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Sample records for acids including serine

  1. The Postnatal Development of d-Serine in the Retinas of Two Mouse Strains, Including a Mutant Mouse with a Deficiency in d-Amino Acid Oxidase and a Serine Racemase Knockout Mouse

    PubMed Central

    2015-01-01

    d-Serine, an N-methyl d-aspartate receptor coagonist, and its regulatory enzymes, d-amino acid oxidase (DAO; degradation) and serine racemase (SR; synthesis), have been implicated in crucial roles of the developing central nervous system, yet the functional position that they play in regulating the availability of d-serine throughout development of the mammalian retina is not well-known. Using capillary electrophoresis and a sensitive method of enantiomeric amino acid separation, we were able to determine total levels of d-serine at specific ages during postnatal development of the mouse retina in two different strains of mice, one of which contained a loss-of-function point mutation for DAO while the other was a SR knockout line. Each mouse line was tested against conspecific wild type (WT) mice for each genetic strain. The universal trend in all WT and transgenic mice was a large amount of total retinal d-serine at postnatal age 2 (P2), followed by a dramatic decrease as the mice matured into adulthood (P70–80). SR knockout mice retinas had 41% less d-serine than WT retinas at P2, and 10 times less as an adult. DAO mutant mice retinas had significantly elevated levels of d-serine when compared to WT retinas at P2 (217%), P4 (223%), P8 (194%), and adulthood (227%). PMID:25083578

  2. Adaptational modification of serine and threonine metabolism in the liver to essential amino acid deficiency in rats.

    PubMed

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

    2009-03-01

    It is known that plasma serine and threonine concentrations are elevated in rats chronically fed an essential amino acid deficient diet, but the underlying mechanisms including related gene expressions or serine and threonine concentrations in liver remained to be elucidated. We fed rats lysine or valine deficient diet for 4 weeks and examined the mRNA expressions of serine synthesising (3-phosphoglycerate dehydrogenase, PHGDH) and serine/threonine degrading enzymes (serine dehydratase, SDS) in the liver. Dietary deficiency induced marked elevation of hepatic serine and threonine levels associated with enhancement of PHGDH mRNA expression and repression of SDS mRNA expression. Increases in plasma serine and threonine levels due to essential amino acid deficiency in diet were caused by marked increases in hepatic serine and threonine levels. Proteolytic responses to the amino acid deficiency may be lessened by storing amino radicals as serine and inducing anorexia through elevation of threonine. PMID:18584286

  3. Pharmacokinetics of Oral d-Serine in d-Amino Acid Oxidase Knockout Mice

    PubMed Central

    Rais, Rana; Thomas, Ajit G.; Wozniak, Krystyna; Wu, Ying; Jaaro-Peled, Hanna; Sawa, Akira; Strick, Christine A.; Engle, Sandra J.; Brandon, Nicholas J.; Rojas, Camilo; Slusher, Barbara S.

    2012-01-01

    d-Amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-amino acids including d-serine, a full agonist at the glycine modulatory site of the N-methyl-d-aspartate (NMDA) receptor. To evaluate the significance of DAAO-mediated metabolism in the pharmacokinetics of oral d-serine, plasma d-serine levels were measured in both wild-type mice and transgenic mice lacking DAAO. Although d-serine levels were rapidly diminished in wild-type mice (t½ = 1.2 h), sustained drug levels over the course of 4 h (t½ > 10 h) were observed in mice lacking DAAO. Coadministration of d-serine with 6-chlorobenzo[d]isoxazol-3-ol (CBIO), a small-molecule DAAO inhibitor, in wild-type mice resulted in the enhancement of plasma d-serine levels, although CBIO seems to have only temporary effects on the plasma d-serine levels due to glucuronidation of the key hydroxyl group. These findings highlight the predominant role of DAAO in the clearance of d-serine from the systemic circulation. Thus, a potent DAAO inhibitor with a longer half-life should be capable of maintaining high plasma d-serine levels over a sustained period of time and might have therapeutic implications for the treatment of schizophrenia. PMID:22837388

  4. Partial amino acid sequence of human factor D:homology with serine proteases.

    PubMed Central

    Volanakis, J E; Bhown, A; Bennett, J C; Mole, J E

    1980-01-01

    Human factor D purified to homogeneity by a modified procedure was subjected to NH2-terminal amino acid sequence analysis by using a modified automated Beckman sequencer. We identified 48 of the first 57 NH2-terminal amino acids in a single sequencer run, using microgram quantities of factor D. The deduced amino acid sequence represents approximately 25% of the primary structure of factor D. This extended NH2-terminal amino acid sequence of factor D was compared to that of other trypsin-related serine proteases. By visual inspection, strong homologies (33--50% identity) were observed with all the serine proteases included in the comparison. Interestingly, factor D showed a higher degree of homology to serine proteases of pancreatic origin than to those of serum origin. Images PMID:6987665

  5. Free Amino Acids in Serine-Antagonized Cells of Tetrahymena pyriformis1

    PubMed Central

    Wragg, June B.; Reynolds, Howard; Pelczar, Michael J.

    1965-01-01

    Wragg, June B. (Agricultural Research Service, Beltsville, Md.), Howard Reynolds, and Michael J. Pelczar, Jr. Free amino acids in serine-antagonized cells of Tetrahymena pyriformis. J. Bacteriol. 90:748–754. 1965.—Growth inhibition of Tetrahymena pyriformis by l-serine in a chemically defined medium was reversed by l-arginine in a manner which resembled competitive antagonism. Composition of the free amino acid pools from cells grown in either a balanced amino acid mixture or a mixture with serine concentrations which inhibited growth suggested an antagonism by serine with energy-yielding reactions. Growth in media with excess serine resulted in the accumulation of higher concentrations of free cellular amino acids and an apparent increase in the rate of conversion of arginine to ornithine, as compared with growth in the balanced medium. The results suggested that serine or a metabolic product of serine interferes with the formation of pyruvic acid. In the presence of high levels of serine, arginine appeared to be metabolized more rapidly and to be spared when alanine, aspartic acid, or glutamic acid was added to the unbalanced medium. PMID:16562077

  6. Serine biosynthesis and transport defects.

    PubMed

    El-Hattab, Ayman W

    2016-07-01

    l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. PMID:27161889

  7. Transport of D-serine via the amino acid transporter ATB(0,+) expressed in the colon.

    PubMed

    Hatanaka, Takahiro; Huang, Wei; Nakanishi, Takeo; Bridges, Christy C; Smith, Sylvia B; Prasad, Puttur D; Ganapathy, Malliga E; Ganapathy, Vadivel

    2002-02-22

    D-Serine, synthesized endogenously in the brain, is an important modulator of glutamatergic neurotransmission. Since colonic bacteria produce D-serine, we asked the question whether there are transport mechanisms in the colon that might make this exogenously produced D-serine available to the host. Here we identify for the first time an amino acid transporter in the intestine for high-affinity active transport of D-serine. This transporter, called ATB(0,+), is a Na(+)- and Cl(-)-coupled transporter for L-enantiomers of neutral and cationic amino acids. Here we demonstrate that ATB(0,+) is also capable of mediating the Na(+)- and Cl(-)-coupled transport of D-serine. The affinity of ATB(0,+) for L-serine and D-serine is similar, the K(t) value for the two enantiomers being approximately 150 microM. In addition to D-serine, ATB(0,+) transports D-alanine, D-methionine, D-leucine, and D-tryptophan. However, several other neutral and cationic amino acids that are transportable substrates for ATB(0,+) as L-enantiomers are not transported when presented as D-enantiomers. ATB(0,+) is expressed in the intestinal tract, interestingly not in the proximal intestine but in the distal intestine. Expression is most predominant in the colon where the transporter is localized to the luminal membrane of colonocytes, making this transporter uniquely suitable for absorption of bacteria-derived D-serine. PMID:11846403

  8. Amino acid sequence of the serine-repeat antigen (SERA) of Plasmodium falciparum determined from cloned cDNA.

    PubMed

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1988-09-01

    We report the isolation of cDNA clones for a Plasmodium falciparum gene that encodes the complete amino acid sequence of a previously identified exported blood stage antigen. The Mr of this antigen protein had been determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis analysis, by different workers, to be 113,000, 126,000, and 140,000. We show, by cDNA nucleotide sequence analysis, that this antigen gene encodes a 989 amino acid protein (111 kDa) that contains a potential signal peptide, but not a membrane anchor domain. In the FCR3 strain the serine content of the protein was 11%, of which 57% of the serine residues were localized within a 201 amino acid sequence that included 35 consecutive serine residues. The protein also contained three possible N-linked glycosylation sites and numerous possible O-linked glycosylation sites. The mRNA was abundant during late trophozoite-schizont parasite stages. We propose to identity this antigen, which had been called p126, by the acronym SERA, serine-repeat antigen, based on its complete structure. The usefulness of the cloned cDNA as a source of a possible malaria vaccine is considered in view of the previously demonstrated ability of the antigen to induce parasite-inhibitory antibodies and a protective immune response in Saimiri monkeys. PMID:2847041

  9. Combined Skin Moisturization of Liposomal Serine Incorporated in Hydrogels Prepared with Carbopol ETD 2020, Rhesperse RM 100 and Hyaluronic Acid.

    PubMed

    Kim, Hyeongmin; Ro, Jieun; Barua, Sonia; Hwang, Deuk Sun; Na, Seon-Jeong; Lee, Ho Sung; Jeong, Ji Hoon; Woo, Seulki; Kim, Hyewon; Hong, Bomi; Yun, Gyiae; Kim, Joong-Hark; Yoon, Young-Ho; Park, Myung-Gyu; Kim, Jia; Sohn, Uy Dong; Lee, Jaehwi

    2015-11-01

    We investigated the combined moisturizing effect of liposomal serine and a cosmeceutical base selected in this study. Serine is a major amino acid consisting of natural moisturizing factors and keratin, and the hydroxyl group of serine can actively interact with water molecules. Therefore, we hypothesized that serine efficiently delivered to the stratum corneum (SC) of the skin would enhance the moisturizing capability of the skin. We prepared four different cosmeceutical bases (hydrogel, oil-in-water (O/W) essence, O/W cream, and water-in-oil (W/O) cream); their moisturizing abilities were then assessed using a Corneometer®. The hydrogel was selected as the optimum base for skin moisturization based on the area under the moisture content change-time curves (AUMCC) values used as a parameter for the water hold capacity of the skin. Liposomal serine prepared by a reverse-phase evaporation method was then incorporated in the hydrogel. The liposomal serine-incorporated hydrogel (serine level=1%) showed an approximately 1.62~1.77 times greater moisturizing effect on the skin than those of hydrogel, hydrogel with serine (1%), and hydrogel with blank liposome. However, the AUMCC values were not dependent on the level of serine in liposomal serine-loaded hydrogels. Together, the delivery of serine to the SC of the skin is a promising strategy for moisturizing the skin. This study is expected to be an important step in developing highly effective moisturizing cosmeceutical products. PMID:26557021

  10. Combined Skin Moisturization of Liposomal Serine Incorporated in Hydrogels Prepared with Carbopol ETD 2020, Rhesperse RM 100 and Hyaluronic Acid

    PubMed Central

    Kim, Hyeongmin; Ro, Jieun; Barua, Sonia; Hwang, Deuk Sun; Na, Seon-Jeong; Lee, Ho Sung; Jeong, Ji Hoon; Woo, Seulki; Kim, Hyewon; Hong, Bomi; Yun, Gyiae; Kim, Joong-Hark; Yoon, Young-Ho; Park, Myung-Gyu; Kim, Jia; Sohn, Uy Dong

    2015-01-01

    We investigated the combined moisturizing effect of liposomal serine and a cosmeceutical base selected in this study. Serine is a major amino acid consisting of natural moisturizing factors and keratin, and the hydroxyl group of serine can actively interact with water molecules. Therefore, we hypothesized that serine efficiently delivered to the stratum corneum (SC) of the skin would enhance the moisturizing capability of the skin. We prepared four different cosmeceutical bases (hydrogel, oil-in-water (O/W) essence, O/W cream, and water-in-oil (W/O) cream); their moisturizing abilities were then assessed using a Corneometer®. The hydrogel was selected as the optimum base for skin moisturization based on the area under the moisture content change-time curves (AUMCC) values used as a parameter for the water hold capacity of the skin. Liposomal serine prepared by a reverse-phase evaporation method was then incorporated in the hydrogel. The liposomal serine-incorporated hydrogel (serine level=1%) showed an approximately 1.62~1.77 times greater moisturizing effect on the skin than those of hydrogel, hydrogel with serine (1%), and hydrogel with blank liposome. However, the AUMCC values were not dependent on the level of serine in liposomal serine-loaded hydrogels. Together, the delivery of serine to the SC of the skin is a promising strategy for moisturizing the skin. This study is expected to be an important step in developing highly effective moisturizing cosmeceutical products. PMID:26557021

  11. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival.

    PubMed

    Kugadas, Abirami; Lamont, Elise A; Bannantine, John P; Shoyama, Fernanda M; Brenner, Evan; Janagama, Harish K; Sreevatsan, Srinand

    2016-01-01

    The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc(2) 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted. PMID:27597934

  12. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

    PubMed Central

    Kugadas, Abirami; Lamont, Elise A.; Bannantine, John P.; Shoyama, Fernanda M.; Brenner, Evan; Janagama, Harish K.; Sreevatsan, Srinand

    2016-01-01

    The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted. PMID:27597934

  13. Catalysis of the Oligomerization of O-Phospho-Serine, Aspartic Acid, or Glutamic Acid by Cationic Micelles

    NASA Technical Reports Server (NTRS)

    Boehler, Christof; Hill, Aubrey R., Jr.; Orgel, Leslie E.

    1996-01-01

    Treatment of relatively concentrated aqueous solutions of O-phospho-serine (50 mM), aspartic acid (100 mM) or glutamic acid (100 mM) with carbonyldiimidazole leads to the formation of an activated intermediate that oligomerizes efficiently. When the concentration of amino acid is reduced tenfold, few long oligomers can be detected. Positively-charged cetyltrimethyl ammonium bromide micelles concentrate the negatively-charged activated intermediates of the amino acids at their surfaces and catalyze efficient oligomerization even from dilute solutions.

  14. Catalysis of the Oligomerization of O-Phospho-Serine, Aspartic Acid, or Glutamic Acid by Cationic Micelles

    NASA Technical Reports Server (NTRS)

    Bohler, Christof; Hill, Aubrey R., Jr.; Orgel, Leslie E.

    1996-01-01

    Treatment of relatively concentrated aqueous solutions of 0-phospho-serine (50 mM), aspartic acid (100 mM) or glutamic acid (100 mM) with carbonyldiimidazole leads to the formation of an activated intermediate that oligomerizes efficiently. When the concentration of amino acid is reduced tenfold, few long oligomers can be detected. Positively-charged cetyltrimethyl ammonium bromide micelles concentrate the negatively-charged activated intermediates of the amino acids at their surfaces and catalyze efficient oligomerization even from dilute solutions.

  15. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation.

    PubMed

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNA(Ala) with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of L-serine into human proteins. We also report that this misincorporation can be inhibited by L-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  16. The Non-Protein Amino Acid BMAA Is Misincorporated into Human Proteins in Place of l-Serine Causing Protein Misfolding and Aggregation

    PubMed Central

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNAAla with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of l-serine into human proteins. We also report that this misincorporation can be inhibited by l-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  17. Pharmacological PPARα Activation Markedly Alters Plasma Turnover of the Amino Acids Glycine, Serine and Arginine in the Rat

    PubMed Central

    Ericsson, Anette; Turner, Nigel; Hansson, Göran I.; Wallenius, Kristina; Oakes, Nicholas D.

    2014-01-01

    The current study extends previously reported PPARα agonist WY 14,643 (30 µmol/kg/day for 4 weeks) effects on circulating amino acid concentrations in rats fed a 48% saturated fat diet. Steady-state tracer experiments were used to examine in vivo kinetic mechanisms underlying altered plasma serine, glycine and arginine levels. Urinary urea and creatinine excretion were measured to assess whole-body amino acid catabolism. WY 14,643 treated animals demonstrated reduced efficiency to convert food consumed to body weight gain while liver weight was increased compared to controls. WY 14,643 raised total amino acid concentration (38%), largely explained by glycine, serine and threonine increases. 3H-glycine, 14C-serine and 14C-arginine tracer studies revealed elevated rates of appearance (Ra) for glycine (45.5±5.8 versus 17.4±2.7 µmol/kg/min) and serine (21.0±1.4 versus 12.0±1.0) in WY 14,643 versus control. Arginine was substantially decreased (−62%) in plasma with estimated Ra reduced from 3.1±0.3 to 1.2±0.2 µmol/kg/min in control versus WY 14,643. Nitrogen excretion over 24 hours was unaltered. Hepatic arginase activity was substantially decreased by WY 14,643 treatment. In conclusion, PPARα agonism potently alters metabolism of several specific amino acids in the rat. The changes in circulating levels of serine, glycine and arginine reflected altered fluxes into the plasma rather than changes in clearance or catabolism. This suggests that PPARα has an important role in modulating serine, glycine and arginine de novo synthesis. PMID:25486018

  18. High-casein diet suppresses guanidinoacetic acid-induced hyperhomocysteinemia and potentiates the hypohomocysteinemic effect of serine in rats.

    PubMed

    Ohuchi, Seiya; Matsumoto, Yuko; Morita, Tatsuya; Sugiyama, Kimio

    2008-12-01

    To determine the effect of dietary protein level on experimental hyperhomocysteinemia, rats were fed 10% casein (10C) and 40% casein (40C) diets with or without 0.5% guanidinoacetic acid (GAA) for 14 d. In addition, rats were fed 10C + 0.75% methionine (10CM) and 40C + 0.75% methionine (40CM) diets with or without 2.5% serine for 14 d to determine the relationship between the dietary protein level and intensity of the hypohomocysteinemic effect of serine. GAA supplementation markedly increased the plasma homocysteine concentration in rats fed with the 10C diet, whereas it did not increase the plasma homocysteine concentration in rats fed with the 40C diet. Although serine supplementation significantly suppressed the methionine-induced enhancement of plasma homocysteine concentration, the decreased plasma homocysteine concentration was significantly lower in rats fed with the 40CM diet than in rats fed with the 10CM diet. The hepatic cystathionine beta-synthase and betaine-homocysteine S-methyltransferase activities were significantly higher in rats fed with the 40C or 40CM diet than in rats fed with the 10C or 10CM diet, irrespective of supplementation with GAA and serine. These results indicate that the high-casein diet was effective for both suppressing GAA-induced hyperhomocysteinemia and potentiating the hypohomocysteinemic effect of serine, probably through the enhanced activity of homocysteine-metabolizing enzymes. PMID:19060401

  19. Discovery of a Cyclic Boronic Acid β-Lactamase Inhibitor (RPX7009) with Utility vs Class A Serine Carbapenemases.

    PubMed

    Hecker, Scott J; Reddy, K Raja; Totrov, Maxim; Hirst, Gavin C; Lomovskaya, Olga; Griffith, David C; King, Paula; Tsivkovski, Ruslan; Sun, Dongxu; Sabet, Mojgan; Tarazi, Ziad; Clifton, Matthew C; Atkins, Kateri; Raymond, Amy; Potts, Kristy T; Abendroth, Jan; Boyer, Serge H; Loutit, Jeffrey S; Morgan, Elizabeth E; Durso, Stephanie; Dudley, Michael N

    2015-05-14

    The increasing dissemination of carbapenemases in Gram-negative bacteria has threatened the clinical usefulness of the β-lactam class of antimicrobials. A program was initiated to discover a new series of serine β-lactamase inhibitors containing a boronic acid pharmacophore, with the goal of finding a potent inhibitor of serine carbapenemase enzymes that are currently compromising the utility of the carbapenem class of antibacterials. Potential lead structures were screened in silico by modeling into the active sites of key serine β-lactamases. Promising candidate molecules were synthesized and evaluated in biochemical and whole-cell assays. Inhibitors were identified with potent inhibition of serine carbapenemases, particularly the Klebsiella pneumoniae carbapenemase (KPC), with no inhibition of mammalian serine proteases. Studies in vitro and in vivo show that RPX7009 (9f) is a broad-spectrum inhibitor, notably restoring the activity of carbapenems against KPC-producing strains. Combined with a carbapenem, 9f is a promising product for the treatment of multidrug resistant Gram-negative bacteria. PMID:25782055

  20. Acid stability of the kinetically stable alkaline serine protease possessing polyproline II fold.

    PubMed

    Rohamare, Sonali; Javdekar, Vaishali; Dalal, Sayli; Nareddy, Pavan Kumar; Swamy, Musti J; Gaikwad, Sushama M

    2015-02-01

    The kinetically stable alkaline serine protease from Nocardiopsis sp.; NprotI, possessing polyproline II fold (PPII) was characterized for its pH stability using proteolytic assay, fluorescence and Circular Dichroism (CD) spectroscopy, and Differential Scanning Calorimetry (DSC). NprotI was found to be functionally stable when incubated at pH 1.0, even after 24 h, while after incubation at pH 10.0, drastic loss in the activity was observed. The enzyme showed enhanced activity after incubation at pH 1.0 and 3.0, at higher temperature (50-60 °C). NprotI maintained the overall PPII fold in broad pH range as seen using far UV CD spectroscopy. The PPII fold of NprotI incubated at pH 1.0 remained fairly intact up to 70 °C. Based on the isodichroic point and Tm values revealed by secondary structural transitions, different modes of thermal denaturation at pH 1.0, 5.0 and 10.0 were observed. DSC studies of NprotI incubated at acidic pH (pH 1.0-5.0) showed Tm values in the range of 74-76 °C while significant decrease in Tm (63.8 °C) was observed at pH 10.0. NprotI could be chemically denatured at pH 5.0 (stability pH) only with guanidine thiocynate. NprotI can be classified as type III protein among the three acid denatured states. Acid tolerant and thermostable NprotI can serve as a potential candidate for biotechnological applications. PMID:25576306

  1. Methionine and serine synergistically suppress hyperhomocysteinemia induced by choline deficiency, but not by guanidinoacetic acid, in rats fed a low casein diet.

    PubMed

    Liu, Yi-qun; Liu, Ying; Morita, Tatsuya; Sugiyama, Kimio

    2011-01-01

    The effects of dietary supplementation with 0.5% methionine, 2.5% serine, or both on hyperhomocysteinemia induced by deprivation of dietary choline or by dietary addition of 0.5% guanidinoacetic acid (GAA) were investigated in rats fed a 10% casein diet. Hyperhomocysteinemia induced by choline deprivation was not suppressed by methionine alone and was only partially suppressed by serine alone, whereas it was completely suppressed by a combination of methionine and serine, suggesting a synergistic effect of methionine and serine. Fatty liver was also completely prevented by the combination of methionine and serine. Compared with methionine alone, the combination of methionine and serine decreased hepatic S-adenosylhomocysteine and homocysteine concentrations and increased hepatic betaine and serine concentrations and betaine-homocysteine S-methyltransferase activity. GAA-induced hyperhomocysteinemia was partially suppressed by methionine alone, but no interacting effect of methionine and serine was detected. In contrast, GAA-induced fatty liver was completely prevented by the combination of methionine and serine. These results indicate that a combination of methionine and serine is effective in suppressing both hyperhomocysteinemia and fatty liver induced by choline deprivation, and that methionine alone is effective in suppressing GAA-induced hyperhomocysteinemia partially. PMID:22146711

  2. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    SciTech Connect

    Lim, Yong-Whan; Yoon, Seung-Yong; Choi, Jung-Eun; Kim, Sang-Min; Lee, Hui-Sun; Choe, Han; Lee, Seung-Chul; Kim, Dong-Hou

    2010-04-30

    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

  3. Targeting class A and C serine β-lactamases with a broad-spectrum boronic acid derivative.

    PubMed

    Tondi, Donatella; Venturelli, Alberto; Bonnet, Richard; Pozzi, Cecilia; Shoichet, Brian K; Costi, Maria Paola

    2014-06-26

    Production of β-lactamases (BLs) is the most widespread resistance mechanism adopted by bacteria to fight β-lactam antibiotics. The substrate spectrum of BLs has become increasingly broad, posing a serious health problem. Thus, there is an urgent need for novel BL inhibitors. Boronic acid transition-state analogues are able to reverse the resistance conferred by class A and C BLs. We describe a boronic acid analogue possessing interesting and potent broad-spectrum activity vs class A and C serine-based BLs. Starting from benzo(b)thiophene-2-boronic acid (BZBTH2B), a nanomolar non-β-lactam inhibitor of AmpC that can potentiate the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria, we designed a novel broad-spectrum nanomolar inhibitor of class A and C BLs. Structure-based drug design (SBDD), synthesis, enzymology data, and X-ray crystallography results are discussed. We clarified the inhibitor binding geometry responsible for broad-spectrum activity vs serine-active BLs using double mutant thermodynamic cycle studies. PMID:24882105

  4. The full amino acid repertoire is superior to serine/tyrosine for selection of high affinity immunoglobulin G binders from the fibronectin scaffold.

    PubMed

    Hackel, Benjamin J; Wittrup, K Dane

    2010-04-01

    The design of combinatorial libraries for molecular recognition requires extensive diversity to provide high affinity binding to myriad epitopes while maintaining a high degree of functionality to enable inclusion of binders in the limited screenable library size. In the current work, we directly compare minimal and maximal amino acid diversity libraries in the context of the 10th type III domain of human fibronectin. Libraries with either serine/tyrosine or full 20 amino acid diversity were created, pooled and screened for binding to rabbit and goat immunoglobulin G (IgG), and affinity matured by directed evolution. Multiple picomolar binders to rabbit IgG and nanomolar binders to goat IgG were engineered with peak affinities of 51 +/- 4 pM and 1.2 +/- 0.4 nM, respectively. Sequence analysis reveals that 93% of the selected BC and FG loops, including those from the highest affinity clones, originate from the full diversity library. Thus, with a modest initial library size (approximately 1 x 10(8)) and an efficient affinity maturation scheme, more extensive diversity is superior to a binary serine/tyrosine code for the generation of picomolar to low nanomolar binders in the fibronectin domain. The highest affinity binders demonstrated utility in affinity purification of IgG from serum and as detection reagents in flow cytometry. PMID:20067921

  5. Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes.

    PubMed

    Bachovchin, W W; Wong, W Y; Farr-Jones, S; Shenvi, A B; Kettner, C A

    1988-10-01

    15N NMR spectroscopy was used to examine the active-site histidyl residue of alpha-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. With the better inhibitors of the class this arrangement is stable over the pH range 4.0-10.5. The results are consistent with a putative tetrahedral intermediate like complex involving a negatively charged, tetrahedral boron atom covalently bonded to O gamma of the active-site serine. (2) Boronic acids that are not substrate analogues form complexes in which N epsilon 2 of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N delta 1 of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. Benzeneboronic acid, which falls in this category, forms an adduct with histidine. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field 1H resonances and the 15N-H spin couplings. These results, coupled with the kinetic data of the preceding paper [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry (preceding paper in this issue)], indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed. PMID:3207700

  6. Amino acid sequence alignment of bacterial and mammalian pancreatic serine proteases based on topological equivalences.

    PubMed

    James, M N; Delbaere, L T; Brayer, G D

    1978-06-01

    The three-dimensional structures of the bacterial serine proteases SGPA, SGPB, and alpha-lytic protease have been compared with those of the pancreatic enzymes alpha-chymotrypsin and elastase. This comparison shows that approximately 60% (55-64%) of the alpha-carbon atom positions of the bacterial serine proteases are topologically equivalent to the alpha-carbon atom positions of the pancreatic enzymes. The corresponding value for a comparison of the bacterial enzymes among themselves is approximately 84%. The results of these topological comparisons have been used to deduce an experimentally sound sequence alignment for these several enzymes. This alignment shows that there is extensive tertiary structural homology among the bacteria and pancreatic enzymes without significant primary sequence identity (less than 21%). The acquisition of a zymogen function by the pancreatic enzymes is accompanied by two major changes to the bacterial enzymes' architecture: an insertion of 9 residues to increase the length of the N-terminal loop, and one of 12 residues to a loop near the activation salt bridge. In addition, in these two enzyme families, the methionine loop (residues 164-182) adopts very different comformations which are associated with their altered substrate specificities. PMID:96920

  7. Evolution of nuclear retinoic acid receptor alpha (RARα) phosphorylation sites. Serine gain provides fine-tuned regulation.

    PubMed

    Samarut, Eric; Amal, Ismail; Markov, Gabriel V; Stote, Roland; Dejaegere, Annick; Laudet, Vincent; Rochette-Egly, Cécile

    2011-07-01

    The human nuclear retinoic acid (RA) receptor alpha (hRARα) is a ligand-dependent transcriptional regulator, which is controlled by a phosphorylation cascade. The cascade starts with the RA-induced phosphorylation of a serine residue located in the ligand-binding domain, S(LBD), allowing the recruitment of the cdk7/cyclin H/MAT1 subcomplex of TFIIH through the docking of cyclin H. It ends by the subsequent phosphorylation by cdk7 of an other serine located in the N-terminal domain, S(NTD). Here, we show that this cascade relies on an increase in the flexibility of the domain involved in cyclin H binding, subsequently to the phosphorylation of S(LBD). Owing to the functional importance of RARα in several vertebrate species, we investigated whether the phosphorylation cascade was conserved in zebrafish (Danio rerio), which expresses two RARα genes: RARα-A and RARα-B. We found that in zebrafish RARαs, S(LBD) is absent, whereas S(NTD) is conserved and phosphorylated. Therefore, we analyzed the pattern of conservation of the phosphorylation sites and traced back their evolution. We found that S(LBD) is most often absent outside mammalian RARα and appears late during vertebrate evolution. In contrast, S(NTD) is conserved, indicating that the phosphorylation of this functional site has been under ancient high selection constraint. This suggests that, during evolution, different regulatory circuits control RARα activity. PMID:21297158

  8. The importance of serine metabolism in cancer.

    PubMed

    Mattaini, Katherine R; Sullivan, Mark R; Vander Heiden, Matthew G

    2016-08-01

    Serine metabolism is frequently dysregulated in cancers; however, the benefit that this confers to tumors remains controversial. In many cases, extracellular serine alone is sufficient to support cancer cell proliferation, whereas some cancer cells increase serine synthesis from glucose and require de novo serine synthesis even in the presence of abundant extracellular serine. Recent studies cast new light on the role of serine metabolism in cancer, suggesting that active serine synthesis might be required to facilitate amino acid transport, nucleotide synthesis, folate metabolism, and redox homeostasis in a manner that impacts cancer. PMID:27458133

  9. Crystal versus solution structure of enzymes: NMR spectroscopy of a peptide boronic acid-serine protease complex in the crystalline state.

    PubMed

    Farr-Jones, S; Smith, S O; Kettner, C A; Griffin, R G; Bachovchin, W W

    1989-09-01

    The effectiveness of boronic acids as inhibitors of serine proteases has been widely ascribed to the ability of the boronyl group to form a tetrahedral adduct with the active-site serine that closely mimics the putative tetrahedral intermediate or transition state formed with substrates. However, recent 15N NMR studies of alpha-lytic protease (EC 3.4.21.12) in solution have shown that some boronic acids and peptide boronic acids form adducts with the active-site histidine instead of with the serine. Such histidine-boron adducts have not thus far been reported in x-ray diffraction studies of boronic acid-serine protease complexes. Here, we report an 15N NMR study of the MeOSuc-Ala-Ala-Pro-boroPhe complex of alpha-lytic protease in the crystalline state using magic-angle spinning. Previous 15N NMR studies have shown this complex involves the formation of a histidine-boron bond in solution. The 15N NMR spectra of the crystalline complex are essentially identical to those of the complex in solution, thereby showing that the structure of this complex is the same in solution and in the crystal and that both involve formation of a histidine-boron adduct. PMID:2780549

  10. Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes

    SciTech Connect

    Bachovchin, W.W.; Wong, W.Y.L.; Farr-Jones, S. ); Shenvi, A.B.; Kettner, C.A. )

    1988-10-04

    {sup 15}N NMR spectroscopy was used to examine the active-site histidyl residue of {alpha}-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. (2) Boronic acids that are not substrate analogues form complexes in which N{sup {epsilon}2} of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N{sup {delta}1} of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field {sup 1}H resonances and the {sup 15}N-H spin couplings. These results indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed.

  11. Effects of dietary soybean stachyose and phytic acid on gene expressions of serine proteases in Japanese flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Mi, Haifeng; Mai, Kangsen; Zhang, Wenbing; Wu, Chenglong; Cai, Yinghua

    2011-09-01

    Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder ( Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively. Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY ( P<0.05). The same level of dietary SBS significantly decreased the gene expression of poEL. In comparison with the control group (ANF-free), dietary PA (0.2% and 0.8%) significantly decreased the gene expression of poTRY, poCTRY and poEL ( P<0.05). However, excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions ( P>0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes' expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

  12. Serine utilization by Klebsiella aerogenes.

    PubMed Central

    Vining, L C; Magasanik, B

    1981-01-01

    Klebsiella aerogenes was found to contain a specific L-serine dehydrase that was induced by threonine, glycine or leucine, but not by its substrate. Cellular concentrations were sensitive to carbon rather than nitrogen sources in the growth medium. A nonspecific isoleucine-sensitive L-threonine dehydrase supplemented the specific L-serine dehydrase activity. K. aerogenes also contains a leucine-inducible L-threonine dehydrogenase which probably initiated a threonine-utilization pathway in which the serine-specific dehydrate participated. Strains that were altered in their ability to metabolize serine differed in either L-serine dehydrase or L-threonine dehydrase activity. Thus, K. aerogenes growing on L-serine as a sole nitrogen source relies upon two enzymes that metabolize the amino acid as subsidiary functions. PMID:6783624

  13. Identification of serine and histidine adducts in complexes of trypsin and trypsinogen with peptide and nonpeptide boronic acid inhibitors by 1H NMR spectroscopy.

    PubMed

    Tsilikounas, E; Kettner, C A; Bachovchin, W W

    1992-12-29

    We have previously shown, in 15N NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N epsilon 2 of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field 1H NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27, 7689-7697]. Here we have used low-field 1H NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1463754

  14. Contributions of the D-serine pathway to schizophrenia.

    PubMed

    Labrie, Viviane; Wong, Albert H C; Roder, John C

    2012-03-01

    The glutamate neurotransmitter system is one of the major candidate pathways for the pathophysiology of schizophrenia, and increased understanding of the pharmacology, molecular biology and biochemistry of this system may lead to novel treatments. Glutamatergic hypofunction, particularly at the NMDA receptor, has been hypothesized to underlie many of the symptoms of schizophrenia, including psychosis, negative symptoms and cognitive impairment. This review will focus on D-serine, a co-agonist at the NMDA receptor that in combination with glutamate, is required for full activation of this ion channel receptor. Evidence implicating D-serine, NMDA receptors and related molecules, such as D-amino acid oxidase (DAO), G72 and serine racemase (SRR), in the etiology or pathophysiology of schizophrenia is discussed, including knowledge gained from mouse models with altered D-serine pathway genes and from preliminary clinical trials with D-serine itself or compounds modulating the D-serine pathway. Abnormalities in D-serine availability may underlie glutamatergic dysfunction in schizophrenia, and the development of new treatments acting through the D-serine pathway may significantly improve outcomes for many schizophrenia patients. PMID:21295046

  15. Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase.

    PubMed

    Evans, D J; Jones, R; Woodley, P R; Wilborn, J R; Robson, R L

    1991-09-01

    Nucleotide sequence was obtained for a region of 7,099 bp spanning the nifU, nifS, nifV, nifW, nifZ, and nifM genes from Azotobacter chroococcum. Chromosomal mutations constructed at several sites within the locus confirmed a requirement for this region for expression of the molybdenum nitrogenase in this organism. The genes are tightly clustered and ordered as in Klebsiella pneumoniae except for two additional open reading frames (ORFs) between nifV and nifW. The arrangement of genes in A. chroococcum closely matches that described for Azotobacter vinelandii. The polypeptide encoded by ORF4 immediately downstream from nifV is 41% identical over 186 amino acids to the product of the cysE gene from Escherichia coli, which encodes serine acetyltransferase (SAT), a key enzyme in cysteine biosynthesis. Plasmids which potentially express ORF4 complemented E. coli JM39, a cysteine auxotroph which lacks SAT. SAT activity was detected in crude extracts of one such complemented strain. A strain of A. chroococcum carrying a chromosomal disruption of ORF4 grew normally with ammonium as the N source but more slowly than the parental strain when N2 was the sole N source. These data suggest that ORF4 encodes a nif-specific SAT required for optimizing expression of nitrogenase activity. ORF4 was assigned the name nifP. nifP may be required to boost rates of synthesis or intracellular concentrations of cysteine or methionine. Sequence identity between nifV and leuA gene products suggests that nifV may catalyze a condensation reaction analogous to that carried out by isopropylmalate synthase (LEUA) but in which acetyl coenzyme and alpha-ketoglutarate are substrates for the formation of homocitrate, the proposed product of NIFV activity. PMID:1885524

  16. Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase.

    PubMed Central

    Evans, D J; Jones, R; Woodley, P R; Wilborn, J R; Robson, R L

    1991-01-01

    Nucleotide sequence was obtained for a region of 7,099 bp spanning the nifU, nifS, nifV, nifW, nifZ, and nifM genes from Azotobacter chroococcum. Chromosomal mutations constructed at several sites within the locus confirmed a requirement for this region for expression of the molybdenum nitrogenase in this organism. The genes are tightly clustered and ordered as in Klebsiella pneumoniae except for two additional open reading frames (ORFs) between nifV and nifW. The arrangement of genes in A. chroococcum closely matches that described for Azotobacter vinelandii. The polypeptide encoded by ORF4 immediately downstream from nifV is 41% identical over 186 amino acids to the product of the cysE gene from Escherichia coli, which encodes serine acetyltransferase (SAT), a key enzyme in cysteine biosynthesis. Plasmids which potentially express ORF4 complemented E. coli JM39, a cysteine auxotroph which lacks SAT. SAT activity was detected in crude extracts of one such complemented strain. A strain of A. chroococcum carrying a chromosomal disruption of ORF4 grew normally with ammonium as the N source but more slowly than the parental strain when N2 was the sole N source. These data suggest that ORF4 encodes a nif-specific SAT required for optimizing expression of nitrogenase activity. ORF4 was assigned the name nifP. nifP may be required to boost rates of synthesis or intracellular concentrations of cysteine or methionine. Sequence identity between nifV and leuA gene products suggests that nifV may catalyze a condensation reaction analogous to that carried out by isopropylmalate synthase (LEUA) but in which acetyl coenzyme and alpha-ketoglutarate are substrates for the formation of homocitrate, the proposed product of NIFV activity. PMID:1885524

  17. Amino acid composition, including key derivatives of eccrine sweat: potential biomarkers of certain atopic skin conditions.

    PubMed

    Mark, Harker; Harding, Clive R

    2013-04-01

    The free amino acid (AA) composition of eccrine sweat is different from other biological fluids, for reasons which are not properly understood. We undertook the detailed analysis of the AA composition of freshly isolated pure human eccrine sweat, including some of the key derivatives of AA metabolism, to better understand the key biological mechanisms governing its composition. Eccrine sweat was collected from the axillae of 12 healthy subjects immediately upon formation. Free AA analysis was performed using an automatic AA analyser after ninhydrin derivatization. Pyrrolidine-5-carboxylic acid (PCA) and urocanic acid (UCA) levels were determined using GC/MS. The free AA composition of sweat was dominated by the presence of serine accounting for just over one-fifth of the total free AA composition. Glycine was the next most abundant followed by PCA, alanine, citrulline and threonine, respectively. The data obtained indicate that the AA content of sweat bears a remarkable similarity to the AA composition of the epidermal protein profilaggrin. This protein is the key source of free AAs and their derivatives that form a major part of the natural moisturizing factor (NMF) within the stratum corneum (SC) and plays a major role in maintaining the barrier integrity of human skin. As perturbations in the production of NMF can lead to abnormal barrier function and can arise as a consequence of filaggrin genotype, we propose the quantification of AAs in sweat may serve as a non-invasive diagnostic biomarker for certain atopic skin conditions, that is, atopic dermatitis (AD). PMID:23075272

  18. Periodic density functional theory study of the high-pressure behavior of crystalline L-serine-L-ascorbic acid.

    PubMed

    Chen, Limin; Liu, Chunsheng; Fang, Henan; Xie, Qiyun; Kong, Chengkai; Ji, Guanghan; Xiang, Ziyue

    2016-01-01

    A detailed study of the structural, electronic and absorption properties of crystalline L-serine-L-ascorbic acid (SAA) in the pressure range of 0-300 GPa was performed by density-functional theory (DFT) calculations in this work. Our results show that the compressible crystal of SAA is anisotropic. Furthermore, specific analysis of the variation tendencies of bond lengths and bond angles under different pressures show that the main structural transformations occur at pressures of 40, 50, 70, 100, 130 and 150 GPa, accompanied by repeated formations and disconnections of covalent bonds between O2(P1) and C2(P2) as well as C3(P1) and O1(P2), and a newly formed five-atom ring at 100 GPa. In addition, from 40 to 230 GPa, complex hydrogen bond transformations occur in SAA under compression, while from 240 to 300 GPa, the curve of lattice constants, bond lengths and bond angles of SAA barely changes, suggesting structural stability after 230 GPa. Then, by analyzing the band gap and density of states of SAA, it was found that the crystal undergoes a phase transformation from insulator to semiconductor at 150 GPa and it becomes more sensitive under compression. In addition, a relatively high optical activity with the pressure increases of SAA was seen from the absorption spectra, and two obvious changes of absorption coefficients were also observed at 50 GPa and 130 GPa, respectively, indicating that structural transformations occur here. Graphical abstract Structural formation and breaking of the five-atom ring O1(P2)-C2(P2)-O2(P1)-C2(P1)-C3(P1) with increasing pressure. PMID:26711816

  19. Peptidyl inverse esters of p-methoxybenzoic acid: a novel class of potent inactivator of the serine proteases.

    PubMed Central

    Lynas, J; Walker, B

    1997-01-01

    A series of novel synthetic peptides, containing a C-terminal beta-amino alcohol linked to p-methoxybenzoic acid via an ester linkage, have been prepared and tested as inhibitors against typical members of the serine protease family. For example, the sequences Ac-Val-Pro-NH-CH-(CH2-C6H5)-CH2O-CO-C6H4-OCH3 (I) and Ac-Val-Pro-NH-CH-[CH-(CH3)2]-CH2O-CO-C6H4-OCH3 (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and elastase respectively, have been found to behave as exceptionally potent irreversible inactivators of their respective target protease. Thus I was found to inactivate chymotrypsin with an overall second-order rate constant (k2/Ki) of approx. 6.6x10(6) M-1. s-1, whereas II is an even more potent inactivator of human neutrophil elastase, exhibiting a second-order rate constant of inactivation of approx. 1.3x10(7) M-1.s-1. These values represent the largest rate constants ever reported for the inactivation of these proteases with synthetic peptide-based inactivators. On prolonged incubation in substrate-containing buffers, samples of the inactivated proteases were found to regain activity slowly. The first-order rate constants for the regeneration of enzymic activity from chymotrypsin and human neutrophil elastase inactivated by I and II respectively were determined to be approx. 5.8x10(-5) s-1 and approx. 4.3x10(-4) s-1. We believe that the most likely mechanism for the inactivation and regeneration of enzymic activity involves the formation and subsequent slow hydrolysis of long-lived acyl enzyme intermediates. PMID:9271079

  20. L-serine synthesis in the central nervous system: a review on serine deficiency disorders.

    PubMed

    Tabatabaie, L; Klomp, L W; Berger, R; de Koning, T J

    2010-03-01

    The de novo synthesis of the amino acid L-serine plays an essential role in the development and functioning of the central nervous system (CNS). L-serine displays many metabolic functions during different developmental stages; among its functions providing precursors for amino acids, protein synthesis, nucleotide synthesis, neurotransmitter synthesis and L-serine derived lipids. Patients with congenital defects in the L-serine synthesizing enzymes present with severe neurological abnormalities and underscore the importance of this synthetic pathway. In this review, we will discuss the cellular functions of the L-serine pathway, structure and enzymatic properties of the enzymes involved and genetic defects associated with this pathway. PMID:19963421

  1. L-Serine Deficiency Elicits Intracellular Accumulation of Cytotoxic Deoxysphingolipids and Lipid Body Formation.

    PubMed

    Esaki, Kayoko; Sayano, Tomoko; Sonoda, Chiaki; Akagi, Takumi; Suzuki, Takeshi; Ogawa, Takuya; Okamoto, Masahiro; Yoshikawa, Takeo; Hirabayashi, Yoshio; Furuya, Shigeki

    2015-06-01

    L-serine is required to synthesize membrane lipids such as phosphatidylserine and sphingolipids. Nevertheless, it remains largely unknown how a diminished capacity to synthesize L-serine affects lipid homeostasis in cells and tissues. Here, we show that deprivation of external L-serine leads to the generation of 1-deoxysphingolipids (doxSLs), including 1-deoxysphinganine, in mouse embryonic fibroblasts (KO-MEFs) lacking D-3-phosphoglycerate dehydrogenase (Phgdh), which catalyzes the first step in the de novo synthesis of L-serine. A novel mass spectrometry-based lipidomic approach demonstrated that 1-deoxydihydroceramide was the most abundant species of doxSLs accumulated in L-serine-deprived KO-MEFs. Among normal sphingolipid species in KO-MEFs, levels of sphinganine, dihydroceramide, ceramide, and hexosylceramide were significantly reduced after deprivation of external L-serine, whereas those of sphingomyelin, sphingosine, and sphingosine 1-phosphate were retained. The synthesis of doxSLs was suppressed by supplementing the culture medium with L-serine but was potentiated by increasing the ratio of L-alanine to L-serine in the medium. Unlike with L-serine, depriving cells of external L-leucine did not promote the occurrence of doxSLs. Consistent with results obtained from KO-MEFs, brain-specific deletion of Phgdh in mice also resulted in accumulation of doxSLs in the brain. Furthermore, L-serine-deprived KO-MEFs exhibited increased formation of cytosolic lipid bodies containing doxSLs and other sphingolipids. These in vitro and in vivo studies indicate that doxSLs are generated in the presence of a high ratio of L-alanine to L-serine in cells and tissues lacking Phgdh, and de novo synthesis of L-serine is necessary to maintain normal sphingolipid homeostasis when the external supply of this amino acid is limited. PMID:25903138

  2. D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1/SLC1A4 and ASCT2/SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures

    PubMed Central

    Foster, Alan C.; Farnsworth, Jill; Lind, Genevieve E.; Li, Yong-Xin; Yang, Jia-Ying; Dang, Van; Penjwini, Mahmud; Viswanath, Veena; Staubli, Ursula; Kavanaugh, Michael P.

    2016-01-01

    N-methyl-D-aspartate (NMDA) receptors play critical roles in synaptic transmission and plasticity. Activation of NMDA receptors by synaptically released L-glutamate also requires occupancy of co-agonist binding sites in the tetrameric receptor by either glycine or D-serine. Although D-serine appears to be the predominant co-agonist at synaptic NMDA receptors, the transport mechanisms involved in D-serine homeostasis in brain are poorly understood. In this work we show that the SLC1 amino acid transporter family members SLC1A4 (ASCT1) and SLC1A5 (ASCT2) mediate homo- and hetero-exchange of D-serine with physiologically relevant kinetic parameters. In addition, the selectivity profile of D-serine uptake in cultured rat hippocampal astrocytes is consistent with uptake mediated by both ASCT1 and ASCT2. Together these data suggest that SLC1A4 (ASCT1) may represent an important route of Na-dependent D-serine flux in the brain that has the ability to regulate extracellular D-serine and thereby NMDA receptor activity. PMID:27272177

  3. L-serine in disease and development.

    PubMed Central

    de Koning, Tom J; Snell, Keith; Duran, Marinus; Berger, Ruud; Poll-The, Bwee-Tien; Surtees, Robert

    2003-01-01

    The amino acid L-serine, one of the so-called non-essential amino acids, plays a central role in cellular proliferation. L-Serine is the predominant source of one-carbon groups for the de novo synthesis of purine nucleotides and deoxythymidine monophosphate. It has long been recognized that, in cell cultures, L-serine is a conditional essential amino acid, because it cannot be synthesized in sufficient quantities to meet the cellular demands for its utilization. In recent years, L-serine and the products of its metabolism have been recognized not only to be essential for cell proliferation, but also to be necessary for specific functions in the central nervous system. The findings of altered levels of serine and glycine in patients with psychiatric disorders and the severe neurological abnormalities in patients with defects of L-serine synthesis underscore the importance of L-serine in brain development and function. This paper reviews these recent insights into the role of L-serine and the pathways of L-serine utilization in disease and during development, in particular of the central nervous system. PMID:12534373

  4. Iron uptake in Pseudomonas aeruginosa mediated by N-(2,3-dihydroxybenzoyl)-L-serine and 2,3-dihydroxybenzoic acid.

    PubMed

    Screen, J; Moya, E; Blagbrough, I S; Smith, A W

    1995-03-15

    Pseudomonas aeruginosa is known to have an inducible uptake system for the enterobacterial siderophore enterobactin. In this work we have examined iron transport mediated by the biosynthetic precursor 2,3-dihydroxybenzoic acid and N-(2,3-dihydroxybenzoyl)-L-serine, a breakdown product of enterobactin. Iron complexed with 2,3-dihydroxybenzoyl-L-serine was transported into P. aeruginosa IA1 via a transport system which is energy-dependent and iron-repressible. The rate of transport was not altered by growing the cells in the presence of either pyoverdin or pyochelin, which have been shown previously to induce transport via that system. Growth of the cells in the presence of enterobactin did cause an increase in the rate of transport, indicating that the complex can be transported by the inducible enterobactin uptake system, but also that a separate system must exist. In contrast, transport of iron complexed with 2,3-dihydroxybenzoic acid was neither iron-repressible nor strongly energy-dependent, from which we conclude that there must be a novel mode of transport not characteristic of iron-siderophore transport systems. PMID:7737477

  5. Serine proteases of parasitic helminths.

    PubMed

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-02-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  6. Serine Proteases of Parasitic Helminths

    PubMed Central

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-01-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  7. Simultaneous and selective decarboxylation of L-serine and deamination of L-phenylalanine in an amino acid mixture--a means of separating amino acids for synthesizing biobased chemicals.

    PubMed

    Teng, Yinglai; Scott, Elinor L; Witte-van Dijk, Susan C M; Sanders, Johan P M

    2016-01-25

    Amino acids (AAs) obtained from the hydrolysis of biomass-derived proteins are interesting feedstocks for the chemical industry. They can be prepared from the byproduct of biofuel production and agricultural wastes. They are rich in functionalities needed in petrochemicals, providing the opportunity to save energy, reagents, and process steps. However, their separation is required before they can be applied for further applications. Electrodialysis (ED) is a promising separation method, but its efficiency needs to be improved when separating AAs with similar isoelectric points. Thus, specific conversions are required to form product with different charges. Here we studied the enzymatic conversions which can be used as a means to aid the ED separation of neutral AAs. A model mixture containing L-serine, L-phenylalanine and L-methionine was used. The reactions of L-serine decarboxylase and L-phenylalanine ammonia-lyase were employed to specifically convert serine and phenylalanine into ethanolamine and trans-cinnamic acid. At the isoelectric point of methionine (pH 5.74), the charge of ethanolamine and trans-cinnamic acid are +1 and -1, therefore facilitating potential separation into three different streams by electrodialysis. Here the enzyme kinetics, specificity, inhibition and the operational stabilities were studied, showing that both enzymes can be applied simultaneously to aid the ED separation of neutral AAs. PMID:25976628

  8. Structural Insight into Serine Protease Rv3671c that Protects M. tuberculosis from Oxidative and Acidic Stress

    SciTech Connect

    Biswas, Tapan; Small, Jennifer; Vandal, Omar; Odaira, Toshiko; Deng, Haiteng; Ehrt, Sabine; Tsodikov, Oleg V.

    2010-11-15

    Rv3671c, a putative serine protease, is crucial for persistence of Mycobacterium tuberculosis in the hostile environment of the phagosome. We show that Rv3671c is required for M. tuberculosis resistance to oxidative stress in addition to its role in protection from acidification. Structural and biochemical analyses demonstrate that the periplasmic domain of Rv3671c is a functional serine protease of the chymotrypsin family and, remarkably, that its activity increases on oxidation. High-resolution crystal structures of this protease in an active strained state and in an inactive relaxed state reveal that a solvent-exposed disulfide bond controls the protease activity by constraining two distant regions of Rv3671c and stabilizing it in the catalytically active conformation. In vitro biochemical studies confirm that activation of the protease in an oxidative environment is dependent on this reversible disulfide bond. These results suggest that the disulfide bond modulates activity of Rv3671c depending on the oxidative environment in vivo.

  9. Structural insight into serine protease Rv3671c that protects M. tuberculosis from oxidative and acidic stress

    PubMed Central

    Biswas, Tapan; Small, Jennifer; Vandal, Omar; Odaira, Toshiko; Deng, Haiteng; Ehrt, Sabine; Tsodikov, Oleg V.

    2010-01-01

    Summary Rv3671c, a putative serine protease, is crucial for persistence of M. tuberculosis in the hostile environment of the phagosome. We show that Rv3671c is required for M. tuberculosis resistance to oxidative stress in addition to its role in protection from acidification. Structural and biochemical analyses demonstrate that the periplasmic domain of Rv3671c is a functional serine protease of the chymotrypsin family and, remarkably, that its activity increases upon oxidation. High-resolution crystal structures of this protease in an active strained state and in an inactive relaxed state reveal that a solvent-exposed disulfide bond controls the protease activity by constraining two distant regions of Rv3671c and stabilizing it in the catalytically active conformation. In vitro biochemical studies confirm that activation of the protease in an oxidative environment is dependent on this reversible disulfide bond. These results suggest that the disulfide bond modulates activity of Rv3671c depending on the oxidative environment in vivo. PMID:20947023

  10. Extraterrestrial Amino Acids in Ureilites Including Almahata Sitta

    NASA Technical Reports Server (NTRS)

    Burton, A. S.; Glavin, D. P.; Callahan, M. P.; Dworkin, J. P.

    2011-01-01

    Ureilites are a class of meteorites that lack chondrules (achondrites) but have relatively high carbon abundances, averaging approx.3 wt %. Using highly sensitive liquid chromatography coupled with UV fluorescence and time-of-flight mass spectrometry (LC-FD/ToF-MS), it was recently determined that there are amino acids in. fragment 94 of the Almahata Sitta ureilite[l]. Based on the presence of amino acids that are rare in the Earth's biosphere, as well as the near-racemic enantiomeric ratios of marry of the more common amino acids, it was concluded that most of the detected amino acids were indigenous to the meteorite. Although the composition of the Almahata Sitta ureilite appears to be unlike other recovered ureilites, the discovery of amino acids in this meteorite raises the question of whether other ureilites rnav also contain amino acids. Herein we present the results of LC-FDlTo.F-MS analyses of: a sand sample from the Almahata Sitta strewn held, Almahata Sitta fragments 425 (an ordinary H5 chondrite) and 427 (ureilite), as well as an Antarctic ureilite (Allan lulls, ALHA 77257).

  11. Cleavage of peptide bonds bearing ionizable amino acids at P{sub 1} by serine proteases with hydrophobic S{sub 1} pocket

    SciTech Connect

    Qasim, Mohammad A.; Song, Jikui; Markley, John L.; Laskowski, Michael

    2010-10-01

    Research highlights: {yields} Large pK shifts in ionizable groups when buried in the protein interior. {yields} Substrate dependent shifts in pH optimum for serine proteases. {yields} Lys side chain is a stronger acid in serine protease S{sub 1} pocket than Asp side chain. -- Abstract: Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, and {approx}10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S{sub 1} pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.

  12. An essential role for de novo biosynthesis of L-serine in CNS development.

    PubMed

    Furuya, Shigeki

    2008-01-01

    L-serine plays a versatile role in intermediary metabolism in eukaryotic cells. The physiological significance of its de novo biosynthesis, however, remains largely unexplored. We demonstrated previously that neurons lose the ability to synthesize L-serine after their final differentiation and thus depend on astrocytes to supply this amino acid. This is due to a lack of neuronal expression of 3-phosphoglycerate dehydrogenase (Phgdh), which initiates de novo L-serine synthesis via the phosphorylated pathway from the glycolytic intermediate 3-phosphoglycerate. In rodent brain, Phgdh is expressed exclusively by the neuroepithelium/radial glia/astrocyte lineage. In humans, serine deficiency disorders can result from a deficiency of Phgdh or other enzymes involved in serine biosynthesis in the phosphorylated pathway. Patients with such disorders have lower serine levels in plasma and cerebrospinal fluid; they exhibit severe neurological symptoms including congenital microcephaly, feeding disabilities, and psychomotor retardation. L-serine supplementation can attenuate developmental defects in these patients. To define the physiological importance of de novo L-serine production, we generated Phgdh knockout mice using targeted gene disruption technique. Phgdh deletion drastically reduced serine and glycine levels in the body. Phgdh knockout mice exhibited overall growth retardation with severe brain malformation, culminating in embryonic lethality. These observations highlight the vital role of de novo L-serine synthesis in the formation and function of the mammalian central nervous system. Furthermore, the embryonic lethal phenotype of Phgdh knockouts indicates that L-serine must be synthesized endogenously in mouse (and probably humans) during embryonic development. PMID:18296366

  13. Polymerization of serine guanylate in the presence of montmorillonite

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1981-01-01

    Serine guanylate was prepared and its polymerization studied in the presence of montmorillonite and in its absence. In water, without clay, serine guanylate polymerizes in the same way as does serine adenylate. In the presence of montmorillonite, serine guanylate polymerizes to a lesser extent and produces also lower degrees of polymerization than does serine adenylate. It is postulated that the reason for this difference in behavior might lie in the fact that guanylic acid is much more acidic than adenylic acid; hence would bind much more strongly to the edges of montmorillonite and thus, by blocking these sites, would inhibit the catalytic activity of the clay.

  14. Conformers of Gaseous Serine.

    PubMed

    He, Kedan; Allen, Wesley D

    2016-08-01

    The myriad conformers of the neutral form of natural amino acid serine (Ser) have been investigated by systematic computations with reliable electronic wave function methods. A total of 85 unique conformers were located using the MP2/cc-pVTZ level of theory. The 12 lowest-energy conformers of serine fall within a 8 kJ mol(-1) window, and for these species, geometric structures, precise relative energies, equilibrium and vibrationally averaged rotational constants, anharmonic vibrational frequencies, infrared intensities, quartic and sextic centrifugal distortion constants, dipole moments, and (14)N nuclear quadrupole coupling constants were computed. The relative energies were refined through composite focal-point analyses employing basis sets as large as aug-cc-pV5Z and correlation treatments through CCSD(T). The rotational constants for seven conformers measured by Fourier-transform microwave spectroscopy are in good agreement with the vibrationally averaged rotational constants computed in this study. Our anharmonic vibrational frequencies are compared to the large number of experimental vibrational absorptions attributable to at least six conformers. PMID:27294314

  15. D-Serine and Serine Racemase Are Associated with PSD-95 and Glutamatergic Synapse Stability.

    PubMed

    Lin, Hong; Jacobi, Ariel A; Anderson, Stewart A; Lynch, David R

    2016-01-01

    D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs), synthesized by serine racemase (SR) through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking α7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR) are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1), in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) and 7-chlorokynurenic acid (7-CK), a specific antagonist at the glycine site of NMDARs, demonstrating

  16. D-Serine and Serine Racemase Are Associated with PSD-95 and Glutamatergic Synapse Stability

    PubMed Central

    Lin, Hong; Jacobi, Ariel A.; Anderson, Stewart A.; Lynch, David R.

    2016-01-01

    D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs), synthesized by serine racemase (SR) through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking α7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR) are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1), in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) and 7-chlorokynurenic acid (7-CK), a specific antagonist at the glycine site of NMDARs, demonstrating

  17. Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

    SciTech Connect

    Arasappan, Ashok; Njoroge, F. George; Parekh, Tejal N.; Yang, Xiaozheng; Pichardo, John; Butkiewicz, Nancy; Prongay, Andrew; Yao, Nanhua; Girijavallabhan, Viyyoor

    2008-06-30

    Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. Inhibitors derived from this new P2 core exhibited activity in the low {micro}M range. X-ray structure of an inhibitor, 15c bound to the protease is presented.

  18. Serine and glycine metabolism in cancer☆

    PubMed Central

    Amelio, Ivano; Cutruzzolá, Francesca; Antonov, Alexey; Agostini, Massimiliano; Melino, Gerry

    2014-01-01

    Serine and glycine are biosynthetically linked, and together provide the essential precursors for the synthesis of proteins, nucleic acids, and lipids that are crucial to cancer cell growth. Moreover, serine/glycine biosynthesis also affects cellular antioxidative capacity, thus supporting tumour homeostasis. A crucial contribution of serine/glycine to cellular metabolism is through the glycine cleavage system, which refuels one-carbon metabolism; a complex cyclic metabolic network based on chemical reactions of folate compounds. The importance of serine/glycine metabolism is further highlighted by genetic and functional evidence indicating that hyperactivation of the serine/glycine biosynthetic pathway drives oncogenesis. Recent developments in our understanding of these pathways provide novel translational opportunities for drug development, dietary intervention, and biomarker identification of human cancers. PMID:24657017

  19. Structure-activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps.

    PubMed

    Sundermann, Tom; Hanekamp, Walburga; Lehr, Matthias

    2016-08-01

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA2α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA2α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA2α and FAAH, respectively. PMID:26153239

  20. In Vivo d-Serine Hetero-Exchange through Alanine-Serine-Cysteine (ASC) Transporters Detected by Microelectrode Biosensors

    PubMed Central

    2013-01-01

    d-Serine, a co-agonist of N-methyl d-aspartate (NMDA) receptors, has been implicated in neurological and psychiatric disorders such as cerebral ischemia, lateral amyotrophic sclerosis, or schizophrenia. d-Serine signaling represents an important pharmacological target for treating these diseases; however, the biochemical mechanisms controlling extracellular d-serine levels in vivo are still unclear. d-Serine heteroexchange through small neutral amino acid transporters has been shown in cell cultures and brain slices and could provide a biochemical mechanism for the control of d-serine extracellular concentration in vivo. Alternatively, exocytotic d-serine release has also been proposed. In this study, the dynamics of d-serine release and clearance were explored in vivo on a second-by-second time scale using microelectrode biosensors. The rate of d-serine clearance in the rat frontal cortex after a microionophoretic injection revealed a transporter-mediated uptake mechanism. d-Serine uptake was blocked by small neutral l-amino acids, implicating alanine-serine-cysteine (ASC) transporters, in particular high affinity Asc-1 and low affinity ASCT2 transporters. Interestingly, changes in alanine, serine, or threonine levels resulted in d-serine release through ASC transporters. Asc-1, but not ASCT2, appeared to release d-serine in response to changes in amino acid concentrations. Finally, neuronal silencing by tetrodotoxin increased d-serine extracellular concentration by an ASC-transporter-dependent mechanism. Together, these results indicate that d-serine heteroexchange through ASC transporters is present in vivo and may constitute a key component in the regulation of d-serine extracellular concentration. PMID:23581544

  1. Purification and characterization of a new serine proteinase from Bacillus subtilis with specificity for amino acids at P1 and P2 positions.

    PubMed

    Yamagata, A; Yoshida, N; Noda, K; Ito, A

    1995-12-01

    A proteinase was purified 230-fold to apparent homogeneity from culture filtrates of Bacillus subtilis by a series of column chromatographies on DE52, DEAE-Toyopearl, Cellulofine GC200M, and Mono-Q, using Boc-Ala-Ala-Pro-Ser-pNA as a substrate. The molecular weight of the proteinase was estimated to be 42,000 by SDS-PAGE in the presence of 2-mercaptoethanol. Studies on the substrate specificity with peptide p-nitroanilides and natural peptides revealed that this proteinase preferentially hydrolyzed the peptide bond on the carboxyl-terminal side of either serine or alanine residues at the P1 position and hydrophobic bulky amino acids at P2. It was most active at pH 9.5 for the hydrolysis of Boc-Ala-Ala-Pro-Ser-pNA. The enzyme was inactivated by diisopropyl fluorophosphate (DFP), but not by tosyl-L-phenylalanine chloromethylketone (TPCK) or by EDTA. Based on the reactivity toward substrates and inhibitors, this enzyme differs from elastase- or subtilisin-like proteinase, hence it is a new type of proteinase with specificity for amino acids at P1 and P2 positions. PMID:8519806

  2. Phosphorylation of Simian Cytomegalovirus Assembly Protein Precursor (pAPNG.5) and Proteinase Precursor (pAPNG1): Multiple Attachment Sites Identified, Including Two Adjacent Serines in a Casein Kinase II Consensus Sequence

    PubMed Central

    Plafker, Scott M.; Woods, Amina S.; Gibson, Wade

    1999-01-01

    The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homologs in other herpesviruses, functions at several key steps during the process of capsid formation. This protein, and the genetically related maturational proteinase, is distinguished from the other capsid proteins by posttranslational modifications, including phosphorylation. The objective of this study was to identify sites at which pAP is phosphorylated so that the functional significance of this modification and the enzyme(s) responsible for it can be determined. In the work reported here, we used peptide mapping, mass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated. The other site(s) is in a different domain of the protein, is phosphorylated less frequently than the CKII site, does not require preceding CKII-site phosphorylation, and causes an electrophoretic mobility shift when phosphorylated. Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the proteinase or on the substrate behavior of pAP. Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications can be made by a cellular enzyme(s). Apparent compartmental differences in phosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modifications. PMID:10516011

  3. Characterization of mycosporine-serine-glycine methyl ester, a major mycosporine-like amino acid from dinoflagellates: a mass spectrometry study.

