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

  12. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

    PubMed

    Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

    2016-06-01

    Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria. PMID:27183143

  13. Degradation signals in the lysine-asparagine sequence space.

    PubMed

    Suzuki, T; Varshavsky, A

    1999-11-01

    The N-degrons, a set of degradation signals recognized by the N-end rule pathway, comprise a protein's destabilizing N-terminal residue and an internal lysine residue. We show that the strength of an N-degron can be markedly increased, without loss of specificity, through the addition of lysine residues. A nearly exhaustive screen was carried out for N-degrons in the lysine (K)-asparagine (N) sequence space of the 14-residue peptides containing either K or N (16 384 different sequences). Of these sequences, 68 were found to function as N-degrons, and three of them were at least as active and specific as any of the previously known N-degrons. All 68 K/N-based N-degrons lacked the lysine at position 2, and all three of the strongest N-degrons contained lysines at positions 3 and 15. The results support a model of the targeting mechanism in which the binding of the E3-E2 complex to the substrate's destabilizing N-terminal residue is followed by a stochastic search for a sterically suitable lysine residue. Our strategy of screening a small library that encompasses the entire sequence space of two amino acids should be of use in many settings, including studies of protein targeting and folding. PMID:10545113

  14. Absence of Btn1p in the yeast model for juvenile Batten disease may cause arginine to become toxic to yeast cells.

    PubMed

    Vitiello, Seasson Phillips; Wolfe, Devin M; Pearce, David A

    2007-05-01

    Lymphoblast cell lines established from individuals with juvenile Batten disease (JNCL) bearing mutations in CLN3 and yeast strains lacking Btn1p (btn1-Delta), the homolog to CLN3, have decreased intracellular levels of arginine and defective lysosomal/vacuolar transport of arginine. It is important to establish the basis for this decrease in arginine levels and whether restoration of arginine levels would be of therapeutic value for Batten disease. Previous studies have suggested that synthesis and degradation of arginine are unaltered in btn1-Delta. Using the yeast model for the Batten disease, we have determined that although btn1-Delta results in decreased intracellular arginine levels, it does not result from altered arginine uptake, arginine efflux or differences in arginine incorporation into peptides. However, expression of BTN1 is dependent on arginine and Gcn4p, the master regulator of amino acid biosynthesis. Moreover, deletion of GCN4 (gcn4-Delta), in combination with btn1-Delta, results in a very specific growth requirement for arginine. In addition, increasing the intracellular levels of arginine through overexpression of Can1p, the plasma membrane basic amino acid permease, results in increased cell volume and a severe growth defect specific to basic amino acid availability for btn1-Delta, but not wild-type cells. Therefore, elevation of intracellular levels of arginine in btn1-Delta cells is detrimental and is suggestive that btn1-Delta and perhaps mutation of CLN3 predispose cells to keep arginine levels lower than normal.

  15. Characterization and expression profile of complete functional domain of granulysin/NK-lysin homologue (buffalo-lysin) gene of water buffalo (Bubalus bubalis).

    PubMed

    Kandasamy, Sukumar; Mitra, Abhijit

    2009-04-15

    Granulysin (GNLY)/NK-lysin (NKL) is an effector antimicrobial cationic peptide expressed in the cytotoxic and natural killer lymphocytes. We report here cDNA sequence (405bp) encoding the complete functional domain of buffalo-lysin (bu-lysin), and its expression profile in the various tissues. The nucleotide sequence of bu-lysin exhibited >85% identity with the bovine lysin. Comparison of the deduced amino acid sequence of bu-lysin with those of GNLY/NKL of different species revealed the conservation of six cysteine (Cys) residues and five alpha helices. Unlike the homologues in other species, bu-lysin composed of 11 positively charged Lys residues as in equine. The expression of bu-lysin mRNA in the in vitro cultured lymphocytes was inducible and increased markedly (p<0.05) in a dose dependant manner when incubated with Concanavalin A (ConA). The expression of bu-lysin mRNA in the different tissues was variable: comparatively higher in the spleen and lymph node, moderate in the uterine endometrium and low in the liver and kidney. These results indicate the existence and active expression of GNLY/NKL homologue in water buffalo having a significant influence in immune response.

  16. Mutagenicity of Maillard reaction products from D-glucose-amino acid mixtures and possible roles of active oxygens in the mutagenicity.

    PubMed

    Kim, S B; Kim, I S; Yeum, D M; Park, Y H

    1991-01-01

    The mutagenicity for Salmonella typhimurium TA100 without S9 mix of Maillard reaction products (MRP) obtained from equimolar amounts of glucose and amino acids under different pHs was investigated. MRP derived from arginine and lysine exhibited the strongest mutagenicity, and weaker mutagenicity was shown by the mixtures with alanine, serine, threonine and monosodium glutamate. MRP from proline and cysteine had no detectable mutagenicity. Furthermore, glucose-arginine and glucose-lysine reaction mixtures, which presented a marked mutagenicity, showed pH- and browning intensity-dependent expression of their mutagenic activities. The mutagenicity of MRP, especially glucose-arginine and glucose-lysine mixtures, was significantly suppressed by active oxygen scavengers such as cysteine, mannitol, alpha-tocopherol, catalase and superoxide dismutase (SOD) and reducing agents such as sodium bisulfite and glutathione. Among these desmutagenic factors tested, cysteine, catalase, sodium bisulfite and glutathione had higher desmutagenic activities than the others. Accordingly, it is assumed that the mutagenicity of MRP is due to the direct action of low-molecular-weight compounds such as carbonyls and heterocyclics produced by the Maillard reaction and is enhanced by active oxygens, especially singlet oxygen and hydrogen peroxide derived from their autoxidation. PMID:1986274

  17. Selective Deletion of the Internal Lysine Residue from the Peptide Sequence by Collisional Activation

    NASA Astrophysics Data System (ADS)

    Banerjee, Shibdas; Mazumdar, Shyamalava

    2012-11-01

    The gas-phase peptide ion fragmentation chemistry is always the center of attraction in proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we describe the formation of an anomalous fragment ion, which corresponds to the selective deletion of the internal lysine residue from a series of lysine containing peptides upon collisional activation in the ion trap. We detected several water-loss fragment ions and the maximum number of water molecules lost from a particular fragment ion was equal to the number of lysine residues in that fragment. As a consequence of this water-loss phenomenon, internal lysine residues were found to be deleted from the peptide ion. The N,N-dimethylation of all the amine functional groups of the peptide stopped the internal lysine deletion reaction, but selective N-terminal α-amino acetylation had no effect on this process indicating involvement of the side chains of the lysine residues. The detailed mechanism of the lysine deletion was investigated by multistage CID of the modified and unmodified peptides, by isotope labeling and by energy resolved CID studies. The results suggest that the lysine deletion might occur through a unimolecular multistep mechanism involving a seven-membered cyclic imine intermediate formed by the loss of water from a lysine residue in the protonated peptide. This intermediate subsequently undergoes degradation reaction to deplete the interior imine ring from the peptide backbone leading to the deletion of an internal lysine residue.

  18. Arginine of retinoic acid receptor beta which coordinates with the carboxyl group of retinoic acid functions independent of the amino acid residues responsible for retinoic acid receptor subtype ligand specificity.

    PubMed

    Zhang, Zeng Ping; Hutcheson, Juliet M; Poynton, Helen C; Gabriel, Jerome L; Soprano, Kenneth J; Soprano, Dianne Robert

    2003-01-15

    The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, RARbeta, and RARgamma) and retinoid X receptors (RXRalpha, RXRbeta, and RXRgamma). Consistent with the X-ray crystal structures of RARalpha and RARgamma, site-directed mutagenesis studies have demonstrated the importance of a conserved Arg residue (alphaArg(276), betaArg(269), and gammaArg(278)) for coordination with the carboxyl group of RA. However, mutation of Arg(269) to Ala in RARbeta causes only a 3- to 6-fold increase in the K(d) for RA and EC(50) in RA-dependent transcriptional transactivation assays while the homologous mutation in either RARalpha or RARgamma causes a 110-fold and a 45-fold increase in EC(50) value, respectively. To further investigate the nature of this difference, we prepared mutant RARs to determine the effect of conversion of betaR269A to a mutant which mimics either RARalpha ligand selectivity (betaA225S/R269A) or RARgamma ligand selectivity (betaI263M/R269A/V338A). Our results demonstrate that in RARbeta mutants that acquire either RARalpha or RARgamma ligand specificity the Arg(269) position responsible for coordination with the carboxyl group of retinoids continued to function like that of RARbeta. Furthermore, three mutant receptors (betaA225S/R269A, betaA225S/F279, and alphaF286A) were found to have a greater than wild-type affinity for the RARalpha-selective ligand Am580. Finally, a homology-based computer model of the ligand binding domain (LBD) of RARbeta and the X-ray crystal structures of the LBD of both RARalpha and RARgamma are used to describe potential mechanisms responsible for the increased affinity of some mutants for Am580 and for the difference in the effect of mutation of Arg(269) in RARbeta compared to its homologous Arg in RARalpha and RARgamma.

  19. Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase.

    PubMed

    Chen, Dawei; Tanem, Justinn; Frey, Perry A

    2007-02-01

    l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.

  20. 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.

  1. Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

    SciTech Connect

    Rice, E.A.; Bannon, G.A.; Glenn, K.C.; Jeong, S.S.; Sturman, E.J.; Rydel, T.J.

    2008-11-21

    The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysine revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.

  2. Accurate determination of the amino acid content of selected feedstuffs.

    PubMed

    Rutherfurd, Shane M

    2009-01-01

    The accurate determination of the amino acid content is important. In the present study, a least-squares non-linear regression model of the amino acid content determined over multiple hydrolysis times was used to accurately determine the content of amino acids in five different feedstuffs. These values were compared with 24-h hydrolysis values determined for the same feedstuffs. Overall, approximately two-thirds of the amino acids determined in this study (aspartic acid, threonine, glutamic acid, proline, glycine, alanine, leucine, tyrosine, phenylalanine and arginine) using 24-h hydrolysis were in good agreement (<3% difference). When examined across feedstuffs, the concentration of serine was underestimated by the 24-h hydrolysis method by 4.8%, while the concentrations of histidine and lysine were overestimated by 3.9% and 3.1%, respectively.

  3. Possible evidence of amide bond formation between sinapinic acid and lysine-containing bacterial proteins by matrix-assisted laser desorption/ionization (MALDI) at 355 nm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, HdeB and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALD...

  4. Electrokinetic characterization of magnetite nanoparticles functionalized with amino acids.

    PubMed

    Viota, J L; Arroyo, F J; Delgado, A V; Horno, J

    2010-04-01

    The synthesis of nanoparticles consisting of a magnetite core coated with one or more layers of amino acid (L-arginine, L-lysine, glycine, and L-glutamine) is described in this paper. For all the amino acids it is found that adsorption increases with concentration in solution in the range 0.5-10 mg/mL. The adsorption, however, differs substantially from one amino acid to another, depending on the length of the hydrocarbon chain and the polarity and charge of the side group. Thus, for given concentration and pH, adsorption is found to increase in the order L-arginine < L-lysine < L-glutamine < glycine. This order corresponds roughly to amino acids with decreasing chain length; in addition, the presence of the less polarizable guanidine group in the arginine molecule may explain why this amino acid is slightly less adsorbed than lysine. The pH dependence of the adsorption of each amino acid is reasonably explained considering the surface charge of magnetite and the charge of the amino acid molecules for different pHs, indicating a significant role of electrostatics in adsorption. This is further checked by means of determinations of the electrophoretic mobility of amino acid-coated magnetite as a function of pH: the results indicate a shift of the isoelectric point of the raw magnetite toward more basic pHs, an indication of adsorption of positive species, as confirmed by the tendency of the mobility of amino acid-coated magnetite toward more positive values below neutral pH. The electrophoretic mobility of coated particles was also measured as a function of the concentration of amino acid, and it was found that for low concentrations the four amino acids provoke charge inversion and overcharging of the magnetite surface at pH 6. Finally, the dependence of the electrophoretic mobility on the ionic strength indicated that from an electrophoretic point of view, the functionalized magnetite-amino acid particles do not behave as soft particles, and that the amino acid

  5. Changes in arginine metabolism during sepsis and critical illness in children.

    PubMed

    de Betue, Carlijn T I; Deutz, Nicolaas E P

    2013-01-01

    Arginine is an important amino acid during disease and healing because of functions in the immune system and as precursor of nitric oxide (NO). In critically ill adults and children, plasma arginine and citrulline concentrations are substantially decreased, indicating an arginine-deficient state. Arginine availability is reduced because of increased arginine disposal in combination with reduced de novo arginine synthesis. The latter is most likely caused by reduced citrulline availability. As a result, NO synthesis may be impaired, which might compromise microcirculation. These metabolic changes seem to be dependent on the severity of inflammation. Arginine or citrulline supplementation in severe inflammation might therefore be beneficial. Possibly, the use of protein-energy-enriched formulas may be a first step to improve arginine availability and NO synthesis. In critically ill children, arginine metabolism and supplementation is however a virtually unexplored field. Since pediatric sepsis is a significant health problem, which differs in epidemiology and pathophysiology from sepsis in adults, and because of the scarcity of data in this population, studies focused on pathophysiological mechanisms and possible interventions in arginine metabolism in pediatric critical illness are warranted.

  6. Lysine nutrition in swine and the related monogastric animals: muscle protein biosynthesis and beyond.

    PubMed

    Liao, Shengfa F; Wang, Taiji; Regmi, Naresh

    2015-01-01

    Improving feed efficiency of pigs with dietary application of amino acids (AAs) is becoming increasingly important because this practice can not only secure the plasma AA supply for muscle growth but also protect the environment from nitrogen discharge with feces and urine. Lysine, the first limiting AA in typical swine diets, is a substrate for generating body proteins, peptides, and non-peptide molecules, while excess lysine is catabolized as an energy source. From a regulatory standpoint, lysine is at the top level in controlling AA metabolism, and lysine can also affect the metabolism of other nutrients. The effect of lysine on hormone production and activities is reflected by the change of plasma concentrations of insulin and insulin-like growth factor 1. Lysine residues in peptides are important sites for protein post-translational modification involved in epigenetic regulation of gene expression. An inborn error of a cationic AA transporter in humans can lead to a lysinuric protein intolerance condition. Dietary deficiency of lysine will impair animal immunity and elevate animal susceptibility to infectious diseases. Because lysine deficiency has negative impact on animal health and growth performance and it appears that dietary lysine is non-toxic even at a high dose of supplementation, nutritional emphasis should be put on lysine supplementation to avoid its deficiency rather than toxicity. Improvement of muscle growth of monogastric animals such as pigs via dietary lysine supply may be due to a greater increase in protein synthesis rather than a decrease in protein degradation. Nevertheless, the underlying metabolic and molecular mechanisms regarding lysine effect on muscle protein accretion merits further clarification. Future research undertaken to fully elucidate the metabolic and regulatory mechanisms of lysine nutrition could provide a sound scientific foundation necessary for developing novel nutritional strategies to enhance the muscle growth and

  7. 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.

  8. Severe dietary lysine restriction affects growth and body composition and hepatic gene expression for nitrogen metabolism in growing rats.

    PubMed

    Kim, J; Lee, K S; Kwon, D-H; Bong, J J; Jeong, J Y; Nam, Y S; Lee, M S; Liu, X; Baik, M

    2014-02-01

    Dietary lysine restriction may differentially affect body growth and lipid and nitrogen metabolism, depending on the degree of lysine restriction. This study was conducted to examine the effect of dietary lysine restriction on growth and lipid and nitrogen metabolism with two different degree of lysine restriction. Isocaloric amino acid-defined diets containing 1.4% lysine (adequate), 0.70% lysine (50% moderate lysine restriction) and 0.35% lysine (75% severe lysine restriction) were fed from the age of 52 to 77 days for 25 days in male Sprague-Dawley rats. The 75% severe lysine restriction increased (p < 0.05) food intake, but retarded (p < 0.05) growth, increased (p < 0.05) liver and muscle lipid contents and abdominal fat accumulation, increased (p < 0.05) blood urea nitrogen levels and mRNA levels of the serine-synthesizing 3-phosphoglycerate dehydrogenase gene, but decreased (p < 0.05) urea cycle arginase gene mRNA levels. In contrast, the 50% lysine restriction did not significantly (p > 0.05) affect body growth and lipid and nitrogen metabolism. Our results demonstrate that severe 75% lysine restriction has detrimental effects on body growth and deregulate lipid and nitrogen metabolism. PMID:23441935

  9. Recombinant arginine-degrading enzymes in metabolic anticancer therapy and bioanalytics.

    PubMed

    Stasyk, Oleh V; Boretsky, Yuriy R; Gonchar, Mykhailo V; Sibirny, Andriy A

    2015-03-01

    Tumor cells often exhibit specific metabolic defects due to the aberrations in oncogene-dependent regulatory and/or signaling pathways that distinguish them from normal cells. Among others, many malignant cells are deficient in biosynthesis of certain amino acids and concomitantly exhibit elevated sensitivity to deprivation of these amino acids. Although the underlying causes of such metabolic changes are still not fully understood, this feature of malignant cells is exploited in metabolic enzymotherapies based on single amino acid, e.g., arginine, deprivation. To achieve efficient arginine depletion in vivo, two recombinant enzymes, bacterial arginine deiminase and human arginase I have been evaluated and are undergoing further development. This review is aimed to summarize the current knowledge on the application of arginine-degrading enzymes as anticancer agents and as bioanalytical tools for arginine assays. The problems that have to be solved to optimize this therapy for clinical application are discussed. PMID:25231409

  10. Free amino acid profiling in the giant puffball mushroom (Calvatia gigantea) using UPLC-MS/MS.

    PubMed

    Kıvrak, İbrahim; Kıvrak, Şeyda; Harmandar, Mansur

    2014-09-01

    Wild edible and medicinal mushroom, Calvatia gigantea, was quantitatively analyzed for the determination of its free amino acids using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The concentrations of total free amino acids, essential and non-essential amino acids were 199.65 mg/100 g, 113.69 mg/100 g, and 85.96 mg/100 g in C. gigantea, respectively. This study showed that C. gigantea, so called a giant puffball mushroom, has free amino acids content. The essential amino acids: tryptophan, isoleucine, valine, phenylalanine, leucine, threonine, lysine, histidine, methionine, and the non-essential amino acids: tyrosine, 4-hyrdroxy proline, arginine, proline, glycine, serine, alanine, glutamine, glutamic acid, aspargine, aspartic acid were detected.

  11. Arginine and citrulline do not stimulate growth of two Oenococcus oeni strains in wine.

    PubMed

    Terrade, Nicolas; Mira de Orduña, Ramón

    2009-01-01

    Arginine metabolism by wine lactic acid bacteria (LAB) may lead to wine quality degradation. While arginine is essential for growth of the wine relevant LAB Oenococcus oeni, it remains unclear whether it also stimulates its growth. This study evaluated the effect of arginine and citrulline, the partially metabolized intermediate of the arginine deiminase pathway, on the growth of two commercial O. oeni strains in comparison with a Lactobacillus buchneri strain in wine and at wine pH values. Neither arginine nor citrulline increased growth of both O. oeni strains in comparison with the L. buchneri strain. However, arginine and citrulline were partially degraded in all incubations. The extent of citrulline degradation correlated with lower pH values in oenococcal cultivations but with higher pH values in those of the L. buchneri strain. The degradation kinetics of O. oeni and L. buchneri for malic acid and arginine differed and the latter grew in sterile filtered post-malolactic fermentation wine. This study shows that arginine and citrulline did not stimulate growth of the two O. oeni strains studied, and that their physiological role differed among the wine LAB considered. While arginine may play a role in wine microbiological stability, other nutrients should be investigated for their suitability to create a selective ecological advantage for O. oeni strains in wine. PMID:19025576

  12. 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

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

    PubMed

    Bakshi, Nitya; Morris, Claudia R

    2016-01-01

    Sickle cell disease (SCD) is the most common hemoglobinopathy in the US, affecting approximately 100,000 individuals in the US and millions worldwide. Pain is the hallmark of SCD, and a subset of patients experience pain virtually all of the time. Of interest, the arginine metabolome is associated with several pain mechanisms highlighted in this review. Since SCD is an arginine deficiency syndrome, the contribution of the arginine metabolome to acute and chronic pain in SCD is a topic in need of further attention. Normal arginine metabolism is impaired in SCD through various mechanisms that contribute to endothelial dysfunction, vaso-occlusion, pulmonary complications, risk of leg ulcers, and early mortality. Arginine is a semiessential amino acid that serves as a substrate for protein synthesis and is the precursor to nitric oxide (NO), polyamines, proline, glutamate, creatine, and agmatine. Since arginine is involved in multiple metabolic processes, a deficiency of this amino acid has the potential to disrupt many cellular and organ functions. NO is a potent vasodilator that is depleted in SCD and may contribute to vaso-occlusive pain. As the obligate substrate for NO production, arginine also plays a mechanistic role in SCD-related pain, although its contribution to pain pathways likely extends beyond NO. Low global arginine bioavailability is associated with pain severity in both adults and children with SCD as well as other non-SCD pain syndromes. Preliminary clinical studies of arginine therapy in SCD demonstrate efficacy in treating acute vaso-occlusive pain, as well as leg ulcers and pulmonary hypertension. Restoration of arginine bioavailability through exogenous supplementation of arginine is, therefore, a promising therapeutic target. Phase II clinical trials of arginine therapy for sickle-related pain are underway and a Phase III randomized controlled trial is anticipated in the near future. PMID:27099528

  14. Estimation of the standardized ileal digestible valine to lysine ratio required for 25- to 120-kilogram pigs fed low crude protein diets supplemented with crystalline amino acids.

    PubMed

    Liu, X T; Ma, W F; Zeng, X F; Xie, C Y; Thacker, P A; Htoo, J K; Qiao, S Y

    2015-10-01

    Four 28-d experiments were conducted to determine the standardized ileal digestible (SID) valine (Val) to lysine (Lys) ratio required for 26- to 46- (Exp. 1), 49- to 70- (Exp. 2), 71- to 92- (Exp. 3), and 94- to 119-kg (Exp. 4) pigs fed low CP diets supplemented with crystalline AA. The first 3 experiments utilized 150 pigs (Duroc × Landrace × Large White), while Exp. 4 utilized 90 finishing pigs. Pigs in all 4 experiments were randomly allocated to 1 of 5 diets with 6 pens per treatment (3 pens of barrows and 3 pens of gilts) and 5 pigs per pen for the first 3 experiments and 3 pigs per pen for Exp. 4. Diets for all experiments were formulated to contain SID Val to Lys ratios of 0.55, 0.60, 0.65, 0.70, or 0.75. In Exp. 1 (26 to 46 kg), ADG increased (linear, = 0.039; quadratic, = 0.042) with an increasing dietary Val:Lys ratio. The SID Val:Lys ratio to maximize ADG was 0.62 using a linear broken-line model and 0.71 using a quadratic model. In Exp. 2 (49 to 70 kg), ADG increased (linear, = 0.021; quadratic, = 0.042) as the SID Val:Lys ratio increased. G:F improved (linear, = 0.039) and serum urea nitrogen (SUN) decreased (linear, = 0.021; quadratic, = 0.024) with an increased SID Val:Lys ratio. The SID Val:Lys ratios to maximize ADG as well as to minimize SUN levels were 0.67 and 0.65, respectively, using a linear broken-line model and 0.72 and 0.71, respectively, using a quadratic model. In Exp. 3 (71 to 92 kg), ADG increased (linear, = 0.007; quadratic, = 0.022) and SUN decreased (linear, = 0.011; quadratic, = 0.034) as the dietary SID Val:Lys ratio increased. The SID Val:Lys ratios to maximize ADG as well as to minimize SUN levels were 0.67 and 0.67, respectively, using a linear broken-line model and 0.72 and 0.74, respectively, using a quadratic model. In Exp. 4 (94 to 119 kg), ADG increased (linear, = 0.041) and G:F was improved (linear, = 0.004; quadratic, = 0.005) as the dietary SID Val:Lys ratio increased. The SID Val:Lys ratio to maximize G:F was 0

  15. 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

  16. Poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): a bioactive copolymer for specific targeting to folate receptor-positive cancer cells.

    PubMed

    Chen, Yin; Cao, Wenbin; Zhou, Junli; Pidhatika, Bidhari; Xiong, Bin; Huang, Lu; Tian, Qian; Shu, Yiwei; Wen, Weijia; Hsing, I-Ming; Wu, Hongkai

    2015-02-01

    In this study, we present the preparation, characterization and application of a novel bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), which has a specific interaction with folate receptor (FR)-positive cancer cells. Glass surface immobilized with PLL-g-PMOXA-c-FA was demonstrated to be adhesive to FR-positive cancer cells (HeLa, JEG-3) while nonadhesive to FR-negative ones (MCF-7, HepG2) in 3 h. The specific interaction between conjugated FA on the substrate and FRs on the cells could hardly be inhibited unless a high concentration (5 mM) of free FA was used due to the multivalent nature of it. The FA functionality ratio of the copolymer on the substrate had a significant influence on the adhesion of HeLa cells, and our experiments revealed that the affinity of the substrate to the cells declined dramatically with the decrease of functionality ratio. This was believed to be caused by the polydispersity of PMOXA tethers, as supported by GPC and ToF-SIMS data. As a proof of concept in the application of our material, we demonstrated successful recovery of HeLa cells from mixture with MCF-7 (1:100) on the copolymer-coated glass, and our results showed that both high sensitivity (95.6 ± 13.3%) and specificity (24.3 ± 8.6%) were achieved.

  17. Lysine carboxylation in proteins: OXA-10 beta-lactamase.

    PubMed

    Li, Jie; Cross, Jason B; Vreven, Thom; Meroueh, Samy O; Mobashery, Shahriar; Schlegel, H Bernhard

    2005-11-01

    An increasing number of proteins are being shown to have an N(zeta)-carboxylated lysine in their structures, a posttranslational modification of proteins that proceeds without the intervention of a specific enzyme. The role of the carboxylated lysine in these proteins is typically structural (hydrogen bonding or metal coordination). However, carboxylated lysines in the active sites of OXA-10 and OXA-1 beta-lactamases and the sensor domain of BlaR signal-transducer protein serve in proton transfer events required for the functions of these proteins. These examples demonstrate the utility of this unusual amino acid in acid-base chemistry, in expansion of function beyond those of the 20 standard amino acids. In this study, the ONIOM quantum-mechanical/molecular-mechanical (QM/MM) method is used to study the carboxylation of lysine in the OXA-10 beta-lactamase. Lys-70 and the active site of the OXA-10 beta-lactamase were treated with B3LYP/6-31G(d,p) density functional calculations and the remainder of the enzyme with the AMBER molecular mechanics force field. The barriers for unassisted carboxylation of neutral lysine by carbon dioxide or bicarbonate are high. However, when the reaction with CO2 is catalyzed by a molecule of water in the active site, it is exothermic by about 13 kcal/mol, with a barrier of approximately 14 kcal/mol. The calculations show that the carboxylation and decarboxylation of Lys-70 are likely to be accompanied by deprotonation and protonation of the carbamate, respectively. The analysis may also be relevant for other proteins with carboxylated lysines, a feature that may be more common in nature than previously appreciated.

  18. Carbohydrate and amino acid metabolism of Spironucleus vortens.

    PubMed

    Millet, Coralie O M; Lloyd, David; Coogan, Michael P; Rumsey, Joanna; Cable, Joanne

    2011-09-01

    The metabolism of Spironucleus vortens, a parasitic, diplomonad flagellate related to Giardia intestinalis, was investigated using a combination of membrane inlet mass spectrometry, (1)H NMR, (13)C NMR, bioscreen continuous growth monitoring, and ion exchange chromatography. The products of glucose-fuelled and endogenous metabolism were identified by (1)H NMR and (13)C NMR as ethanol, acetate, alanine and lactate. Mass spectrometric monitoring of gas metabolism in buffered cell suspensions showed that glucose and ethanol could be used by S. vortens as energy-generating substrates, but bioscreen automated monitoring of growth in culture medium, as well as NMR analyses, suggested that neither of these compounds are the substrates of choice for this organism. Ion-exchange chromatographic analyses of free amino-acid and amino-acid hydrolysate of growth medium revealed that, despite the availability of large pools of free amino-acids in the medium, S. vortens hydrolysed large amounts of proteins during growth. The organism produced alanine and aspartate, and utilised lysine, arginine, leucine, cysteine and urea. However, mass spectrometric and bioscreen investigations showed that addition of the utilised amino acids to diluted culture medium did not induce any significant increase in metabolic or growth rates. Moreover, as no significant amounts of ornithine were produced, and addition of arginine under aerobic conditions did not generate NO production, there was no evidence of the presence of an energy-generating, arginine dihydrolase pathway in S. vortens under in vitro conditions.

  19. Application of PCDA/SPH/CHO/Lysine vesicles to detect pathogenic bacteria in chicken.

    PubMed

    de Oliveira, Taíla V; Soares, Nilda de F F; de Andrade, Nélio J; Silva, Deusanilde J; Medeiros, Eber Antônio A; Badaró, Amanda T

    2015-04-01

    During the course of infection, Salmonella must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments, as lysine decarboxylation to cadaverine. The idea of Salmonella defenses responses could be employed in systems as polydiacetylene (PDA) to detect this pathogen so important to public health system. Beside that PDA is an important substance because of the unique optical property; that undergoes a colorimetric transitions by various external stimuli. Therefore 10,12-pentacosadyinoic acid (PCDA)/Sphingomyelin(SPH)/Cholesterol(CHO)/Lysine system was tested to determine the colorimetric response induced by Salmonella choleraesuis. PCDA/SPH/CHO/Lysine vesicles showed a colour change even in low S. choleraesuis concentration present in laboratory conditions and in chicken meat. Thus, this work showed a PCDA/SPH/CHO/Lysine vesicle application to simplify routine analyses in food industry, as chicken meat industry.

  20. Biodegradable Tri-Block Copolymer Poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)(PLA-PEG-PLL) as a Non-Viral Vector to Enhance Gene Transfection

    PubMed Central

    Fu, Chunhua; Sun, Xiaoli; Liu, Donghua; Chen, Zhijing; Lu, Zaijun; Zhang, Na

    2011-01-01

    Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid)-poly(ethylene glycol)-poly(l-lysine) (PLA-PEG-PLL) copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z)-NCA onto amine-terminated NH2-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs) possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ɛ-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs) were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells) than PEI/DNA complexes (14.01% and 24.22%). These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo. PMID:21541064

  1. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  2. Lysine fortification: past, present, and future.

    PubMed

    Pellett, Peter L; Ghosh, Shibani

    2004-06-01

    Fortification with lysine to improve the protein value of human diets that are heavily based on cereals has received support from the results of these recent studies [1,2]. Support also comes from examination of average food and nutrient availability data derived from food balance sheets. Whereas nutritional status is influenced by the nutrient content of foods consumed in relation to need, the requirements for protein and amino acids are influenced by many additional factors [10, 12, 14, 28, 29]. These include age, sex, body size, physical activity, growth, pregnancy and lactation, infection, and the efficiency of nutrient utilization. Even if the immune response was influenced by the added lysine, adequate water and basic sanitation would remain essential. Acute and chronic undernutrition and most micronutrient deficiencies primarily affect poor and deprived people who do not have access to food of adequate nutritional value, live in unsanitary environments without access to clean water and basic services, and lack access to appropriate education and information [30]. A further variable is the possible interaction between protein and food energy availability [31]. This could affect the protein value of diets when food energy is limiting to a significant degree. Thus, the additional effects of food energy deficiency on protein utilization could well be superimposed on the very poorest. The improvement of dietary diversity must be the long-term aim, with dietary fortification considered only a short-term solution. The former should take place as wealth improves and the gaps between rich and poor diminish. Although such changes are taking place, they are highly uneven. Over the last several decades, increases have occurred in the availability of food energy, total protein, and animal protein for both developed and developing countries. However, for the very poorest developing countries over the same period, changes have been almost nonexistent, and the values for

  3. Characterization of the fibrinogen binding domain of bacteriophage lysin from Streptococcus mitis.

    PubMed

    Seo, Ho Seong; Sullam, Paul M

    2011-09-01

    The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis SF100 is mediated in part by a lysin encoded by the lysogenic bacteriophage SM1. In addition to its role in the phage life cycle, lysin mediates the binding of S. mitis to human platelets via its interaction with fibrinogen on the platelet surface. To better define the region of lysin mediating fibrinogen binding, we tested a series of purified lysin truncation variants for their abilities to bind this protein. These studies revealed that the fibrinogen binding domain of lysin is contained within the region spanned by amino acid residues 102 to 198 (lysin(102-198)). This region has no sequence homology to other known fibrinogen binding proteins. Lysin(102-198) bound fibrinogen comparably to full-length lysin and with the same selectivity for the fibrinogen Aα and Bβ chains. Lysin(102-198) also inhibited the binding in vitro of S. mitis to human fibrinogen and platelets. When assessed by platelet aggregometry, the disruption of the lysin gene in SF100 resulted in a significantly longer time to the onset of aggregation of human platelets than that of the parent strain. The preincubation of platelets with purified lysin(102-198) also delayed the onset of aggregation by SF100. These results indicate that the binding of lysin to fibrinogen is mediated by a specific domain of the phage protein and that this interaction is important for both platelet binding and aggregation by S. mitis. PMID:21690235

  4. Integrin αvβ3 as a Promising Target to Image Neoangiogenesis Using In-House Generator-Produced Positron Emitter (68)Ga-Labeled DOTA-Arginine-Glycine-Aspartic Acid (RGD) Ligand.

    PubMed

    Vatsa, Rakhee; Bhusari, Priya; Kumar, Sunil; Chakraborty, Sudipta; Dash, Ashutosh; Singh, Gurpreet; Dhawan, Devinder Kumar; Shukla, Jaya; Mittal, Bhagwant Rai

    2015-06-01

    For the growth and spread of a tumor beyond 2 mm, angiogenesis plays a crucial role, and association of various integrins with angiogenesis is evidential. The aim of the study was radiolabeling of DOTA-chelated RGD (arginine-glycine-aspartic acid) peptide with (68)Ga for PET imaging in locally advanced breast carcinoma. DOTA-RGD was incubated with (68)GaCl3, eluted in 0.05 m HCl. Elution volume, peptide amount, and reaction pH were studied. Radio-ITLC, gas chromatography, endotoxin, and sterility testing were performed. Serial (n=3) and whole-body (n=2) PET/CT imaging was done on patients post i.v. injection of 111-185 MBq of (68)Ga-DOTA-RGD. Maximum radiolabeling yield was achieved with 3 mL elution volume of 15-20 μg peptide at pH 3.5-4.0 with 10 minutes of incubation at 95°C. Product samples were sterile having 99.5% radiochemical purity with residual ethanol content and endotoxins in injectable limits. Intense radiotracer uptake was noticed in the tumor with SUVmax 15.3 at 45 minutes in serial images. Physiological radiotracer uptake was seen in the liver, spleen, ventricles, and thyroid with excretion through the kidneys. The authors concluded that (68)Ga-DOTA-RGD has the potential for imaging α,vβ3 integrin-expressing tumors. PMID:26083951

  5. Integrin αvβ3 as a Promising Target to Image Neoangiogenesis Using In-House Generator-Produced Positron Emitter (68)Ga-Labeled DOTA-Arginine-Glycine-Aspartic Acid (RGD) Ligand.

    PubMed

    Vatsa, Rakhee; Bhusari, Priya; Kumar, Sunil; Chakraborty, Sudipta; Dash, Ashutosh; Singh, Gurpreet; Dhawan, Devinder Kumar; Shukla, Jaya; Mittal, Bhagwant Rai

    2015-06-01

    For the growth and spread of a tumor beyond 2 mm, angiogenesis plays a crucial role, and association of various integrins with angiogenesis is evidential. The aim of the study was radiolabeling of DOTA-chelated RGD (arginine-glycine-aspartic acid) peptide with (68)Ga for PET imaging in locally advanced breast carcinoma. DOTA-RGD was incubated with (68)GaCl3, eluted in 0.05 m HCl. Elution volume, peptide amount, and reaction pH were studied. Radio-ITLC, gas chromatography, endotoxin, and sterility testing were performed. Serial (n=3) and whole-body (n=2) PET/CT imaging was done on patients post i.v. injection of 111-185 MBq of (68)Ga-DOTA-RGD. Maximum radiolabeling yield was achieved with 3 mL elution volume of 15-20 μg peptide at pH 3.5-4.0 with 10 minutes of incubation at 95°C. Product samples were sterile having 99.5% radiochemical purity with residual ethanol content and endotoxins in injectable limits. Intense radiotracer uptake was noticed in the tumor with SUVmax 15.3 at 45 minutes in serial images. Physiological radiotracer uptake was seen in the liver, spleen, ventricles, and thyroid with excretion through the kidneys. The authors concluded that (68)Ga-DOTA-RGD has the potential for imaging α,vβ3 integrin-expressing tumors.