    PubMed

    Carignan, Mario O; Carreto, José I

    2013-08-01

    Several unknown mycosporine-like amino acids (MAAs) have been previously isolated from some cultured species of toxic dinoflagellates of the Alexandrium genus (Dinophyceae). One of them, originally called M-333, was tentatively identified as a shinorine methyl ester, but the precise nature of this compound is still unknown. Using a high-resolution reversed-phase liquid chromatography mass spectrometry analyses (HPLC/MS), we found that natural populations of the red tide dinoflagellate Prorocentrum micans Ehrenberg showed a net dominance of M-333 together with lesser amounts of other MAAs. We also documented the isolation and characterization of this MAA from natural dinoflagellate populations and from Alexandrium tamarense (Lebour) Balech cultures. Using a comparative fragmentation study in electrospray mass spectrometry between deuterated and non-deuterated M-333 compounds and synthesized mono and dimethyl esters of shinorine, this novel compound was characterized as mycosporine-serine-glycine methyl ester, a structure confirmed by nuclear magnetic resonance. These isobaric compounds can be differentiated by their fragmentation patterns in MS(3) experiments because the extension and the specific site of the methylation changed the fragmentation pathway. PMID:27007200

  4. Crystal growth and preliminary X-ray study of glutamic acid specific serine protease from Bacillus intermedius

    NASA Astrophysics Data System (ADS)

    Kuranova, I. P.; Blagova, E. V.; Levdikov, V. M.; Rudenskaya, G. N.; Balaban, N. P.; Shakirov, E. V.

    1999-01-01

    The glutamic acid specific protease (glutamyl-endopeptidase) from Bacillus intermedius, strain 3-19, was isolated and purified using ion exchange chromatography on CM-cellulose and Mono-S FPLC column. The conditions for crystallization of the enzyme have been discussed. The crystals of enzyme were grown using hanging-drop vapor-diffusion technique. Crystals belong to the space group C2 with unit cell parameters of a=61.62 Å, b=55.84 Å, c=60.40 Å, β=117.6° X-ray diffraction data to 1.68 Å resolution were collected using synchrotron radiation (EMBL, Hamburg) and an imaging plate scanner.

  5. Urinary loss of glucose, phosphate, and protein by diffusion into proximal straight tubules injured by D-serine and maleic acid

    SciTech Connect

    Carone, F.A.; Nakamura, S.; Goldman, B.

    1985-06-01

    In several models of acute renal failure leakage of glomerular filtrate out of the tubule is an important pathogenetic mechanism; however, bidirectional diffusion of solute to account for certain pathophysiologic features of acute renal failure has received meager attention. Using micropuncture and clearance methods, the authors assessed sequentially leakage of solutes and inulin across proximal straight tubules (PST) injured by two nephrotoxins. In d-serine-treated rats with extensive necrosis of PST, the basis for glucosuria and tubular leakage of inulin was studied. Glucose absorption by the proximal convoluted tubule and glucose delivery to the PST were normal, but glucose delivery to the distal tubule was increased nearly 8-fold, indicating diffusion of glucose from interstitial to tubular luminal fluid across the necrotic PST. Total kidney inulin clearance was greatly reduced, but single nephron glomerular filtration rate, based on proximal convoluted tubule samples, was normal, indicating tubular loss of inulin. Urinary recovery of (/sup 14/C)inulin infused into tubular lumina revealed that proximal convoluted tubule and distal tubule were impermeable to inulin and that inulin diffused out of the necrotic PST. The progressive return over 6 days of tubular impermeability for inulin correlated with relining of PST with new cells. In maleic acid-treated rats the site and extent of tubular necrosis and the nature of urinary loss of solutes were studied. Microdissection revealed that maleic acid caused limited necrosis of PST which averaged 7.4% of total proximal tubular length. Increased urinary excretion of protein, phosphate, and glucose and increased tubular permeability to microinfused (/sup 14/C)inulin occurred with the onset of PST necrosis, and return of these abnormalities to normal correlated with the degree of cellular repair of the PST.

  6. Catalysis of the Carbonylation of Alcohols to Carboxylic Acids Including Acetic Acid Synthesis from Methanol.

    ERIC Educational Resources Information Center

    Forster, Denis; DeKleva, Thomas W.

    1986-01-01

    Monsanto's highly successful synthesis of acetic acid from methanol and carbon monoxide illustrates use of new starting materials to replace pretroleum-derived ethylene. Outlines the fundamental aspects of the acetic acid process and suggests ways of extending the synthesis to higher carboxylic acids. (JN)

  7. D-serine transporter in Staphylococcus saprophyticus identified.

    PubMed

    Marlinghaus, Lennart; Huß, Melanie; Korte-Berwanger, Miriam; Sakinc-Güler, Türkan; Gatermann, Sören G

    2016-07-01

    Among staphylococci Staphylococcus saprophyticus is the only species that is typically uropathogenic and an important cause of urinary tract infections in young women. The amino acid D-serine occurs in relatively high concentrations in human urine and has a bacteriostatic or toxic effect on many bacteria. In uropathogenic Escherichia coli and S. saprophyticus, the amino acid regulates the expression of virulence factors and can be used as a nutrient. The ability of uropathogens to respond to or to metabolize D-serine has been suggested as a factor that enables colonization of the urinary tract. Until now nothing is known about D-serine transport in S. saprophyticus We generated mutants of putative transporter genes in S. saprophyticus 7108 that show homology to the D-serine transporter cycA of E. coli and tested them in a D-serine depletion assay to analyze the D-serine uptake rate of the cells. The mutant of SPP1070 showed a strong decrease in D-serine uptake. Therefore, SSP1070 was identified as a major D-serine transporter in S. saprophyticus 7108 and was named D-serine transporter A (DstA). D-serine caused a prolonged lag phase of S. saprophyticus in a chemically defined medium. This negative effect was dependent on the presence of DstA. PMID:27252156

  8. Serine deprivation enhances antineoplastic activity of biguanides.

    PubMed

    Gravel, Simon-Pierre; Hulea, Laura; Toban, Nader; Birman, Elena; Blouin, Marie-José; Zakikhani, Mahvash; Zhao, Yunhua; Topisirovic, Ivan; St-Pierre, Julie; Pollak, Michael

    2014-12-15

    Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism. PMID:25377470

  9. Polymerase chain reaction system using magnetic beads for analyzing a sample that includes nucleic acid

    SciTech Connect

    Nasarabadi, Shanavaz

    2011-01-11

    A polymerase chain reaction system for analyzing a sample containing nucleic acid includes providing magnetic beads; providing a flow channel having a polymerase chain reaction chamber, a pre polymerase chain reaction magnet position adjacent the polymerase chain reaction chamber, and a post pre polymerase magnet position adjacent the polymerase chain reaction chamber. The nucleic acid is bound to the magnetic beads. The magnetic beads with the nucleic acid flow to the pre polymerase chain reaction magnet position in the flow channel. The magnetic beads and the nucleic acid are washed with ethanol. The nucleic acid in the polymerase chain reaction chamber is amplified. The magnetic beads and the nucleic acid are separated into a waste stream containing the magnetic beads and a post polymerase chain reaction mix containing the nucleic acid. The reaction mix containing the nucleic acid flows to an analysis unit in the channel for analysis.

  10. Un-catalyzed peptide bond formation between two monomers of glycine, alanine, serine, threonine, and aspartic acid in gas phase: a density functional theory study

    NASA Astrophysics Data System (ADS)

    Bhunia, Snehasis; Singh, Ajeet; Ojha, Animesh K.

    2016-05-01

    In the present report, un-catalyzed peptide bond formation between two monomers of glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), and aspartic acid (Asp) has been investigated in gas phase via two steps reaction mechanism and concerted mechanism at B3LYP/6-31G(d,p) and M062X/6-31G(d,p) level of theories. The peptide bond is formed through a nucleophilic reaction via transition states, TS1 and TS2 in stepwise mechanism. The TS1 reveals formation of a new C-N bond while TS2 illustrate the formation of C=O bond. In case of concerted mechanism, C-N bond is formed by a single four-centre transition state (TS3). The energy barrier is used to explain the involvement of energy at each step of the reaction. The energy barrier (20-48 kcal/mol) is required for the transformation of reactant state R1 to TS1 state and intermediate state I1 to TS2 state. The large value of energy barrier is explained in terms of distortion and interaction energies for stepwise mechanism. The energy barrier of TS3 in concerted mechanism is very close to the energy barrier of the first transition state (TS1) of the stepwise mechanism for the formation of Gly-Gly and Ala-Ala di- peptide. However, in case of Ser-Ser, Thr-Thr and Asp-Asp di-peptide, the energy barrier of TS3 is relatively high than that of the energy barrier of TS1 calculated at B3LYP/6-31G(d,p) and M062X/6-31G(d,p) level of theories. In both the mechanisms, the value of energy barrier calculated at B3LYP/6-31G(d,p) level of theory is greater than that of the value calculated at M062X/6-31G(d,p) level of theory.

  11. Bioinformatics analysis of the serine and glycine pathway in cancer cells

    PubMed Central

    Morello, Maria; Minieri, Marilena; Melino, Gerry; Amelio, Ivano

    2014-01-01

    Serine and glycine are amino acids that provide the essential precursors for the synthesis of proteins, nucleic acids and lipids. Employing 3 subsequent enzymes, phosphoglycerate dehydrogenase (PHGDH), phosphoserine phosphatase (PSPH), phosphoserine aminotransferase 1 (PSAT1), 3-phosphoglycerate from glycolysis can be converted in serine, which in turn can by converted in glycine by serine methyl transferase (SHMT). Besides proving precursors for macromolecules, serine/glycine biosynthesis is also required for the maintenance of cellular redox state. Therefore, this metabolic pathway has a pivotal role in proliferating cells, including cancer cells. In the last few years an emerging literature provides genetic and functional evidences that hyperactivation of serine/glycine biosynthetic pathway drives tumorigenesis. Here, we extend these observations performing a bioinformatics analysis using public cancer datasets. Our analysis highlighted the relevance of PHGDH and SHMT2 expression as prognostic factor for breast cancer, revealing a substantial ability of these enzymes to predict patient survival outcome. However analyzing patient datasets of lung cancer our analysis reveled that some other enzymes of the pathways, rather than PHGDH, might be associated to prognosis. Although these observations require further investigations they might suggest a selective requirement of some enzymes in specific cancer types, recommending more cautions in the development of novel translational opportunities and biomarker identification of human cancers. PMID:25436979

  12. Serine incorporation into the selenocysteine moiety of glutathione peroxidase

    SciTech Connect

    Sunde, R.A.; Evenson, J.K.

    1987-01-15

    The selenium in mammalian glutathione peroxidase is present as a selenocysteine ((Se)Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the (Se)Cys moiety, we perfused isolated rat liver with /sup 14/C- or /sup 3/H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the (Se)Cys in GSH peroxidase to carboxymethylselenocysteine ((Se)Cys(Cm)), and determined the amino acid specific activity. Perfusion with (/sup 14/C)cystine resulted in (/sup 14/C)cystine incorporation into GSH peroxidase without labeling (Se)Cys(Cm), indicating that cysteine is not a direct precursor for (Se)Cys. (/sup 14/C)Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and (Se)Cys(Cm) in purified GSH peroxidase, whereas (3-3H)serine perfusion only labeled serine and (Se)Cys(Cm), thus demonstrating that the (Se)Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and (Se)Cys(Cm) strongly suggest that the precursor pool of serine used for (Se) Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.

  13. 11B NMR spectroscopy of peptide boronic acid inhibitor complexes of alpha-lytic protease. Direct evidence for tetrahedral boron in both boron-histidine and boron-serine adduct complexes.

    PubMed

    Tsilikounas, E; Kettner, C A; Bachovchin, W W

    1993-11-30

    We have previously shown, using 15N and 1H NMR spectroscopy, that MeOSuc-Ala-Ala-Pro-boroPhe and certain other boronic acid inhibitors form boron-histidine adducts with alpha-lytic protease instead of transition-state-like tetrahedral boron-serine adducts as is generally supposed [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. (1988) Biochemistry 27, 7689-7697]. An X-ray crystallographic study of the MeOSuc-Ala-Ala-Pro-boroPhe complex with alpha-lytic protease [Bone, R., Frank, D., Kettner, C. A., & Agard, D. A. (1989) Biochemistry 28, 7600-7609] has confirmed the existence of the boron-histidine bond but has concluded that the boron atom is trigonal rather than tetrahedral. Here we report a 11B NMR study at 160.46 MHz of this histidine adduct complex and of two other complexes known to be serine adducts: alpha-lytic protease with MeOSuc-Ala-Ala-Pro-boroVal and chymotrypsin with MeOSucAla-Ala-Pro-boroPhe. The 11B NMR chemical shifts demonstrate that the boron atom is tetrahedral in both the histidine and serine adduct complexes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8251483

  14. Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

    NASA Astrophysics Data System (ADS)

    Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

    2016-08-01

    β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as `transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

  15. Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

    PubMed Central

    Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

    2016-01-01

    β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as ‘transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs. PMID:27499424

  16. Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates.

    PubMed

    Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A; Clifton, Ian J; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B; Spencer, James; Fishwick, Colin W G; Schofield, Christopher J

    2016-01-01

    β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as 'transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs. PMID:27499424

  17. Hexachlorobenzene-induced alterations on neutral and acidic sphingomyelinases and serine palmitoyltransferase activities. A time course study in two strains of rats.

    PubMed

    Billi de Catabbi, S C; Setton-Advruj, C P; Sterin-Speziale, N; San Martín de Viale, L C; Cochón, A C

    2000-08-21

    Hexachlorobenzene (HCB) induces porphyria both in humans and rodents, and hepatocarcinoma in rodents. In a previous work we observed that HCB produces a continuous decrease in hepatic sphingomyelin (SM) content in Wistar rats. A distinguishing characteristic of sphingolipids breakdown products is their participation in anti-proliferative and apoptotic processes and in the suppression of oncogenesis. As a first step to elucidate the role of SM decrease in the hepatotoxicity induced by HCB, the present study evaluates the metabolic causes of the continuous decrease in hepatic SM content observed in Wistar rats with HCB intoxication, and its relation with porphyria development. For this purpose, the time-course (3, 7, 15, 21 and 28 days) of the effects of HCB on hepatic SM levels and on some of the enzymes of SM synthesis (serine palmitoyltransferase, SPT) and catabolism (sphingomyelinases, SMases) was followed, using two strains of rats differing in their susceptibility to acquire porphyria: Chbb THOM (low) and Wistar (high). HCB (1 g kg(-1) b.w. per day) was administered by gastric intubation as an aqueous suspension. After 5 days of HCB treatment, animals were allowed a 2-day recovery period without HCB administration. Two phases in the HCB-induced damages to sphingolipid metabolism were observed. The first stage (7 days of treatment), common to both strains of rats, was characterized by a decrease in hepatic SM levels (17-25%) and in SPT activity (50-43%), while strain differences were found for the later stage. In Chbb THOM rats, hepatic SM content was restored to normal values concomitantly with an increase in SPT activity (44%, at day 28), and without any increase in SM catabolism. In addition, the level of the other phospholipids was not altered. In Wistar rats, hepatic SM levels decreased continuously throughout the experiment, accompanied by increases in SPT, acidic sphingomyelinase (A-SMase) and neutral sphingomyelinase (N-SMase) activities (86, 28.5 and 78

  18. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    NASA Astrophysics Data System (ADS)

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  19. On the phenotypic spectrum of serine biosynthesis defects.

    PubMed

    El-Hattab, Ayman W; Shaheen, Ranad; Hertecant, Jozef; Galadari, Hassan I; Albaqawi, Badi S; Nabil, Amira; Alkuraya, Fowzan S

    2016-05-01

    L-serine is a non-essential amino acid that is de novo synthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, L-serine is a precursor of a number of important compounds. Serine biosynthesis defects result from deficiencies in PGDH, PSAT, or PSP and have a broad phenotypic spectrum ranging from Neu-Laxova syndrome, a lethal multiple congenital anomaly disease at the severe end to a childhood disease with intellectual disability at the mild end, with infantile growth deficiency, and severe neurological manifestations as an intermediate phenotype. In this report, we present three subjects with serine biosynthesis effects. The first was a stillbirth with Neu-Laxova syndrome and a homozygous mutation in PHGDH. The second was a neonate with growth deficiency, microcephaly, ichthyotic skin lesions, seizures, contractures, hypertonia, distinctive facial features, and a homozygous mutation in PSAT1. The third subject was an infant with growth deficiency, microcephaly, ichthyotic skin lesions, anemia, hypertonia, distinctive facial features, low serine and glycine in plasma and CSF, and a novel homozygous mutation in PHGDH gene. Herein, we also review previous reports of serine biosynthesis defects and mutations in the PHGDH, PSAT1, and PSPH genes, discuss the variability in the phenotypes associated with serine biosynthesis defects, and elaborate on the vital roles of serine and the potential consequences of its deficiency. PMID:26960553

  20. Serine protease inhibitors of parasitic helminths.

    PubMed

    Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

    2012-05-01

    Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships. PMID:22310379

  1. Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis.

    PubMed

    Manuse, Sylvie; Fleurie, Aurore; Zucchini, Laure; Lesterlin, Christian; Grangeasse, Christophe

    2016-01-01

    Bacteria possess a repertoire of versatile protein kinases modulating diverse aspects of their physiology by phosphorylating proteins on various amino acids including histidine, cysteine, aspartic acid, arginine, serine, threonine and tyrosine. One class of membrane serine/threonine protein kinases possesses a catalytic domain sharing a common fold with eukaryotic protein kinases and an extracellular mosaic domain found in bacteria only, named PASTA for 'Penicillin binding proteins And Serine/Threonine kinase Associated'. Over the last decade, evidence has been accumulating that these protein kinases are involved in cell division, morphogenesis and developmental processes in Firmicutes and Actinobacteria. However, observations differ from one species to another suggesting that a general mechanism of activation of their kinase activity is unlikely and that species-specific regulation of cell division is at play. In this review, we survey the latest research on the structural aspects and the cellular functions of bacterial serine/threonine kinases with PASTA motifs to illustrate the diversity of the regulatory mechanisms controlling bacterial cell division and morphogenesis. PMID:26429880

  2. Paradoxical roles of serine racemase and D-serine in the G93A mSOD1 mouse model of ALS

    PubMed Central

    Thompson, Misty; Marecki, John C.; Marinesco, Stephane; Labrie, Viviane; Roder, John C.; Barger, Steven W.; Crow, John P.

    2012-01-01

    D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A SOD1 mice – the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the transporter Asc-1. Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the presymptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator. PMID:22117694

  3. Fatty Acid Composition of Egg Yolk from Chickens Fed a Diet including Marigold (Tagetes erecta L.)

    PubMed Central

    Altuntaş, A.; Aydin, R.

    2014-01-01

    The objective of this study was to determine the effects of diet supplemented with marigold on egg yolk fatty acid composition and egg quality parameters. Sixty hens were assigned into three groups and fed diets supplemented with 0 (control), 10 g kg−1, or 20 g kg−1 marigold for 42 days. Eggs collected at the 6th week of the study were analyzed for fatty acid analysis. Laying performance, egg quality parameters, and feed intake were also evaluated. Yolk color scores in the group fed the 20 g kg−1 marigold-supplemented diet were found greater than control (10.77 versus 9.77). Inclusion of 20 g kg−1 marigold in diet influenced egg weights adversely compared to the control. Diet supplemented with 10 g kg−1 or 20 g kg−1 marigold increased the levels of C16:0 and C18:0 and decreased levels of C16:1 (n-7) and C18:1 (n-9) in the egg yolk. Also, diet including marigold increased total saturated fatty acids (SFA) and decreased monounsaturated fatty acids (MUFA) in the egg yolk. PMID:25587451

  4. New L-Serine Derivative Ligands as Cocatalysts for Diels-Alder Reaction

    PubMed Central

    Sousa, Carlos A. D.; Rodríguez-Borges, José E.; Freire, Cristina

    2013-01-01

    New L-serine derivative ligands were prepared and tested as cocatalyst in the Diels-Alder reactions between cyclopentadiene (CPD) and methyl acrylate, in the presence of several Lewis acids. The catalytic potential of the in situ formed complexes was evaluated based on the reaction yield. Bidentate serine ligands showed good ability to coordinate medium strength Lewis acids, thus boosting their catalytic activity. The synthesis of the L-serine ligands proved to be highly efficient and straightforward. PMID:24383009

  5. Neuroserpin, an axonally secreted serine protease inhibitor.

    PubMed Central

    Osterwalder, T; Contartese, J; Stoeckli, E T; Kuhn, T B; Sonderegger, P

    1996-01-01

    We have identified and chromatographically purified an axonally secreted glycoprotein of CNS and PNS neurons. Several peptides derived from it were microsequenced. Based on these sequences, a fragment of the corresponding cDNA was amplified and used as a probe to isolate a full length cDNA from a chicken brain cDNA library. Because the deduced amino acid sequence qualified the protein as a novel member of the serpin family of serine protease inhibitors, we called it neuroserpin. Analysis of the primary structural features further characterized neuroserpin as a heparin-independent, functional inhibitor of a trypsin-like serine protease. In situ hybridization revealed a predominantly neuronal expression during the late stages of neurogenesis and in the adult brain in regions which exhibit synaptic plasticity. Thus, neuroserpin might function as an axonally secreted regulator of the local extracellular proteolysis involved in the reorganization of the synaptic connectivity during development and synapse plasticity in the adult. Images PMID:8670795

  6. Does the autoantibody immunodominant region on thyroid peroxidase include amino acid residues 742-771?

    PubMed

    Xiong, Z; Farilla, L; Guo, J; McLachlan, S; Rapoport, B

    2001-03-01

    Identification of the thyroid peroxidase (TPO) amino acid residues that comprise the autoantibody immunodominant region is an important goal that has proven difficult because of the conformational nature of the epitopes involved. Recent data suggest that the immunodominant region has been located. Thus, by autoantibody recognition of tryptic fragments of native TPO, as well as of conformational portions of TPO expressed as cell-free translates, the autoantibody immunodominant region appears to include amino acid residues 742-771, near the C terminus of the ectodomain. To evaluate this deduction, we expressed as cell-free translates the full TPO ectodomain, as well as TPO truncated after residues 741 and 771. The epitopic integrity of these molecules was first confirmed by immunoprecipitation by patient sera containing TPO autoantibodies. However, autoantibody recognition could involve a minority of TPO autoantibodies with the individual sera, not fulfilling the strict criteria for immunodominance. In order to obtain definitive data, we performed immunoprecipitations on these TPO variants with four recombinant human monoclonal autoantibodies that define the immunodominant region. All four monoclonal autoantibodies immunoprecipitated TPO 1-741 to the same extent as they did TPO 1-771 and the full TPO ectodomain, indicating that the immunodominant region comprises (at least in large part) amino acid residues upstream of residue 741. PMID:11327613

  7. Pro-soft Val-boroPro: a strategy for enhancing in vivo performance of boronic acid inhibitors of serine proteases.

    PubMed

    Poplawski, Sarah E; Lai, Jack H; Sanford, David G; Sudmeier, James L; Wu, Wengen; Bachovchin, William W

    2011-04-14

    Val-boroPro, 1, is a potent, but relatively nonspecific inhibitor of the prolyl peptidases. It has antihyperglycemic activity from inhibition of DPPIV but also striking anticancer activity and a toxicity for which the mechanisms are unknown. 1 cyclizes at physiological pH, which attenuates its inhibitory potency >100-fold, which is a "soft drug" effect. Here we show that this phenomenon can be exploited to create prodrugs with unique properties and potential for selective in vivo targeting. Enzyme-mediated release delivers 1 to the target in the active form at physiological pH; cyclization attenuates systemic pharmacological effects from subsequent diffusion. This "pro-soft" design is demonstrated with a construct activated by and targeted to DPPIV, including in vivo results showing improved antihyperglycemic activity and reduced toxicity relative to 1. Pro-soft derivatives of 1 can help to illuminate the mechanisms underlying the three biological activities, or to help localize 1 at a tumor and thereby lead to improved anticancer agents with reduced toxicity. The design concept can also be applied to a variety of other boronic acid inhibitors. PMID:21388136

  8. Involvement of fub4, a putative serine hydrolase, in fusaric acid biosynthesis in the cotton pathogen Fusarium oxysporum f. sp. vasinfectum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous work has determined that fusaric acid is required for virulence in the Australian isolate of Fusarium oxysporum f. sp. vasinfectum (Fov), which produce copious amounts of fusaric acid. Race 4 isolates, identified in the San Joaquin Valley of California, has caused serious losses and is a p...

  9. Synthesis of Branched Methyl Hydroxy Stearates Including an Ester from Bio-Based Levulinic Acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the synthesis of 5 useful branched methyl alpha-hydroxy oleate esters from commercially available methyl oleate and common organic acids. Of special interest is the synthesis utilizing the natural byproduct, levulinic acid. The other common organic acids used herein were propionic acid, ...

  10. Covalent structure of human haptoglobin: a serine protease homolog.

    PubMed Central

    Kurosky, A; Barnett, D R; Lee, T H; Touchstone, B; Hay, R E; Arnott, M S; Bowman, B H; Fitch, W M

    1980-01-01

    The complete amino acid sequences and the disulfide arrangements of the two chains of human haptoglobin 1-1 were established. The alpha 1 and beta chains of haptoglobin contain 83 and 245 residues, respectively. Comparison of the primary structure of haptoglobin with that of the chymotrypsinogen family of serine proteases revealed a significant degree of chemical similarity. The probability was less than 10(-5) that the chemical similarity of the beta chain of haptoglobin to the proteases was due to chance. The amino acid sequence of the beta chain of haptoglobin is 29--33% identical to bovine trypsin, bovine chymotrypsin, porcine elastase, human thrombin, or human plasmin. Comparison of haptoglobin alpha 1 chain to activation peptide regions of the zymogens revealed an identity of 25% to the fifth "kringle" region of the activation peptide of plasminogen. The probability was less than 0.014 that this similarity was due to chance. These results strongly indicate haptoglobin to be a homolog of the chymotrypsinogen family of serine proteases. Alignment of the beta-chain sequence of haptoglobin to the serine proteases is remarkably consistent except for an insertion of 16 residues in the region corresponding to the methionyl loop of the serine proteases. The active-site residues typical of the serine proteases, histidine-57 and serine-195, are replaced in haptoglobin by lysine and alanine, respectively; however, aspartic acid-102 and the trypsin specificity, residue, aspartic acid-189, do occur in haptoglobin. Haptoglobin and the serine proteases represent a striking example of homologous proteins with different biological functions. PMID:6997877

  11. The effect of D-serine administration on cognition and mood in older adults

    PubMed Central

    Madeira, Caroline; Vargas-Lopes, Charles; Marques, Priscila; Dantas, Camila; Manhães, Alex C.; Leite, Homero; Panizzutti, Rogerio

    2016-01-01

    Background D-serine is an endogenous co-agonist of the N-Methyl D-Aspartate Receptor (NMDAR) that plays a crucial role in cognition including learning processes and memory. Decreased D-serine levels have been associated with age-related decline in mechanisms of learning and memory in animal studies. Here, we asked whether D-serine administration in older adults improves cognition. Results D-serine administration improved performance in the Groton Maze learning test of spatial memory and learning and problem solving (F(3, 38)= 4.74, p = 0.03). Subjects that achieved higher increases in plasma D-serine levels after administration improved more in test performance (r2=−0.19 p = 0.009). D-serine administration was not associated with any significant changes in the other cognitive tests or in the mood of older adults (p > 0.05). Methods Fifty healthy older adults received D-serine and placebo in a randomized, double blind, placebo-controlled, crossover design study. We studied the effect of D-serine administration on the performance of cognitive tests and an analogue mood scale. We also collected blood samples to measure D-serine, L-serine, glutamate and glutamine levels. Conclusions D-serine administration may be a strategy to improve spatial memory, learning and problem solving in healthy older adults. Future studies should evaluate the impact of long-term D-serine administration on cognition in older adults. PMID:26933803

  12. Ischemic Acute Kidney Injury Perturbs Homeostasis of Serine Enantiomers in the Body Fluid in Mice: Early Detection of Renal Dysfunction Using the Ratio of Serine Enantiomers

    PubMed Central

    Sasabe, Jumpei; Suzuki, Masataka; Miyoshi, Yurika; Tojo, Yosuke; Okamura, Chieko; Ito, Sonomi; Konno, Ryuichi; Mita, Masashi; Hamase, Kenji; Aiso, Sadakazu

    2014-01-01

    The imbalance of blood and urine amino acids in renal failure has been studied mostly without chiral separation. Although a few reports have shown the presence of D-serine, an enantiomer of L-serine, in the serum of patients with severe renal failure, it has remained uncertain how serine enantiomers are deranged in the development of renal failure. In the present study, we have monitored serine enantiomers using a two-dimensional HPLC system in the serum and urine of mice after renal ischemia-reperfusion injury (IRI), known as a mouse model of acute kidney injury. In the serum, the level of D-serine gradually increased after renal IRI in parallel with that of creatinine, whereas the L-serine level decreased sharply in the early phase after IRI. The increase of D-serine was suppressed in part by genetic inactivation of a D-serine-degrading enzyme, D-amino acid oxidase (DAO), but not by disruption of its synthetic enzyme, serine racemase, in mice. Renal DAO activity was detected exclusively in proximal tubules, and IRI reduced the number of DAO-positive tubules. On the other hand, in the urine, D-serine was excreted at a rate nearly triple that of L-serine in mice with sham operations, indicating that little D-serine was reabsorbed while most L-serine was reabsorbed in physiological conditions. IRI significantly reduced the ratio of urinary D−/L-serine from 2.82±0.18 to 1.10±0.26 in the early phase and kept the ratio lower than 0.5 thereafter. The urinary D−/L-serine ratio can detect renal ischemia earlier than kidney injury molecule-1 (KIM-1) or neutrophil gelatinase-associated lipocalin (NGAL) in the urine, and more sensitively than creatinine, cystatin C, or the ratio of D−/L-serine in the serum. Our findings provide a novel understanding of the imbalance of amino acids in renal failure and offer a potential new biomarker for an early detection of acute kidney injury. PMID:24489731

  13. Inactivation of the 3-phosphoglycerate dehydrogenase gene in mice: changes in gene expression and associated regulatory networks resulting from serine deficiency.

    PubMed

    Furuya, Shigeki; Yoshida, Kazuyuki; Kawakami, Yuriko; Yang, Jyung Hoon; Sayano, Tomoko; Azuma, Norihiro; Tanaka, Hideyuki; Kuhara, Satoru; Hirabayashi, Yoshio

    2008-08-01

    D-3-Phosphoglycerate dehydrogenase (Phgdh) is a necessary enzyme for de novo L-serine biosynthesis. Mutations in the human PHGDH cause serine deficiency disorders characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. We showed previously that targeted disruption of Phgdh in mice causes overall growth retardation with severe brain microcephaly and leads to embryonic lethality. Here, amino acid analysis of Phgdh knockout (KO) mouse embryos demonstrates that free serine and glycine concentrations are decreased markedly in head samples, reflecting the metabolic changes of serine deficiency found in human patients. To understand the pathogenesis of serine deficiency disorders at the molecular level, we have exploited this animal model to identify altered gene expression patterns using a microarray technology. Comparative microarray analysis of the Phgdh KO and wild-type head at gestational day 13.5 revealed an upregulation of genes involved in transfer RNA aminoacylation, amino acid metabolism, amino acid transport, transcriptional regulation, and translation, and a downregulation of genes involved in transcription in neuronal progenitors and muscle and cartilage development. A computational network analysis software was used to construct transcriptional regulatory networks operative in the Phgdh KO embryos in vivo. These observations suggest that Phgdh inactivation alters transcriptional programs in several regulatory networks. PMID:18228065

  14. Fatty acid composition including cis-9, trans-11 CLA of cooked ground lamb

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little information is available on effect of cooking on beneficial fatty acids such as conjugated linoleic acid (CLA) and n-3 polyunsaturated fatty acids (PUFA). The objective of this study was to examine impact of cooking on the FA composition of ground lamb of two different muscles. Samples were p...

  15. The role of D-serine in peripheral tissues.

    PubMed

    Montesinos Guevara, Camila; Mani, Ali R

    2016-06-01

    A considerable level of D-serine (a free D-amino acid) was discovered, surprisingly, in the mammalian brain in the early 1990s. Since then, D-serine has been considered to be a co-agonist of glutamate at the glycine site of NMDA receptors. D-serine is synthetized by racemization of L-serine in most neural and non-neural cells, and modulates a variety of physiological functions in mammals. In addition to the central nervous system, NMDA receptors have an important function in the modulation of physiological processes in peripheral tissues. Thus, investigations on the functions of D-serine in the peripheral nervous system, as well as the visceral organs, have gained attention in recent years. In this review we summarize the current knowledge on the role of D-serine in the kidneys, skeletal system, skin as well as on the non-adrenergic, non-cholinergic transmission within the autonomic nervous system. PMID:27038518

  16. Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding.

    PubMed

    Medeiros, Ane H; Mingossi, Fabiana B; Dias, Renata O; Franco, Flávia P; Vicentini, Renato; Mello, Marcia O; Moura, Daniel S; Silva-Filho, Marcio C

    2016-01-01

    Sugarcane's (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory. PMID:27598134

  17. Identification of Novel Peptidyl Serine α-Galactosyltransferase Gene Family in Plants*♦

    PubMed Central

    Saito, Fumie; Suyama, Akiko; Oka, Takuji; Yoko-o, Takehiko; Matsuoka, Ken; Jigami, Yoshifumi; Shimma, Yoh-ichi

    2014-01-01

    In plants, serine residues in extensin, a cell wall protein, are glycosylated with O-linked galactose. However, the enzyme that is involved in the galactosylation of serine had not yet been identified. To identify the peptidyl serine O-α-galactosyltransferase (SGT), we chose Chlamydomonas reinhardtii as a model. We established an assay system for SGT activity using C. reinhardtii and Arabidopsis thaliana cell extracts. SGT protein was partially purified from cell extracts of C. reinhardtii and analyzed by tandem mass spectrometry to determine its amino acid sequence. The sequence matched the open reading frame XP_001696927 in the C. reinhardtii proteome database, and a corresponding DNA fragment encoding 748 amino acids (BAL63043) was cloned from a C. reinhardtii cDNA library. The 748-amino acid protein (CrSGT1) was produced using a yeast expression system, and the SGT activity was examined. Hydroxylation of proline residues adjacent to a serine in acceptor peptides was required for SGT activity. Genes for proteins containing conserved domains were found in various plant genomes, including A. thaliana and Nicotiana tabacum. The AtSGT1 and NtSGT1 proteins also showed SGT activity when expressed in yeast. In addition, knock-out lines of AtSGT1 and knockdown lines of NtSGT1 showed no or reduced SGT activity. The SGT1 sequence, which contains a conserved DXD motif and a C-terminal membrane spanning region, is the first example of a glycosyltransferase with type I membrane protein topology, and it showed no homology with known glycosyltransferases, indicating that SGT1 belongs to a novel glycosyltransferase gene family existing only in the plant kingdom. PMID:24914209

  18. Oxidative Deselenization of Selenocysteine: Applications for Programmed Ligation at Serine.

    PubMed

    Malins, Lara R; Mitchell, Nicholas J; McGowan, Sheena; Payne, Richard J

    2015-10-19

    Despite the unique chemical properties of selenocysteine (Sec), ligation at Sec is an under-utilized methodology for protein synthesis. We describe herein an unprecedented protocol for the conversion of Sec to serine (Ser) in a single, high-yielding step. When coupled with ligation at Sec, this transformation provides a new approach to programmed ligations at Ser residues. This new reaction is compatible with a wide range of functionality, including the presence of unprotected amino acid side chains and appended glycans. The utility of the methodology is demonstrated in the rapid synthesis of complex glycopeptide fragments of the epithelial glycoproteins MUC5AC and MUC4 and through the total synthesis of the structured, cysteine (Cys)-free protein eglin C. PMID:26384718

  19. Development of Trypsin-Like Serine Protease Inhibitors as Therapeutic Agents: Opportunities, Challenges, and their Unique Structure-Based Rationales.

    PubMed

    Liang, Guyan; Bowen, J Phillip

    2016-01-01

    There has been a revolution in the development of effective, small-molecule anticoagulants and antiplatelet agents. Numerous trypsin-like serine proteases have been under active pursuit as therapeutic targets. Important examples include thrombin, factor VIIa, factor Xa, and β-tryptase with indications ranging from thrombosis and inflammation to asthma and chronic obstructive pulmonary disease (COPD). Trypsin-like serine proteases exhibit a highly similar tertiary folding pattern, especially for the region near the substrate binding pocket that includes the conserved catalytic triad consisting of histidine 57, aspartic acid 102, and serine 195. A rich collection of X-ray structures for many trypsin-like serine proteases is available, which greatly facilitated the optimization of small organic inhibitors as therapeutic agents. The present review surveyed those inhibitors disclosed in peer-reviewed scientific journals and patent publications with a special focus on structural features and protein-inhibitor interactions that implicated the inhibitor optimization process. The role played by the residue 190 of trypsin-like serine proteases is critical. While many inhibitors without a basic group have progressed into the clinic for ones with alanine 190, the task for those with serine 190 remains extremely challenging, if not impossible. In addition to warfarin, heparin, and low molecular weight heparins (LMWHs), treatment options have expanded with the development and approval of the new oral anticoagulants (NOACs). The NOACs are superior to vitamin K antagonists in terms of rapid onset, pharmacokinetics, drug/food interactions, and regular coagulation monitoring; but one serious drawback is the lack of an effective antidote at this time. Apixaban (Eliquis), rivaroxaban (Xarelto), and edoxaban (Savaysa) are the new Xa inhibitors that have been recently approved by the U.S. FDA and are in current clinical practice. These drugs bind to the active site of factor Xa (f

  20. Influence of Fatty Acid Precursors, Including Food Preservatives, on the Growth and Fatty Acid Composition of Listeria monocytogenes at 37 and 10°C ▿

    PubMed Central

    Julotok, Mudcharee; Singh, Atul K.; Gatto, Craig; Wilkinson, Brian J.

    2010-01-01

    Listeria monocytogenes is a food-borne pathogen that grows at refrigeration temperatures and increases its content of anteiso-C15:0 fatty acid, which is believed to be a homeoviscous adaptation to ensure membrane fluidity, at these temperatures. As a possible novel approach for control of the growth of the organism, the influences of various fatty acid precursors, including branched-chain amino acids and branched- and straight-chain carboxylic acids, some of which are also well-established food preservatives, on the growth and fatty acid composition of the organism at 37°C and 10°C were studied in order to investigate whether the organism could be made to synthesize fatty acids that would result in impaired growth at low temperatures. The results indicate that the fatty acid composition of L. monocytogenes could be modulated by the feeding of branched-chain amino acid, C4, C5, and C6 branched-chain carboxylic acid, and C3 and C4 straight-chain carboxylic acid fatty acid precursors, but the growth-inhibitory effects of several preservatives were independent of effects on fatty acid composition, which were minor in the case of preservatives metabolized via acetyl coenzyme A. The ability of a precursor to modify fatty acid composition was probably a reflection of the substrate specificities of the first enzyme, FabH, in the condensation of primers of fatty acid biosynthesis with malonyl acyl carrier protein. PMID:20048057

  1. The pharmacological landscape and therapeutic potential of serine hydrolases.

    PubMed

    Bachovchin, Daniel A; Cravatt, Benjamin F

    2012-01-01

    Serine hydrolases perform crucial roles in many biological processes, and several of these enzymes are targets of approved drugs for indications such as type 2 diabetes, Alzheimer's disease and infectious diseases. Despite this, most of the human serine hydrolases (of which there are more than 200) remain poorly characterized with respect to their physiological substrates and functions, and the vast majority lack selective, in vivo-active inhibitors. Here, we review the current state of pharmacology for mammalian serine hydrolases, including marketed drugs, compounds that are under clinical investigation and selective inhibitors emerging from academic probe development efforts. We also highlight recent methodological advances that have accelerated the rate of inhibitor discovery and optimization for serine hydrolases, which we anticipate will aid in their biological characterization and, in some cases, therapeutic validation. PMID:22212679

  2. The Pharmacological Landscape and Therapeutic Potential of Serine Hydrolases

    PubMed Central

    Bachovchin, Daniel A.; Cravatt, Benjamin F.

    2013-01-01

    Serine hydrolases play critical roles in many biological processes, and several are targets of approved drugs for indications such as type 2 diabetes, Alzheimer’s disease, and infectious disease. Despite this, most of the 200+ human serine hydrolases remain poorly characterized with respect to their physiological substrates and functions, and the vast majority lack selective, in vivo-active inhibitors. Here, we review the current state of pharmacology for mammalian serine hydrolases, including marketed drugs, compounds under clinical investigation, and selective inhibitors emerging from academic probe development efforts. We also highlight recent methodological advances that have accelerated the rate of inhibitor discovery and optimization for serine hydrolases, which we anticipate will aid in their biological characterization and, in some cases, therapeutic validation. PMID:22212679

  3. [Metabolic flux analysis of L-serine synthesis by Corynebacterium glutamicum SYPS-062].

    PubMed

    Zhang, Xiaomei; Dou, Wenfang; Xu, Hongyu; Xu, Zhenghong

    2010-10-01

    Corynebacterium glutamicum SYPS-062 was an L-serine producing strain stored at our lab and could produce L-serine directly from sugar. We studied the effects of cofactors in one carbon unit metabolism-folate and VB12 on the cell growth, sucrose consumption and L-serine production by SYPS-062. In the same time, the metabolic flux distribution was determined in different conditions. The supplementation of folate or VB12 enhanced the cell growth, energy synthesis, and finally increased the flux of pentose phosphate pathway (HMP), whereas the carbon flux to L-serine was decreased. The addition of VB12 not only increased the ratio of L-serine synthesis pathway on G3P joint, but also caused the insufficiency of tricarboxylic acid cycle (TCA) flux, which needed more anaplerotic reaction flux to replenish TCA cycle, that was an important limiting factor for the further increasing of the L-serine productivity. PMID:21218623

  4. Gene characterization of two digestive serine proteases in orange blossom wheat midge (Sitodiplosis mosellana)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two full length cDNA sequences, encoding digestive serine proteases (designated as SmPROT-1 and SmPROT-2), were recovered from the midgut of the wheat midge, Sitodiplosis mosellana in an ongoing EST project. The deduced amino acid sequences shared homology with digestive serine proteases from insect...

  5. Substrate properties of C1 inhibitor Ma (alanine 434----glutamic acid). Genetic and structural evidence suggesting that the P12-region contains critical determinants of serine protease inhibitor/substrate status.

    PubMed

    Skriver, K; Wikoff, W R; Patston, P A; Tausk, F; Schapira, M; Kaplan, A P; Bock, S C

    1991-05-15

    The serine protease inhibitor (serpin) C1 inhibitor inactivates enzymes involved in the regulation of vascular permeability. A patient from the Ma family with the genetic disorder hereditary angioedema inherited a dysfunctional C1 inhibitor allele. Relative to normal plasma, the patients's plasma contained an additional C1 inhibitor immunoreactive band, which comigrated with normal C1 inhibitor cleaved by plasma kallikrein, C1s, or factor XIIa. C1 inhibitor Ma did not react with a monoclonal antibody to a neoepitope that is present in complexed and cleaved normal C1 inhibitor, suggesting conformational differences between cleaved normal C1- inhibitor and cleaved C1 inhibitor Ma. Molecular cloning and sequencing of exon 8 of the C1 inhibitor Ma allele revealed a single C to A mutation, changing alanine 434 to glutamic acid. Ala 434 of C1 inhibitor aligns with the P12 residue of the prototypical serpin alpha 1-antitrypsin. The P12 amino acid of all inhibitory serpins is alanine, and it is present in a highly conserved region on the amino-terminal side of the serpin-reactive center loop. Whereas normal C1 inhibitor expressed by transfected COS-1 cells formed complexes with and was cleaved by kallikrein, fXIIa, and C1s, COS-1-expressed Ala434---Glu C1 inhibitor was cleaved by these enzymes but did not form complexes with them. These results, together with evidence from other studies, suggest that serpin protease inhibitor activity is the result of protein conformational change that occurs when the P12 region of a serpin moves from a surface location, on the reactive site loop of the native molecule, to an internal location within sheet A of the complexed inhibitor. PMID:2026621

  6. Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase.

    PubMed

    Sahonero-Canavesi, Diana X; Sohlenkamp, Christian; Sandoval-Calderón, Mario; Lamsa, Anne; Pogliano, Kit; López-Lara, Isabel M; Geiger, Otto

    2015-09-01

    Phospholipids are well known for their membrane-forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD-deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin-like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003-deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth. PMID:25711932

  7. Real-time monitoring of matrix acidizing including the effects of diverting agents

    SciTech Connect

    Hill, A.D.; Zhu, D.

    1996-05-01

    Real-time monitoring of the injection rate and pressure during matrix acidizing provides operators with a way to determine the changing skin factor as stimulation proceeds. Current methods are based either on the assumption of steady-state flow in the region around the wellbore affected by acid injection or on computer solution of the transient flow equations describing the unsteady reservoir flow process occurring during acidizing. In this paper, a new method for real-time monitoring of matrix acidizing, the inverse injectivity vs. superposition time function plot, is presented. This new method can be applied with a spreadsheet computer program or a programmable calculator and accounts for the transient flow effects occurring during matrix acidizing at multiple rates and injection pressures. The evolving skin factor during a matrix treatment is readily obtained from the diagnostic plot. Hypothetical examples show how the inverse injectivity plot can be used to assess the efficiency of stimulation and diversion. Comparisons with previously presented field cases show the new method to be a simple and accurate means of monitoring the evolving skin factor during matrix acidizing.

  8. 2.8-A structure of yeast serine carboxypeptidase.

    PubMed

    Endrizzi, J A; Breddam, K; Remington, S J

    1994-09-20

    The structure of monomeric serine carboxypeptidase from Saccharomyces cerevisiae (CPD-Y), deglycosylated by an efficient new procedure, has been determined by multiple isomorphous replacement and crystallographic refinement. The model contains 3333 non-hydrogen atoms, all 421 amino acids, 3 of 4 carbohydrate residues, 5 disulfide bridges, and 38 water molecules. The standard crystallographic R-factor is 0.162 for 10,909 reflections observed between 20.0- and 2.8-A resolution. The model has rms deviations from ideality of 0.016 A for bond lengths and 2.7 degrees for bond angles and from restrained thermal parameters of 7.9 A2. CPD-Y, which exhibits a preference for hydrophobic peptides, is distantly related to dimeric wheat serine carboxypeptidase II (CPD-WII), which has a preference for basic peptides. Comparison of the two structures suggests that substitution of hydrophobic residues in CPD-Y for negatively charged residues in CPD-WII in the binding site is largely responsible for this difference. Catalytic residues are in essentially identical configurations in the two molecules, including strained main-chain conformational angles for three active site residues (Ser 146, Gly 52, and Gly 53) and an unusual hydrogen bond between the carboxyl groups of Glu 145 and Glu 65. The binding of an inhibitor, benzylsuccinic acid, suggests that the C-terminal carboxylate binding site for peptide substrates is Asn 51, Gly 52, Glu 145, and His 397 and that the "oxyanion hole" consists of the amides of Gly 53 and Tyr 147. A surprising result of the study is that the domains consisting of residues 180-317, which form a largely alpha-helical insertion into the highly conserved cores surrounding the active site, are quite different structurally in the two molecules. It is suggested that these domains have evolved much more rapidly than other parts of the molecule and are involved in substrate recognition. PMID:7727362

  9. Regulation of GluA1 α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptor Function by Protein Kinase C at Serine-818 and Threonine-840

    PubMed Central

    Jenkins, Meagan A.; Wells, Gordon; Bachman, Julia; Snyder, James P.; Jenkins, Andrew; Huganir, Richard L.; Oswald, Robert E.

    2014-01-01

    Three residues within the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor subunit GluA1 C terminus (Ser818, Ser831, Thr840) can be phosphorylated by Ca2+/phospholipid-dependent protein kinase (PKC). Here, we show that PKC phosphorylation of GluA1 Ser818 or Thr840 enhances the weighted mean channel conductance without altering the response time course or agonist potency. These data support the idea that these residues constitute a hyper-regulatory domain for the AMPA receptor. Introduction of phosphomimetic mutations increases conductance only at these three sites within the proximal C terminus, consistent with a structural model with a flexible linker connecting the distal C-terminal domain to the more proximal domain containing a helix bracketed by Ser831 and Thr840. NMR spectra support this model and raise the possibility that phosphorylation can alter the configuration of this domain. Our findings provide insight into the structure and function of the C-terminal domain of GluA1, which controls AMPA receptor function and trafficking during synaptic plasticity in the central nervous system. PMID:24452473

  10. Phosphorylation of serine residue modulates cotton Di19-1 and Di19-2 activities for responding to high salinity stress and abscisic acid signaling

    PubMed Central

    Qin, Li-Xia; Nie, Xiao-Ying; Hu, Rong; Li, Gang; Xu, Wen-Liang; Li, Xue-Bao

    2016-01-01

    Di19 (drought-induced protein 19) family is a novel type of Cys2/His2 zinc-finger proteins. In this study, we demonstrated that cotton Di19-1 and Di19-2 (GhDi19-1/-2) proteins could be phosphorylated in vitro by the calcium-dependent protein kinase (CDPK). Mutation of Ser to Ala in N-terminus of GhDi19-1/-2 led to the altered subcellular localization of the two proteins, but the constitutively activated form (Ser was mutated to Asp) of GhDi19-1/-2 still showed the nuclear localization. GhDi19-1/-2 overexpression transgenic Arabidopsis seedlings displayed the hypersensitivity to high salinity and abscisic acid (ABA). However, Ser site-mutated GhDi19-1(S116A) and GhDi19-2(S114A), and Ser and Thr double sites-mutated GhDi19-1(S/T-A/A) and GhDi19-2(S/T-A/A) transgenic Arabidopsis did not show the salt- and ABA-hypersensitive phenotypes. In contrast, overexpression of Thr site-mutated GhDi19-1(T114A) and GhDi19-2(T112A) in Arabidopsis still resulted in salt- and ABA-hypersensitivity phenotypes, like GhDi19-1/-2 transgenic lines. Overexpression of GhDi19-1/-2 and their constitutively activated forms in Atcpk11 background could recover the salt- and ABA-insensitive phenotype of the mutant. Thus, our results demonstrated that Ser phosphorylation (not Thr phosphorylation) is crucial for functionally activating GhDi19-1/-2 in response to salt stress and ABA signaling during early plant development, and GhDi19-1/-2 proteins may be downstream targets of CDPKs in ABA signal pathway. PMID:26829353

  11. Kinetic model of water disinfection using peracetic acid including synergistic effects.

    PubMed

    Flores, Marina J; Brandi, Rodolfo J; Cassano, Alberto E; Labas, Marisol D

    2016-01-01

    The disinfection efficiencies of a commercial mixture of peracetic acid against Escherichia coli were studied in laboratory scale experiments. The joint and separate action of two disinfectant agents, hydrogen peroxide and peracetic acid, were evaluated in order to observe synergistic effects. A kinetic model for each component of the mixture and for the commercial mixture was proposed. Through simple mathematical equations, the model describes different stages of attack by disinfectants during the inactivation process. Based on the experiments and the kinetic parameters obtained, it could be established that the efficiency of hydrogen peroxide was much lower than that of peracetic acid alone. However, the contribution of hydrogen peroxide was very important in the commercial mixture. It should be noted that this improvement occurred only after peracetic acid had initiated the attack on the cell. This synergistic effect was successfully explained by the proposed scheme and was verified by experimental results. Besides providing a clearer mechanistic understanding of water disinfection, such models may improve our ability to design reactors. PMID:26819382

  12. Case Studies in Systems Chemistry. Final Report. [Includes Complete Case Study, Carboxylic Acid Equilibria

    ERIC Educational Resources Information Center

    Fleck, George

    This publication was produced as a teaching tool for college chemistry. The book is a text for a computer-based unit on the chemistry of acid-base titrations, and is designed for use with FORTRAN or BASIC computer systems, and with a programmable electronic calculator, in a variety of educational settings. The text attempts to present computer…

  13. Phosphoramidates as novel activity-based probes for serine proteases.

    PubMed

    Haedke, Ute R; Frommel, Sandra C; Hansen, Fabian; Hahne, Hannes; Kuster, Bernhard; Bogyo, Matthew; Verhelst, Steven H L

    2014-05-26

    Activity-based probes (ABPs) are small molecules that exclusively form covalent bonds with catalytically active enzymes. In the last decade, they have especially been used in functional proteomics studies of proteases. Here, we present phosphoramidate peptides as a novel type of ABP for serine proteases. These molecules can be made in a straightforward manner by standard Fmoc-based solid-phase peptide synthesis, allowing rapid diversification. The resulting ABPs covalently bind different serine proteases, depending on the amino acid recognition element adjacent to the reactive group. A reporter tag enables downstream gel-based analysis or LC-MS/MS-mediated identification of the targeted proteases. Overall, we believe that these readily accessible probes will provide new avenues for the functional study of serine proteases in complex proteomes. PMID:24817682

  14. d-serine levels in Alzheimer's disease: implications for novel biomarker development

    PubMed Central

    Madeira, C; Lourenco, M V; Vargas-Lopes, C; Suemoto, C K; Brandão, C O; Reis, T; Leite, R E P; Laks, J; Jacob-Filho, W; Pasqualucci, C A; Grinberg, L T; Ferreira, S T; Panizzutti, R

    2015-01-01

    Alzheimer's disease (AD) is a severe neurodegenerative disorder still in search of effective methods of diagnosis. Altered levels of the NMDA receptor co-agonist, d-serine, have been associated with neurological disorders, including schizophrenia and epilepsy. However, whether d-serine levels are deregulated in AD remains elusive. Here, we first measured D-serine levels in post-mortem hippocampal and cortical samples from nondemented subjects (n=8) and AD patients (n=14). We next determined d-serine levels in experimental models of AD, including wild-type rats and mice that received intracerebroventricular injections of amyloid-β oligomers, and APP/PS1 transgenic mice. Finally, we assessed d-serine levels in the cerebrospinal fluid (CSF) of 21 patients with a diagnosis of probable AD, as compared with patients with normal pressure hydrocephalus (n=9), major depression (n=9) and healthy controls (n=10), and results were contrasted with CSF amyloid-β/tau AD biomarkers. d-serine levels were higher in the hippocampus and parietal cortex of AD patients than in control subjects. Levels of both d-serine and serine racemase, the enzyme responsible for d-serine production, were elevated in experimental models of AD. Significantly, d-serine levels were higher in the CSF of probable AD patients than in non-cognitively impaired subject groups. Combining d-serine levels to the amyloid/tau index remarkably increased the sensitivity and specificity of diagnosis of probable AD in our cohort. Our results show that increased brain and CSF d-serine levels are associated with AD. CSF d-serine levels discriminated between nondemented and AD patients in our cohort and might constitute a novel candidate biomarker for early AD diagnosis. PMID:25942042

  15. Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis

    PubMed Central

    Pompeo, Frédérique; Foulquier, Elodie; Galinier, Anne

    2016-01-01

    Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them. PMID:27148245

  16. Biochemical features, molecular biology and clinical relevance of the human 15-domain serine proteinase inhibitor LEKTI.

    PubMed

    Walden, Michael; Kreutzmann, Peter; Drögemüller, Katrin; John, Harald; Forssmann, Wolf-Georg; Hans-Jürgen, Mägert

    2002-01-01

    Based on the isolation of a 55 amino acid peptide from human hemofiltrate, we cloned the cDNA for a novel human 15-domain serine proteinase inhibitor termed LEKTI. A trypsin-inhibiting activity was demonstrated for three different domains. High levels of expression of the corresponding gene were detected in oral mucosa, followed by the tonsils, parathyroid glands, thymus, and trachea. Hovnanian and coworkers recently found that certain mutations within the LEKTI gene are linked to the severe congenital disease Netherton syndrome and atopic manifestations (including asthma). Thus, a future therapeutic use of LEKTI is conceivable. PMID:12437098

  17. Serine, glycine and the one-carbon cycle: cancer metabolism in full circle

    PubMed Central

    Locasale, Jason W

    2013-01-01

    One carbon metabolism involving the folate and methionine cycle integrates carbon units from amino acids, including serine and glycine, and generates diverse outputs, such as the biosynthesis of lipids, nucleotides and proteins, the maintenance of redox status, and the substrates for methylation reactions. Long considered a ‘housekeeping’ process, this pathway has been recently shown to have additional complexity. Recent genetic and functional evidence also suggests that hyperactivation of this pathway is a possible driver of oncogenesis and establishes links to cellular epigenetic status. Given the wealth of clinically available agents that target one carbon metabolism, these new findings could present opportunities for translation into precision cancer medicine. PMID:23822983

  18. Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

    SciTech Connect

    Vanderslice, P.; Ballinger, S.M., Tam, E.K.; Goldstein, S.M.; Craik, C.S.; Caughey, G.H. )

    1990-05-01

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the {approx}1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5{prime} regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family.

  19. Longitudinal distributions of dicarboxylic acids, ω-oxoacids, pyruvic acid, α-dicarbonyls, and fatty acids in the marine aerosols from the central Pacific including equatorial upwelling

    NASA Astrophysics Data System (ADS)

    Hoque, Mir Md. Mozammal; Kawamura, Kimitaka

    2016-03-01

    Remote marine aerosol samples (total suspended particles) were collected during a cruise in the central Pacific from Japan to Mexico (1°59'N-35°N and 171°54'E-90°58'W). The aerosol samples were analyzed for dicarboxylic acids (C2-C11), ω-oxoacids, pyruvic acid, α-dicarbonyls, and fatty acids as well as organic and elemental carbon, water-soluble organic carbon, and total nitrogen (WSTN). During the study, diacids were the most abundant compound class followed by fatty acids, ω-oxoacids, and α-dicarbonyls. Molecular compositions of diacids showed a predominance of oxalic (C2) acid followed by malonic (C3) and succinic (C4) acids. Oxalic acid comprises 74% of total diacids. This result suggests that photochemical production of oxalic acid is significant over the central Pacific. Spatial distributions of diacids, ω-oxoacids, pyruvic acid, α-dicarbonyls, and fatty acids together with total carbon and WSTN showed higher abundances in the eastern equatorial Pacific where the upwelling of high-nutrient waters followed by high biological productivity is common, indicating that their in situ production is important in the warmer central Pacific through photochemical oxidation from their gaseous and particulate precursors. This study demonstrates that there is a strong linkage in biogeochemical cycles of carbon in the sea-air interface via ocean upwelling, phytoplankton productivity, sea-to-air emissions of organic matter, and formation of secondary organic aerosols in the eastern equatorial Pacific.

  20. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE.

    PubMed

    Van de Wouwer, Dorien; Vanholme, Ruben; Decou, Raphaël; Goeminne, Geert; Audenaert, Dominique; Nguyen, Long; Höfer, René; Pesquet, Edouard; Vanholme, Bartel; Boerjan, Wout

    2016-09-01

    Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization. PMID:27485881

  1. Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases in silico docked to different inhibitors

    PubMed Central

    2010-01-01

    Background Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. Results We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR) for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB) does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI), ecotine and ovomucoid third domain inhibitor. The table (matrix) of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. Conclusions The serine proteases interfaces prefer polar (including glycine) residues (with some exceptions). Charged residues were found to be uniquely prevalent at the interfaces between the

  2. Prediction and Analysis of Post-Translational Pyruvoyl Residue Modification Sites from Internal Serines in Proteins

    PubMed Central

    Jiang, Yang; Li, Bi-Qing; Zhang, Yuchao; Feng, Yuan-Ming; Gao, Yu-Fei; Zhang, Ning; Cai, Yu-Dong

    2013-01-01

    Most of pyruvoyl-dependent proteins observed in prokaryotes and eukaryotes are critical regulatory enzymes, which are primary targets of inhibitors for anti-cancer and anti-parasitic therapy. These proteins undergo an autocatalytic, intramolecular self-cleavage reaction in which a covalently bound pyruvoyl group is generated on a conserved serine residue. Traditional detections of the modified serine sites are performed by experimental approaches, which are often labor-intensive and time-consuming. In this study, we initiated in an attempt for the computational predictions of such serine sites with Feature Selection based on a Random Forest. Since only a small number of experimentally verified pyruvoyl-modified proteins are collected in the protein database at its current version, we only used a small dataset in this study. After removing proteins with sequence identities >60%, a non-redundant dataset was generated and was used, which contained only 46 proteins, with one pyruvoyl serine site for each protein. Several types of features were considered in our method including PSSM conservation scores, disorders, secondary structures, solvent accessibilities, amino acid factors and amino acid occurrence frequencies. As a result, a pretty good performance was achieved in our dataset. The best 100.00% accuracy and 1.0000 MCC value were obtained from the training dataset, and 93.75% accuracy and 0.8441 MCC value from the testing dataset. The optimal feature set contained 9 features. Analysis of the optimal feature set indicated the important roles of some specific features in determining the pyruvoyl-group-serine sites, which were consistent with several results of earlier experimental studies. These selected features may shed some light on the in-depth understanding of the mechanism of the post-translational self-maturation process, providing guidelines for experimental validation. Future work should be made as more pyruvoyl-modified proteins are found and the method

  3. Bioanalytical method for the simultaneous determination of D- and L-serine in human plasma by LC/MS/MS.

    PubMed

    Sugimoto, Hiroshi; Kakehi, Masaaki; Jinno, Fumihiro

    2015-10-15

    D-Serine is an endogenous modulator of N-methyl-D-aspartate (NMDA) receptors. Plasma concentrations of D-serine and the ratio of D-serine to total serine may be used as clinically-translatable biomarkers in NMDA receptor-related disease. We developed a highly sensitive and specific method using high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the D- and L-isomers of serine in human plasma. Since D- and L-serine are endogenous components, phosphate buffered saline was used as the surrogate matrix. D- and L-serine in human plasma and PBS were treated by cationic exchange solid phase extraction. D-Serine (m/z 106.1 > 60.0), L-serine (m/z 106.1 > 60.1) and DL-serine-d3 (m/z 109.1 > 63.0) were detected using a multiple reaction monitoring. The enantiomer separation of D- and L-serine was successfully achieved without any derivatization step using tandemly-arranged and ice-cold CROWNPAK CR-I(+) columns with an isocratic mobile phase comprised of 0.3% trifluoroacetic acid in 10% acetonitrile. The standard curves were linear throughout the calibration range with 0.01-10 μg/mL (D-serine) and 0.1-100 μg/mL (L-serine), respectively. Intra-day and inter-day precision and accuracy of the quality control samples were within relative standard deviations of less than 15%. The endogenous concentrations of D- and L-serine in human plasma were 0.124-0.199 and 7.97-13.1 μg/mL, respectively. PMID:26205585

  4. Inferring selection in the Anopheles gambiae species complex: an example from immune-related serine protease inhibitors

    PubMed Central

    Obbard, Darren J; Welch, John J; Little, Tom J

    2009-01-01

    Background Mosquitoes of the Anopheles gambiae species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect Plasmodium development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution. Methods Three serine protease inhibitors have previously been identified as candidate immune system genes mediating mosquito-Plasmodium interaction, and serine protease inhibitors have been identified as hot-spots of adaptive evolution in other taxa. Population-genetic tests for selection, including a recent multi-gene extension of the McDonald-Kreitman test, were applied to 16 serine protease inhibitors and 16 other genes sampled from the An. gambiae species complex in both East and West Africa. Results Serine protease inhibitors were found to show a marginally significant trend towards higher levels of amino acid diversity than other genes, and display extensive genetic structuring associated with the 2La chromosomal inversion. However, although serpins are candidate targets for strong parasite-mediated selection, no evidence was found for rapid adaptive evolution in these genes. Conclusion It is well known that phylogenetic and population history in the An. gambiae complex can present special problems for the application of standard population-genetic tests for selection, and this may explain the failure of this study to detect selection acting on serine protease inhibitors. The pitfalls of uncritically applying these tests in this species complex are highlighted, and the future prospects for detecting selection acting on the An. gambiae genome are discussed. PMID:19497100

  5. Heterogeneity of the serine synthetic pathway in Entamoeba species.

    PubMed

    Chiba, Yoko; Makiuchi, Takashi; Jeelani, Ghulam; Nozaki, Tomoyoshi

    2016-06-01

    Phosphoserine phosphatase (PSP) catalyzes the third step of the phosphorylated serine biosynthetic pathway, and occurred multiple times in evolution, while enzymes catalyzing the first and second steps in the pathway have single respective origins. In the present study, we examined the existence of PSP among genus Entamoeba including a human enteric parasite, Entamoeba histolytica. E. histolytica as well as majority of Entamoeba species have the first and second enzymes, but lacks PSP. In contrast, a reptilian enteric parasite, Entamoeba invadens possesses canonical PSP. Thus, there are variations in the existence of the serine biosynthetic ability among Entamoeba species. PMID:27268730

  6. Production, purification, and properties of serine carboxypeptidase from Paecilomyces carneus.

    PubMed

    Umetsu, H; Hishinuma, K; Wake, H; Ichishima, E

    1996-07-01

    Seventeen strains of the genus Paecilomyces were examined for their ability to produce serine carboxypeptidase. Paecilomyces carneus IFO 7012 exhibited the highest potency for serine carboxypeptidase production. A maximum yield of serine carboxypeptidase was obtained by koji culture of the strain at 22 degrees C for 7 days. The serine carboxypeptidase was purified to homogeneity from an extract of the koji culture. The molecular weight of the enzyme was estimated to be 47,000 by HPLC. The isoelectric point of the enzyme was determined to be 4.0, and the optimum pH was 4.0 toward benzyloxycarbonyl-L-glutamyl-L-tyrosine (Z-Glu-Tyr) and benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala), respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and p-chloromercurybenzoate. Relative hydrolysis rates of N-acylpeptides and kinetic studies indicated that the enzyme preferred substrates having bulky amino acids in the penultimate position from their carboxy-termini. PMID:8661688

  7. A serine sensor for multicellularity in a bacterium

    PubMed Central

    Subramaniam, Arvind R; DeLoughery, Aaron; Bradshaw, Niels; Chen, Yun; O’Shea, Erin; Losick, Richard; Chai, Yunrong

    2013-01-01

    We report the discovery of a simple environmental sensing mechanism for biofilm formation in the bacterium Bacillus subtilis that operates without the involvement of a dedicated RNA or protein. Certain serine codons, the four TCN codons, in the gene for the biofilm repressor SinR caused a lowering of SinR levels under biofilm-inducing conditions. Synonymous substitutions of these TCN codons with AGC or AGT impaired biofilm formation and gene expression. Conversely, switching AGC or AGT to TCN codons upregulated biofilm formation. Genome-wide ribosome profiling showed that ribosome density was higher at UCN codons than at AGC or AGU during biofilm formation. Serine starvation recapitulated the effect of biofilm-inducing conditions on ribosome occupancy and SinR production. As serine is one of the first amino acids to be exhausted at the end of exponential phase growth, reduced translation speed at serine codons may be exploited by other microbes in adapting to stationary phase. DOI: http://dx.doi.org/10.7554/eLife.01501.001 PMID:24347549

  8. An update on serine deficiency disorders.

    PubMed

    van der Crabben, S N; Verhoeven-Duif, N M; Brilstra, E H; Van Maldergem, L; Coskun, T; Rubio-Gozalbo, E; Berger, R; de Koning, T J

    2013-07-01

    Serine deficiency disorders are caused by a defect in one of the three synthesising enzymes of the L-serine biosynthesis pathway. Serine deficiency disorders give rise to a neurological phenotype with psychomotor retardation, microcephaly and seizures in newborns and children or progressive polyneuropathy in adult patients. There are three defects that cause serine deficiency of which 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency, the defect affecting the first step in the pathway, has been reported most frequently. The other two disorders in L-serine biosynthesis phosphoserine aminotransferase (PSAT) deficiency and phosphoserine phosphatase (PSP) deficiency have been reported only in a limited number of patients. The biochemical hallmarks of all three disorders are low concentrations of serine in cerebrospinal fluid and plasma. Prompt recognition of affected patients is important, since serine deficiency disorders are treatable causes of neurometabolic disorders. The use of age-related reference values for serine in CSF and plasma can be of great help in establishing a correct diagnosis of serine deficiency, in particular in newborns and young children. PMID:23463425

  9. Phosphoserine phosphatase activity is elevated and correlates negatively with plasma d-serine concentration in patients with schizophrenia.

    PubMed

    Ozeki, Yuji; Sekine, Masae; Fujii, Kumiko; Watanabe, Takashi; Okayasu, Hiroaki; Takano, Yumiko; Shinozaki, Takahiro; Aoki, Akiko; Akiyama, Kazufumi; Homma, Hiroshi; Shimoda, Kazutaka

    2016-03-30

    The pathophysiology of schizophrenia may involve N-methyl-D-aspartate receptor (NMDAR) hypofunction. D-3serine and glycine are endogenous l-serine-derived NMDAR co-agonists. We hypothesized that the l-serine synthesis pathway could be involved in schizophrenia. We measured the activity of phosphoserine phosphatase (PSP), a rate-limiting enzyme in l-serine synthesis, in peripheral blood mononuclear cells of 54 patients with schizophrenia and 49 normal control subjects. Plasma amino acid (l-serine, d-serine, glycine, glutamine, and glutamate) levels were measured by high performance liquid chromatography. Peripheral blood mRNA expression levels of PHGDH, PSAT1, PSP, and SR, determined by quantitative real-time PCR were compared between patients and controls. PSP activity was higher in patients than in controls, especially in male patients. In male patients, the plasma l-serine concentration was higher than that in controls. In patients, PSP activity was negatively correlated with plasma d-serine and glycine levels. Furthermore, PSP activity was positively correlated with plasma l-serine concentration. These results were statistically significant only in male patients. PSP, PSAT1, and PHGDH mRNA levels were lower in patients than in controls, except when the PHGDH expression level was compared with ACTB expression. In summary, we found the l-serine synthesis system to be altered in patients with schizophrenia, especially in male patients. PMID:26804975

  10. A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate.

    PubMed

    Pacold, Michael E; Brimacombe, Kyle R; Chan, Sze Ham; Rohde, Jason M; Lewis, Caroline A; Swier, Lotteke J Y M; Possemato, Richard; Chen, Walter W; Sullivan, Lucas B; Fiske, Brian P; Cho, Steve; Freinkman, Elizaveta; Birsoy, Kıvanç; Abu-Remaileh, Monther; Shaul, Yoav D; Liu, Chieh Min; Zhou, Minerva; Koh, Min Jung; Chung, Haeyoon; Davidson, Shawn M; Luengo, Alba; Wang, Amy Q; Xu, Xin; Yasgar, Adam; Liu, Li; Rai, Ganesha; Westover, Kenneth D; Vander Heiden, Matthew G; Shen, Min; Gray, Nathanael S; Boxer, Matthew B; Sabatini, David M

    2016-06-01

    Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo. PMID:27110680

  11. The N-methyl D-aspartate receptor glycine site and D-serine metabolism: an evolutionary perspective.

    PubMed Central

    Schell, Michael J

    2004-01-01

    The N-methyl D-aspartate (NMDA) type of glutamate receptor requires two distinct agonists to operate. Glycine is assumed to be the endogenous ligand for the NMDA receptor glycine site, but this notion has been challenged by the discovery of high levels of endogenous d-serine in the mammalian forebrain. I have outlined an evolutionary framework for the appearance of a glycine site in animals and the metabolic events leading to high levels of D-serine in brain. Sequence alignments of the glycine-binding regions, along with the scant experimental data available, suggest that the properties of invertebrate NMDA receptor glycine sites are probably different from those in vertebrates. The synthesis of D-serine in brain is due to a pyridoxal-5'-phosphate (B(6))-requiring serine racemase in glia. Although it remains unknown when serine racemase first evolved, data concerning the evolution of B(6) enzymes, along with the known occurrences of serine racemases in animals, point to D-serine synthesis arising around the divergence time of arthropods. D-Serine catabolism occurs via the ancient peroxisomal enzyme d-amino acid oxidase (DAO), whose ontogenetic expression in the hindbrain of mammals is delayed until the postnatal period and absent from the forebrain. The phylogeny of D-serine metabolism has relevance to our understanding of brain ontogeny, schizophrenia and neurotransmitter dynamics. PMID:15306409

  12. Application of Infrared Multiphoton Dissociation Spectroscopy for the Study of Chiral Recognition in the Protonated Serine Clusters: Part II

    NASA Astrophysics Data System (ADS)

    Sunahori, Fumie X.; Kitova, Elena N.; Klassen, John S.; Xu, Yunjie; Yang, Guochun

    2011-06-01

    Serine is an amino acid which has long been known to form the magic-number serine octamer [Ser_8 + H]^+. It has been shown that the serine octamer exhibits strong preference for homochirality. Although a few possible structures for the homochiral serine octamer have been proposed, no definite conclusion has so far been drawn. Last year at this conference, we reported on the study of the protonated serine octamer and dimer as well as the chiral recognition in these clusters using infrared multiphoton dissociation (IRMPD) spectroscopic technique coupled with a Fourier transform ion cyclotron (FTICR) mass spectrometer. Here we present our latest results on the search for the infrared signatures of chiral recognition in the serine octamer and the dimer using a mixture of the deuterated 2,3,3-d_3-L-serine and normal D-serine solution. Using the isotopic labeled species, we could isolate the heterochiral species and obtain their IRMPD spectra which can be directly compared with those of the homochiral species. As an aid to interpret the observed spectra, molecular structures and vibrational frequencies of both homochiral and heterochiral octamer and dimer have been predicted by ab initio calculations. New insights into the hitherto undetermined structure of the serine octamer will be discussed. S. C. Nanita and R. G. Cooks Angew. Chem. Int. Ed. 45, (554), 2006.

  13. Purification and Characterization of a New Serine Protease (VLCII) Isolated from Vipera lebetina Venom: Its Role in Hemostasis.

    PubMed

    Amel, Kadi-Saci; Fatima, Laraba-Djebari

    2015-08-01

    Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG-75, DEAE-Sephadex A-50, and reversed-phase high-performance liquid chromatography (RP-HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). VLCII as a thrombin-like enzyme was able to hydrolyze Nα-CBZ L-arginine-p-nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10-phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bβ chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration-dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway. PMID:25917874

  14. Thermodynamic characteristics of protolytic equilibria of L-serine in aqueous solutions

    NASA Astrophysics Data System (ADS)

    Kochergina, L. A.; Volkov, A. V.; Khokhlova, E. A.; Krutova, O. N.

    2011-05-01

    The heat effects of the reaction of aqueous solution of L-serine with aqueous solutions of HNO3 and KOH were determined by calorimetry at temperatures of 288.15, 298.15, and 308.15 K, and ionic strength values of 0.2, 0.5, and 1.0 (background electrolyte, KNO3). Standard thermodynamic characteristics (Δr H o, Δr G o, Δr S o, Δ C {/p o}) of the acid-base reactions in aqueous solutions of L-serine were calculated. The effect of the concentration of background electrolyte and temperature on the heats of dissociation of amino acid was considered. The combustion energy of L-serine by bomb calorimetry in the medium of oxygen was determined. The standard combustion and formation enthalpies of crystalline L-serine were calculated. The heats of dissolution of crystalline L-serine in water and solutions of potassium hydroxide at 298.15 K were measured by direct calorimetry. The standard enthalpies of formation of L-serine and products of its dissociation in aqueous solution were calculated.

  15. Influence of cold stress on contents of soluble sugars, vitamin C and free amino acids including gamma-aminobutyric acid (GABA) in spinach (Spinacia oleracea).

    PubMed

    Yoon, Young-Eun; Kuppusamy, Saranya; Cho, Kye Man; Kim, Pil Joo; Kwack, Yong-Bum; Lee, Yong Bok

    2017-01-15

    The contents of soluble sugars (sucrose, fructose, glucose, maltose and raffinose), vitamin C and free amino acids (34 compounds, essential and non-essential) were quantified in open-field and greenhouse-grown spinaches in response to cold stress using liquid chromatography. In general, greenhouse cultivation produced nutritionally high value spinach in a shorter growing period, where the soluble sugars, vitamin C and total amino acids concentrations, including essential were in larger amounts compared to those grown in open-field scenarios. Further, low temperature exposure of spinach during a shorter growth period resulted in the production of spinach with high sucrose, ascorbate, proline, gamma-aminobutyric acid, valine and leucine content, and these constitute the most important energy/nutrient sources. In conclusion, cultivation of spinach in greenhouse at a low temperature (4-7°C) and exposure for a shorter period (7-21days) before harvest is recommended. This strategy will produce a high quality product that people can eat. PMID:27542466

  16. Neutral serine proteases of neutrophils.

    PubMed

    Kettritz, Ralph

    2016-09-01

    Neutrophil serine proteases (NSPs) exercise tissue-degrading and microbial-killing effects. The spectrum of NSP-mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase-3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small-vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life-threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications. PMID:27558338

  17. Interactions of “Bora-Penicilloates” with Serine β-Lactamases and DD-Peptidases

    PubMed Central

    2015-01-01

    Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial β-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in β-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C β-lactamases but, very unexpectedly, not inhibitors of class A β-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of β-lactam antibiotics, where deacylation of β-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions. PMID:25302576

  18. Serine-rich protein is a novel positive regulator for silicon accumulation in mangrove.

    PubMed

    Sahebi, Mahbod; Hanafi, Mohamed M; Siti Nor Akmar, A; Rafii, Mohd Y; Azizi, Parisa; Idris, A S

    2015-02-10

    Silicon (Si) plays an important role in reducing plant susceptibility against a variety of different biotic and abiotic stresses; and also has an important regulatory role in soil to avoid heavy metal toxicity and providing suitable growing conditions for plants. A full-length cDNAs of 696bp of serine-rich protein was cloned from mangrove plant (Rhizophora apiculata) by amplification of cDNA ends from an expressed sequence tag homologous to groundnut (Arachis hypogaea), submitted to NCBI (KF211374). This serine-rich protein gene encodes a deduced protein of 223 amino acids. The transcript titre of the serine-rich protein was found to be strongly enriched in roots compared with the leaves of two month old mangrove plants and expression level of this serine-rich protein was found to be strongly induced when the mangrove seedlings were exposed to SiO2. Expression of the serine-rich protein transgenic was detected in transgenic Arabidopsis thaliana, where the amount of serine increased from 1.02 to 37.8mg/g. The same trend was also seen in Si content in the roots (14.3%) and leaves (7.4%) of the transgenic A. thaliana compared to the wild-type plants under Si treatment. The biological results demonstrated that the accumulation of the serine amino acid in the vegetative tissues of the transgenic plants enhanced their ability to absorb and accumulate more Si in the roots and leaves and suggests that the serine-rich protein gene has potential for use in genetic engineering of different stress tolerance characteristics. PMID:25479011

  19. Impact of hedonic evaluation on consumers' preferences for beef attributes including its enrichment with n-3 and CLA fatty acids.

    PubMed

    Baba, Yasmina; Kallas, Zein; Costa-Font, Montserrat; Gil, José María; Realini, Carolina E

    2016-01-01

    The impact of hedonic evaluation on consumers' preferences for beef attributes was evaluated (origin, animal diet, fat content, color, price) including its enrichment with omega-3 (n-3) and conjugated linoleic acid (CLA) fatty acids. One group of consumers (n=325) received information about n-3 and CLA, while the other group (n=322) received no information. Consumers conducted a Discrete Choice Experiment (DCE), using the recently developed Generalized Multinomial Logit model; followed by a blind hedonic evaluation of beef samples, which were identified after tasting, and finally repeated the DCE. Results showed that hedonic evaluation had a significant impact on consumers' preferences, which were similar after tasting for all consumers, with less emphasis on the fat content, color, and origin attributes and greater emphasis on animal diet. Preference for n-3 enriched beef increased, while preference for CLA enriched beef was still not significant after tasting. The information provided had a significant effect on consumers' beef preferences, but no significant impact on beef liking scores. PMID:26331961

  20. A Spider-Derived Kunitz-Type Serine Protease Inhibitor That Acts as a Plasmin Inhibitor and an Elastase Inhibitor

    PubMed Central

    Wan, Hu; Lee, Kwang Sik; Kim, Bo Yeon; Zou, Feng Ming; Yoon, Hyung Joo; Je, Yeon Ho; Li, Jianhong; Jin, Byung Rae

    2013-01-01

    Kunitz-type serine protease inhibitors are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis, and inflammation. While spider-derived Kunitz-type proteins show activity in trypsin or chymotrypsin inhibition and K+ channel blocking, no additional role for these proteins has been elucidated. In this study, we identified the first spider (Araneus ventricosus) Kunitz-type serine protease inhibitor (AvKTI) that acts as a plasmin inhibitor and an elastase inhibitor. AvKTI possesses a Kunitz domain consisting of a 57-amino-acid mature peptide that displays features consistent with Kunitz-type inhibitors, including six conserved cysteine residues and a P1 lysine residue. Recombinant AvKTI, expressed in baculovirus-infected insect cells, showed a dual inhibitory activity against trypsin (Ki 7.34 nM) and chymotrypsin (Ki 37.75 nM), defining a role for AvKTI as a spider-derived Kunitz-type serine protease inhibitor. Additionally, AvKTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, AvKTI inhibited plasmin (Ki 4.89 nM) and neutrophil elastase (Ki 169.07 nM), indicating that it acts as an antifibrinolytic factor and an antielastolytic factor. These findings constitute molecular evidence that AvKTI acts as a plasmin inhibitor and an elastase inhibitor and also provide a novel view of the functions of a spider-derived Kunitz-type serine protease inhibitor. PMID:23308198

  1. An automated efficient conformation search of L-serine by the scaled hypersphere search method

    NASA Astrophysics Data System (ADS)

    Kishimoto, Naoki; Harayama, Manami; Ohno, Koichi

    2016-05-01

    Stable conformers of L-serine were automatically explored by applications of the scaled hypersphere search (SHS) method to equilibrium structures maintaining the chemical bond skeletons of serine. Energy barriers for conformational changes of L-serine were estimated from the heights of obtained transition structures. Zero-point-corrected electronic energies and Gibbs free energies of the 24 lowest energy conformers and 21 transition structures were calculated at 100, 298, and 400 K by a composite quantum chemistry method (Gaussian-4). Relative populations of 24 conformers including nine new conformers were calculated from the Gibbs energies assuming thermal equilibrium.

  2. New members of the brachyurins family in lobster include a trypsin-like enzyme with amino acid substitutions in the substrate-binding pocket.

    PubMed

    Perera, Erick; Pons, Tirso; Hernandez, Damir; Moyano, Francisco J; Martínez-Rodríguez, Gonzalo; Mancera, Juan M

    2010-09-01

    Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed. PMID:20649906

  3. Comprehensive Analysis of a Vibrio parahaemolyticus Strain Extracellular Serine Protease VpSP37

    PubMed Central

    Bennici, Carmelo; Quatrini, Paola; Catania, Valentina; Mazzola, Salvatore; Ghersi, Giulio; Cuttitta, Angela

    2015-01-01

    Proteases play an important role in the field of tissue dissociation combined with regenerative medicine. During the years new sources of proteolytic enzymes have been studied including proteases from different marine organisms both eukaryotic and prokaryotic. Herein we have purified a secreted component of an isolate of Vibrio parahaemolyticus, with electrophoretic mobilities corresponding to 36 kDa, belonging to the serine proteases family. Sequencing of the N-terminus enabled the in silico identification of the whole primary structure consisting of 345 amino acid residues with a calculated molecular mass of 37.4 KDa. The purified enzyme, named VpSP37, contains a Serine protease domain between residues 35 and 276 and a canonical Trypsin/Chimotrypsin 3D structure. Functional assays were performed to evaluate protease activity of purified enzyme. Additionally the performance of VpSP37 was evaluated in tissue dissociations experiments and the use of such enzyme as a component of enzyme blend for tissue dissociation procedures is strongly recommended. PMID:26162075

  4. Substitution of aspartic acid for glycine at position 310 in type II collagen produces achondrogenesis II, and substitution of serine at position 805 produces hypochondrogenesis: analysis of genotype-phenotype relationships.

    PubMed

    Bonaventure, J; Cohen-Solal, L; Ritvaniemi, P; Van Maldergem, L; Kadhom, N; Delezoide, A L; Maroteaux, P; Prockop, D J; Ala-Kokko, L

    1995-05-01

    Two different mutations were found in two unrelated probands with lethal chondrodysplasias, one with achondrogenesis type II and the other with the less severe phenotype of hypochondrogenesis. The mutations in the COL2A1 gene were identified by denaturing gradient gel electrophoresis analysis of genomic DNA followed by dideoxynucleotide sequencing and restriction site analysis. The proband with achondrogenesis type II had a heterozygous single-base mutation that substituted aspartate for glycine at position 310 of the alpha 1(II) chain of type II procollagen. The proband with hypochondrogenesis had a heterozygous single-base mutation that substituted serine for glycine at position 805. Type II collagen extracted from cartilage from the probands demonstrated the presence of type I collagen and a delayed electrophoretic mobility, indicating post-translational overmodifications. Analysis of CNBr peptides showed that, in proband 1, the entire peptides were overmodified. Examination of chondrocytes cultured in agarose or alginate indicated that there was a delayed secretion of type II procollagen. In addition, type II collagen synthesized by cartilage fragments from the probands demonstrated a decreased thermal stability. The melting temperature of the type II collagen containing the aspartate-for-glycine substitution was reduced by 4 degrees C, and that of the collagen containing the serine-for-glycine substitution was reduced by 2 degrees C. Electron microscopy of the extracellular matrix from the chondrocyte cultures showed a decreased density of matrix and the presence of unusually short and thin fibrils. Our results indicate that glycine substitutions in the N-terminal region of the type II collagen molecule can produce more severe phenotypes than mutations in the C-terminal region. The aspartate-for-glycine substitution at position 310, which was associated with defective secretion and a probable increased degradation of collagen, is the most destabilizing

  5. Substitution of aspartic acid for glycine at position 310 in type II collagen produces achondrogenesis II, and substitution of serine at position 805 produces hypochondrogenesis: analysis of genotype-phenotype relationships.

    PubMed Central

    Bonaventure, J; Cohen-Solal, L; Ritvaniemi, P; Van Maldergem, L; Kadhom, N; Delezoide, A L; Maroteaux, P; Prockop, D J; Ala-Kokko, L

    1995-01-01

    Two different mutations were found in two unrelated probands with lethal chondrodysplasias, one with achondrogenesis type II and the other with the less severe phenotype of hypochondrogenesis. The mutations in the COL2A1 gene were identified by denaturing gradient gel electrophoresis analysis of genomic DNA followed by dideoxynucleotide sequencing and restriction site analysis. The proband with achondrogenesis type II had a heterozygous single-base mutation that substituted aspartate for glycine at position 310 of the alpha 1(II) chain of type II procollagen. The proband with hypochondrogenesis had a heterozygous single-base mutation that substituted serine for glycine at position 805. Type II collagen extracted from cartilage from the probands demonstrated the presence of type I collagen and a delayed electrophoretic mobility, indicating post-translational overmodifications. Analysis of CNBr peptides showed that, in proband 1, the entire peptides were overmodified. Examination of chondrocytes cultured in agarose or alginate indicated that there was a delayed secretion of type II procollagen. In addition, type II collagen synthesized by cartilage fragments from the probands demonstrated a decreased thermal stability. The melting temperature of the type II collagen containing the aspartate-for-glycine substitution was reduced by 4 degrees C, and that of the collagen containing the serine-for-glycine substitution was reduced by 2 degrees C. Electron microscopy of the extracellular matrix from the chondrocyte cultures showed a decreased density of matrix and the presence of unusually short and thin fibrils. Our results indicate that glycine substitutions in the N-terminal region of the type II collagen molecule can produce more severe phenotypes than mutations in the C-terminal region. The aspartate-for-glycine substitution at position 310, which was associated with defective secretion and a probable increased degradation of collagen, is the most destabilizing

  6. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells.

    PubMed

    Maddocks, Oliver D K; Labuschagne, Christiaan F; Adams, Peter D; Vousden, Karen H

    2016-01-21

    Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. PMID:26774282

  7. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

    PubMed Central

    Maddocks, Oliver D.K.; Labuschagne, Christiaan F.; Adams, Peter D.; Vousden, Karen H.

    2016-01-01

    Summary Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. PMID:26774282

  8. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

    The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354

  9. The Cryptic dsdA Gene Encodes a Functional D-Serine Dehydratase in Pseudomonas aeruginosa PAO1.

    PubMed

    Li, Guoqing; Lu, Chung-Dar

    2016-06-01

    D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on D-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the D-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative D-serine dehydratase was subjected to further characterization. Growth on D-serine as the sole source of nitrogen was retained in the ∆dsdA mutant and was abolished completely in the ∆dadA and ∆dadA-∆dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on D-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of D-serine into pyruvate and ammonia. Among D- and L-amino acids tested, only D-serine and D-threonine could serve as the substrates of DsdA, and the Km of DsdA with D-serine was calculated to be 330 μM. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a D-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional D-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous D-serine might be due to the loss of regulatory elements for gene activation during evolution. PMID:26957519

  10. Crystal Structure of Serine Racemase that Produces Neurotransmitter d-Serine for Stimulation of the NMDA Receptor

    NASA Astrophysics Data System (ADS)

    Goto, Masaru

    d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5’-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. We have determined the structures of three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe. Lys57 and Ser82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique lysino-d-alanyl residue at the active site, also binds the substrate serine in the active site, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as Lys57 of the wild type enzyme.

  11. DEVELOPMENTAL TOXICITY AND STRUCTURE-ACTIVITY RELATIONSHIPS OF ALIPHATIC ACIDS, INCLUDING DOSE-RESPONSE ASSESSMENT OF VALPROIC ACID IN MICE AND RATS

    EPA Science Inventory

    The anticonvulsant valproic acid (VPA), or 2-propylpentanoic acid, is a short-chain aliphatic acid that is teratogenic in humans and rodents. PA and 14 related using the Chernoff/Kavlock assay Sprague-Dawley rats were gavaged with the test agent in corn oil once daily organogenes...

  12. Functional Characterization of the Serine-Rich Tract of Varicella-Zoster Virus IE62

    PubMed Central

    Shakya, Akhalesh K.; Kim, Seongman; O'Callaghan, Dennis J.

    2015-01-01

    ABSTRACT The immediate early 62 protein (IE62) of varicella-zoster virus (VZV), a major viral trans-activator, initiates the virus life cycle and is a key component of pathogenesis. The IE62 possesses several domains essential for trans-activation, including an acidic trans-activation domain (TAD), a serine-rich tract (SRT), and binding domains for USF, TFIIB, and TATA box binding protein (TBP). Transient-transfection assays showed that the VZV IE62 lacking the SRT trans-activated the early VZV ORF61 promoter at only 16% of the level of the full-length IE62. When the SRT of IE62 was replaced with the SRT of equine herpesvirus 1 (EHV-1) IEP, its trans-activation activity was completely restored. Herpes simplex virus 1 (HSV-1) ICP4 that lacks a TAD very weakly (1.5-fold) trans-activated the ORF61 promoter. An IE62 TAD-ICP4 chimeric protein exhibited trans-activation ability (10.2-fold), indicating that the IE62 TAD functions with the SRT of HSV-1 ICP4 to trans-activate viral promoters. When the serine and acidic residues of the SRT were replaced with Ala, Leu, and Gly, trans-activation activities of the modified IE62 proteins IE62-SRTΔSe and IE62-SRTΔAc were reduced to 46% and 29% of wild-type activity, respectively. Bimolecular complementation assays showed that the TAD of IE62, EHV-1 IEP, and HSV-1 VP16 interacted with Mediator 25 in human melanoma MeWo cells. The SRT of IE62 interacted with the nucleolar-ribosomal protein EAP, which resulted in the formation of globular structures within the nucleus. These results suggest that the SRT plays an important role in VZV viral gene expression and replication. IMPORTANCE The immediate early 62 protein (IE62) of varicella-zoster virus (VZV) is a major viral trans-activator and is essential for viral growth. Our data show that the serine-rich tract (SRT) of VZV IE62, which is well conserved within the alphaherpesviruses, is needed for trans-activation mediated by the acidic trans-activation domain (TAD). The TADs of IE62

  13. Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy

    PubMed Central

    2008-01-01

    Background Bacterial penicillin-binding proteins and β-lactamases (PBP-βLs) constitute a large family of serine proteases that perform essential functions in the synthesis and maintenance of peptidoglycan. Intriguingly, genes encoding PBP-βL homologs occur in many metazoan genomes including humans. The emerging role of LACTB, a mammalian mitochondrial PBP-βL homolog, in metabolic signaling prompted us to investigate the evolutionary history of metazoan PBP-βL proteins. Results Metazoan PBP-βL homologs including LACTB share unique structural features with bacterial class B low molecular weight penicillin-binding proteins. The amino acid residues necessary for enzymatic activity in bacterial PBP-βL proteins, including the catalytic serine residue, are conserved in all metazoan homologs. Phylogenetic analysis indicated that metazoan PBP-βL homologs comprise four alloparalogus protein lineages that derive from α-proteobacteria. Conclusion While most components of the peptidoglycan synthesis machinery were dumped by early eukaryotes, a few PBP-βL proteins were conserved and are found in metazoans including humans. Metazoan PBP-βL homologs are active-site-serine enzymes that probably have distinct functions in the metabolic circuitry. We hypothesize that PBP-βL proteins in the early eukaryotic cell enabled the degradation of peptidoglycan from ingested bacteria, thereby maximizing the yield of nutrients and streamlining the cell for effective phagocytotic feeding. PMID:18226203

  14. Cyclopropane derivatives as potential human serine racemase inhibitors: unveiling novel insights into a difficult target.

    PubMed

    Beato, Claudia; Pecchini, Chiara; Cocconcelli, Chiara; Campanini, Barbara; Marchetti, Marialaura; Pieroni, Marco; Mozzarelli, Andrea; Costantino, Gabriele

    2016-08-01

    d-Serine is the co-agonist of NMDA receptors and binds to the so-called glycine site. d-Serine is synthesized by human serine racemase (SR). Over activation of NMDA receptors is involved in many neurodegenerative diseases and, therefore, the inhibition of SR might represent a novel strategy for the treatment of these pathologies. SR is a very difficult target, with only few compounds so far identified exhibiting weak inhibitory activity. This study was aimed at the identification of novel SR inhibitor by mimicking malonic acid, the best-known SR inhibitor, with a cyclopropane scaffold. We developed, synthesized, and tested a series of cyclopropane dicarboxylic acid derivatives, complementing the synthetic effort with molecular docking. We identified few compounds that bind SR in high micromolar range with a lack of significant correlation between experimental and predicted binding affinities. The thorough analysis of the results can be exploited for the development of more potent SR inhibitors. PMID:26133542

  15. Identification of the active-site serine in human lecithin: cholesterol acyltransferase

    SciTech Connect

    Farooqui, J.; Wohl, R.C.; Kezdy, F.J.; Scanu, A.M.

    1987-05-01

    Lecithin:cholesterol acyltransferase (LCAT) from human plasma reacts stoichiometrically with diisopropylphosphorofluoridate (DFP) resulting in the complete loss of transacylase activity. Purified LCAT was covalently labeled with (TH) DFP and the labeled protein was reduced and carboxymethylated. Cyanogen bromide cleavage followed by gel permeation chromatography yielded a peptide of 4-5 KDa (LCAT CNBr-III) containing most of the radioactive label. Preliminary studies comparing the amino acid composition of the LCAT-CNBr-III with the sequence of LCAT indicate that this peptide corresponds to fragment 168-220. Automated Edman degradation of the radioactive peptide recovered a radioactive PTC-amino acid at cycle 14. Of all predicted CNBr fragments only peptide 168-220 contained a serine at residue 14 from the amino terminus of the peptide. The authors conclude that serine 181 is the active site serine of LCAT.

  16. Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

    PubMed Central

    Ekici, Özlem Doğan; Paetzel, Mark; Dalbey, Ross E.

    2008-01-01

    Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called “classical” catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of “nonclassical” serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class. PMID:18824507

  17. D-serine in the midbrain periaqueductal gray contributes to morphine tolerance in rats

    PubMed Central

    Cao, Song; Sun, Mengjie; Li, Youyan

    2016-01-01

    Background The N-methyl-D-aspartate subtype of glutamate receptor plays a critical role in morphine tolerance. D-serine, a co-agonist of N-methyl-D-aspartate receptor, participates in many physiological and pathophysiological processes via regulating N-methyl-D-aspartate receptor activation. The purinergic P2X7 receptor activation can induce the D-serine release in the central nervous system. This study aimed to investigate the role of the ventrolateral midbrain periaqueductal gray D-serine in the mechanism of morphine tolerance in rats. The development of morphine tolerance was induced in normal adult male Sprague–Dawley rats through subcutaneous injection of morphine (10 mg/kg). The analgesic effect of morphine (5 mg/kg, i.p.) was assessed by measuring mechanical withdrawal thresholds in rats with an electronic von Frey anesthesiometer. The D-serine concentration and serine racemase expression levels in the ventrolateral midbrain periaqueductal gray were evaluated through enzyme-linked immunosorbent assay and Western blot analysis, respectively. The effects of intra-ventrolateral midbrain periaqueductal gray injections of the D-serine degrading enzyme D-amino acid oxidase and antisense oligodeoxynucleotide targeting the P2X7 receptor on chronic morphine-treated rats were also explored. Results We found that repeated morphine administrations decreased the antinociceptive potency of morphine evidenced by the percent changes in mechanical pain threshold in rats. By contrast, the D-serine contents and the expression levels of the serine racemase protein were upregulated in the ventrolateral midbrain periaqueductal gray in morphine-tolerant rats. The development of morphine tolerance was markedly alleviated by intra-ventrolateral midbrain periaqueductal gray injections of D-amino acid oxidase or antisense oligodeoxynucleotide targeting the P2X7 receptor. Conclusions Our data indicate that the development of antinociceptive tolerance to morphine is partially

  18. Biochemical and Structural Studies of Uncharacterized Protein PA0743 from Pseudomonas aeruginosa Revealed NAD+-dependent l-Serine Dehydrogenase*

    PubMed Central

    Tchigvintsev, Anatoli; Singer, Alexander; Brown, Greg; Flick, Robert; Evdokimova, Elena; Tan, Kemin; Gonzalez, Claudio F.; Savchenko, Alexei; Yakunin, Alexander F.

    2012-01-01

    The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD+-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2–2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD+ complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases. PMID:22128181

  19. GH inhibition of lipogenesis and stimulation of lipolysis in sheep adipose tissue: involvement of protein serine phosphorylation and dephosphorylation and phospholipase C.

    PubMed

    Vernon, R G

    1996-07-01

    The intracellular signalling systems involved in the chronic insulin-antagonistic, anti-lipogenic effects and also the lipolytic effect of GH have been investigated in sheep adipose tissue in an in vitro tissue culture system. During culture, chronic exposure to GH decreased the rate of lipogenesis and prevented the increase in lipogenesis induced by insulin. GH also increased glycerol release into the culture medium. GH had no acute, insulin-like effect on lipogenesis in sheep adipose tissue. Pretreatment with phorbol ester to down-regulate isoforms of protein kinase C or addition of the protein serine kinase inhibitor staurosporine decreased the anti-lipogenic effect of GH while the protein serine kinase inhibitor H7 eliminated it completely. Pretreatment with phorbol ester or addition of H7 also decreased the insulin-antagonistic effect of GH on lipogenesis. Addition of the protein serine phosphatase inhibitor okadaic acid or the phosphatidyl choline phospholipase C inhibitor D609 both diminished the anti-lipogenic and insulin-antagonistic effects of GH. Chronic exposure of adipose tissue to GH had no effect on the total activity of acetyl CoA carboxylase or its activation status but it did diminish the increase in activation status induced by insulin. H7 and okadaic acid also diminished the increase in activation status of acetyl CoA carboxylase induced by insulin but did not alter the effect of GH on this variable. Okadaic acid decreased total acetyl CoA carboxylase activity. Pretreatment with phorbol ester or the addition of H7, staurosporine or okadaic acid increased glycerol release into the culture medium to the same extent as GH itself; the effects of GH and these various agents were not additive. These studies suggest that the anti-lipogenic, insulin-antagonistic effects of GH involve both protein serine kinases and phosphatases, possibly including one or more isoforms of protein kinase C, and a phosphatidyl choline-specific phospholipase C. Comparison

  20. Fatty acid composition, including CLA's isomers and cholesterol content of m. longissimus lumborum and m. semimebranosus of Katahdin, Suffolk, Katahdin x Suffolk, and Suffolk x Katahdin lambs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipids in meat products have important human health implications. Muscle tissues from Katahdin (KK), Suffolk (SS), Katahdin x Suffolk (KS), and Suffolk x Katahdin (SS) lambs were analyzed to determine the effect of breed-type on muscle fatty acid composition, including conjugated linoleic acid (CLA)...

  1. D-serine in the developing human central nervous system.

    PubMed

    Fuchs, Sabine A; Dorland, Lambertus; de Sain-van der Velden, Monique G; Hendriks, Margriet; Klomp, Leo W J; Berger, Ruud; de Koning, Tom J

    2006-10-01

    To elucidate the role of D-serine in human central nervous system, we analyzed D-serine, L-serine, and glycine concentrations in cerebrospinal fluid of healthy children and children with a defective L-serine biosynthesis (3-phosphoglycerate dehydrogenase deficiency). Healthy children showed high D-serine concentrations immediately after birth, both absolutely and relative to glycine and L-serine, declining to low values at infancy. D-Serine concentrations were almost undetectable in untreated 3-phosphoglycerate dehydrogenase-deficient patients. In one patient treated prenatally, D-serine concentration was nearly normal at birth and the clinical phenotype was normal. These observations suggest a pivotal role for D-serine in normal and aberrant human brain development. PMID:17068790

  2. Fractalkine (CX3CL1) enhances hippocampal N-methyl-d-aspartate receptor (NMDAR) function via d-serine and adenosine receptor type A2 (A2AR) activity

    PubMed Central

    2013-01-01

    Background N-Methyl-d-aspartate receptors (NMDARs) play fundamental roles in basic brain functions such as excitatory neurotransmission and learning and memory processes. Their function is largely regulated by factors released by glial cells, including the coagonist d-serine. We investigated whether the activation of microglial CX3CR1 induces the release of factors that modulate NMDAR functions. Methods We recorded the NMDAR component of the field excitatory postsynaptic potentials (NMDA-fEPSPs) elicited in the CA1 stratum radiatum of mouse hippocampal slices by Shaffer collateral stimulation and evaluated d-serine content in the extracellular medium of glial primary cultures by mass spectrometry analysis. Results We demonstrated that CX3CL1 increases NMDA-fEPSPs by a mechanism involving the activity of the adenosine receptor type A2 (A2AR) and the release of the NMDAR coagonist d-serine. Specifically (1) the selective A2AR blocker 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261) and the genetic ablation of A2AR prevent CX3CL1 action while the A2AR agonist 5-(6-amino-2-(phenethylthio)-9H-purin-9-yl)-N-ethyl-3,4-dihydroxytetrahydrofuran-2-carboxamide (VT7) mimics CX3CL1 effect, and (2) the selective blocking of the NMDAR glycine (and d-serine) site by 5,7-dicholorokynurenic acid (DCKA), the enzymatic degradation of d-serine by d-amino acid oxidase (DAAO) and the saturation of the coagonist site by d-serine, all block the CX3CL1 effect. In addition, mass spectrometry analysis demonstrates that stimulation of microglia and astrocytes with CX3CL1 or VT7 increases d-serine release in the extracellular medium. Conclusions CX3CL1 transiently potentiates NMDAR function though mechanisms involving A2AR activity and the release of d-serine. PMID:23981568

  3. Rapid identification of triterpenoid sulfates and hydroxy fatty acids including two new constituents from Tydemania expeditionis by LC-MS

    PubMed Central

    Zhang, Jian-Long; Kubanek, Julia; Hay, Mark E.; Aalbersberg, William; Ye, Wen-Cai; Jiang, Ren-Wang

    2011-01-01

    Tydemania expeditionis Weber-van Bosse (Udoteaceae) is a weakly calcified green alga. In the present paper, liquid chromatography coupled with photodiode array detection and electrospray mass spectrometry was developed to identify the fingerprint components. A total of four triterpenoid sulfates and three hydroxy fatty acids in the ethyl acetate fraction of the crude extract were structurally characterized on the basis of retention time, online UV spectrum and mass fragmentation pattern. Furthermore, detailed LC-MS analysis revealed two new hydroxy fatty acids, which were then prepared and characterized by extensive NMR analyses. The proposed method provides a scientific and technical platform for the rapid identification of triterpenoid sulfates and hydroxy fatty acids in similar marine algae and terrestrial plants. PMID:21915955

  4. Significance of the D-serine-deaminase and D-serine metabolism of Staphylococcus saprophyticus for virulence.

    PubMed

    Korte-Berwanger, Miriam; Sakinc, Türkan; Kline, Kimberly; Nielsen, Hailyn V; Hultgren, Scott; Gatermann, Sören G

    2013-12-01

    Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a D-serine-deaminase (DsdA). As D-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the D-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that D-serine-deaminase or the ability to respond to or to metabolize D-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular D-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high D-serine concentrations; however, its D-serine metabolism has not been described. The activity of the D-serine-deaminase was verified by analyzing the formation of pyruvate from D-serine in different strains with and without D-serine-deaminase. Cocultivation experiments were performed to show that D-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of D-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ΔdsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of D-serine. In addition, we show that S. saprophyticus is able to use D-serine as the sole carbon source, but interestingly, D-serine had a negative effect on growth when glucose was also present. Taken together, D-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of D-serine and a ΔdsdA mutant was attenuated in virulence murine model of urinary tract infection. PMID:24082071

  5. Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors.

    PubMed

    Adibekian, Alexander; Martin, Brent R; Wang, Chu; Hsu, Ku-Lung; Bachovchin, Daniel A; Niessen, Sherry; Hoover, Heather; Cravatt, Benjamin F

    2011-07-01

    Serine hydrolases are a diverse enzyme class representing ∼1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,β-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes. PMID:21572424

  6. Subtilases: the superfamily of subtilisin-like serine proteases.

    PubMed Central

    Siezen, R. J.; Leunissen, J. A.

    1997-01-01

    Subtilases are members of the clan (or superfamily) of subtilisin-like serine proteases. Over 200 subtilases are presently known, more than 170 of which with their complete amino acid sequence. In this update of our previous overview (Siezen RJ, de Vos WM, Leunissen JAM, Dijkstra BW, 1991, Protein Eng 4:719-731), details of more than 100 new subtilases discovered in the past five years are summarized, and amino acid sequences of their catalytic domains are compared in a multiple sequence alignment. Based on sequence homology, a subdivision into six families is proposed. Highly conserved residues of the catalytic domain are identified, as are large or unusual deletions and insertions. Predictions have been updated for Ca(2+)-binding sites, disulfide bonds, and substrate specificity, based on both sequence alignment and three-dimensional homology modeling. PMID:9070434

  7. Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin.

    PubMed Central

    Bellis, S L; Perrotta, J A; Curtis, M S; Turner, C E

    1997-01-01

    Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both in vitro and in vivo. By using glutathione S-transferase-paxillin fusion proteins in precipitation-kinase assays in vitro we observed that a fusion protein spanning amino acid residues 54-313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to residues 168-191 of paxillin. To assess the physiological relevance of these sites phosphorylated in vitro, the profile of paxillin phosphorylation in vivo stimulated by seeding fibroblasts on fibronectin was characterized. As expected, plating cells on fibronectin enhanced the tyrosine phosphorylation of paxillin. However, 96% of the phosphorylation of paxillin occurred on serine residues. Comparison by two-dimensional phosphopeptide analyses indicated that the major sites of tyrosine and serine phosphorylation detected in the assays in vitro co-migrate with phosphopeptides derived from paxillin phosphorylated in vivo in response to plating cells on fibronectin. These findings support a role for both tyrosine and serine kinases in the signal transduction pathway linking integrin activation to paxillin phosphorylation. PMID:9230116

  8. Competitive Activity-Based Protein Profiling Identifies Aza-β-Lactams as a Versatile Chemotype for Serine Hydrolase Inhibition

    PubMed Central

    Zuhl, Andrea M.; Mohr, Justin T.; Bachovchin, Daniel A.; Niessen, Sherry; Hsu, Ku-Lung; Berlin, Jacob M.; Dochnahl, Maximilian; López-Alberca, María P.; Fu, Gregory C.; Cravatt, Benjamin F.

    2012-01-01

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine hydrolases lack selective inhibitors, which are needed for assigning functions to these enzymes. We recently discovered a set of aza-β-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme’s serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine hydrolases, including the uncharacterized enzyme alpha, beta-hydrolase-10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC50 value ~ 30 nM) and selectively inactivated ABHD10 in vitro and in living cells. A comparison of optimized inhibitors for PME-1 and ABHD10 indicates that modest structural changes that alter steric bulk can tailor the ABL to selectively react with distinct, sequence-unrelated serine hydrolases. Our findings, taken together, designate the ABL as a versatile reactive group for creating first-in-class serine hydrolase inhibitors. PMID:22400490

  9. Competitive activity-based protein profiling identifies aza-β-lactams as a versatile chemotype for serine hydrolase inhibition.

    PubMed

    Zuhl, Andrea M; Mohr, Justin T; Bachovchin, Daniel A; Niessen, Sherry; Hsu, Ku-Lung; Berlin, Jacob M; Dochnahl, Maximilian; López-Alberca, María P; Fu, Gregory C; Cravatt, Benjamin F

    2012-03-21

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine hydrolases lack selective inhibitors, which are valuable probes for assigning functions to these enzymes. We recently discovered a set of aza-β-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme's serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine hydrolases, including the uncharacterized enzyme α,β-hydrolase domain-containing 10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC(50) ≈ 30 nM) and selectively inactivated ABHD10 in vitro and in living cells. A comparison of optimized inhibitors for PME-1 and ABHD10 indicates that modest structural changes that alter steric bulk can tailor the ABL to selectively react with distinct, distantly related serine hydrolases. Our findings, taken together, designate the ABL as a versatile reactive group for creating first-in-class serine hydrolase inhibitors. PMID:22400490

  10. Expression and location of phospho-Artemis (Serine516) in hair follicles during induced growth of mouse hair.

    PubMed

    Wu, Xian-Jie; Zhu, Jian-Wei; Liu, Hai; Lu, Zhong-Fa; Zheng, Min

    2012-05-01

    Artemis has been implicated in having a role in NHEJ, and it is also a multifunctional protein. Previous studies have found Omenn syndrome-like phenotype due to Artemis mutations and associated with alopecia. As Artemis phosphorylation in its c-terminus including Serine516 is prerequisite for the Artemis endonuclease reaction, we postulate that Artemis (Serine516) may be expressed in hair follicle and relate to hair cycling. In this study, hair growth in C57BL/6 mice was induced by plucking the telogen hair on the back. Expression of Artemis (Serine516) in hair follicles during the hair growth cycle was evaluated by immunofluorescence using cryosections and a specific polyclonal anti-Artemis (Serine516) immunoglobulin G (IgG) antibody. It was detected in germ cells, cap, and club hair adjoining the epidermis in telogen. In anagen II, intense staining for Artemis (Serine516) was found in the whole interfollicular epidermis, and in strand keratinocytes. In anagen IV, intense staining for Artemis (Serine516) was detected in basal cells and upper of outer root sheath (ORS) and inner root sheath (IRS). But only upper ORS and lower medulla were stained positive in anagen VI. Upper ORS and lower cortex were positively stained with Artemis (Serine516) in catagen. Based on the phenomenon that the expression of Artemis (Serine516) in mid-anagen and mature anagen was stronger than that in telogen and catagen, we suggest it may take roles in induced growth of mouse hair. PMID:22476261

  11. 21 CFR 582.5701 - Serine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Serine. 582.5701 Section 582.5701 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  12. 21 CFR 582.5701 - Serine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Serine. 582.5701 Section 582.5701 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  13. Inhibition of homocysteine-induced endoplasmic reticulum stress and endothelial cell damage by l-serine and glycine.

    PubMed

    Sim, Woo-Cheol; Han, Inhoi; Lee, Wonseok; Choi, You-Jin; Lee, Kang-Yo; Kim, Dong Gwang; Jung, Seung-Hwan; Oh, Seon-Hee; Lee, Byung-Hoon

    2016-08-01

    Hyperhomocysteinemia is an independent risk factor for several cardiovascular diseases. The use of vitamins to modulate homocysteine metabolism substantially lowers the risk by reducing plasma homocysteine levels. In this study, we evaluated the effects of l-serine and related amino acids on homocysteine-induced endoplasmic reticulum (ER) stress and endothelial cell damage using EA.hy926 human endothelial cells. Homocysteine treatment decreased cell viability and increased apoptosis, which were reversed by cotreatment with l-serine. l-Serine inhibited homocysteine-induced ER stress as verified by decreased glucose-regulated protein 78kDa (GRP78) and C/EBP homologous protein (CHOP) expression as well as X-box binding protein 1 (xbp1) mRNA splicing. The effects of l-serine on homocysteine-induced ER stress are not attributed to intracellular homocysteine metabolism, but instead to decreased homocysteine uptake. Glycine exerted effects on homocysteine-induced ER stress, apoptosis, and cell viability that were comparable to those of l-serine. Although glycine did not affect homocysteine uptake or export, coincubation of homocysteine with glycine for 24h reduced the intracellular concentration of homocysteine. Taken together, l-serine and glycine cause homocysteine-induced endothelial cell damage by reducing the level of intracellular homocysteine. l-Serine acts by competitively inhibiting homocysteine uptake in the cells. However, the mechanism(s) by which glycine lowers homocysteine levels are unclear. PMID:27064126

  14. Effects of phosphorylation on the intrinsic propensity of backbone conformations of serine/threonine.

    PubMed

    He, Erbin; Yan, Guanghui; Zhang, Jian; Wang, Jun; Li, Wenfei

    2016-03-01

    Each amino acid has its intrinsic propensity for certain local backbone conformations, which can be further modulated by the physicochemical environment and post-translational modifications. In this work, we study the effects of phosphorylation on the intrinsic propensity for different local backbone conformations of serine/threonine by molecular dynamics simulations. We showed that phosphorylation has very different effects on the intrinsic propensity for certain local backbone conformations for the serine and threonine. The phosphorylation of serine increases the propensity of forming polyproline II, whereas that of threonine has the opposite effect. Detailed analysis showed that such different responses to phosphorylation mainly arise from their different perturbations to the backbone hydration and the geometrical constraints by forming side-chain-backbone hydrogen bonds due to phosphorylation. Such an effect of phosphorylation on backbone conformations can be crucial for understanding the molecular mechanism of phosphorylation-regulated protein structures/dynamics and functions. PMID:26759163

  15. Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers

    PubMed Central

    Mullarky, Edouard; Lucki, Natasha C.; Beheshti Zavareh, Reza; Anglin, Justin L.; Gomes, Ana P.; Nicolay, Brandon N.; Wong, Jenny C. Y.; Christen, Stefan; Takahashi, Hidenori; Singh, Pradeep K.; Blenis, John; Fendt, Sarah-Maria; Asara, John M.; DeNicola, Gina M.; Lyssiotis, Costas A.; Lairson, Luke L.; Cantley, Lewis C.

    2016-01-01

    Cancer cells reprogram their metabolism to promote growth and proliferation. The genetic evidence pointing to the importance of the amino acid serine in tumorigenesis is striking. The gene encoding the enzyme 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first committed step of serine biosynthesis, is overexpressed in tumors and cancer cell lines via focal amplification and nuclear factor erythroid-2-related factor 2 (NRF2)-mediated up-regulation. PHGDH-overexpressing cells are exquisitely sensitive to genetic ablation of the pathway. Here, we report the discovery of a selective small molecule inhibitor of PHGDH, CBR-5884, identified by screening a library of 800,000 drug-like compounds. CBR-5884 inhibited de novo serine synthesis in cancer cells and was selectively toxic to cancer cell lines with high serine biosynthetic activity. Biochemical characterization of the inhibitor revealed that it was a noncompetitive inhibitor that showed a time-dependent onset of inhibition and disrupted the oligomerization state of PHGDH. The identification of a small molecule inhibitor of PHGDH not only enables thorough preclinical evaluation of PHGDH as a target in cancers, but also provides a tool with which to study serine metabolism. PMID:26831078

  16. Experimental sink removal induces stress responses, including shifts in amino acid and phenylpropanoid metabolism, in soybean leaves

    PubMed Central

    Turner, Glenn W.; Cuthbertson, Daniel J.; Voo, Siau Sie; Settles, Matthew L.; Grimes, Howard D.

    2012-01-01

    The repeated removal of flower, fruit, or vegetative buds is a common treatment to simulate sink limitation. These experiments usually lead to the accumulation of specific proteins, which are degraded during later stages of seed development, and have thus been designated as vegetative storage proteins. We used oligonucleotide microarrays to assess global effects of sink removal on gene expression patterns in soybean leaves and found an induction of the transcript levels of hundreds of genes with putative roles in the responses to biotic and abiotic stresses. In addition, these data sets indicated potential changes in amino acid and phenylpropanoid metabolism. As a response to sink removal we detected an induced accumulation of γ-aminobutyric acid, while proteinogenic amino acid levels decreased. We also observed a shift in phenylpropanoid metabolism with an increase in isoflavone levels, concomitant with a decrease in flavones and flavonols. Taken together, we provide evidence that sink removal leads to an up-regulation of stress responses in distant leaves, which needs to be considered as an unintended consequence of this experimental treatment. PMID:22109846

  17. Evaluation of oxidative stress in D-serine induced nephrotoxicity.

    PubMed

    Orozco-Ibarra, Marisol; Medina-Campos, Omar Noel; Sánchez-González, Dolores Javier; Martínez-Martínez, Claudia María; Floriano-Sánchez, Esaú; Santamaría, Abel; Ramirez, Victoria; Bobadilla, Norma A; Pedraza-Chaverri, José

    2007-01-01

    It has been suggested that oxidative stress is involved in d-serine-induced nephrotoxicity. The purpose of this study was to assess if oxidative stress is involved in this experimental model using several approaches including (a) the determination of several markers of oxidative stress and the activity of some antioxidant enzymes in kidney and (b) the use of compounds with antioxidant or prooxidant effects. Rats were sacrificed at several periods of time (from 3 to 24h) after a single i.p. injection of d-serine (400mg/kg). Control rats were injected with l-serine (400mg/kg) and sacrificed 24h after. The following markers were used to assess the temporal aspects of renal damage: (a) urea nitrogen (BUN) and creatinine in blood serum, (b) kidney injury molecule (KIM-1) mRNA levels, and (c) tubular necrotic damage. In addition, creatinine clearance, proteinuria, and urinary excretion of N-acetyl-beta-d-glucosaminidase (NAG) were measured 24h after d-serine injection. Protein carbonyl content, malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), fluorescent products of lipid peroxidation, reactive oxygen species (ROS), glutathione (GSH) content, and heme oxygenase-1 (HO-1) expression were measured as markers of oxidative stress in the kidney. Additional experiments were performed using the following compounds with antioxidant or pro-oxidant effects before d-serine injection: (a) alpha-phenyl-tert-butyl-nitrone (PBN), a spin trapping agent; (b) 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) (FeTPPS), a soluble complex able to metabolize peroxynitrite; (c) aminotriazole (ATZ), a catalase (CAT) inhibitor; (d) stannous chloride (SnCl(2)), an HO-1 inductor; (e) tin mesoporphyrin (SnMP), an HO inhibitor. In the time-course study, serum creatinine and BUN increased significantly on 15-24 and 20-24h, respectively, and KIM-1 mRNA levels increased significantly on 6-24h. Histological analyses revealed tubular necrosis at 12h. The activity of antioxidant enzymes

  18. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    PubMed Central

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

    2016-01-01

    Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4–fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester. PMID:27150669

  19. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  20. Structural basis of substrate specificity in the serine proteases.

    PubMed Central

    Perona, J. J.; Craik, C. S.

    1995-01-01

    Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system. PMID:7795518

  1. Fatty acid composition of ruminal digesta and longissimus muscle from lambs fed silage mixtures including red clover, sainfoin, and timothy.

    PubMed

    Campidonico, L; Toral, P G; Priolo, A; Luciano, G; Valenti, B; Hervás, G; Frutos, P; Copani, G; Ginane, C; Niderkorn, V

    2016-04-01

    This work investigated the effects of feeding silage mixtures of a plant containing polyphenol oxidase (PPO; red clover [; RC]), a plant containing tannins (sainfoin [; SF]), and a grass species not containing these compounds (timothy [; T]) on ruminal and intramuscular (i.m.) fatty acids of lambs. Forty 4-mo-old castrated male Romane lambs, divided into 5 groups, received 1 of the following silages: 1) T (100%), 2) a binary mixture of timothy and tannin-containing sainfoin ( cv. Perly; 50:50 [T-SF]), 3) a binary mixture of timothy and PPO-containing red clover ( cv. Mervius; 50:50 [T-RC]), 4) a ternary mixture of timothy, sainfoin, and red clover containing both tannins and PPO (50:25:25, respectively [T-SF-RC]), and 5) a binary mixture of tannin-containing sainfoin and PPO-containing red clover (50:50 [SF-RC]). In the rumen digesta, the partial or total replacement of T with forage legumes was associated with greater concentrations of PUFA ( < 0.001) and 1esser concentrations of MUFA ( < 0.001). The inclusion of forage legumes in the silage favored the accumulation of 18:3 -3 ( < 0.001), with the greatest concentrations being observed in SF-RC. This latter diet also led to the greatest percentage of 18:2 -6 ( < 0.001). Forage legumes decreased the -11 18:1 to 30% of T in rumen digesta ( < 0.001). Forage legumes decreased the total concentration of branched-chain fatty acids in the rumen digesta (on average, -28%; < 0.001), this effect being less marked (-17%; = 0.014) in T-RC in comparison with T. The dietary treatment tended to affect the proportion of MUFA ( = 0.081) and of PUFA ( = 0.079) in the i.m. fat of the LM, respectively, at the highest and lowest numerical value in the T group. The sum of -3 fatty acids was less in the T and T-SF groups compared with the mixture of legumes without T (SF-RC; < 0.001 and < 0.008, respectively). The latter group had also a lesser -6-to--3 ratio than the T-SF group ( = 0.01). -11 18:1 was greater ( < 0.03) in lambs given T

  2. The role of D-serine as co-agonist of NMDA receptors in the nucleus accumbens: relevance to cocaine addiction.

    PubMed

    D'Ascenzo, Marcello; Podda, Maria Vittoria; Grassi, Claudio

    2014-01-01

    Cocaine addiction is characterized by compulsive drug use despite adverse consequences and high rate of relapse during periods of abstinence. Increasing consensus suggests that addiction to drugs of abuse usurps learning and memory mechanisms normally related to natural rewards, ultimately producing long-lasting neuroadaptations in the mesocorticolimbic system. This system, formed in part by the ventral tegmental area and nucleus accumbens (NAc), has a central role in the development and expression of addictive behaviors. In addition to a broad spectrum of changes that affect morphology and function of NAc excitatory circuits in cocaine-treated animals, impaired N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity is a typical feature. D-serine, a D-amino acid that has been found at high levels in mammalian brain, binds with high affinity the co-agonist site of NMDAR and mediates, along with glutamate, several important processes including synaptic plasticity. Here we review recent literature focusing on cocaine-induced impairment in synaptic plasticity mechanisms in the NAc and on the fundamental role of D-serine as co-agonist of NMDAR in functional and dysfunctional synaptic plasticity within this nucleus. The emerging picture is that reduced D-serine levels play a crucial role in synaptic plasticity relevant to cocaine addiction. This finding opens new perspectives for therapeutic approaches to treat this addictive state. PMID:25076900

  3. The role of D-serine as co-agonist of NMDA receptors in the nucleus accumbens: relevance to cocaine addiction

    PubMed Central

    D’Ascenzo, Marcello; Podda, Maria Vittoria; Grassi, Claudio

    2014-01-01

    Cocaine addiction is characterized by compulsive drug use despite adverse consequences and high rate of relapse during periods of abstinence. Increasing consensus suggests that addiction to drugs of abuse usurps learning and memory mechanisms normally related to natural rewards, ultimately producing long-lasting neuroadaptations in the mesocorticolimbic system. This system, formed in part by the ventral tegmental area and nucleus accumbens (NAc), has a central role in the development and expression of addictive behaviors. In addition to a broad spectrum of changes that affect morphology and function of NAc excitatory circuits in cocaine–treated animals, impaired N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity is a typical feature. D-serine, a D-amino acid that has been found at high levels in mammalian brain, binds with high affinity the co-agonist site of NMDAR and mediates, along with glutamate, several important processes including synaptic plasticity. Here we review recent literature focusing on cocaine-induced impairment in synaptic plasticity mechanisms in the NAc and on the fundamental role of D-serine as co-agonist of NMDAR in functional and dysfunctional synaptic plasticity within this nucleus. The emerging picture is that reduced D-serine levels play a crucial role in synaptic plasticity relevant to cocaine addiction. This finding opens new perspectives for therapeutic approaches to treat this addictive state. PMID:25076900

  4. Liquid chromatographic resolution of amino acid esters of acyclovir including racemic valacyclovir on crown ether-based chiral stationary phases.

    PubMed

    Ahn, Seong Ae; Hyun, Myung Ho

    2015-03-01

    Valacyclovir, a potential prodrug for the treatment of patients with herpes simplex and herpes zoster, and its analogs were resolved on two chiral stationary phases (CSPs) based on (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 covalently bonded to silica gel. In order to find out an appropriate mobile phase condition, various mobile phases consisting of various organic modifiers in water containing various acidic modifiers were applied to the resolution of valacyclovir and its analogs. When 30% acetonitrile in water containing any of 0.05 M, 0.10 M, or 0.15 M perchloric acid was used as a mobile phase, valacyclovir and its analogs were resolved quite well on the two CSPs with the separation factors (α) in the range of 2.49 ~ 6.35 and resolutions (RS ) in the range of 2.95 ~ 12.21. Between the two CSPs, the CSP containing residual silanol protecting n-octyl groups on the silica surface was found to be better than the CSP containing residual silanol groups. PMID:25626672

  5. Skin aging and photoaging alter fatty acids composition, including 11,14,17-eicosatrienoic acid, in the epidermis of human skin.

    PubMed

    Kim, Eun Ju; Kim, Min-Kyoung; Jin, Xing-Ji; Oh, Jang-Hee; Kim, Ji Eun; Chung, Jin Ho

    2010-06-01

    We investigated the alterations of major fatty acid components in epidermis by natural aging and photoaging processes, and by acute ultraviolet (UV) irradiation in human skin. Interestingly, we found that 11,14,17-eicosatrienoic acid (ETA), which is one of the omega-3 polyunsaturated acids, was significantly increased in photoaged human epidermis in vivo and also in the acutely UV-irradiated human skin in vivo, while it was significantly decreased in intrinsically aged human epidermis. The increased ETA content in the epidermis of photoaged human skin and acute UV-irradiated human skin is associated with enhanced expression of human elongase 1 and calcium-independent phosphodiesterase A(2). We demonstrated that ETA inhibited matrix metalloproteinase (MMP)-1 expression after UV-irradiation, and that inhibition of ETA synthesis using EPTC and NA-TCA, which are elongase inhibitors, increased MMP-1 expression. Therefore, our results suggest that the UV increases the ETA levels, which may have a photoprotective effect in the human skin. PMID:20514327

  6. Skin Aging and Photoaging Alter Fatty Acids Composition, Including 11,14,17-eicosatrienoic Acid, in the Epidermis of Human Skin

    PubMed Central

    Kim, Eun Ju; Kim, Min-Kyoung; Jin, Xing-Ji; Oh, Jang-Hee; Kim, Ji Eun

    2010-01-01

    We investigated the alterations of major fatty acid components in epidermis by natural aging and photoaging processes, and by acute ultraviolet (UV) irradiation in human skin. Interestingly, we found that 11,14,17-eicosatrienoic acid (ETA), which is one of the omega-3 polyunsaturated acids, was significantly increased in photoaged human epidermis in vivo and also in the acutely UV-irradiated human skin in vivo, while it was significantly decreased in intrinsically aged human epidermis. The increased ETA content in the epidermis of photoaged human skin and acute UV-irradiated human skin is associated with enhanced expression of human elongase 1 and calcium-independent phophodiesterase A2. We demonstrated that ETA inhibited matrix metalloproteinase (MMP)-1 expression after UV-irradiation, and that inhibition of ETA synthesis using EPTC and NA-TCA, which are elongase inhibitors, increased MMP-1 expression. Therefore, our results suggest that the UV increases the ETA levels, which may have a photoprotective effect in the human skin. PMID:20514327

  7. Comparative Mitogenomics of Plant Bugs (Hemiptera: Miridae): Identifying the AGG Codon Reassignments between Serine and Lysine

    PubMed Central

    Wang, Pei; Song, Fan; Cai, Wanzhi

    2014-01-01

    Insect mitochondrial genomes are very important to understand the molecular evolution as well as for phylogenetic and phylogeographic studies of the insects. The Miridae are the largest family of Heteroptera encompassing more than 11,000 described species and of great economic importance. For better understanding the diversity and the evolution of plant bugs, we sequence five new mitochondrial genomes and present the first comparative analysis of nine mitochondrial genomes of mirids available to date. Our result showed that gene content, gene arrangement, base composition and sequences of mitochondrial transcription termination factor were conserved in plant bugs. Intra-genus species shared more conserved genomic characteristics, such as nucleotide and amino acid composition of protein-coding genes, secondary structure and anticodon mutations of tRNAs, and non-coding sequences. Control region possessed several distinct characteristics, including: variable size, abundant tandem repetitions, and intra-genus conservation; and was useful in evolutionary and population genetic studies. The AGG codon reassignments were investigated between serine and lysine in the genera Adelphocoris and other cimicomorphans. Our analysis revealed correlated evolution between reassignments of the AGG codon and specific point mutations at the antidocons of tRNALys and tRNASer(AGN). Phylogenetic analysis indicated that mitochondrial genome sequences were useful in resolving family level relationship of Cimicomorpha. Comparative evolutionary analysis of plant bug mitochondrial genomes allowed the identification of previously neglected coding genes or non-coding regions as potential molecular markers. The finding of the AGG codon reassignments between serine and lysine indicated the parallel evolution of the genetic code in Hemiptera mitochondrial genomes. PMID:24988409

  8. Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family

    SciTech Connect

    Hanks, S.K.

    1987-01-01

    Mixed /sup 32/P-labeled oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identify with rabbit skeletal muscle phosphorylase kinase ..gamma..-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.