  6. Recombinant bacteriophage lysins as antibacterials

    PubMed Central

    Fenton, Mark; Ross, Paul; McAuliffe, Olivia; O'Mahony, Jim

    2010-01-01

    With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential. PMID:21327123

  7. [Absorption of amino acids from the perfused ovine rumen].

    PubMed

    L' Leng; Tomás, J; Várady, J; Szányiová, M

    1978-06-01

    The experiments with extracoroporeal perfusion of sheep rumen were performed [Leng et al., 1977]. Bovine plasma, diluted in a 1:1ratio with an isotonic solution of sodium chloride, was used for four perfusions, and autologous blood was used for two perfusions in the course of 150 minutes. After 60 minutes perfusion 20 g enzymatic casein hydrolyzate were applied to the rumen. The levels of free amino acids in the perfusate were recorded after 60 minutes' perfusion [the first phase of perfusion] and at the end of the experiment [the second phase]. The levels of lysine, aspartic acid and glutamic acid increased after perfusions with bovine plasma during the first phase, the levels of glutamic acid, phenylalanine, and in one case of alanine, increased after perfusions with autologus blood. Simultaneously the level of valine decreased after perfusions with bovine plasma, and after perfusions with blood the levels of arginine and valine, and/or lysine, dropped. During the second phase of perfusion, the levels of all the observed amino acids except methionine [bovine plasma], and/or orginine and methionine [blood] rose in the perfusate. The experiments showed that the level of amino acids in the rumen content presented a decisive factor affecting amino acid absorption from the rumen into the blood. Transformation of the amino acids during their passage through the remen wall may be assumed, and glutamic acid is one of the chief products of this process.

  8. Effects of amino acids on the physiochemical properties of potato starch.

    PubMed

    Cui, Min; Fang, Ling; Zhou, Hongxian; Yang, Hong

    2014-05-15

    The objective of this study was to evaluate effects of different amino acid additives (phenylalanine (Phe), methionine (Met), lysine (Lys), arginine (Arg), aspartic acid (Asp) and glutamic acid (Glu)) on the physicochemical properties of potato starch gels. Charge-carrying amino acids (Lys, Arg, Asp and Glu) significantly decreased the swelling power, solubility, light transmittance, L(∗) value and gel strength of potato starch, but increased syneresis during freeze-thaw treatment, while neutral amino acids (Phe and Met) did not cause modifications in starch gels. During heating, potato starch with fortified charge-carrying amino acids showed a lower peak G' (storage modulus), when compared with Phe and Met. Results showed that charge-carrying amino acids could modify physicochemical properties and improve the nutritional values of starch-based products.

  9. 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.

  10. Role of basic amino acids in the interaction of bindin with sulfated fucans.

    PubMed

    DeAngelis, P L; Glabe, C G

    1988-10-18

    Bindin, the acrosomal sperm adhesion protein of the sea urchin Strongylocentrotus purpuratus, binds specifically and with high affinity (Kd = 10(-8) M) to egg sulfated fucans in the high ionic strength milieu of seawater (0.55 M salt). Previous studies indicated that the negatively charged sulfate groups of the polysaccharide are critical for binding which suggested a binding mechanism involving basic residues of bindin. We found that the binding of fucan to bindin or polyarginine is stable at the ionic strength of seawater, whereas the binding of fucan to polylysine or polyhistidine is inhibited by 50% or more at this ionic strength. Group-specific modification of either arginine, lysine, or histidine residues of bindin results in a substantial inactivation of fucan binding activity. Preincubation of bindin with fucan can almost completely protect bindin from inactivation by arginine-specific reagents, butanedione and phenylglyoxal, but only moderately slowed the inactivation by the histidine reagent diethyl pyrocarbonate. In contrast, prior fucan binding could not prevent loss of activity by the reaction of citraconic anhydride with lysine residues. Other sulfated polysaccharides which do not interact strongly with bindin did not protect binding from phenylglyoxal-mediated inactivation when 800-3000-fold more polysaccharide than fucan was used during the preincubation before modification. We found that the larger and more hydrophobic arginine-modifying reagents, camphorquinone-10-sulfonic acid and cyclohexanedione, fail to inactivate fucan binding, suggesting that essential arginine residues may reside in an environment with restricted accessibility to these reagents. Parallel kinetic studies monitoring [14C]phenylglyoxal incorporation and fucan binding inactivation indicate that several of the four total arginine residues may be critical for fucan binding.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Intramuscular bioavailability of ketoprofen lysine salt in horses.

    PubMed

    Anfossi, P; Villa, R; Montesissa, C; Carli, S

    1997-06-01

    Lysine salts are often used in human pharmaceuticals to increase the solubility and absorption of acidic drugs when these are administered parenterally. In this study the intramuscular bioavailability of ketoprofen administered as the lysine salt was evaluated in horses (n = 5) treated intravenously and intramuscularly (2.2 mg/kg active substance) in a cross-over study. The absorption rate of ketoprofen administered as the lysine salt was rather low: the mean residence time increased from 31.7 min after IV injection to 128.9 min (after IM injection), and the bioavailability was high (mean 92.4%). The calculated steady state plasma concentrations of ketoprofen during multiple dosage were much higher after intramuscular (0.106 g/ml) than after intravenous (0.066 microgram/ml) administration. Intramuscular injections of the ketoprofen lysine salt can therefore be given to horses, which are particularly prone to develop soft tissue reactions, since use of the lysine salt markedly reduced local irritation at the injection site.

  12. Site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance substrate specificity towards maltodextrin for enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G).

    PubMed

    Han, Ruizhi; Liu, Long; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Chen, Jian

    2013-07-01

    In this work, the site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase towards maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) by CGTase. When using maltodextrin as glycosyl donor, four mutants K47F (lysine→ phenylalanine), K47L (lysine→ leucine), K47V (lysine→ valine) and K47W (lysine→ tryptophan) showed higher AA-2G yield as compared with that produced by the wild-type CGTase. The transformation conditions (temperature, pH and the mass ratio of L-ascorbic acid to maltodextrin) were optimized and the highest titer of AA-2G produced by the mutant K47L could reach 1.97 g/l, which was 64.2% higher than that (1.20 g/l) produced by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, the four mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. The mechanism responsible for the enhanced substrate specificity was further explored by structure modeling and it was indicated that the enhancement of maltodextrin specificity may be due to the short residue chain and the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at -3 subsite. Here the obtained mutant CGTases, especially the K47L, has a great potential in the production of AA-2G with maltodextrin as a cheap and easily soluble substrate.

  13. The CASTOR proteins are arginine sensors for the mTORC1 pathway

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-05-01

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

  15. 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.

  16. The microbiome, intestinal function, and arginine metabolism of healthy Indian women are different from those of American and Jamaican women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Indian women have slower arginine flux during pregnancy compared with American and Jamaican women. Arginine is a semi-essential amino acid that becomes essential during periods of rapid lean tissue deposition. It is synthesized only from citrulline, a nondietary amino acid produced mainly in the gut...

  17. Targeting histone lysine demethylases — Progress, challenges, and the future☆

    PubMed Central

    Thinnes, Cyrille C.; England, Katherine S.; Kawamura, Akane; Chowdhury, Rasheduzzaman; Schofield, Christopher J.; Hopkinson, Richard J.

    2014-01-01

    N-Methylation of lysine and arginine residues has emerged as a major mechanism of transcriptional regulation in eukaryotes. In humans, Nε-methyllysine residue demethylation is catalysed by two distinct subfamilies of demethylases (KDMs), the flavin-dependent KDM1 subfamily and the 2-oxoglutarate- (2OG) dependent JmjC subfamily, which both employ oxidative mechanisms. Modulation of histone methylation status is proposed to be important in epigenetic regulation and has substantial medicinal potential for the treatment of diseases including cancer and genetic disorders. This article provides an introduction to the enzymology of the KDMs and the therapeutic possibilities and challenges associated with targeting them, followed by a review of reported KDM inhibitors and their mechanisms of action from kinetic and structural perspectives. This article is part of a Special Issue entitled: Methylation: A Multifaceted Modification — looking at transcription and beyond. PMID:24859458

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

    PubMed

    Marc Rhoads, J; Wu, Guoyao

    2009-05-01

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

  19. 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

  20. pH dependent growth of poly( L-lysine)/poly( L-glutamic) acid multilayer films and their cell adhesion properties

    NASA Astrophysics Data System (ADS)

    Richert, Ludovic; Arntz, Youri; Schaaf, Pierre; Voegel, Jean-Claude; Picart, Catherine

    2004-10-01

    The short-term interaction of chondrosarcoma cells with (PGA/PLL) polyelectrolyte multilayers was investigated in a serum-containing medium for films built at different pHs and subsequently exposed to the culture medium. The buildup of the films and their stability was first investigated by means of optical waveguide lightmode spectroscopy, quartz crystal microbalance, streaming potential measurements and atomic force microscopy. While film growth is linear at all pHs, after a few layers have been deposited the growth is much larger for the films built at basic pH and even more pronounced for those built at acidic pH. However, these latter films remain stable in the culture medium only if they have been crosslinked prior to the ionic strength and pH jumps. The films built at acidic pH were found to swell in water by about 200% whereas those built at other pHs did not swell in a physiological buffer. For thin films (≈20 nm) built at pH = 7.4, the detachment forces were dependent on the outermost layer, the forces being significantly higher on PLL-ending films than on PGA-ending ones. In contrast, for the thick films built at pH = 4.4 and at pH = 10.4 (thickness of the order of few hundred of nanometers), the detachment forces were independent of the outermost layer of the film. The films built at pH = 10.4, which shrink in contact with salt containing solutions, were highly cell adhesive whereas those built at acidic pH were highly cell resistant. Protein adsorption and film roughness (as measured by AFM) could not explain these striking differences. The high adhesion observed on the film built at pH 10.4 may rather be related to the secondary structure of the film and to its relatively low swellability in water, whereas the cell resistance of the films built at pH 4.4 may be linked to their high swellability. Therefore, for the PGA/PLL films, the cell adhesion properties can be tuned depending on the deposition pH of the polyelectrolyte solutions. This study

  1. Metabolism of Arginine by Aging and 7 Day Old Pumpkin Seedlings

    PubMed Central

    Splittstoesser, Walter E.

    1969-01-01

    The metabolism of arginine by etiolated pumpkin (Cucurbita moschata) seedlings was studied over various time and age intervals by injecting arginine-U-14C into the cotyledons. At most, 25% of the 14C was transported from the cotyledon to the axis tissue and the amount of this transport decreased with increasing age of the seedlings. The cotyledons of 25 day old plants contained 60% of the administered 14C as unmetabolized arginine. Little 14C was in sugars and it appeared that arginine was the primary translocation product. Time course studies showed that arginine was extensively metabolized and the labeling patterns suggest that different pathways were in operation in the axis and cotyledons. The amount of arginine incorporated into cotyledonary protein show that synthesis and turnover were occurring at rapid rate. Only 25% of the label incorporated into protein by 1.5 hr remained after 96 hr. The label in protein was stable in the axis tissue. By 96 hr 50% of the administered label occurred as 14CO2 and it appeared that arginine was metabolized, through glutamate, by the citrio acid cycle in the cotyledons. The experiments showed that an extensive conversion of arginine carbon into other amino acids did not occur. PMID:16657070

  2. Characterization of arginine decarboxylase from Dianthus caryophyllus.

    PubMed

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

    2004-04-01

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

  3. Root uptake of cationic amino acids by Arabidopsis depends on functional expression of amino acid permease 5.

    PubMed

    Svennerstam, Henrik; Ganeteg, Ulrika; Näsholm, Torgny

    2008-01-01

    * Specific transporters mediate uptake of amino acids by plant roots. Earlier studies have indicated that the lysine histidine transporter 1 and amino acid permease 1 participate in this process, but although plant roots have been shown to absorb cationic amino acids with high affinity, neither of these transporters seems to mediate transport of L-arginine (L-Arg) or L-lysine (L-Lys). * Here, a collection of T-DNA knockout mutants were screened for alterations in Arabidopsis root uptake rates of L-Arg and it was found that only the AAP5 mutant displayed clear phenotypic divergence on high concentrations of L-Arg. A second screen using low concentrations of (15)N-labelled L-Arg in the growth media also identified AAP5 as being involved in L-Arg acquisition. * Momentaneous root uptake of basic amino acids was strongly affected in AAP5 mutant lines, but their uptake of other types of amino acids was only marginally affected. Comparisons of the root uptake characteristics of AAP5 and LHT1 mutants corroborated the hypothesis that the two transporters have distinct affinity spectra in planta. * Root uptake of all tested amino acids, except L-aspartic acid (L-Asp), was significantly affected in double AAP5*LHT1 mutants, suggesting that these two transporters account for a major proportion of roots' uptake of amino acids at low concentrations. PMID:18681934

  4. Towards the absolute proton affinities of 20 α-amino acids

    NASA Astrophysics Data System (ADS)

    Maksić, Z. B.; Kovačević, B.

    1999-07-01

    The absolute proton affinities (APA) of 20 α-amino acids, as obtained by the MP2(fc)/6-311+G ∗∗//HF/6-31G ∗ + ZPVE(HF/6-31G ∗) and the scaled Hartree-Fock (HFsc) models, are presented. It is shown that the α-NH 2 group is protonated in all but four cases: lysine ( K), proline ( P), histidine ( H), and arginine ( R). There is a good overall agreement with experimental data measured by the kinetic method. However, there are some notable exceptions such as glutamine ( Q) and lysine ( K), where strong hydrogen bonds in the protonated forms occur. It is suggested that the present results and theoretical models employed could be useful for resolving such experimental ambiguities. Furthermore, it appears that the HFsc model provides an efficient tool for elucidating APAs of artificial α-AAs, derivatives of natural α-AAs and their oligomers.

  5. Evaluation of Drosophila metabolic labeling strategies for in vivo quantitative proteomic analyses with applications to early pupa formation and amino acid starvation.

    PubMed

    Chang, Ying-Che; Tang, Hong-Wen; Liang, Suh-Yuen; Pu, Tsung-Hsien; Meng, Tzu-Ching; Khoo, Kay-Hooi; Chen, Guang-Chao

    2013-05-01

    Although stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was first developed as a cell culture-based technique, stable isotope-labeled amino acids have since been successfully introduced in vivo into select multicellular model organisms by manipulating the feeding diets. An earlier study by others has demonstrated that heavy lysine labeled Drosophila melanogaster can be derived by feeding with an exclusive heavy lysine labeled yeast diet. In this work, we have further evaluated the use of heavy lysine and/or arginine for metabolic labeling of fruit flies, with an aim to determine its respective quantification accuracy and versatility. In vivo conversion of heavy lysine and/or heavy arginine to several nonessential amino acids was observed in labeled flies, leading to distorted isotope pattern and underestimated heavy to light ratio. These quantification defects can nonetheless be rectified at protein level using the normalization function. The only caveat is that such a normalization strategy may not be suitable for every biological application, particularly when modified peptides need to be individually quantified at peptide level. In such cases, we showed that peptide ratios calculated from the summed intensities of all isotope peaks are less affected by the heavy amino acid conversion and therefore less sequence-dependent and more reliable. Applying either the single Lys8 or double Lys6/Arg10 metabolic labeling strategy to flies, we quantitatively mapped the proteomic changes during the onset of metamorphosis and upon amino acid deprivation. The expression of a number of steroid hormone 20-hydroxyecdysone regulated proteins was found to be changed significantly during larval-pupa transition, while several subunits of the V-ATPase complex and components regulating actomyosin were up-regulated under starvation-induced autophagy conditions.

  6. The feeding route (enteral or parenteral) affects the plasma response of the dipetide Ala-Gln and the amino acids glutamine, citrulline and arginine, with the administration of Ala-Gln in preoperative patients.

    PubMed

    Melis, Gerdien C; Boelens, Petra G; van der Sijp, Joost R M; Popovici, Theodora; De Bandt, Jean-Pascal; Cynober, Luc; van Leeuwen, Paul A M

    2005-07-01

    Enhancement of depressed plasma concentrations of glutamine and arginine is associated with better clinical outcome. Supplementation of glutamine might be a way to provide the patient with glutamine, and also arginine, because glutamine provides the kidney with citrulline, from which the kidney produces arginine when plasma levels of arginine are low. The aim of the present study was to investigate the parenteral and enteral response of the administered dipeptide Ala-Gln, glutamine, citrulline and arginine. Therefore, seven patients received 20 g Ala-Gln, administered over 4 h, parenterally or enterally, on two separate occasions. Arterial blood samples were taken before and during the administration of Ala-Gln. ANOVA and a paired t test were used to test differences (P<0.05). Ala-Gln was undetectable with enteral administration, whereas Ala-Gln remained stable at a plasma concentration of 268 micromol/l throughout parenteral infusion and rapidly decreased towards zero after infusion was stopped. The highest level of glutamine was observed with parenteral infusion of the dipeptide, although enteral infusion also significantly increased plasma levels of glutamine. The highest plasma response of citrulline was observed with the enteral administration of the dipeptide, although parenteral administration also increased plasma levels of citrulline. Plasma arginine increased significantly with parenteral infusion, but not with enteral administration of Ala-Gln. In conclusion, administrations of Ala-Gln, parenteral or enteral, resulted in an increased plasma glutamine response, as compared with baseline. Interestingly, in spite of the high availability of citrulline with enteral administration of the dipeptide, only parenteral infusion of Ala-Gln increased plasma arginine concentration.

  7. Structural insights into the recovery of aldolase activity in N-acetylneuraminic acid lyase by replacement of the catalytically active lysine with γ-thialysine by using a chemical mutagenesis strategy.

    PubMed

    Timms, Nicole; Windle, Claire L; Polyakova, Anna; Ault, James R; Trinh, Chi H; Pearson, Arwen R; Nelson, Adam; Berry, Alan

    2013-03-01

    Chemical modification has been used to introduce the unnatural amino acid γ-thialysine in place of the catalytically important Lys165 in the enzyme N-acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing γ-thialysine regained 17 % of the wild-type activity. To understand the reason for this lower activity, we solved X-ray crystal structures of the wild-type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165-γ-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ-thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH-activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine-containing enzyme showed almost 30 % of the activity of the wild-type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid-containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK(a) of the residue at position 165 is perturbed by replacement with γ-thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.

  8. Rumen-protected methionine and lysine: effects on milk production and plasma amino acids of dairy cows with reference to metabolisable protein status.