  9. Excess of threonine compared with serine promotes threonine aldolase activity in Lactococcus lactis IL1403.

    PubMed

    Aller, Kadri; Adamberg, Kaarel; Reile, Indrek; Timarova, Veronica; Peebo, Karl; Vilu, Raivo

    2015-05-01

    Lactococcus lactis is an important lactic acid starter for food production as well as a cell factory for production of food grade additives, among which natural flavour production is one of the main interests of food producers. Flavour production is associated with the degradation of amino acids and comprehensive studies are required to elucidate mechanisms behind these pathways. In this study using chemically defined medium, labelled substrate and steady-state cultivation, new data for the catabolism of threonine in Lc. lactis have been obtained. The biosynthesis of glycine in this organism is associated with the catabolic pathways of glucose and serine. Nevertheless, if threonine concentration in the growth environment exceeds that of serine, threonine becomes the main source for glycine biosynthesis and the utilization of serine decreases. Also, the conversion of threonine to glycine was initiated by a threonine aldolase and this was the principal pathway used for threonine degradation. As in Streptococcus thermophilus, serine hydroxymethyltransferase in Lc. lactis may possess a secondary activity as threonine aldolase. Other catabolic pathways of threonine (e.g. threonine dehydrogenase and threonine dehydratase) were not detected. PMID:25743155

  10. Pharmacokinetic and Pharmacodynamic Interaction of Boswellic Acids and Andrographolide with Glyburide in Diabetic Rats: Including Its PK/PD Modeling.

    PubMed

    Samala, Sujatha; Veeresham, Ciddi

    2016-03-01

    The effect of boswellic acids (BA) and andrographolide (AD) on the pharmacokinetics and pharmacodynamics of glyburide in normal as well as in streptozotocin-induced diabetic rats was studied. In normal and diabetic rats, the combination of glyburide with BA or AD increased significantly (p < 0.01) all the pharmacokinetic parameters, such as Cmax, AUC0-n, AUCtotal, t1/2, and mean residence time, and decreased the clearance, Vd, markedly as compared with the control group. In rat liver, microsomes BA and AD have shown CYP3A4 inhibitory activity significantly (p < 0.01), compared with the vehicle group. The increase in hypoglycemic action by concomitant administration of glyburide with BA or AD was more in diabetic rats than when the drugs were used singly and with the control group, which suggests the enhancement of glucose reduction capacity of glyburide in diabetic rats along with BA or AD. In PK/PD modeling of BA and AD with glyburide, the predicted PK and PD parameters are in line with the observed PK and PD parameters. The results revealed that BA and AD led to the PK/PD changes because of glyburide-increased bioavailability and because of the inhibition of CYP3A4 enzyme. In conclusion, add-on preparations containing BA or AD may increase the bioavailability of glyburide, and hence the dose should be monitored. PMID:26762235

  11. Dynamic regulation of d-serine release in the vertebrate retina

    PubMed Central

    Gustafson, Eric G; Stevens, Eric S; Miller, Robert F

    2015-01-01

    The present study aimed to investigate the functional properties of NMDA receptor coagonist release and to specifically evaluate whether light-evoked release mechanisms contribute to the availability of the coagonist d-serine. Two different methods were involved in our approach: (i) whole-cell recordings from identified retinal ganglion cells in the tiger salamander were used to study light adaptation with positive and negative contrast stimuli over a range of ± 1 log unit against a steady background illumination and (ii) the mechanisms for intensity encoding to a range of light intensities covering 6 log10 units were investigated. This latter study employed extracellular recordings of the proximal negative field potential, pharmacologically manipulated to generate a pure NMDA mediated response. For the adaptation study, we examined the light-evoked responses under control conditions, followed by light stimuli presented in the presence of d-serine, followed by light stimulation in the presence of dichlorokynurenic acid to block the coagonist site of NMDA receptors. For the brightness encoding studies, we examined the action of d-serine on each intensity used and then applied the enzyme d-serine deaminase to remove significant levels of d-serine. These studies provided new insights into the mechanisms that regulate coagonist availability in the vertebrate retina. Our results strongly support the idea that light-evoked coagonist release, a major component of which is d-serine, is needed to provide the full range of coagonist availability for optimal activation of NMDA receptors. PMID:25480802

  12. Retinoic Acid Induced 1, RAI1: A Dosage Sensitive Gene Related to Neurobehavioral Alterations Including Autistic Behavior

    PubMed Central

    Carmona-Mora, Paulina; Walz, Katherina

    2010-01-01

    Genomic structural changes, such as gene Copy Number Variations (CNVs) are extremely abundant in the human genome. An enormous effort is currently ongoing to recognize and catalogue human CNVs and their associations with abnormal phenotypic outcomes. Recently, several reports related neuropsychiatric diseases (i.e. autism spectrum disorders, schizophrenia, mental retardation, behavioral problems, epilepsy) with specific CNV. Moreover, for some conditions, both the deletion and duplication of the same genomic segment are related to the phenotype. Syndromes associated with CNVs (microdeletion and microduplication) have long been known to display specific neurobehavioral traits. It is important to note that not every gene is susceptible to gene dosage changes and there are only a few dosage sensitive genes. Smith-Magenis (SMS) and Potocki-Lupski (PTLS) syndromes are associated with a reciprocal microdeletion and microduplication within chromosome 17p11.2. in humans. The dosage sensitive gene responsible for most phenotypes in SMS has been identified: the Retinoic Acid Induced 1 (RAI1). Studies on mouse models and humans suggest that RAI1 is likely the dosage sensitive gene responsible for clinical features in PTLS. In addition, the human RAI1 gene has been implicated in several neurobehavioral traits as spinocerebellar ataxia (SCA2), schizophrenia and non syndromic autism. In this review we discuss the evidence of RAI1 as a dosage sensitive gene, its relationship with different neurobehavioral traits, gene structure and mutations, and what is known about its molecular and cellular function, as a first step in the elucidation of the mechanisms that relate dosage sensitive genes with abnormal neurobehavioral outcomes. PMID:21629438

  13. Impaired neurogenesis in embryonic spinal cord of Phgdh knockout mice, a serine deficiency disorder model.

    PubMed

    Kawakami, Yuriko; Yoshida, Kazuyuki; Yang, Jung Hoon; Suzuki, Takeshi; Azuma, Norihiro; Sakai, Kazuhisa; Hashikawa, Tsutomu; Watanabe, Masahiko; Yasuda, Kaori; Kuhara, Satoru; Hirabayashi, Yoshio; Furuya, Shigeki

    2009-03-01

    Mutations in the d-3-phosphoglycerate dehydrogenase (PHGDH; EC 1.1.1.95) gene, which encodes an enzyme involved in de novol-serine biosynthesis, are shown to cause human serine deficiency disorder. This disorder has been characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. Our previous work demonstrated that targeted disruption of mouse Phgdh leads to a marked decrease in serine and glycine, severe growth retardation of the central nervous system, and lethality after embryonic day 13.5. To clarify how a serine deficiency causes neurodevelopmental defects, we characterized changes in metabolites, gene expression and morphological alterations in the spinal cord of Phgdh knockout mice. BeadChip microarray analysis revealed significant dysregulation of genes involved in the cell cycle. Ingenuity Pathway Analysis also revealed a significant perturbation of regulatory networks that operate in the cell cycle progression. Moreover, morphological examinations of the knockout spinal cord demonstrated a marked deficit in dorsal horn neurons. Radial glia cells, native neural stem/progenitor cells, accumulated in the dorsal ventricular zone, but they did not proceed to a G(0)-like quiescent state. The present integrative study provides in vivo evidence that normal cell cycle progression and subsequent neurogenesis of radial glia cells are severely impaired by serine deficiency. PMID:19114063

  14. Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins

    PubMed Central

    Thomas, Marshall P.; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-01-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. Here we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein (RBP) targets, while adding RNA to recombinant RBP substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Pre-incubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (CATG). During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps (NETs), which bind NE and CATG. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and NETs in a DNA-dependent manner. Thus, high affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

  15. Distribution and evolution of the serine/aspartate racemase family in invertebrates.

    PubMed

    Uda, Kouji; Abe, Keita; Dehara, Yoko; Mizobata, Kiriko; Sogawa, Natsumi; Akagi, Yuki; Saigan, Mai; Radkov, Atanas D; Moe, Luke A

    2016-02-01

    Free D-amino acids have been found in various invertebrate phyla, while amino acid racemase genes have been identified in few species. The purpose of this study is to elucidate the distribution, function, and evolution of amino acid racemases in invertebrate animals. We searched the GenBank databases, and found 11 homologous serine racemase genes from eight species in eight different invertebrate phyla. The cloned genes were identified based on their maximum activity as Acropora millepora (Cnidaria) serine racemase (SerR) and aspartate racemase (AspR), Caenorhabditis elegans (Nematoda) SerR, Capitella teleta (Annelida) SerR, Crassostrea gigas (Mollusca) SerR and AspR, Dugesia japonica (Platyhelminthes) SerR, Milnesium tardigradum (Tardigrada) SerR, Penaeus monodon (Arthropoda) SerR and AspR and Strongylocentrotus purpuratus (Echinodermata) AspR. We found that Acropora, Aplysia, Capitella, Crassostrea and Penaeus had two amino acid racemase paralogous genes and these paralogous genes have evolved independently by gene duplication at their recent ancestral species. The transcriptome analyses using available SRA data and enzyme kinetic data suggested that these paralogous genes are expressed in different tissues and have different functions in vivo. Phylogenetic analyses clearly indicated that animal SerR and AspR are not separated by their particular racemase functions and form a serine/aspartate racemase family cluster. Our results revealed that SerR and AspR are more widely distributed among invertebrates than previously known. Moreover, we propose that the triple serine loop motif at amino acid positions 150-152 may be responsible for the large aspartate racemase activity and the AspR evolution from SerR. PMID:26352274

  16. Activity-Based Protein Profiling of Organophosphorus and Thiocarbamate Pesticides Reveals Multiple Serine Hydrolase Targets in Mouse Brain

    PubMed Central

    NOMURA, DANIEL K.; CASIDA, JOHN E.

    2010-01-01

    Organophosphorus (OP) and thiocarbamate (TC) agrochemicals are used worldwide as insecticides, herbicides, and fungicides, but their safety assessment in terms of potential off-targets remains incomplete. In this study, we used a chemoproteomic platform, termed activity-based protein profiling, to broadly define serine hydrolase targets in mouse brain of a panel of 29 OP and TC pesticides. Among the secondary targets identified, enzymes involved in degradation of endocannabinoid signaling lipids, monoacylglycerol lipase and fatty acid amide hydrolase, were inhibited by several OP and TC pesticides. Blockade of these two enzymes led to elevations in brain endocannabinoid levels and dysregulated brain arachidonate metabolism. Other secondary targets include enzymes thought to also play important roles in the nervous system and unannotated proteins. This study reveals a multitude of secondary targets for OP and TC pesticides and underscores the utility of chemoproteomic platforms in gaining insights into biochemical pathways that are perturbed by these toxicants. PMID:21341672

  17. Omega-3 fatty acid concentrate from Dunaliella salina possesses anti-inflammatory properties including blockade of NF-κB nuclear translocation.

    PubMed

    Chitranjali, T; Anoop Chandran, P; Muraleedhara Kurup, G

    2015-02-01

    The health benefits of omega-3 polyunsaturated fatty acids (ω-3 PUFA), mainly eicosapentaenoic acid (EPA 20:5) and docosahexaenoic acid (DHA, 22:6), have been long known. Although various studies have demonstrated the health benefits of ω-3 PUFA, the mechanisms of action of ω-3 PUFAs are still not completely understood. While the major commercial source is marine fish oil, in this study we suggest the marine micro algae, Dunaliella salina as an alternate source of omega-3 fatty acids. Treatment with this algal omega-3 fatty acid concentrate (Ds-ω-3 FA) resulted in significant down-regulation of LPS-induced production of TNF-α and IL-6 by peripheral blood mononuclear cells (PBMCs). The concentrate was also found to be a potent blocker of cyclooxygenase (COX-2) and matrix metalloproteinase (MMP-2 and MMP-9) expression. The present study reveals the anti-inflammatory properties of Ds-ω-3 FA concentrate including the inhibition of NF-κB translocation. PMID:25391558

  18. New CE-ESI-MS analytical method for the separation, identification and quantification of seven phenolic acids including three isomer compounds in virgin olive oil.

    PubMed

    Nevado, Juan José Berzas; Peñalvo, Gregorio Castañeda; Robledo, Virginia Rodríguez; Martínez, Gabriela Vargas

    2009-10-15

    A sensitive and expeditious CE-ESI-MS analytical method for the separation, identification and determination of seven selected antioxidants (cinnamic and benzoic acids), including three isomers of coumaric acid (ortho-, meta- and para-) has been developed. In order to obtain the analytical separation, capillary electrophoresis and CE-MS interface parameters (e.g., buffer pH and composition, sheath liquid and gas flow rates, sheath liquid composition, electrospray voltage, etc.) were carefully optimized. The polar fraction containing the selected phenolic acids was obtained using a previously optimized SPE pretreatment. An MS detector in order to extract structural information about the target compounds and facilitate their qualitative analysis was used in the negative ion mode. The proposed off-line SPE CE-ESI-MS method was validated by assessing its precision, LODs and LOQs, linearity range and accuracy. The optimized and validated method was used in order to quantify the selected antioxidants in various samples of virgin olive oil and extra-virgin olive oil obtained from the main olive varieties cropped in Castilla-La Mancha, Spain. Salicylic acid was used as internal standard throughout in order to ensure reproducibility in the quantitative analysis of the oil samples. The results confirmed the presence of hydroxyphenyl acetic, p-coumaric, ferulic and vanillic acids in substantial amounts (microg g(-1) level) in all samples. PMID:19635353

  19. Fatal cerebral edema associated with serine deficiency in CSF.

    PubMed

    Keularts, Irene M L W; Leroy, Piet L J M; Rubio-Gozalbo, Estela M; Spaapen, Leo J M; Weber, Biene; Dorland, Bert; de Koning, Tom J; Verhoeven-Duif, Nanda M

    2010-12-01

    Two young girls without a notable medical history except for asthma presented with an acute toxic encephalopathy with very low serine concentrations both in plasma and cerebrospinal fluid (CSF) comparable to patients with 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency. Clinical symptoms and enzyme measurement (in one patient) excluded 3-PGDH deficiency. Deficiencies in other serine biosynthesis enzymes were highly unlikely on clinical grounds. On basis of the fasting state, ketone bodies and lactate in plasma, urine and CSF, we speculate that reduced serine levels were due to its use as gluconeogenic substrate, conversion to pyruvate by brain serine racemase or decreased L-serine production because of a lack of glucose. These are the first strikingly similar cases of patients with a clear secondary serine deficiency associated with a toxic encephalopathy. PMID:20300853

  20. Reducing the serine availability complements the inhibition of the glutamine metabolism to block leukemia cell growth

    PubMed Central

    Polet, Florence; Corbet, Cyril; Pinto, Adan; Rubio, Laila Illan; Martherus, Ruben; Bol, Vanesa; Drozak, Xavier; Grégoire, Vincent; Riant, Olivier; Feron, Olivier

    2016-01-01

    Leukemia cells are described as a prototype of glucose-consuming cells with a high turnover rate. The role of glutamine in fueling the tricarboxylic acid cycle of leukemia cells was however recently identified confirming its status of major anaplerotic precursor in solid tumors. Here we examined whether glutamine metabolism could represent a therapeutic target in leukemia cells and whether resistance to this strategy could arise. We found that glutamine deprivation inhibited leukemia cell growth but also led to a glucose-independent adaptation maintaining cell survival. A proteomic study revealed that glutamine withdrawal induced the upregulation of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT), two enzymes of the serine pathway. We further documented that both exogenous and endogenous serine were critical for leukemia cell growth and contributed to cell regrowth following glutamine deprivation. Increase in oxidative stress upon inhibition of glutamine metabolism was identified as the trigger of the upregulation of PHGDH. Finally, we showed that PHGDH silencing in vitro and the use of serine-free diet in vivo inhibited leukemia cell growth, an effect further increased when glutamine metabolism was blocked. In conclusion, this study identified serine as a key pro-survival actor that needs to be handled to sensitize leukemia cells to glutamine-targeting modalities. PMID:26625201

  1. Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts.

    PubMed

    Ngo, Phong D; Mansoorabadi, Steven O; Frey, Perry A

    2016-08-01

    Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties. PMID:27387593

  2. Human prostate-specific antigen: structural and functional similarity with serine proteases.

    PubMed Central

    Watt, K W; Lee, P J; M'Timkulu, T; Chan, W P; Loor, R

    1986-01-01

    The complete amino acid sequence of the prostate-specific antigen (PA) from human seminal plasma has been determined from analyses of the peptides generated by cyanogen bromide, hydroxylamine, endoproteinases Arg-C and Lys-C. The single polypeptide chain of PA contains 240-amino acid residues and has a calculated Mr of 26,496. An N-linked carbohydrate side chain is predicted at asparagine-45, and O-linked carbohydrate side chains are possibly attached to serine-69, threonine-70, and serine-71. The primary structure of PA shows a high degree of sequence homology with other serine proteases of the kallikrein family. The active site residues of histidine, aspartic acid, and serine comprising the charge-relay system of typical serine proteases were found in similar positions in PA (histidine-41, aspartic acid-96, and serine-192). At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant interleukin 2, and--to a lesser extent--gelatin, myoglobin, ovalbumin, and fibrinogen. The cleavage sites of these proteins by PA were chemically analyzed as the alpha-carboxyl side of some hydrophobic residues, tyrosine, leucine, valine, and phenylalanine, and of basic residues histidine, lysine, and arginine. The chymotrypsin-like activity of PA exhibited with the chromogenic substrate N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide yielded a specific activity of 9.21 microM per min per mg of PA and Km and kcat values of 15.3 mM and 0.075s-1, respectively. "Trypsin-like" activity of PA was also detected with N alpha-benzoyl-DL-arginine p-nitroanilide and gave a specific activity of 1.98 microM per min per mg of PA. Protease inhibitors such as phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, L-1-tosylamido-2-phenylethyl chloromethyl ketone, aprotinin, leupeptin, soybean trypsin inhibitor as well as Zn2+ and spermidine were effective inhibitors of PA enzymatic activity. PMID:2422647

  3. Serine O-sulfation probed by IRMPD spectroscopy.

    PubMed

    Paciotti, Roberto; Coletti, Cecilia; Re, Nazzareno; Scuderi, Debora; Chiavarino, Barbara; Fornarini, Simonetta; Crestoni, Maria Elisa

    2015-10-21

    The sulfation of amino acids is a frequent post-translational modification. It is highly labile, though, and characterizing it by mass spectrometry, an otherwise powerful and widely exploited tool in analytical proteomics, is a challenge. The presently reported study is aimed at revealing the O-sulfation of l-serine and elucidating the effects of protonation and deprotonation on the structure and stability of the ensuing ionic species, [sSer + H](+) and [sSer - H](-). These ions are obtained as gaseous, isolated species by electrospray ionization, trapped in a Paul ion-trap, and sampled by IR multiple photon dissociation (IRMPD) spectroscopy in either the 750-1900 cm(-1) fingerprint range, or the 2900 and 3700 cm(-1) range encompassing the N-H and O-H stretching modes. The recorded IRMPD spectra present diagnostic signatures of the sulfate modification which are missing in the spectra of the native serine ions, [Ser + H](+) and [Ser - H](-). The experimental IRMPD features have been interpreted by comparison with the linear IR spectra of the lowest energy structures that are likely candidates for the sampled ions, calculated at the M06-2X/6-311+G(d,p) level of theory. Evidence is gathered that the most stable conformations of [sSer + H](+) are stabilized by hydrogen bonding interactions between the protonated amino group and both the carbonyl and sulfate oxygens. [sSer - H](-) ions possess a negatively charged sulfate group involved in either a S=O···HN or a S=O···HO hydrogen bond. The experimental IRMPD spectra are consistent with the presence of multiple low-lying structures in a thermally equilibrated population of several species particularly in the case of [sSer - H](-) ions, where the high structural flexibility combined with the presence of a negative charge favors the co-existence of several different H-bonding motifs. PMID:26027702

  4. High-speed civil transport impact: Role of sulfate, nitric acid trihydrate, and ice aerosols studied with a two-dimensional model including aerosol physics

    SciTech Connect

    Pitari, G.; Ricciardulli, L.; Visconti, G.; Rizi, V.

    1993-12-20

    The authors discuss a two-dimensional model used to study the atmospheric interactions of ozone with exhaust gases from high speed civil transport (HSCT) fleets. Their model encompases the stratosphere and troposphere, includes photochemical reactions as part of the sulfur cycle, and models sulfuric acid aerosols. The inclusion of heterogeneous chemistry effects tempers the impact of nitrogen oxide emissions from HSCT on ozone depletion, in support of previous work from other studies.

  5. Imidazopyridine and Pyrazolopiperidine Derivatives as Novel Inhibitors of Serine Palmitoyl Transferase.

    PubMed

    Genin, Michael J; Gonzalez Valcarcel, Isabel C; Holloway, William G; Lamar, Jason; Mosior, Marian; Hawkins, Eric; Estridge, Thomas; Weidner, Jeffrey; Seng, Thomas; Yurek, David; Adams, Lisa A; Weller, Jennifer; Reynolds, Vincent L; Brozinick, Joseph T

    2016-06-23

    To develop novel treatments for type 2 diabetes and dyslipidemia, we pursued inhibitors of serine palmitoyl transferase (SPT). To this end compounds 1 and 2 were developed as potent SPT inhibitors in vitro. 1 and 2 reduce plasma ceramides in rodents, have a slight trend toward enhanced insulin sensitization in DIO mice, and reduce triglycerides and raise HDL in cholesterol/cholic acid fed rats. Unfortunately these molecules cause a gastric enteropathy after chronic dosing in rats. PMID:27213958

  6. Extension of a PBPK model for ethylene glycol and glycolic acid to include the competitive formation and clearance of metabolites associated with kidney toxicity in rats and humans

    SciTech Connect

    Corley, R.A.; Saghir, S.A.; Bartels, M.J.; Hansen, S.C.; Creim, J.; McMartin, K.E.; Snellings, W.M.

    2011-02-01

    A previously developed PBPK model for ethylene glycol and glycolic acid was extended to include glyoxylic acid, oxalic acid, and the precipitation of calcium oxalate that is associated with kidney toxicity in rats and humans. The development and evaluation of the PBPK model was based upon previously published pharmacokinetic studies coupled with measured blood and tissue partition coefficients and rates of in vitro metabolism of glyoxylic acid to oxalic acid, glycine and other metabolites using primary hepatocytes isolated from male Wistar rats and humans. Precipitation of oxalic acid with calcium in the kidneys was assumed to occur only at concentrations exceeding the thermodynamic solubility product for calcium oxalate. This solubility product can be affected by local concentrations of calcium and other ions that are expressed in the model using an ion activity product estimated from toxicity studies such that calcium oxalate precipitation would be minimal at dietary exposures below the NOAEL for kidney toxicity in the sensitive male Wistar rat. The resulting integrated PBPK predicts that bolus oral or dietary exposures to ethylene glycol would result in typically 1.4-1.6-fold higher peak oxalate levels and 1.6-2-fold higher AUC's for calcium oxalate in kidneys of humans as compared with comparably exposed male Wistar rats over a dose range of 1-1000 mg/kg. The converse (male Wistar rats predicted to have greater oxalate levels in the kidneys than humans) was found for inhalation exposures although no accumulation of calcium oxalate is predicted to occur until exposures are well in excess of the theoretical saturated vapor concentration of 200 mg/m{sup 3}. While the current model is capable of such cross-species, dose, and route-of-exposure comparisons, it also highlights several areas of potential research that will improve confidence in such predictions, especially at low doses relevant for most human exposures.

  7. Human milk nonprotein nitrogen components: changing patterns of free amino acids and urea in the course of early lactation.

    PubMed

    Harzer, G; Franzke, V; Bindels, J G

    1984-08-01

    Free amino acids and urea were analyzed in 78 human milk samples obtained during the first 5 wk of lactation from 10 mothers delivering at term. Significant differences (p less than 0.05) in the concentrations between colostral and mature milk were found for glutamic acid, glutamine, alanine, glycine, cystine, and phosphoethanolamine which increased, and with serine, phosphoserine, aspartic acid + asparagine, arginine, lysine, isoleucine, phenylalanine, proline, methionine, tryptophan, and beta-alanine which decreased. Some of these changes occurred within the first 5 days of lactation, so that differences between transitional and mature milk became negligible (glutamic acid, alanine, and serine, aspartic acid + asparagine, lysine, isoleucine, methionine, tryptophan, respectively). No significant differences between any of the three stages of lactation were found regarding the concentrations of total free amino acids, urea, taurine, threonine, valine, leucine, histidine, and tyrosine. Possible relevances for free amino acids, including nonprotein ones, in human milk are discussed. PMID:6147084

  8. Modulation of glycine sites enhances social memory in rats using PQQ combined with d-serine.

    PubMed

    Zhou, Xingqin; Liu, Dong; Zhang, Rongjun; Peng, Ying; Qin, Xiaofeng; Mao, Shishi

    2016-07-15

    The aim of study was to investigate the effects of pyrroloquinoline quinone (PQQ) combined with d-serine on the modulation of glycine sites in the brain of rats using social recognition test. Rats were divided into seven groups (n=10) and given repeated intraperitoneal (ip) injections of saline, MK-801 (0.5mg/kg), clozapine (1mg/kg), haloperidol (0.1mg/kg), d-serine (0.8g/kg), PQQ (2.0μg/kg), or d-serine (0.4g/kg) combined with PQQ (1.0μg/kg) for seven days. A social recognition test, including assessment of time-dependent memory impairment, was performed. A non-competitive NMDA receptor antagonist, MK-801, significantly impaired social memory, and this impairment was significantly repaired with an atypical antipsychotic (clozapine) but not with a typical antipsychotic (haloperidol). Likewise, d-serine combined with PQQ significantly improved MK-801-disrupted cognition in naïve rats, whereas haloperidol was ineffective. The present results show that the co-agonist NMDA receptor treated with PQQ and d-serine enhances social memory and may be an effective approach for treating the cognitive dysfunction observed in schizophrenic patients. PQQ stimulates glycine modulatory sites by which it may antagonize indirectly by removing glycine from the synaptic cleft or by binding the unsaturated site with d-serine in the brain, providing the insights into future research of central nervous system and drug discovery. PMID:27109337

  9. In Vitro Antibacterial and Antibiofilm Activities of Chlorogenic Acid against Clinical Isolates of Stenotrophomonas maltophilia including the Trimethoprim/Sulfamethoxazole Resistant Strain

    PubMed Central

    Karunanidhi, Arunkumar; Thomas, Renjan; van Belkum, Alex; Neela, Vasanthakumari

    2013-01-01

    The in vitro antibacterial and antibiofilm activity of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia was investigated through disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill and biofilm assays. A total of 9 clinical S. maltophilia isolates including one isolate resistant to trimethoprim/sulfamethoxazole (TMP/SMX) were tested. The inhibition zone sizes for the isolates ranged from 17 to 29 mm, while the MIC and MBC values ranged from 8 to 16 μg mL−1 and 16 to 32 μg mL−1. Chlorogenic acid appeared to be strongly bactericidal at 4x MIC, with a 2-log reduction in viable bacteria at 10 h. In vitro antibiofilm testing showed a 4-fold reduction in biofilm viability at 4x MIC compared to 1x MIC values (0.085 < 0.397 A 490 nm) of chlorogenic acid. The data from this study support the notion that the chlorogenic acid has promising in vitro antibacterial and antibiofilm activities against S. maltophilia. PMID:23509719

  10. A novel Na(+) -Independent alanine-serine-cysteine transporter 1 inhibitor inhibits both influx and efflux of D-Serine.

    PubMed

    Sakimura, Katsuya; Nakao, Kenji; Yoshikawa, Masato; Suzuki, Motohisa; Kimura, Haruhide

    2016-10-01

    NMDA receptor dysfunctions are hypothesized to underlie the pathophysiology of schizophrenia, and treatment with D-serine (D-Ser), an NMDA receptor coagonist, may improve the clinical symptoms of schizophrenia. Thus, upregulating the synaptic D-Ser level is a novel strategy for schizophrenia treatment. Na(+) -independent alanine-serine-cysteine transporter 1 (asc-1) is a transporter responsible for regulating the extracellular D-Ser levels in the brain. In this study, we discovered a novel asc-1 inhibitor, (+)-amino(1-(3,5-dichlorophenyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (ACPP), and assessed its pharmacological profile. ACPP inhibited the D-[(3) H]Ser uptake in human asc-1-expressing CHO cells and rat primary neurons with IC50 values of 0.72 ± 0.13 and 0.89 ± 0.30 μM, respectively. In accordance with the lower asc-1 expression levels in astrocytes, ACPP did not inhibit D-Ser uptake in rat primary astrocytes. In a microdialysis study, ACPP dose dependently decreased the extracellular D-Ser levels in the rat hippocampus under the same conditions in which the asc-1 inhibitor S-methyl-L-cysteine (SMLC) increased it. To obtain insights into this difference, we conducted a D-[(3) H]Ser efflux assay using asc-1-expressing CHO cells. ACPP inhibited D-[(3) H]Ser efflux, whereas SMLC increased it. These results suggest that ACPP is a novel inhibitor of asc-1. © 2016 Wiley Periodicals, Inc. PMID:27302861

  11. Nuclear Compartmentalization of Serine Racemase Regulates D-Serine Production: IMPLICATIONS FOR N-METHYL-D-ASPARTATE (NMDA) RECEPTOR ACTIVATION.

    PubMed

    Kolodney, Goren; Dumin, Elena; Safory, Hazem; Rosenberg, Dina; Mori, Hisashi; Radzishevsky, Inna; Radzishevisky, Inna; Wolosker, Herman

    2015-12-25

    D-Serine is a physiological co-agonist that activates N-methyl D-aspartate receptors (NMDARs) and is essential for neurotransmission, synaptic plasticity, and behavior. D-Serine may also trigger NMDAR-mediated neurotoxicity, and its dysregulation may play a role in neurodegeneration. D-Serine is synthesized by the enzyme serine racemase (SR), which directly converts L-serine to D-serine. However, many aspects concerning the regulation of D-serine production under physiological and pathological conditions remain to be elucidated. Here, we investigate possible mechanisms regulating the synthesis of D-serine by SR in paradigms relevant to neurotoxicity. We report that SR undergoes nucleocytoplasmic shuttling and that this process is dysregulated by several insults leading to neuronal death, typically by apoptotic stimuli. Cell death induction promotes nuclear accumulation of SR, in parallel with the nuclear translocation of GAPDH and Siah proteins at an early stage of the cell death process. Mutations in putative SR nuclear export signals (NESs) elicit SR nuclear accumulation and its depletion from the cytosol. Following apoptotic insult, SR associates with nuclear GAPDH along with other nuclear components, and this is accompanied by complete inactivation of the enzyme. As a result, extracellular D-serine concentration is reduced, even though extracellular glutamate concentration increases severalfold. Our observations imply that nuclear translocation of SR provides a fail-safe mechanism to prevent or limit secondary NMDAR-mediated toxicity in nearby synapses. PMID:26553873

  12. Targeted mutation of Δ12 and Δ15 desaturase genes in hemp produce major alterations in seed fatty acid composition including a high oleic hemp oil.

    PubMed

    Bielecka, Monika; Kaminski, Filip; Adams, Ian; Poulson, Helen; Sloan, Raymond; Li, Yi; Larson, Tony R; Winzer, Thilo; Graham, Ian A

    2014-06-01

    We used expressed sequence tag library and whole genome sequence mining to identify a suite of putative desaturase genes representing the four main activities required for production of polyunsaturated fatty acids in hemp seed oil. Phylogenetic-based classification and developing seed transcriptome analysis informed selection for further analysis of one of seven Δ12 desaturases and one of three Δ15 desaturases that we designate CSFAD2A and CSFAD3A, respectively. Heterologous expression of corresponding cDNAs in Saccharomyces cerevisiae showed CSFAD2A to have Δx+3 activity, while CSFAD3A activity was exclusively at the Δ15 position. TILLING of an ethyl methane sulphonate mutagenized population identified multiple alleles including non-sense mutations in both genes and fatty acid composition of seed oil confirmed these to be the major Δ12 and Δ15 desaturases in developing hemp seed. Following four backcrosses and sibling crosses to achieve homozygosity, csfad2a-1 was grown in the field and found to produce a 70 molar per cent high oleic acid (18:1(Δ9) ) oil at yields similar to wild type. Cold-pressed high oleic oil produced fewer volatiles and had a sevenfold increase in shelf life compared to wild type. Two low abundance octadecadienoic acids, 18:2(Δ6,9) and 18:2(Δ9,15), were identified in the high oleic oil, and their presence suggests remaining endogenous desaturase activities utilize the increased levels of oleic acid as substrate. Consistent with this, CSFAD3A produces 18:2(Δ9,15) from endogenous 18:1(Δ9) when expressed in S. cerevisiae. This work lays the foundation for the development of additional novel oil varieties in this multipurpose low input crop. PMID:24506492

  13. Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii.

    PubMed

    Yadav, Subhash C; Pande, Monu; Jagannadham, M V

    2006-07-01

    A serine protease, named as "Milin" was purified to homogeneity from the latex of Euphorbia milii, a medicinal plant of Euphorbiaceae family. The molecular mass (SDS-PAGE), optimum pH and temperature of the enzyme were 51kDa, pH 8.0 and 60 degrees C, respectively. Milin retains full proteolytic activity over a wide range of pH (5.5-12) and temperature (up to 65 degrees C) with casein and azoalbumin as substrates. The activity of milin is inhibited by serine proteases inhibitors like PMSF, APMSF and DFP, but not by any other protease inhibitors such as E-64 and PCMB. Like the other serine proteases from the genus Euphorbia, the activity of milin was not inhibited by the proteinaceous inhibitor soyabean trypsin inhibitor (SBTI) even at very high concentrations that is naturally present in plants. The specific extinction coefficient (epsilon(280 nm)(1%)), molar extinction coefficient (a(m)) and isoelectric point of the enzyme were found to be 29, 152,500 M(-1) cm(-1) and pH 7.2, respectively. The enzyme is a glycoprotein with detectable carbohydrate moiety (7-8%) in its constitution, which is essential for the activity. The numbers of tryptophan, tyrosine and cysteine residues in the sequence of milin were estimated chemically and are 23, 14 and 14, respectively. Of the 14-cysteine residues, 12 constituted 6-disulfide linkages while two are free cysteines. The N-terminal sequence (first 12 amino acid residues) was determined and does not match with any sequence of known plant serine proteases. Perturbation studies by temperature, pH and chaotropes of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Thus, this serine protease may have potential applications in food industry. PMID:16839575

  14. An unorthodox sensory adaptation site in the Escherichia coli serine chemoreceptor.

    PubMed

    Han, Xue-Sheng; Parkinson, John S

    2014-02-01

    The serine chemoreceptor of Escherichia coli contains four canonical methylation sites for sensory adaptation that lie near intersubunit helix interfaces of the Tsr homodimer. An unexplored fifth methylation site, E502, lies at an intrasubunit helix interface closest to the HAMP domain that controls input-output signaling in methyl-accepting chemotaxis proteins. We analyzed, with in vivo Förster resonance energy transfer (FRET) kinase assays, the serine thresholds and response cooperativities of Tsr receptors with different mutationally imposed modifications at sites 1 to 4 and/or at site 5. Tsr variants carrying E or Q at residue 502, in combination with unmodifiable D and N replacements at adaptation sites 1 to 4, underwent both methylation and demethylation/deamidation, although detection of the latter modifications required elevated intracellular levels of CheB. These Tsr variants could not mediate a chemotactic response to serine spatial gradients, demonstrating that adaptational modifications at E502 alone are not sufficient for Tsr function. Moreover, E502 is not critical for Tsr function, because only two amino acid replacements at this residue abrogated serine chemotaxis: Tsr-E502P had extreme kinase-off output and Tsr-E502I had extreme kinase-on output. These large threshold shifts are probably due to the unique HAMP-proximal location of methylation site 5. However, a methylation-mimicking glutamine at any Tsr modification site raised the serine response threshold, suggesting that all sites influence signaling by the same general mechanism, presumably through changes in packing stability of the methylation helix bundle. These findings are consistent with control of input-output signaling in Tsr through dynamic interplay of the structural stabilities of the HAMP and methylation bundles. PMID:24272777

  15. Interleukin-4-induced transcriptional activation by stat6 involves multiple serine/threonine kinase pathways and serine phosphorylation of stat6.

    PubMed

    Pesu, M; Takaluoma, K; Aittomäki, S; Lagerstedt, A; Saksela, K; Kovanen, P E; Silvennoinen, O

    2000-01-15

    Stat6 transcription factor is a critical mediator of IL-4-specific gene responses. Tyrosine phosphorylation is required for nuclear localization and DNA binding of Stat6. The authors investigated whether Stat6-dependent transcriptional responses are regulated through IL-4-induced serine/threonine phosphorylation. In Ramos B cells, the serine/threonine kinase inhibitor H7 inhibited IL-4-induced expression of CD23. Treatment with H7 did not affect IL-4R-mediated immediate signaling events such as tyrosine phosphorylation of Jak1, Jak3, insulin receptor substrate (IRS)-1 and IRS-2, or tyrosine phosphorylation and DNA binding of Stat6. To analyze whether the H7-sensitive pathway was regulating Stat6-activated transcription, we used reporter constructs containing different IL-4 responsive elements. H7 abrogated Stat6-, as well as Stat5-, mediated reporter gene activation and partially reduced C/EBP-dependent reporter activity. By contrast, IL-4-induced transcription was not affected by wortmannin, an inhibitor of the phosphatidyl-inositol 3'-kinase pathway. Phospho-amino acid analysis and tryptic phosphopeptide maps revealed that IL-4 induced phosphorylation of Stat6 on serine and tyrosine residues in Ramos cells and in 32D cells lacking endogenous IRS proteins. However, H7 treatment did not inhibit the phosphorylation of Stat6. Instead, H7 inhibited the IL-4-induced phosphorylation of RNA polymerase II. These results indicate that Stat6-induced transcription is dependent on phosphorylation events mediated by H7-sensitive kinase(s) but that it also involves serine phosphorylation of Stat6 by an H7-insensitive kinase independent of the IRS pathway. (Blood. 2000;95:494-502) PMID:10627454

  16. Induction of a heparin-stimulated serine proteinase in sex accessory gland tumors of the Lobund-Wistar rat.

    PubMed

    Wilson, Michael J; Lind, Jeremy; Sinha, Akhouri A

    2015-08-01

    Induction of new proteinase activities that may process growth factors, modify cell surface receptors, cleave extracellular matrix proteins, etc. is considered fundamental in carcinogenesis. The purpose of this study was to characterize a novel proteinase activity induced in sex accessory gland cancers (about 70% in seminal vesicles) of adult male Lobund-Wistar rats by a single injection of N-nitroso-N-methylurea (NMU; 25mg/kg) followed by implanted testosterone propionate (45mg in silastic tubing every 2months) treatment for 10-14months. A 28kDa proteinase activity was detected in tumor extracts using SDS-gelatin gel zymography with incubations done without CaCl2. Its activity was stimulated 15 fold by heparin (optimal activity 1.5-3.0μg/lane) added to the tissue extract-SDS sample buffer prior to electrophoresis. No 28kDa heparin-stimulated proteinase (H-SP) was found in the dorsal, lateral and anterior (coagulating gland) prostate lobes or seminal vesicles of untreated adult rats, but there was a 26-30kDa Ca(2+)-independent proteinase activity in the ventral prostate that showed limited heparin stimulation. The 28kDa H-SP was completely inhibited by 1.0mM 4-(2-aminoethyl)benzenesulfonylfluoride (AESBF) indicating that it was a serine-type proteinase. Other types of proteinase inhibitors were without effect, including serine proteinase inhibitors benzamidine, tranexamic acid and ε-aminocaproic acid. Proteinase activities of about 28kDa were found with casein, fibrinogen or carboxymethylated transferrin as substrate, however, these activities were not stimulated by heparin. Similar levels of activities of the 28kDa H-SP were found in primary tumors and their metastases, but little/no activity was detected in serum, even from rats with large tumor volume and metastases. These data demonstrate overexpression of a heparin-stimulated 28kDa serine proteinase in the primary tumors of sex accessory gland cancers and their metastases. This proteinase either does not

  17. Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus: cDNA cloning and primary structure of the enzymes

    PubMed Central

    Rudenskaya, Galina N; Kislitsin, Yuri A; Rebrikov, Denis V

    2004-01-01

    Background In this paper, we describe cDNA cloning of a new anionic trypsin and a collagenolytic serine protease from king crab Paralithodes camtschaticus and the elucidation of their primary structures. Constructing the phylogenetic tree of these enzymes was undertaken in order to prove the evolutionary relationship between them. Results The mature trypsin PC and collagenolytic protease PC contain 237 (Mcalc 24.8 kDa) and 226 amino acid residues (Mcalc 23.5 kDa), respectively. Alignments of their amino acid sequences revealed a high degree of the trypsin PC identity to the trypsin from Penaeus vannamei (approximately 70%) and of the collagenolytic protease PC identity to the collagenase from fiddler crab Uca pugilator (76%). The phylogenetic tree of these enzymes was constructed. Conclusions Primary structures of the two mature enzymes from P. camtschaticus were obtained and compared with those of other proteolytic proteins, including some enzymes from brachyurans. A phylogenetic analysis was also carried out. These comparisons revealed that brachyurins are closely related to their vertebrate and bacterial congeners, occupy an intermediate position between them, and their study significantly contributes to the understanding of the evolution and function of serine proteases. PMID:14731305

  18. Highly potent fibrinolytic serine protease from Streptomyces.

    PubMed

    Uesugi, Yoshiko; Usuki, Hirokazu; Iwabuchi, Masaki; Hatanaka, Tadashi

    2011-01-01

    We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis. PMID:22112764

  19. l-Ala-γ-d-Glu-meso-diaminopimelic Acid (DAP) Interacts Directly with Leucine-rich Region Domain of Nucleotide-binding Oligomerization Domain 1, Increasing Phosphorylation Activity of Receptor-interacting Serine/Threonine-protein Kinase 2 and Its Interaction with Nucleotide-binding Oligomerization Domain 1*

    PubMed Central

    Laroui, Hamed; Yan, Yutao; Narui, Yoshie; Ingersoll, Sarah A.; Ayyadurai, Saravanan; Charania, Moiz A.; Zhou, Feimeng; Wang, Binghe; Salaita, Khalid; Sitaraman, Shanthi V.; Merlin, Didier

    2011-01-01

    The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-d-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a Kd value of 34.5 μm. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a Kd value of 4.13 μm. However, NOD1/RICK binding was of higher affinity (Kd of 3.26 μm) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity. PMID:21757725

  20. Broad Spectrum Activity of a Lectin-Like Bacterial Serine Protease Family on Human Leukocytes

    PubMed Central

    Ayala-Lujan, Jorge Luis; Vijayakumar, Vidhya; Gong, Mei; Smith, Rachel; Santiago, Araceli E.; Ruiz-Perez, Fernando

    2014-01-01

    The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system. PMID:25251283

  1. Broad spectrum activity of a lectin-like bacterial serine protease family on human leukocytes.

    PubMed

    Ayala-Lujan, Jorge Luis; Vijayakumar, Vidhya; Gong, Mei; Smith, Rachel; Santiago, Araceli E; Ruiz-Perez, Fernando

    2014-01-01

    The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system. PMID:25251283

  2. Asymmetric Synthesis, Structure, and Reactivity of Unexpectedly Stable Spiroepoxy-β-Lactones Including Facile Conversion to Tetronic Acids: Application to (+)-Maculalactone A

    PubMed Central

    Duffy, Richard J.; Morris, Kay A.; Vallakati, Ravikrishna; Zhang, Wei; Romo, Daniel

    2009-01-01

    A novel class of small spirocyclic heterocycles, spiroepoxy-β-lactones (1,4-dioxaspiro[2.3]-hexan-5-ones), is described that exhibit a number of interesting reactivity patterns. These spiroheterocycles, including an optically active series, are readily synthesized by epoxidation of ketene dimers (4-alkylidene-2-oxetanones) available from homo- or heteroketene dimerization. An analysis of bond lengths in these systems by X-ray crystallography and comparison to data for known spirocycles and those determined computationally, suggest that anomeric effects in these systems may be more pronounced due to their rigidity and may contribute to their surprising stability. The synthetic utility of spiroepoxy-β-lactones was explored and one facile rearrangement identified under several conditions provides a 3-step route from acid chlorides to optically active tetronic acids, ubiquitous heterocycles in bioactive natural products. The addition of various nucleophiles to these spirocycles leads primarily to addition at C5 and C2. The utility of an optically active spiroepoxy-β-lactone was demonstrated in the concise, enantioselective synthesis of the anti-fouling agent, (+)-maculalactone A, which proceeds in 5 steps from hydrocinnamoyl chloride by way of a tetronic acid intermediate. PMID:19453152

  3. Asymmetric synthesis, structure, and reactivity of unexpectedly stable spiroepoxy-beta-lactones including facile conversion to tetronic acids: application to (+)-maculalactone A.

    PubMed

    Duffy, Richard J; Morris, Kay A; Vallakati, Ravikrishna; Zhang, Wei; Romo, Daniel

    2009-07-01

    A novel class of small spirocyclic heterocycles, spiroepoxy-beta-lactones (1,4-dioxaspiro[2.3]-hexan-5-ones), is described that exhibit a number of interesting reactivity patterns. These spiroheterocycles, including an optically active series, are readily synthesized by epoxidation of ketene dimers (4-alkylidene-2-oxetanones) available from homo- or heteroketene dimerization. An analysis of bond lengths in these systems by X-ray crystallography and comparison to data for known spirocycles and those determined computationally suggest that anomeric effects in these systems may be more pronounced due to their rigidity and may contribute to their surprising stability. The synthetic utility of spiroepoxy-beta-lactones was explored, and one facile rearrangement identified under several conditions provides a three-step route from acid chlorides to optically active tetronic acids, ubiquitous heterocycles in bioactive natural products. The addition of various nucleophiles to these spirocycles leads primarily to addition at C5 and C2. The utility of an optically active spiroepoxy-beta-lactone was demonstrated in the concise, enantioselective synthesis of the antifouling agent, (+)-maculalactone A, which proceeds in five steps from hydrocinnamoyl chloride by way of a tetronic acid intermediate. PMID:19453152

  4. Serine protease EpiP from Staphylococcus epidermidis catalyzes the processing of the epidermin precursor peptide.

    PubMed Central

    Geissler, S; Götz, F; Kupke, T

    1996-01-01

    The function of serine protease EpiP in epidermin biosynthesis was investigated. Epidermin is synthesized as a 52-amino-acid precursor peptide, EpiA, which is posttranslationally modified and processed to the mature 22-amino-acid peptide antibiotic. epiP was expressed in Staphylococcus carnosus with xylose-regulated expression vector pCX15. The cleavage of the unmodified EpiA precursor peptide to leader peptide and proepidermin by EpiP-containing culture filtrates of S. carnosus (pCX15epiP) was followed by reversed-phase chromatography and subsequent electrospray mass spectrometry. PMID:8550430

  5. Design of a Selective Substrate and Activity Based Probe for Human Neutrophil Serine Protease 4.

    PubMed

    Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J; Lin, S Jack; Kirchhofer, Daniel; Salvesen, Guy S; Drag, Marcin

    2015-01-01

    Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs. PMID:26172376

  6. RAF protein-serine/threonine kinases: Structure and regulation

    SciTech Connect

    Roskoski, Robert

    2010-08-27

    Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.

  7. Selective synthesis of 2',3'-cyclic nucleotide 3'-phosphodiesterase isoform 2 and identification of specifically phosphorylated serine residues.

    PubMed

    O'Neill, R C; Braun, P E

    2000-02-01

    2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is a protein found abundantly in the cytoplasmic compartments of CNS myelin. Two isoforms of this protein, CNP1 and CNP2, are detectable. They differ by a 20-amino acid extension exclusive to CNP2. Additionally, CNP2 is essentially the only isoform to be phosphorylated in vivo. In this study, we examine the phosphorylation of CNP2 in transfected cells. CNP2 was selectively expressed ectopically in 293T cells and labeled with 32P. Immunoprecipitation of labeled CNP2 and tryptic phosphopeptide mapping analyses identified serines 9 and 22 as the major sites of phosphorylation. Only serine 22 was phosphorylated initially in oligodendrocyte-enriched cultures of neonatal rat brain glial cells. However, 4beta-phorbol 12,13-dibutyrate (PDB) induced the phosphorylation of serine 9, thereby producing the same pattern seen in 293T cells. These results suggest that serine 9 is phosphorylated by a PDB-sensitive kinase, likely protein kinase C, and that serine 22 appears to be constitutively phosphorylated. PMID:10646504

  8. Survey analysis and chemical characterization of solid inhomogeneous samples using a general homogenization procedure including acid digestion, drying, grinding and briquetting together with X-ray fluorescence.

    PubMed

    Sahlin, Eskil; Magnusson, Bertil

    2012-08-15

    A survey analysis and chemical characterization methodology for inhomogeneous solid waste samples of relatively large samples (typically up to 100g) using X-ray fluorescence following a general homogenization procedure is presented. By using a combination of acid digestion and grinding various materials can be homogenized e.g. pure metals, alloys, salts, ores, plastics, organics. In the homogenization step, solid material is fully or partly digested in a mixture of nitric acid and hydrochloric acid in an open vessel. The resulting mixture is then dried, grinded, and finally pressed to a wax briquette. The briquette is analyzed using wave-length dispersive X-ray fluorescence with fundamental parameters evaluation. The recovery of 55 elements were tested by preparing samples with known compositions using different alloys, pure metals or elements, oxides, salts and solutions of dissolved compounds. It was found that the methodology was applicable to 49 elements including Na, Mg, Al, Si, P, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag, Cd, In, Sn, Sb, Te, Cs, Ba, La, Ce, Ta, W, Re, Ir, Pt, Au, Tl, Pb, Bi, and Th, that all had recoveries >0.8. 6 elements were lost by volatilization, including Br, I, Os, and Hg that were completely lost, and S and Ge that were partly lost. Since all lanthanides are chemically similar to La and Ce, all actinides are chemically similar to Th, and Hf is chemically similar to Zr, it is likely that the method is applicable to 77 elements. By using an internal standard such as strontium, added as strontium nitrate, samples containing relatively high concentrations of elements not measured by XRF (hydrogen to fluorine), e.g. samples containing plastics, can be analyzed. PMID:22841048

  9. Design of activated serine-containing catalytic triads with atomic level accuracy

    PubMed Central

    Rajagopalan, Sridharan; Wang, Chu; Yu, Kai; Kuzin, Alexandre P.; Richter, Florian; Lew, Scott; Miklos, Aleksandr E.; Matthews, Megan L.; Seetharaman, Jayaraman; Su, Min; Hunt, John. F.; Cravatt, Benjamin F.; Baker, David

    2014-01-01

    A challenge in the computational design of enzymes is that multiple properties must be simultaneously optimized -- substrate-binding, transition state stabilization, and product release -- and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads, and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate-reactivity. Following optimization by yeast-display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest the designs could provide the basis for a new class of organophosphate captures agents. PMID:24705591

  10. Evidence for possible involvement of an elastolytic serine protease in aspergillosis.

    PubMed Central

    Kolattukudy, P E; Lee, J D; Rogers, L M; Zimmerman, P; Ceselski, S; Fox, B; Stein, B; Copelan, E A

    1993-01-01

    A number of isolates of Aspergillus fumigatus obtained from the hospital environment produced extracellular elastolytic activity. This activity was found to be catalyzed by a single 33-kDa protein which was purified and characterized to be a serine protease. A. fumigatus, when grown on the insoluble structural material obtained from murine and bovine lung, produced the same extracellular 33-kDa elastolytic protease, indicating that this enzyme is likely to be produced when the organism infects the lung. Polymerase chain reaction with an oligonucleotide primer based on the N-terminal amino acid sequence of the elastolytic enzyme yielded a cDNA which was cloned and sequenced. The active serine motif showed more similarity to subtilisin than to mammalian elastase. The amino acid sequence showed 80% identity to the alkaline protease from Aspergillus oryzae. Screening of hospital isolates of Aspergillus flavus showed great variation in the production of elastolytic activity and a much lower level of activity than that produced by A. fumigatus. The elastolytic protease from A. flavus was shown to be a serine protease susceptible to modification and inactivation by active serine and histidine-directed reagents. This protease cross-reacted with the antibodies prepared against the elastolytic protease from A. fumigatus. Immunogold localization of the elastolytic enzyme showed that A. fumigatus germinating and penetrating into the lungs of neutropenic mice secreted the elastolytic protease. An elastase-deficient mutant generated from a highly virulent isolate of A. fumigatus caused drastically reduced mortality when nasally introduced into the lung of neutropenic mice. All of the evidence suggests that extracellular elastolytic protease is a significant virulence factor in invasive aspergillosis. Images PMID:8500876

  11. Utilization of glycine and serine as nitrogen sources in the roots of Zea mays and Chamaegigas intrepidus.

    PubMed

    Hartung, W; Ratcliffe, R G

    2002-12-01

    Glycine and serine are potential sources of nitrogen for the aquatic resurrection plant Chamaegigas intrepidus Dinter in the rock pools that provide its natural habitat. The pathways by which these amino acids might be utilized were investigated by incubating C. intrepidus roots and maize (Zea mays) root tips with [(15)N]glycine, [(15)N]serine and [2-(13)C]glycine. The metabolic fate of the label was followed using in vivo NMR spectroscopy, and the results were consistent with the involvement of the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) in the utilization of glycine. In contrast, the labelling patterns provided no evidence for the involvement of serine:glyoxylate aminotransferase in the metabolism of glycine by the root tissues. The key observations were: (i) the release of [(15)N]ammonium during [(15)N]-labelling experiments; and (ii) the detection of a characteristic set of serine isotopomers in the [2-(13)C]glycine experiments. The effects of aminoacetonitrile, amino-oxyacetate, and isonicotinic acid hydrazide, all of which inhibit GDC and SHMT to some extent, and of methionine sulphoximine, which inhibited the reassimilation of the ammonium, supported the conclusion that GDC and SHMT were essential for the metabolism of glycine. C. intrepidus was observed to metabolize serine more readily than the maize root tips and this may be an adaptation to its nitrogen-deficient habitat. Overall, the results support the emerging view that GDC is an essential component of glycine catabolism in non-photosynthetic tissues. PMID:12432023

  12. Multi-Biomarkers for Early Detection of Type 2 Diabetes, Including 10- and 12-(Z,E)-Hydroxyoctadecadienoic Acids, Insulin, Leptin, and Adiponectin

    PubMed Central

    Umeno, Aya; Yoshino, Kohzoh; Hashimoto, Yoshiko; Shichiri, Mototada; Kataoka, Masatoshi; Yoshida, Yasukazu

    2015-01-01

    We have previously found that fasting plasma levels of totally assessed 10- and 12-(Z,E)-hydroxyoctadecadienoic acid (HODE) correlated well with levels of glycated hemoglobin (HbA1c) and glucose during oral glucose tolerance tests (OGTT); these levels were determined via liquid chromatography—mass spectrometry after reduction and saponification. However, 10- and 12-(Z,E)-HODE alone cannot perfectly detect early impaired glucose tolerance (IGT) and/or insulin resistance, which ultimately lead to diabetes. In this study, we randomly recruited healthy volunteers (n = 57) who had no known history of any diseases, and who were evaluated using the OGTT, the HODE biomarkers, and several additional proposed biomarkers, including retinol binding protein 4 (RBP4), adiponectin, leptin, insulin, glycoalbumin, and high sensitivity-C-reactive protein. The OGTT revealed that our volunteers included normal individuals (n = 44; Group N), “high-normal” individuals (fasting plasma glucose 100–109 mg/dL) with IGT (n = 11; Group HN+IGT), and diabetic individuals (n = 2; Group D). We then used these groups to evaluate the potential biomarkers for the early detection of type 2 diabetes. Plasma levels of RBP4 and glycoalbumin were higher in Group HN+IGT, compared to those in Group N, and fasting levels of 10- and 12-(Z,E)-HODE/linoleic acids were significantly correlated with levels of RBP4 (p = 0.003, r = 0.380) and glycoalbumin (p = 0.006, r = 0.316). Furthermore, we developed a stepwise multiple linear regression models to predict the individuals’ insulin resistance index (the Matsuda Index 3). Fasting plasma levels of 10- and 12-(Z,E)-HODE/linoleic acids, glucose, insulin, and leptin/adiponectin were selected as the explanatory variables for the models. The risks of type 2 diabetes, early IGT, and insulin resistance were perfectly predicted by comparing fasting glucose levels to the estimated Matsuda Index 3 (fasting levels of 10- and 12-(Z,E)-HODE/linoleic acids, insulin

  13. Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta.

    PubMed

    Cao, Xiaolong; He, Yan; Hu, Yingxia; Zhang, Xiufeng; Wang, Yang; Zou, Zhen; Chen, Yunru; Blissard, Gary W; Kanost, Michael R; Jiang, Haobo

    2015-07-01

    Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., in preparation), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sushi, Wonton, TSP, CUB, Frizzle, and SR domains. There are nineteen groups of genes evolved from relatively recent gene duplication and sequence divergence. Thirty-five SPs and seven SPHs contain 1, 2 or 5 clip domains. Multiple sequence alignment and molecular modeling of the 54 clip domains have revealed structural diversity of these regulatory modules. Sequence comparison with their homologs in Drosophila melanogaster, Anopheles gambiae and Tribolium castaneum allows us to classify them into five subfamilies: A are SPHs with 1 or 5 group-3 clip domains, B are SPs with 1 or 2 group-2 clip domains, C, D1 and D2 are SPs with a single clip domain in group-1a, 1b and 1c, respectively. We have classified into six categories the 125 expression profiles of SP-related proteins in fat body, brain, midgut, Malpighian tubule, testis, and ovary at different stages, suggesting that they participate in various physiological processes. Through RNA-Seq-based gene annotation and expression profiling, as well as intragenomic sequence comparisons, we have established a framework of information for future biochemical research of nondigestive SPs and SPHs in this model species. PMID:25530503

  14. Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain.

    PubMed

    Stojanoski, Vlatko; Adamski, Carolyn J; Hu, Liya; Mehta, Shrenik C; Sankaran, Banumathi; Zwart, Peter; Prasad, B V Venkataram; Palzkill, Timothy

    2016-05-01

    Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. In this study, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine with glycine on protein stability in serine β-lactamases. Six different enzymes comprising representatives from each of the three classes of serine β-lactamases were examined, including TEM-1, CTX-M-14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutant were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to that of the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48, and OXA-163 did not alter stability, suggesting removal of the Cβ atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. Additionally, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intrahelical hydrogen bond network compared to those of the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. These findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the

  15. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    SciTech Connect

    Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2011-09-01

    Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: > Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. > Bt-VSP activates prothrombin. > Bt-VSP directly degrades fibrinogen into fibrin degradation products. > Bt-VSP is a hemostatically active protein that is a potent clinical agent.

  16. Enteric Bacterial Metabolites Propionic and Butyric Acid Modulate Gene Expression, Including CREB-Dependent Catecholaminergic Neurotransmission, in PC12 Cells - Possible Relevance to Autism Spectrum Disorders

    PubMed Central

    Nankova, Bistra B.; Agarwal, Raj; MacFabe, Derrick F.; La Gamma, Edmund F.

    2014-01-01

    Alterations in gut microbiome composition have an emerging role in health and disease including brain function and behavior. Short chain fatty acids (SCFA) like propionic (PPA), and butyric acid (BA), which are present in diet and are fermentation products of many gastrointestinal bacteria, are showing increasing importance in host health, but also may be environmental contributors in neurodevelopmental disorders including autism spectrum disorders (ASD). Further to this we have shown SCFA administration to rodents over a variety of routes (intracerebroventricular, subcutaneous, intraperitoneal) or developmental time periods can elicit behavioral, electrophysiological, neuropathological and biochemical effects consistent with findings in ASD patients. SCFA are capable of altering host gene expression, partly due to their histone deacetylase inhibitor activity. We have previously shown BA can regulate tyrosine hydroxylase (TH) mRNA levels in a PC12 cell model. Since monoamine concentration is known to be elevated in the brain and blood of ASD patients and in many ASD animal models, we hypothesized that SCFA may directly influence brain monoaminergic pathways. When PC12 cells were transiently transfected with plasmids having a luciferase reporter gene under the control of the TH promoter, PPA was found to induce reporter gene activity over a wide concentration range. CREB transcription factor(s) was necessary for the transcriptional activation of TH gene by PPA. At lower concentrations PPA also caused accumulation of TH mRNA and protein, indicative of increased cell capacity to produce catecholamines. PPA and BA induced broad alterations in gene expression including neurotransmitter systems, neuronal cell adhesion molecules, inflammation, oxidative stress, lipid metabolism and mitochondrial function, all of which have been implicated in ASD. In conclusion, our data are consistent with a molecular mechanism through which gut related environmental signals such as

  17. Synthetic sulfoglycolipids targeting the serine-threonine protein kinase Akt.

    PubMed

    Costa, Barbara; Dangate, Milind; Vetro, Maria; Donvito, Giulia; Gabrielli, Luca; Amigoni, Loredana; Cassinelli, Giuliana; Lanzi, Cinzia; Ceriani, Michela; De Gioia, Luca; Filippi, Giulia; Cipolla, Laura; Zaffaroni, Nadia; Perego, Paola; Colombo, Diego

    2016-08-15

    The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a β-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-β-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors. PMID:27316541

  18. New serine-derived gemini surfactants as gene delivery systems.

    PubMed

    Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; de Lima, Maria C Pedroso; Jurado, Amália S

    2015-01-01

    Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties characteristic of the gemini surfactants with high biocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amide and ester), were evaluated for their ability to mediate gene delivery either per se or in combination with helper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic diameter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, able to transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA charge ratios (1/1-2/1). The most efficient complexes presented sizes suitable for intravenous administration and negative surface charge, a feature known to preclude potentially adverse interactions with serum components. This work brings forward a new family of gemini surfactants with great potential as gene delivery systems. PMID:25513958

  19. Regulation of Sulfate Assimilation by Light and O-Acetyl-l-Serine in Lemna minor L. 1

    PubMed Central

    Neuenschwander, Urs; Suter, Marianne; Brunold, Christian

    1991-01-01

    The effect of 0.5 millimolar O-acetyl-l-serine added to the nutrient solution on sulfate assimilation of Lemna minor L., cultivated in the light or in the dark, or transferred from light to the dark, was examined. During 24 hours after transfer from light to the dark the extractable activity of adenosine 5′-phosphosulfate sulfotransferase, a key enzyme of sulfate assimilation, decreased to 10% of the light control. Nitrate reductase (EC 1.7.7.1.) activity, measured for comparison, decreased to 40%. Adenosine 5′-triphosphate (ATP) sulfurylase (EC 2.7.7.4.) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8.) activities were not affected by the transfer. When O-acetyl-l-serine was added to the nutrient solution at the time of transfer to the dark, adenosine 5′-phosphosulfate sulfotransferase activity was still at 50% of the light control after 24 hours, ATP sulfurylase and O-acetyl-l-serine sulfhydrylase activity were again not affected, and nitrate reductase activity decreased as before. Addition of O-acetyl-l-serine at the time of the transfer caused a 100% increase in acid-soluble SH compounds after 24 hours in the dark. In continuous light the corresponding increase was 200%. During 24 hours after transfer to the dark the assimilation of 35SO42− into organic compounds decreased by 80% without O-acetyl-l-serine but was comparable to light controls in its presence. The addition of O-acetyl-l-serine to Lemna minor precultivated in the dark for 24 hours induced an increase in adenosine 5′-phosphosulfate sulfotransferase activity so that a constant level of 50% of the light control was reached after an additional 9 hours. Cycloheximide as well as 6-methyl-purine inhibited this effect. In the same type of experiment O-acetyl-l-serine induced a 100-fold increase in the incorporation of label from 35SO42− into cysteine after additional 24 hours in the dark. Taken together, these results show that exogenous O-acetyl-l-serine has a regulatory effect on

  20. Functional and Structural Characterization of Vibrio cholerae Extracellular Serine Protease B, VesB*

    PubMed Central

    Gadwal, Shilpa; Korotkov, Konstantin V.; Delarosa, Jaclyn R.; Hol, Wim G. J.; Sandkvist, Maria

    2014-01-01

    The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser. Using synthetic peptides, VesB efficiently cleaved a trypsin substrate, but not chymotrypsin and elastase substrates. The reversible serine protease inhibitor, benzamidine, inhibited VesB and served as an immobilized ligand for VesB affinity purification, further indicating its relationship with trypsin-like enzymes. Consistent with this family of serine proteases, N-terminal sequencing implied that the propeptide is removed in the secreted form of VesB. Separate mutagenesis of the activation site and catalytic serine rendered VesB inactive, confirming the importance of these features for activity, but not for secretion. Similar to trypsin but, in contrast to thrombin and other coagulation factors, Na+ did not stimulate the activity of VesB, despite containing the Tyr250 signature. The crystal structure of catalytically inactive pro-VesB revealed that the protease domain is structurally similar to trypsinogen. The C-terminal domain of VesB was found to adopt an immunoglobulin (Ig)-fold that is structurally homologous to Ig-folds of other extracellular Vibrio proteins. Possible roles of the Ig-fold domain in stability, substrate specificity, cell surface association, and type II secretion of VesB, the first bacterial multidomain trypsin-like protease with known structure, are discussed. PMID:24459146

  1. Serine carboxypeptidase-like acyltransferases from plants.

    PubMed

    Mugford, Sam T; Milkowski, Carsten

    2012-01-01

    Serine carboxypeptidase-like (SCPL) acyltransferases facilitate transacylation reactions using energy-rich 1-O-β-glucose esters in the synthesis of an array of bioactive compounds and are associated with the diversification of plant natural products. SCPL acyltransferases have evolved from a hydrolytic ancestor by adapting functional elements of the proteases such as the catalytic triad, oxyanion hole, and substrate recognition H-bond network to their new function. As vacuolar proteins, SCPL acyltransferases define an alternative cellular route of transacylation spatially separated from the cytoplasmic enzymes of the BAHD acyltransferase family named according to the first characterized members (BEAT, AHCT, HCBT, and DAT). Recent efforts in cloning and characterization led to the identification of diagnostic peptides for SCPL acyltransferases, enabling the detection of candidate genes in several plant genomes. Detailed biochemical analysis of SCPL acyltransferases is strongly dependent on comprehensive heterologous expression systems, efficient protein purification protocols, and the supply of appropriate substrates. This chapter describes some useful techniques and strategies for identification and characterization of SCPL acyltransferases. PMID:23034234

  2. Serine 220 phosphorylation of the Merkel cell polyomavirus large T antigen crucially supports growth of Merkel cell carcinoma cells.