    PubMed

    Awawdeh, Mofleh S

    2016-05-01

    Two experiments were conducted to study the effects of rumen-protected Met (RPM) alone or with rumen-protected Lys (RPL) on milk yield and plasma amino acids of dairy cows. In experiment 1, 24 multiparous Holstein cows (154 DIM) were assigned to one of 3 groups where each cow received 0 g/d of RPM and RPL (C), 30 g/d of RPM (M), or 30 g/d of RPM plus 25 g of RPL (ML). The study lasted for 8 weeks where milk yield and composition were determined weekly. Daily milk yield averaged 28·0, 27·8, and 29·7 kg/cow for the C, M, and ML groups, respectively. Dietary treatments had no effects (P ≥ 0·54) on milk contents of fat, lactose, solid non-fat or total solids. Milk protein content in the ML group was greater (P < 0·05) than the C and M groups. Plasma levels of all AA were not significantly (P ≥ 0·09) affected by supplemental RPL and/or RPM. In experiment 2, 30 multiparous Holstein cows (100 DIM) were assigned to one of 3 groups where each cow received 0 g/d of RPM and RPL (C), 50 g/d of RPM (M), or 50 g/d of RPM plus 25 g/d of RPL (ML). The study lasted for 5 weeks. Cows in the M (30·5 kg) and ML (31·4 kg) groups produced (P < 0·05) more milk than those of the C group (29·1 kg). Under conditions of this study, RPM plus RPL improved milk yield and protein contents of dairy cows and was better than supplying RPM alone. Response in milk yield to RPM and RPL was affected by the MP status of cows which deserves further investigation.

  9. Bioavailability of lysine in selected foods by rat growth assay.

    PubMed

    McDonough, F E; Bodwell, C E; Hitchins, A D; Staples, R S

    1989-01-01

    Lysine bioavailabilities in reference protein and 16 test protein diets were estimated using 10 day rat growth assays. A standard growth curve was obtained by feeding 5 diets containing casein, zein and synthetic amino acids ranging in total lysine concentration from 0.3 to 0.7%. Experimental foods were added to the basal diet at the expense of zein and/or synthetic amino acids to provide 2 specific lysine concentrations, i.e., 0.4 and 0.6%. Availabilities were established by comparing growth responses from the test food diets to the regression line of the standard growth data. Availabilities were over 88% for 13 of 16 products. Utilization was poor in pinto beans (73%), rice-wheat gluten cereal (70%), and skim milk powder heated to 100 degrees C for 12 h (66%). Addition of excess lysine (700 mg/100 g diet) to the pinto bean diet did not improve growth response; thus poor digestibility or some unidentified growth inhibitor is indicated. PMID:2496403

  10. Effects of free amino acids on cytokine secretion and proliferative activity of feline T cells in an in vitro study using the cell line MYA-1.

    PubMed

    Paßlack, Nadine; Doherr, Marcus G; Zentek, Jürgen

    2016-10-01

    In vitro studies might be an interesting screening method for targeted in vivo studies in the field of immunonutrition and help to reduce and refine animal studies. As the role of amino acids for immune function of cats has not been evaluated in detail so far, the present study aimed at investigating the effects of eight different amino acids (arginine, leucine, isoleucine, valine, glutamine, lysine, threonine and tryptophan) in six concentrations each (0, 0.25, 0.5, 1, 2 and 8x the cat blood level) on cytokine secretion and proliferative activity of feline T cells (MYA-1) in vitro. The results demonstrated that high doses of arginine increased IL-4, IL-10 and TNF-α secretion of T cells, while increasing concentrations of lysine increased IL-10 secretion and proliferative activity of the T cells. High doses of leucine enhanced GM-CSF and IL-10 secretion, while concentrations of threonine in the cell culture media greater than blood concentration also increased GM-CSF and additionally TNF-α secretion of the cells. The effects of glutamine and isoleucine on T cell function were only small. In conclusion, the present in vitro study could evaluate the immunomodulating potential of specific amino acids for feline T cell function. High doses of arginine, lysine, leucine and threonine had a significant impact on cytokine secretion and proliferative activity of the T cells. Targeted in vivo studies should investigate the clinical relevance of dietary supplementation of those amino acids in healthy and diseased cats as a next step. PMID:27510653

  11. Effects of free amino acids on cytokine secretion and proliferative activity of feline T cells in an in vitro study using the cell line MYA-1.

    PubMed

    Paßlack, Nadine; Doherr, Marcus G; Zentek, Jürgen

    2016-10-01

    In vitro studies might be an interesting screening method for targeted in vivo studies in the field of immunonutrition and help to reduce and refine animal studies. As the role of amino acids for immune function of cats has not been evaluated in detail so far, the present study aimed at investigating the effects of eight different amino acids (arginine, leucine, isoleucine, valine, glutamine, lysine, threonine and tryptophan) in six concentrations each (0, 0.25, 0.5, 1, 2 and 8x the cat blood level) on cytokine secretion and proliferative activity of feline T cells (MYA-1) in vitro. The results demonstrated that high doses of arginine increased IL-4, IL-10 and TNF-α secretion of T cells, while increasing concentrations of lysine increased IL-10 secretion and proliferative activity of the T cells. High doses of leucine enhanced GM-CSF and IL-10 secretion, while concentrations of threonine in the cell culture media greater than blood concentration also increased GM-CSF and additionally TNF-α secretion of the cells. The effects of glutamine and isoleucine on T cell function were only small. In conclusion, the present in vitro study could evaluate the immunomodulating potential of specific amino acids for feline T cell function. High doses of arginine, lysine, leucine and threonine had a significant impact on cytokine secretion and proliferative activity of the T cells. Targeted in vivo studies should investigate the clinical relevance of dietary supplementation of those amino acids in healthy and diseased cats as a next step.

  12. Correlation of carnitine levels to methionine and lysine intake.

    PubMed

    Krajcovicová-Kudlácková, M; Simoncic, R; Béderová, A; Babinská, K; Béder, I

    2000-01-01

    Plasma carnitine levels were measured in two alternative nutrition groups--strict vegetarians (vegans) and lactoovovegetarians (vegetarians consuming limited amounts of animal products such as milk products and eggs). The results were compared to an average sample of probands on mixed nutrition (omnivores). Carnitine levels were correlated with the intake of essential amino acids, methionine and lysine (as substrates of its endogenous synthesis), since the intake of carnitine in food is negligible in the alternative nutrition groups (the highest carnitine content is in meat, lower is in milk products, while fruit, cereals and vegetables contain low or no carnitine at all). An average carnitine level in vegans was significantly reduced with hypocarnitinemia present in 52.9% of probands. Similarly, the intake of methionine and lysine was significantly lower in this group due to the exclusive consumption of plant proteins with reduced content of these amino acids. Carnitine level in lactoovovegetarians was also significantly reduced, but the incidence of values below 30 micromol/l was lower than in vegans representing 17.8% vs. 3.3% in omnivores. Intake of methionine and lysine was also significantly reduced in this group, but still higher compared to vegans (73% of protein intake covered by plant proteins). Significant positive correlation of carnitine levels with methionine and lysine intake in alternative nutrition groups indicates that a significant portion of carnitine requirement is covered by endogenous synthesis. Approximately two thirds of carnitine requirement in omnivores comes from exogenous sources. The results demonstrate the risks of alternative nutrition with respect to the intake of essential amino acids, methionine and lysine, and with respect to the intake and biosynthesis of carnitine. PMID:11043928

  13. Correlation of carnitine levels to methionine and lysine intake.

    PubMed

    Krajcovicová-Kudlácková, M; Simoncic, R; Béderová, A; Babinská, K; Béder, I

    2000-01-01

    Plasma carnitine levels were measured in two alternative nutrition groups--strict vegetarians (vegans) and lactoovovegetarians (vegetarians consuming limited amounts of animal products such as milk products and eggs). The results were compared to an average sample of probands on mixed nutrition (omnivores). Carnitine levels were correlated with the intake of essential amino acids, methionine and lysine (as substrates of its endogenous synthesis), since the intake of carnitine in food is negligible in the alternative nutrition groups (the highest carnitine content is in meat, lower is in milk products, while fruit, cereals and vegetables contain low or no carnitine at all). An average carnitine level in vegans was significantly reduced with hypocarnitinemia present in 52.9% of probands. Similarly, the intake of methionine and lysine was significantly lower in this group due to the exclusive consumption of plant proteins with reduced content of these amino acids. Carnitine level in lactoovovegetarians was also significantly reduced, but the incidence of values below 30 micromol/l was lower than in vegans representing 17.8% vs. 3.3% in omnivores. Intake of methionine and lysine was also significantly reduced in this group, but still higher compared to vegans (73% of protein intake covered by plant proteins). Significant positive correlation of carnitine levels with methionine and lysine intake in alternative nutrition groups indicates that a significant portion of carnitine requirement is covered by endogenous synthesis. Approximately two thirds of carnitine requirement in omnivores comes from exogenous sources. The results demonstrate the risks of alternative nutrition with respect to the intake of essential amino acids, methionine and lysine, and with respect to the intake and biosynthesis of carnitine.

  14. Amperometric biosensor based on diamond paste for the enantioanalysis of L-lysine.

    PubMed

    Stefan-van Staden, Raluca-Ioana; Nejem, R'afat Mahmoud; van Staden, Jacobus Frederick; Aboul-Enein, Hassan Y

    2012-05-15

    An amperometric biosensor was proposed for the enantioanalysis of L-lysine. The biosensor is based on the impregnation of L-lysine oxidase in diamond paste. The potential used for the determination of l-lysine was 650 mV. The biosensor exhibited a linear concentration range between 1 and 100 nmol/L with a limit of detection of 4 pmol/L. The selectivity of the biosensor is high over other amino acids, such as L-serine, L-leucine, L-aspartic acid, L-glutamic acid, histamine, glycine. The proposed biosensor can be applied for the determination of L-lysine in serum samples and pharmaceutical compounds.

  15. Enteral Arginine Does Not Increase Superior Mesenteric Arterial Blood Flow but Induces Mucosal Growth in Neonatal Pigs123

    PubMed Central

    Puiman, Patrycja J.; Stoll, Barbara; van Goudoever, Johannes B.; Burrin, Douglas G.

    2011-01-01

    Arginine is an essential amino acid in neonates synthesized by gut epithelial cells and a precursor for NO that regulates vasodilatation and blood flow. Arginine supplementation has been shown to improve intestinal integrity in ischemia-reperfusion models and low plasma levels are associated with necrotizing enterocolitis. We hypothesized that enteral arginine is a specific stimulus for neonatal intestinal blood flow and mucosal growth under conditions of total parenteral nutrition (TPN) or partial enteral nutrition (PEN). We first tested the dose dependence and specificity of acute (3 h) enteral arginine infusion on superior mesenteric artery (SMA) blood flow in pigs fed TPN or PEN. We then determined whether chronic (4 d) arginine supplementation of PEN increases mucosal growth and if this was affected by treatment with the NO synthase inhibitor, NG-nitro-l-arginine methyl ester (L-NAME). Acute enteral arginine infusion increased plasma arginine dose dependently in both TPN and PEN groups, but the plasma response was markedly higher (100–250%) in the PEN group than in the TPN group at the 2 highest arginine doses. Baseline SMA blood flow was 90% higher in the PEN (2.37 ± 0.32 L⋅kg−1⋅h−1) pigs than in the TPN pigs (1.23 ± 0.17 L⋅kg−1⋅h−1), but was not affected by acute infusion individually of arginine, citrulline, or other major gut fuels. Chronic dietary arginine supplementation in PEN pigs induced mucosal growth in the intestine, but this effect was not prevented by treatment with L-NAME. Intestinal crypt cell proliferation, protein synthesis, and phosphorylation of mammalian target of rapamycin and p70S6 kinase were not affected by dietary arginine. We conclude that partial enteral feeding, but not acute enteral arginine, increases SMA blood flow in the neonatal pig. Furthermore, supplementing arginine in partial enteral feeding modestly increases intestinal mucosal growth and was NO independent. PMID:21106927

  16. Aminocella lysinolytica gen. nov., sp. nov., a L-lysine-degrading, strictly anaerobic bacterium in the class Clostridia isolated from a methanogenic reactor of cattle farms.

    PubMed

    Ueki, Atsuko; Shibuya, Toru; Kaku, Nobuo; Ueki, Katsuji

    2015-01-01

    A strictly anaerobic bacterial strain (WN037(T)) was isolated from a methanogenic reactor. Cells were Gram-positive rods. Strain WN037(T) was asaccharolytic. The strain fermented L-lysine in the presence of B-vitamin mixture or vitamin B12 and produced acetate and butyrate. L-arginine and casamino acids poorly supported the growth. Strain WN037(T) used neither other amino acids nor organic acids examined. The strain had C18:1 ω7c, C16:0 and C18:1 ω7c DMA as the predominant cellular fatty acids. The genomic DNA G + C content was 44.2 mol %. Phylogenetic analysis based on the 16S rRNA gene sequence placed strain WN037(T) in the family Eubacteriaceae in the class Clostridia. The closest relative was Eubacterium pyruvativorans (sequence similarity, 92.8 %). Based on the comprehensive analyses, the novel genus and species, Aminocella lysinolytica gen. nov., sp. nov. was proposed to accommodate the strain. The type strain is WN037(T) (= JCM 19863(T) = DSM 28287(T)).

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

    SciTech Connect

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

    1988-11-01

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

  18. Transition state stabilization by six arginines clustered in the active site of creatine kinase.

    PubMed

    Jourden, Michael J; Geiss, Paul R; Thomenius, Michael J; Horst, Lindsay A; Barty, Melissa M; Brym, Melissa J; Mulligan, Guy B; Almeida, Ryan M; Kersteen, Betsy A; Myers, Nichole R; Snider, Mark J; Borders, Charles L; Edmiston, Paul L

    2005-08-10

    Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.

  19. Measurements of radiological data of some amino acids in the energy range 0.122-1.330MeV

    NASA Astrophysics Data System (ADS)

    Lokhande, Rajkumar M.; More, Chaitali V.; Surung, Bharat S.; Raut, Siddheshwar D.; Pawar, Pravina P.

    2016-05-01

    Radiological parameters such as μm, σt, σe, Zeff and Neff of amino acids, namely Lysine (C6H15N2O2), Histidine (C6H9N3O2) and Arginine (C6H15N4O2), were measured using NaI (Tl)-based gamma spectrometry. Radioactive sources used in the study are 57Co, 133Ba, 137Cs, 54Mn, 60Co and 22Na. Gamma ray transmission method in a narrow beam good geometry set up was used in the study. The measured data were compared against XCOM-based data. The agreement is within 2%.

  20. Bioactive products of arginine in sepsis: tissue and plasma composition after LPS and iNOS blockade.

    PubMed

    Lortie, M J; Ishizuka, S; Schwartz, D; Blantz, R C

    2000-06-01

    Blockade or gene deletion of inducible nitric oxide synthase (iNOS) fails to fully abrogate all the sequelae leading to the high morbidity of septicemia. An increase in substrate uptake may be necessary for the increased production of nitric oxide (NO), but arginine is also a precursor for other bioactive products. Herein, we demonstrate an increase in alternate arginine products via arginine and ornithine decarboxylase in rats given lipopolysaccharide (LPS). The expression of iNOS mRNA in renal tissue was evident 60 but not 30 min post-LPS, yet a rapid decrease in blood pressure was obtained within 30 min that was completely inhibited by selective iNOS blockade. Plasma levels of arginine and ornithine decreased by at least 30% within 60 min of LPS administration, an effect not inhibited by the iNOS blocker L-N(6)(1-iminoethyl)lysine (L-NIL). Significant increases in plasma nitrates and citrulline occurred only 3-4 h post-LPS, an effect blocked by L-NIL pretreatment. The intracellular composition of organs harvested 6 h post-LPS reflected tissue-specific profiles of arginine and related metabolites. Tissue arginine concentration, normally an order of magnitude higher than in plasma, did not decrease after LPS. Pretreatment with L-NIL had a significant impact on the disposition of tissue arginine that was organ specific. These data demonstrate changes in arginine metabolism before and after de novo iNOS activity. Selective blockade of iNOS did not prevent uptake and can deregulate the production of other bioactive arginine metabolites.

  1. Nitrogen fertilizer factory effects on the amino acid and nitrogen content in the needles of Scots pine.

    PubMed

    Kupsinskiene, E

    2001-12-01

    The aim of the research was to evaluate the content of amino acids in the needles of Pinus sylvestris growing in the area affected by a nitrogen fertilizer factory and to compare them with other parameters of needles, trees, and sites. Three young-age stands of Scots pine were selected at a distance of 0.5 km, 5 km, and 17 km from the factory. Examination of the current-year needles in winter of the year 2000 revealed significant (p < 0.05) differences between the site at a 0.5-km distance from the factory and the site at a 17-km distance from the factory--with the site closest to the factory showing the highest concentrations of protein (119%), total arginine (166%), total other amino acids (depending on amino acid, the effect ranged between 119 and 149%), free arginine (771%), other free amino acids (glutamic acid, threonine, serine, lysine--depending on amino acid, the effect ranged between 162 and 234%), also the longest needles, widest diameter, largest surface area, and heaviest dry weight (respectively, 133, 110, 136, and 169%). The gradient of nitrogen concentration in the needles was assessed on the selected plots over the period of 1995-2000, with the highest concentration (depending on year, 119 to 153%) documented in the site located 0.5 km from the factory. Significant correlations were determined between the total amino acid contents (r = 0.448 -0.939, p < 0.05), some free amino acid (arginine, aspartic acid, glutamic acid, lysine, threonine, and serine) contents (r = 0.418 - 0.975, p < 0.05), and air pollutant concentration at the sites, the distance between the sites and the factory, and characteristics of the needles. No correlation was found between free or total arginine content and defoliation or retention of the needles. In conclusion, it was revealed that elevated mean monthly concentration of ammonia (26 microg m(-3)) near the nitrogen fertilizer factory caused changes in nitrogen metabolism, especially increasing (nearly eight times

  2. Nitrogen fertilizer factory effects on the amino acid and nitrogen content in the needles of Scots pine.

    PubMed

    Kupsinskiene, E

    2001-12-04

    The aim of the research was to evaluate the content of amino acids in the needles of Pinus sylvestris growing in the area affected by a nitrogen fertilizer factory and to compare them with other parameters of needles, trees, and sites. Three young-age stands of Scots pine were selected at a distance of 0.5 km, 5 km, and 17 km from the factory. Examination of the current-year needles in winter of the year 2000 revealed significant (p < 0.05) differences between the site at a 0.5-km distance from the factory and the site at a 17-km distance from the factory--with the site closest to the factory showing the highest concentrations of protein (119%), total arginine (166%), total other amino acids (depending on amino acid, the effect ranged between 119 and 149%), free arginine (771%), other free amino acids (glutamic acid, threonine, serine, lysine--depending on amino acid, the effect ranged between 162 and 234%), also the longest needles, widest diameter, largest surface area, and heaviest dry weight (respectively, 133, 110, 136, and 169%). The gradient of nitrogen concentration in the needles was assessed on the selected plots over the period of 1995-2000, with the highest concentration (depending on year, 119 to 153%) documented in the site located 0.5 km from the factory. Significant correlations were determined between the total amino acid contents (r = 0.448 -0.939, p < 0.05), some free amino acid (arginine, aspartic acid, glutamic acid, lysine, threonine, and serine) contents (r = 0.418 - 0.975, p < 0.05), and air pollutant concentration at the sites, the distance between the sites and the factory, and characteristics of the needles. No correlation was found between free or total arginine content and defoliation or retention of the needles. In conclusion, it was revealed that elevated mean monthly concentration of ammonia (26 microg m(-3)) near the nitrogen fertilizer factory caused changes in nitrogen metabolism, especially increasing (nearly eight times

  3. Stimulation of growth and polyamine biosynthesis of the ciliated protozoan Tetrahymena thermophila. Regulation by L-arginine.

    PubMed

    Eichler, W

    1989-10-01

    Tetrahymena thermophila cells grown in a synthetic nutrient medium for 9 h removed 97% of the free L-arginine but less than 50% of any of the other essential amino acids. The major portion of the arginine was degraded rapidly (76-92%) whereas 5-15% was conserved as intact and only 2.5-10% were incorporated into protein. However, if bovine serum albumin (BSA) was present in the medium as a macromolecular arginine source the incorporation of free arginine into protein was reduced to less than 1% but the degraded fraction was increased. Apparently, the uptake mode of arginine determines its fate: arginine taken up by phagocytosis is bound for protein biosynthesis, arginine taken up by membrane receptors is chanelled to degradation. Media without arginine did not support growth of Tetrahymena. Citrulline and ornithine, the precursors of arginine biosynthesis in yeast and vertebrates, were not able to substitute for arginine. Pronounced morphological changes, e.g. greatly reduced ribosome content, were observed in Tetrahymena cells after 24 h of arginine starvation in otherwise complete medium, but not in cells starved in water, salt solution, or buffer. Thus, arginine is an essential nutrient component for Tetrahymena and the rapid degradation of this compound involving the enzymes arginine deiminase (ADI) and citrulline hydrolase (CH) might be of regulatory importance for the unicellular, as it is the case with acetylcholine and catecholamines in mammalian organisms. Since the product of these enzymes, L-ornithine, is the substrate for the regulatory key enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), the effects of the presence of absence of arginine on the activities of each particular enzyme of the pathway were studied, including ODC and the enzyme ornithine-oxo-acid aminotransferase (O delta T), which is a competitor of ODC for the common substrate. The arginine-degradative pathway was stimulated by extracellular free but not by peptide

  4. Utilization of arginine by Klebsiella aerogenes.

    PubMed

    Friedrich, B; Magasanik, B

    1978-02-01

    Klebsiella aerogenes utilized arginine as the sole source of carbon or nitrogen for growth. Arginine was degraded to 2-ketoglutarate and not to succinate, since a citrate synthaseless mutant grows on arginine as the only nitrogen source. When glucose was the energy source, all four nitrogen atoms of arginine were utilized. Three of them apparently did not pass through ammonia but were transferred by transamination, since a mutant unable to produce glutamate by glutamate synthase or glutamate dehydrogenase utilized three of four nitrogen atoms of arginine. Urea was not involved as intermediate, since a unreaseless mutant did not accumulate urea and grew on arginine as efficiently as the wild-type strain. Ornithine appeared to be an intermediate, because cells grown either on glucose and arginine or arginine alone could convert arginine in the presence of hydroxylamine to ornithine. This indicates that an amidinotransferase is the initiating enzyme of arginine breakdown. In addition, the cells contained a transaminase specific for ornithine. In contrast to the hydroxylamine-dependent reaction, this activity could be demonstrated in extracts. The arginine-utilizing system (aut) is apparently controlled like the enzymes responsible for the degradation of histidine (hut) through induction, catabolite repression, and activation by glutamine synthetase.

  5. Metabolic fate of L-arginine in relation to microbiostatic capability of murine macrophages.

    PubMed Central

    Granger, D L; Hibbs, J B; Perfect, J R; Durack, D T

    1990-01-01

    L-arginine is required for the fungistatic action of murine macrophages in vitro. To further investigate this requirement, L-arginine metabolism by macrophages was measured under conditions where fungistasis either succeeded or failed. Macrophage fungistasis correlated with metabolism of L-arginine to citrulline, nitrite, and nitrate. The metabolic rate was dependent on extracellular L-arginine concentration, reaching a maximum of 67 nmol nitrite/h per mg protein. It accounted for one-third of arginine consumed by fungistatic macrophages. Equimolar amounts of citrulline and total nitrite plus nitrate accumulated in medium. This was consistent with the hypothesis that one of the equivalent guanidino nitrogens of L-arginine was oxidized to both nitrite and nitrate leaving L-citrulline as the amino acid reaction product. The analogue, NG-mono-methyl-L-arginine, selectively inhibited nitrogen oxidation and it was shown previously that it inhibited fungistatic capability. Resident macrophages were not fungistatic and their nitrogen oxidation was low. Once macrophages began producing nitrite/nitrate, protein synthesis was not required during the next 8 h for either fungistasis or nitrogen oxidation. Two-thirds of L-arginine consumption was due to macrophage arginase yielding L-ornithine and urea, which accumulated in medium. This activity was dissociated from macrophage fungistasis. Nitrogen oxidation metabolism by macrophages is linked to a mechanism that inhibits proliferation of fungi. This may involve synthesis of an intermediate compound(s) that has antimicrobial properties. PMID:2404026

  6. Effect alteration of methamphetamine by amino acids or their salts on ambulatory activity in mice.

    PubMed

    Kuribara, H; Tadokoro, S

    1983-02-01

    Effect alterations of methamphetamine by pretreatment of amino acids or their salts on ambulatory activity in mice were investigated to confirm a fact that certain amino acids, particularly monosodium L-glutamate, are added to methamphetamine by the street users, and that the amino acids augment the effect of methamphetamine. The ambulatory activity of mouse was measured by a tilting-type round activity cage of 25 cm in diameter. The amino acids or their salts tested were monosodium L-glutamate, monosodium L-aspartate, gamma-amino-butyric acid, L-alanine, L-lysine hydrochloride and L-arginine hydrochloride. A single administration of each chemical at doses of 1 and 2 g/kg i.p. did not induce a marked change in the ambulatory activity in mice. Methamphetamine 2 mg/kg s.c. induced an increase in the ambulatory activity with a peak at 40 min after the administration, and the increased ambulatory activity persisted for 3 hr. The ambulation-increasing effect of methamphetamine was augmented by the pretreatment of monosodium L-glutamate and monosodium L-aspartate at 30 min before the methamphetamine administration, while attenuated by the pretreatment of L-lysine hydrochloride and L-arginine hydrochloride in a dose-dependent manner. Gamma-aminobutyric acid and L-alanine did not affect the effect of methamphetamine. Similar augmentation and attenuation in the ambulation-increasing effect of methamphetamine were induced by the pretreatment of sodium bicarbonate 0.9 g/kg i.p. (urinary alkalizer) and ammonium chloride 0.07 g/kg i.p. (urinary acidifier), respectively. The urinary pH level was elevated by the administration of monosodium L-glutamate, monosodium L-aspartate and sodium bicarbonate, and decreased by L-lysine hydrochloride, L-arginine hydrochloride and ammonium chloride. Gamma-aminobutyric acid and L-alanine did not elicit a marked change in the urinary pH level. The present experiment confirms the fact in human that monosodium L-glutamate augments the effect of

  7. The effect of sugar, amino acid, metal ion, and NaCl on model Maillard reaction under pH control.

    PubMed

    Kwak, E-J; Lim, S-I

    2004-08-01

    The color intensities was determined of Maillard reaction products (MRPs) prepared by heating each of five sugars (maltose, fructose, glucose, arabinose, and xylose) with each of 12 amino acids (aspartic acid, glutamic acid, alanine, leucine, isoleucine, valine, proline, serine, cysteine, phenylalanine, arginine, and lysine). The remaining percentages of glucose and rate of change of color intensity due to the addition of a metal ion and NaCl were monitored for nine MRPs that had been formed between glucose and each of nine amino acids (aspartic acid, glutamic acid, alanine, valine, serine, cysteine, phenylalanine, arginine, and lysine). Model MRPs were prepared in a block heater at 100 degrees C for 1-12 h with the pH value controlled at 6.5. The resulting color intensity of each MRPs formed from the basic amino acids was greater due to the higher reactivity than those from the acidic amino acids. The remaining percentage of glucose in each MRPs from the basic amino acids was lower than those from the acidic amino acids. The MRPs from the nonpolar amino acids showed an intermediate color intensity and remaining percentages of glucose between those formed from the basic and acidic amino acids. Browning tended to be accelerated in the presence of metal ions, especially Fe2+ and Cu2+, although it was affected by the property of the amino acid and heating time as well as by the type of metal ion. On the other hand, browning was greatly inhibited by a high concentration of NaCl.

  8. Identification, expression and antibacterial activities of an antimicrobial peptide NK-lysin from a marine fish Larimichthys crocea.

    PubMed

    Zhou, Qi-Jia; Wang, Jun; Liu, Min; Qiao, Ying; Hong, Wan-Shu; Su, Yong-Quan; Han, Kun-Huang; Ke, Qiao-Zhen; Zheng, Wei-Qiang

    2016-08-01

    As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion. PMID:27238427

  9. 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

  10. Investigation of Lysine-Functionalized Dendrimers as Dichlorvos Detoxification Agents.

    PubMed

    Durán-Lara, Esteban F; Marple, Jennifer L; Giesen, Joseph A; Fang, Yunlan; Jordan, Jacobs H; Godbey, W Terrence; Marican, Adolfo; Santos, Leonardo S; Grayson, Scott M

    2015-11-01

    Lysine-containing polymers have seen broad application due to their amines' inherent ability to bind to a range of biologically relevant molecules. The synthesis of multiple generations of polyester dendrimers bearing lysine groups on their periphery is described in this report. Their hydrolytic stabilities with respect to pH and time, their toxicity to a range of cell lines, and their possible application as nano-detoxification agents of organophosphate compounds are all investigated. These zeroth-, first-, and second-generation water-soluble dendrimers have been designed to bear exactly 4, 8, and 16 lysine groups, respectively, on their dendritic periphery. Such monodisperse bioactive polymers show potential for a range of applications including drug delivery, gene delivery, heavy metal binding, and the sequestration of organic toxins. These monodisperse bioactive dendrimers were synthesized using an aliphatic ester dendritic core (prepared from pentaerythritol) and protected amino acid moieties. This library of lysine-conjugated dendrimers showed the ability to efficiently capture the pesticide dichlorvos, confirming the potential of dendrimer-based antidotes to maintain acetylcholinesterase activity in response to poisoning events. PMID:26460283

  11. Protein lysine methylation by seven-β-strand methyltransferases.

    PubMed

    Falnes, Pål Ø; Jakobsson, Magnus E; Davydova, Erna; Ho, Angela; Małecki, Jędrzej

    2016-07-15

    Methylation of biomolecules is a frequent biochemical reaction within the cell, and a plethora of highly specific methyltransferases (MTases) catalyse the transfer of a methyl group from S-adenosylmethionine (AdoMet) to various substrates. The posttranslational methylation of lysine residues, catalysed by numerous lysine (K)-specific protein MTases (KMTs), is a very common and important protein modification, which recently has been subject to intense studies, particularly in the case of histone proteins. The majority of KMTs belong to a class of MTases that share a defining 'SET domain', and these enzymes mostly target lysines in the flexible tails of histones. However, the so-called seven-β-strand (7BS) MTases, characterized by a twisted beta-sheet structure and certain conserved sequence motifs, represent the largest MTase class, and these enzymes methylate a wide range of substrates, including small metabolites, lipids, nucleic acids and proteins. Until recently, the histone-specific Dot1/DOT1L was the only identified eukaryotic 7BS KMT. However, a number of novel 7BS KMTs have now been discovered, and, in particular, several recently characterized human and yeast members of MTase family 16 (MTF16) have been found to methylate lysines in non-histone proteins. Here, we review the status and recent progress on the 7BS KMTs, and discuss these enzymes at the levels of sequence/structure, catalytic mechanism, substrate recognition and biological significance. PMID:27407169

  12. The regulation of ER export and Golgi retention of ST3Gal5 (GM3/GM4 synthase) and B4GalNAcT1 (GM2/GD2/GA2 synthase) by arginine/lysine-based motif adjacent to the transmembrane domain.

    PubMed

    Uemura, Satoshi; Shishido, Fumi; Kashimura, Madoka; Inokuchi, Jin-ichi

    2015-12-01

    In the Golgi maturation model, the Golgi cisternae dynamically mature along a secretory pathway. In this dynamic process, glycosyltransferases are transported from the endoplasmic reticulum (ER) to the Golgi apparatus where they remain and function. The precise mechanism behind this maturation process remains unclear. We investigated two glycosyltransferases, ST3Gal5 (ST3G5) and B4GalNAcT1 (B4GN1), involved in ganglioside synthesis and examined their signal sequences for ER export and Golgi retention. Reports have suggested that the [R/K](X)[R/K] motif functions as an ER exporting signal; however, this signal sequence is insufficient in stably expressed, full-length ST3G5. Through further analysis, we have clarified that the (2)R(3)R(X)(5) (9)K(X)(3) (13)K sequence in ST3G5 is essential for ER export. We have named the sequence the R/K-based motif. On the other hand, for ER export of B4GN1, the homodimer formation in addition to the R/K-based motif is required for ER export suggesting the importance of unidentified lumenal side interaction. We found that ST3G5 R2A/R3A and K9A/K13A mutants localized not only in Golgi apparatus but also in endosomes. Furthermore, the amounts of mature type asparagine-linked (N)-glycans in ST3G5 R2A/R3A and K9A/K13A mutants were decreased compared with those in wild-type proteins, and the stability of the mutants was lower. These results suggest that the R/K-based motif is necessary for the Golgi retention of ST3G5 and that the retention is involved in the maturation of N-glycans and in stability. Thus, several basic amino acids located on the cytoplasmic tail of ST3G5 play important roles in both ER export and Golgi retention.

  13. The regulation of ER export and Golgi retention of ST3Gal5 (GM3/GM4 synthase) and B4GalNAcT1 (GM2/GD2/GA2 synthase) by arginine/lysine-based motif adjacent to the transmembrane domain.

    PubMed

    Uemura, Satoshi; Shishido, Fumi; Kashimura, Madoka; Inokuchi, Jin-ichi

    2015-12-01

    In the Golgi maturation model, the Golgi cisternae dynamically mature along a secretory pathway. In this dynamic process, glycosyltransferases are transported from the endoplasmic reticulum (ER) to the Golgi apparatus where they remain and function. The precise mechanism behind this maturation process remains unclear. We investigated two glycosyltransferases, ST3Gal5 (ST3G5) and B4GalNAcT1 (B4GN1), involved in ganglioside synthesis and examined their signal sequences for ER export and Golgi retention. Reports have suggested that the [R/K](X)[R/K] motif functions as an ER exporting signal; however, this signal sequence is insufficient in stably expressed, full-length ST3G5. Through further analysis, we have clarified that the (2)R(3)R(X)(5) (9)K(X)(3) (13)K sequence in ST3G5 is essential for ER export. We have named the sequence the R/K-based motif. On the other hand, for ER export of B4GN1, the homodimer formation in addition to the R/K-based motif is required for ER export suggesting the importance of unidentified lumenal side interaction. We found that ST3G5 R2A/R3A and K9A/K13A mutants localized not only in Golgi apparatus but also in endosomes. Furthermore, the amounts of mature type asparagine-linked (N)-glycans in ST3G5 R2A/R3A and K9A/K13A mutants were decreased compared with those in wild-type proteins, and the stability of the mutants was lower. These results suggest that the R/K-based motif is necessary for the Golgi retention of ST3G5 and that the retention is involved in the maturation of N-glycans and in stability. Thus, several basic amino acids located on the cytoplasmic tail of ST3G5 play important roles in both ER export and Golgi retention. PMID:26362868

  14. Lys63-linked polyubiquitination of BRAF at lysine 578 is required for BRAF-mediated signaling

    PubMed Central

    An, Lei; Jia, Wei; Yu, Yang; Zou, Ning; Liang, Li; Zhao, Yanling; Fan, Yihui; Cheng, Jin; Shi, Zhongcheng; Xu, Gufeng; Li, Grace; Yang, Jianhua; Zhang, Hong

    2013-01-01

    The RAF kinase family is essential in mediating signal transduction from RAS to ERK. BRAF constitutively active mutations correlate with human cancer development. However, the precise molecular regulation of BRAF activation is not fully understood. Here we report that BRAF is modified by Lys63-linked polyubiquitination at lysine 578 within its kinase domain once it is activated by gain of constitutively active mutation or epidermal growth factor (EGF) stimulation. Substitution of BRAF lysine 578 with arginine (K578R) inhibited BRAF-mediated ERK activation. Furthermore, ectopic expression of BRAF K578R mutant inhibited anchorage-independent colony formation of MCF7 breast cancer cell line. Our studies have identified a previously unrecognized regulatory role of Lys63-linked polyubiquitination in BRAF-mediated normal and oncogenic signalings. PMID:23907581

  15. Formation and reduction of 5-hydroxymethylfurfural at frying temperature in model system as a function of amino acid and sugar composition.

    PubMed

    Kavousi, Parviz; Mirhosseini, Hamed; Ghazali, Hasanah; Ariffin, Abdul Azis

    2015-09-01

    5-Hydroxymethylfurfural (HMF) is formed during heat treatment of carbohydrate-containing foods, especially in a deep-fat frying process. This study aimed to investigate the effect of amino acids on the formation and reduction of HMF from glucose, fructose and sucrose at frying temperature in model systems containing binary mixtures of an amino acid and a sugar in equal concentrations (0.3M). The results revealed that the formation of HMF from sugars accelerated in the presence of acidic amino acids (i.e. glutamic and aspartic acids). Conversely, the presence of basic amino acids (i.e. lysine, arginine and histidine) led to reduced concentrations of HMF to non-detectable levels in model systems. The results showed that both pH and heating time significantly affected the formation of HMF from fructose in the presence of glutamic acid. In this regard, a higher amount of HMF was formed at lower pH.