    PubMed

    Schrama, David; Hesbacher, Sonja; Angermeyer, Sabrina; Schlosser, Andreas; Haferkamp, Sebastian; Aue, Annemarie; Adam, Christian; Weber, Alexandra; Schmidt, Marc; Houben, Roland

    2016-03-01

    Merkel cell polyomavirus (MCPyV) is regarded as a major causal factor for Merkel cell carcinoma (MCC). Indeed, tumor cell growth of MCPyV-positive MCC cells is dependent on the expression of a truncated viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. Here we determined the phosphorylation pattern of a truncated MCPyV-LT characteristically for MCC by mass spectrometry revealing MCPyV-LT as multi-phospho-protein phosphorylated at several serine and threonine residues. Remarkably, disruption of most of these phosphorylation sites did not affect its ability to rescue knockdown of endogenous T antigens in MCC cells indicating that phosphorylation of the respective amino acids is not essential for the growth promoting function of MCPyV-LT. However, alteration of serine 220 to alanine completely abolished the ability of MCPyV-LT to support proliferation of MCC cells. Conversely, mimicking the phosphorylated state by mutation of serine 220 to glutamic acid resulted in a fully functional LT. Moreover, MCPyV-LT(S220A) demonstrated reduced binding to RB in co-immunoprecipitation experiments as well as weaker induction of RB target genes in MCC cells. In conclusion, we provide evidence that phosphorylation of serine 220 is required for efficient RB inactivation in MCC and may therefore be a potential target for future therapeutic approaches. PMID:26383606

  3. An alkaline serine-proteinase from a bacterium isolated from bat feces: purification and characterization.

    PubMed

    Tanskul, Somporn; Hiraga, Kazumi; Takada, Katsumi; Rungratchote, Suchart; Suntinanalert, Prasert; Oda, Kohei

    2009-11-01

    An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 degrees C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H(2)O(2), 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P(1) position, in descending order. The k(cat), K(m) and k(cat)/K(m) values for Z-Val-Lys-Met-MCA were 16.8+/-0.14 min(-1), 5.1+/-0.28 microM, and 3.3+/-0.28 microM(-1) min(-1) respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS. PMID:19897920

  4. Serine/Threonine/Tyrosine Protein Kinase Phosphorylates Oleosin, a Regulator of Lipid Metabolic Functions1[OA

    PubMed Central

    Parthibane, Velayoudame; Iyappan, Ramachandiran; Vijayakumar, Anitha; Venkateshwari, Varadarajan; Rajasekharan, Ram

    2012-01-01

    Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A2 activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A2 activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca2+ inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation. PMID:22434039

  5. An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family1[w

    PubMed Central

    Fraser, Christopher M.; Rider, Lance W.; Chapple, Clint

    2005-01-01

    The Arabidopsis (Arabidopsis thaliana) genome encodes a family of 51 proteins that are homologous to known serine carboxypeptidases. Based on their sequences, these serine carboxypeptidase-like (SCPL) proteins can be divided into several major clades. The first group consists of 21 proteins which, despite the function implied by their annotation, includes two that have been shown to function as acyltransferases in plant secondary metabolism: sinapoylglucose:malate sinapoyltransferase and sinapoylglucose:choline sinapoyltransferase. A second group comprises 25 SCPL proteins whose biochemical functions have not been clearly defined. Genes encoding representatives from both of these clades can be found in many plants, but have not yet been identified in other phyla. In contrast, the remaining SCPL proteins include five members that are similar to serine carboxypeptidases from a variety of organisms, including fungi and animals. Reverse transcription PCR results suggest that some SCPL genes are expressed in a highly tissue-specific fashion, whereas others are transcribed in a wide range of tissue types. Taken together, these data suggest that the Arabidopsis SCPL gene family encodes a diverse group of enzymes whose functions are likely to extend beyond protein degradation and processing to include activities such as the production of secondary metabolites. PMID:15908604

  6. The phosphorylation of serine 492 of perilipin a directs lipid droplet fragmentation and dispersion.

    PubMed

    Marcinkiewicz, Amy; Gauthier, Denise; Garcia, Anne; Brasaemle, Dawn L

    2006-04-28

    Perilipin A is a key regulator of triacylglycerol storage and hydrolysis in adipocytes; phosphorylation of perilipin A by protein kinase A facilitates maximal lipolysis. Chronic stimulation of lipolysis in 3T3-L1 adipocytes causes large perinuclear lipid droplets to fragment into myriad dispersed perilipin A-covered microlipid droplets. In cultured fibroblasts stably expressing ectopic perilipin A, clustered lipid droplets disperse throughout the cytoplasm upon incubation of the cells with forskolin and isobutylmethylxanthine (IBMX) to elevate levels of cAMP and activate protein kinase A, mirroring events observed in adipocytes. Furthermore, diethylum-belliferyl phosphate inhibits stimulated lipolysis but not the dispersion of lipid droplets, suggesting that products of lipolysis are not required for this remodeling process. We hypothesized that protein kinase A-mediated phosphorylation of perilipin A triggers the remodeling of lipid droplets. The mutation of serine 492 of perilipin A to alanine prevented the dispersion of clustered lipid droplets in fibroblasts stably expressing the mutated perilipin upon incubation with forskolin and IBMX. In contrast, the substitution of serines 81, 222, 276, or 433 with alanine, either singly or in combinations, did not affect the protein kinase A-mediated remodeling of lipid droplets. Interestingly, substitution of serines 433, 492, and 517 of perilipin A with glutamic acid residues blocked the dispersion of clustered lipid droplets in cells incubated with forskolin and IBMX, indicating that the addition of a negative charge does not mimic a phosphate group. We conclude that protein kinase A-mediated phosphorylation of serine 492 of perilipin A drives the fragmentation and dispersion of lipid droplets. PMID:16488886

  7. Negative Role of RIG-I Serine 8 Phosphorylation in the Regulatin of Interferon-beta Production

    SciTech Connect

    E Nistal-Villan; M Gack; G Martinez-Delgado; N Maharaj; K Inn; H Yang; R Wang; A Aggarwal; J Jung; A Garcia-Sastre

    2011-12-31

    RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-beta mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-beta production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-beta or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.

  8. Regulation of Serine, Glycine, and One-Carbon Biosynthesis.

    PubMed

    Stauffer, George V

    2004-12-01

    The biosynthesis of serine, glycine, and one-carbon (C1) units constitutes a major metabolic pathway in Escherichia coli and Salmonella enterica serovar Typhimurium. C1 units derived from serine and glycine are used in the synthesis of purines, histidine, thymine, pantothenate, and methionine and in the formylation of the aminoacylated initiator fMet-TRNAfMet used to start translation in E. coli and serovar Typhimurium. The need for serine, glycine, and C1 units in many cellular functions makes it necessary for the genes encoding enzymes for their synthesis to be carefully regulated to meet the changing demands of the cell for these intermediates. This review discusses the regulation of the following genes: serA, serB, and serC; gly gene; gcvTHP operon; lpdA; gcvA and gcvR; and gcvB genes. Threonine utilization (the Tut cycle) constitutes a secondary pathway for serine and glycine biosynthesis. L-Serine inhibits the growth of E. coli cells in GM medium, and isoleucine releases this growth inhibition. The E. coli glycine transport system (Cyc) has been shown to transport glycine, D-alanine, D-serine, and the antibiotic D-cycloserine. Transport systems often play roles in the regulation of gene expression, by transporting effector molecules into the cell, where they are sensed by soluble or membrane-bound regulatory proteins. PMID:26443363

  9. Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden

    PubMed Central

    Martin, Erik W.; Buzza, Marguerite S.; Driesbaugh, Kathryn H.; Liu, Shihui; Fortenberry, Yolanda M.; Leppla, Stephen H.; Antalis, Toni M.

    2015-01-01

    The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent. PMID:26392335

  10. ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

    EPA Science Inventory

    D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

  11. Conversion of human 5-lipoxygenase to a 15-lipoxygenase by a point mutation to mimic phosphorylation at Serine-663

    SciTech Connect

    Gilbert, Nathaniel C.; Rui, Zhe; Neau, David B.; Waight, Maria T.; Bartlett, Sue G.; Boeglin, William E.; Brash, Alan R.; Newcomer, Marcia E.

    2012-08-31

    The enzyme 5-lipoxygenase (5-LOX) initiates biosynthesis of the proinflammatory leukotriene lipid mediators and, together with 15-LOX, is also required for synthesis of the anti-inflammatory lipoxins. The catalytic activity of 5-LOX is regulated through multiple mechanisms, including Ca{sup 2+}-targeted membrane binding and phosphorylation at specific serine residues. To investigate the consequences of phosphorylation at S663, we mutated the residue to the phosphorylation mimic Asp, providing a homogenous preparation suitable for catalytic and structural studies. The S663D enzyme exhibits robust 15-LOX activity, as determined by spectrophotometric and HPLC analyses, with only traces of 5-LOX activity remaining; synthesis of the anti-inflammatory lipoxin A4 from arachidonic acid is also detected. The crystal structure of the S663D mutant in the absence and presence of arachidonic acid (in the context of the previously reported Stable-5-LOX) reveals substantial remodeling of helices that define the active site so that the once fully encapsulated catalytic machinery is solvent accessible. Our results suggest that phosphorylation of 5-LOX at S663 could not only down-regulate leukotriene synthesis but also stimulate lipoxin production in inflammatory cells that do not express 15-LOX, thus redirecting lipid mediator biosynthesis to the production of proresolving mediators of inflammation.

  12. A highly acid-resistant novel strain of Lactobacillus johnsonii No. 1088 has antibacterial activity, including that against Helicobacter pylori, and inhibits gastrin-mediated acid production in mice

    PubMed Central

    Aiba, Yuji; Nakano, Yasuhiro; Koga, Yasuhiro; Takahashi, Kenji; Komatsu, Yasuhiko

    2015-01-01

    A novel strain of Lactobacillus johnsonii No. 1088 was isolated from the gastric juice of a healthy Japanese male volunteer, and characterized for its effectiveness in the stomach environment. Lactobacillus johnsonii No. 1088 was found to have the strongest acid resistance among several lactobacilli examined (>10% of cells survived at pH 1.0 after 2 h), and such a high acid resistance property was a specific characteristic of this strain of L. johnsonii. When cultured with various virulent bacteria, L. johnsonii No. 1088 inhibited the growth of Helicobacter pylori,Escherichia coli O-157, Salmonella Typhimurium, and Clostridium difficile, in which case its effectiveness was more potent than that of a type strain of L. johnsonii,JCM2012. In addition to its effect in vitro, L. johnsonii No. 1088 inhibited the growth of H. pylori in human intestinal microbiota-associated mice in both its live and lyophilized forms. Moreover, L. johnsonii No. 1088 suppressed gastric acid secretion in mice via decreasing the number of gastrin-positive cells in the stomach. These results taken together suggest that L. johnsonii No. 1088 is a unique lactobacillus having properties beneficial for supporting H. pylori eradication by triple therapy including the use of a proton pump inhibitor (PPI) and also for prophylaxis of gastroesophageal reflux disease possibly caused after H. pylori eradication as a side effect of PPI. PMID:25771812

  13. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation.

    PubMed

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N; Bridges, Dave; Park, Edwards A; Cole, Robert N; Elam, Marshall B; Raghow, Rajendra

    2016-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c-SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCF(Fbw7) ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  14. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation

    PubMed Central

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N.; Bridges, Dave; Park, Edwards A.; Cole, Robert N.; Elam, Marshall B.; Raghow, Rajendra

    2015-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c–SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCFFbw7 ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  15. 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

  16. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA)

    PubMed Central

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S.; Harlow, Mark L.

    2015-01-01

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5′ ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission. PMID:26446566

  17. Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6.

    PubMed

    Kantyka, Tomasz; Fischer, Jan; Wu, Zhihong; Declercq, Wim; Reiss, Karina; Schröder, Jens-Michael; Meyer-Hoffert, Ulf

    2011-06-01

    Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK family and caspase-14. Using chromogenic substrates, SPINK6 exhibited inhibitory activity against KLK12 and KLK13 with K(i) around 1nM, KLK4 with K(i)=27.3nM, KLK6 with K(i)=140nM, caspase-14 with a K(i) approximating 1μM and no activity against KLK1, KLK3 and KLK11. Taken together, SPINK6 is a potent inhibitor of distinct KLKs members. PMID:21439340

  18. Characterization of a membrane-associated serine protease in Escherichia coli

    SciTech Connect

    Palmer, S.M.; St. John, A.C.

    1987-04-01

    Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively. The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. They termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using (/sup 3/H)diisopropylfluorophosphate as an active-site labeling reagent, they determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, an endogenous, periplasmic inhibitor of trypsin.

  19. Purification and biochemical characterization of an extracellular serine peptidase from Aspergillus terreus.

    PubMed

    Biaggio, Rafael Tage; Silva, Ronivaldo Rodrigues da; Rosa, Nathalia Gonsales da; Leite, Rodrigo Simões Ribeiro; Arantes, Eliane Candiani; Cabral, Tatiana Pereira de Freitas; Juliano, Maria A; Juliano, Luiz; Cabral, Hamilton

    2016-04-01

    Peptidases are important because they play a central role in pharmaceutical, food, environmental, and other industrial processes. A serine peptidase from Aspergillus terreus was isolated after two chromatography steps that showed a yield of 15.5%. Its molecular mass was determined to be 43 kD, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This peptidase was active between pH 5.0 to 8.0 and had maximum activity at pH 7.0, at 45°C. When exposited with 1 M of urea, the enzyme maintained 100% activity and used azocasein as substrate. The N-terminal (first 15 residues) showed 33% identity with the serine peptidase of Aspergillus clavatus ES1. The kinetics assays showed that subsite S2 did not bind polar basic amino acids (His and Arg) nonpolar acidic amino acids (Asp and Glu). The subsite S1 showed higher catalytic efficiency than the S2 and S3 subsites. PMID:25830777

  20. Constitutively signaling fragments of Tsr, the Escherichia coli serine chemoreceptor.

    PubMed Central

    Ames, P; Parkinson, J S

    1994-01-01

    Tsr, the serine chemoreceptor of Escherichia coli, has two signaling modes. One augments clockwise (CW) flagellar rotation, and the other augments counterclockwise (CCW) rotation. To identify the portion of the Tsr molecule responsible for these activities, we isolated soluble fragments of the Tsr cytoplasmic domain that could alter the flagellar rotation patterns of unstimulated wild-type cells. Residues 290 to 470 from wild-type Tsr generated a CW signal, whereas the same fragment with a single amino acid replacement (alanine 413 to valine) produced a CCW signal. The soluble components of the chemotaxis phosphorelay system needed for expression of these Tsr fragment signals were identified by epistasis analysis. Like full-length receptors, the fragments appeared to generate signals through interactions with the CheA autokinase and the CheW coupling factor. CheA was required for both signaling activities, whereas CheW was needed only for CW signaling. Purified Tsr fragments were also examined for effects on CheA autophosphorylation activity in vitro. Consistent with the in vivo findings, the CW fragment stimulated CheA, whereas the CCW fragment inhibited CheA. CheW was required for stimulation but not for inhibition. These findings demonstrate that a 180-residue segment of the Tsr cytoplasmic domain can produce two active signals. The CCW signal involves a direct contact between the receptor and the CheA kinase, whereas the CW signal requires participation of CheW as well. The correlation between the in vitro effects of Tsr signaling fragments on CheA activity and their in vivo behavioral effects lends convincing support to the phosphorelay model of chemotactic signaling. Images PMID:7929006

  1. Metabolic evidence of vitamin B-12 deficiency, including high homocysteine and methylmalonic acid and low holotranscobalamin, is more pronounced in older adults with elevated plasma folate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: An analysis of data from the National Health and Nutrition Examination Survey indicated that in older adults exposed to folic acid fortification, the combination of low serum vitamin B-12 and elevated folate is associated with higher concentrations of homocysteine and methylmalonic acid ...

  2. The prebiotic synthesis of amino acids - interstellar vs. atmospheric mechanisms

    NASA Astrophysics Data System (ADS)

    Meierhenrich, U. J.; Muñoz Caro, G. M.; Schutte, W. A.; Barbier, B.; Arcones Segovia, A.; Rosenbauer, H.; Thiemann, W. H.-P.; Brack, A.

    2002-11-01

    Until very recently, prebiotic amino acids were believed to have been generated in the atmosphere of the early Earth, as successfully simulated by the Urey-Miller experiments. Two independent studies now identified ice photochemistry in the interstellar medium as a possible source of prebiotic amino acids. Ultraviolet irradiation of ice mixtures containing identified interstellar molecules (such as H2O, CO2, CO, CH3OH, and NH3) in the conditions of vacuum and low temperature found in the interstellar medium generated amino acid structures including glycine, alanine, serine, valine, proline, and aspartic acid. After warmup, hydrolysis and derivatization, our team was able to identify 16 amino acids as well as furans and pyrroles. Enantioselective analyses of the amino acids showed racemic mixtures. A prebiotic interstellar origin of amino acid structures is now discussed to be a plausible alternative to the Urey-Miller mechanism.

  3. Phosphorylation of Serine Residues in the C-terminal Cytoplasmic Tail of Connexin43 Regulates Proliferation of Ovarian Granulosa Cells

    PubMed Central

    Dyce, Paul W.; Norris, Rachael P.; Lampe, Paul D.; Kidder, Gerald M.

    2013-01-01

    Connexin43 (Cx43) forms 22 s gap junctions that couple the granulosa cells of ovarian follicles. In Cx43 knockout mice, follicle growth is restricted due to impaired granulosa cell proliferation. We have used these mice to examine the importance of specific Cx43 phosphorylation sites in follicle growth. Serines at residues 255, 262, 279 and 282 are MAP kinase substrates that, when phosphorylated, reduce junctional conductance. Mutant forms of Cx43 were constructed with these serines replaced with amino acids that cannot be phosphorylated. These mutants were transduced into Cx43 knockout ovarian somatic cells which were combined with wildtype oocytes and grafted into immunocompromised female mice permitting follicle growth in vivo. Despite residues 255 or 262 being mutated to prevent their being phosphorylated, recombinant ovaries constructed with these mutants were able to rescue the null phenotype, restoring complete folliculogenesis. In contrast, Cx43 with serine to alanine mutations at both residues 279 and 282 or at all four residues failed to rescue folliculogenesis; the mutant molecules were largely confined to intracellular sites, with few gap junctions. Using an in vitro proliferation assay, we confirmed a decrease in proliferation of granulosa cells expressing the double mutant construct. These results indicate that Cx43 phosphorylation by MAP kinase at serines 279 and 282 occurs in granulosa cells of early follicles and that this is involved in regulating follicle development. PMID:22729691

  4. Participation of D-serine in the development and reproduction of the silkworm Bombyx mori.

    PubMed

    Tanigawa, Minoru; Suzuki, Chihiro; Niwano, Kimio; Kanekatsu, Rensuke; Tanaka, Hiroyuki; Horiike, Kihachiro; Hamase, Kenji; Nagata, Yoko

    2016-04-01

    The silkworm Bombyx mori contains high concentrations of free D-serine, an optical isomer of L-serine. To elucidate its function, we first investigated the localization of D-serine in various organs of silkworm larvae, pupae, and adult moths. Using immunohistochemical analysis with an anti-D-serine antibody, we found D-serine in the microvilli of midgut goblet and cylindrical cells and in peripheral matrix components of testicular and ovarian cells. By spectrophotometric analysis, D-serine was also found in the hemolymph and fat body. D-Alanine was not detected in the various organs by immunohistochemistry. Serine racemase, which catalyzes the inter-conversion of L- and D-serine, was found to co-localize with D-serine, and D-serine production from L-serine by intrinsic serine racemase was suggested. O-Phospho-L-serine is an inhibitor of serine racemase, and it was administered to the larvae to reduce the D-serine level. This reagent decreased the midgut caspase-3 level and caused a delay in spermatogenesis and oogenesis. The reagent also decreased mature sperm and egg numbers, suggesting D-serine participation in these processes. D-Serine administration induced an increase in pyruvate levels in testis, midgut, and fat body, indicating conversion of D-serine to pyruvate. On the basis of these results, together with our previous investigation of ATP biosynthesis in testis, we consider the possible involvement of D-serine in ATP synthesis for metamorphosis and reproduction. PMID:26828952

  5. Isotopologue profiling of Legionella pneumophila: role of serine and glucose as carbon substrates.

    PubMed

    Eylert, Eva; Herrmann, Vroni; Jules, Matthieu; Gillmaier, Nadine; Lautner, Monika; Buchrieser, Carmen; Eisenreich, Wolfgang; Heuner, Klaus

    2010-07-16

    Legionella pneumophila (Lp) is commonly found in freshwater habitats but is also the causative agent of Legionnaires' disease when infecting humans. Although various virulence factors have been reported, little is known about the nutrition and the metabolism of the bacterium. Here, we report the application of isotopologue profiling for analyzing the metabolism of L. pneumophila. Cultures of Lp were supplied with [U-(13)C(3)]serine, [U-(13)C(6)]glucose, or [1,2-(13)C(2)]glucose. After growth, (13)C enrichments and isotopologue patterns of protein-derived amino acids and poly-3-hydroxybutyrate were determined by mass spectrometry and/or NMR spectroscopy. The labeling patterns detected in the experiment with [U-(13)C(3)]serine showed major carbon flux from serine to pyruvate and from pyruvate to acetyl-CoA, which serves as a precursor of poly-3-hydroxybutyrate or as a substrate of a complete citrate cycle with Si specificity of the citrate synthase. Minor carbon flux was observed between pyruvate and oxaloacetate/malate by carboxylation and decarboxylation, respectively. The apparent lack of label in Val, Ile, Leu, Pro, Phe, Met, Arg, and Tyr confirmed that L. pneumophila is auxotrophic for these amino acids. Experiments with [(13)C]glucose showed that the carbohydrate is also used as a substrate to feed the central metabolism. The specific labeling patterns due to [1,2-(13)C(2)]glucose identified the Entner-Doudoroff pathway as the predominant route for glucose utilization. In line with these observations, a mutant lacking glucose-6-phosphate dehydrogenase (Delta zwf) did not incorporate label from glucose at significant levels and was slowly outcompeted by the wild type strain in successive rounds of infection in Acanthamoeba castellanii, indicating the importance of this enzyme and of carbohydrate usage in general for the life cycle of Lp. PMID:20442401

  6. Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

    PubMed

    Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

    2016-02-19

    Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli. PMID:26544153

  7. Functional Suppression of HAMP Domain Signaling Defects in the E. coli Serine Chemoreceptor

    PubMed Central

    Lai, Run-Zhi; Parkinson, John S.

    2014-01-01

    HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric E. coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA, bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: Unmodified E residues promote kinase-off output; methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer three of the HAMP bundle and followed in vivo CheA activity with a FRET-based assay. We found that an alanine or serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells, but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E, S255A) shifted Tsr output toward the kinase-off state, but two others (S255G, L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations. PMID:25134756

  8. Identification of a small molecule inhibitor of serine 276 phosphorylation of the p65 subunit of NF-κB using in silico molecular docking

    PubMed Central

    Law, Mary; Corsino, Patrick; Parker, Nicole Teoh; Law, Brian K.

    2009-01-01

    NF-κB is activated in many types of cancer. Phosphorylation of p65 at serine 276 is required for the expression of a subset of NF-κB regulated genes, including vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8). Thus, inhibition of serine 276 phosphorylation may prevent metastasis and angiogenesis in certain tumor types. Using in silico molecular docking, small molecules that are predicted to bind to a structural pocket near serine 276 were identified. One compound, NSC-127102, hinders serine 276 phosphorylation and the expression of IL-8 and VCAM-1. Small molecules such as NSC-127102 may be optimized for the future treatment of cancer. PMID:19910110

  9. A general method for making peptide therapeutics resistant to serine protease degradation: application to dipeptidyl peptidase IV substrates.

    PubMed

    Heard, Kathryn R; Wu, Wengen; Li, Youhua; Zhao, Peng; Woznica, Iwona; Lai, Jack H; Beinborn, Martin; Sanford, David G; Dimare, Matthew T; Chiluwal, Amrita K; Peters, Diane E; Whicher, Danielle; Sudmeier, James L; Bachovchin, William W

    2013-11-14

    Bioactive peptides have evolved to optimally fulfill specific biological functions, a fact which has long attracted attention for their use as therapeutic agents. While there have been some recent commercial successes fostered in part by advances in large-scale peptide synthesis, development of peptides as therapeutic agents has been significantly impeded by their inherent susceptibility to protease degradation in the bloodstream. Here we report that incorporation of specially designed amino acid analogues at the P1' position, directly C-terminal of the enzyme cleavage site, renders peptides, including glucagon-like peptide-1 (7-36) amide (GLP-1) and six other examples, highly resistant to serine protease degradation without significant alteration of their biological activity. We demonstrate the applicability of the method to a variety of proteases, including dipeptidyl peptidase IV (DPP IV), dipeptidyl peptidase 8 (DPP8), fibroblast activation protein α (FAPα), α-lytic protease (αLP), trypsin, and chymotrypsin. In summary, the "P1' modification" represents a simple, general, and highly adaptable method of generating enzymatically stable peptide-based therapeutics. PMID:24044354

  10. Characterization of a serine protease-mediated cell death program activated in human leukemia cells

    SciTech Connect

    O'Connell, A.R.; Holohan, C.; Torriglia, A.; Lee, B.F.; Stenson-Cox, C. . E-mail: catherine.stenson@nuigalway.ie

    2006-01-01

    Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis.

  11. p38 MAPK regulates PKAα and CUB-serine protease in Amphibalanus amphitrite cyprids.

    PubMed

    Zhang, Gen; He, Li-Sheng; Him Wong, Yue; Xu, Ying; Zhang, Yu; Qian, Pei-Yuan

    2015-01-01

    The MKK3-p38 MAPK pathway has been reported to mediate larval settlement in Amphibalanus (=Balanus) amphitrite. To clarify the underlying molecular mechanism, we applied label-free proteomics to analyze changes in the proteome of cyprids treated with a p38 MAPK inhibitor. The results showed that the expression levels of 80 proteins were significantly modified (p < 0.05). These differentially expressed proteins were assigned to 15 functional groups according to the KOG database and 9 pathways were significantly enriched. Further analysis revealed that p38 MAPK might regulate the energy supply and metamorphosis. Two potential regulatory proteins, CUB-serine protease and PKAα, were both down-regulated in expression. CUB-serine protease localized to postaxial seta 2 and 3, as well as the 4 subterminal sensilla in the antennule. Importantly, it was co-localized with the neuron transmitter serotonin in the sections, suggesting that the CUB-serine protease was present in the neural system. PKAα was highly expressed during the cyprid and juvenile stages, and it was co-localized with phospho-p38 MAPK (pp38 MAPK) to the cement gland, suggesting that PKAα might have some functions in cement glands. Overall, p38 MAPK might regulate multiple functions in A. amphitrite cyprids, including the energy supply, metamorphosis, neural system and cement glands. PMID:26434953

  12. p38 MAPK regulates PKAα and CUB-serine protease in Amphibalanus amphitrite cyprids

    PubMed Central

    Zhang, Gen; He, Li-Sheng; Him Wong, Yue; Xu, Ying; Zhang, Yu; Qian, Pei-Yuan

    2015-01-01

    The MKK3-p38 MAPK pathway has been reported to mediate larval settlement in Amphibalanus (=Balanus) amphitrite. To clarify the underlying molecular mechanism, we applied label-free proteomics to analyze changes in the proteome of cyprids treated with a p38 MAPK inhibitor. The results showed that the expression levels of 80 proteins were significantly modified (p < 0.05). These differentially expressed proteins were assigned to 15 functional groups according to the KOG database and 9 pathways were significantly enriched. Further analysis revealed that p38 MAPK might regulate the energy supply and metamorphosis. Two potential regulatory proteins, CUB-serine protease and PKAα, were both down-regulated in expression. CUB-serine protease localized to postaxial seta 2 and 3, as well as the 4 subterminal sensilla in the antennule. Importantly, it was co-localized with the neuron transmitter serotonin in the sections, suggesting that the CUB-serine protease was present in the neural system. PKAα was highly expressed during the cyprid and juvenile stages, and it was co-localized with phospho-p38 MAPK (pp38 MAPK) to the cement gland, suggesting that PKAα might have some functions in cement glands. Overall, p38 MAPK might regulate multiple functions in A. amphitrite cyprids, including the energy supply, metamorphosis, neural system and cement glands. PMID:26434953

  13. Stimulation of MC38 tumor growth by insulin analog X10 involves the serine synthesis pathway.

    PubMed

    Hvid, Henning; Fendt, Sarah-Maria; Blouin, Marie-José; Birman, Elena; Voisin, Gregory; Svendsen, Angela Manegold; Frank, Russell; Vander Heiden, Matthew G; Stephanopoulos, Gregory; Hansen, Bo Falck; Pollak, Michael

    2012-08-01

    Recent evidence suggests that type II diabetes is associated with increased risk and/or aggressive behavior of several cancers, including those arising from the colon. Concerns have been raised that endogenous hyperinsulinemia and/or exogenous insulin and insulin analogs might stimulate proliferation of neoplastic cells. However, the mechanisms underlying possible growth-promoting effects of insulin and insulin analogs in cancer cells in vivo, such as changes in gene expression, are incompletely described. We observed that administration of the insulin analog X10 significantly increased tumor growth and proliferation in a murine colon cancer model (MC38 cell allografts). Insulin and X10 altered gene expression in MC38 tumors in a similar fashion, but X10 was more potent in terms of the number of genes influenced and the magnitude of changes in gene expression. Many of the affected genes were annotated to metabolism, nutrient uptake, and protein synthesis. Strikingly, expression of genes encoding enzymes in the serine synthesis pathway, recently shown to be critical for neoplastic proliferation, was increased following treatment with insulin and X10. Using stable isotopic tracers and mass spectrometry, we confirmed that insulin and X10 increased glucose contribution to serine synthesis in MC38 cells. The data demonstrate that the tumor growth-promoting effects of insulin and X10 are associated with changes in expression of genes involved in cellular energy metabolism and reveal previously unrecognized effects of insulin and X10 on serine synthesis. PMID:22685267

  14. Structure of haptoglobin heavy chain and other serine protease homologs by comparative model building

    SciTech Connect

    Grer, J.

    1980-10-01

    Proteins often occur in families whose structure is closely similar, even though the proteins may come from widely different sources and have quite distinct functions. It would be useful to be able to construct the three-dimensional structure of these proteins from the known structure of one or more of them without having to solve the structure of each protein ab initio. We have been using comparative model building to derive the structure of an unusual protein of the trypsin-like serine protease family. We have recently extended this comparison to include other serine protease homologs for which a primary structure is available. To generate structures for the different members of the serine protease family, it is necessary to extract the common structural features of the molecule. Fortunately, three independently determined protein structures are available: schymotrypsin, trypsin, and elastase. These three structures were compared in detail and the structurally conserved regions in all three, mainly the BETA-sheet and the ..cap alpha..-helix, were identified. The variable portions occur in the loops on the surface of the molecule. By using these structures, the primary sequences of these three proteins were aligned. From this alignment, it is clear that sequence homology between the proteins occurs mainly in the structurally conserved regions of the molecule, while the variable portions show very little sequence homology.

  15. Distinct and Site-Specific Phosphorylation of the Retinoblastoma Protein at Serine 612 in Differentiated Cells

    PubMed Central

    Hattori, Takayuki; Uchida, Chiharu; Takahashi, Hirotaka; Yamamoto, Naoki; Naito, Mikihiko; Taya, Yoichi

    2014-01-01

    The retinoblastoma susceptibility protein (pRB) is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated in vivo. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB. PMID:24466208

  16. Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase*

    PubMed Central

    Maenpuen, Somchart; Amornwatcharapong, Watcharee; Krasatong, Pasupat; Sucharitakul, Jeerus; Palfey, Bruce A.; Yuthavong, Yongyuth; Chitnumsub, Penchit; Leartsakulpanich, Ubolsree; Chaiyen, Pimchai

    2015-01-01

    Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes a hydroxymethyl group transfer from l-serine to tetrahydrofolate (H4folate) to yield glycine and 5,10-methylenetetrahydrofolate (CH2-H4folate). SHMT is crucial for deoxythymidylate biosynthesis and a target for antimalarial drug development. Our previous studies indicate that PvSHMT catalyzes the reaction via a ternary complex mechanism. To define the kinetic mechanism of this catalysis, we explored the PvSHMT reaction by employing various methodologies including ligand binding, transient, and steady-state kinetics as well as product analysis by rapid-quench and HPLC/MS techniques. The results indicate that PvSHMT can bind first to either l-serine or H4folate. The dissociation constants for the enzyme·l-serine and enzyme·H4folate complexes were determined as 0.18 ± 0.08 and 0.35 ± 0.06 mm, respectively. The amounts of glycine formed after single turnovers of different preformed binary complexes were similar, indicating that the reaction proceeds via a random-order binding mechanism. In addition, the rate constant of glycine formation measured by rapid-quench and HPLC/MS analysis is similar to the kcat value (1.09 ± 0.05 s−1) obtained from the steady-state kinetics, indicating that glycine formation is the rate-limiting step of SHMT catalysis. This information will serve as a basis for future investigation on species-specific inhibition of SHMT for antimalarial drug development. PMID:25678710

  17. Malonate-based inhibitors of mammalian serine racemase: kinetic characterization and structure-based computational study.

    PubMed

    Vorlová, Barbora; Nachtigallová, Dana; Jirásková-Vaníčková, Jana; Ajani, Haresh; Jansa, Petr; Rezáč, Jan; Fanfrlík, Jindřich; Otyepka, Michal; Hobza, Pavel; Konvalinka, Jan; Lepšík, Martin

    2015-01-01

    Overactivation of NMDA receptors has been implicated in various neuropathological conditions, including brain ischaemia, neurodegenerative disorders and epilepsy. Production of d-serine, an NMDA receptor co-agonist, from l-serine is catalyzed in vivo by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine racemase. Specific inhibition of this enzyme has been proposed as a promising strategy for treatment of neurological conditions caused by NMDA receptor dysfunction. Here we present the synthesis and activity analysis of a series of malonate-based inhibitors of mouse serine racemase (mSR). The compounds possessed IC50 values ranging from 40 ± 11 mM for 2,2-bis(hydroxymethyl)malonate down to 57 ± 1 μM for 2,2-dichloromalonate, the most effective competitive mSR inhibitor known to date. The structure-activity relationship of the whole series in the human orthologue (hSR) was interpreted using Glide docking, WaterMap analysis of hydration and quantum mechanical calculations based on the X-ray structure of the hSR/malonate complex. Docking into the hSR active site with three thermodynamically favourable water molecules was able to discern qualitatively between good and weak inhibitors. Further improvement in ranking was obtained using advanced PM6-D3H4X/COSMO semiempirical quantum mechanics-based scoring which distinguished between the compounds with IC50 better/worse than 2 mM. We have thus not only found a new potent hSR inhibitor but also worked out a computer-assisted protocol to rationalize the binding affinity which will thus aid in search for more effective SR inhibitors. Novel, potent hSR inhibitors may represent interesting research tools as well as drug candidates for treatment of diseases associated with NMDA receptor overactivation. PMID:25462239

  18. Increased Sensitivity to Inflammatory Pain Induced by Subcutaneous Formalin Injection in Serine Racemase Knock-Out Mice

    PubMed Central

    Tabata-Imai, Ayako; Inoue, Ran; Mori, Hisashi

    2014-01-01

    D-Serine, an endogenous coagonist of the N-methyl-D-aspartate receptor (NMDAR), is widely distributed in the central nervous system and is synthesized from L-serine by serine racemase (SR). NMDAR plays an important role in pain processing including central sensitization that eventually causes hyperalgesia. To elucidate the roles of D-serine and SR in pain transmission, we evaluated the behavioral changes and spinal nociceptive processing induced by formalin using SR knock-out (KO) mice. We found that SR is mainly distributed in lamina II of the dorsal horn of the spinal cord in wild-type (WT) mice. Although the formalin injected subcutaneously induced the biphasic pain response of licking in SR-KO and WT mice, the time spent on licking was significantly longer in the SR-KO mice during the second phase of the formalin test. The number of neurons immunopositive for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK), which are molecular pain markers, in laminae I-II of the ipsilateral dorsal horn was significantly larger in the SR-KO mice. Immunohistochemical staining revealed that the distribution of SR changed from being broad to being concentrated in cell bodies after the formalin injection. On the other hand, the expression level of the cytosolic SR in the ipsilateral dorsal horn significantly decreased. Oral administration of 10 mM D-serine in drinking water for one week cancelled the difference in pain behaviors between WT and SR-KO mice in phase 2 of the formalin test. These findings demonstrate that the SR-KO mice showed increased sensitivity to inflammatory pain and the WT mice showed translocation of SR and decreased SR expression levels after the formalin injection, which suggest a novel antinociceptive mechanism via SR indicating an important role of D-serine in pain transmission. PMID:25133605

  19. The emerging roles of serine protease cascades in the epidermis.

    PubMed

    Ovaere, Petra; Lippens, Saskia; Vandenabeele, Peter; Declercq, Wim

    2009-09-01

    It has become clear in recent years that serine proteases have an important role in epidermal homeostasis, and the signaling cascades are gradually being identified. For example, matriptase, prostasin and furin are implicated in a cascade that could activate ENaC, leading to epidermal barrier formation and hydration, probably in part through their involvement in filaggrin processing. Kallikreins can form a signaling cascade to coordinate corneocyte desquamation. Knowledge is also emerging about how endogenous inhibitors, calcium and pH control these cascades. It is becoming clear that some skin pathologies are associated with deregulated serine protease activity. Therefore, a deeper knowledge of the regulation of these serine protease cascades could form the basis for development of appropriate treatments for skin disorders such as Netherton syndrome. PMID:19726197

  20. Elevational Variation in Soil Amino Acid and Inorganic Nitrogen Concentrations in Taibai Mountain, China.

    PubMed

    Cao, Xiaochuang; Ma, Qingxu; Zhong, Chu; Yang, Xin; Zhu, Lianfeng; Zhang, Junhua; Jin, Qianyu; Wu, Lianghuan

    2016-01-01

    Amino acids are important sources of soil organic nitrogen (N), which is essential for plant nutrition, but detailed information about which amino acids predominant and whether amino acid composition varies with elevation is lacking. In this study, we hypothesized that the concentrations of amino acids in soil would increase and their composition would vary along the elevational gradient of Taibai Mountain, as plant-derived organic matter accumulated and N mineralization and microbial immobilization of amino acids slowed with reduced soil temperature. Results showed that the concentrations of soil extractable total N, extractable organic N and amino acids significantly increased with elevation due to the accumulation of soil organic matter and the greater N content. Soil extractable organic N concentration was significantly greater than that of the extractable inorganic N (NO3--N + NH4+-N). On average, soil adsorbed amino acid concentration was approximately 5-fold greater than that of the free amino acids, which indicates that adsorbed amino acids extracted with the strong salt solution likely represent a potential source for the replenishment of free amino acids. We found no appreciable evidence to suggest that amino acids with simple molecular structure were dominant at low elevations, whereas amino acids with high molecular weight and complex aromatic structure dominated the high elevations. Across the elevational gradient, the amino acid pool was dominated by alanine, aspartic acid, glycine, glutamic acid, histidine, serine and threonine. These seven amino acids accounted for approximately 68.9% of the total hydrolyzable amino acid pool. The proportions of isoleucine, tyrosine and methionine varied with elevation, while soil major amino acid composition (including alanine, arginine, aspartic acid, glycine, histidine, leucine, phenylalanine, serine, threonine and valine) did not vary appreciably with elevation (p>0.10). The compositional similarity of many

  1. Elevational Variation in Soil Amino Acid and Inorganic Nitrogen Concentrations in Taibai Mountain, China

    PubMed Central

    Yang, Xin; Zhu, Lianfeng; Zhang, Junhua; Jin, Qianyu; Wu, Lianghuan

    2016-01-01

    Amino acids are important sources of soil organic nitrogen (N), which is essential for plant nutrition, but detailed information about which amino acids predominant and whether amino acid composition varies with elevation is lacking. In this study, we hypothesized that the concentrations of amino acids in soil would increase and their composition would vary along the elevational gradient of Taibai Mountain, as plant-derived organic matter accumulated and N mineralization and microbial immobilization of amino acids slowed with reduced soil temperature. Results showed that the concentrations of soil extractable total N, extractable organic N and amino acids significantly increased with elevation due to the accumulation of soil organic matter and the greater N content. Soil extractable organic N concentration was significantly greater than that of the extractable inorganic N (NO3−-N + NH4+-N). On average, soil adsorbed amino acid concentration was approximately 5-fold greater than that of the free amino acids, which indicates that adsorbed amino acids extracted with the strong salt solution likely represent a potential source for the replenishment of free amino acids. We found no appreciable evidence to suggest that amino acids with simple molecular structure were dominant at low elevations, whereas amino acids with high molecular weight and complex aromatic structure dominated the high elevations. Across the elevational gradient, the amino acid pool was dominated by alanine, aspartic acid, glycine, glutamic acid, histidine, serine and threonine. These seven amino acids accounted for approximately 68.9% of the total hydrolyzable amino acid pool. The proportions of isoleucine, tyrosine and methionine varied with elevation, while soil major amino acid composition (including alanine, arginine, aspartic acid, glycine, histidine, leucine, phenylalanine, serine, threonine and valine) did not vary appreciably with elevation (p>0.10). The compositional similarity of many

  2. Primary structure of human corticosteroid binding globulin, deduced from hepatic and pulmonary cDNAs, exhibits homology with serine protease inhibitors.

    PubMed Central

    Hammond, G L; Smith, C L; Goping, I S; Underhill, D A; Harley, M J; Reventos, J; Musto, N A; Gunsalus, G L; Bardin, C W

    1987-01-01

    We have isolated and sequenced cDNAs for corticosteroid binding globulin (CBG) prepared from human liver and lung mRNAs. Our results indicate that CBG mRNA is relatively abundant in the liver but is also present in the lung, testis, and kidney. The liver CBG cDNA contains an open reading frame for a 405-amino acid (Mr 45,149) polypeptide. This includes a predominantly hydrophobic, leader sequence of 22 residues that precedes the known NH2-terminal sequence of human CBG. We, therefore, predict that the mature protein is composed of 383 amino acids and is a polypeptide of Mr 42,646. A second, in-frame, 72-base-pair cistron of unknown significance exists between the TAA termination codon for CBG and a possible polyadenylylation signal (AATAAA) located 16 nucleotides before the polyadenylylation site. The deduced amino acid sequence of mature CBG contains two cysteine residues and consensus sequences for the attachment of six possible N-linked oligosaccharide chains. The sequences of the human lung and liver CBG cDNAs differ by only one nucleotide within the proposed leader sequence, and we attribute this to a point mutation. No sequence homology was found between CBG and other steroid binding proteins, but there is a remarkable similarity between the amino acid sequences of CBG and of alpha 1-antitrypsin, and this extends to other members of the serpin (serine protease inhibitor) superfamily. Images PMID:3299377

  3. An evaluation of chromogenic substrates for characterization of serine protease produced by pathogenic Vibrio alginolyticus.

    PubMed

    Chen, F R; Liu, P C; Lee, K K

    1999-01-01

    Four chromogenic substrates for characterizing serine protease of Vibrio alginolyticus were evaluated. The protease activity of bacterial extracellular products, or the fractions of 33 kD protease purified by the AKTA purifier system with various columns, was completely inhibited by ethylenediamine tetra-acetic acid, ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), antipain and phenylmethylsulphonyl fluoride (PMSF) using water-soluble substrates (azoalbumin and azocasein). It was only completely inhibited by antipain and PMSF using water-insoluble substrates (azocoll and hide powder azure). The protease activity was not, or only partially, inhibited by 1,10-phenanthroline and sodium dodecyl sulphate (SDS) using all four substrates. Since chelating agents and 1,10-phenanthroline are commonly employed as inhibitors to identify metalloprotease, the two water-soluble substrates may not be appropriate for this purpose, except for using 1,10-phenanthroline as an inhibitor. Chelating agents may be still applicable as inhibitors using water-insoluble substrates and 1,10-phenanthroline is highly recommended in the characterization for metalloprotease to avoid confusion. In the present study, the 33 kD protease was further confirmed as an SDS-resistant serine protease and not a metalloprotease. PMID:10413876

  4. African swine fever virus encodes a serine protein kinase which is packaged into virions.

    PubMed Central

    Baylis, S A; Banham, A H; Vydelingum, S; Dixon, L K; Smith, G L

    1993-01-01

    Nucleotide sequencing of the SalI j region of the virulent Malawi (LIL20/1) strain of African swine fever virus (ASFV) identified an open reading frame (ORF), designated j9L, with extensive similarity to the family of protein kinases. This ORF encodes a 35.1-kDa protein of 299 amino acids which shares 24.6% amino acid identity with the human pim-1 proto-oncogene and 21.0% identity with the vaccinia virus B1R-encoded protein kinase. The ASFV ORF contains the motifs characteristic of serine-threonine protein kinases, with the exception of the presumed ATP-binding site, which is poorly conserved. The ORF was expressed to high levels in Escherichia coli, and the recombinant enzyme phosphorylated a calf thymus histone protein on serine residues in vitro. An antibody raised to an amino-terminal peptide of the ASFV protein kinase was reactive with the recombinant protein in Western immunoblot analyses and was used to demonstrate the presence of the protein kinase in ASF virions. Images PMID:8331722

  5. Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii.

    PubMed

    Pszenny, V; Angel, S O; Duschak, V G; Paulino, M; Ledesma, B; Yabo, M I; Guarnera, E; Ruiz, A M; Bontempi, E J

    2000-04-15

    A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host. PMID:10779600

  6. Serine protease autotransporters of enterobacteriaceae (SPATEs): biogenesis and function.

    PubMed

    Dautin, Nathalie

    2010-06-01

    Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins. PMID:22069633

  7. Sequence analysis and enzyme kinetics of the L2 serine beta-lactamase from Stenotrophomonas maltophilia.

    PubMed Central

    Walsh, T R; MacGowan, A P; Bennett, P M

    1997-01-01

    The L2 serine active-site beta-lactamase from Stenotrophomonas maltophilia has been classified as a clavulanic acid-sensitive cephalosporinase. The gene encoding this enzyme from S. maltophilia 1275 IID has been cloned on a 3.3-kb fragment into pK18 under the control of a Ptac promoter to generate recombinant plasmid pUB5840; when expressed in Escherichia coli, this gene confers resistance to cephalosporins and penicillins. Sequence analysis has revealed an open reading frame (ORF) of 909 bp with a GC content of 71.6%, comparable to that of the L1 metallo-beta-lactamase gene (68.4%) from the same bacterium. The ORF encodes an unmodified protein of 303 amino acids with a predicted molecular mass of 31.5 kDa, accommodating a putative leader peptide of 27 amino acids. Comparison of the amino acid sequence with those of other beta-lactamases showed it to be most closely related (54% identity) to the BLA-A beta-lactamase from Yersinia enterocolitica. Sequence identity is most obvious near the STXK active-site motif and the SDN loop motif common to all serine active-site penicillinases. Sequences outside the conserved regions display low homology with comparable regions of other class A penicillinases. Kinetics of the enzyme from the cloned gene demonstrated an increase in activity with cefotaxime but markedly less activity with imipenem than previously reported. Hence, the S. maltophilia L2 beta-lactamase is an inducible Ambler class A beta-lactamase which would account for the sensitivity to clavulanic acid. PMID:9210666

  8. AND-34/BCAR3 Regulates Adhesion-Dependent p130Cas Serine Phosphorylation and Breast Cancer Cell Growth Pattern

    PubMed Central

    Makkinje, Anthony; Near, Richard I.; Infusini, Giuseppe; Borre, Pierre Vanden; Bloom, Alexander; Cai, Dongpo; Costello, Catherine E.; Lerner, Adam

    2009-01-01

    NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS PAGE while NSP-1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype. PMID:19454314

  9. Serine/Threonine Phosphatase Stp1 Mediates Post-transcriptional Regulation of Hemolysin, Autolysis, and Virulence of Group B Streptococcus*

    PubMed Central

    Burnside, Kellie; Lembo, Annalisa; Harrell, Maria Isabel; Gurney, Michael; Xue, Liang; BinhTran, Nguyen-Thao; Connelly, James E.; Jewell, Kelsea A.; Schmidt, Byron Z.; de los Reyes, Melissa; Tao, Weiguo Andy; Doran, Kelly S.; Rajagopal, Lakshmi

    2011-01-01

    Elucidating how serine/threonine phosphatases regulate kinase function and bacterial virulence is critical for our ability to combat these infections. Group B streptococci (GBS) are β-hemolytic Gram-positive bacteria that cause invasive infections in humans. To adapt to environmental changes, GBS encodes signaling mechanisms comprising two component systems and eukaryotic-like enzymes. We have previously described the importance of the serine/threonine kinase Stk1 to GBS pathogenesis. However, how the presence or absence of the cognate serine/threonine phosphatase Stp1 affects Stk1 function and GBS virulence is not known. Here, we show that GBS deficient only in Stp1 expression are markedly reduced for their ability to cause systemic infections, exhibit decreased β-hemolysin/cytolysin activity, and show increased sensitivity to autolysis. Although transcription of genes important for β-hemolysin/cytolysin expression and export is similar to the wild type (WT), 294 genes (excluding stp1) showed altered expression in the stp1 mutant and included autolysin genes. Furthermore, phosphopeptide enrichment analysis identified that 35 serine/threonine phosphopeptides, corresponding to 27 proteins, were unique to the stp1 mutant. This included phosphorylation of ATP synthase, DNA and RNA helicases, and proteins important for cell division and protein synthesis. Collectively, our results indicate that Stp1 is important for appropriate regulation of Stk1 function, hemolysin activity, autolysis, and GBS virulence. PMID:22081606

  10. Permanent farnesylation of lamin A mutants linked to progeria impairs its phosphorylation at serine 22 during interphase.

    PubMed

    Moiseeva, Olga; Lopes-Paciencia, Stéphane; Huot, Geneviève; Lessard, Frédéric; Ferbeyre, Gerardo

    2016-02-01

    Mutants of lamin A cause diseases including the Hutchinson-Gilford progeria syndrome (HGPS) characterized by premature aging. Lamin A undergoes a series of processing reactions, including farnesylation and proteolytic cleavage of the farnesylated C-terminal domain. The role of cleavage is unknown but mutations that affect this reaction lead to progeria. Here we show that interphase serine 22 phosphorylation of endogenous mutant lamin A (progerin) is defective in cells from HGPS patients. This defect can be mimicked by expressing progerin in human cells and prevented by inhibition of farnesylation. Furthermore, serine 22 phosphorylation of non-farnesylated progerin was enhanced by a mutation that disrupts lamin A head to tail interactions. The phosphorylation of lamin A or non-farnesylated progerin was associated to the formation of spherical intranuclear lamin A droplets that accumulate protein kinases of the CDK family capable of phosphorylating lamin A at serine 22. CDK inhibitors compromised the turnover of progerin, accelerated senescence of HGPS cells and reversed the effects of FTI on progerin levels. We discuss a model of progeria where faulty serine 22 phosphorylation compromises phase separation of lamin A polymers, leading to accumulation of functionally impaired lamin A structures. PMID:26922519

  11. Permanent farnesylation of lamin A mutants linked to progeria impairs its phosphorylation at serine 22 during interphase

    PubMed Central

    Moiseeva, Olga; Lopes-Paciencia, Stéphane; Huot, Geneviève; Lessard, Frédéric; Ferbeyre, Gerardo

    2016-01-01

    Mutants of lamin A cause diseases including the Hutchinson-Gilford progeria syndrome (HGPS) characterized by premature aging. Lamin A undergoes a series of processing reactions, including farnesylation and proteolytic cleavage of the farnesylated C-terminal domain. The role of cleavage is unknown but mutations that affect this reaction lead to progeria. Here we show that interphase serine 22 phosphorylation of endogenous mutant lamin A (progerin) is defective in cells from HGPS patients. This defect can be mimicked by expressing progerin in human cells and prevented by inhibition of farnesylation. Furthermore, serine 22 phosphorylation of non-farnesylated progerin was enhanced by a mutation that disrupts lamin A head to tail interactions. The phosphorylation of lamin A or non-farnesylated progerin was associated to the formation of spherical intranuclear lamin A droplets that accumulate protein kinases of the CDK family capable of phosphorylating lamin A at serine 22. CDK inhibitors compromised the turnover of progerin, accelerated senescence of HGPS cells and reversed the effects of FTI on progerin levels. We discuss a model of progeria where faulty serine 22 phosphorylation compromises phase separation of lamin A polymers, leading to accumulation of functionally impaired lamin A structures. PMID:26922519

  12. Arsenic Metabolites, Including N-Acetyl-4-hydroxy-m-arsanilic Acid, in Chicken Litter from a Roxarsone-Feeding Study Involving 1600 Chickens.

    PubMed

    Yang, Zonglin; Peng, Hanyong; Lu, Xiufen; Liu, Qingqing; Huang, Rongfu; Hu, Bin; Kachanoski, Gary; Zuidhof, Martin J; Le, X Chris

    2016-07-01

    The poultry industry has used organoarsenicals, such as 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, ROX), to prevent disease and to promote growth. Although previous studies have analyzed arsenic species in chicken litter after composting or after application to agricultural lands, it is not clear what arsenic species were excreted by chickens before biotransformation of arsenic species during composting. We describe here the identification and quantitation of arsenic species in chicken litter repeatedly collected on days 14, 24, 28, 30, and 35 of a Roxarsone-feeding study involving 1600 chickens of two strains. High performance liquid chromatography separation with simultaneous detection by both inductively coupled plasma mass spectrometry and electrospray ionization tandem mass spectrometry provided complementary information necessary for the identification and quantitation of arsenic species. A new metabolite, N-acetyl-4-hydroxy-m-arsanilic acid (N-AHAA), was identified, and it accounted for 3-12% of total arsenic. Speciation analyses of litter samples collected from ROX-fed chickens on days 14, 24, 28, 30, and 35 showed the presence of N-AHAA, 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), inorganic arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), and ROX. 3-AHPAA accounted for 3-19% of the total arsenic. Inorganic arsenicals (the sum of As(III) and As(V)) comprised 2-6% (mean 3.5%) of total arsenic. Our results on the detection of inorganic arsenicals, methylarsenicals, 3-AHPAA, and N-AHAA in the chicken litter support recent findings that ROX is actually metabolized by the chicken or its gut microbiome. The presence of the toxic metabolites in chicken litter is environmentally relevant as chicken litter is commonly used as fertilizer. PMID:26876684

  13. A low-barrier hydrogen bond in the catalytic triad of serine proteases? Theory versus experiment.

    PubMed

    Ash, E L; Sudmeier, J L; De Fabo, E C; Bachovchin, W W

    1997-11-01

    Cleland and Kreevoy recently advanced the idea that a special type of hydrogen bond (H-bond), termed a low-barrier hydrogen bond (LBHB), may account for the "missing" transition state stabilization underlying the catalytic power of many enzymes, and Frey et al. have proposed that the H-bond between aspartic acid 102 and histidine 57 in the catalytic triad of serine proteases is an example of a catalytically important LBHB. Experimental facts are here considered regarding the aspartic acid-histidine and cis-urocanic H-bonds that are inconsistent with fundamental tenets of the LBHB hypothesis. The inconsistencies between theory and experiment in these paradigm systems cast doubt on the existence of LBHBs, as currently defined, within enzyme active sites. PMID:9353195

  14. Radiation-induced destruction of hydroxyl-containing amino acids and dipeptides

    NASA Astrophysics Data System (ADS)

    Sladkova, А. А.; Sosnovskaya, А. А.; Edimecheva, I. P.; Shadyro, О. I.

    2012-12-01

    The yields of molecular products resulting from radiolysis of hydroxyl-containing amino acids and dipeptides under various conditions were determined. The possibility of a new radiation-induced destruction pathway has been shown for serine and threonine, as well as for the dipeptides having residues of these amino acids at the N-terminal part of the respective molecule. This process includes formation of N-centered radicals from the starting molecules followed by their decomposition with elimination of side substituents. On radiolysis, serine and threonine were also shown to undergo free-radical destruction to form acetaldehyde and acetone, respectively. A mechanism has been proposed including consecutive stages of fragmentation of α-hydroxyl-containing carbon-centered radicals with elimination of ammonia and decomposition of the secondary radicals with elimination of CO2. The yields of CO2 obtained on radiolysis of serine and threonine were significantly higher (except for solutions at pH 12) than those for alanine and valine, which have no hydroxyl groups in their structures. The obtained data indicate that the hydroxyl-containing amino acids occupy a special place among other amino acids as regards the variety of radiation-induced reactions which they may undergo due to their structural features.

  15. Contribution of Eukaryotic-Type Serine/Threonine Kinase to Stress Response and Virulence of Streptococcus suis

    PubMed Central

    Zhu, Haodan; Zhou, Junming; Ni, Yanxiu; Yu, Zhengyu; Mao, Aihua; Hu, Yiyi; Wang, Wei; Zhang, Xuehan; Wen, Libin; Li, Bin; Wang, Xiaomin; Yu, Yang; Lv, Lixin; Guo, Rongli; Lu, Chengping; He, Kongwang

    2014-01-01

    Streptococcus suis serotype 2 (SS2) is an important swine and human pathogen responsible for septicemia and meningitis. The bacterial homologues of eukaryotic-type serine/threonine kinases (ESTKs) have been reported to play critical roles in various cellular processes. To investigate the role of STK in SS2, an isogenic stk mutant strain (Δstk) and a complemented strain (CΔstk) were constructed. The Δstk showed a significant decrease in adherence to HEp-2 cells, compared with the wild-type strain, and a reduced survival ratio in whole blood. In addition, the Δstk exhibited a notable reduced tolerance of environmental stresses including high temperature, acidic pH, oxidative stress, and high osmolarity. More importantly, the Δstk was attenuated in both the CD1 mouse and piglet models of infection. The results of quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that the expressions of a few genes involving in adherence, stress response and virulence were clearly decreased in the Δstk mutant strain. Our data suggest that SsSTK is required for virulence and stress response in SS2. PMID:24637959

  16. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    TECHNICAL ABSTRACT A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated the enzyme had...

  17. Clues to tRNA Evolution from the Distribution of Class II tRNAs and Serine Codons in the Genetic Code.

    PubMed

    Bernhardt, Harold S

    2016-01-01

    We have previously proposed that tRNA(Gly) was the first tRNA and glycine was the first amino acid incorporated into the genetic code. The next two amino acids incorporated would have been the other two small hydrophilic amino acids serine and aspartic acid, which occurred through the duplication of the tRNA(Gly) sequence, followed by mutation of its anticodon by single C to U transition mutations, possibly through spontaneous deamination. Interestingly, however, tRNA(Ser) has a different structure than most other tRNAs, possessing a long variable arm; because of this tRNA(Ser) is classified as a class II tRNA. Also, serine codons are found not only in the bottom right-hand corner of the genetic code table next to those for glycine and aspartic acid, but also in the top row of the table, next to those for two of the most hydrophobic amino acids, leucine and phenylalanine. In the following, I propose that the class II tRNA structure of tRNA(Ser) and the arrangement of serine codons in the genetic code provide clues to the early evolution of tRNA and the genetic code. In addition, I address Di Giulio's recent criticism of our proposal that tRNA(Gly) was the first tRNA, and discuss how early peptides produced from a restricted amino acid alphabet of glycine, serine and aspartic acid might have possessed proteolytic activity, which is possibly important for the early recycling of amino acid monomers. PMID:26927183

  18. Clues to tRNA Evolution from the Distribution of Class II tRNAs and Serine Codons in the Genetic Code

    PubMed Central

    Bernhardt, Harold S.

    2016-01-01

    We have previously proposed that tRNAGly was the first tRNA and glycine was the first amino acid incorporated into the genetic code. The next two amino acids incorporated would have been the other two small hydrophilic amino acids serine and aspartic acid, which occurred through the duplication of the tRNAGly sequence, followed by mutation of its anticodon by single C to U transition mutations, possibly through spontaneous deamination. Interestingly, however, tRNASer has a different structure than most other tRNAs, possessing a long variable arm; because of this tRNASer is classified as a class II tRNA. Also, serine codons are found not only in the bottom right-hand corner of the genetic code table next to those for glycine and aspartic acid, but also in the top row of the table, next to those for two of the most hydrophobic amino acids, leucine and phenylalanine. In the following, I propose that the class II tRNA structure of tRNASer and the arrangement of serine codons in the genetic code provide clues to the early evolution of tRNA and the genetic code. In addition, I address Di Giulio’s recent criticism of our proposal that tRNAGly was the first tRNA, and discuss how early peptides produced from a restricted amino acid alphabet of glycine, serine and aspartic acid might have possessed proteolytic activity, which is possibly important for the early recycling of amino acid monomers. PMID:26927183

  19. Hypochlorous acid via peroxynitrite activates protein kinase Cθ and insulin resistance in adipocytes

    PubMed Central

    Zhou, Jun; Wang, Qilong; Ding, Ye; Zou, Ming-Hui

    2015-01-01

    We recently reported that genetic deletion of myeloperoxidase (MPO) alleviates obesity-related insulin resistance in mice in vivo. How MPO impairs insulin sensitivity in adipocytes is poorly characterized. As hypochlorous acid (HOCl) is a principal oxidant product generated by MPO, we evaluated the effects of HOCl on insulin signaling in adipocytes differentiated from 3T3-L1 cells. Exposure of 3T3-L1 adipocytes to exogenous HOCl (200 μmol/l) attenuated insulin-stimulated 2-deoxyglucose uptake, GLUT4 translocation, and insulin signals, including tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and phosphorylation of Akt. Furthermore, treatment with HOCl induced phosphorylation of IRS1 at serine 307, inhibitor κB kinase (IKK), c-Jun NH2-terminal kinase (JNK), and phosphorylation of PKCθ (PKCθ). In addition, genetic and pharmacological inhibition of IKK and JNK abolished serine phosphorylation of IRS1 and impairment of insulin signaling by HOCl. Furthermore, knockdown of PKCθ using siRNA transfection suppressed phosphorylation of IKK and JNK and consequently attenuated the HOCl-impaired insulin signaling pathway. Moreover, activation of PKCθ by peroxynitrite was accompanied by increased phosphorylation of IKK, JNK, and IRS1-serine 307. In contrast, ONOO− inhibitors abolished HOCl-induced phosphorylation of PKCθ, IKK, JNK, and IRS1-serine 307, as well as insulin resistance. Finally, high-fat diet (HFD)-induced insulin resistance was associated with enhanced phosphorylation of PKCθ, IKK, JNK, and IRS1 at serine 307 in white adipose tissues from WT mice, all of which were not found in Mpo knockout mice fed HFDs. We conclude that HOCl impairs insulin signaling pathway by increasing ONOO− mediated phosphorylation of PKCθ, resulting in phosphorylation of IKK/JNK and consequent serine phosphorylation of IRS1 in adipocytes. PMID:25381390

  20. Association Study of Serine Racemase Gene with Methamphetamine Psychosis

    PubMed Central

    Yokobayashi, E; Ujike, H; Kotaka, T; Okahisa, Y; Takaki, M; Kodama, M; Inada, T; Uchimura, N; Yamada, M; Iwata, N; Iyo, M; Sora, I; Ozaki, N; Kuroda, S

    2011-01-01

    Experimental studies have demonstrated that not only dopaminergic signaling but also glutamatergic/NMDA receptor signaling play indispensable roles in the development of methamphetamine psychosis. Our recent genetic studies provided evidence that genetic variants of glutamate-related genes such as DTNBP1, GLYT1, and G72, which are involved in glutamate release and regulation of co-agonists for NMDA receptors, conferred susceptibility to methamphetamine psychosis. Serine racemase converts l-serine to d-serine, which is an endogenous co-agonist for NMDA receptors. Three single nucleotide polymorphisms (SNPs) in the promoter region of the serine racemase gene (SRR), rs224770, rs3760229, and rs408067, were proven to affect the transcription activity of SRR. Therefore, we examined these SNPs in 225 patients with methamphetamine psychosis and 291 age- and sex-matched controls. There was no significant association between methamphetamine psychosis and any SNP examined or between the disorder and haplotypes comprising the three SNPs. However, rs408067 was significantly associated with the prognosis for methamphetamine psychosis and multi-substance abuse status. The patients with C-positive genotypes (CC or CG) of rs408067 showed better prognosis of psychosis after therapy and less abuse of multiple substances than the patients with GG genotypes. Because the C allele of rs408067 reduces the expression of SRR, a lower d-serine level or reduced NMDA receptor activation may affect the prognosis of methamphetamine psychosis and multiple substance abuse. Our sample size is, however, not large enough to eliminate the possibility of a type I error, our findings must be confirmed by replicate studies with larger samples. PMID:21886585

  1. Effect of including carob pulp in the diet of fattening pigs on the fatty acid composition and oxidative stability of pork.

    PubMed

    Inserra, L; Luciano, G; Bella, M; Scerra, M; Cilione, C; Basile, P; Lanza, M; Priolo, A

    2015-02-01

    The effect of feeding pigs with carob pulp on meat quality was investigated. Nine pigs were finished on a conventional concentrate-based diet (control), while two groups received a diet comprising of the same ingredients with the inclusion of 8% or 15% carob pulp (Carob 8% and Carob 15%, respectively). Feeding carob-containing diets reduced the concentration of saturated fatty acids in the muscle, increased the concentration of monounsaturated fatty acids in meat (P < 0.01) and of n-3 polyunsaturated fatty acids (PUFAs) and reduced the n-6/n-3 PUFA ratio (P < 0.001). The meat underwent slow oxidative deterioration over 9 days of storage. However, the Carob 15% treatment increased meat susceptibility to lipid oxidation across storage (P = 0.03), while the dietary treatment did not affect meat colour stability. In conclusion, feeding pigs with carob pulp could represent a strategy,in the Mediterranean areas, to naturally improve meat nutritional value and to promote the exploitation of this local feed resource. PMID:25460134

  2. Serine versus threonine glycosylation with α-O-GalNAc: unexpected selectivity in their molecular recognition with lectins.

    PubMed

    Madariaga, David; Martínez-Sáez, Nuria; Somovilla, Víctor J; García-García, Laura; Berbis, M Álvaro; Valero-Gónzalez, Jessika; Martín-Santamaría, Sonsoles; Hurtado-Guerrero, Ramon; Asensio, Juan L; Jiménez-Barbero, Jesús; Avenoza, Alberto; Busto, Jesús H; Corzana, Francisco; Peregrina, Jesús M

    2014-09-22

    The molecular recognition of several glycopeptides bearing Tn antigen (α-O-GalNAc-Ser or α-O-GalNAc-Thr) in their structure by three lectins with affinity for this determinant has been analysed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the molecular recognition. In fact, while Soybean agglutinin and Vicia villosa agglutinin lectins prefer Tn-threonine, Helix pomatia agglutinin shows a higher affinity for the glycopeptides carrying Tn-serine. The different conformational behaviour of the two Tn biological entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topology of the binding site of the lectins are at the origin of these differences. PMID:25111627

  3. Difference in the dynamic properties of chiral and racemic crystals of serine studied by Raman spectroscopy at 3-295 K.

    PubMed

    Kolesov, B A; Boldyreva, E V

    2007-12-27

    Single-crystal polarized Raman spectra (60-4000 cm(-1) at 3 < or = T < or = 295 K) were measured for chiral L- and racemic DL-serine, alpha-amino-beta-hydroxypropionic acid, (NH3)+CH(CH2OH)(COO)-. The Raman spectra of dl-serine do not show any striking changes with temperature or on storage. In contrast to that, the dynamical properties of L-serine change at about 140 K. These changes can be interpreted as the reorientation of the side chain -CH2OH fragments of the zwitterions with respect to the backbone C-C bonds, resulting in the positional disorder of the O-H...O intermolecular H-bonds. The redistribution in the intensities of the Raman spectra of the crystals of L-serine stored for a long time (about a year) indicates the changes in the orientation of the molecular fragments in the direction normal to the axes of the head-to-tail chains. The difference in the thermodynamic functions of L- and DL-serine reported previously [Drebushchak, V. A.; Kovalevskaya, Yu. A.; Paukov, I. E.; Boldyreva, E. V. J. Therm. Anal. Calorim. 2007, 89 (2), 649-654] is explained by the difference in the spectra of external vibrations of the crystals. PMID:18052147

  4. Phosphorylation of HPV-16 E2 at Serine 243 Enables Binding to Brd4 and Mitotic Chromosomes

    PubMed Central

    Chang, Szu-Wei; Liu, Wei-Chen; Liao, Kuan-Yu; Tsao, Yeou-Ping; Hsu, Pang-Hung; Chen, Show-Li

    2014-01-01

    The papillomavirus E2 protein is involved in the maintenance of persistent infection and known to bind either to cellular factors or directly to mitotic chromosomes in order to partition the viral genome into the daughter cells. However, how the HPV-16 E2 protein acts to facilitate partitioning of the viral genome remains unclear. In this study, we found that serine 243 of HPV-16 E2, located in the hinge region, is crucial for chromosome binding during mitosis. Bromodomain protein 4 (Brd4) has been identified as a cellular binding target through which the E2 protein of bovine papillomavirus type 1 (BPV-1) tethers the viral genome to mitotic chromosomes. Mutation analysis showed that, when the residue serine 243 was substituted by glutamic acid or aspartic acid, whose negative charges mimic the effect of constitutive phosphorylation, the protein still can interact with Brd4 and colocalize with Brd4 in condensed metaphase and anaphase chromosomes. However, substitution by the polar uncharged residues asparagine or glutamine abrogated Brd4 and mitotic chromosome binding. Moreover, following treatment with the inhibitor JQ1 to release Brd4 from the chromosomes, Brd4 and E2 formed punctate foci separate from the chromosomes, further supporting the hypothesis that the association of the HPV-16 E2 protein with the chromosomes is Brd4-dependent. In addition, the S243A E2 protein has a shorter half-life than the wild type, indicating that phosphorylation of the HPV-16 E2 protein at serine 243 also increases its half-life. Thus, phosphorylation of serine 243 in the hinge region of HPV-16 E2 is essential for interaction with Brd4 and required for host chromosome binding. PMID:25340539

  5. The DEG15 Serine Protease Cleaves Peroxisomal Targeting Signal 2-Containing Proteins in Arabidopsis1[OA

    PubMed Central

    Schuhmann, Holger; Huesgen, Pitter F.; Gietl, Christine; Adamska, Iwona

    2008-01-01

    Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. Recently, candidates for the responsible processing protease were identified from plants (DEG15) and mammals (TYSND1). We demonstrate that plants lacking DEG15 show an expressed phenotype potentially linked to reduced β-oxidation, indicating the impact of protein processing on peroxisomal functions in higher eukaryotes. Mutational analysis of Arabidopsis (Arabidopsis thaliana) DEG15 revealed that conserved histidine, aspartic acid, and serine residues are essential for the proteolytic activity of this enzyme in vitro. This indicates that DEG15 and related enzymes are trypsin-like serine endopeptidases. Deletion of a plant-specific stretch present in the protease domain diminished, but did not abolish, the proteolytic activity of DEG15 against the PTS2-containing glyoxysomal malate dehydrogenase. Fluorescence microscopy showed that a DEG15-green fluorescent protein fusion construct is targeted to peroxisomes in planta. In vivo studies with isolated homozygous deg15 knockout mutants and complemented mutant lines suggest that this enzyme mediates general processing of PTS2-containing proteins. PMID:18952862

  6. Contribution of the Serine 129 of Histone H2A to Chromatin Structure▿

    PubMed Central

    Fink, Michel; Imholz, Daniela; Thoma, Fritz

    2007-01-01

    Phosphorylation of a yeast histone H2A at C-terminal serine 129 has a central role in double-strand break repair. Mimicking H2A phosphorylation by replacement of serine 129 with glutamic acid (hta1-S129E) suggested that phosphorylation destabilizes chromatin structures and thereby facilitates the access of repair proteins. Here we have tested chromatin structures in hta1-S129 mutants and in a C-terminal tail deletion strain. We show that hta1-S129E affects neither nucleosome positioning in minichromosomes and genomic loci nor supercoiling of minichromosomes. Moreover, hta1-S129E has no effect on chromatin stability measured by conventional nuclease digestion, nor does it affect DNA accessibility and repair of UV-induced DNA lesions by nucleotide excision repair and photolyase in vivo. Similarly, deletion of the C-terminal tail has no effect on nucleosome positioning and stability. These data argue against a general role for the C-terminal tail in chromatin organization and suggest that phosphorylated H2A, γ-H2AX in higher eukaryotes, acts by recruitment of repair components rather than by destabilizing chromatin structures. PMID:17353265

  7. Analysis of the serine protease function of porcine factor I produced by liver cells for xenotransplantation.

    PubMed

    Nakahata, Kengo; Matsunami, Katsuyoshi; Kobayashi, Chizuko; Omori, Takeshi; Xu, Hengjie; Firdawes, Sabere; Fukuzawa, Masahiro; Miyagawa, Shuji

    2008-04-01

    The use of a bioartificial liver with pig liver cells in the treatment of fulminant hepatic failure has already begun as a clinical trial in several countries. Therefore, studies on plasma complement regulatory proteins of the pig are necessary, because the liver produces them. Complement factor I is a serine protease that cleaves C3b and C4b. The DNA sequences of factor I have been reported in many species, with the noted exception of pigs. In this study, porcine factor I was cloned and an open reading frame of 1794 base pairs were identified. The predicted amino acid sequence maintained a relatively high homology compared to those of other mammals, especially in the serine protease (SP) region. The cell membrane-bound forms of the porcine and the human SP domain of factor I were constructed. Amelioration of complement-mediated cell lysis by these molecules was then tested, using several kinds of sera and Chinese hamster ovary (CHO) cell transfectants. Both molecules had a suppressing effect on pig, human and dog complements, indicating little species-specificity. Further studies of other plasma complement regulatory proteins produced from pig liver cells will need to be considered as the primary feature of the pig bioartificial liver. PMID:18346635

  8. A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex.

    PubMed

    Siritapetawee, Jaruwan; Thumanu, Kanjana; Sojikul, Punchapat; Thammasirirak, Sompong

    2012-07-01

    A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis. PMID:22579962

  9. A tri-serine tri-lactone scaffold for the quantification of citrate in urine.

    PubMed

    Akdeniz, Ali; Caglayan, Mehmet Gokhan; Anzenbacher, Pavel

    2016-01-31

    Tri-serine tri-lactone based C3 symmetry fluorescent sensors were synthesized. Citrate is shown to bind to sensors, while displaying an increase in fluorescence intensity for the sensor with thiourea and a quenching for the sensor with sulfonamide. Information-rich responses of the sensors enable us to discriminate structurally similar anions, including mono-, di- and tri-carboxylates with 100% correct classification. A simple two-sensor array enables the determination of the concentration of citrate in urine without any sample preparation with high accuracy (error < 2%). PMID:26669653

  10. Silicone hydrogels grafted with natural amino acids for ophthalmological application.

    PubMed

    Xu, Chen; He, Ruiyu; Xie, Binbin; Ismail, Muhammad; Yao, Chen; Luan, Jie; Li, Xinsong

    2016-09-01

    In this report, protein repelling silicone hydrogels with improved hydrophilicity were prepared by photo-polymerization of silicone-containing monomer and glycidyl methacrylate followed by grafting zwitterionic amino acids. The grafted silicone hydrogels possessed excellent hydrophilic surfaces due to the enrichment of amino acids, which was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, contact angle, and equilibrium water content measurements. Remarkable resistance to bovine serum albumin and lysozyme fouling was observed for the silicone hydrogels immobilized with neutrally charged amino acids because of the formation of zwitterionic surfaces with pairs of protonated secondary ammonium cations and deprotonated carboxyl anions. Meanwhile, the silicone hydrogels grafted with positively or negatively charged amino acids were able to repulse same charged protein with reduced deposition and attract oppositely charged protein with increased adsorption. Preliminary cytotoxicity test indicated that the zwitterionic silicone hydrogels were non-cytotoxic. Similarly, three types of natural amino acids, including serine, aspartic acid and histidine, modified silicone hydrogel contact lenses exhibited excellent hydrophilicity and non-damage to the rabbit's eyes, but only serine modified zwitterionic contact lens showed superior protein fouling resistance compared with the current commercial hydrogel contact lens, which may have great potential application in ophthalmology. PMID:27297564

  11. Sensitive determination of D-amino acids in mammals and the effect of D-amino-acid oxidase activity on their amounts.

    PubMed

    Hamase, Kenji; Konno, Ryuichi; Morikawa, Akiko; Zaitsu, Kiyoshi

    2005-09-01

    The determination of small amounts of D-amino acids in mammalian tissues is still a challenging theme in the separation sciences. In this review, various gas-chromatographic and high-performance liquid chromatographic methods are discussed including highly selective and sensitive column-switching procedures. Based on these methods, the distributions of D-aspartic acid, D-serine, D-alanine, D-leucine and D-proline have been clarified in the mouse brain. As the regulation mechanisms of D-amino acid amounts in mammals, we focused on the D-amino-acid oxidase, which catalyzes the degradation of D-amino acids. Using the mutant mouse strain lacking D-amino-acid oxidase activity, the effects of the enzymatic activity on the amounts and distributions of various D-amino acids have been investigated. PMID:16141519

  12. Amino-terminal analysis of tryptophan hydroxylase: protein kinase phosphorylation occurs at serine-58.

    PubMed

    Kumer, S C; Mockus, S M; Rucker, P J; Vrana, K E

    1997-10-01

    Tryptophan hydroxylase (TPH) catalyzes the rate-limiting and committed step in serotonin biosynthesis. Within this enzyme, two distinct domains have been hypothesized to exist, an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. In the present experiments, the functional boundary between the putative domains was defined using deletion mutagenesis. A full-length cDNA clone for rabbit TPH was engineered for expression in bacteria. Five amino-terminal deletions were constructed using PCR, i.e., Ndelta50, Ndelta60, Ndelta90, Ndelta106, and Ndelta116 (referring to the number of amino acids deleted from the amino terminus). Enzymatic activity was determined for each mutant after expression in bacteria. Whereas deletion of 116 amino acids (Ndelta116) abolished enzyme activity, all of the other amino-terminal deletions exhibited increased specific activity relative to the recombinant wild-type TPH. The ability of the cyclic AMP-dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined. Deletion of the first 60 amino-terminal residues abolished the ability of the enzyme to serve as a substrate for PKA, yet the native and Ndelta50 enzymes were phosphorylated. Moreover, a serine-58 point mutant (S58A) was not phosphorylated by PKA. In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine-58. In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase. PMID:9326303

  13. Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

    PubMed

    Beuvink, I; Hess, D; Flotow, H; Hofsteenge, J; Groner, B; Hynes, N E

    2000-04-01

    The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration. PMID:10744710

  14. Fetal serine fluxes across fetal liver, hindlimb, and placenta in late gestation.

    PubMed

    Cetin, I; Fennessey, P V; Sparks, J W; Meschia, G; Battaglia, F C

    1992-10-01

    Eleven studies of fetal serine fluxes were performed in chronically catheterized fetal lambs by continuous infusion of [1-13C]- and [U-14C]serine into a fetal brachial vein. At tracer serine steady state, samples were collected from the fetal abdominal aorta, umbilical vein, fetal hepatic vein, and fetal femoral vein and from the maternal femoral artery and uterine vein. Analyses were performed for plasma serine and glycine concentration, for serine and glycine 13C mole percent enrichment, and for whole blood 14CO2 and O2 concentrations. Uterine and umbilical blood flows were also measured. The placenta had a significant net uptake of fetal serine (2.1 +/- 0.5 mumol.min-1.kg-1, P < 0.01). Fetal plasma serine disposal rate (DR) was 42.5 +/- 3.9 mumol.min-1.kg-1.CO2 production from decarboxylation of fetal plasma serine represented 7.9 +/- 0.5% of DR, or 10.1 +/- 1.2 mumol CO2.min-1.kg-1. Fetal plasma glycine enrichment was 59.7 +/- 4.9% of fetal plasma serine enrichment. There was a significant loss of tracer serine from the fetal circulation into the placenta accounting for approximately 45% of infused tracer. Fifteen percent of this was converted to glycine and released into the umbilical circulation. There was a significant uptake of tracer serine by both fetal liver and fetal hindlimb with a significant CO2 production by both sites with serine oxidation predominantly in the carcass. These results indicate a high fetal serine disposal rate in the lamb, with rapid fetoplacental serine exchange, resulting in a net uptake of fetal serine by the placenta.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1415701

  15. Amino acid profile during exercise and training in Standardbreds.

    PubMed

    Westermann, C M; Dorland, L; Wijnberg, I D; de Sain-van der Velden, M G M; van Breda, E; Barneveld, A; de Graaf-Roelfsema, E; Keizer, H A; van der Kolk, J H

    2011-08-01

    The objective of this study is to assess the influence of acute exercise, training and intensified training on the plasma amino acid profile. In a 32-week longitudinal study using 10 Standardbred horses, training was divided into four phases, including a phase of intensified training for five horses. At the end of each phase, a standardized exercise test, SET, was performed. Plasma amino acid concentrations before and after each SET were measured. Training significantly reduced mean plasma aspartic acid concentration, whereas exercise significantly increased the plasma concentrations of alanine, taurine, methionine, leucine, tyrosine and phenylalanine and reduced the plasma concentrations of glycine, ornithine, glutamine, citrulline and serine. Normally and intensified trained horses differed not significantly. It is concluded that amino acids should not be regarded as limiting training performance in Standardbreds except for aspartic acid which is the most likely candidate for supplementation. PMID:20863542

  16. Plasmodium falciparum: an epitope within a highly conserved region of the 47-kDa amino-terminal domain of the serine repeat antigen is a target of parasite-inhibitory antibodies.

    PubMed

    Fox, B A; Xing-Li, P; Suzue, K; Horii, T; Bzik, D J

    1997-02-01

    Previously, the Plasmodium falciparum serine repeat antigen has been shown to be protective in primate models of malaria immunity and also to be a target of in vitro parasite-inhibitory antibodies. To further define parasite-inhibitory epitopes a series of deletions from the amino-terminal 47-kDa domain of the serine repeat antigen (SERA) were constructed as glutathione-S-transferase fusion proteins. Several GST-SERA fusion proteins were used to vaccinate mice with Freund's adjuvant and the resulting immune sera were used to assay for the inhibition of P. falciparum invasion of erythrocytes in vitro. The minimal epitope shown to be the target of invasion-blocking antibodies was SERA amino acids 17-165. Additional GST-SERA deletion constructs of the 47-kDa domain were developed and evaluated for reactivity, by Western immunoblot analysis, with a parasite-inhibitory murine monoclonal antibody (mAb 43E5), a parasite-inhibitory pooled goat polyclonal sera, and a pooled human Nigerian immune serum. The parasite-inhibitory epitope defined by mAb 43E5 was mapped to SERA amino acids 17-110 and, at least, part of the epitope was defined to include amino acids in the region of amino acids 59-72. The parasite-inhibitory epitope recognized by mAb 43E5 appears to be well conserved between diverse geographical isolates of P. falciparum. The results have relevance for malaria vaccine development and suggest that an appropriately designed recombinant SERA antigen produced from a synthetic gene in Escherichia coli may be an effective component of a candidate malaria vaccine. PMID:9030663

  17. Ultraviolet-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 2. Oxidation of serine in the 23-kDa NH/sub 2/-terminal tryptic peptide

    SciTech Connect

    Cremo, C.R.; Grammer, J.C.; Yount, R.G.

    1988-11-01

    Myosin subfragment 1 (S1) can be specifically photomodified at the active site without polypeptide chain cleavage by irradiating the stable MgADP-orthovanadate-S1 complex with UV light above 300 nm. Here, the UV spectral properties of photomodified S1 were used to determined the nature and location of the photomodified residue(s) within S1. By comparison of the unusual pH dependence of the UV absorption spectrum of the photomodified S1 to that of the S1-MgADP-V/sub i/ complex as a control the photomodified residue(s) was (were) localized to the 23-kDa NH/sub 2/-terminal tryptic peptide of the heavy chain. NaBH/sub 4/ reduced the photomodified S1, but not the control, to regenerate the original spectral properties and ATPase activities of the unmodified S1. Amino acid analysis of photomodified S1 reduced with NaB/sup 3/H/sub 4/ gave only (/sup 3/H)serine, suggesting the hydroxyl group of serine had been oxidized to a serine aldehyde. The pH dependence of the absorption spectrum of the photomodified enzyme can be explained by an equilibrium between a chromophoric enolate anion of the serine aldehyde (favored in base) and less chromophoric keto and enol forms (favored in acid). The oxidized serine(s) was (were) shown to be directly involved with the vanadate-dependent photocleavage of the S1 heavy chain previously described by Grammer et al. (1988). This serine(s) is (are) likely to be important to the binding and hydrolysis of the ..gamma..-PO/sub 4/ of ATP at the active site of S1.

  18. Evolution of serine carboxypeptidase-like acyltransferases in the monocots

    PubMed Central

    Mugford, Sam T

    2010-01-01

    The serine carboxypeptidases are a large family of proteases. in higher plants some members of this family have diversified and adopted new functions as acyltransferases required for the synthesis of natural products. we recently reported the first serine carboxypeptidase-like (scpl) acyltransferase enzyme to be characterized from monocotyledonous plants.1 This enzyme, AsSCPL1, is required for acylation of antimicrobial terpenes (avenacins) that are produced in the roots of oat (Avena spp.) and that provide protection against soil-borne pathogens. The SCPL acyltransferase enzyme family has undergone substantial expansion following the divergence of monocots and dicots. Here we discuss the evolution of this SCPL enzyme family in monocots, their contribution to metabolic diversity, and the roles of these enzymes in biotic and abiotic stress tolerance. PMID:20173416

  19. Probing protein stability with unnatural amino acids

    SciTech Connect

    Mendel, D.; Ellman, J.A.; Zhiyuh Chang; Veenstra, D.L.; Kollman, P.A.; Schultz, P.G. )

    1992-06-26

    Unnatural amino acid mutagenesis, in combination with molecular modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (1) leucine (wt), norvaline, ethylglycine, and alanine to measure the cost of stepwise removal of methyl groups from the hydrophobic core, (2) norvaline and O-methyl serine to evaluate the effects of side chain solvation, and (3) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing density and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.

  20. Dynamics simulation of the interaction between serine and water

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Zhang, Peng; Lu, Ying-Bo; Han, Sheng-Hao; Yu, Hui

    2013-05-01

    Using the first principles density functional theory (DFT), we simulated the neutron scattering spectra of the hydration dynamics of serine. Experimental data analyses have shown that dissociative H2O molecules were more likely to form hydrogen bonds (H-bonds) with an -OH group in monohydrated serine and easily shift to a -NH_3 ^ + group at a higher hydration level [P. Zhang, Y. Zhang, S. H. Han, Q. W. Yan, R. C. Ford, and J. C. Li, J. Phys. Chem. A 110, 5000 (2006), 10.1021/jp0569741]. We set the 1:1 ratio hydrated compounds at the two positions and found that the H2O could be optimized to form H-bonds with -OH and -NH3+ separately. When the simulated phonon signals of the -OH…H2O and -NH3+…H2O combinations were summed on a 3:1 scale, the calculating spectra were in good agreement with the experimental results, especially for the peak at 423 cm-1 of the -OH…H2O combination and the peak at 367 cm-1 of the -NH3+…H2O combination, which mutually complemented the real spectrum. We confirm that H2O may break the intermolecular H-bonds of the interlaced binding -OH to form a new structure, and that with the skeleton deformation of serine, H2O forms stronger H-bonds more often with the -NH3+ side indicating the flexible dynamic mechanism of the serine hydration process.

  1. Serine racemase: a key player in apoptosis and necrosis

    PubMed Central

    Canu, Nadia; Ciotti, Maria Teresa; Pollegioni, Loredano

    2014-01-01

    A fine balance between cell survival and cell death is required to sculpt the nervous system during development. However, an excess of cell death can occur following trauma, exposure to neurotoxins or alcohol, and some developmental and neurodegenerative diseases, such as Alzheimer's disease (AD). N-Methyl-D-aspartate receptors (NMDARs) support synaptic plasticity and survival of many neuronal populations whereas inappropriate activation may promote various forms of cell death, apoptosis, and necrosis representing the two extremes of a continuum of cell death processes both “in vitro” and “in vivo.” Hence, by identifying the switches controlling pro-survival vs. apoptosis and apoptosis vs. pro-excitotoxic outcome of NMDAR stimulation, NMDAR modulators could be developed that selectively block the cell death enhancing pro-survival signaling or synaptic plasticity mediated by NMDAR. Among these modulators, a role is emerging for the enzyme serine racemase (SR) that synthesizes D-serine, a key co-agonist with glutamate at NMDAR. This review summarizes the experimental evidence from “in vitro” neuronal cultures—with special emphasis on cerebellar granule neurons (CGNs)—and “in vivo” models of neurodegeneration, where the dual role of the SR/D-serine pathway as a master regulator of apoptosis and the apoptosis-necrosis shift will be discussed. PMID:24795622

  2. Structural Basis for Catalytic Activation of a Serine Recombinase

    SciTech Connect

    Keenholtz, Ross A.; Rowland, Sally-J.; Boocock, Martin R.; Stark, W. Marshall; Rice, Phoebe A.

    2014-10-02

    Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 {angstrom} crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.

  3. Inhibition of Bcr serine kinase by tyrosine phosphorylation.

    PubMed Central

    Liu, J; Wu, Y; Ma, G Z; Lu, D; Haataja, L; Heisterkamp, N; Groffen, J; Arlinghaus, R B

    1996-01-01

    The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired. PMID:8622703

  4. Osteogenesis from Dental Pulp Derived Stem Cells: A Novel Conditioned Medium Including Melatonin within a Mixture of Hyaluronic, Butyric, and Retinoic Acids

    PubMed Central

    Maioli, Margherita; Basoli, Valentina; Santaniello, Sara; Cruciani, Sara; Delitala, Alessandro Palmerio; Pinna, Roberto; Milia, Egle; Grillari-Voglauer, Regina; Fontani, Vania; Rinaldi, Salvatore; Muggironi, Roberta; Pigliaru, Gianfranco; Ventura, Carlo

    2016-01-01

    Human dental pulp stem cells (hDPSCs) have shown relevant potential for cell therapy in the orthopedic and odontoiatric fields. The optimization of their osteogenic potential is currently a major challenge. Vascular endothelial growth factor A (VEGF A) has been recently reported to act as a major conductor of osteogenesis in vitro and in vivo. Here, we attempted to prime endogenous VEGF A expression without the need for viral vector mediated gene transfer technologies. We show that hDPSCs exposure to a mixture of hyaluronic, butyric, and retinoic acids (HA + BU + RA) induced the transcription of a gene program of osteogenesis and the acquirement of an osteogenic lineage. Such response was also elicited by cell exposure to melatonin, a pleiotropic agent that recently emerged as a remarkable osteogenic inducer. Interestingly, the commitment to the osteogenic fate was synergistically enhanced by the combinatorial exposure to a conditioned medium containing both melatonin and HA + BU + RA. These in vitro results suggest that in vivo osteogenesis might be improved and further studies are needed. PMID:26880937

  5. Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2.

    PubMed

    Chen, Lei-Lei; Liu, Li-Jun; Shi, Mei; Song, Xiao-Yan; Zheng, Chang-Ying; Chen, Xiu-Lan; Zhang, Yu-Zhong

    2009-10-01

    Trichoderma pseudokoningii SMF2 is a biocontrol fungus with inhibitory ability against phytopathogenic fungi. Here, a crude extract of strain SMF2 in a solid ferment exhibited strong nematicidal activity against Meloidogyne incognita, and a novel serine protease SprT with nematicidal activity was purified from the crude extract. Protease SprT has a molecular mass of 31 kDa, a pH optimum of 8.5, and a temperature optimum of 60-65 degrees C. It had good thermostability, and was stable in an alkaline environment. SprT could degrade bovine serum albumin, lysozyme, and gelatin, and its activity was enhanced by many metal ions. The cuticles of nematodes treated by protease SprT obviously crimpled. Purified protease SprT could kill juveniles of M. incognita and inhibit egg hatch, suggesting that it is involved in the nematicidal process of T. pseudokoningii SMF2. The full-length cDNA gene-encoding protease SprT was cloned by rapid amplification of cDNA ends. Sequence analysis showed that SprT is a monodomain subtilase containing 284 amino acid residues. It had higher identities and a closer relation to the nematicidal serine proteases (59-69%) from nematode parasitic fungi than to the serine proteases (<50%) from Trichoderma. Protease SprT represents the first well-characterized subtilase with nematicidal activity from Trichoderma. PMID:19702879

  6. Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom.

    PubMed

    He, Ying-Ying; Liu, Shu-Bai; Lee, Wen-Hui; Qian, Jin-Qiao; Zhang, Yun

    2008-10-01

    Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities. PMID:18582511

  7. Zn(2+) and Cd(2+) cationized serine complexes: infrared multiple photon dissociation spectroscopy and density functional theory investigations.

    PubMed

    Coates, Rebecca A; Boles, Georgia C; McNary, Christopher P; Berden, Giel; Oomens, Jos; Armentrout, P B

    2016-08-10

    The gas-phase structures of zinc and cadmium dications bound to serine (Ser) are investigated by infrared multiple photon dissociation (IRMPD) action spectroscopy using the free electron laser FELIX, in combination with ab initio calculations. To identify the structures of the experimentally observed species, [Zn(Ser-H)CH3CN](+) and CdCl(+)(Ser), the measured action spectra are compared to linear absorption spectra calculated at the B3LYP/6-311+G(d,p) level for Zn(2+) containing complexes and B3LYP/def2-TZVP levels for Cd(2+) containing complexes. Good agreement between the observed IRMPD spectra and the predicted spectra allows identification of the isomers present. The intact amino acid interacting with cadmium chloride adopts a tridentate chelation involving the amino acid backbone amine and carbonyl groups as well as the hydroxyl group of the side-chain, [N,CO,OH]. The presence of two low-energy conformers is observed for the deprotonated serine-zinc complex, with the same tridentate coordination as for the cadmium complex but proton loss occurs at both the hydroxyl side-chain, [N,CO,O(-)], and the carboxylic acid of the amino acid backbone, [N,CO(-),OH]. These results are profitably compared with the analogous results previously obtained for comparable complexes with cysteine. PMID:27465924

  8. Perturbation response scanning specifies key regions in subtilisin serine protease for both function and stability.

    PubMed

    Abdizadeh, Haleh; Guven, Gokce; Atilgan, Ali Rana; Atilgan, Canan

    2015-12-01

    Can one infer the amino acids of the enzymes that are responsible for the stability or the level of the catalytic activity by computationally experimenting on the inhibited enzyme in the enzyme-inhibitor complex? In this article, we answer this question positively both by designing molecular dynamics simulations and by devising coarse-grained methodologies on the subtilisin serine protease. Both methodologies are based on the cross-correlations of the fluctuations of the residues, obtained either by monitoring the trajectories from the simulation or by constructing the inverse Laplacian of the elastic network model, of the complex. A perturbation scanning is applied to the complex using these correlations. The results indicate that the two methods almost point out the same regions on the flexible of the enzyme. These regions are: (i) 50-61, (ii) 155-164 and (iii) 192-194, all of which are designated to be important by experimental studies in the literature. PMID:25643757

  9. Molecular dynamic and docking interaction study of Heterodera glycines serine proteinase with Vigna mungo proteinase inhibitor.

    PubMed

    Prasad, C V S Siva; Gupta, Saurabh; Gaponenko, Alex; Tiwari, Murlidhar

    2013-08-01

    Many plants do produce various defense proteins like proteinase inhibitors (PIs) to protect them against various pests. PIs function as pseudosubstrates of digestive proteinase, which inhibits proteolysis in pests and leads to amino acid deficiency-based mortality. This work reports the structural interaction studies of serine proteinase of Heterodera glycines (SPHG) with Vigna mungo proteinase inhibitor (VMPI). 3D protein structure modeling, validation of SPHG and VMPI, and their putative protein-protein binding sites were predicted. Protein-protein docking followed by molecular dynamic simulation was performed to find the reliable confirmation of SPHG-VMPI complex. Trajectory analysis of each successive conformation concludes better interaction of first loop in comparison with second loop. Lysine residues of first loop were actively participating in complex formation. Overall, this study discloses the structural aspects and interaction mechanisms of VMPI with SPHG, and it would be helpful in the development of pest-resistant genetically modified crops. PMID:23813339

  10. A fluorescence-based demonstration of intestinal villi and epithelial cell in chickens fed dietary silicic acid powder including bamboo vinegar compound liquid.

    PubMed

    Ruttanavut, J; Matsumoto, Y; Yamauchi, K

    2012-10-01

    This study investigates the combined effect of silicic acid and bamboo vinegar compound liquid (SPV) on the growth and intestinal histological alterations in poultry. Forty-eight 7-day-old male Sanuki Cochin chickens were fed a commercial mash diet supplemented with SPV at 0, 0.1, 0.2, and 0.3% level ad libitum for 112 days. Body weight gain tended to improve with increased concentrations of dietary SPV, although these results were not statistically significant (P<0.1). Tissue observation by light microscopy revealed that the jejunal villus height (P<0.01) and duodenal and jejunal villus area (P<0.05) increased in the 0.2 and 0.3% SPV groups, respectively, compared with the control. Cell mitosis within the duodenum and jejunum also increased in the 0.2 and 0.3% SPV groups. Scanning electron microscopy revealed a prominent increase in the number of protuberant cells on the villus apical surface of the duodenum and jejunum for the 0.2 and 0.3% SPV groups compared with the control. Poultry in the 0.3% SPV group had the highest body weight gain and hypertrophied histological alterations of intestinal villi. Fluorescent microscopic images of cell mitosis and protuberant cells in the duodenal crypt clearly confirmed positive reactions for the activator protein 2α (AP-2α) and proliferating cell nuclear antigen (PCNA), compared with the control. The present results indicate that dietary SPV stimulates adsorption by the epithelial cells, which activate cell proliferation and self-renewal and regulate the expression of cell cycle regulators AP-2α and PCNA, resulting in higher body weight gain. Thus, we can conclude that a concentration of 0.3% dietary SPV is ideal for promoting growth in poultry. PMID:22936452

  11. Dihydropyrimidinone positive modulation of delta-subunit-containing gamma-aminobutyric acid type A receptors, including an epilepsy-linked mutant variant.

    PubMed

    Lewis, Ryan W; Mabry, John; Polisar, Jason G; Eagen, Kyle P; Ganem, Bruce; Hess, George P

    2010-06-15

    Gamma-aminobutyric acid type A receptors (GABA(A) receptors) are ligand-gated chloride channels that play a central role in signal transmission within the mammalian central nervous system. Compounds that modulate specific GABA(A) receptor subtypes containing the delta-subunit are scarce but would be valuable research tools and starting points for potential therapeutic agents. Here we report a class of dihydropyrimidinone (DHPM) heterocycles that preferentially potentiate peak currents of recombinant GABA(A) receptor subtypes containing the delta-subunit expressed in HEK293T cells. Using the three-component Biginelli reaction, 13 DHPMs with structural features similar to those of the barbiturate phenobarbital were synthesized; one DHPM used (monastrol) is commercially available. An up to approximately 3-fold increase in the current from recombinant alpha1beta2delta receptors was observed with the DHPM compound JM-II-43A or monastrol when co-applied with saturating GABA concentrations, similar to the current potentiation observed with the nonselective potentiating compounds phenobarbital and tracazolate. No agonist activity was observed for the DHPMs at the concentrations tested. A kinetic model was used in conjunction with dose-dependent measurements to calculate apparent dissociation constant values for JM-II-43A (400 muM) and monastrol (200 microM) at saturating GABA concentrations. We examined recombinant receptors composed of combinations of subunits alpha1, alpha4, alpha5, alpha6, beta2, beta3, gamma2L, and delta with JM-II-43A to demonstrate the preference for potentiation of delta-subunit-containing receptors. Lastly, reduced currents from receptors containing the mutated delta(E177A) subunit, described by Dibbens et al. [(2004) Hum. Mol. Genet. 13, 1315-1319] as a heritable susceptibility allele for generalized epilepsy with febrile seizures plus, are also potentiated by these DHPMs. PMID:20450160

  12. A Trigger Residue for Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor

    PubMed Central

    Kitanovic, Smiljka; Ames, Peter

    2015-01-01

    ABSTRACT The transmembrane Tsr protein of Escherichia coli mediates chemotactic responses to environmental serine gradients. Serine binds to the periplasmic domain of the homodimeric Tsr molecule, promoting a small inward displacement of one transmembrane helix (TM2). TM2 piston displacements, in turn, modulate the structural stability of the Tsr-HAMP domain on the cytoplasmic side of the membrane to control the autophosphorylation activity of the signaling CheA kinase bound to the membrane-distal cytoplasmic tip of Tsr. A five-residue control cable segment connects TM2 to the AS1 helix of HAMP and transmits stimulus and sensory adaptation signals between them. To explore the possible role of control cable helicity in transmembrane signaling by Tsr, we characterized the signaling properties of mutant receptors with various control cable alterations. An all-alanine control cable shifted Tsr output toward the kinase-on state, whereas an all-glycine control cable prevented Tsr from reaching either a fully on or fully off output state. Restoration of the native isoleucine (I214) in these synthetic control cables largely alleviated their signaling defects. Single amino acid replacements at Tsr-I214 shifted output toward the kinase-off (L, N, H, and R) or kinase-on (A and G) states, whereas other control cable residues tolerated most amino acid replacements with little change in signaling behavior. These findings indicate that changes in control cable helicity might mediate transitions between the kinase-on and kinase-off states during transmembrane signaling by chemoreceptors. Moreover, the Tsr-I214 side chain plays a key role, possibly through interaction with the membrane interfacial environment, in triggering signaling changes in response to TM2 piston displacements. IMPORTANCE The Tsr protein of E. coli mediates chemotactic responses to environmental serine gradients. Stimulus signals from the Tsr periplasmic sensing domain reach its cytoplasmic kinase control

  13. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by. beta. -chloroalanine

    SciTech Connect

    Medlock, K.A.; Merrill, A.H. Jr.

    1988-09-06

    The effects of ..beta..-chloroalanine (..beta..-Cl-alanine) on the serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid, irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with ..beta..-Cl-L-alanine and was blocked by L-serine. These are characteristics of mechanism-based (suicide) inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with ..beta..-Cl-alanine and this treatment inhibited (/sup 14/C)serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of ..beta..-Cl-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, (/sup 14/C)serine uptake was not blocked, total lipid biosynthesis from (/sup 14/C)acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and (/sup 3/H)thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of (/sup 3/H)leucine, which was only decreased by 14%. Although ..beta..-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that ..beta..-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.

  14. Ligand Discovery for the Alanine-Serine-Cysteine Transporter (ASCT2, SLC1A5) from Homology Modeling and Virtual Screening

    PubMed Central

    Gameiro, Armanda; Albers, Thomas; Singh, Kurnvir; Shere, Helen; Bonomi, Massimiliano; Holst, Jeff; Schlessinger, Avner

    2015-01-01

    The Alanine-Serine-Cysteine transporter ASCT2 (SLC1A5) is a membrane protein that transports neutral amino acids into cells in exchange for outward movement of intracellular amino acids. ASCT2 is highly expressed in peripheral tissues such as the lung and intestines where it contributes to the homeostasis of intracellular concentrations of neutral amino acids. ASCT2 also plays an important role in the development of a variety of cancers such as melanoma by transporting amino acid nutrients such as glutamine into the proliferating tumors. Therefore, ASCT2 is a key drug target with potentially great pharmacological importance. Here, we identify seven ASCT2 ligands by computational modeling and experimental testing. In particular, we construct homology models based on crystallographic structures of the aspartate transporter GltPh in two different conformations. Optimization of the models’ binding sites for protein-ligand complementarity reveals new putative pockets that can be targeted via structure-based drug design. Virtual screening of drugs, metabolites, fragments-like, and lead-like molecules from the ZINC database, followed by experimental testing of 14 top hits with functional measurements using electrophysiological methods reveals seven ligands, including five activators and two inhibitors. For example, aminooxetane-3-carboxylate is a more efficient activator than any other known ASCT2 natural or unnatural substrate. Furthermore, two of the hits inhibited ASCT2 mediated glutamine uptake and proliferation of a melanoma cancer cell line. Our results improve our understanding of how substrate specificity is determined in amino acid transporters, as well as provide novel scaffolds for developing chemical tools targeting ASCT2, an emerging therapeutic target for cancer and neurological disorders. PMID:26444490

  15. [Evaluation of ten fish species to be included as part of renal diet, due to their protein, phosphorus and fatty acids content].

    PubMed

    Castro-González, Maria Isabel; Maafs-Rodríguez, Ana Gabriela; Pérez-Gil Romo, Fernando

    2012-06-01

    Because renal disease is highly complex, its nutritional treatment is complicated and many foods are restricted, including fish because its phosphorus content. The aim of the present study was to analyze ten fillet fish species, commonly consumed in Mexico (Cyprinus carpio carpio, Ophichthus rex, Symphurus elongatus, Eucinostomus entomelas, Chirostoma patzcuaro, Bairdiella chrysoura, Salmo salar Oreochromis urolepis hornorum, Sphyraena guachancho, Istiophorus albicans), to determine their phosphorus (P), protein (Pr), cholesterol, sodium, potassium, vitamins D3 and E, and n-3 PUFA (EPA+DHA) according to the AOAC techniques, in order to identify which species could be included in renal diet; particularly because of their risk:benefit relations (calculated with those results). Protein values ranged from 16.5 to 33.5g/100 g of fillet; the specie with the highest phosphorus contest was Salmo salar, and with the lowest, Symphurus elongatus. EPA+DHA quantity ranged from 79.64 mg/100 g to 1,381.53 mg/100 g. Considering de P/Pr relation recommended to renal patients, all analyzed species (except Salmo salar, Ophichthus rex and Istiophorus albicans) could be included in their diet. As for the P/EPA+DHA relation, the species most recommended to renal patients are Symphurus elongatus, Bairdiella chrysoura and Sphyraena guachancho. PMID:23610899

  16. Two novel PRPF31 premessenger ribonucleic acid processing factor 31 homolog mutations including a complex insertion-deletion identified in Chinese families with retinitis pigmentosa

    PubMed Central

    Dong, Bing; Chen, Jieqiong; Zhang, Xiaohui; Pan, Zhe; Bai, Fengge

    2013-01-01

    Objective To identify the causative mutations in two Chinese families with retinitis pigmentosa (RP), and to describe the associated phenotype. Methods Individuals from two unrelated families underwent full ophthalmic examinations. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Linkage analysis was performed on the known genetic loci for autosomal dominant retinitis pigmentosa with a panel of polymorphic markers in the two families, and then all coding exons of the PRP31 premessenger ribonucleic acid processing factor 31 homolog (PRPF31) gene were screened for mutations with direct sequencing of PCR-amplified DNA fragments. Allele-specific PCR was used to validate a substitution in all available family members and 100 normal controls. A large deletion was detected with real-time quantitative PCR (RQ-PCR) using a panel of primers from regions around the PRPF31 gene. Long-range PCR, followed by DNA sequencing, was used to define the breakpoints. Results Clinical examination and pedigree analysis revealed two four-generation families (RP24 and RP106) with autosomal dominant retinitis pigmentosa. A significant two-point linkage odd disequilibrium score was generated at marker D19S926 (Zmax=3.55, θ=0) for family RP24 and D19S571 (Zmax=3.21, θ=0) for family RP106, and further linkage and haplotype studies confined the disease locus to chromosome 19q13.42 where the PRPF31 gene is located. Mutation screening of the PRPF31 gene revealed a novel deletion c.1215delG (p.G405fs+7X) in family RP106. The deletion cosegregated with the family’s disease phenotype, but was not found in 100 normal controls. No disease-causing mutation was detected in family RP24 with PCR-based sequencing analysis. RQ-PCR and long-range PCR analysis revealed a complex insertion-deletion (indel) in the patients of family RP24. The deletion is more than 19 kb and encompasses part of the PRPF31 gene (exons 1–3), together with three adjacent

  17. The temporal and spatial pattern of histone H3 phosphorylation at serine 28 and serine 10 is similar in plants but differs between mono- and polycentric chromosomes.

    PubMed

    Gernand, D; Demidov, D; Houben, A

    2003-01-01

    Immunolabeling using site-specific antibodies against phosphorylated histone H3 at serine 10 or serine 28 revealed in plants an almost similar temporal and spatial pattern of both post-translational modification sites at mitosis and meiosis. During the first meiotic division the entire chromosomes are highly H3 phosphorylated. In the second meiotic division, like in mitosis, the chromosomes contain high phosphorylation levels in the pericentromeric region and very little H3 phosphorylation along the arms of monocentric species. In the polycentric plant Luzula luzuloides phosphorylation at both serine positions occurs along the whole chromosomes, whereas in monocentric species, only the pericentromeric regions showed strong signals from mitotic prophase to telophase. No phosphorylated serine 10 or serine 28 was detectable on single chromatids at anaphase II resulting from equational segregation of rye B chromosome univalents during the preceding anaphase I. In addition, we found a high level of serine 28 as well as of serine 10 phosphorylation along the entire mitotic monocentric chromosomes after treatment of mitotic cells using the phosphatase inhibitor cantharidin. These observations suggest that histone H3 phosphorylation at serine 10 and 28 is an evolutionarily conserved event and both sites are likely to be involved in the same process, such as sister chromatid cohesion. PMID:14610360

  18. Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase type 5 (PP5)

    NASA Technical Reports Server (NTRS)

    Swingle, Mark R.; Ciszak, Ewa M.; Honkanen, Richard E.

    2004-01-01

    Serine/threonine protein phosphatase-5 (PP5) is a member of the PPP-gene family of protein phosphatases that is widely expressed in mammalian tissues and is highly conserved among eukaryotes. PP5 associates with several proteins that affect signal transduction networks, including the glucocorticoid receptor (GR)-heat shock protein-90 (Hsp90)-heterocomplex, the CDC16 and CDC27 subunits of the anaphase-promoting complex, elF2alpha kinase, the A subunit of PP2A, the G12-alpha / G13-alpha subunits of heterotrimeric G proteins and DNA-PK. The catalytic domain of PP5 (PP5c) shares 35-45% sequence identity with the catalytic domains of other PPP-phosphatases, including protein phosphatase-1 (PP1), -2A (PP2A), -2B / calcineurin (PP2B), -4 (PP4), -6 (PP6), and -7 (PP7). Like PP1, PP2A and PP4, PP5 is also sensitive to inhibition by okadaic acid, microcystin, cantharidin, tautomycin, and calyculin A. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 angstroms. From this structure we propose a mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1):M(sub 2)-W(sup 1)-His(sup 304)-Asp(sup 274) catalytic motif. The structure of PP5c provides a possible structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

  19. The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite.

    PubMed

    Putrianti, Elyzana D; Schmidt-Christensen, Anja; Arnold, Iris; Heussler, Volker T; Matuschewski, Kai; Silvie, Olivier

    2010-06-01

    Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs. PMID:20039882

  20. Prediction of individual milk proteins including free amino acids in bovine milk using mid-infrared spectroscopy and their correlations with milk processing characteristics.

    PubMed

    McDermott, A; Visentin, G; De Marchi, M; Berry, D P; Fenelon, M A; O'Connor, P M; Kenny, O A; McParland, S

    2016-04-01

    The aim of this study was to evaluate the effectiveness of mid-infrared spectroscopy in predicting milk protein and free amino acid (FAA) composition in bovine milk. Milk samples were collected from 7 Irish research herds and represented cows from a range of breeds, parities, and stages of lactation. Mid-infrared spectral data in the range of 900 to 5,000 cm(-1) were available for 730 milk samples; gold standard methods were used to quantify individual protein fractions and FAA of these samples with a view to predicting these gold standard protein fractions and FAA levels with available mid-infrared spectroscopy data. Separate prediction equations were developed for each trait using partial least squares regression; accuracy of prediction was assessed using both cross validation on a calibration data set (n=400 to 591 samples) and external validation on an independent data set (n=143 to 294 samples). The accuracy of prediction in external validation was the same irrespective of whether undertaken on the entire external validation data set or just within the Holstein-Friesian breed. The strongest coefficient of correlation obtained for protein fractions in external validation was 0.74, 0.69, and 0.67 for total casein, total β-lactoglobulin, and β-casein, respectively. Total proteins (i.e., total casein, total whey, and total lactoglobulin) were predicted with greater accuracy then their respective component traits; prediction accuracy using the infrared spectrum was superior to prediction using just milk protein concentration. Weak to moderate prediction accuracies were observed for FAA. The greatest coefficient of correlation in both cross validation and external validation was for Gly (0.75), indicating a moderate accuracy of prediction. Overall, the FAA prediction models overpredicted the gold standard values. Near-unity correlations existed between total casein and β-casein irrespective of whether the traits were based on the gold standard (0.92) or mid

  1. The PE16 (Rv1430) of Mycobacterium tuberculosis Is an Esterase Belonging to Serine Hydrolase Superfamily of Proteins

    PubMed Central

    Sultana, Rafiya; Vemula, Mani Harika; Banerjee, Sharmishta; Guruprasad, Lalitha

    2013-01-01

    The PE and PPE multigene families, first discovered during the sequencing of M. tuberculosis H37Rv genome are responsible for antigenic variation and have been shown to induce increased humoral and cell mediated immune response in the host. Using the bioinformatics tools, we had earlier reported that the 225 amino acid residue PE-PPE domain (Pfam: PF08237) common to some PE and PPE proteins has a “serine α/β hydrolase” fold and conserved Ser, Asp and His catalytic triad characteristic of lipase, esterase and cutinase activities. In order to prove experimentally that PE-PPE domain is indeed a serine hydrolase, we have cloned the full-length Rv1430 and its PE-PPE domain into pET-28a vector, expressed the proteins in E. coli and purified to homogeneity. The activity assays of both purified proteins were carried out using p-nitrophenyl esters of aliphatic carboxylic acids with varying chain length (C2–C16) to study the substrate specificity. To characterize the active site of the PE-PPE domain, we mutated the Ser199 to Ala. The activity of the protein in the presence of serine protease inhibitor- PMSF and the mutant protein were measured. Our results reveal that Rv1430 and its PE-PPE domain possess esterase activity and hydrolyse short to medium chain fatty acid esters with the highest specific activity for pNPC6 at 37°C, 38°C and pH 7.0, 8.0. The details of this work and the observed results are reported in this manuscript. PMID:23383323

  2. Gas-Phase Acidities of Phosphorylated Amino Acids.

    PubMed

    Stover, Michele L; Plummer, Chelsea E; Miller, Sean R; Cassady, Carolyn J; Dixon, David A

    2015-11-19

    Gas-phase acidities and heats of formation have been predicted at the G3(MP2)/SCRF-COSMO level of theory for 10 phosphorylated amino acids and their corresponding amides, including phospho-serine (pSer), -threonine (pThr), and -tyrosine (pTyr), providing the first reliable set of these values. The gas-phase acidities (GAs) of the three named phosphorylated amino acids and their amides have been determined using proton transfer reactions in a Fourier transform ion cyclotron mass spectrometer. Excellent agreement was found between the experimental and predicted GAs. The phosphate group is the deprotonation site for pSer and pThr and deprotonation from the carboxylic acid generated the lowest energy anion for pTyr. The infrared spectra were calculated for six low energy anions of pSer, pThr, and pTyr. For deprotonated pSer and pThr, good agreement is found between the experimental IRMPD spectra and the calculated spectra for our lowest energy anion structure. For pTyr, the IR spectra for a higher energy phosphate deprotonated structure is in good agreement with experiment. Additional experiments tested electrospray ionization (ESI) conditions for pTyr and determined that variations in solvent, temperature, and voltage can result in a different experimental GA value, indicating that ESI conditions affect the conformation of the pTyr anion. PMID:26492552

  3. The Origin of Amino Acids in Lunar Regolith Samples

    NASA Technical Reports Server (NTRS)

    Cook, Jamie E.; Callahan, Michael P.; Dworkin, Jason P.; Glavin, Daniel P.; McLain, Hannah L.; Noble, Sarah K.; Gibson, Everett K., Jr.

    2016-01-01

    We analyzed the amino acid content of seven lunar regolith samples returned by the Apollo 16 and Apollo 17 missions and stored under NASA curation since collection using ultrahigh-performance liquid chromatography with fluorescence detection and time-of-flight mass spectrometry. Consistent with results from initial analyses shortly after collection in the 1970s, we observed amino acids at low concentrations in all of the curated samples, ranging from 0.2 parts-per-billion (ppb) to 42.7 ppb in hot-water extracts and 14.5 ppb to 651.1 ppb in 6M HCl acid-vapor-hydrolyzed, hot-water extracts. Amino acids identified in the Apollo soil extracts include glycine, D- and L-alanine, D- and L-aspartic acid, D- and L-glutamic acid, D- and L-serine, L-threonine, and L-valine, all of which had previously been detected in lunar samples, as well as several compounds not previously identified in lunar regoliths: -aminoisobutyric acid (AIB), D-and L-amino-n-butyric acid (-ABA), DL-amino-n-butyric acid, -amino-n-butyric acid, -alanine, and -amino-n-caproic acid. We observed an excess of the L enantiomer in most of the detected proteinogenic amino acids, but racemic alanine and racemic -ABA were present in some samples.

  4. Protein kinase A phosphorylation at serine-2808 of the cardiac Ca2+-release channel (ryanodine receptor) does not dissociate 12.6-kDa FK506-binding protein (FKBP12.6).

    PubMed

    Xiao, Bailong; Sutherland, Cindy; Walsh, Michael P; Chen, S R Wayne

    2004-03-01

    Dissociation of FKBP12.6 from the cardiac Ca2+-release channel (RyR2) as a consequence of protein kinase A (PKA) hyperphosphorylation of RyR2 at a single amino acid residue, serine-2808, has been proposed as an important mechanism underlying cardiac dysfunction in heart failure. However, the issue of whether PKA phosphorylation of RyR2 can dissociate FKBP12.6 from RyR2 is controversial. To additionally address this issue, we investigated the effect of PKA phosphorylation and mutations at serine-2808 of RyR2 on recombinant or native FKBP12.6-RyR2 interaction. Site-specific antibodies, which recognize the serine-2808 phosphorylated or nonphosphorylated form of RyR2, were used to unambiguously correlate the phosphorylation state of RyR2 at serine-2808 with its ability to bind FKBP12.6. We found that FKBP12.6 can bind to both the serine-2808 phosphorylated and nonphosphorylated forms of RyR2. The S2808D mutant thought to mimic constitutive phosphorylation also retained the ability to bind FKBP12.6. Complete phosphorylation at serine-2808 by exogenous PKA disrupted neither the recombinant nor native FKBP12.6-RyR2 complex. Furthermore, binding of site-specific antibodies to the serine-2808 phosphorylation site did not dissociate FKBP12.6 from or prevent FKBP12.6 from binding to RyR2. Taken together, our results do not support the notion that PKA phosphorylation at serine-2808 dissociates FKBP12.6 from RyR2. PMID:14715536

  5. Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement.

    PubMed

    Homa, Joanna; Ortmann, Weronika; Kolaczkowska, Elzbieta

    2016-01-01

    Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates. PMID:27416067

  6. Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement

    PubMed Central

    Ortmann, Weronika; Kolaczkowska, Elzbieta

    2016-01-01

    Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates. PMID:27416067

  7. A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation

    PubMed Central

    Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

    2016-01-01

    Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and –beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function. DOI: http://dx.doi.org/10.7554/eLife.12345.001 PMID:26779719

  8. PIM serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers

    PubMed Central

    Brault, Laurent; Gasser, Christelle; Bracher, Franz; Huber, Kilian; Knapp, Stefan; Schwaller, Jürg

    2010-01-01

    The identification as cooperating targets of Proviral Integrations of Moloney virus in murine lymphomas suggested early on that PIM serine/threonine kinases play an important role in cancer biology. Whereas elevated levels of PIM1 and PIM2 were mostly found in hematologic malignancies and prostate cancer, increased PIM3 expression was observed in different solid tumors. PIM kinases are constitutively active and their activity supports in vitro and in vivo tumor cell growth and survival through modification of an increasing number of common as well as isoform-specific substrates including several cell cycle regulators and apoptosis mediators. PIM1 but not PIM2 seems also to mediate homing and migration of normal and malignant hematopoietic cells by regulating chemokine receptor surface expression. Knockdown experiments by RNA interference or dominant-negative acting mutants suggested that PIM kinases are important for maintenance of a transformed phenotype and therefore potential therapeutic targets. Determination of the protein structure facilitated identification of an increasing number of potent small molecule PIM kinase inhibitors with in vitro and in vivo anticancer activity. Ongoing efforts aim to identify isoform-specific PIM inhibitors that would not only help to dissect the kinase function but hopefully also provide targeted therapeutics. Here, we summarize the current knowledge about the role of PIM serine/threonine kinases for the pathogenesis and therapy of hematologic malignancies and solid cancers, and we highlight structural principles and recent progress on small molecule PIM kinase inhibitors that are on their way into first clinical trials. PMID:20145274

  9. 3-(1H-Indol-3-yl)-2-(2-nitro­benzene­sulfonamido)­propanoic acid including an unknown solvate

    PubMed Central

    Khan, Islam Ullah; Mubashar-ur-Rehman, Hafiz; Aziz, Salman; Harrison, William T. A.

    2012-01-01

    In the title compound, C17H15N3O6S, which crystallized with highly disordered methanol and/or water solvent mol­ecules, the dihedral angle between the the indole and benzene ring systems is 5.3 (2)°, which allows for the formation of intra­molecular π–π stacking inter­actions [centroid–centroid separations = 3.641 (3) and 3.694 (3) Å] and an approximate overall U-shape for the mol­ecule. In the crystal, dimers linked by pairs of Ns—H⋯Oc (s = sulfonamide and c = carboxyl­ate) hydrogen bonds generate R 2 2(10) loops, whereas Ni—H⋯π (i = indole) inter­actions lead to chains propagating in [100] or [010]. Together, these lead to a three-dimensional network in which the solvent voids are present as inter­secting (two-dimensional) systems of [100] and [010] channels. The title compound was found to contain a heavily disordered solvent mol­ecule, which could be methanol or water or a mixture of the two. Due to its uncertain nature and the unresolvable disorder, the data were processed with the SQUEEZE option in PLATON [Spek (2009 ▶). Acta Cryst. D65, 148–155], which revealed 877.8 Å3 of solvent-accessible volume per unit cell and 126 electron-units of scattering density or 109.7 Å3 (16 electron units) per organic mol­ecule.. This was not included in the calculations of overall formula weight, density and absorption coefficient. PMID:22807845

  10. Purification and characterization of a collagenolytic serine proteinase from the skeletal muscle of red sea bream (Pagrus major).

    PubMed

    Wu, Guo-Ping; Chen, Su-Hua; Liu, Guang-Ming; Yoshida, Asami; Zhang, Ling-Jing; Su, Wen-Jin; Cao, Min-Jie

    2010-03-01

    A collagenolytic serine proteinase (CSP) was purified from red sea bream (Pagrus major) skeletal muscle to homogeneity by ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Phenyl Sepharose and Hydroxyapatite. The molecular mass of CSP was approximately 85 kDa as estimated by SDS-PAGE and gel filtration. Optimum temperature and pH of CSP were 40 degrees C and 8.0, respectively. CSP was specifically inhibited by serine proteinase inhibitors, while inhibitors to other type proteinases did not show much inhibitory effects. The K(m) and k(cat) values of CSP for Boc-Leu-Lys-Arg-MCA were 3.58 microM and 0.13 s(-1) at 37 degrees C, respectively. Furthermore, CSP hydrolyzed gelatin and native type I collagen effectively though its degradation on myosin heavy chain (MHC) was not significant, suggesting its involvement in the texture tenderization of fish muscle during the post-mortem stage. PMID:19945542

  11. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    SciTech Connect

    Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

    1988-02-01

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage lambdagt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16/sup +/ natural killer cells and CD3/sup +/, CD16/sup -/ T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells.

  12. Development and validation of a robust and sensitive assay for the discovery of selective inhibitors for serine/threonine protein phosphatases PP1α (PPP1C) and PP5 (PPP5C).

    PubMed

    Swingle, Mark R; Honkanen, Richard E

    2014-10-01

    Protein phosphatase types 1 α (PP1α/PPP1C) and 5 (PP5/PPP5C) are members of the PPP family of serine/threonine protein phosphatases. PP1 and PP5 share a common catalytic mechanism, and several natural compounds, including okadaic acid, microcystin, and cantharidin, act as strong inhibitors of both enzymes. However, to date there have been no reports of compounds that can selectively inhibit PP1 or PP5, and specific or highly selective inhibitors for either PP1 or PP5 are greatly desired by both the research and pharmaceutical communities. Here we describe the development and optimization of a sensitive and robust (representative PP5C assay data: Z'=0.93; representative PP1Cα assay data: Z'=0.90) fluorescent phosphatase assay that can be used to simultaneously screen chemical libraries and natural product extracts for the presence of catalytic inhibitors of PP1 and PP5. PMID:25383722

  13. Development and Validation of a Robust and Sensitive Assay for the Discovery of Selective Inhibitors for Serine/Threonine Protein Phosphatases PP1α (PPP1C) and PP5 (PPP5C)

    PubMed Central

    Swingle, Mark R.

    2014-01-01

    Abstract Protein phosphatase types 1 α (PP1α/PPP1C) and 5 (PP5/PPP5C) are members of the PPP family of serine/threonine protein phosphatases. PP1 and PP5 share a common catalytic mechanism, and several natural compounds, including okadaic acid, microcystin, and cantharidin, act as strong inhibitors of both enzymes. However, to date there have been no reports of compounds that can selectively inhibit PP1 or PP5, and specific or highly selective inhibitors for either PP1 or PP5 are greatly desired by both the research and pharmaceutical communities. Here we describe the development and optimization of a sensitive and robust (representative PP5C assay data: Z′=0.93; representative PP1Cα assay data: Z′=0.90) fluorescent phosphatase assay that can be used to simultaneously screen chemical libraries and natural product extracts for the presence of catalytic inhibitors of PP1 and PP5. PMID:25383722

  14. Characterization of an extracellular serine protease of Leishmania (Leishmania) amazonensis.

    PubMed

    Silva-Lopez, R E; Coelho, M G Pinto; De Simone, S G

    2005-07-01

    A serine protease was purified 942-fold from culture supernatant of L. amazonensis promastigotes using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose and continuous elution electrophoresis by Prep Cell, yielding a total recovery of 61%. The molecular mass of the active enzyme estimated by SDS-PAGE under conditions of reduction was 56 kDa and 115 kDa under conditions of non-reduction, suggesting that the protease is a dimeric protein. Additionally, it was found to be a non-glycosylated enzyme, with a pI of 5.0. The optimal pH and temperature of the enzyme were 7.5 and 28 degrees C respectively, using alpha-N-rho-tosyl-L-arginine-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 61% of the enzyme activity was preserved after 1 h of pre-treatment at 42 degrees C. Haemoglobin, bovine serum albumin (BSA), ovalbumin, fibrinogen, collagen, gelatin and peptide substrates containing arginine in an ester bond and amide substrates containing hydrophobic residues at the P1 site were hydrolysed by this extracellular protease. The insulin beta-chain was also hydrolysed by the enzyme and many peptidic bonds were susceptible to the protease action, and 4 of them (L11-V12, E3-A14, L15-Y16 and Y16-L17) were identified. Inhibition studies suggested that the enzyme belongs to the serine protease class inhibited by calcium and manganese and activated by zinc. These findings show that this enzyme of L. amazonensis is a novel serine protease, which differs from all known flagellate proteases characterized. PMID:16038400

  15. Insulin resistance and muscle insulin receptor substrate‐1 serine hyperphosphorylation

    PubMed Central

    Stuart, Charles A.; Howell, Mary E. A.; Cartwright, Brian M.; McCurry, Melanie P.; Lee, Michelle L.; Ramsey, Michael W.; Stone, Michael H.

    2014-01-01

    Abstract Insulin resistance in metabolic syndrome subjects is profound in spite of muscle insulin receptor and insulin‐responsive glucose transporter (GLUT4) expression being nearly normal. Insulin receptor tyrosine kinase phosphorylation of insulin receptor substrate‐1 (IRS‐1) at Tyr896 is a necessary step in insulin stimulation of translocation of GLUT4 to the cell surface. Serine phosphorylation of IRS‐1 by some kinases diminishes insulin action in mice. We evaluated the phosphorylation status of muscle IRS‐1 in 33 subjects with the metabolic syndrome and seventeen lean controls. Each underwent euglycemic insulin clamps and a thigh muscle biopsy before and after 8 weeks of either strength or endurance training. Muscle IRS‐1 phosphorylation at six sites was quantified by immunoblots. Metabolic syndrome muscle IRS‐1 had excess phosphorylation at Ser337 and Ser636 but not at Ser307, Ser789, or Ser1101. Ser337 is a target for phosphorylation by glycogen synthase kinase 3 (GSK3) and Ser636 is phosphorylated by c‐Jun N‐terminal kinase 1 (JNK1). Exercise training without weight loss did not change the IRS‐1 serine phosphorylation. These data suggest that baseline hyperphosphorylation of at least two key serines within muscle IRS‐1 diminishes the transmission of the insulin signal and thereby decreases the insulin‐stimulated translocation of GLUT4. Excess fasting phosphorylation of muscle IRS‐1 at Ser636 may be a major cause of the insulin resistance seen in obesity and might prevent improvement in insulin responsiveness when exercise training is not accompanied by weight loss. PMID:25472611

  16. The Structure and Phase Diagram of Chiral Alkyl-Serine Monolayers on Mercury

    SciTech Connect

    L Tamam; D Medina; T Menahem; Y Mastai; E Sloutskin; S Yefet; M Deutsch

    2011-12-31

    The structure of liquid-mercury-supported Langmuir films (LFs) of chiral serine-modified fatty acid molecules was studied as a function of length, n = 8-22 carbons, temperature, T = 5-25 C, and surface coverage, A {approx} 40-200 {angstrom}{sup 2} per molecule, for both homochiral and heterochiral compounds. Using surface pressure {pi}-area A isotherms and surface-specific synchrotron X-ray diffraction methods the phase diagram was determined in detail. No lateral order was found for phases comprising surface-parallel molecules, in contrast with unmodified fatty acid LFs on mercury. For phases comprising standing-up molecules, long range lateral order was found for n {>=} 12, but no order for n = 8. The molecules in the ordered phases are extended, and tilt rigidly by {approx}40{sup o} from the surface normal. The homochiral LFs pack in an oblique, single-molecule, unit cell. The heterochiral LFs pack in a body-centered rectangular unit cell, containing two molecules. Unlike unmodified fatty acid LFs, the structure of the standing-up phase does not vary with n, T or A. The interactions underlying these characteristics, and the role of chirality, are discussed.