  16. Formation and reduction of 5-hydroxymethylfurfural at frying temperature in model system as a function of amino acid and sugar composition.

    PubMed

    Kavousi, Parviz; Mirhosseini, Hamed; Ghazali, Hasanah; Ariffin, Abdul Azis

    2015-09-01

    5-Hydroxymethylfurfural (HMF) is formed during heat treatment of carbohydrate-containing foods, especially in a deep-fat frying process. This study aimed to investigate the effect of amino acids on the formation and reduction of HMF from glucose, fructose and sucrose at frying temperature in model systems containing binary mixtures of an amino acid and a sugar in equal concentrations (0.3M). The results revealed that the formation of HMF from sugars accelerated in the presence of acidic amino acids (i.e. glutamic and aspartic acids). Conversely, the presence of basic amino acids (i.e. lysine, arginine and histidine) led to reduced concentrations of HMF to non-detectable levels in model systems. The results showed that both pH and heating time significantly affected the formation of HMF from fructose in the presence of glutamic acid. In this regard, a higher amount of HMF was formed at lower pH. PMID:25842323

  17. Coacervate-like microspheres from lysine-rich proteinoid

    NASA Technical Reports Server (NTRS)

    Rohlfing, D. L.

    1975-01-01

    Microspheres form isothermally from lysine-rich proteinoid when the ionic strength of the solution is increased with NaCl or other salts. Studies with different monovalent anions and with polymers of different amino acid composition indicate that charge neutralization and hydrophobic bonding contribute to microsphere formation. The particles also form in sea water, especially if heated or made slightly alkaline. The microspheres differ from those made from acidic proteinoid but resemble coacervate droplets in some ways (isothermal formation, limited stability, stabilization by quinone, uptake of dyes). Because the constituent lysine-rich proteinoid is of simulated prebiotic origin, the study is interpreted to add emphasis to and suggest an evolutionary continuity for coacervation phenomena.

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

    PubMed

    Yoshizawa, Shunsuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2016-10-01

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

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

    PubMed

    Yoshizawa, Shunsuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2016-10-01

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

  20. Enteral arginine does not increase superior mesenteric arterial blood flow and but induces mucosal growth in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arginine is an essential amino acid in neonates synthesized by gut epithelial cells and a precursor for nitric oxide (NO) that regulates vasodilatation and blood flow. Arginine supplementation has been shown to improve intestinal integrity in ischemia-reperfusion models and low plasma levels are ass...

  1. Dietary L-arginine supplementation during mouse gestation enhances reproductive performance and Vegfr2 transcription activity in the fetoplacental unit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Regarded as one of the most versatile amino acids, arginine serves as a precursor for many molecules and has been reported to improve the reproductive performance of rats and pigs. To this end, we sought to determine if dietary L-arginine alters fetoplacental vascular endothelial growth factor recep...

  2. Acetylation of Conserved Lysines in Bovine Papillomavirus E2 by p300

    PubMed Central

    Quinlan, Edward J.; Culleton, Sara P.; Wu, Shwu-Yuan; Chiang, Cheng-Ming

    2013-01-01

    The p300, CBP, and pCAF lysine acetyltransferase (KAT) proteins have been reported to physically interact with bovine (BPV) and human (HPV) papillomavirus E2 proteins. While overexpression of these KAT proteins enhances E2-dependent transcription, the mechanism has not been determined. Using RNA interference (RNAi) to deplete these factors, we demonstrated that E2 transcriptional activity requires physiological levels of p300, CBP, and pCAF. Each protein appears to have a unique function in E2-dependent transcription, since overexpression of one KAT failed to compensate for RNAi knockdown of another KAT. Using an in vitro acetylation assay, we identified highly conserved lysines that are targeted by p300 for acetylation. The conservative changes of lysines at positions 111 and 112 to arginine were of particular interest. The K111R and the K111R/K112R mutants showed reduced transcriptional activity that was not responsive to p300 overexpression, while the K112R mutant retained activity. p300 and CBP were detected at the viral promoter; however, pCAF was not. We propose a model by which E2 transcriptional activity is controlled by p300-mediated acetylation of lysine 111. This model represents a novel mechanism regulating papillomavirus gene expression. PMID:23152516

  3. Histone lysine methyltransferases as anti-cancer targets for drug discovery

    PubMed Central

    Liu, Qing; Wang, Ming-wei

    2016-01-01

    Post-translational epigenetic modification of histones is controlled by a number of histone-modifying enzymes. Such modification regulates the accessibility of DNA and the subsequent expression or silencing of a gene. Human histone methyltransferases (HMTs)constitute a large family that includes histone lysine methyltransferases (HKMTs) and histone/protein arginine methyltransferases (PRMTs). There is increasing evidence showing a correlation between HKMTs and cancer pathogenesis. Here, we present an overview of representative HKMTs, including their biological and biochemical properties as well as the profiles of small molecule inhibitors for a comprehensive understanding of HKMTs in drug discovery. PMID:27397541

  4. Arginine nutrition and fetal brown adipose tissue development in diet-induced obese sheep.

    PubMed

    Carey Satterfield, M; Dunlap, Kathrin A; Keisler, Duane H; Bazer, Fuller W; Wu, Guoyao

    2012-10-01

    The global incidence of human obesity has more than doubled over the past three decades. An ovine model of obesity was developed to determine effects of maternal obesity and arginine supplementation on maternal, placental, and fetal parameters of growth, health, and well being. One-hundred-twenty days prior to embryo transfer, ewes were fed either ad libitum (n = 10) to induce obesity or 100% National Research Council-recommended nutrient requirements (n = 10) as controls. Embryos from superovulated ewes with normal body condition were transferred to the uterus of control-fed and obese ewes on day 5.5 post-estrus to generate genetically similar singleton pregnancies. Beginning on day 100 of gestation, obese ewes received intravenous administration of saline or L-arginine-HCl three times daily (81 mg arginine/kg body weight/day) to day 125, whereas control-fed ewes received saline. Fetal growth was assessed at necropsy on day 125. Maternal obesity increased (1) percentages of maternal and fetal carcass lipids and (2) concentrations of leptin, insulin, glucose, glutamate, leucine, lysine and threonine in maternal plasma while reducing (1) concentrations of progesterone, glycine and serine in maternal plasma and (2) amniotic and allantoic fluid volumes. Administration of L-arginine to obese ewes increased arginine and ornithine concentrations in maternal and fetal plasma, amniotic fluid volume, protein content in maternal carcass, and fetal brown adipose tissue (+60%), while reducing maternal lipid content and circulating leptin levels. Fetal or placental weight did not differ among treatments. Results indicate that arginine treatment beneficially reduces maternal adiposity and enhances fetal brown adipose tissue development in obese ewes.

  5. Administration of Exogenous Growth Hormone Is Associated with Changes in Plasma and Intracellular Mammary Amino Acid Profiles and Abundance of the Mammary Gland Amino Acid Transporter SLC3A2 in Mid-Lactation Dairy Cows

    PubMed Central

    Sciascia, Quentin L.; Pacheco, David; McCoard, Susan A.

    2015-01-01

    The objectives of this study were to (1) identify changes in plasma and mammary intracellular amino acid (AA) profiles in dairy cows treated with growth hormone (GH), and (2) evaluate the expression of mammary gland genes involved in the transport of AA identified in (1). Eight non-pregnant (n = 4 per group) lactating dairy cows were treated with a single subcutaneous injection of either a slow-release formulation of commercially available GH (Lactotropin 500 mg) or physiological saline solution. Six days after treatment, cows were milked and blood collected from the jugular vein for the analysis of free AA in the plasma. Cows were euthanized and mammary tissue harvested. Treatment with GH increased milk, protein, fat and lactose yields, with no effect on dry matter intake. Plasma concentrations of lysine and group I AA decreased significantly, and arginine, methionine, tyrosine and arginine-family AA tended to decrease in GH-treated cows. Concentrations of intracellular glycine, serine and glutamate increased significantly, with a trend for decreased arginine observed in the mammary gland of GH-treated cows. A trend for increased concentrations of intracellular total AA, NEAA and arginine-family AA were observed in the mammary gland of GH-treated cows. Variance in the concentration of plasma methionine, tyrosine, valine, alanine, ornithine, BCAA, EAA was significantly different between treatments. Variance in the concentration of intracellular lysine, valine, glutamine, EAA and group II was significantly different between treatments. AA changes were associated with increased mRNA abundance of the mammary gland AA transporter SLC3A2. We propose that these changes occur to support increased milk protein and fatty acid production in the mammary gland of GH-treated cows via potential mTOR pathway signaling. PMID:26226162

  6. Segregation for endosperm lysine in F2, F 3 and F 4 progeny from a cross of in vitro-selected and unselected cultivar of rice.

    PubMed

    Schaeffer, G W; Sharpe, F T; Dudley, J T

    1989-02-01

    Lysine is a limiting amino acid for optimal nutritional quality in rice grain. In vitro selections using inhibitory levels of lysine plus threonine or s-aminoethylcysteine allow the predictable recovery of variants with elevated levels of lysine and protein. These methods may generate useful starting germplasm for plant breeders. This study was conducted to define the genetics of lysine mutants in progeny from crosses of mutants derived from cells cultured in vitro in the presence of inhibitory levels of lysine plus threonine and s-(2-aminoethyl)-cysteine. In vitro selections produce a wide range of mutants, including endosperm mutants with elevated lysine and protein levels as well as mutants for high and low seed weights. Mutants were analyzed for lysine content by the endosperm half-seed method in which the halves without the embryo were ground and acid hydrolyzed for amino acid determinations. The halves with the embryos were preserved for later germination. In two different F2 populations derived from a cross of a selected mutant x M-101, a parental marker, there was an inverse relationship between seed weight and percent lysine in endosperm protein (R(2) 0.52 and 0.56). The F2 segregation patterns show that elevated lysine is inherited as a recessive gene and that increased lysine is correlated with decreased seed size. F3 and F4 data provide evidence for the transmission of high lysine genes to advanced germplasm in rice. This work supports our earlier conclusions that high lysine phenotypes can be recovered predictably from in vitro selections. The elevated lysine phenotypes are frequently, but not exclusively, associated with opaque seed. Some segregants from crosses produced increased lysine in plants with near normal seed weight and good fertility. PMID:24232525

  7. Arginine-based biodegradable ether-ester polymers with low cytotoxicity as potential gene carriers.

    PubMed

    Memanishvili, Tamar; Zavradashvili, Nino; Kupatadze, Nino; Tugushi, David; Gverdtsiteli, Marekh; Torchilin, Vladimir P; Wandrey, Christine; Baldi, Lucia; Manoli, Sagar S; Katsarava, Ramaz

    2014-08-11

    The success of gene therapy depends on safe and effective gene carriers. Despite being widely used, synthetic vectors based on poly(ethylenimine) (PEI), poly(l-lysine) (PLL), or poly(l-arginine) (poly-Arg) are not yet fully satisfactory. Thus, both improvement of established carriers and creation of new synthetic vectors are necessary. A series of biodegradable arginine-based ether-ester polycations was developed, which consists of three main classes: amides, urethanes, and ureas. Compared to that of PEI, PLL, and poly-Arg, much lower cytotoxicity was achieved for the new cationic arginine-based ether-ester polymers. Even at polycation concentrations up to 2 mg/mL, no significant negative effect on cell viability was observed upon exposure of several cell lines (murine mammary carcinoma, human cervical adenocarcinoma, murine melanoma, and mouse fibroblast) to the new polymers. Interaction with plasmid DNA yielded compact and stable complexes. The results demonstrate the potential of arginine-based ether-ester polycations as nonviral carriers for gene therapy applications. PMID:24963693

  8. CPLM: a database of protein lysine modifications

    PubMed Central

    Liu, Zexian; Wang, Yongbo; Gao, Tianshun; Pan, Zhicheng; Cheng, Han; Yang, Qing; Cheng, Zhongyi; Guo, Anyuan; Ren, Jian; Xue, Yu

    2014-01-01

    We reported an integrated database of Compendium of Protein Lysine Modifications (CPLM; http://cplm.biocuckoo.org) for protein lysine modifications (PLMs), which occur at active ε-amino groups of specific lysine residues in proteins and are critical for orchestrating various biological processes. The CPLM database was updated from our previously developed database of Compendium of Protein Lysine Acetylation (CPLA), which contained 7151 lysine acetylation sites in 3311 proteins. Here, we manually collected experimentally identified substrates and sites for 12 types of PLMs, including acetylation, ubiquitination, sumoylation, methylation, butyrylation, crotonylation, glycation, malonylation, phosphoglycerylation, propionylation, succinylation and pupylation. In total, the CPLM database contained 203 972 modification events on 189 919 modified lysines in 45 748 proteins for 122 species. With the dataset, we totally identified 76 types of co-occurrences of various PLMs on the same lysine residues, and the most abundant PLM crosstalk is between acetylation and ubiquitination. Up to 53.5% of acetylation and 33.1% of ubiquitination events co-occur at 10 746 lysine sites. Thus, the various PLM crosstalks suggested that a considerable proportion of lysines were competitively and dynamically regulated in a complicated manner. Taken together, the CPLM database can serve as a useful resource for further research of PLMs. PMID:24214993

  9. Arginine deprivation therapy for malignant melanoma

    PubMed Central

    Yoon, Jung-Ki; Frankel, Arthur E; Feun, Lynn G; Ekmekcioglu, Suhendan; Kim, Kevin B

    2013-01-01

    Despite recent development of promising immunotherapeutic and targeted drugs, prognosis in patients with advanced melanoma remains poor, and a cure for this disease remains elusive in most patients. The success of melanoma therapy depends on a better understanding of the biology of melanoma and development of drugs that effectively target the relevant genes or proteins essential for tumor cell survival. Melanoma cells frequently lack argininosuccinate synthetase, an essential enzyme for arginine synthesis, and as a result they become dependent on the availability of exogenous arginine. Accordingly, a therapeutic approach involving depletion of available arginine has been shown to be effective in preclinical studies. Early clinical studies have demonstrated sufficient antitumor activity to give rise to cautious optimism. In this article, the rationale for arginine deprivation therapy is discussed. Additionally, various strategies for depleting arginine are discussed and the preclinical and clinical investigations of arginine deprivation therapy in melanoma are reviewed. PMID:23293541

  10. Hemoglobin Labeled by Radioactive Lysine

    DOE R&D Accomplishments Database

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.

    1949-12-08

    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  11. Unusual Arginine Formations in Protein Function and Assembly: Rings, Strings and Stacks

    PubMed Central

    Neves, Marco A. C.; Yeager, Mark; Abagyan, Ruben

    2012-01-01

    Protein-protein interfaces are often stabilized by a small number of dominant contacts, exemplified by the overrepresentation of arginine residues at oligomerization interfaces. Positively charged arginines are most commonly involved in ion pairs of opposite charge; however, previous work of Scheraga and coworkers described the stable, close range interaction between guanidinium pairs in a solvated environment. To extend this work, we searched over 70 thousand protein structures and complexes for unusual formations of arginine residues supported by the electron density. Symmetry transformations were used to generate full assemblies. Clusters of four to eight arginine residues with Cζ-Cζ distances < 5 Å, organized as rings with 4 to 8 members, stacks of two arginines, and strings of stacked arginines, are commonly located at the interfaces of oligomeric proteins. The positive charge is properly balanced by negatively charged counter ions in about 90% of the cases. We also observed planar stacking of guanidinium groups, bridged by hydrogen bonds and interactions with water molecules. The guanidinium groups are commonly involved in 5 hydrogen bonds with water molecules and acceptor groups from surrounding amino acids. Water molecules have a bridging effect on the arginine pairs, but in some cases small molecular weight chemicals in the crystallization buffer may be misinterpreted as water molecules. In summary, despite electrostatic repulsion, arginines do form various clusters that are exposed to interact with and potentially be controlled or switched by charged metabolites, membrane lipids, nucleic acids or side chains of other proteins. Control of the stability of arginine clusters may play an important role in protein-protein oligomerization, molecular recognition and ligand binding. PMID:22497303

  12. Amino Acid Racemization in Pseudomonas putida KT2440

    PubMed Central

    Radkov, Atanas D.

    2013-01-01

    d-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the kcat/Km values with l- and d-lysine were 3 orders of magnitude greater than the kcat/Km values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher kcat/Km values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species. PMID:23995642

  13. Spectrophotometric assays for L-lysine alpha-oxidase and gamma-glutamylamine cyclotransferase.

    PubMed

    Danson, Jedidah W; Trawick, Mary Lynn; Cooper, Arthur J L

    2002-04-15

    A new assay for l-lysine alpha-oxidase is described. In this assay, the oxidized product generated from l-lysine is reacted with semicarbazide to form alpha-keto-epsilon-aminocaproate semicarbazone. Formation of the alpha-keto acid semicarbazone is continuously monitored spectrophotometrically at 248 nm (epsilon 10,160 +/- 240 M(-1) cm(-1)). The method was adapted to provide a new assay for gamma-glutamylamine cyclotransferase. This enzyme catalyzes the conversion of many l-gamma-glutamylamines to 5-oxo-l-proline and free amine. A biologically important substrate is N(epsilon)-(gamma-l-glutamyl)-l-lysine, which is converted to 5-oxo-l-proline and l-lysine by the action of gamma-glutamylamine cyclotransferase. The l-lysine generated from N(epsilon)-(gamma-l-glutamyl)-l-lysine in an endpoint assay is converted to alpha-keto epsilon-aminocaproate semicarbazone in the presence of semicarbazide, excess l-lysine alpha-oxidase, and catalase. The methods were applied to the determination of gamma-glutamylamine cyclotransferase activity of partially purified preparations of the bovine kidney enzyme and to detect gamma-glutamylamine cyclotransferase activity in rat kidney and liver homogenates. PMID:11950211

  14. Structural Basis for l-Lysine Feedback Inhibition of Homocitrate Synthase

    SciTech Connect

    Bulfer, Stacie L.; Scott, Erin M.; Pillus, Lorraine; Trievel, Raymond C.