  17. Involvement of a Serpin serine protease inhibitor (OoSerpin) from mollusc Octopus ocellatus in antibacterial response.

    PubMed

    Wei, Xiumei; Xu, Jie; Yang, Jianmin; Liu, Xiangquan; Zhang, Ranran; Wang, Weijun; Yang, Jialong

    2015-01-01

    Serpin is an important member of serine protease inhibitors (SPIs), which is capable of regulating proteolytic events and involving in a variety of physiological processes. In present study, a Serpin homolog was identified from Octopus ocellatus (designated as OoSerpin). Full-length cDNA of OoSerpin was of 1735 bp, containing a 5' untranslated region of 214 bp, a 3' UTR of 282 bp, and an open reading frame of 1239 bp. The open reading frame encoded a polypeptide of 412 amino acids which has a predicted molecular weight of 46.5 kDa and an isoelectric point of 8.52. The OoSerpin protein shares 37% sequence identity with other Serpins from Mus musculus (NP_941373) and Ixodes scapularis (XP_002407493). The existence of a conserved SERPIN domain strongly suggested that OoSerpin was a member of the Serpin subfamily. Expression patterns of OoSerpin, both in tissues and towards bacterial stimulation, were then characterized. The mRNA of OoSerpin was constitutively expressed at different levels in all tested tissues of untreated O. ocellatus, including mantle (lowest), muscle, renal sac, gill, hemocyte, gonad, systemic heart, and hepatopancreas (highest). The transcriptional level of OoSerpin was significantly up-regulated (P<0.01) in O. ocellatus upon bacterial challenges with Vibrio anguillarum and Micrococcus luteus, indicating its involvement in the antibacterial immune response. Furthermore, rOoSerpin, the recombinant protein of OoSerpin, exhibited strong abilities to inhibit proteinase activities of trypsin and chymotrypsin as well as the growth of Escherichia coli. Our results demonstrate that OoSerpin is a potential antibacterial factor involved in the immune response of O. ocellatus against bacterial infection. PMID:25449372

  18. Direct NMR observation and pKa determination of the Asp102 side chain in a serine protease.

    PubMed

    Everill, Paul; Sudmeier, James L; Bachovchin, William W

    2012-02-01

    The pK(a) value of aspartic acid in the catalytic triad of serine proteases has been a pivotal element in essentially every mechanism proposed for these enzymes over the past 40 years, but has, until now, eluded direct determination. Here, we have used the multinuclear 3D-NMR pulse programs HCACO and HCCH-TOCSY to directly identify and study the side-chain resonances of the aspartate and glutamate residues in uniformly (13)C-labeled α-lytic protease. Resonances from four of the six residues were detected and assigned, including that of Asp(102), which is notably the weakest of the four. pH titrations have shown all of the carboxylate (13)C signals to have unusually low pK(a) values: 2.0, 3.2, and 1.7 for Glu(129), Glu(174), and Glu(229), respectively, and an upper limit of 1.5 for Asp(102). The multiple H-bonds to Asp(102), long known from X-ray crystal studies, probably account for its unusually low pK(a) value through preferential stabilization of its anionic form. These H-bonds probably also contribute to the weakness of the NMR resonances of Asp(102) by restricting its mobility. The Asp(102)(13)C(γ) atom responds to the ionization of His(57) in the resting enzyme and to the inhibitor-derived oxyanion in a chloromethyl ketone complex, observations that strongly support the assignment. The low pK(a) value of Asp(102) would appear to be incompatible with mechanisms involving strong Asp(102)-His(57) H-bonds or high pK(a) values, but is compatible with mechanisms involving normal Asp(102)-His(57) H-bonds and moving His(57) imidazole rings, such as the reaction-driven ring flip. PMID:22229736

  19. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.

    PubMed Central

    Grakoui, A; McCourt, D W; Wychowski, C; Feinstone, S M; Rice, C M

    1993-01-01

    Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. Images PMID:8386278

  20. Production and partial characterization of serine and metallo peptidases secreted by Aspergillus fumigatus Fresenius in submerged and solid state fermentation.

    PubMed

    da Silva, Ronivaldo Rodrigues; de Freitas Cabral, Tatiana Pereira; Rodrigues, André; Cabral, Hamilton

    2013-01-01

    Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 °C with an inoculum of 1 × 10(6) spores and yielded 1500 active units (U/mL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 °C and yielded 40 U/mL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 °C, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase. PMID:24159310

  1. Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation

    SciTech Connect

    Marti, T.; Otto, H.; Mogi, T.; Roesselet, S.J.H.; Heyn, M.P.; Khorana, H.G. )

    1991-04-15

    To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.

  2. Structural and functional adaptation of vancomycin resistance VanT serine racemases

    DOE PAGESBeta

    Meziane-Cherif, Djalal; Stogios, Peter J.; Evdokimova, Elena; Egorova, Olga; Savchenko, Alexei; Courvalin, Patrice

    2015-08-11

    Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl–D-alanine target of peptidoglycan precursors with D-alanyl–D-lactate or D-alanyl–D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 wasmore » determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for theL-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of D-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria.« less

  3. Structural and functional adaptation of vancomycin resistance VanT serine racemases

    SciTech Connect

    Meziane-Cherif, Djalal; Stogios, Peter J.; Evdokimova, Elena; Egorova, Olga; Savchenko, Alexei; Courvalin, Patrice

    2015-08-11

    Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl–D-alanine target of peptidoglycan precursors with D-alanyl–D-lactate or D-alanyl–D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for theL-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of D-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria.

  4. Characterization of a novel arginine/serine-rich splicing factor in Arabidopsis.

    PubMed Central

    Lopato, S; Waigmann, E; Barta, A

    1996-01-01

    Many splicing factors in vertebrate nuclei belong to a class of evolutionarily conserved proteins containing arginine/serine (RS) or serine/arginine (SR) domains. Previously, we demonstrated the existence of SR splicing factors in plants. In this article, we report on a novel member of this splicing factor family from Arabidopsis designated atRSp31. It has one N-terminal RNA recognition motif and a C-terminal RS domain highly enriched in arginines. The RNA recognition motif shows significant homology to all animal SR proteins identified to date, but the intermediate region does not show any homology to any other known protein. Subsequently, we characterized two cDNAs from Arabidopsis that are highly homologous to atRSp31 (designated atRSp35 and atRSp41). Their deduced amino acid sequences indicate that these proteins constitute a new family of RS domain splicing factors. Purified recombinant atRSp31 is able to restore splicing in SR protein-deficient human S100 extracts. This indicates that atRSp31 is a true plant splicing factor and plays a crucial role in splicing, similar to that of other RS splicing factors. All of the three genes are differentially expressed in a tissue-specific manner. The isolation of this new plant splicing factor family enlarges the essential group of RS domain splicing factors. Furthermore, because no animal equivalent to this protein family has been identified to date, our results suggest that these proteins play key roles in constitutive and alternative splicing in plants. PMID:8989882

  5. Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases

    PubMed Central

    Meziane-Cherif, Djalal; Stogios, Peter J.; Evdokimova, Elena; Egorova, Olga

    2015-01-01

    ABSTRACT Vancomycin resistance in Gram-positive bacteria results from the replacement of the d-alanyl–d-alanine target of peptidoglycan precursors with d-alanyl–d-lactate or d-alanyl–d-serine (d-Ala-d-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of d-Ala-d-Ser-terminating precursors by converting l-Ser to d-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in l-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5′-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the l-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against l-Ser versus l-Ala implied that this enzyme relies on its membrane-bound domain for l-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria. PMID:26265719

  6. A quantitative study of the interactive effects of glycine and serine with threonine and choline on growth performance in broilers.

    PubMed

    Siegert, W; Ahmadi, H; Helmbrecht, A; Rodehutscord, M

    2015-07-01

    Differences in the optimal dietary concentrations of Gly (glycine) and Ser (serine) in broiler diets may be due to levels of endogenous Gly precursors that differ in literature. Therefore, we measured the extent of the interactive effects between equivalents of Gly and Ser (Glyequi) and the endogenous Gly precursors choline and Thr (threonine) on growth performance. A fractional central composite design included concentrations of 15-25 g/kg DM, 0.6-2.0 g/kg DM, and 6.4-10.4 g/kg DM for Glyequi, choline, and Thr, respectively, in 5 levels each. The various concentrations were achieved by adding Gly, choline chloride, and l-Thr to a basal mix. Except for 20 replicates of the central diet, all treatments were tested with 5 replicates, each with 10 birds. Food was provided for ad libitum consumption throughout the experiment. The data were evaluated using artificial neural networks. Digestibility was studied for selected diets using separate birds. Since average daily feed intake (ADFI) varied between replicates, the intake of prececal digestible Glyequi, choline, and prececal digestible Thr were more adequate independent variables than the dietary concentration of each amino acid. From d 1 to d 7, no treatment effects on G:F and average daily gain (ADG) were detected; subsequent results refer to the period from d 7 to d 21. Increasing prececal digestible Thr intake considerably decreased the need for prececal digestible Glyequi to achieve certain levels of G:F and ADG. The extent of this effect cannot be explained only by the endogenous metabolism of Thr to Gly. Since essential amino acids were present above the recommended levels, Thr probably limited performance, and excessive intake of other essential amino acids prompted a Gly-dissipating process. Choline exerted a considerable effect on the required intake of prececal digestible Glyequi and prececal digestible Thr to achieve certain levels of G:F and ADG. The results of this study partly explain the previously

  7. Breaking the low barrier hydrogen bond in a serine protease.

    PubMed Central

    Kidd, R. D.; Sears, P.; Huang, D. H.; Witte, K.; Wong, C. H.; Farber, G. K.

    1999-01-01

    The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water-miscible organic co-solvent such as N,N-dimethylformamide (DMF). However, in 50% DMF, the k(cat) for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the k(cat) for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397+1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 A resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 180 degrees around the Cbeta-Cgamma bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co-solvents. PMID:10048334

  8. Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

    SciTech Connect

    Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.; Fairlie, W. Douglas; Colman, Peter M.; Crabb, Brendan S.; Smith, Brian J.

    2009-08-28

    The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.

  9. Remarkable enhancement in PLD activity from Streptoverticillium cinnamoneum by substituting serine residue into the GG/GS motif.

    PubMed

    Ogino, Chiaki; Daido, Hidenori; Ohmura, Yuka; Takada, Namiko; Itou, Yoshiki; Kondo, Akihiko; Fukuda, Hideki; Shimizu, Nobuaki

    2007-06-01

    The gene that encodes phospholipase D (PLD) from Streptoverticillium cinnamoneum contains three consensus regions (Region I, II and IV as shown in Fig. 1A) that are conserved among the PLD superfamily. The glycine-glycine (GG) motif in Region I and the glycine-serine (GS) motif in Region IV are also conserved in the PLD superfamily. These (GG and GS) motifs are located 7 residues downstream from each HKD motif. In an investigation of fifteen GG/GS motif mutants, generated as fusion proteins with maltose-binding protein (MBP-PLDs), three highly active mutants were identified. Three of the mutants (G215S, G216S, and G216S-S489G) contained a serine residue in the GG motif, and exhibited approximately a 9-27-fold increased transphosphatidylation activity to DPPC compared with recombinant wild type MBP-PLD. When heat stability was compared between three mutants and the recombinant wild type, only G216S-S489G showed heat labile properties. It appears that the 489th serine residue in the GS motif also contributes to the thermal stability of the enzyme. In addition, the GG/GS motif was very close to the active center residue, including two HKD motifs, as shown by computer modeling. The findings suggest that the GG/GS motif of PLD is a key motif that affects catalytic function and enzymatic stability. PMID:17499030

  10. Enzymatic oxidation by frog epidermis tyrosinase of 4-methylcatechol and p-cresol. Influence of L-serine.

    PubMed

    García-Carmona, F; Cabanes, J; García-Cánovas, F

    1987-08-01

    A study has been made of the kinetics of cresolase and catecholase activities of tyrosinase on the p-cresol/4-methyl-catechol pair in the presence of L-serine. For this, a spectrophotometer assay for both activities based on the spectrophotometric and stoichiometric characteristics of the chemical reactions in the evolution of 4-methyl-o-benzoquinone is proposed. The presence of L-serine in the reaction medium caused a modification in the lag period and the steady-state rate expressed during the cresolase activity of tyrosinase, but no modification was observed during the expression of catecholase activity. These results can be explained taking into account the complex mechanism proposed for tyrosinase which included the chemical steps involved in the process. Furthermore, a singular form of regulation of enzymatic activity by L-serine has been clarified, not by any direct interaction between the protein molecule and the nucleophile, but by modification of the chemical reactions involved in the mechanism of tyrosinase. PMID:3111538

  11. Enantioselective inhibition of D-serine transport by (S)-ketamine

    PubMed Central

    Singh, Nagendra S; Bernier, Michel; Camandola, Simonetta; Khadeer, Mohammed A; Moaddel, Ruin; Mattson, Mark P; Wainer, Irving W

    2015-01-01

    Background and Purpose Patients with major depressive disorder receiving racemic ketamine, (R,S)-ketamine, experience transient increases in Clinician-Administered Dissociative States Scale scores and a coincident drop in plasma d-serine levels. The results suggest that (R,S)-ketamine produces an immediate, concentration-dependent pharmacological effect on d-serine plasma concentrations. One potential source of this effect is (R,S)-ketamine-induced inhibition of the transporter ASCT2, which regulates intracellular d-serine concentrations. In this study, we tested this hypothesis by examining the effect of (S)- and (R)-ketamine on ASCT2-mediated transport of d-serine in PC-12 and 1321N1 cells and primary neuronal cells in culture. Experimental Approach Intracellular and extracellular d-serine levels were determined using capillary electrophoresis–laser-induced fluorescence and liquid chromatography–mass spectrometry respectively. Expression of ASCT2, Asc-1 and serine racemase was determined utilizing Western blotting. Key Results (S)-Ketamine produced a concentration-dependent increase in intracellular d-serine and reduced extracellular d-serine accumulation. In contrast, (R)-ketamine decreased both intracellular and extracellular d-serine levels. The ASCT2 inhibitor, benzyl-d-serine (BDS), and ASCT2 gene knockdown mimicked the action of (S)-ketamine on d-serine in PC-12 cells, while the Asc-1 agonist d-isoleucine reduced intracellular d-serine and increased extracellular d-serine accumulation. This response to d-isoleucine was not affected by BDS or (S)-ketamine. Primary cultures of rat neuronal cells expressed ASCT2 and were responsive to (S)-ketamine and BDS. (S)- and (R)-ketamine increased the expression of monomeric serine racemase in all the cells studied, with (S)-ketamine having the greatest effect. Conclusions and Implications (S)-Ketamine decreased cellular export of d-serine via selective inhibition of ASCT2, and this could represent a possible source

  12. Cloning and sequence analysis of partial genomic DNA coding for HtrA-type serine protease of Wolbachia from human lymphatic filarial parasite, Wuchereria bancrofti

    PubMed Central

    Dhamodharan, R; Hoti, SL; Sivapragasam, G; Das, MK

    2011-01-01

    Background: Periplasmic serine proteases of HtrA type of Wolbachia have been shown to play a role in the pathogenesis of filarial disease. Aims: This study was aimed to sequence Wb-HtrA serine protease and analyze its phylogenetic position by comparing with other filarial and non-filarial nematode homologs. Materials and Methods: Partial HtrA gene fragment was amplified from DNA isolated from periodic and sub-periodic Wuchereria bancrofti parasites collected from Pondicherry and Nicobar islands, respectively. The amplicons were sequenced, and sequence homology and phylogenetic relationship with other filarial and non-filarial nematodes were analyzed. Results: Partial orthologue of HtrA-type serine protease from Wolbachia of W. bancrofti was amplified, cloned and sequenced. The deduced amino acid sequence exhibited 87%, 81% and 74% identity with the homologous Wolbachia proteases identified from Brugia malayi, Onchocerca volvulus and Drosophila melanogaster, respectively. The Wb-HtrA has arthologues in several proteobacteria with very high homology and hence is highly conserved not only among Wolbachia of filarial parasites but also across proteobacteria. The phylogenetic tree constructed using Neighbor-Joining method showed two main clusters: cluster-I containing bacteria that dwell in diverse habitats such as soil, fresh and marine waters and plants and cluster-II comprising Anaplasma sp. and Erlichia, and Wolbachia endosymbionts of insects and nematodes, in distinct groups. Conclusions: HtrA-type serine protease from Wolbachia of W. bancrofti is highly conserved among filarial parasites. It will be of interest to know whether filarial Wolbachia HtrA type of serine protease might influence apoptosis and lymphatic epithelium, thereby playing a role in the filarial pathogenesis. Such information will be useful for identifying targets for the development of newer drugs for filariasis treatment, especially for preventing lymphatic pathology. PMID:23508470

  13. Characterization of a new oxidant-stable serine protease isolated by functional metagenomics.

    PubMed

    Biver, Sophie; Portetelle, Daniel; Vandenbol, Micheline

    2013-01-01

    A novel serine protease gene, SBcas3.3, was identified by functional screening of a forest-soil metagenomic library on agar plates supplemented with AZCL-casein. Overproduction in Escherichia coli revealed that the enzyme is produced as a 770-amino-acid precursor which is processed to a mature protease of ~55 kDa. The latter was purified by affinity chromatography for characterization with the azocasein substrate. The enzyme proved to be an alkaline protease showing maximal activity between pH 9 and 10 and at 50°C. Treatment with the chelating agent ethylenediaminetetraacetic acid irreversibly denatured the protease, whose stability was found to depend strictly on calcium ions. The enzyme appeared relatively resistant to denaturing and reducing agents, and its activity was enhanced in the presence of 10 ml/l nonionic detergent (Tween 20, Tween 80, or Triton X-100). Moreover, SBcas3.3 displayed oxidant stability, a feature particularly sought in the detergent and bleaching industries. SBcas3.3 was activated by hydrogen peroxide at concentrations up to 10 g/l and it still retained 30% of activity in 50 g/l H2O2. PMID:24024096

  14. Brain-specific Phgdh deletion reveals a pivotal role for L-serine biosynthesis in controlling the level of D-serine, an N-methyl-D-aspartate receptor co-agonist, in adult brain.

    PubMed

    Yang, Jung Hoon; Wada, Akira; Yoshida, Kazuyuki; Miyoshi, Yurika; Sayano, Tomoko; Esaki, Kayoko; Kinoshita, Masami O; Tomonaga, Shozo; Azuma, Norihiro; Watanabe, Masahiko; Hamase, Kenji; Zaitsu, Kiyoshi; Machida, Takeo; Messing, Albee; Itohara, Shigeyoshi; Hirabayashi, Yoshio; Furuya, Shigeki

    2010-12-31

    In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L- and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus. PMID:20966073

  15. Purification and partial characterization of an elastolytic serine protease of Prevotella intermedia.

    PubMed Central

    Shibata, Y; Fujimura, S; Nakamura, T

    1993-01-01

    Elastolytic strains of Prevotella intermedia were isolated from pus samples of adult periodontal lesions. Elastase was found to associate with envelope, and it could be solubilized with guanidine-HCl. The enzyme was purified to homogeneity by sequential procedures including ion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. This elastase was a serine protease, and its mass was 31 kDa. It hydrolyzed elastin powder, but collagen and azodye-conjugated proteins were not degraded by this enzyme. Both synthetic substrates for human pancreatic (glutaryl-L-alanyl-L-alanyl-L-prolyl-L-leucine p-nitroanilide) and leukocyte elastase (methoxy succinyl-L-alanyl-alanyl-L-prolyl-L-valine p-nitroanilide) were hydrolyzed. Images PMID:8357246

  16. Glycolytic flux controls d-serine synthesis through glyceraldehyde-3-phosphate dehydrogenase in astrocytes

    PubMed Central

    Suzuki, Masataka; Sasabe, Jumpei; Miyoshi, Yurika; Kuwasako, Kanako; Muto, Yutaka; Hamase, Kenji; Matsuoka, Masaaki; Imanishi, Nobuaki; Aiso, Sadakazu

    2015-01-01

    d-Serine is an essential coagonist with glutamate for stimulation of N-methyl-d-aspartate (NMDA) glutamate receptors. Although astrocytic metabolic processes are known to regulate synaptic glutamate levels, mechanisms that control d-serine levels are not well defined. Here we show that d-serine production in astrocytes is modulated by the interaction between the d-serine synthetic enzyme serine racemase (SRR) and a glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In primary cultured astrocytes, glycolysis activity was negatively correlated with d-serine level. We show that SRR interacts directly with GAPDH, and that activation of glycolysis augments this interaction. Biochemical assays using mutant forms of GAPDH with either reduced activity or reduced affinity to SRR revealed that GAPDH suppresses SRR activity by direct binding to GAPDH and through NADH, a product of GAPDH. NADH allosterically inhibits the activity of SRR by promoting the disassociation of ATP from SRR. Thus, astrocytic production of d-serine is modulated by glycolytic activity via interactions between GAPDH and SRR. We found that SRR is expressed in astrocytes in the subiculum of the human hippocampus, where neurons are known to be particularly vulnerable to loss of energy. Collectively, our findings suggest that astrocytic energy metabolism controls d-serine production, thereby influencing glutamatergic neurotransmission in the hippocampus. PMID:25870284

  17. A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii

    PubMed Central

    Menyhárd, Dóra K.; Kiss-Szemán, Anna; Tichy-Rács, Éva; Hornung, Balázs; Rádi, Krisztina; Szeltner, Zoltán; Domokos, Klarissza; Szamosi, Ilona; Náray-Szabó, Gábor; Polgár, László; Harmat, Veronika

    2013-01-01

    Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated “check-in” system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic β-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states. PMID:23632025

  18. L-serine capped ZnS:Mn nanocrystals for plant cell biological studies and as a growth enhancing agent for micropropagation of Bacopa monnieri Linn. (Brahmi:Scrophulariaceae)

    SciTech Connect

    Augustine, M. Sajimol; Mathew, Lizzy; Alex, Roselin; Deepa, G. D.; Jayalekshmi, S.

    2014-01-28

    In the present work, the prospects of ZnS:Mn nanocrystals capped with L- serine, a bio-compatible amino acid, synthesized by wet chemical route, as efficient fluorescent probes for plant cell biological studies have been investigated. The present synthesis route using bio-compatible material is a low cost and easy to control method. The colloidal stability of the capped nano crystals is very good as they remain stable without settling down for long time. It is observed that L- serine significantly modifies the structural and optical characteristics of the ZnS:Mn nanocrystals and hence is suitable as a bio-compatible capping agent. The structural properties of L- serine capped nanocrystals were investigated by XRD technique. The size of the L- serine capped ZnS:Mn nanocrystals is found to be around 2 nm . The optical characterization of the nanocrystals was carried out on the basis of photoluminescence (PL) spectroscopic studies. The intense photoluminescence emission observed around 597nm for L-serine capped ZnS:Mn offers high prospects of applications in bio-imaging fields. The unique optical properties of nanoparticles make them appealing as in vivo and in vitro fluorophores in a variety of biological investigations. In the present study, L-serine capped ZnS:Mn nanocrystals were used as a staining dye in fluorescent microscope for observing cell division, cell structure etc. These nanocrystals were also incorporated into the culture media along with the normal auxin- cytokinin hormone combinations in Murashige and Skoog (MS) medium for micropropagation of Bacopa monnieri Linn. (Brahmi:Scrophulariaceae), an Ayurvedic medicine. The results suggest that L-serine capped ZnS:Mn nanocrystals can act as efficient enhancers towards quick callusing and shoot proliferation.

  19. L-serine capped ZnS:Mn nanocrystals for plant cell biological studies and as a growth enhancing agent for micropropagation of Bacopa monnieri Linn. (Brahmi:Scrophulariaceae)

    NASA Astrophysics Data System (ADS)

    Augustine, M. Sajimol; Mathew, Lizzy; Alex, Roselin; Deepa, G. D.; Jayalekshmi, S.

    2014-01-01

    In the present work, the prospects of ZnS:Mn nanocrystals capped with L- serine, a bio-compatible amino acid, synthesized by wet chemical route, as efficient fluorescent probes for plant cell biological studies have been investigated. The present synthesis route using bio-compatible material is a low cost and easy to control method. The colloidal stability of the capped nano crystals is very good as they remain stable without settling down for long time. It is observed that L- serine significantly modifies the structural and optical characteristics of the ZnS:Mn nanocrystals and hence is suitable as a bio-compatible capping agent. The structural properties of L- serine capped nanocrystals were investigated by XRD technique. The size of the L- serine capped ZnS:Mn nanocrystals is found to be around 2 nm . The optical characterization of the nanocrystals was carried out on the basis of photoluminescence (PL) spectroscopic studies. The intense photoluminescence emission observed around 597nm for L-serine capped ZnS:Mn offers high prospects of applications in bio-imaging fields. The unique optical properties of nanoparticles make them appealing as in vivo and in vitro fluorophores in a variety of biological investigations. In the present study, L-serine capped ZnS:Mn nanocrystals were used as a staining dye in fluorescent microscope for observing cell division, cell structure etc. These nanocrystals were also incorporated into the culture media along with the normal auxin- cytokinin hormone combinations in Murashige and Skoog (MS) medium for micropropagation of Bacopa monnieri Linn. (Brahmi:Scrophulariaceae), an Ayurvedic medicine. The results suggest that L-serine capped ZnS:Mn nanocrystals can act as efficient enhancers towards quick callusing and shoot proliferation.

  20. Biochemical Analysis of the NAD+-Dependent Malate Dehydrogenase, a Substrate of Several Serine/Threonine Protein Kinases of Mycobacterium tuberculosis

    PubMed Central

    Wang, Xiao Ming; Soetaert, Karine; Peirs, Priska; Kalai, Michaël; Fontaine, Véronique; Dehaye, Jean Paul; Lefèvre, Philippe

    2015-01-01

    PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions. PMID:25860441

  1. Autotransported serine protease A of Neisseria meningitidis: an immunogenic, surface-exposed outer membrane, and secreted protein.

    PubMed

    Turner, David P J; Wooldridge, Karl G; Ala'Aldeen, Dlawer A A

    2002-08-01

    Several autotransporter proteins have previously been identified in Neisseria meningitidis. Using molecular features common to most members of the autotransporter family of proteins, we have identified an additional novel ca. 112-kDa autotransporter protein in the meningococcal genomic sequence data. This protein, designated autotransported serine protease A (AspA), has significant N-terminal homology to the secreted serine proteases (subtilases) from several organisms and contains a serine protease catalytic triad. The amino acid sequence of AspA is well-conserved in serogroup A, B, and C meningococci. In Neisseria gonorrhoeae, the AspA homologue appears to be a pseudogene. The gene encoding AspA was cloned and expressed from meningococcal strain MC58 (B15:P1.16b). Anti-AspA antibodies were detected in patients' convalescent-phase sera, suggesting that AspA is expressed in vivo during infection and is immunogenic and cross-reactive. Rabbit polyclonal monospecific anti-AspA serum was used to probe whole-cell proteins from a panel of wild-type meningococcal strains and two AspA mutant strains. Expression of the ca. 112-kDa precursor polypeptide was detected in 12 of 20 wild-type meningococcal strains examined, suggesting that AspA expression is phase variable. Immunogold electron microscopy and cellular fractionation studies showed that the AspA precursor is transported to the outer membrane and remains surface exposed. Western blot experiments confirmed that smaller, ca. 68- or 70-kDa components of AspA (AspA68 and AspA70, respectively) are then secreted into the meningococcal culture supernatant. Site-directed mutagenesis of S426 abolished secretion of both rAspA68 and rAspA70 in Escherichia coli, confirming that AspA is an autocleaved autotransporter protein. In conclusion, we characterized a novel, surface-exposed and secreted, immunogenic, meningococcal autotransporter protein. PMID:12117956

  2. Role of Serine Proteases in the Regulation of Interleukin-877 during the Development of Bronchopulmonary Dysplasia in Preterm Ventilated Infants

    PubMed Central

    Chakraborty, Mallinath; McGreal, Eamon P.; Williams, Andrew; Davies, Philip L.; Powell, Wendy; Abdulla, Salima; Voitenok, Nikolai N.; Hogwood, John; Gray, Elaine; Spiller, Brad; Chambers, Rachel C.; Kotecha, Sailesh

    2014-01-01

    Rationale The chemokine interleukin-8 is implicated in the development of bronchopulmonary dysplasia in preterm infants. The 77-amino acid isoform of interleukin-8 (interleukin-877) is a less potent chemoattractant than other shorter isoforms. Although interleukin-877 is abundant in the preterm circulation, its regulation in the preterm lung is unknown. Objectives To study expression and processing of pulmonary interleukin-877 in preterm infants who did and did not develop bronchopulmonary dysplasia. Methods Total interleukin-8 and interleukin-877 were measured in bronchoalveolar lavage fluid from preterm infants by immunoassay. Neutrophil serine proteases were used to assess processing. Neutrophil chemotaxis assays and degranulation of neutrophil matrix metalloproteinase-9 were used to assess interleukin-8 function. Main Results Peak total interleukin-8 and interleukin-877 concentrations were increased in infants who developed bronchopulmonary dysplasia compared to those who did not. Shorter forms of interleukin-8 predominated in the preterm lung (96.3% No-bronchopulmonary dysplasia vs 97.1% bronchopulmonary dysplasia, p>0.05). Preterm bronchoalveolar lavage fluid significantly converted exogenously added interleukin-877 to shorter isoforms (p<0.001). Conversion was greater in bronchopulmonary dysplasia infants (p<0.05). This conversion was inhibited by α-1 antitrypsin and antithrombin III (p<0.01). Purified neutrophil serine proteases efficiently converted interleukin-877 to shorter isoforms in a time- and dose-dependent fashion; shorter interleukin-8 isoforms were primarily responsible for neutrophil chemotaxis (p<0.001). Conversion by proteinase-3 resulted in significantly increased interleukin-8 activity in vitro (p<0.01). Conclusions Shorter, potent, isoforms interleukin-8 predominate in the preterm lung, and are increased in infants developing bronchopulmonary dysplasia, due to conversion of interleukin-877 by neutrophil serine proteases and thrombin

  3. Prebiotic feeding elevates central brain derived neurotrophic factor, N-methyl-D-aspartate receptor subunits and D-serine.

    PubMed

    Savignac, Helene M; Corona, Giulia; Mills, Henrietta; Chen, Li; Spencer, Jeremy P E; Tzortzis, George; Burnet, Philip W J

    2013-12-01

    The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we examined whether plasma from prebiotic treated rats released BDNF from human SH-SY5Y neuroblastoma cells, to provide an initial indication of mechanism of action. Rats were gavaged with fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) or water for five weeks, prior to measurements of brain BDNF, NMDAR subunits and amino acids associated with glutamate neurotransmission (glutamate, glutamine, and serine and alanine enantiomers). Prebiotics increased hippocampal BDNF and NR1 subunit expression relative to controls. The intake of GOS also increased hippocampal NR2A subunits, and frontal cortex NR1 and d-serine. Prebiotics did not alter glutamate, glutamine, l-serine, l-alanine or d-alanine concentrations in the brain, though GOSfeeding raised plasma d-alanine. Elevated levels of plasma peptide YY (PYY) after GOS intake was observed. Plasma from GOS rats increased the release of BDNF from SH-SY5Y cells, but not in the presence of PYY antisera. The addition of synthetic PYY to SH-SY5Y cell cultures, also elevated BDNF secretion. We conclude that prebiotic-mediated proliferation of gut microbiota in rats, like probiotics, increases brain BDNF expression, possibly through the involvement of gut hormones. The effect of GOS on components of central NMDAR signalling was greater than FOS, and may reflect the proliferative potency of GOS on microbiota. Our data therefore, provide a sound basis to further investigate the utility of prebiotics in the maintenance of brain health and

  4. In vivo sulfhydryl modification of the ligand-binding site of Tsr, the Escherichia coli serine chemoreceptor.

    PubMed Central

    Iwama, T; Kawagishi, I; Gomi, S; Homma, M; Imae, Y

    1995-01-01

    The Escherichia coli chemoreceptor Tsr mediates an attractant response to serine. We substituted Cys for Thr-156, one of the residues involved in serine sensing. The mutant receptor Tsr-T156C retained serine- and repellent-sensing abilities. However, it lost serine-sensing ability when it was treated in vivo with sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). Serine protected Tsr-T156C from these reagents. We showed that [3H]NEM bound to Tsr-T156C and that binding decreased in the presence of serine. By pretreating cells with serine and cold NEM, Tsr-T156C was selectively labeled with radioactive NEM. These results are consistent with the location of Thr-156 in the serine-binding site. Chemical modification of the Tsr ligand-binding site provides a basis for simple purification and should assist further in vivo and in vitro investigations of this chemoreceptor protein. PMID:7721714

  5. IrSPI, a tick serine protease inhibitor involved in tick feeding and Bartonella henselae infection.

    PubMed

    Liu, Xiang Ye; de la Fuente, Jose; Cote, Martine; Galindo, Ruth C; Moutailler, Sara; Vayssier-Taussat, Muriel; Bonnet, Sarah I

    2014-07-01

    Ixodes ricinus is the most widespread and abundant tick in Europe, frequently bites humans, and is the vector of several pathogens including those responsible for Lyme disease, Tick-Borne Encephalitis, anaplasmosis, babesiosis and bartonellosis. These tick-borne pathogens are transmitted to vertebrate hosts via tick saliva during blood feeding, and tick salivary gland (SG) factors are likely implicated in transmission. In order to identify such tick factors, we characterized the transcriptome of female I. ricinus SGs using next generation sequencing techniques, and compared transcriptomes between Bartonella henselae-infected and non-infected ticks. High-throughput sequencing of I. ricinus SG transcriptomes led to the generation of 24,539 isotigs. Among them, 829 and 517 transcripts were either significantly up- or down-regulated respectively, in response to bacterial infection. Searches based on sequence identity showed that among the differentially expressed transcripts, 161 transcripts corresponded to nine groups of previously annotated tick SG gene families, while the others corresponded to genes of unknown function. Expression patterns of five selected genes belonging to the BPTI/Kunitz family of serine protease inhibitors, the tick salivary peptide group 1 protein, the salp15 super-family, and the arthropod defensin family, were validated by qRT-PCR. IrSPI, a member of the BPTI/Kunitz family of serine protease inhibitors, showed the highest up-regulation in SGs in response to Bartonella infection. IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs. This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector. This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its

  6. Abrogation of IFN-γ mediated epithelial barrier disruption by serine protease inhibition

    PubMed Central

    Willemsen, LEM; Hoetjes, JP; Van Deventer, SJH; Van Tol, EAF

    2005-01-01

    The intestinal barrier function is often impaired in a variety of diseases including chronic inflammatory bowel disease. Increased intestinal permeability during episodes of active disease correlates with destruction or rearrangement of the tight junction protein complex. IFN-γ has been widely studied for its effect on barrier function and tight junction structures but its mode of action remains unclear. Since the claudin family of tight junction proteins is proposed to be involved in barrier maintenance we studied the effect of IFN-γ on claudin expression in relation to epithelial barrier function. Cycloheximide and protease inhibitors were used to study mechanisms of IFN-γ mediated barrier disruption. Intestinal epithelial cells were exposed to IFN-γ and permeability was evaluated by horse radish peroxidase (HRP) and 4 kD FITC-dextran fluxes. Occludin and claudin-1, -2, -3, and -4 tight junction protein expression was determined by Western blotting. Occludin and claudin-2 protein expression was dramatically reduced after IFN-γ exposure, which correlated with increased permeability for HRP and FITC-dextran. Interestingly, cleavage of claudin-2 was observed after incubation with IFN-γ. Serine protease inhibitor AEBSF completely abrogated IFN-γ mediated barrier disruption which was associated with preservation of claudin-2 expression. Moreover, IFN-γ induced loss of barrier integrity was found to affect claudin-2 and occludin expression through different mechanisms. Since inhibition of serine protease activity abrogates IFN-γ mediated barrier disruption this may be an important target for therapeutic intervention. PMID:16232214

  7. New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina

    PubMed Central

    2010-01-01

    Background Subtilisin-like serine proteases play an important role in pathogenic fungi during the penetration and colonization of their hosts. In this study, we perform an evolutionary analysis of the subtilisin-like serine protease genes of subphylum Pezizomycotina to find if there are similar pathogenic mechanisms among the pathogenic fungi with different life styles, which utilize subtilisin-like serine proteases as virulence factors. Within Pezizomycotina, nematode-trapping fungi are unique because they capture soil nematodes using specialized trapping devices. Increasing evidence suggests subtilisin-like serine proteases from nematode-trapping fungi are involved in the penetration and digestion of nematode cuticles. Here we also conduct positive selection analysis on the subtilisin-like serine protease genes from nematode-trapping fungi. Results Phylogenetic analysis of 189 subtilisin-like serine protease genes from Pezizomycotina suggests five strongly-supported monophyletic clades. The subtilisin-like serine protease genes previously identified or presumed as endocellular proteases were clustered into one clade and diverged the earliest in the phylogeny. In addition, the cuticle-degrading protease genes from entomopathogenic and nematode-parasitic fungi were clustered together, indicating that they might have overlapping pathogenic mechanisms against insects and nematodes. Our experimental bioassays supported this conclusion. Interestingly, although they both function as cuticle-degrading proteases, the subtilisin-like serine protease genes from nematode-trapping fungi and nematode-parasitic fungi were not grouped together in the phylogenetic tree. Our evolutionary analysis revealed evidence for positive selection on the subtilisin-like serine protease genes of the nematode-trapping fungi. Conclusions Our study provides new insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. Pezizomycotina subtilisins most likely evolved

  8. Serpin-serine protease binding kinetics: alpha 2-antiplasmin as a model inhibitor.

    PubMed

    Longstaff, C; Gaffney, P J

    1991-01-29

    We have examined in detail the kinetics of binding of the serpin alpha 2-antiplasmin to the serine proteases alpha-chymotrypsin and plasmin. These represent model systems for serpin binding. We find, in contrast to earlier published results with alpha 2-antiplasmin and plasmin, that binding is reversible, and slow binding kinetics can be observed, under appropriate conditions. Binding follows a two-step process with both enzymes, with the formation of an initial loose complex which then proceeds to a tightly bound complex. In the absence of lysine and analogues, equilibrium between alpha 2-antiplasmin and plasmin is achieved rapidly, with an overall inhibition constant (Ki') of 0.3 pM. In the presence of tranexamic acid or 6-aminohexanoic acid, lysine analogues that mimic the effects of fibrin, plasmin binding kinetics are changed such that equilibrium is reached slowly following a lag phase after mixing of enzyme and inhibitor. The Ki' is also affected, rising to 2 pM in the presence of 6-aminohexanoic acid concentrations above 15 mM. Thus extrapolation to the in vivo situation indicates that complex formation in the presence of fibrin will be delayed, allowing a burst of enzyme activity following plasmin generation, but a tight, pseudoirreversible complex will result eventually. Chymotrypsin is more weakly inhibited by alpha 2-antiplasmin, exhibiting an overall Ki' of 0.1 nM, after two-stage complex formation. The inhibition constant for the initial loose complex (Ki) is very similar for both enzymes. The difference in binding strength between the two enzymes is accounted for by the dissociation rate constant of the second step of complex formation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1703440

  9. Aspartic acid

    MedlinePlus

    ... also called asparaginic acid. Aspartic acid helps every cell in the body work. It plays a role in: Hormone production and release Normal nervous system function Plant sources of aspartic acid include: Legumes such as ...

  10. Exogenous γ-aminobutyric acid treatment affects citrate and amino acid accumulation to improve fruit quality and storage performance of postharvest citrus fruit.

    PubMed

    Sheng, Ling; Shen, Dandan; Luo, Yi; Sun, Xiaohua; Wang, Jinqiu; Luo, Tao; Zeng, Yunliu; Xu, Juan; Deng, Xiuxin; Cheng, Yunjiang

    2017-02-01

    The loss of organic acids during postharvest storage is one of the major factors that reduces the fruit quality and economic value of citrus. Citrate is the most important organic acid in citrus fruits. Molecular evidence has proved that γ-aminobutyric acid (GABA) shunt plays a key role in citrate metabolism. Here, we investigated the effects of exogenous GABA treatment on citrate metabolism and storage quality of postharvest citrus fruit. The content of citrate was significantly increased, which was primarily attributed to the inhibition of the expression of glutamate decarboxylase (GAD). Amino acids, including glutamate, alanine, serine, aspartate and proline, were also increased. Moreover, GABA treatment decreased the fruit rot rate. The activities of antioxidant enzymes and the content of energy source ATP were affected by the treatment. Our results indicate that GABA treatment is a very effective approach for postharvest quality maintenance and improvement of storage performance in citrus production. PMID:27596402

  11. Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper.

    PubMed

    Pérez, A V; Rucavado, A; Sanz, L; Calvete, J J; Gutiérrez, J M

    2008-01-01

    A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 microg) and fibrinogen (minimum coagulant dose = 4.2 microg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 microg). In addition, when injected intravenously in mice at doses of 5 and 10 microg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the ;gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms. PMID:17994164

  12. O-xylosylation in a recombinant protein is directed at a common motif on glycine-serine linkers.

    PubMed

    Spencer, David; Novarra, Shabazz; Zhu, Liang; Mugabe, Sheila; Thisted, Thomas; Baca, Manuel; Depaz, Roberto; Barton, Christopher

    2013-11-01

    Glycine-serine (GS) linkers are commonly used in recombinant proteins to connect domains. Here, we report the posttranslational O-glycosylation of a GS linker in a novel fusion protein. The structure of the O-glycan moiety is a xylose-based core substituted with hexose and sulfated hexauronic acid residues. The total level of O-xylosylation was approximately 30% in the material expressed in HEK-293 cell lines. There was an approximate 10-fold reduction in O-xylosylation levels when the material was expressed in Chinese hamster ovary cell lines. Similar O-glycan structures have been reported for human urinary thrombomodulin and represent the initial building block for proteoglycans such as chondroitin sulfate and heparin. The sites of attachment, determined by electron transfer dissociation mass spectrometry, were localized to serine in the linker regions of the recombinant fusion protein. This attachment could be attributed, in part, to the inherent xylosyltransferase motif present in GS linkers. Elimination of the O-glycan moiety was achieved with modified linkers containing only glycine residues. The aggregation and fragmentation behavior of the GGG construct were comparable to the GSG-linked material during thermal stress. The O-xylosylation reported has implications for the manufacturing consistency of recombinant proteins containing GS linkers. PMID:24105735

  13. Identification, purification and characterization of a novel collagenolytic serine protease from fig (Ficus carica var. Brown Turkey) latex.

    PubMed

    Raskovic, Brankica; Bozovic, Olga; Prodanovic, Radivoje; Niketic, Vesna; Polovic, Natalija

    2014-12-01

    A novel collagenolytic serine protease was identified and then purified (along with ficin) to apparent homogeneity from the latex of fig (Ficus carica, var. Brown Turkey) by two step chromatographic procedure using gel and covalent chromatography. The enzyme is a monomeric protein of molecular mass of 41 ± 9 kDa as estimated by analytical gel filtration chromatography. It is an acidic protein with a pI value of approximately 5 and optimal activity at pH 8.0-8.5 and temperature 60°C. The enzymatic activity was strongly inhibited by PMSF and Pefabloc SC, indicating that the enzyme is a serine protease. The enzyme showed specificity towards gelatin and collagen (215 GDU/mg and 24.8 CDU/mg, respectively) and non-specific protease activity (0.18 U/mg against casein). The enzyme was stable and retained full activity over a broad range of pH and temperature. The fig latex collagenolytic protease is potentially useful as a non-microbial enzyme with collagenolytic activity for various applications in the fields of biochemistry, biotechnology and medicine. PMID:24982021

  14. Trichinella spiralis newborn larvae: characterization of a stage specific serine proteinase expression, NBL1, using monoclonal antibodies.

    PubMed

    Yang, Yong; Lacour, Sandrine A; Lainé-Prade, Véronique; Versillé, Nicolas; Grasset-Chevillot, Aurélie; Feng, Shuang; Liu, Ming Yuan; Boireau, Pascal; Vallée, Isabelle

    2015-05-01

    Trichinella spiralis is an intracellular parasitic nematode of mammalian skeletal muscle, causing a serious zoonotic disease in humans and showing a high economic impact mainly in pig breeding. Serine proteinases of T. spiralis play important roles in the host-parasite interactions mediating host invasion. In this study, we have focused on newborn larvae (NBL-1), the first identified serine proteinase from the NBL stage of T. spiralis. Five monoclonal antibodies (mAbs) directed against the C-terminal part of NBL1, were produced. These mAbs were IgG1κ isotype and specifically recognized as a common motif of 10 amino acids (PSSGSRPTYP). Selected mAbs were further characterized using antigens from various developmental stages of T. spiralis. Western blot revealed that selected mAbs reacted with the native NBL1 at Mr 50 kDa in the adult and NBL mixed antigens and NBL stage alone. Indirect immunofluorescence analysis revealed that selected mAbs intensely stained only the embryos within the gravid females and the NBL. Thus, the produced mAbs are useful tools for the characterization of NBL1 as a major antigen of Trichinella involved in the invasion of the host but also for the development of new serological tests with an early detection of T. spiralis infection. PMID:25597315

  15. Testis-specific serine/threonine protein kinase 4 (Tssk4) phosphorylates Odf2 at Ser-76

    PubMed Central

    Wang, Xiaoli; Li, Han; Fu, Guolong; Wang, Yunfu; Du, Shiming; Yu, Long; Wei, Youheng; Chen, Shi

    2016-01-01

    As a member of the testis-specific serine/threonine protein kinase (TSSK) family, Tssk4 is exclusively expressed in the testis and plays an essential role in male fertility. We previously reported that Tssk4 can associate with and phosphorylate Odf2, but the phosphorylation site is still unknown. Here we confirm that the C-terminal region (amino acids 214-638) of Odf2 is required for association with Tssk4. Furthermore, to identify the site at which Tssk4 phosphorylates Odf2, we generated several Odf2 point mutants (Ser/Thr/Lys to Ala) and identified serine 76 of Odf2 as one of the phosphorylation sites. In vivo, phosphorylated Odf2 was evaluated in mouse sperm using a specific phospho-Ser-76 Odf2 antibody and LC-MS/MS. These findings are the first to demonstrate the phosphorylation site in Odf2 by Tssk4, providing essential clues regarding the function of Tssk4 in regulating sperm motility and/or structure and thus male fertility. PMID:26961893

  16. Serine- and threonine/valine-dependent activation of PDK and Tor orthologs converge on Sch9 to promote aging.

    PubMed

    Mirisola, Mario G; Taormina, Giusi; Fabrizio, Paola; Wei, Min; Hu, Jia; Longo, Valter D

    2014-02-01

    Dietary restriction extends longevity in organisms ranging from bacteria to mice and protects primates from a variety of diseases, but the contribution of each dietary component to aging is poorly understood. Here we demonstrate that glucose and specific amino acids promote stress sensitization and aging through the differential activation of the Ras/cAMP/PKA, PKH1/2 and Tor/S6K pathways. Whereas glucose sensitized cells through a Ras-dependent mechanism, threonine and valine promoted cellular sensitization and aging primarily by activating the Tor/S6K pathway and serine promoted sensitization via PDK1 orthologs Pkh1/2. Serine, threonine and valine activated a signaling network in which Sch9 integrates TORC1 and Pkh signaling via phosphorylation of threonines 570 and 737 and promoted intracellular relocalization and transcriptional inhibition of the stress resistance protein kinase Rim15. Because of the conserved pro-aging role of nutrient and growth signaling pathways in higher eukaryotes, these results raise the possibility that similar mechanisms contribute to aging in mammals. PMID:24516402

  17. Serine- and Threonine/Valine-Dependent Activation of PDK and Tor Orthologs Converge on Sch9 to Promote Aging

    PubMed Central

    Fabrizio, Paola; Wei, Min; Hu, Jia; Longo, Valter D.

    2014-01-01

    Dietary restriction extends longevity in organisms ranging from bacteria to mice and protects primates from a variety of diseases, but the contribution of each dietary component to aging is poorly understood. Here we demonstrate that glucose and specific amino acids promote stress sensitization and aging through the differential activation of the Ras/cAMP/PKA, PKH1/2 and Tor/S6K pathways. Whereas glucose sensitized cells through a Ras-dependent mechanism, threonine and valine promoted cellular sensitization and aging primarily by activating the Tor/S6K pathway and serine promoted sensitization via PDK1 orthologs Pkh1/2. Serine, threonine and valine activated a signaling network in which Sch9 integrates TORC1 and Pkh signaling via phosphorylation of threonines 570 and 737 and promoted intracellular relocalization and transcriptional inhibition of the stress resistance protein kinase Rim15. Because of the conserved pro-aging role of nutrient and growth signaling pathways in higher eukaryotes, these results raise the possibility that similar mechanisms contribute to aging in mammals. PMID:24516402

  18. Identification of homologues to the pathogenicity factor Pat-1, a putative serine protease of Clavibacter michiganensis subsp. michiganensis.

    PubMed

    Burger, Annette; Gräfen, Ines; Engemann, Jutta; Niermann, Erik; Pieper, Martina; Kirchner, Oliver; Gartemann, Karl-Heinz; Eichenlaub, Rudolf

    2005-01-01

    Hybridization of Clavibacter michiganensis subsp. michiganensis total DNA against the pathogenicity gene pat-1 indicated the presence of pat-1 homologous nucleotide sequences on the chromosome and on plasmid pCM2. Isolation of the corresponding DNA fragments and nucleotide sequence determination showed that there are three pat-1 homologous genes: chpA (chromosome) and phpA and phpB (plasmid pCM2). The gene products share common characteristics, i.e. a signal sequence for Sec-dependent secretion, a serine protease motif, and six cysteine residues at conserved positions. Gene chpA located on the chromosome is a pseudogene since it contains a translational stop codon after 97 of 280 amino acids. In contrast to pat-1, cloning of the plasmid encoded homologs phpA and phpB into the avirulent plasmid free Cmm strain CMM100 did not result in a virulent phenotype. So far, no proteolytic activity could be demonstrated for Pat-1, however, site specific mutagenesis of pat-1 showed that the serine residue in the motif GDSGG is required for the virulent phenotype of pat-1 and thus Pat-1 could be a functional protease. PMID:16255147

  19. Concordance between isolated cleft palate in mice and alterations within a region including the gene encoding the [beta][sub 3] subunit of the type A [gamma]-aminobutyric acid receptor

    SciTech Connect

    Culiat, C.T.; Stubbs, L.; Nicholls, R.D.; Montgomery, C.S.; Russell, L.B.; Johnson, D.K. ); Rinchik, E.M. Univ. of Florida, Gainesville )

    1993-06-01

    Genetic and molecular analyses of a number of radiation-induced deletion mutations of the pink-eyed dilution (p) locus in mouse chromosome 7 have identified a specific interval on the genetic map associated with a neonatally lethal mutation that results in cleft palate. This interval, closely linked and distal to p, and bracketed by the genes encoding the [alpha][sub 5] and [beta][sub 3] subunits of the type A [gamma]-aminobutyric acid receptor (Gabra5 and Gabrb3, respectively), contains a gene(s) (cp1; cleft palate 1) necessary for normal palate development. The cp1 interval extends from the distal breakpoint of the prenatally lethal p[sup 83FBFo] deletion to the Gabrb3 locus. Among 20 p deletions tested, there was complete concordance between alterations at the Gabrb3 transcription unit and inability to complement the cleft-palate defect. These mapping data, along with previously described in vivo and in vitro teratological effects of [gamma]-aminobutyric acid or its agonists on palate development, suggest the possibility that a particular type A [gamma]-aminobutyric acid receptor that includes the [beta][sub 3] subunit may be necessary for normal palate development. The placement of the cp1 gene within a defined segment of the larger D15S12h (p)-D15S9h-1 interval in the mouse suggests that the highly homologous region of the human genome, 15q11-q13, be evaluated for a role(s) in human fetal facial development. 29 refs., 4 figs., 1 tab.

  20. Chemiluminescence Detection of Serine, Proline, Glycine, Asparagine, Leucine, and Histidine by Using Corresponding Aminoacyl-tRNA Synthetases as Recognition Elements.

    PubMed

    Kugimiya, Akimitsu; Fukada, Rie

    2015-06-01

    Analysis of the concentration of free amino acids in biological samples is useful in clinical diagnostics. However, currently available methods are time consuming, potentially delaying diagnosis. Therefore, the development of more rapid analytical tools is needed. In this study, a chemiluminescence detection method for amino acids was developed, and the conditions for the enzyme reaction and assay were examined. For the recognition of each amino acid (here, serine, proline, glycine, asparagine, leucine, and histidine), the corresponding aminoacyl-tRNA synthetase (aaRS) was employed, and multiple enzymatic reactions were combined with a luminol chemiluminescence reaction. This method provided selective quantification from 1 to 20 μM for serine, proline, glycine, and leucine; 1 to 60 μM for asparagine; and 1 to 150 μM for histidine. This assay, which utilized aaRSs for the detection of amino acids, could be useful for simple and rapid analysis of amino acids in clinical diagnostics. PMID:25935222

  1. D-Serine and D-Cycloserine Reduce Compulsive Alcohol Intake in Rats.

    PubMed

    Seif, Taban; Simms, Jeffrey A; Lei, Kelly; Wegner, Scott; Bonci, Antonello; Messing, Robert O; Hopf, F Woodward

    2015-09-01

    There is considerable interest in NMDAR modulators to enhance memory and treat neuropsychiatric disorders such as addiction, depression, and schizophrenia. D-serine and D-cycloserine, the NMDAR activators at the glycine site, are of particular interest because they have been used in humans without serious adverse effects. Interestingly, D-serine also inhibits some NMDARs active at hyperpolarized potentials (HA-NMDARs), and we previously found that HA-NMDARs within the nucleus accumbens core (NAcore) are critical for promoting compulsion-like alcohol drinking, where rats consume alcohol despite pairing with an aversive stimulus such as quinine, a paradigm considered to model compulsive aspects of human alcohol use disorders (AUDs). Here, we examined the impact of D-serine and D-cycloserine on this aversion-resistant alcohol intake (that persists despite adulteration with quinine) and consumption of quinine-free alcohol. Systemic D-serine reduced aversion-resistant alcohol drinking, without altering consumption of quinine-free alcohol or saccharin with or without quinine. Importantly, D-serine within the NAcore but not the dorsolateral striatum also selectively reduced aversion-resistant alcohol drinking. In addition, D-serine inhibited EPSCs evoked at -70 mV in vitro by optogenetic stimulation of mPFC-NAcore terminals in alcohol-drinking rats, similar to reported effects of the NMDAR blocker AP5. Further, D-serine preexposure occluded AP5 inhibition of mPFC-evoked EPSCs, suggesting that D-serine reduced EPSCs by inhibiting HA-NMDARs. Systemic D-cycloserine also selectively reduced intake of quinine-adulterated alcohol, and D-cycloserine inhibited NAcore HA-NMDARs in vitro. Our results indicate that HA-NMDAR modulators can reduce aversion-resistant alcohol drinking, and support testing of D-serine and D-cycloserine as immediately accessible, FDA-approved drugs to treat AUDs. PMID:25801502

  2. Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins

    PubMed Central

    Haynes, Chad; Iakoucheva, Lilia M.

    2006-01-01

    Serine/arginine-rich (SR) splicing factors play an important role in constitutive and alternative splicing as well as during several steps of RNA metabolism. Despite the wealth of functional information about SR proteins accumulated to-date, structural knowledge about the members of this family is very limited. To gain a better insight into structure-function relationships of SR proteins, we performed extensive sequence analysis of SR protein family members and combined it with ordered/disordered structure predictions. We found that SR proteins have properties characteristic of intrinsically disordered (ID) proteins. The amino acid composition and sequence complexity of SR proteins were very similar to those of the disordered protein regions. More detailed analysis showed that the SR proteins, and their RS domains in particular, are enriched in the disorder-promoting residues and are depleted in the order-promoting residues as compared to the entire human proteome. Moreover, disorder predictions indicated that RS domains of SR proteins were completely unstructured. Two different classification methods, the charge-hydropathy measure and the cumulative distribution function (CDF) of the disorder scores, were in agreement with each other, and they both strongly predicted members of the SR protein family to be disordered. This study emphasizes the importance of the disordered structure for several functions of SR proteins, such as for spliceosome assembly and for interaction with multiple partners. In addition, it demonstrates the usefulness of order/disorder predictions for inferring protein structure from sequence. PMID:16407336

  3. [Conformational stability of serine proteinase inhibitor from the sea anemone Heteractis crispa].

    PubMed

    Vakorina, T I; Gladkikh, I N; Monastyrnaia, M M; Kozlovskaia, E P

    2011-01-01

    The influence of different environmental values of the pH and temperature on the spatial organization of serine proteinase inhibitor from the sea anemone Heteractis crispa (=Radianthus macrodactylus) on the level of tertiary and secondary structure was studied by CD spectroscopy. The molecule InhVJ was shown to possess a high conformational thermo- and pH-stability. We determined the point of conformational thermotransition of polypeptide (70 degrees C) after which the molecule gets denaturational stable state with conservation of 80% proteinase inhibitory activity. The significant partial reversible changes of molecule spatial organization were established to occur at the level of tertiary structure in the process of acid-base titration in the range of pH 11.0-13.0. This can be explained by of ionization of tyrosine residues. The molecule InhVJ is conformationally stable at the low pH values (2.0). The quenching of tyrosine residues by acrylamide showed that two of these residues are accessible to the quencher in full, while the third part is available. PMID:21899045

  4. Serine protease activity and residual LEKTI expression determine phenotype in Netherton syndrome.

    PubMed

    Hachem, Jean-Pierre; Wagberg, Fredrik; Schmuth, Matthias; Crumrine, Debra; Lissens, Willy; Jayakumar, Arumugam; Houben, Evi; Mauro, Theodora M; Leonardsson, Göran; Brattsand, Maria; Egelrud, Torbjorn; Roseeuw, Diane; Clayman, Gary L; Feingold, Kenneth R; Williams, Mary L; Elias, Peter M

    2006-07-01

    Mutations in the SPINK5 gene encoding the serine protease (SP) inhibitor, lymphoepithelial-Kazal-type 5 inhibitor (LEKTI), cause Netherton syndrome (NS), a life-threatening disease, owing to proteolysis of the stratum corneum (SC). We assessed here the basis for phenotypic variations in nine patients with "mild", "moderate", and "severe" NS. The magnitude of SP activation correlated with both the barrier defect and clinical severity, and inversely with residual LEKTI expression. LEKTI co-localizes within the SC with kallikreins 5 and 7 and inhibits both SP. The permeability barrier abnormality in NS was further linked to SC thinning and proteolysis of two lipid hydrolases (beta-glucocerebrosidase and acidic sphingomyelinase), with resultant disorganization of extracellular lamellar membranes. SC attenuation correlated with phenotype-dependent, SP activation, and loss of corneodesmosomes, owing to desmoglein (DSG)1 and desmocollin (DSC)1 degradation. Although excess SP activity extended into the nucleated layers in NS, degrading desmosomal mid-line structures with loss of DSG1/DSC1, the integrity of the nucleated epidermis appears to be maintained by compensatory upregulation of DSG3/DSC3. Maintenance of sufficient permeability barrier function for survival correlated with a compensatory acceleration of lamellar body secretion, providing a partial permeability barrier in NS. These studies provide a mechanistic basis for phenotypic variations in NS, and describe compensatory mechanisms that permit survival of NS patients in the face of unrelenting SP attack. PMID:16601670

  5. Building a Molecular Trap for a Serine Protease from Aptamer and Peptide Modules.

    PubMed

    Dupont, Daniel M; Bjerregaard, Nils; Verpaalen, Ben; Andreasen, Peter A; Jensen, Jan K

    2016-04-20

    In drug development, molecular intervention strategies are usually based on interference with a single protein function, such as enzyme activity or receptor binding. However, in many cases, protein drug targets are multifunctional, with several molecular functions contributing to their pathophysiological actions. Aptamers and peptides are interesting synthetic building blocks for the design of multivalent molecules capable of modulating multiple functions of a target protein. Here, we report a molecular trap with the ability to interfere with the activation, catalytic activity, receptor binding, etc. of the serine protease urokinase-type plasminogen activator (uPA) by a rational combination of two RNA aptamers and a peptide with different inhibitory properties. The assembly of these artificial inhibitors into one molecule enhanced the inhibitory activity between 10- and 10,000-fold toward several functions of uPA. The study highlights the potential of multivalent designs and illustrates how they can easily be constructed from aptamers and peptides using nucleic acid engineering, chemical synthesis, and bioconjugation chemistry. By aptamer to aptamer and aptamer to peptide conjugation, we created, to the best of our knowledge, the first trivalent molecule which combines three artificial inhibitors binding to three different sites in a protein target. We hypothesize that by simultaneously preventing all of the functional interactions and activities of the target protein, this approach may represent an alternative to siRNA technology for a functional knockout. PMID:26926041

  6. Identification and characterization of alkaline serine protease from goat skin surface metagenome.

    PubMed

    Pushpam, Paul Lavanya; Rajesh, Thangamani; Gunasekaran, Paramasamy

    2011-01-01

    Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively. PMID:21906326

  7. Characterization of serine/cysteine protease inhibitor alpha1-antitripsin from meconium-instilled rabbit lungs.

    PubMed

    Zagariya, A M; Bhat, R; Zhabotynsky, E; Chari, G; Navale, S; Xu, Q; Keiderling, T A; Vidyasagar, D

    2005-09-01

    We have recently purified from meconium-instilled rabbit lungs a novel serine proteinase inhibitor, with an apparent molecular mass of 50 kDa, which we assign to be alpha1-antitripsin. We hypothesize that serpin may attenuate pulmonary inflammation and improve surfactant function after meconium aspiration. Alpha1-antitripsin is a member of the proteinase inhibitor (serpin) superfamily and inhibitor of neutrophil elastase, and it can be identified as a member of the family by its amino acid sequence due to the high degree of conserved residues. Alpha1-antitripsin is synthesized by epithelial cells, macrophages, monocytes, and neutrophils. Deficiency in alpha1-antitripsin leads to exposure of lungs to uncontrolled proteolytic attack from neutrophil elastase or other damaging factors culminating in lung destruction and cell apoptosis. We hypothesize that accumulation of alpha1-antitripsin in the lungs serves as a predisposed protection against meconium-induced lung injury. In this paper, we show how this knowledge can lead to the development of novel therapeutic approaches for treatment of MAS. PMID:15962329

  8. Astacin Family Metallopeptidases and Serine Peptidase Inhibitors in Spider Digestive Fluid

    PubMed Central

    Foradori, Matthew J.; Tillinghast, Edward K.; Smith, J. Stephen; Townley, Mark A.; Mooney, Robert E.

    2006-01-01

    Digestive fluid of the araneid spider Argiope aurantia is known to contain zinc metallopeptidases. Using anion-exchange chromatography, size-exclusion chromatography, sucrose density gradient centrifugation, and gel electrophoresis, we isolated two lower-molecular-mass peptidases, designated p16 and p18. The N-terminal amino acid sequences of p16 (37 residues) and p18 (20 residues) are 85% identical over the first 20 residues and are most similar to the N-terminal sequences of the fully active form of meprin (β subunits) from several vertebrates (47–52% and 50–60% identical, respectively). Meprin is a peptidase in the astacin (M12A) subfamily of the astacin (M12) family. Additionally, a 66-residue internal sequence obtained from p16 aligns with the conserved astacin subfamily domain. Thus, at least some spider digestive peptidases appear related to astacin of decapod crustaceans. However, important differences between spider and crustacean metallopeptidases with regard to isoelectric point and their susceptibility to hemolymph-borne inhibitors are demonstrated. Anomalous behavior of the lower-molecular-mass Argiope peptidases during certain fractionation procedures indicates that these peptidases may take part in reversible associations with each other or with other proteins. A. aurantia digestive fluid also contains inhibitory activity effective against insect digestive peptidases. Here we present evidence for at least thirteen, heat-stable serine peptidase inhibitors ranging in molecular mass from about 15 to 32 kDa. PMID:16458560

  9. The synthesis of peptidylfluoromethanes and their properties as inhibitors of serine proteinases and cysteine proteinases.