    2010-09-02

    The {alpha}-aminoadipate pathway of lysine biosynthesis is modulated at the transcriptional and biochemical levels by feedback inhibition. The first enzyme in the {alpha}-aminoadipate pathway, homocitrate synthase (HCS), is the target of the feedback regulation and is strongly inhibited by L-lysine. Here we report the structure of Schizosaccharomyces pombe HCS (SpHCS) in complex with L-lysine. The structure illustrates that the amino acid directly competes with the substrate 2-oxoglutarate for binding within the active site of HCS. Differential recognition of the substrate and inhibitor is achieved via a switch position within the ({alpha}/{beta}){sub 8} TIM barrel of the enzyme that can distinguish between the C5-carboxylate group of 2-oxoglutarate and the {epsilon}-ammonium group of L-lysine. In vitro and in vivo assays demonstrate that mutations of the switch residues, which interact with the L-lysine {epsilon}-ammonium group, abrogate feedback inhibition, as do substitutions of residues within the C-terminal domain that were identified in a previous study of L-lysine-insensitive HCS mutants in Saccharomyces cerevisiae. Together, these results yield new insights into the mechanism of feedback regulation of an enzyme central to lysine biosynthesis.

  15. Genome-Wide Analysis of the Lysine Biosynthesis Pathway Network during Maize Seed Development

    PubMed Central

    Liu, Yuwei; Xie, Shaojun; Yu, Jingjuan

    2016-01-01

    Lysine is one of the most limiting essential amino acids for humans and livestock. The nutritional value of maize (Zea mays L.) is reduced by its poor lysine content. To better understand the lysine biosynthesis pathway in maize seed, we conducted a genome-wide analysis of the genes involved in lysine biosynthesis. We identified lysine biosynthesis pathway genes (LBPGs) and investigated whether a diaminopimelate pathway variant exists in maize. We analyzed two genes encoding the key enzyme dihydrodipicolinate synthase, and determined that they contribute differently to lysine synthesis during maize seed development. A coexpression network of LBPGs was constructed using RNA-sequencing data from 21 developmental stages of B73 maize seed. We found a large set of genes encoding ribosomal proteins, elongation factors and zein proteins that were coexpressed with LBPGs. The coexpressed genes were enriched in cellular metabolism terms and protein related terms. A phylogenetic analysis of the LBPGs from different plant species revealed different relationships. Additionally, six transcription factor (TF) families containing 13 TFs were identified as the Hub TFs of the LBPGs modules. Several expression quantitative trait loci of LBPGs were also identified. Our results should help to elucidate the lysine biosynthesis pathway network in maize seed. PMID:26829553

  16. Light-activated amino acid transport in Halobacterium halobium envelope vesicles

    NASA Technical Reports Server (NTRS)

    Macdonald, R. E.; Lanyi, J. K.

    1977-01-01

    Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na+ gradient. On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids: arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.

  17. Compartmentalization of amino acids in surfactant aggregates - Partitioning between water and aqueous micellar sodium dodecanoate and between hexane and dodecylammonium propionate trapped water in hexane

    NASA Technical Reports Server (NTRS)

    Fendler, J. H.; Nome, F.; Nagyvary, J.

    1975-01-01

    The partitioning of amino acids (glycine, alanine, leucine, phenylalanine, histidine, aspartic acid, glutamic acid, lysine, isoleucine, threonine, serine, valine, proline, arginine) in aqueous and nonaqueous micellar systems was studied experimentally. Partitioning from neat hexane into dodecylammonium propionate trapped water in hexane was found to be dependent on both electrostatic and hydrophobic interactions, which implies that the interior of dodecylammonium propionate aggregates is negatively charged and is capable of hydrogen bonding in addition to providing a hydrophobic environment. Unitary free energies of transfer of amino acid side chains from hexane to water were determined and solubilities of amino acids in neat hexane substantiated the amino acid hydrophobicity scale. The relevance of the experiments to prebiotic chemistry was examined.

  18. Stimulation of L-ornithine uptake and L-citrulline and urea biosynthesis by D-arginine.

    PubMed

    Saavedra-Molina, A; Piña, E

    1991-05-01

    The action of D-arginine on isolated cells and mitochondria obtained from rat liver was studied. The D-amino acid at 200 microM stimulated by 40% the rate of urea biosynthesis by isolated hepatocytes. Citrulline formation was increased 60-70% in rat liver mitochondria incubated with 10 microM D-arginine. In these mitochondria, ornithine uptake was enhanced 204% with 1 microM D-arginine. Inhibition in urea and citrulline synthesis and in ornithine uptake was recorded with high concentrations of the D-amino acid. Respiratory control in liver mitochondria with glutamate-malate was inhibited 32% by 100 microM D-arginine. In isolated mitochondria loaded with Fluo-3-acetoxymethyl (AM) ester, 50 microM D-arginine diminished the matrix free calcium concentration. PMID:1930251

  19. Depletion of arginine in yeast cells decreases the resistance to hydrostatic pressure

    NASA Astrophysics Data System (ADS)

    Nomura, Kazuki; Iwahashi, Hitoshi; Iguchi, Akinori; Shigematsu, Toru

    2015-07-01

    High hydrostatic pressure (HP) inhibits growth and inactivates microorganisms by destabilizing non-covalent molecular interactions. Arginine contributes to stress resistance because it has a guanidine side chain, which assists in the refolding of aggregated proteins. We attempted to analyze the contribution of arginine to high HP stress using a pressure-sensitive mutant strain of Saccharomyces cerevisiae and a metabolomics approach. Our results showed that the content of 136 out of 250 detected metabolites differed in the mutant and parent strains. Decreased metabolites were involved in the tricarboxylic acid cycle and arginine biosynthesis. The expression of genes contributing to arginine biosynthesis was significantly lower in the mutant strain than in the parent strain. When arginine was supplemented to the medium, the mutant strain showed more tolerance to pressure. These results suggest that yeast cells survived due to the contribution of arginine to high pressure resistance. This indicates that depletion of arginine caused by decreased activity of the biosynthesis pathway confers sensitivity to HP.

  20. Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity

    PubMed Central

    Carroll, Bernadette; Maetzel, Dorothea; Maddocks, Oliver DK; Otten, Gisela; Ratcliff, Matthew; Smith, Graham R; Dunlop, Elaine A; Passos, João F; Davies, Owen R; Jaenisch, Rudolf; Tee, Andrew R; Sarkar, Sovan; Korolchuk, Viktor I

    2016-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) is the key signaling hub that regulates cellular protein homeostasis, growth, and proliferation in health and disease. As a prerequisite for activation of mTORC1 by hormones and mitogens, there first has to be an available pool of intracellular amino acids. Arginine, an amino acid essential during mammalian embryogenesis and early development is one of the key activators of mTORC1. Herein, we demonstrate that arginine acts independently of its metabolism to allow maximal activation of mTORC1 by growth factors via a mechanism that does not involve regulation of mTORC1 localization to lysosomes. Instead, arginine specifically suppresses lysosomal localization of the TSC complex and interaction with its target small GTPase protein, Rheb. By interfering with TSC-Rheb complex, arginine relieves allosteric inhibition of Rheb by TSC. Arginine cooperates with growth factor signaling which further promotes dissociation of TSC2 from lysosomes and activation of mTORC1. Arginine is the main amino acid sensed by the mTORC1 pathway in several cell types including human embryonic stem cells (hESCs). Dependence on arginine is maintained once hESCs are differentiated to fibroblasts, neurons, and hepatocytes, highlighting the fundamental importance of arginine-sensing to mTORC1 signaling. Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signaling. DOI: http://dx.doi.org/10.7554/eLife.11058.001 PMID:26742086

  1. The MurE synthetase from Thermotoga maritima is endowed with an unusual D-lysine adding activity.

    PubMed

    Boniface, Audrey; Bouhss, Ahmed; Mengin-Lecreulx, Dominique; Blanot, Didier

    2006-06-01

    The peptidoglycan of Thermotoga maritima, an extremely thermophilic eubacterium, was shown to contain no diaminopimelic acid and approximate amounts of both enantiomers of lysine (Huber, R., Langworthy, T. A., König, H., Thomm, M., Woese, C. R., Sleytr, U. B., and Stetter, K. O. (1986) Arch. Microbiol. 144, 324-333). To assess the possible involvement of the MurE activity in the incorporation of D-lysine, the murE gene from this organism was cloned in Escherichia coli, and the corresponding protein was purified as the C-terminal His6-tagged form. In vitro assays showed that D-lysine and meso-diaminopimelic acid were added to UDP-N-acetylmuramoyl-dipeptide with 25 and 10% efficiencies, respectively, relative to L-lysine. The purified enzyme was used to synthesize the L- and D-lysine-containing UDP-N-acetylmuramoyl-tripeptides; chemical analysis revealed an unusual structure for the D-lysine-containing nucleotide, namely acylation of the epsilon-amino function of D-lysine by the D-glutamyl residue. In vitro assays with MurF and MraY enzymes from T. maritima showed that this novel nucleotide was not a substrate for MurF but that it could be directly processed into tripeptide lipid I by MraY, thereby substantiating the role of MurE in the incorporation of D-lysine into peptidoglycan.

  2. The Construction and Expression of Lysine-Rich Gene in the Mammary Gland of Transgenic Mice

    PubMed Central

    Ma, Xin; Zhang, Peng; Song, Guangqi; Chen, Yue; Wang, Zhongwei; Yin, Yupeng; Kong, Delong; Zhang, Sheng; Zhao, Zhihui; Ouyang, Hongsheng

    2012-01-01

    Lysine is the limiting amino acid in cereal grains, which represent a major source of human food and animal feed worldwide, and is considered the most important of the essential amino acids. In this study, β-casein, αS2-casein, and lactotransferrin cDNA clone fragments encoding lysine-rich peptides were fused together to generate a lysine-rich (LR) gene and the mammary gland-specific expression vector pBC1-LR-NEOr was constructed. Transgenic mice were generated by pronuclear microinjection of the linearized expression vectors harboring the LR transgene. The transgenic mice and their offspring were examined using multiplex polymerase chain reaction (PCR), Southern blotting, reverse transcriptase–PCR, in situ hybridization, and Western blotting techniques. Our results showed that the LR gene was successfully integrated into the mouse genome and was transmitted stably. The specific LR gene expression was restricted to the mammary gland, active alveoli of the transgenic female mice during lactation. The lysine level of the two transgenic lines was significantly higher than that of nontransgenic controls (p<0.05). In addition, the growth performance of transgenic pups was enhanced by directly feeding them the LR protein-enriched transgenic milk. Our results demonstrated that lysine-rich gene was successfully constructed and expressed in mammary gland of transgenic mice. This study will provide a better understanding of how mammary gland expression systems that increase the lysine content of milk can be applied to other mammals, such as cows. PMID:22577831

  3. Na/sup +/-dependent transport of /sup 14/C-L-lysine across bullfrog alveolar epithelium

    SciTech Connect

    Kim, K.J.; Crandall, E.D.

    1986-03-01

    Transepithelial transport of the basic amino acid L-lysine has been studied utilizing the isolated intact bullfrog lung mounted in the Ussing chamber. Lungs were excised from doubly pithed bullfrogs and sandwiched between two hemichambers. /sup 14/C-(U)-L-lysine was added to the upstream reservoir of amphibian Ringer solution, while the tissue was short-circuited. Two lungs from the same animal were used simultaneously to determine the two opposite unidirectional fluxes. Downstream and upstream radioactivities were assayed and used to estimate the apparent permeability (P) of the labeled lysine. Results indicate that the apparent P of /sup 14/C-L-lysine measured in the alveolar (M) to the pleural (S) direction is 19.06 (+- 2.84) x 10/sup -7/ cm/s and P in the S to M direction is 3.29 (+- 0.02) x 10/sup -7/ cm/s. When the 100 mM NaCl in the bath was replaced by 110 mM choline chloride, the flux of /sup 14/C-L-lysine from the alveolar to the pleural side decreased to the same value as that in the opposite direction. The flux from the pleural to the alveolar direction in the absence of Na/sup +/ did not change. These results suggest that the alveolar epithelium exhibits Na/sup +/-dependent amino acid (L-lysine) transport in the M->S, but not in the S->M, direction.

  4. Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids.

    PubMed

    Mohapatra, Sridev; Minocha, Rakesh; Long, Stephanie; Minocha, Subhash C

    2010-04-01

    The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and gamma-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra x maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and gamma-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.

  5. Engineering a Lysine-ON Riboswitch for Metabolic Control of Lysine Production in Corynebacterium glutamicum.

    PubMed

    Zhou, Li-Bang; Zeng, An-Ping

    2015-12-18

    Riboswitches are natural RNA elements that regulate gene expression by binding a ligand. Here, we demonstrate the possibility of altering a natural lysine-OFF riboswitch from Eschericia coli (ECRS) to a synthetic lysine-ON riboswitch and using it for metabolic control. To this end, a lysine-ON riboswitch library was constructed using tetA-based dual genetic selection. After screening the library, the functionality of the selected lysine-ON riboswitches was examined using a report gene, lacZ. Selected lysine-ON riboswitches were introduced into the lysE gene (encoding a lysine transport protein) of Corynebacterium glutamicum and used to achieve dynamic control of lysine transport in a recombinant lysine-producing strain, C. glutamicum LPECRS, which bears a deregulated aspartokinase and a lysine-OFF riboswitch for dynamic control of the enzyme citrate synthase. Batch fermentation results of the strains showed that the C. glutamicum LPECRS strain with an additional lysine-ON riboswitch for the control of lysE achieved a 21% increase in the yield of lysine compared to that of the C. glutamicum LPECRS strain and even a 89% increase in yield compared to that of the strain with deregulated aspartokinase. This work provides a useful approach to generate lysine-ON riboswitches for C. glutamicum metabolic engineering and demonstrates for the first time a synergetic effect of lysine-ON and -OFF riboswitches for improving lysine production in this industrially important microorganism. The approach can be used to dynamically control other genes and can be applied to other microorganisms. PMID:26300047

  6. European Sea Bass (Dicentrarchus labrax) Immune Status and Disease Resistance Are Impaired by Arginine Dietary Supplementation.

    PubMed

    Azeredo, Rita; Pérez-Sánchez, Jaume; Sitjà-Bobadilla, Ariadna; Fouz, Belén; Tort, Lluis; Aragão, Cláudia; Oliva-Teles, Aires; Costas, Benjamín

    2015-01-01

    Infectious diseases and fish feeds management are probably the major expenses in the aquaculture business. Hence, it is a priority to define sustainable strategies which simultaneously avoid therapeutic procedures and reinforce fish immunity. Currently, one preferred approach is the use of immunostimulants which can be supplemented to the fish diets. Arginine is a versatile amino acid with important mechanisms closely related to the immune response. Aiming at finding out how arginine affects the innate immune status or improve disease resistance of European seabass (Dicentrarchus labrax) against vibriosis, fish were fed two arginine-supplemented diets (1% and 2% arginine supplementation). A third diet meeting arginine requirement level for seabass served as control diet. Following 15 or 29 days of feeding, fish were sampled for blood, spleen and gut to assess cell-mediated immune parameters and immune-related gene expression. At the same time, fish from each dietary group were challenged against Vibrio anguillarum and survival was monitored. Cell-mediated immune parameters such as the extracellular superoxide and nitric oxide decreased in fish fed arginine-supplemented diets. Interleukins and immune-cell marker transcripts were down-regulated by the highest supplementation level. Disease resistance data were in accordance with a generally depressed immune status, with increased susceptibility to vibriosis in fish fed arginine supplemented diets. Altogether, these results suggest a general inhibitory effect of arginine on the immune defences and disease resistance of European seabass. Still, further research will certainly clarify arginine immunomodulation pathways thereby allowing the validation of its potential as a prophylactic strategy.

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

    PubMed Central

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

    2016-01-01

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

  8. European Sea Bass (Dicentrarchus labrax) Immune Status and Disease Resistance Are Impaired by Arginine Dietary Supplementation

    PubMed Central

    Azeredo, Rita; Pérez-Sánchez, Jaume; Sitjà-Bobadilla, Ariadna; Fouz, Belén; Tort, Lluis; Aragão, Cláudia; Oliva-Teles, Aires; Costas, Benjamín

    2015-01-01

    Infectious diseases and fish feeds management are probably the major expenses in the aquaculture business. Hence, it is a priority to define sustainable strategies which simultaneously avoid therapeutic procedures and reinforce fish immunity. Currently, one preferred approach is the use of immunostimulants which can be supplemented to the fish diets. Arginine is a versatile amino acid with important mechanisms closely related to the immune response. Aiming at finding out how arginine affects the innate immune status or improve disease resistance of European seabass (Dicentrarchus labrax) against vibriosis, fish were fed two arginine-supplemented diets (1% and 2% arginine supplementation). A third diet meeting arginine requirement level for seabass served as control diet. Following 15 or 29 days of feeding, fish were sampled for blood, spleen and gut to assess cell-mediated immune parameters and immune-related gene expression. At the same time, fish from each dietary group were challenged against Vibrio anguillarum and survival was monitored. Cell-mediated immune parameters such as the extracellular superoxide and nitric oxide decreased in fish fed arginine-supplemented diets. Interleukins and immune-cell marker transcripts were down-regulated by the highest supplementation level. Disease resistance data were in accordance with a generally depressed immune status, with increased susceptibility to vibriosis in fish fed arginine supplemented diets. Altogether, these results suggest a general inhibitory effect of arginine on the immune defences and disease resistance of European seabass. Still, further research will certainly clarify arginine immunomodulation pathways thereby allowing the validation of its potential as a prophylactic strategy. PMID:26447480

  9. Amino acid concentrations in human embryological fluids.

    PubMed

    Jauniaux, E; Sherwood, R A; Jurkovic, D; Boa, F G; Campbell, S

    1994-06-01

    The concentrations of amino acids in samples of coelomic fluid (n = 15), amniotic fluid (n = 9) and maternal serum (n = 15) obtained from normal pregnancies between 7 and 12 weeks of gestation were measured using reversed-phase chromatography with pre-column derivatization. The total molar concentration of the 18 amino acids measured was 2.3 times higher in coelomic fluid than in maternal serum. All amino acids except serine and tryptophan were present in significantly higher concentrations in coelomic fluid than in maternal serum. Significant correlations between maternal serum and coelomic fluid were only found for proline, tyrosine, and tryptophan, suggesting that levels of the other amino acids are mainly influenced by placental synthesis and do not directly depend on maternal amino acid metabolism. Levels of all amino acids were significantly higher in coelomic fluid compared to amniotic fluid. Compared to maternal serum, the amniotic fluid contained significantly higher levels of arginine, lysine, alanine and tyrosine and lower levels of serine, glutamine and tryptophan. The total molar amino acid concentration decreased significantly with gestational age in both coelomic fluid and maternal serum. These results suggest that amino acids accumulate in coelomic fluid to support the metabolism of the secondary yolk sac, and that the exocoelomic cavity is the reservoir for most nutrients needed by the embryo and early fetus in the first trimester of human pregnancy.

  10. 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

  11. Arginine kinase from Myzostoma cirriferum, a basal member of annelids.

    PubMed

    Yano, Daichi; Mimura, Sayo; Uda, Kouji; Suzuki, Tomohiko

    2016-08-01

    We assembled a phosphagen kinase gene from the Expressed Sequence Tags database of Myzostoma cirriferum, a basal member of annelids. The assembled gene sequence was synthesized using an overlap extension polymerase chain reaction method and was expressed in Escherichia coli. The recombinant enzyme (355 residues) exhibited monomeric behavior on a gel filtration column and showed strong activity only for l-arginine. Thus, the enzyme was identified as arginine kinase (AK). The two-substrate kinetic parameters were obtained and compared with other AKs. Phylogenetic analysis of amino acid sequences of phosphagen kinases indicated that the Myzostoma AK gene lineage differed from that of the polychaete Sabellastarte spectabilis AK, which is a dimer of creatine kinase (CK) origin. It is likely that the Myzostoma AK gene lineage was lost at an early stage of annelid evolution and that Sabellastarte AK evolved secondarily from the CK gene. This work contributes to our understanding of the evolution of phosphagen kinases of annelids with marked diversity.

  12. Hydrogenosome-localization of arginine deiminase in Trichomonas vaginalis.

    PubMed

    Morada, Mary; Smid, Ondrej; Hampl, Vladimir; Sutak, Robert; Lam, Brian; Rappelli, Paola; Dessì, Daniele; Fiori, Pier L; Tachezy, Jan; Yarlett, Nigel

    2011-03-01

    The arginine dihydrolase (ADH) pathway has an analogous function to the urea cycle in mitochondria-containing cells, by removing nitrogen from amino acids and generating ATP. Subcellular localization of the ADH pathway enzymes in Trichomonas vaginalis revealed that arginine deiminase (ADI) localizes to the hydrogenosome, a mitochondrion-like organelle of anaerobic protists. However the other enzymes of the ADH pathway, ornithine carbamyltransferase and carbamate kinase localize to the cytosol. Three gene sequences of T. vaginalis ADI (ADI 1-3) were identified in the T. vaginalis genome, all having putative mitochondrial targeting sequences. The ADI sequences were cloned and used to probe T. vaginalis using a carboxyterminal di-hemogglutinin epitope tag which demonstrated co-localization with malic enzyme confirming the hydrogenosome localization of this enzyme.

  13. Streptococcus pyogenes arginine and citrulline catabolism promotes infection and modulates innate immunity.

    PubMed

    Cusumano, Zachary T; Watson, Michael E; Caparon, Michael G

    2014-01-01

    A bacterium's ability to acquire nutrients from its host during infection is an essential component of pathogenesis. For the Gram-positive pathogen Streptococcus pyogenes, catabolism of the amino acid arginine via the arginine deiminase (ADI) pathway supplements energy production and provides protection against acid stress in vitro. Its expression is enhanced in murine models of infection, suggesting an important role in vivo. To gain insight into the function of the ADI pathway in pathogenesis, the virulence of mutants defective in each of its enzymes was examined. Mutants unable to use arginine (ΔArcA) or citrulline (ΔArcB) were attenuated for carriage in a murine model of asymptomatic mucosal colonization. However, in a murine model of inflammatory infection of cutaneous tissue, the ΔArcA mutant was attenuated but the ΔArcB mutant was hyperattenuated, revealing an unexpected tissue-specific role for citrulline metabolism in pathogenesis. When mice defective for the arginine-dependent production of nitric oxide (iNOS(-/-)) were infected with the ΔArcA mutant, cutaneous virulence was rescued, demonstrating that the ability of S. pyogenes to utilize arginine was dispensable in the absence of nitric oxide-mediated innate immunity. This work demonstrates the importance of arginine and citrulline catabolism and suggests a novel mechanism of virulence by which S. pyogenes uses its metabolism to modulate innate immunity through depletion of an essential host nutrient.

  14. Decreased miR122 in hepatocellular carcinoma leads to chemoresistance with increased arginine

    PubMed Central

    Kishikawa, Takahiro; Otsuka, Motoyuki; Tan, Poh Seng; Ohno, Motoko; Sun, Xiaochen; Yoshikawa, Takeshi; Shibata, Chikako; Takata, Akemi; Kojima, Kentaro; Takehana, Kenji; Ohishi, Maki; Ota, Sana; Noyama, Tomoyuki; Kondo, Yuji; Sato, Masaya; Soga, Tomoyoshi; Hoshida, Yujin; Koike, Kazuhiko

    2015-01-01

    Reduced expression of microRNA122 (miR122), a liver-specific microRNA, is frequent in hepatocellular carcinoma (HCC). However, its biological significances remain poorly understood. Because deregulated amino acid levels in cancers can affect their biological behavior, we determined the amino acid levels in miR122-silenced mouse liver tissues, in which intracellular arginine levels were significantly increased. The increased intracellular arginine levels were through upregulation of the solute carrier family 7 (SLC7A1), a transporter of arginine and a direct target of miR122. Arginine is the substrate for nitric oxide (NO) synthetase, and intracellular NO levels were increased in miR122-silenced HCC cells, with increased resistance to sorafenib, a multikinase inhibitor. Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib. Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib. These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset. PMID:25826076

  15. A Jump-from-Cavity Pyrophosphate Ion Release Assisted by a Key Lysine Residue in T7 RNA Polymerase Transcription Elongation

    PubMed Central

    Da, Lin-Tai; E, Chao; Duan, Baogen; Zhang, Chuanbiao; Zhou, Xin; Yu, Jin

    2015-01-01

    Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucleotide addition cycle. The kinetics and energetics of the process as well as how it proceeds with substantial conformational changes of the polymerase complex determine the mechano-chemical coupling mechanism of the transcription elongation. Here we investigated detailed dynamics of the PPi release process in a single-subunit RNA polymerase (RNAP) from bacteriophage T7, implementing all-atom molecular dynamics (MD) simulations. We obtained a jump-from-cavity kinetic model of the PPi release utilizing extensive nanosecond MD simulations. We found that the PPi release in T7 RNAP is initiated by the PPi dissociation from two catalytic aspartic acids, followed by a comparatively slow jump-from-cavity activation process. Combining with a number of microsecond long MD simulations, we also found that the activation process is hindered by charged residue associations as well as by local steric and hydrogen bond interactions. On the other hand, the activation is greatly assisted by a highly flexible lysine residue Lys472 that swings its side chain to pull PPi out. The mechanism can apply in general to single subunit RNA and DNA polymerases with similar molecular structures and conserved key residues. Remarkably, the flexible lysine or arginine residue appears to be a universal module that assists the PPi release even in multi-subunit RNAPs with charge facilitated hopping mechanisms. We also noticed that the PPi release is not tightly coupled to opening motions of an O-helix on the fingers domain of T7 RNAP according to the microsecond MD simulations. Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of the single-subunit polymerase. PMID:26599007

  16. Role of a helix B lysine residue in the photoactive site in channelrhodopsins.

    PubMed

    Li, Hai; Govorunova, Elena G; Sineshchekov, Oleg A; Spudich, John L

    2014-04-15

    In most studied microbial rhodopsins two conserved carboxylic acid residues (the homologs of Asp-85 and Asp-212 in bacteriorhodopsin) and an arginine residue (the homolog of Arg-82) form a complex counterion to the protonated retinylidene Schiff base, and neutralization of the negatively charged carboxylates causes red shifts of the absorption maximum. In contrast, the corresponding neutralizing mutations in some relatively low-efficiency channelrhodopsins (ChRs) result in blue shifts. These ChRs do not contain a lysine residue in the second helix, conserved in higher efficiency ChRs (Lys-132 in the crystallized ChR chimera). By action spectroscopy of photoinduced channel currents in HEK293 cells and absorption spectroscopy of detergent-purified pigments, we found that in tested ChRs the Lys-132 homolog controls the direction of spectral shifts in the mutants of the photoactive site carboxylic acid residues. Analysis of double mutants shows that red spectral shifts occur when this Lys is present, whether naturally or by mutagenesis, and blue shifts occur when it is replaced with a neutral residue. A neutralizing mutation of the Lys-132 homolog alone caused a red spectral shift in high-efficiency ChRs, whereas its introduction into low-efficiency ChR1 from Chlamydomonas augustae (CaChR1) caused a blue shift. Taking into account that the effective charge of the carboxylic acid residues is a key factor in microbial rhodopsin spectral tuning, these findings suggest that the Lys-132 homolog modulates their pKa values. On the other hand, mutation of the Arg-82 homolog that fulfills this role in bacteriorhodopsin caused minimal spectral changes in the tested ChRs. Titration revealed that the pKa of the Asp-85 homolog in CaChR1 lies in the alkaline region unlike in most studied microbial rhodopsins, but is substantially decreased by introduction of a Lys-132 homolog or neutralizing mutation of the Asp-212 homolog. In the three ChRs tested the Lys-132 homolog also alters

  17. Ascorbic acid-induced crosslinking of lens proteins: evidence supporting a Maillard reaction.

    PubMed

    Ortwerth, B J; Olesen, P R

    1988-08-31

    The incubation of calf lens extracts with 20 mM ascorbic acid under sterile conditions for 8 weeks caused extensive protein crosslinking, which was not observed with either 20 mM sorbitol or 20 mM glucose. While no precipitation was observed, ascorbic acid did induce the formation of high-molecular-weight protein aggregates as determined by Agarose A-5m chromatography. Proteins modified by ascorbic acid bound strongly to a boronate affinity column, however, crosslinked proteins were present mainly in the unbound fraction. These observations suggest that the cis-diol groups of ascorbic acid were present in the primary adduct, but were either lost during the crosslinking reaction or sterically hindered from binding to the column matrix. The amino acid composition of the ascorbic acid-modified proteins was identical to controls except for a 15% decrease in lysine. Amino acid analysis after borohydride reduction, however, showed a 25% decrease in lysine, a 7% decrease in arginine and an additional peak which eluted between phenylalanine and histidine. Extensive browning occurred during the ascorbic acid-modification reaction. This resulted in protein-bound chromophores with a broad absorption spectrum from 300 to 400 nm, and protein-bound fluorophores with excitation/emission maxima of 350/450 nm. A 4 week incubation of dialyzed crude lens extract with [1-14C]ascorbic acid showed increased incorporation for 2 weeks, followed by a decrease over the next 2 weeks as crosslinking was initiated. The addition of cyanoborohydride to the reaction mixture completely inhibited crosslinking and increased [1-14C]ascorbic acid incorporation to a plateau value of 180 nmol per mg protein. Amino acid analysis showed a 50% loss of lysine, and 8% decrease in arginine and the presence of a new peak which eluted slightly earlier than methionine. These data are consistent with the non-enzymatic glycation of lens proteins by either ascorbic acid or an oxidation product of ascorbic acid via

  18. A Reversible Association between Smc Coiled Coils Is Regulated by Lysine Acetylation and Is Required for Cohesin Association with the DNA.

    PubMed

    Kulemzina, Irina; Ang, Keven; Zhao, Xiaodan; Teh, Jun-Thing; Verma, Vikash; Suranthran, Sasikala; Chavda, Alap P; Huber, Roland G; Eisenhaber, Birgit; Eisenhaber, Frank; Yan, Jie; Ivanov, Dmitri

    2016-09-15

    Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes. PMID:27618487

  19. Colorimetric sensor array based on gold nanoparticles and amino acids for identification of toxic metal ions in water.

    PubMed

    Sener, Gulsu; Uzun, Lokman; Denizli, Adil

    2014-01-01

    A facile colorimetric sensor array for detection of multiple toxic heavy metal ions (Hg(2+), Cd(2+), Fe(3+), Pb(2+), Al(3+), Cu(2+), and Cr(3+)) in water is demonstrated using 11-mercaptoundecanoic acid (MUA)-capped gold nanoparticles (AuNPs) and five amino acids (lysine, cysteine, histidine, tyrosine, and arginine). The presence of amino acids (which have functional groups that can form complexes with metal ions and MUA) regulates the aggregation of MUA-capped particles; it can either enhance or diminish the particle aggregation. The combinatorial colorimetric response of all channels of the sensor array (i.e., color change in each of AuNP and amino acid couples) enables naked-eye discrimination of all of the metal ions tested in this study with excellent selectivity.

  20. Colorimetric sensor array based on gold nanoparticles and amino acids for identification of toxic metal ions in water.

    PubMed

    Sener, Gulsu; Uzun, Lokman; Denizli, Adil

    2014-01-01

    A facile colorimetric sensor array for detection of multiple toxic heavy metal ions (Hg(2+), Cd(2+), Fe(3+), Pb(2+), Al(3+), Cu(2+), and Cr(3+)) in water is demonstrated using 11-mercaptoundecanoic acid (MUA)-capped gold nanoparticles (AuNPs) and five amino acids (lysine, cysteine, histidine, tyrosine, and arginine). The presence of amino acids (which have functional groups that can form complexes with metal ions and MUA) regulates the aggregation of MUA-capped particles; it can either enhance or diminish the particle aggregation. The combinatorial colorimetric response of all channels of the sensor array (i.e., color change in each of AuNP and amino acid couples) enables naked-eye discrimination of all of the metal ions tested in this study with excellent selectivity. PMID:25330256

  1. The development and amino acid binding ability of nano-materials based on azo derivatives: theory and experiment.

    PubMed

    Shang, Xuefang; Du, Jinge; Yang, Wancai; Liu, Yun; Fu, Zhiyuan; Wei, Xiaofang; Yan, Ruifang; Yao, Ningcong; Guo, Yaping; Zhang, Jinlian; Xu, Xiufang

    2014-05-01

    Two nano-material-containing azo groups have been designed and developed, and the binding ability of nano-materials with various amino acids has been characterized by UV-vis and fluorescence titrations. Results indicated that two nano-materials showed the strongest binding ability for homocysteine among twenty normal kinds of amino acids (alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, arginine, glycine, serine, threonine, asparagine, phenylalanine, histidine, tryptophan, proline, lysine, glutamine, tyrosine and homocysteine). The reason for the high sensitivity for homocysteine was that two nano-materials containing an aldehyde group reacted with SH in homocysteine and afforded very stable thiazolidine derivatives. Theoretical investigation further illustrated the possible binding mode in host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. Thus, the two nano-materials can be used as optical sensors for the detection of homocysteine.

  2. Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): study of enzymes and nitrogen-containing compounds.

    PubMed

    Varisi, Vanderlei A; Camargos, Liliane S; Aguiar, Leandro F; Christofoleti, Renata M; Medici, Leonardo O; Azevedo, Ricardo A

    2008-01-01

    Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-l-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound.

  3. Partitioning of amino-acid analogues in a five-slab membrane model

    SciTech Connect

    Sengupta, D; Smith, Jeremy C; Ullmann, G. Matthias

    2008-09-01

    The positional preferences of the twenty amino-acid residues in a phospholipid bilayer are investigated by calculating the solvation free energy of the corresponding side chain analogues using a five-slab continuum electrostatic model. The side-chain analogues of the aromatic residues tryptophan and tyrosine are found to partition in the head-group region, due to compensation between the increase of the non-polar component of the solvation free energy at the boundary with the aqueous region and the decrease in the electrostatic component. The side chain analogue of phenylalanine differs from the other aromatic molecules by being able to partition in both the head-group region and the membrane core. This finding is consistent with experimental findings of the position of phenylalanine in membrane helices. Interestingly, the charged side-chain analogues of arginine and lysine are shown to prefer the head-group region in an orientation that allows the charged moiety to interact with the aqueous layer. The orientation adopted is similar to the 'snorkelling' effect seen in lysine and arginine residues in membrane helices. In contrast, the preference of the charged side-chain analogues of histidine (protonated) and aspartate (deprotonated) for the aqueous layer is shown to be due to a steep decrease in the electrostatic component of the solvation free energy at the boundary to the aqueous region. The calculations allow an understanding of the origins of side chain positioning in membranes and are thus useful in understanding membrane-protein:lipid thermodynamics.

  4. Lysine fortification of wheat flour improves selected indices of the nutritional status of predominantly cereal-eating families in Pakistan.

    PubMed

    Hussain, Tajammal; Abbas, Shaid; Khan, Mushtaq A; Scrimshaw, Nevin S

    2004-06-01

    Wheat provides more than 50% of the protein and calorie intake of the population of Pakistan. Legumes and animal protein that could complement the amino acid pattern of wheat, in which lysine is the first limiting amino acid for utilization of protein, are not affordable by members of lower socioeconomic groups in developing countries. The purpose of the study was to determine whether lysine fortification of wheat flour would have a positive impact on populations consuming a predominantly wheat-based diet. A double-blind study was carried out for three months on the outskirts of Peshawar, Pakistan. Forty families received wheat flour fortified with lysine, and 40 families received wheat flour without lysine. Wheat provided 59% of the protein for men, 65% for women, and 58% for children. The weight and height of the children in both groups increased during the study, but the increase was significantly greater in the lysine group. Hemoglobin increased significantly in the women receiving lysine-fortified flour. Transferrin levels increased significantly in men, women, and children in the lysine group as compared with those in the control group. Prealbumin increased significantly in adults receiving additional lysine but decreased in children. Men, women, and children in the lysine-supplemented families had significant increases in CD4, CD8, and complement C3 as compared with controls. These results indicate that lysine fortification of wheat flour can significantly improve sensitive indicators of nutritional status in a population consuming a diet in which 58% to 65% of the protein, depending on age and sex, is supplied by wheat.

  5. AMINO ACIDS AND HEMOGLOBIN PRODUCTION IN ANEMIA

    PubMed Central

    Whipple, G. H.; Robscheit-Robbins, F. S.

    1940-01-01

    Certain individual amino acids when given to standard anemic dogs cause an increase in new hemoglobin production. Occasional negative experiments are recorded. Glycine, glutamic acid, aspartic acid, cystine, histidine, phenylalanine, and proline when given in 1 gm. doses daily for 2 weeks, increase hemoglobin output on the average 23 to 25 gm. above the control level. This reaction amounts to 25 to 30 per cent of the new hemoglobin produced by the feeding of 300 gm. liver daily for 2 weeks—a standard liver test. Alanine, valine, isoleucine, and arginine in the same dosage increase the hemoglobin output on the average 13 to 17 gm. per 2 weeks over the control level. Leucine, methionine, lysine, tryptophane, and tyrosine fall in a middle group with hemoglobin output of about 20 gm. Isovaleric acid, β-hydroxybutyric acid, glutaric acid, and asparagine have shown positive effects and the butyrate is unusually potent for hemoglobin production (Table 2). The isomeric and dl-synthetic forms of the amino acids are as effectively utilized in this reaction as are the natural forms. PMID:19870982

  6. 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 an