    PubMed Central

    Rauber, P; Angliker, H; Walker, B; Shaw, E

    1986-01-01

    A synthesis of peptidylfluoromethanes is described that utilizes the conversion of phthaloyl amino acids into their fluoromethane derivatives. These can be deblocked and elongated. The inactivation of chymotrypsin by Cbz-Phe-CH2F (benzyloxycarbonylphenylalanylfluoromethane) was found to be considerably slower than that of the analogous chloromethane. The fluoromethane analogue inactivates chymotrypsin with an overall rate constant that is 2% of that observed for the inactivation of the enzyme with the chloromethane. However, the result is the same. The reagent complexes in a substrate-like manner, with Ki = 1.4 X 10(-4) M, and alkylates the active-centre histidine residue. Cbz-Phe-Phe-CH2F and Cbz-Phe-Ala-CH2F were investigated as inactivators of the cysteine proteinase cathepsin B. The difference in reactivity between fluoromethyl ketones and chloromethyl ketones is less pronounced in the case of the cysteine proteinase than for the serine proteinase. Covalent bond formation takes place in this case also, as demonstrated by the use of a radiolabelled reagent. PMID:3827817

  10. Identification of folding intermediates of streblin, the most stable serine protease: biophysical analysis.

    PubMed

    Kumar, Reetesh; Tripathi, Pinki; de Moraes, Fabio Rogerio; Caruso, Icaro P; Jagannadham, Medicherla V

    2014-01-01

    Streblin, a serine proteinase from plant Streblus asper, has been used to investigate the conformational changes induced by pH, temperature, and chaotropes. The near/far UV circular dichroism activities under fluorescence emission spectroscopy and 8-aniline-1-naphthalene sulfonate (ANS) binding have been carried out to understand the unfolding of the protein in the presence of denaturants. Spectroscopic studies reveal that streblin belongs to the α+β class of proteins and exhibits stability towards chemical denaturants, guanidine hydrochloride (GuHCl). The pH-induced transition of this protein is noncooperative for transition phases between pH 0.5 and 2.5 (midpoint, 1.5) and pH 2.5 and 10.0 (midpoint, 6.5). At pH 1.0 or lower, the protein unfolds to form acid-unfolded state, and for pH 7.5 and above, protein turns into an alkaline denatured state characterized by the absence of ANS binding. At pH 2.0 (1 M GuHCl), streblin exists in a partially unfolded state with characteristics of a molten globule state. The protein is found to exhibit strong and predominant ANS binding. In total, six different intermediate states has been identified to show protein folding pathways. PMID:24108566

  11. Alteration by phosphatidyl serine of tension responses and 45Ca distribution in aortic smooth muscle.

    PubMed

    Goodman, F R; Weiss, G B; Goth, A

    1976-07-01

    The effects of phosphatidyl serine (PS) on 45Ca distribution, 45Ca movements and contractions were examined in rabbit aortic smooth muscle. Contractile responses to submaximal concentrations of norepinephrine and histamine were potentiated by prior exposure to PS, but equivalent responses to potassium were unaffected. Addition of PS to the incubation solution decreased 45Ca uptake; exposure of aortic strips to PS during washout of either 45Ca or promethium (147Pm) resulted in maintained increases in efflux. These PS-induced alterations in net loss of 45Ca or 147Pm can be attributed to a decreased membrane reuptake and/or rebinding. However, the presence of PS during the washout significantly reduced the increases in 45Ca efflux rate elicited with either 0.05 mM concentrations of Ca++ or ethylenediamine tetraacetic acid. Thus, in rabbit aortic smooth muscle, exogenous PS can alter the availability and/or exchangeability of a membrane-bound Ca++ fraction. By specifically increasing the affinity for Ca++ at relevant membrane sites or stores. PS may enhance the ability of vascular smooth muscle to respond to stimulatory agents that mobilize Ca++ from these sites and, in this manner, potentiate contractile responses. PMID:933004

  12. Lipoprotein(a) binds to fibronectin and has serine proteinase activity capable of cleaving it.

    PubMed Central

    Salonen, E M; Jauhiainen, M; Zardi, L; Vaheri, A; Ehnholm, C

    1989-01-01

    The plasma concentration of human lipoprotein(a) [Lp(a)] is correlated with the risk of heart disease. A distinct feature of the Lp(a) particle is the apolipoprotein (a) [apo(a)], which is associated with apoB-100, the main protein component of low-density lipoprotein. We now report that apo(a), which has extensive homology to plasminogen, binds to immobilized fibronectin. The binding of Lp(a) was localized to the C-terminal heparin-binding domain of fibronectin. Incubation of Lp(a) with fibronectin resulted in fragmentation of fibronectin. The cleavage pattern, as visualized by gel electrophoresis and immunoblotting, was reproducibly obtained with Lp(a) purified from five different individuals and was distinct from that obtained upon proteolysis of fibronectin by plasmin or kallikrein. The use of synthetic peptide substrates demonstrated that the amino acid specificity for Lp(a) was arginine rather than lysine. The proteolytic activity of Lp(a) was localized to apo(a) and experiments with inhibitors indicated that the proteolytic activity was of serine proteinase-type. Images PMID:2531657

  13. Identification and characterization of alkaline serine protease from goat skin surface metagenome

    PubMed Central

    2011-01-01

    Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively. PMID:21906326

  14. Study on A.C. electrical properties of pure and L-serine doped ADP crystals

    NASA Astrophysics Data System (ADS)

    Joshi, J. H.; Dixit, K. P.; Joshi, M. J.; Parikh, K. D.

    2016-05-01

    Ammonium Dihydrogen Phosphate (ADP) crystals have a wide range of applications in integrated and nonlinear optics. Amino acids having significant properties like molecular chirality, zwitter ionic nature, etc. attracted many researchers to dope them in various NLO crystals. In the present study, pure and different weight percentage L-serine doped ADP crystals were grown by slow solvent evaporation technique at room temperature. The A.C. electrical study was carried out for palletized samples at room temperature. The Nyquist plot showed two semi circles for pure ADP indicated the effect of grain and grain boundary, whereas the doped ADP samples exhibited the single semi circle suggesting the effect of grain. The values resistance and capacitance for grain and grain boundary were calculated. The effect of doping was clearly seen in the grain capacitance and resistance values. The dielectric constant and dielectric loss decreased with increase in frequency for all samples. The Jonscher power law was applied for A.C. conductivity for pure and doped ADP samples. The imaginary part of modulus and impedance versus frequency were drawn and the value of stretch exponent (β) was calculated for all the samples.

  15. Rapid development of a potent photo-triggered inhibitor of the serine hydrolase RBBP9.

    PubMed

    Liu, Xiaodan; Dix, Melissa; Speers, Anna E; Bachovchin, Daniel A; Zuhl, Andrea M; Cravatt, Benjamin F; Kodadek, Thomas J

    2012-09-24

    The serine hydrolases constitute a large class of enzymes that play important roles in physiology. There is great interest in the development of potent and selective pharmacological inhibitors of these proteins. Traditional active-site inhibitors often have limited selectivity within this superfamily and are tedious and expensive to discover. Using the serine hydrolase RBBP9 as a model target, we designed a rapid and relatively inexpensive route to highly selective peptoid-based inhibitors that can be activated by visible light. This technology provides rapid access to photo-activated tool compounds capable of selectively blocking the function of particular serine hydrolases. PMID:22907802

  16. Rapid Development of a Potent Photo-Triggered Inhibitor of the Serine Hydrolase RBBP9

    PubMed Central

    Liu, Xiaodan; Dix, Melissa; Speers, Anna E.; Bachovchin, Daniel A.; Zuhl, Andrea M.

    2013-01-01

    The serine hydrolases constitute a large class of enzymes that play important roles in physiology. There is great interest in the development of potent and selective pharmacological inhibitors to these proteins. Traditional active site inhibitors often have limited selectivity within this superfamily and are tedious and expensive to discover. Using the serine hydrolase RBBP9 as a model target, we report here a rapid and relatively inexpensive route to highly selective peptoid-based inhibitors that can be activated with visible light. This technology provides rapid access to photo-activated tool compounds capable of selectively blocking the function of particular serine hydrolases. PMID:22907802

  17. Isolation and complete amino acid sequence of two fibrinolytic proteinases from the toxic Saturnid caterpillar Lonomia achelous.

    PubMed

    Amarant, T; Burkhart, W; LeVine, H; Arocha-Pinango, C L; Parikh, I

    1991-08-30

    The major toxic and fibrinolytic activity of the saliva and hemolymph of the larval form of Lonomia achelous was purified to homogeneity by a combination of metal chelate and affinity chromatography. Two apparent isozymes, Achelase I (213 amino acids, pIcalc = 10.55) and Achelase II (214 amino acids, pIcalc = 8.51), were sequenced by automated Edman degradation, and their C-termini confirmed by Fourier-transform mass spectrometry. The calculated molecular weights (22,473 and 22,727) correspond well to Mr estimates of 24,000 by SDS-PAGE. No carbohydrate was detected during sequencing. The enzymes degraded all three chains of fibrin, alpha greater than beta much greater than gamma, yielding a fragmentation pattern indistinguishable from that produced by trypsin. Chromogenic peptides S-2222 (Factor Xa and trypsin), S-2251 (plasmin), S-2302 (kallikrein) and S-2444 (urokinase) were substrates while S-2288 (broad range of serine proteinases including thrombin) was not hydrolyzed. Among a range of inhibitors Hg+2, aminophenylmercuriacetate, leupeptin, antipain and E-64 but not N-ethylmaleimide or iodoacetate abolished the activity of the purified isozymes against S-2444. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and aprotinin were less effective. The presence of the classic catalytic triad (histidine-41, aspartate-86 and serine-189) suggests that Achelases I and II may be serine proteinases, but with a potentially free cysteine-185 which could react with thiol proteinase-directed reagents. PMID:1911844

  18. Immune Responses Induced by Gene Gun or Intramuscular Injection of DNA Vaccines That Express Immunogenic Regions of the Serine Repeat Antigen from Plasmodium falciparum

    PubMed Central

    Belperron, Alexia A.; Feltquate, David; Fox, Barbara A.; Horii, Toshihiro; Bzik, David J.

    1999-01-01

    The liver- and blood-stage-expressed serine repeat antigen (SERA) of Plasmodium falciparum is a candidate protein for a human malaria vaccine. We compared the immune responses induced in mice immunized with SERA-expressing plasmid DNA vaccines delivered by intramuscular (i.m.) injection or delivered intradermally by Gene Gun immunization. Mice were immunized with a pcdna3 plasmid encoding the entire 47-kDa domain of SERA (amino acids 17 to 382) or the N-terminal domain (amino acids 17 to 110) of SERA. Minimal antibody responses were detected following DNA vaccination with the N-terminal domain of SERA, suggesting that the N-terminal domain alone is not highly immunogenic by this route of vaccine delivery. Immunization of mice by Gene Gun delivery of the 47-kDa domain of SERA elicited a significantly higher serum antibody titer to the antigen than immunization of mice by i.m. injection with the same plasmid did. The predominant isotype subclass of the antibodies elicited to the SERA protein following i.m. and Gene Gun immunizations with SERA plasmid DNA was immunoglobulin G1. Coimmunization of mice with SERA plasmid DNA and a plasmid expressing the hepatitis B surface antigen (pCMV-s) by the i.m. route resulted in higher anti-SERA titers than those generated in mice immunized with the SERA DNA plasmid alone. Vaccination with DNA may provide a viable alternative or may be used in conjunction with protein-based subunit vaccines to maximize the efficacy of a human malaria vaccine that includes immunogenic regions of the SERA protein. PMID:10496891

  19. Immune responses induced by gene gun or intramuscular injection of DNA vaccines that express immunogenic regions of the serine repeat antigen from Plasmodium falciparum.

    PubMed

    Belperron, A A; Feltquate, D; Fox, B A; Horii, T; Bzik, D J

    1999-10-01

    The liver- and blood-stage-expressed serine repeat antigen (SERA) of Plasmodium falciparum is a candidate protein for a human malaria vaccine. We compared the immune responses induced in mice immunized with SERA-expressing plasmid DNA vaccines delivered by intramuscular (i.m.) injection or delivered intradermally by Gene Gun immunization. Mice were immunized with a pcdna3 plasmid encoding the entire 47-kDa domain of SERA (amino acids 17 to 382) or the N-terminal domain (amino acids 17 to 110) of SERA. Minimal antibody responses were detected following DNA vaccination with the N-terminal domain of SERA, suggesting that the N-terminal domain alone is not highly immunogenic by this route of vaccine delivery. Immunization of mice by Gene Gun delivery of the 47-kDa domain of SERA elicited a significantly higher serum antibody titer to the antigen than immunization of mice by i.m. injection with the same plasmid did. The predominant isotype subclass of the antibodies elicited to the SERA protein following i.m. and Gene Gun immunizations with SERA plasmid DNA was immunoglobulin G1. Coimmunization of mice with SERA plasmid DNA and a plasmid expressing the hepatitis B surface antigen (pCMV-s) by the i.m. route resulted in higher anti-SERA titers than those generated in mice immunized with the SERA DNA plasmid alone. Vaccination with DNA may provide a viable alternative or may be used in conjunction with protein-based subunit vaccines to maximize the efficacy of a human malaria vaccine that includes immunogenic regions of the SERA protein. PMID:10496891

  20. Conformation effects on the molecular orbitals of serine

    NASA Astrophysics Data System (ADS)

    Wang, Ke-Dong; Ma, Peng-Fei; Shan, Xu

    2011-03-01

    This paper calculates the five most stable conformers of serine with Hartree—Fock theory, density functional theory (B3LYP), Møller—Plesset perturbation theory (MP4(SDQ)) and electron propagation theory with the 6-311++G(2d,2p) basis set. The calculated vertical ionization energies for the valence molecular orbitals of each conformer are in agreement with the experimental data, indicating that a range of molecular conformations would coexist in an equilibrium sample. Information of the five outer valence molecular orbitals for each conformer is explored in coordinate and momentum spaces using dual space analysis to investigate the conformational processes, which are generated from the global minimum conformer Ser1 by rotation of C2-C3 (Ser4), C1-C2 (Ser5) and C1-O2 (Ser2 and Ser3). Orbitals 28a, 27a and 26a are identified as the fingerprint orbitals for all the conformational processes. Project supported by the Doctoral Research Fund of Henan Normal University, China (Grant No. 525449).

  1. Serine protease inhibitor A3 in atherosclerosis and aneurysm disease.

    PubMed

    Wågsäter, Dick; Johansson, Daniel; Fontaine, Vincent; Vorkapic, Emina; Bäcklund, Alexandra; Razuvaev, Anton; Mäyränpää, Mikko I; Hjerpe, Charlotta; Caidahl, Kenneth; Hamsten, Anders; Franco-Cereceda, Anders; Wilbertz, Johannes; Swedenborg, Jesper; Zhou, Xinghua; Eriksson, Per

    2012-08-01

    Remodeling of extracellular matrix (ECM) plays an important role in both atherosclerosis and aneurysm disease. Serine protease inhibitor A3 (serpinA3) is an inhibitor of several proteases such as elastase, cathepsin G and chymase derived from mast cells and neutrophils. In this study, we investigated the putative role of serpinA3 in atherosclerosis and aneurysm formation. SerpinA3 was expressed in endothelial cells and medial smooth muscle cells in human atherosclerotic lesions and a 14-fold increased expression of serpinA3n mRNA was found in lesions from Apoe-/- mice compared to lesion-free vessels. In contrast, decreased mRNA expression (-80%) of serpinA3 was found in biopsies of human abdominal aortic aneurysm (AAA) compared to non-dilated aortas. Overexpression of serpinA3n in transgenic mice did not influence the development of atherosclerosis or CaCl2-induced aneurysm formation. In situ zymography analysis showed that the transgenic mice had lower cathepsin G and elastase activity, and more elastin in the aortas compared to wild-type mice, which could indicate a more stable aortic phenotype. Differential vascular expression of serpinA3 is clearly associated with human atherosclerosis and AAA but serpinA3 had no major effect on experimentally induced atherosclerosis or AAA development in mouse. However, serpinA3 may be involved in a phenotypic stabilization of the aorta. PMID:22580763

  2. The Nature of D-Serine-Induced Nephrotoxicity

    PubMed Central

    Ganote, Charles E.; Peterson, Darryl R.; Carone, Frank A.

    1974-01-01

    Renal structural changes were studied sequentially between 1 hour and 6 days in rats treated with D-serine. Extensive necrosis of proximal straight tubules was rapid in onset and was followed by complete tubular regeneration 6 days post-treatment. The apparent progression of cellular changes was initial shrinkage, followed either by swelling and loss of apical cytoplasm or immediate lysis of cytoplasmic and nuclear contents. Tubular damage left only the basement membrane as a barrier between interstitial and luminal fluids. In similarly treated rats, proteinuria and glucosuria developed at the onset of tubular necrosis and disappeared when the tubules were completely relined by epithelium suggesting that they are due to diffusion of protein and glucose from interstitium into tubular fluid across the denuded basement membranes and that epithelial cells, under normal conditions, act as a barrier to diffusion of certain substances between the interstitium and tubular fluid. ImagesFig 10Fig 11Fig 1Fig 2Fig 3Fig 12Fig 13Fig 14Fig 15Fig 4Fig 5Fig 6Fig 7Fig 8Fig 9 PMID:4447130

  3. Exploring a new serine protease from Cucumis sativus L.

    PubMed

    Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process. PMID:25577345

  4. Regulating the regulators: serine/arginine-rich proteins under scrutiny.

    PubMed

    Risso, Guillermo; Pelisch, Federico; Quaglino, Ana; Pozzi, Berta; Srebrow, Anabella

    2012-10-01

    Serine/arginine-rich (SR) proteins are among the most studied splicing regulators. They constitute a family of evolutionarily conserved proteins that, apart from their initially identified and deeply studied role in splicing regulation, have been implicated in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export and mRNA translation. Remarkably, this list of SR protein activities seems far from complete, as unexpected functions keep being unraveled. An intriguing aspect that awaits further investigation is how the multiple tasks of SR proteins are concertedly regulated within mammalian cells. In this article, we first discuss recent findings regarding the regulation of SR protein expression, activity and accessibility. We dive into recent studies describing SR protein auto-regulatory feedback loops involving different molecular mechanisms such asunproductive splicing, microRNA-mediated regulation and translational repression. In addition, we take into account another step of regulation of SR proteins, presenting new findings about a variety of post-translational modifications by proteomics approaches and how some of these modifications can regulate SR protein sub-cellular localization or stability. Towards the end, we focus in two recently revealed functions of SR proteins beyond mRNA biogenesis and metabolism, the regulation of micro-RNA processing and the regulation of small ubiquitin-like modifier (SUMO) conjugation. PMID:22941908

  5. Specific domain structures control abscisic acid-, salicylic acid-, and stress-mediated SIZ1 phenotypes.

    PubMed

    Cheong, Mi Sun; Park, Hyeong Cheol; Hong, Mi Ju; Lee, Jiyoung; Choi, Wonkyun; Jin, Jing Bo; Bohnert, Hans J; Lee, Sang Yeol; Bressan, Ray A; Yun, Dae-Jin

    2009-12-01

    SIZ1 (for yeast SAP and MIZ1) encodes the sole ortholog of mammalian PIAS (for protein inhibitor of activated STAT) and yeast SIZ SUMO (for small ubiquitin-related modifier) E3 ligases in Arabidopsis (Arabidopsis thaliana). Four conserved motifs in SIZ1 include SAP (for scaffold attachment factor A/B/acinus/PIAS domain), PINIT (for proline-isoleucine-asparagine-isoleucine-threonine), SP-RING (for SIZ/PIAS-RING), and SXS (for serine-X-serine, where X is any amino acid) motifs. SIZ1 contains, in addition, a PHD (for plant homeodomain) typical of plant PIAS proteins. We determined phenotypes of siz1-2 knockout mutants transformed with SIZ1 alleles carrying point mutations in the predicted domains. Domain SP-RING is required for SUMO conjugation activity and nuclear localization of SIZ1. Salicylic acid (SA) accumulation and SA-dependent phenotypes of siz1-2, such as diminished plant size, heightened innate immunity, and abscisic acid inhibition of cotyledon greening, as well as SA-independent basal thermotolerance were not complemented by the altered SP-RING allele of SIZ1. The SXS domain also controlled SA accumulation and was involved in greening and expansion of cotyledons of seedlings germinated in the presence of abscisic acid. Mutations of the PHD zinc finger domain and the PINIT motif affected in vivo SUMOylation. Expression of the PHD and/or PINIT domain mutant alleles of SIZ1 in siz1-2 promoted hypocotyl elongation in response to sugar and light. The various domains of SIZ1 make unique contributions to the plant's ability to cope with its environment. PMID:19837819

  6. Establishment of an In Vitro d-Cycloserine-Synthesizing System by Using O-Ureido-l-Serine Synthase and d-Cycloserine Synthetase Found in the Biosynthetic Pathway

    PubMed Central

    Uda, Narutoshi; Matoba, Yasuyuki; Kumagai, Takanori; Oda, Kosuke; Noda, Masafumi

    2013-01-01

    We have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic, d-cycloserine. The gene cluster is composed of 10 open reading frames, designated dcsA to dcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization of O-ureidoserine. DcsD is similar to O-acetylserine sulfhydrylase, which generates l-cysteine using O-acetyl-l-serine with sulfide, and therefore, DcsD may be a synthase to generate O-ureido-l-serine using O-acetyl-l-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase converting O-ureido-d-serine into d-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed in Escherichia coli and purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substrates O-acetyl-l-serine and hydroxyurea, synthesis of d-cycloserine was successfully attained. These in vitro studies yield the conclusion that DcsD and DcsG are necessary for the syntheses of O-ureido-l-serine and d-cycloserine, respectively. DcsD was also able to catalyze the synthesis of l-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclic d-amino acid analogs, such as d-homocysteine thiolactone. PMID:23529730

  7. 34 CFR 303.15 - Include; including.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 2 2010-07-01 2010-07-01 false Include; including. 303.15 Section 303.15 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION EARLY INTERVENTION PROGRAM FOR INFANTS AND TODDLERS...

  8. Effects of Chronic D-Serine Elevation on Animal Models of Depression and Anxiety-Related Behavior

    PubMed Central

    Otte, David-Marian; Barcena de Arellano, Maria Luisa; Bilkei-Gorzo, Andras; Albayram, Önder; Imbeault, Sophie; Jeung, Haang; Alferink, Judith; Zimmer, Andreas

    2013-01-01

    NMDA receptors are activated after binding of the agonist glutamate to the NR2 subunit along with a co-agonist, either L-glycine or D-serine, to the NR1 subunit. There is substantial evidence to suggest that D-serine is the most relevant co-agonist in forebrain regions and that alterations in D-serine levels contribute to psychiatric disorders. D-serine is produced through isomerization of L-serine by serine racemase (Srr), either in neurons or in astrocytes. It is released by astrocytes by an activity-dependent mechanism involving secretory vesicles. In the present study we generated transgenic mice (SrrTg) expressing serine racemase under a human GFAP promoter. These mice were biochemically and behaviorally analyzed using paradigms of anxiety, depression and cognition. Furthermore, we investigated the behavioral effects of long-term administration of D-serine added to the drinking water. Elevated brain D-serine levels in SrrTg mice resulted in specific behavioral phenotypes in the forced swim, novelty suppression of feeding and olfactory bulbectomy paradigms that are indicative of a reduced proneness towards depression-related behavior. Chronic dietary D-serine supplement mimics the depression-related behavioral phenotype observed in SrrTg mice. Our results suggest that D-serine supplementation may improve mood disorders. PMID:23805296

  9. Extracellular production of a Serratia marcescens serine protease in Escherichia coli.

    PubMed

    Ohnishi, Y; Horinouchi, S

    1996-10-01

    The Serratia marcescens serine protease (SSP) is one of the extracellular enzymes secreted from this Gram-negative bacterium. When the ssp gene, which encodes a SSP precursor (preproSSP) composed of a typical NH2-terminal signal peptide, a mature enzyme domain, and a large COOH-terminal pro-region, is expressed in Escherichia coli, the mature protease is excreted through the outer membrane into the medium. The COOH-terminal pro-region, which is integrated into the outer membrane, provides the essential function for the export of the mature protein across the outer membrane. This is a very simple pathway, in contrast to the general secretory pathway exemplified by the secretion of a pullulanase from Klebsiella oxytoca, in which many separately encoded accessory proteins are required for the transport through the outer membrane. Moreover, the NH2-terminal region of 71 amino acid residues of the COOH-terminal pro-sequence plays an essential role, as an "intramolecular chaperone," in the folding of the mature enzyme in the medium. In addition to ssp, the S. marcescens strain contains two ssp homologues encoding proteins similar to SSP in amino acid sequence and size, but with no protease activity. Characterization of the homologue proteins and chimeric proteins between the homologues and SSP, all of which are produced in E. coli, has shown that they are membrane proteins that are localized in the outer membrane in the same manner as for SSP. By use of the COOH-terminal domain of SSP, pseudoazurin was exported to the cell surface of E. coli, which proves the usefulness of the SSP secretory system in the export of foreign proteins across the outer membrane. PMID:8987650

  10. A novel locust (Schistocerca gregaria) serine protease inhibitor with a high affinity for neutrophil elastase

    PubMed Central

    Brillard-Bourdet, Michèle; Hamdaoui, Ahmed; Hajjar, Eric; Boudier, Christian; Reuter, Nathalie; Ehret-Sabatier, Laurence; Bieth, Joseph G.; Gauthier, Francis

    2006-01-01

    We have purified to homogeneity two forms of a new serine protease inhibitor specific for elastase/chymotrypsin from the ovary gland of the desert locust Schistocerca gregaria. This protein, greglin, has 83 amino acid residues and bears putative phosphorylation sites. Amino acid sequence alignments revealed no homology with pacifastin insect inhibitors and only a distant relationship with Kazal-type inhibitors. This was confirmed by computer-based structural studies. The most closely related homologue is a putative gene product from Ciona intestinalis with which it shares 38% sequence homology. Greglin is a fast-acting and tight binding inhibitor of human neutrophil elastase (kass=1.2×107 M−1·s−1, Ki=3.6 nM) and subtilisin. It also binds neutrophil cathepsin G, pancreatic elastase and chymotrypsin with a lower affinity (26 nM≤Ki≤153 nM), but does not inhibit neutrophil protease 3 or pancreatic trypsin. The capacity of greglin to inhibit neutrophil elastase was not significantly affected by exposure to acetonitrile, high temperature (90 °C), low or high pH (2.5–11.0), N-chlorosuccinimide-mediated oxidation or the proteolytic enzymes trypsin, papain and pseudolysin from Pseudomonas aeruginosa. Greglin efficiently inhibits the neutrophil elastase activity of sputum supernatants from cystic fibrosis patients. Its biological function in the locust ovary gland is currently unknown, but its physicochemical properties suggest that it can be used as a template to design a new generation of highly resistant elastase inhibitors for treating inflammatory diseases. PMID:16839309

  11. Mutational analysis of the control cable that mediates transmembrane signaling in the Escherichia coli serine chemoreceptor.

    PubMed

    Kitanovic, Smiljka; Ames, Peter; Parkinson, John S

    2011-10-01

    During transmembrane signaling by Escherichia coli Tsr, changes in ligand occupancy in the periplasmic serine-binding domain promote asymmetric motions in a four-helix transmembrane bundle. Piston displacements of the signaling TM2 helix in turn modulate the HAMP bundle on the cytoplasmic side of the membrane to control receptor output signals to the flagellar motors. A five-residue control cable joins TM2 to the HAMP AS1 helix and mediates conformational interactions between them. To explore control cable structural features important for signal transmission, we constructed and characterized all possible single amino acid replacements at the Tsr control cable residues. Only a few lesions abolished Tsr function, indicating that the chemical nature and size of the control cable side chains are not individually critical for signal control. Charged replacements at I214 mimicked the signaling consequences of attractant or repellent stimuli, most likely through aberrant structural interactions of the mutant side chains with the membrane interfacial environment. Prolines at residues 214 to 217 also caused signaling defects, suggesting that the control cable has helical character. However, proline did not disrupt function at G213, the first control cable residue, which might serve as a structural transition between the TM2 and AS1 helix registers. Hydrophobic amino acids at S217, the last control cable residue, produced attractant-mimic effects, most likely by contributing to packing interactions within the HAMP bundle. These results suggest a helix extension mechanism of Tsr transmembrane signaling in which TM2 piston motions influence HAMP stability by modulating the helicity of the control cable segment. PMID:21803986

  12. Structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation of the P2 position of Xaa-boroPro dipeptides.

    PubMed

    Coutts, S J; Kelly, T A; Snow, R J; Kennedy, C A; Barton, R W; Adams, J; Krolikowski, D A; Freeman, D M; Campbell, S J; Ksiazek, J F; Bachovchin, W W

    1996-05-10

    A series of prolineboronic acid (boroPro) containing dipeptides were synthesized and assayed for their ability to inhibit the serine protease dipeptidyl peptidase IV (DPPIV). Inhibitory activity, which requires the (R)-stereoisomer of boroPro in the P1 position, appears to tolerate a variety of L-amino acids in the P2 position. Substitution at the P2 position which is not tolerated include the D-amino acids, alpha,alpha-disubstituted amino acids, and glycine. Specificity against DPPII and proline specific endopeptidase is reported. A correlation between the ability to inhibit DPPIV in cell culture and in the human mixed lymphocyte reaction is demonstrated. A synthesis of prolineboronic acid is reported as well as conditions for generating the fully unprotected boronic acid dipeptides in either their cyclic or acyclic forms. PMID:8642568

  13. Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme.

    PubMed

    Bachovchin, W W

    1985-12-01

    Proton NMR spectra of serine proteases in 1H2O solutions typically show a single resonance at very low magnetic field--i.e., 14-18 ppm from dimethylsilylapentanesulfonate. This resonance has been assigned to the proton hydrogen bonded between aspartic acid-102 and histidine-57 (chymotrypsin numbering system) of the "charge-relay system" or catalytic triad of serine proteases [Robillard, G. & Shulman, R. G. (1972) J. Mol. Biol. 71, 507-511]. Since then, there have been a number of reports that have cast doubt on its correctness. In the present work we have tested this assignment using alpha-lytic protease (EC 3.4.21.12, Myxobacter alpha-lytic proteinase), a bacterial serine protease homologous to elastase, which is specifically labeled with nitrogen-15 at N delta 1 of its single histidine residue. The low-field region of the proton spectra of this labeled enzyme shows a single resonance having the properties reported [Robillard, G. & Shulman, R. G. (1974) J. Mol. Biol. 86, 519-540], which, in addition, exhibits spin-spin splitting to the nitrogen-15 label. The observation of this 15N delta 1-H coupling makes the assignment of this resonance to the charge-relay proton unequivocal. PMID:3934665

  14. Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme

    SciTech Connect

    Bachovchin, W.W.

    1985-12-01

    Proton NMR spectra of serine proteases in /sup 1/H/sub 2/O solutions typically show a single resonance at very low magnetic field i.e., 14-18 ppm from dimethylsilylapentanesulfonate. This resonance has been assigned to the proton hydrogen bonded between aspartic acid-102 and histidine-57 (chymotrypsin numbering system) of the charge-relay system or catalytic triad of serine proteases. There have been a number of reports that have cast doubt on its correctness. In the present work the authors have tested this assignment using ..cap alpha..-lytic protease, a bacterial serine protease homologous to elastase, which is specifically labeled with nitrogen-15 at N/sup delta/sub 1// of its single histidine residue. The low-field region of the proton spectra of this labeled enzyme shows a single resonance having the properties reported which, in addition, exhibits spin-spin splitting to the nitrogen-15 label. The observation of this /sup 15/N-/sup delta/sub 1//-H coupling makes the assignment of this resonance to the charge-relay proton unequivocal.

  15. A unique loop extension in the serine protease domain of haptoglobin is essential for CD163 recognition of the haptoglobin-hemoglobin complex.

    PubMed

    Nielsen, Marianne Jensby; Petersen, Steen Vang; Jacobsen, Christian; Thirup, Søren; Enghild, Jan Johannes; Graversen, Jonas Heilskov; Moestrup, Søren Kragh

    2007-01-12

    Haptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (alpha-chain) and a serine protease domain (beta-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the beta-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin beta-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin. PMID:17102136

  16. Chasing One-Carbon Units to Understand the Role of Serine in Epigenetics.

    PubMed

    Parker, Seth J; Metallo, Christian M

    2016-01-21

    In this issue of Molecular Cell, Maddocks et al. (2016) use stable isotope tracing, mass spectrometry, and nutrient modulation in cancer cells to highlight the role of serine in supporting methylation through maintenance of nucleotide levels. PMID:26799763

  17. A Novel Effect of MARCKS Phosphorylation by Activated PKC: The Dephosphorylation of Its Serine 25 in Chick Neuroblasts

    PubMed Central

    Toledo, Andrea; Zolessi, Flavio R.; Arruti, Cristina

    2013-01-01

    MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25) in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B) inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A) form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS. PMID:23634231

  18. A novel effect of MARCKS phosphorylation by activated PKC: the dephosphorylation of its serine 25 in chick neuroblasts.

    PubMed

    Toledo, Andrea; Zolessi, Flavio R; Arruti, Cristina

    2013-01-01

    MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25) in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B) inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A) form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS. PMID:23634231

  19. Expression of serine proteinase P186 of Arthrobotrys oligospora and analysis of its nematode-degrading activity.

    PubMed

    Zhao, Hailong; Qiao, Jun; Meng, Qingling; Gong, Shasha; Chen, Cheng; Liu, Tianli; Tian, Lulu; Cai, Xuepeng; Luo, Jianxun; Chen, Chuangfu

    2015-12-01

    The nematode-trapping fungi possess a unique capability of predating and invading nematodes. As a representative nematode-trapping fungus, Arthrobotrys oligospora has been widely used to study the interactions between nematode-trapping fungi and their hosts. Serine proteinase is one of the important virulence factors during process of invasion of the nematode-trapping fungi into nematodes. In this study, using reverse transcription polymerase chain reaction, we amplified the gene sequence of serine proteinase 186 from A. oligospora, cloned it into pPIC9K vector and expressed it in the yeast Pichia pastoris. The expressed recombinant serine proteinase186 (reP186) was purified via Ni-affinity chromatography. The in vitro nematode-degrading activity of reP186 was analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis revealed that reP186 with molecular weight of 33 kDa was successfully obtained. ReP186 was capable of degrading a series of protein substrates including casein, gelatin, bovine serum albumin, denatured collagen and nematode cortical layer. The reP186 exhibited the maximal activity at pH 8.0 and 55 °C and was highly sensitive to the inhibitor, phenylmethanesulfonylfluoride. Treatment of Caenorhabditis elegans and Haemonchus contortus with reP186 for 12, 24 and 36 h, respectively, resulted in 62, 88 and 100 % of killing rates for C. elegans, and 52, 65 and 84 % of killing rates for H. contortus, respectively, indicating a relatively strong nematode-degrading bioactivity of reP186. PMID:26419902

  20. Corynebacterium glutamicum as a host for synthesis and export of D-Amino Acids.

    PubMed

    Stäbler, Norma; Oikawa, Tadao; Bott, Michael; Eggeling, Lothar

    2011-04-01

    A number of d-amino acids occur in nature, and there is growing interest in their function and metabolism, as well as in their production and use. Here we use the well-established l-amino-acid-producing bacterium Corynebacterium glutamicum to study whether d-amino acid synthesis is possible and whether mechanisms for the export of these amino acids exist. In contrast to Escherichia coli, C. glutamicum tolerates d-amino acids added extracellularly. Expression of argR (encoding the broad-substrate-specific racemase of Pseudomonas taetrolens) with its signal sequence deleted results in cytosolic localization of ArgR in C. glutamicum. The isolated enzyme has the highest activity with lysine (100%) but also exhibits activity with serine (2%). Upon overexpression of argR in an l-arginine, l-ornithine, or l-lysine producer, equimolar mixtures of the d- and l-enantiomers accumulated extracellularly. Unexpectedly, argR overexpression in an l-serine producer resulted in extracellular accumulation of a surplus of d-serine (81 mM d-serine and 37 mM l-serine) at intracellular concentrations of 125 mM d-serine plus 125 mM l-serine. This points to a nonlimiting ArgR activity for intracellular serine racemization and to the existence of a specific export carrier for d-serine. Export of d-lysine relies fully on the presence of lysE, encoding the exporter for l-lysine, which is apparently promiscuous with respect to the chirality of lysine. These data show that d-amino acids can also be produced with C. glutamicum and that in special cases, due to specific carriers, even a preferential extracellular accumulation of this enantiomer is possible. PMID:21257776

  1. Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris.

    PubMed Central

    Lee, Guan-Chiun; Lee, Li-Chiun; Sava, Vasyl; Shaw, Jei-Fu

    2002-01-01

    The 17 non-universal serine codons (CTG) in the Candida rugosa LIP2 gene have been converted into universal serine codons (TCT) by overlap extension PCR-based multiple site-directed mutagenesis. An active recombinant LIP2 lipase was overexpressed in Pichia pastoris and secreted into the culture medium. The recombinant LIP2 showed distinguishing catalytic activities when compared with recombinant LIP4 and commercial C. rugosa lipase. The purified enzyme showed optimum activity at pH 7 and a broad temperature optimum in the range 30-50 degrees C. The enzyme retained 80% of residual activity after being heated at 70 degrees C for 10 min. Recombinant LIP2 demonstrated high esterase activity towards long-chain (C12-C16) p-nitrophenyl esters. Tributyrin was the preferred substrate among all triacylglycerols tested for lipolysis. Among cholesteryl esters, LIP2 showed highest lipolytic activity towards cholesteryl laurate. The esterification of myristic acid with alcohols of various chain lengths showed that the long-chain n-octadecanol (C18) was the preferred substrate. In contrast, the esterification of n-propanol with fatty acids of various chain lengths showed that the short-chain butyric acid was the best substrate. From comparative modelling analysis, it appears that several amino acid substitutions resulting in greater hydrophobicity in the substrate-binding site might play an important role in the substrate specificity of LIP2. PMID:12020350

  2. Serine-15 is the regulatory seryl-phosphorylation site in C sub 4 -leaf phosphoenolpyruvate carboxylase (PEPCase) from maize

    SciTech Connect

    Jiao, Jinan; Chollet, R. )

    1990-05-01

    The {sup 32}P-labeled regulatory site phosphopeptide was purified from a tryptic digest of in vitro phosphorylated/activated dark-form PEPCase by metal ion affinity and reversed-phase chromatography and subjected to automated Edman degradation analysis. The amino acid sequence of this phosphoseryl peptide is His-His-Ser(P)-Ile-Asp-Ala-Gln-Leu-Arg. This nonapeptide, which corresponds exactly to residues 13-21 in the deduced primary sequence of the maize leaf carboxylase, is far removed from a recently identified active-site cysteine (Cys-553) in the C-terminal region of the primary structure. Comparative analysis of the deduced N-terminal sequences of C{sub 3}, C{sub 4}, and CAM leaf PEPCases suggests that the motif of Lys/Arg-X-X-Ser is an important structural requirement of the C{sub 4}- and CAM-leaf protein-serine kinases.

  3. Enhanced serine production by bone metastatic breast cancer cells stimulates osteoclastogenesis.

    PubMed

    Pollari, Sirkku; Käkönen, Sanna-Maria; Edgren, Henrik; Wolf, Maija; Kohonen, Pekka; Sara, Henri; Guise, Theresa; Nees, Matthias; Kallioniemi, Olli

    2011-01-01

    Since bone metastatic breast cancer is an incurable disease, causing significant morbidity and mortality, an understanding of the underlying molecular mechanisms would be highly valuable. Here, we describe in vitro and in vivo evidences for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Genome-wide gene expression profiling of this isogenic cell line pair revealed that all the three genes involved in the L: -serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L: -serine in mammalian tissues. Consistently, we observed that the proliferation of MDA-MB-231(SA) cells in serine-free conditions was dependent on PSAT1 expression. In addition, we observed that L: -serine is essential for the formation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L: -serine on osteoclastogenesis and cancer cell proliferation indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions. PMID:20352489

  4. Energetics of the molecular interactions of L-cysteine, L-serine, and L-asparagine in aqueous propylene glycol solutions at 298.15 K

    NASA Astrophysics Data System (ADS)

    Mezhevoi, I. N.; Badelin, V. G.

    2015-03-01

    Integral enthalpies of dissolution Δsol H m of L-cysteine, L-serine, and L-asparagine in aqueous solutions of 1,3-propylene glycol at organic solvent concentrations of up to 0.26 mole fraction are measured via the thermochemistry of dissolution. Standard enthalpies of dissolution (Δsol H ○) and transfer (Δtr H ○) of amino acids from water to a mixed solvent are calculated. It is found that the calculated enthalpy coefficients of pair interactions of the amino acids with polyhydric alcohol molecules have positive values. The effect the arrangement of the hydroxyl group in the structure of polyhydric alcohols has on the enthalpy of interaction of amino acids in aqueous solutions is revealed. The effect of different types of interactions in solutions and the structural features of biomolecules and cosolvents on the enthalpy of dissolution of amino acids is analyzed.

  5. Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme.

    PubMed

    Boldrini-França, Johara; Santos Rodrigues, Renata; Santos-Silva, Ludier Kesser; de Souza, Dayane Lorena Naves; Gomes, Mário Sérgio Rocha; Cologna, Camila Takeno; de Pauw, Edwin; Quinton, Loïc; Henrique-Silva, Flávio; de Melo Rodrigues, Veridiana; Arantes, Eliane Candiani

    2015-12-01

    Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous expression of a serine protease from Crotalus durissus collilineatus, named collinein-1, in Pichia pastoris, as well as the enzymatic comparative characterization of the toxin in native and recombinant forms. The complementary DNA (cDNA) encoding collinein-1 was amplified from cDNA library of C. d. collilineatus venom gland and cloned into the pPICZαA vector. The recombinant plasmid was used to transform cells of KM71H P. pastoris. Heterologous expression was induced by methanol and yielded 56 mg of recombinant collinein-1 (rCollinein-1) per liter of culture. The native collinein-1 was purified from C. d. collilineatus venom, and its identity was confirmed by amino acid sequencing. The native and recombinant enzymes showed similar effects upon bovine fibrinogen by releasing preferentially fibrinopeptide A. Although both enzymes have induced plasma coagulation, native Colinein-1 has shown higher coagulant activity. The serine proteases were able to hydrolyze the chromogenic substrates S-2222, S-2238, and S2302. Both enzymes showed high stability on different pH and temperature, and their esterase activities were inhibited in the presence of Zn2+ and Cu2+. The serine proteases showed similar k cat/K m values in enzyme kinetics assays, suggesting no significant differences in efficiency of these proteins to hydrolyze the substrate. These results demonstrated that rCollinein-1 was expressed with functional integrity on the evaluated parameters. The success in producing a functionally active recombinant SVSP may generate perspectives to their future therapeutic applications. PMID:26227411

  6. Regional distribution and postnatal changes of D-amino acids in rat brain.

    PubMed

    Hamase, K; Homma, H; Takigawa, Y; Fukushima, T; Santa, T; Imai, K

    1997-03-15

    Regional distribution of D-amino acids in rat brain was studied by the modified highly sensitive analytical method which was previously developed. The method includes fluorogenic derivatization of each amino acid, isolation of each amino acid by reverse-phase HPLC, followed by enantiomeric separation with Pirkle-type chiral stationary phases. D-Amino acid contents were determined in the cerebrum, cerebellum, hippocampus, medulla oblongata, pituitary gland and pineal gland. D-Aspartic acid was observed in the pineal gland (3524 +/- 263 nmol/g, data are for male rats of 6 weeks of age) and the pituitary gland (80.5 +/- 9.0 nmol/g). D-Serine was found in various regions of the brain except for the cerebellum and medulla oblongata. D-Alanine was observed exclusively in the pituitary gland (25.9 +/- 4.4 nmol/g), whereas D-leucine was found in the pineal gland (3.4 +/- 0.4 nmol/g) and the hippocampus (1.6 +/- 0.07 nmol/g). No other D-amino acids were detected in the brain. The contents of D-aspartic acid in the pituitary gland and D-serine in the pineal gland were higher in female rats. In contrast the contents of D-alanine in the pituitary gland and D-leucine in the pineal gland and the hippocampus were higher in males. Postnatal changes of D-aspartic acid and D-leucine in the pineal gland and D-alanine in the pituitary gland were also investigated. The results described in this paper suggested that distinct regulatory mechanisms exist for individual D-amino acids in the corresponding region of rat brain. PMID:9101716

  7. Serine-71 phosphorylation of Rac1 modulates downstream signaling.

    PubMed

    Schwarz, Janett; Proff, Julia; Hävemeier, Anika; Ladwein, Markus; Rottner, Klemens; Barlag, Britta; Pich, Andreas; Tatge, Helma; Just, Ingo; Gerhard, Ralf

    2012-01-01

    The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways. PMID:22970203

  8. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    SciTech Connect

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  9. Hexokinase 2 from Saccharomyces cerevisiae: regulation of oligomeric structure by in vivo phosphorylation at serine-14.

    PubMed

    Behlke, J; Heidrich, K; Naumann, M; Müller, E C; Otto, A; Reuter, R; Kriegel, T

    1998-08-25

    Homodimeric hexokinase 2 from Saccharomyces cerevisiae is known to have two sites of phosphorylation: for serine-14 the modification in vivo increases with glucose exhaustion [Kriegel et al. (1994) Biochemistry 33, 148-152], while for serine-157 it occurs in vitro with ATP in the presence of nonphosphorylateable five-carbon analogues of glucose [Heidrich et al. (1997) Biochemistry 36, 1960-1964]. We show now by site-directed mutagenesis and sedimentation analysis that serine-14 phosphorylation affects the oligomeric state of hexokinase, its substitution by glutamate causing complete dissociation; glutamate exchange for serine-157 does not. Phosphorylation of wild-type hexokinase at serine-14 likewise causes dissociation in vitro. In view of the higher glucose affinity of monomeric hexokinase and the high hexokinase concentration in yeast [Womack, F., and Colowick, S. P. (1978) Arch. Biochem. Biophys. 191, 742-747; Mayes, E. L., Hoggett, J. G., and Kellett, G. L. (1983) Eur. J. Biochem. 133, 127-134], we speculate that the in vivo phosphorylation at serine-14 as transiently occurring in glucose derepression might provide a mechanism to improve glucose utilization from low level and/or that nuclear localization of the monomer might be involved in the signal transduction whereby glucose causes catabolite repression. PMID:9718324

  10. CSF d-serine concentrations are similar in Alzheimer's disease, other dementias, and elderly controls.

    PubMed

    Biemans, Elisanne A L M; Verhoeven-Duif, Nanda M; Gerrits, Johan; Claassen, Jurgen A H R; Kuiperij, H Bea; Verbeek, Marcel M

    2016-06-01

    Cerebrospinal fluid (CSF) levels of d-serine were recently reported as a potential new biomarker for Alzheimer's disease (AD), showing a perfect distinction between AD patients and healthy controls. In this study, we aimed to confirm these results and extend these previous findings to dementia with Lewy bodies and frontotemporal dementia. d-Serine levels in CSF of 29 AD patients, 8 dementia with Lewy bodies patients, 14 frontotemporal dementia patients, and 28 nondemented controls were measured using ultra-high-performance liquid chromatography-tandem mass spectrometry. In contrast to previous findings, in our study CSF d-serine levels were only slightly increased in AD patients compared with controls. CSF d-serine in AD did not differ from other dementias and was also not correlated to mini-mental state examination-scores. Owing to the large overlap of d-serine levels, we conclude that CSF d-serine is neither a suitable biomarker for AD nor for cognitive decline. PMID:27143438

  11. Ketamine Metabolites Enantioselectively Decrease Intracellular D-Serine Concentrations in PC-12 Cells

    PubMed Central

    Singh, Nagendra S.; Rutkowska, Ewelina; Plazinska, Anita; Khadeer, Mohammed; Moaddel, Ruin; Jozwiak, Krzysztof; Bernier, Michel; Wainer, Irving W.

    2016-01-01

    D-Serine is an endogenous NMDA receptor co-agonist that activates synaptic NMDA receptors modulating neuronal networks in the cerebral cortex and plays a key role in long-term potentiation of synaptic transmission. D-serine is associated with NMDA receptor neurotoxicity and neurodegeneration and elevated D-serine concentrations have been associated with Alzheimer’s and Parkinsons’ diseases and amyotrophic lateral sclerosis. Previous studies have demonstrated that the ketamine metabolites (rac)-dehydronorketamine and (2S,6S)-hydroxynorketamine decrease intracellular D-serine concentrations in a concentration dependent manner in PC-12 cells. In the current study, PC-12 cells were incubated with a series of ketamine metabolites and the IC50 values associated with attenuated intracellular D-serine concentrations were determined. The results demonstrate that structural and stereochemical features of the studied compounds contribute to the magnitude of the inhibitory effect with (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine displaying the most potent inhibition with IC50 values of 0.18 ± 0.04 nM and 0.68 ± 0.09 nM. The data was utilized to construct a preliminary 3D-QSAR/pharmacophore model for use in the design of new and more efficient modulators of D-serine. PMID:27096720

  12. The RNA-Binding Chaperone Hfq Is an Important Global Regulator of Gene Expression in Pasteurella multocida and Plays a Crucial Role in Production of a Number of Virulence Factors, Including Hyaluronic Acid Capsule.

    PubMed

    Mégroz, Marianne; Kleifeld, Oded; Wright, Amy; Powell, David; Harrison, Paul; Adler, Ben; Harper, Marina; Boyce, John D

    2016-05-01

    The Gram-negative bacterium Pasteurella multocida is the causative agent of a number of economically important animal diseases, including avian fowl cholera. Numerous P. multocida virulence factors have been identified, including capsule, lipopolysaccharide (LPS), and filamentous hemagglutinin, but little is known about how the expression of these virulence factors is regulated. Hfq is an RNA-binding protein that facilitates riboregulation via interaction with small noncoding RNA (sRNA) molecules and their mRNA targets. Here, we show that a P. multocida hfq mutant produces significantly less hyaluronic acid capsule during all growth phases and displays reduced in vivo fitness. Transcriptional and proteomic analyses of the hfq mutant during mid-exponential-phase growth revealed altered transcript levels for 128 genes and altered protein levels for 78 proteins. Further proteomic analyses of the hfq mutant during the early exponential growth phase identified 106 proteins that were produced at altered levels. Both the transcript and protein levels for genes/proteins involved in capsule biosynthesis were reduced in the hfq mutant, as were the levels of the filamentous hemagglutinin protein PfhB2 and its secretion partner LspB2. In contrast, there were increased expression levels of three LPS biosynthesis genes, encoding proteins involved in phosphocholine and phosphoethanolamine addition to LPS, suggesting that these genes are negatively regulated by Hfq-dependent mechanisms. Taken together, these data provide the first evidence that Hfq plays a crucial role in regulating the global expression of P. multocida genes, including the regulation of key P. multocida virulence factors, capsule, LPS, and filamentous hemagglutinin. PMID:26883595

  13. Midgut serine proteases and alternative host plant utilization in Pieris brassicae L.

    PubMed Central

    Kumar, Rakesh; Bhardwaj, Usha; Kumar, Pawan; Mazumdar-Leighton, Sudeshna

    2015-01-01

    Pieris brassicae L. is a serious pest of cultivated crucifers in several parts of the world. Larvae of P. brassicae also feed prolifically on garden nasturtium (Tropaeolum majus L., of the family Tropaeolaceae). Proteolytic digestion was studied in larvae feeding on multiple hosts. Fourth instars were collected from cauliflower fields before transfer onto detached, aerial tissues of selected host plants in the lab. Variable levels of midgut proteases were detected in larvae fed on different hosts using protein substrates (casein and recombinant RBCL cloned from cauliflower) and diagnostic, synthetic substrates. Qualitative changes in midgut trypsin activities and quantitative changes in midgut chymotrypsin activities were implicated in physiological adaptation of larvae transferred to T. majus. Midgut proteolytic activities were inhibited to different extents by serine protease inhibitors, including putative trypsin inhibitors isolated from herbivore-attacked and herbivore-free leaves of cauliflower (CfTI) and T. majus (TpTI). Transfer of larvae to T. majus significantly influenced feeding parameters but not necessarily when transferred to different tissues of the same host. Results obtained are relevant for devising sustainable pest management strategies, including transgenic approaches using genes encoding plant protease inhibitors. PMID:25873901

  14. Group B Streptococcal Serine-Rich Repeat Proteins Promote Interaction With Fibrinogen and Vaginal Colonization

    PubMed Central

    Wang, Nai-Yu; Patras, Kathryn A.; Seo, Ho Seong; Cavaco, Courtney K.; Rösler, Berenice; Neely, Melody N.; Sullam, Paul M.; Doran, Kelly S.

    2014-01-01

    Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 “latching” domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr–fibrinogen interactions in the female reproductive tract. PMID:24620021

  15. Picornaviral 3C cysteine proteinases have a fold similar to the chymotrypsin-like serine proteinases

    SciTech Connect

    Allaire,M.; Chernaia, M.; Malcolm, B.; James, M.

    1994-01-01

    The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 {angstrom} resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an inter-molecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

  16. The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    PubMed Central

    Brunati, Martina; Perucca, Simone; Han, Ling; Cattaneo, Angela; Consolato, Francesco; Andolfo, Annapaola; Schaeffer, Céline; Olinger, Eric; Peng, Jianhao; Santambrogio, Sara; Perrier, Romain; Li, Shuo; Bokhove, Marcel; Bachi, Angela; Hummler, Edith; Devuyst, Olivier; Wu, Qingyu; Jovine, Luca; Rampoldi, Luca

    2015-01-01

    Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins. DOI: http://dx.doi.org/10.7554/eLife.08887.001 PMID:26673890

  17. Effect of amino acids on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in creatinine/phenylalanine and creatinine/phenylalanine/4-oxo-2-nonenal reaction mixtures.

    PubMed

    Zamora, Rosario; Alcón, Esmeralda; Hidalgo, Francisco J

    2013-12-15

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) formation in mixtures of creatinine, phenylalanine, amino acids and 4-oxo-2-nonenal was studied, to analyse the role of amino acids on the generation of this heterocyclic aromatic amine. When oxidised lipid was absent, cysteine, serine, aspartic acid, threonine, asparagine, tryptophan, tyrosine, proline, and methionine increased significantly (p < 0.05) the amount of PhIP formed in comparison to the control. When lipid was present, only the addition of methionine, glycine, and serine increased significantly (p < 0.05) the amount of PhIP produced, while histidine, cysteine, lysine, tryptophan, tyrosine, and alanine reduced significantly (p < 0.05) PhIP. These results may be a consequence of the different competitive reactions that occur. Thus, in the absence of lipids, thermal decomposition of the amino acids produced reactive carbonyls that converted phenylalanine into phenylacetaldehyde as a key step in the formation of PhIP. When oxidised lipid was present, amino acids competed with phenylalanine for the lipid, and amino acid degradation products were formed, among which alpha-keto acids seemed to play a role in these reactions. These results suggest that PhIP can be produced by several alternative reaction pathways from all major food components, including amino acids and lipids, in addition to carbohydrates. PMID:23993611

  18. Thermodynamic characteristics of the formation of Cd2+ ion-L-serine complexes in aqueous KNO3 solutions at 288-308 K

    NASA Astrophysics Data System (ADS)

    Kochergina, L. A.; Khokhlova, E. A.; Drobilova, O. M.

    2014-06-01

    The heats of formation of complexes of L-serine and doubly charged cadmium ions are determined by calorimetry. The heat effects of the reaction between an amino acid solution and a cadmium(II) solution and the respective heats of dilution of cadmium nitrate solution are measured at temperatures of 288.15, 298.15, and 308.15 K and ionic strengths of 0.25, 0.50, and 0.75 against the background of KNO3. The standard thermodynamic characteristics of complexation are calculated. Standard enthalpies of formation of mono-, bis-, and tris-coordinated complexes of cadmium(II) in an aqueous solution are found.

  19. STAT3 supports experimental K-RasG12D–induced murine myeloproliferative neoplasms dependent on serine phosphorylation

    PubMed Central

    Gough, Daniel J.; Marié, Isabelle J.; Lobry, Camille; Aifantis, Iannis

    2014-01-01

    Juvenile myelomonocytic leukemia, acute myeloid leukemia (AML), and other myeloproliferative neoplasms (MPNs) are genetically heterogeneous but frequently display activating mutations in Ras GTPases and activation of signal transducer and activator of transcription 3 (STAT3). Altered STAT3 activity is observed in up to 50% of AML correlating with poor prognosis. Activated STAT proteins, classically associated with tyrosine phosphorylation, support tumor development as transcription factors, but alternative STAT functions independent of tyrosine phosphorylation have been documented, including roles for serine-phosphorylated STAT3 in mitochondria supporting transformation by oncogenic Ras. We examined requirements for STAT3 in experimental murine K-Ras–dependent hematopoietic neoplasia. We show that STAT3 is phosphorylated on S727 but not Y705 in diseased animals. Moreover, a mouse with a point mutation abrogating STAT3 S727 phosphorylation displayed delayed onset and decreased disease severity with significantly extended survival. Activated K-Ras required STAT3 for cytokine-independent growth of myeloid progenitors in vitro, and mitochondrially restricted STAT3 and STAT3-Y705F, both transcriptionally inert mutants, supported factor-independent growth. STAT3 was dispensable for growth of normal or K-Ras–mutant myeloid progenitors in response to cytokines. However, abrogation of STAT3-S727 phosphorylation impaired factor-independent malignant growth. These data document that serine-phosphorylated mitochondrial STAT3 supports neoplastic hematopoietic cell growth induced by K-Ras. PMID:25150294

  20. Abnormal serine phosphorylation of insulin receptor substrate 1 is associated with tau pathology in Alzheimer's disease and tauopathies

    PubMed Central

    Yarchoan, Mark; Toledo, Jon B.; Lee, Edward B.; Arvanitakis, Zoe; Kazi, Hala; Han, Li-Ying; Louneva, Natalia; Lee, Virginia M.-Y.; Kim, Sangwon F.; Trojanowski, John Q.; Arnold, Steven E.

    2015-01-01

    Neuronal insulin signaling abnormalities have been associated with Alzheimer's disease (AD). However, the specificity of this association and its underlying mechanisms have been unclear. This study investigated the expression of abnormal serine phosphorylation of insulin receptor substrate 1 (IRS1) in 157 human brain autopsy cases that included AD, tauopathies, α-synucleinopathies, TDP-43 proteinopathies, and normal aging. IRS1-pS616, IRS1-pS312 and downstream target Akt-pS473 measures were most elevated in AD but were also significantly increased in the tauopathies: Pick's disease, corticobasal degeneration and progressive supranuclear palsy. Double immunofluorescence labeling showed frequent co-expression of IRS1-pS616 with pathologic tau in neurons and dystrophic neurites. To further investigate an association between tau and abnormal serine phosphorylation of IRS1, we examined the presence of abnormal IRS1-pS616 expression in pathological tau-expressing transgenic mice and demonstrated that abnormal IRS1-pS616 frequently co-localizes in tangle-bearing neurons. Conversely, we observed increased levels of hyperphosphorylated tau in the high-fat diet-fed mouse, a model of insulin resistance. These results provide confirmation and specificity that abnormal phosphorylation of IRS1 is a pathological feature of AD and other tauopathies, and provide support for an association between insulin resistance and abnormal tau as well as amyloid-β. PMID:25107476

  1. Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens

    PubMed Central

    Wright, David P; Ulijasz, Andrew T

    2014-01-01

    Bacterial eukaryotic-like serine threonine kinases (eSTKs) and serine threonine phosphatases (eSTPs) have emerged as important signaling elements that are indispensable for pathogenesis. Differing considerably from their histidine kinase counterparts, few eSTK genes are encoded within the average bacterial genome, and their targets are pleiotropic in nature instead of exclusive. The growing list of important eSTK/P substrates includes proteins involved in translation, cell division, peptidoglycan synthesis, antibiotic tolerance, resistance to innate immunity and control of virulence factors. Recently it has come to light that eSTK/Ps also directly modulate transcriptional machinery in many microbial pathogens. This novel form of regulation is now emerging as an additional means by which bacteria can alter their transcriptomes in response to host-specific environmental stimuli. Here we focus on the ability of eSTKs and eSTPs in Gram-positive bacterial pathogens to directly modulate transcription, the known mechanistic outcomes of these modifications, and their roles as an added layer of complexity in controlling targeted RNA synthesis to enhance virulence potential. PMID:25603430

  2. Acid Rain.

    ERIC Educational Resources Information Center

    Openshaw, Peter

    1987-01-01

    Provides some background information on acid deposition. Includes a historical perspective, describes some effects of acid precipitation, and discusses acid rain in the United Kingdom. Contains several experiments that deal with the effects of acid rain on water quality and soil. (TW)

  3. Identification and structural analysis of four serine proteases in a monotreme, the platypus, Ornithorhynchus anatinus.

    PubMed

    Poorafshar, M; Aveskogh, M; Munday, B; Hellman, L

    2000-11-01

    To study the emergence of the major subfamilies of serine proteases during vertebrate evolution, we present here the primary structure of four serine proteases expressed in the spleen of a monotreme, the platypus, Ornithorhynchus anatinus. Partial cDNA clones for four serine proteases were isolated by a PCR-based strategy. This strategy is based on the high level of sequence identity between various members of the large gene family of trypsin-related serine proteases, over two highly conserved regions, those of the histidine and the serine of the catalytic triad. The partial cDNA clones were used to isolate full-length or almost full-length cDNA clones for three of these proteases from a platypus spleen cDNA library. By phylogenetic analysis, these three clones were identified as being the platypus homologues of human coagulation factor X, neutrophil elastase, and a protease distantly related to the T-cell granzymes. The remaining partial clone was found to represent a close homologue of human complement factor D (adipsin). The isolation of these four clones shows that several of the major subfamilies of serine proteases had evolved as separate subfamilies long before the radiation of the major mammalian lineages of today, the monotremes, the marsupials, and the placental mammals. Upon comparison of the corresponding proteases of monotremes and eutherian mammals, the coagulation and complement proteases were shown to display a higher degree of conservation compared to the hematopoietic proteases N-elastase and the T-cell granzymes. This latter finding indicates a higher evolutionary pressure to maintain specific functions in the complement and coagulation enzymes compared to many of the hematopoietic serine proteases. PMID:11132153

  4. An Essential Signal Peptide Peptidase Identified in an RNAi Screen of Serine Peptidases of Trypanosoma brucei

    PubMed Central

    Moss, Catherine X.; Brown, Elaine; Hamilton, Alana; Van der Veken, Pieter; Augustyns, Koen; Mottram, Jeremy C.

    2015-01-01

    The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1). This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival. PMID:25816352

  5. Bioinformatic analyses of male and female Amblyomma americanum tick expressed serine protease inhibitors (serpins)

    PubMed Central

    Porter, Lindsay; Radulovic, Zeljko; Kim, Tae; Braz, Gloria R. C.; Da Silva Vaz, Itabajara; Mulenga, Albert

    2014-01-01

    Serine protease inhibitors (serpins) are a diverse family of proteins that is conserved across taxa. The diversity of Amblyomma americanum serpins (AAS) is far more complex than previously thought as revealed by discovery of 57 and 33 AAS transcripts that are respectively expressed in male and female A. americanum ticks, with 30 found in both. While distinct reproductively, both male and female metastriate ticks, such as A. americanum, require a blood meal. Thus, 30 AAS sequences found in both male and female ticks could play important role(s) in regulating tick feeding and thus represent attractive candidates for anti-tick vaccine development. Of significant interest, 19 AAS sequences expressed in male and female ticks are also part of the 48 AAS sequences expressed in fed female tick salivary glands or midguts; two organs through which the tick interacts with host blood and immune response factors. Considered the most important domain for serpin function, the reactive center loop (RCL) is further characterized by a single ‘P1’ site amino acid residue, which is central to determining the protease regulated by the serpin. In this study, a diversity of 17 different P1 site amino acid residues were predicted, suggesting that A. americanum serpins potentially regulate a large number of proteolytic pathways. Our data also indicate that some serpins in this study could regulate target protease common to all tick species, in that more than 40% of AAS show 58–97% inter-species amino acid conservation. Of significance, 24% of AAS showed 62–100% inter-species conservation within the functional RCL domain, with 10 RCLs showing ≥90–100% conservation. In vertebrates, serpins with basic residues at the P1 site regulate key host defense pathways, which the tick must evade to feed successfully. Interestingly, we found that AAS sequences with basic or polar uncharged residues at the putative P1 site are more likely to be conserved across tick species. Another notable

  6. A novel method for predicting post-translational modifications on serine and threonine sites by using site-modification network profiles.

    PubMed

    Wang, Minghui; Jiang, Yujie; Xu, Xiaoyi

    2015-11-01

    Post-translational modifications (PTMs) regulate many aspects of biological behaviours including protein-protein interactions and cellular processes. Identification of PTM sites is helpful for understanding the PTM regulatory mechanisms. The PTMs on serine and threonine sites include phosphorylation, O-linked glycosylation and acetylation. Although a lot of computational approaches have been developed for PTM site prediction, currently most of them generate the predictive models by employing only local sequence information and few of them consider the relationship between different PTMs. In this paper, by adopting the site-modification network (SMNet) profiles that efficiently incorporate in situ PTM information, we develop a novel method to predict PTM sites on serine and threonine. PTM data are collected from various PTM databases and the SMNet is built to reflect the relationship between multiple PTMs, from which SMNet profiles are extracted to train predictive models based on SVM. Performance analysis of the SVM models shows that the SMNet profiles play an important role in accurately predicting PTM sites on serine and threonine. Furthermore, the proposed method is compared with existing PTM prediction approaches. The results from 10-fold cross-validation demonstrate that the proposed method with SMNet profiles performs remarkably better than existing methods, suggesting the power of SMNet profiles in identifying PTM sites. PMID:26344496

  7. Polymerization on the rocks: negatively-charged alpha-amino acids

    NASA Technical Reports Server (NTRS)

    Hill, A. R. Jr; Bohler, C.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1998-01-01

    Oligomers of the negatively-charged amino acids, glutamic acid, aspartic acid, and O-phospho-L-serine are adsorbed by hydroxylapatite and illite with affinities that increase with oligomer length. In the case of oligo-glutamic acids adsorbed on hydroxylapatite, addition of an extra residue results in an approximately four-fold increase in the strength of adsorption. Oligomers much longer than the 7-mer are retained tenaciously by the mineral. Repeated incubation of short oligo-glutamic acids adsorbed on hydroxylapatite or illite with activated monomer leads to the accumulation of oligomers at least 45 units long. The corresponding reactions of aspartic acid and O-phospho-L-serine on hydroxylapatite are less effective in generating long oligomers, while illite fails to accumulate substantial amounts of long oligomers of aspartic acid or of O-phospho-L-serine.

  8. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening.

    PubMed

    Bachovchin, Daniel A; Ji, Tianyang; Li, Weiwei; Simon, Gabriel M; Blankman, Jacqueline L; Adibekian, Alexander; Hoover, Heather; Niessen, Sherry; Cravatt, Benjamin F

    2010-12-01

    Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo. PMID:21084632

  9. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening